EPA-600/1-78-017
February 1978
Environmental Health Effects Research Series
IRON
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, North Carolina 27711
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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clude biomedical instrumentation and health research techniques utilizing ani-
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This document is available to the public through the National Technical Informa-
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EPA-600/1-78-017
February 1978
ron
by
Subcommittee on Iron
Committee on the Medical and Biologic Effects of
Environmental Pollutants
National Research Council
National Academy of Sciences
Washington, D.C.
Contract No. 68-02-1226
Project Officer
Orin Stopinski
Criteria and Special Studies Office
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved
for publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S. Environmental
Protection Agency, nor does mention of trade names or commercial
products consitute endorsement or recommendation for use.
NOTICE
The project that is the subject of this report was approved by the
Governing Board of the National Research Council, whose members are
drawn from the Councils of the National Academy of Sciences, the National
Academy of Engineering, and the Institute of Medicine. The members of
the Committee responsible for the report were chosen for their special
competences and with regard for appropriate balance.
This report has been reviewed by a group other than the authors
according to procedures approved by a Report Review Committee consisting
of members of the National Academy of Sciences, the National Academy of
Engineering, and the Institute of Medicine.
ii
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FOREWORD
The many benefits of our modern, developing, industrial society are
accompanied by certain hazards. Careful assessment of the relative risk of
existing and new man-made environmental hazards is necessary for the estab-
lishment of sound regulatory policy. These regulations serve to enhance
the quality of our environment in order to promote the public health and
welfare and the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects. These
studies address problems in air pollution, non-ionizing radiation, environ-
mental carcinogenesis and the toxicology of pesticides as well as other
chemical pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards. Direct support to the regulatory function
of the Agency is provided in the form of expert testimony and preparation
of affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health.
To aid the Health Effects Research Laboratory to fulfill the functions
listed above, the National Academy of Sciences (NAS) under EPA Contract
No. 68-02-1226 prepares evaluative reports of current knowledge of selected
atmospheric pollutants. These documents serve as background material for
the preparation or revision of criteria documents, scientific and technical
assessment reports, partial bases for EPA decisions and recommendations
for research needs. "Iron" is one of these reports.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
iii
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SUBCOMMITTEE ON IRON
COMMITTEE ON MEDICAL AND BIOLOGIC EFFECTS OF ENVIRONMENTAL POLLUTANTS
CLEMENT A. FINCH, University of Washington School of Medicine, Seattle,
WA, Chairman
ELMER B. BROWN, Washington University School of Medicine, St. Louis, MO
JOHN C. BROWN, Agricultural Research Center, Beltsville, MD
HOWARD E. BUMSTED, U. S. Steel Research Laboratory, Monroeville, PA
MERLE BUNDY, U. S. Steel Corporation, Pittsburgh, PA
JOHN D. HEM, U. S. Geological Survey, Menlo Park, CA
J. B. NEILAHDS, University of California, Berkeley, CA
DARRELL R. VAN CAMPEN, U. S. Plant, Soil, and Nutrition Laboratory, Ithaca,
NY
RAYMOND C. WANTA, Bedford, MA
MUNSEY S. WHEBY, University of Virginia School of Medicine, Charlottesvllle,
VA
DONALD 0. WHUTEMORE, Kansas State University, Manhattan, KS
THOMAS H. BOTHWELL, Johannesburg Medical School, University of the
Witwatersrand, Johannesburg, South Africa, Resource Person
ROBERT W. CHARLTON, Johannesburg Medical School, University of the
Wltwatersrand, Johannesburg, South Africa, Resource Person
REUEL A. STALLONES, School of Public Health, University of Texas,
Houston, Chairman
MARTIN ALEXANDER, Cornell University, Ithaca
ANDREW A. BENSON, Scripps Institution of Oceanography, University of
California, La Jolla
RONALD F. COBURN, University of Pennsylvania School of Medicine, Philadelphia
CLEMENT A. FINCH, University of Washington School of Medicine, Seattle
EVILLE GORHAM, University of Minnesota, Minneapolis
ROBERT I. HENKIN, Georgetown University Medical Center, Washington, D.C.
IAN T. T. HIGGINS, School of Public Health, University of Michigan, Ann Arbor
JOE W. HIGHTOWER, Rice University, Houston
HENRY RAMIK, Duke University Medical Center, Durham, North Carolina
ORVILLB A. LEVANDER, Agricultural Research Center, Beltsville, Maryland
ROGER P. SMITH, Dartmouth Medical School, Hanover, New Hampshire
T. D. BOAZ, JR., Division of Medical Sciences, National Research Council,
Washington, D.C., Executive Director
T. D. BOAZ, JR., National Research Council, Washington, DC, Staff Officer
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CONTENTS
Introduction 1
1 Iron in the Environment 3
2 Microorganisms and Iron 64
3 Iron and Plants 91
4 Iron Metabolism in Humans and Other Mammals 122
5 Iron Deficiency 163
6 Acute Toxicity of Ingested Iron 190
7 Chronic Iron Toxicity 196
8 Inhalation of Iron 234
9 Summary 244
10 Recommendations 253
Appendix: Analysis of Iron in Environmental 257
and Biologic Samples
References 271
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INTRODUCTION
The Subcommittee on Iron has surveyed the effects of organic
and inorganic iron that are relevant to humans and their environment.
It must be recognized that the biology and chemistry of iron are
complex and only partially understood. Iron participates in oxida-
tion reduction processes that not only affect its geochemical mobility,
but also its entrance into biologic systems. Hydrated ferric oxide
surfaces have adsorbent properties and may act as reaction sites and
catalysts. In biologic systems, the iron atom is incorporated into
several protein enzymes that participate in many oxygen and electron
transport reactions.
A first consideration is the quantity and form of iron in the
earth's crust, in the hydrosphere, and in the atmosphere, and the
degree of movement that occurs among them (Chapter 1). Of particular
interest is the interaction between inorganic and organic forms of
the metal. Iron is brought into the organic cycle through sophisti-
cated mechanisms that microorganisms have developed for converting
highly insoluble and unavailable forms of iron into usable ones
(Chapter 2). Similarly, the roots of some plants have capabilities
for retrieving iron from the soil; matching this affinity to the con-
dition of local soils has led to improvement in agriculture (Chapter 3).
Vertebrates in general, despite their high iron requirements for hemo-
globin synthesis, appear to be able to achieve satisfactory iron
balance (Chapter 4). Humans are the outstanding exception—hundreds
of millions of the world's peoples are iron-deficient because of
inadequate amounts of available iron in the diet (Chapter 5). External
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factors are believed to be responsible for this borderline balance, and
deficiency may thus be considered the major iron-related environmental
health problem faced by humans. An account is also presented of a much
smaller population that shows iron overload (Chapter 7). The presence
of large deposits of ferritin and hemosiderin in parenchymal tissues has
been shown to result in damage to several vital organs. Acute iron
toxicity has been reported, but only with the ingestion of large amounts
of iron salts (Chapter 6). Pulmonary inhalation of iron compounds from
industrial exposure has not been shown to be a hazard (Chapters 1 and 8),
and 8).
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CHAPTER 1
IRON IN THE ENVIRONMENT
Iron is element number 26 in the periodic table, and has an atomic
weight of 55.85. The average isotopic composition is 5.8% of mass number
54, 91.7% of 56, 2.2% of 57, and 0.3% of 58. It is the fourth most abun-
dant element in the earth's crust; only oxygen, silicon, and aluminum are
more common. Metallic iron occurs in a few types of rock and is a primary
constituent of the earth's core. However, in most rocks and soils it is
combined in crystal structures either as divalent ferrous or trivalent
o
ferric ions. The effective ionic radius of the ferrous ion is about 0.80A.
The ferric ion has an effective radius of about 0.67A. Fundamental aspects
SRI
of iron chemistry have been described by Nicholls.
Iron occurs in solution in water as Fe(II) or Fe.(Ill), or as inorganic or
organic ferrous or ferric complexes. It also can be found in small quantities
as a stable colloid or hydrosol, most commonly constituted of small dispersed
particles of ferric oxyhydroxide. The terms "ferric oxyhydroxide" and
"hydrated ferric oxide" will be used interchangeably to mean a relatively
fresh precipitate with a poorly organized crystal structure. Its composi-
tion is approximated by the formula Fe(OH)g, and sometimes such materials
are referred to as ferric hydroxide, which does not imply specific stoichio-
metry or crystallinity. Upon aging, this material may achieve the structure
of goethite, a-FeOOH, hematite, ct-Fe203, or other polymorphs.
Because of its fundamental importance to human activities, large amounts
of iron are extracted from ores each year, and converted to the metallic
form. In this form, iron is chemically unstable under earth-surface
-------
conditions and is slowly oxidized and converted to ferrous and ferric
compounds. The human contribution to the geochemical cycle of iron is
94
important on a global scale. Bowen estimated that the annual amount
of iron mined exceeds the amount carried to the ocean by natural rock
weathering by a factor of 8.
The chemical behavior of ..iron in the environment is a function of
the following four characteristics of the element:
• Iron participates readily in chemical oxidation and reduction
processes. These processes influence the geochemical mobility
of the element.
• Iron forms organic and inorganic complexes that affect its
solubility in water and its subsequent chemical reactions.
• Surfaces of hydrated ferric oxide are active in absorbing
other materials and may act as reaction sites and catalysts.
Small particles of the hydrated oxide may form colloidal sus-
pensions in water or air and have large surface to weight and
surface to volume ratios.
• Iron is essential to biologic processes and is present in
all living matter.
The material in this chapter is set forth in terms of properties,
sources, and transport processes that influence the environmental
occurrence and behavior of iron. Both natural and anthropogenic sources
and processes are considered.
NATURAL SOURCES AND THEIR PROPERTIES
The Earth's Crust
Rocks. Iron is common and abundant in igneous, metamorphic, and
sedimentary rocks. In most igneous and metamorphic rocks, more iron
exists in the ferrous than in the ferric state, whereas the reverse is
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true for sedimentary rocks. Data for iron concentrations in the earth's
471
lithlc (lithosphere) and continental crust have been compiled by Lepp
and Parker;602 seiected values from the literature are listed in Table
1-1.
Iron in igneous rocks is found primarily in the ferromagnesian sili-
cates, the most common of which are olivine, (Mg, Fe>2SiO/, pyroxenes,
amphiboles, and iron-containing micas. Important iron-rich
accessory minerals are magnetite, Fe304, ilmenite, FeTi03, and pyrite,
FeS2« Basic (or mafic) igneous rocks contain more of these minerals and
thus more iron, whereas acidic (or felsic) rocks contain less (Table
1-1). The average concentration of iron in the lithic crust ±s close
to that of the average igneous rock, because 95% of the crust is composed
of igneous rocks or metamorphic rocks derived from them. In the conti-
nental section of the crust, the ratio of the amounts of basic to acidic
fin ?
igneous rocks is 1:20 The thinner oceanic crust consists mainly of
basic rocks. Most of the acidic igneous rock outcrops on the continen-
tal crust are granitic; most of the basic rocks are basaltic.
The weathering of igneous rocks oxidizes most ferrous iron to the
ferric state. The ferric iron is then incorporated into clay minerals
or hydrolyzed to ferric oxyhydroxides. During deposition and diagenesis
of sediments derived from the weathering products, iron is often reduced
and precipitated as sulfides. The average content of iron in sedimentary
rocks (including sediments) given in Table 1-1 is close to that of the
average continental igneous rock. The main difference between iron from
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TABLE 1-1
Average Iron Contents of the Earth's Crust and Common Rocks
Crust or Rock Type
Lithospherea
Continental crust
Igneous rocksc
Ultrabasic
Basic
Intermediate
Acidic
Sedimentary rocks
Average sedimentary
rock
Shale6
Sandstone-'
Limestone*?
Average metamorphic rock
Percentage by Weight
Iron (III)
Oxide
2.7
2.4
4.6
4.0
3.4
1.6
3.5
4.2
1.7
1.5
Iron (II)
Oxide
5.1
4.7
8.4
7.1
4.1
1.9
2.6
3.0
1.5
2.9
Total
Iron
5.8
5.4
9.8
8.3
5.6
2.6
4.5
5.3
2.4
0.38
3.3
aAverage of values in Poldervaart and Ronov and Yaroshevsky.
« SQQ £ c/
^Average of values in Pakiser and Robinson and Ronov and Yaroshevsky.
^Average of values in Table 93 of Geochemical Tables.
^From Garrels and Mackenzie.
eFrom Lepp.471
•^From Petti John.614
Clarke.170
"Average of values for crystalline shields in Table III of Geochemical Tables.
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igneous and from sedimentary rocks is the increase in the average ferric
to ferrous ratio, from 0.5 to 1.2. The average sedimentary rock is based
295
on a calculated ratio of 81:11:8 for shale:sandstone:limestone; these
components comprise about 97% of all sedimentary rocks. Approximate relative
abundances of these rocks on continents have been measured as 50% for shale,
26% for sandstone, and 20% for limestone.615 About 75% of the world's
outcrops are composed of sedimentary rocks. Thus they are much more
abundant on land surfaces than igneous rocks. The iron contents of
specific types of shales, sandstones, and limestones have been measured. '
The elemental compositions of metamorphic rocks are generally similar
to the igneous or sedimentary rocks from which they were formed. The
average iron content of metamorphic rocks in the earth's crystalline
shields is between that of intermediate and acidic igneous rocks.
Soils. Iron is primarily ferric in most soils, although the
ferrous state may be predominant in some soils that are flooded and rich
in organic matter. The principal iron-containing minerals in soils are
the ferric oxyhydroxides, which are set forth in Table 1-2. The most
common are amorphous oxyhydroxide, goethite, and hematite. They occur
in soils as small particles, concretions, lateritic crusts, or coatings
199 588
on clays and other minerals. ' The first minerals formed from the
oxidation and by hydrolysis of ferrous iron are geothite and lepidocrocite
(although less often), because of the relatively low nucleation energy
needed. Hematite usually evolves from long-term aging of amorphous material
442
or from dehydration of fine-grained goethite during warm, dry periods.
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TABLE 1-2
Naturally Occurring Ferric Oxyhydroxidesa
Ferric Oxyhydroxide Ideal Formula
Amorphous Indefinite: often represented
as Fe(OH)3
Goethite ot-FeOOH
Akagane'ite g-FeOOH
Lepidocrocite y-FeOOH
Hematite a-Fe20-j
Maghemite Y-Fe0
a 444
Derived from Langmuir and Whittemore.
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The presence of organic compounds in soils retards the
crystallization of amorphous oxyhydroxides. In organic-containing
soil horizons, the ferric oxyhydroxide that forms is generally goethite;
in environments poor in organic matter, the amorphous material alters
faster to hematite. An increase in the organic content in a hematitic
soil could cause the dissolution of hematite through reduction and/or
complexing of iron by organic compounds. Then the dissolved iron
could reprecipitate as poorly crystallized goethite.
Appreciable amounts of ferrous and ferric iron in soils are in-
corporated in clay minerals as an essential or a minor isomorphous
substitute for Mg(H) or Al(III) in the octahedral sites in their structures.
Relatively common soil clays containing iron as an essential constituent
are nontronite (a smectite), chlorites, glauconite and some illites or
149
hydromicas (clay micas), and certain vermiculites.
Iron can also exist in some soils as unweathered ferromagnesium
silicates and in the heavy accessory minerals magnetite and ilmenite.
Soils being formed from shales containing pyrite or marcasite, FeS ,
can become quite acidic, because of the oxidation of the sulfides. The
low pH allows some ferrous iron to remain in soil moisture before it
can oxidize to form ferric oxyhydroxides. .Small amounts of amorphous
or poorly crystalline strengite, FePO^.Zl^O exist in many acidic
soils with high phosphate activity. Under reducing conditions, vivianite,
390
Fe-CPO.K.SH-O, and siderite, FeCO , form in some soils. Iron salts
are generally limited to poorly-drained, acid soils where basic ferric
sulfates may be present as a yellow mottling or yellow surface crust.
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390
The iron content of most soils ranges from 0.7-4.2%. In a
series of U. S. Geological Survey studies of 3,026 soil samples in the
United States, the total iron content ranged from 0.01-13%, with geomet-
174
ric means for the different types of soils ranging from 0.47-4.3%.
Based on samples collected from a depth of 20 cm for 861 soils, Shacklette
et al. determined the geometric means and deviations of the iron con-
tent for the western United States to be 2.0 and 1.90%, and for the
685
East to be 1.5 and 2.76%. Vinogradov reported iron contents from
773
0.59-10.4% for 16 soils in different zones on the East European plain.
Iron may be relatively uniformly distributed throughout soil horizons
as in the Mollisols, the main types of soils of the prairie and Great
Plains grain-growing states. For example, the iron contents of the A,
B,, B-, and C horizons of a loam in South Dakota were 2.7, 2.8, 2.6,
390
and 2.7%, respectively. Where the parent material is low in iron,
or in environments where iron has been leached from the soil, iron
contents can be small. Iron contents averaged approximately 0.5% in
the A and B horizons of 30 uncultivated garden soils in east central
fiftfi
Georgia. In many moist forest soils, especially the Spodosols of the
northeastern United States and northern Michigan and Wisconsin, the
total iron concentration can reach several percent in the B horizon.
Most of it has accumulated as ferric oxyhydroxides and organic complexes,
as shown by an average of 3.6% free iron (dithionite extraction) in the
220
B horizons of 10 northern Appalachian Spodosols.
Under prolonged or intense weathering and leaching (generally in
hot, wet climates) soils known as Oxisols can form. Iron contents
10
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of Oxisols are high, ranging from 15-55%, and mainly in ferric
oxyhydroxides. The higher percentages of iron in soils often referred
to as laterites are generally produced during alternating wet and dry
conditions that form more stable, less soluble, crystalline oxyhydroxides,
and dehydrate goethite to hematite. There are no major areas, however,
where recently formed Oxisols exist in the continental United States.
In poorly drained soils high in organic matter, ferric iron can be
reduced to the ferrous state. Dissolved ferrous iron can be relatively
high in the soil moisture and may be controlled by the solubility of
ferrous carbonates, sulfides, or phosphates, depending on the oxidation
potential and the activities of the anions. Iron contents can vary widely,
390
e.g., from 1-20%, as measured in ash of 5 peats in the United States.
Sediments. Sediments are composed of more ferrous iron than soils
because most soils occur above the water table and thus are aerated,
whereas many sediments are deposited or undergo diagenesis in anaerobic,
subaqueous environments. In a normal depositional environment, in which
the pH is generally 8+1 unit, ferric oxyhydroxides are the most impor-
tant form of precipitated iron. During diagenesis, original amorphous
oxyhydroxides and poorly crystallized goethite generally are converted to
iff\
hematite. Several reviews have discussed the formation of sedimentary
iron minerals, »'» and concentrations of iron in average
sedimentary rocks are listed in Table 1-1.
Ferrous compounds usually form in sediments in a reducing environ-
ment generated by the oxidation of organic matter. Pyrite is the most
common one, although siderite can form in waters where relatively high
11
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iron, low calcium, and very low sulfide activities exist, as in some non-
marine aqueous environments. During diagenesis of sediments with high
organic content, available sulfate, and slow detrital addition, all iron
202
compounds tend to transform into pyrite. Thus, pyrite is common in
many clay sediments high in organic matter and equivalent sedimentary
rocks, and is often found in coals and associated strata.
Hydrous sheet silicates, glauconite, chamosite, and greehalite also
are major constituents of some sediments. They generally form in marine
sediments with pore waters of intermediate redox potential, low sulfide
and carbonate activity, and available silica and alumina. In general,
sediments poorer in iron occur near coasts; iron content of sediments will
increase with ocean depth. Berner and James have summarized the
expected sequences of iron minerals to be found from fresh water to pro-
gressively deeper marine waters. Argillaceous deep-sea sediments tend to
be uniform and moderately high in iron content; the average is 5.8% for
250
total iron on a carbonate and water-free basis. Carbonaceous deep-sea
250 198
sediments and oxide-rich sediments on the East Pacific Rise range
up to 11 and 18% iron, respectively. Ferromanganese oxide nodules on the
ocean floor have from 1-42% iron in them and average 19, 13, and 15% iron
for the Atlantic, Pacific, and Indian Oceans.55'198
Iron ores. Rock that is economical to mine for iron ranges from
20-69% total iron. Ores may be grouped into four classes, according to
whether or not the deposits were formed by sedimentary processes, igneous
activity, hydrothermal solutions, or surface or near-surface enrichment.
The most important ores are the sedimentary iron formations and ironstones,
12
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which are distributed throughout the world. The largest and most abundant
deposits are around the Atlantic and Indian Oceans.^07>^28'601
Sedimentary iron deposits are mainly marine chemical precipitates of
iron weathered and transported from land masses. Most of these ore
deposits are very old, generally of Precambrian or early Paleozoic age.
Some were altered later by metamorphism or enriched by weathering. On the
basis of the predominant iron minerals, the sedimentary ores can be divided
407
into four major facies groups—oxide, silicate, carbonate, and sulfide.
"Iron formation" is the term for a thinly bedded rock with layers of
iron oxides (mainly hematite and magnetite), siderite, and iron silicates
407
(primarily greenalite, a septachlorite) alternating with cherty layers.
471
The average total iron concentration of such rocks is 28%. Iron forma-
tions constitute the greatest single source of iron being mined and the
largest reserves. The biggest deposits of this type occur in the Lake
Superior region of the United States and Canada, the Labrador districts
of Canada, and areas in Brazil, Russia, and Australia. The total iron
concentration of the ores generally ranges from 25-45%, except where the
silica has been leached and replaced by the action of surface and ground
waters. These bodies enriched by secondary processes are known in the
Lake Superior region as "direct-shipping ores," containing soft goethite
and hematite and iron contents of 50-68%, or the "wash ores" or "semi-
/ O Q
taconite." Where the iron formation has been metamorphosed, coarse
grains of hematite and magnetite have formed, and the silicates have
altered primarily to chlorite, minnesotaite—the iron analog of talc,
13
-------
Mg3(Si4010)(OH)2—and stilpnomelane, a sheet silicate with a structure simi-
lar to talc.407 The resulting rock is taconite, the low-grade ore currently
mined in great quantities in the Lake Superior region. Small quantities
of the amphiboles making up the cummingtonite-grunerite series are
found in metamorphosed parts of the iron formations of northern
Minnesota (primarily in the Mesabi Range), and northern Michigan.^
Most of the asbestiform fibers in the tailings dumped by a mining
company into Lake Superior and found in the water system of Duluth,
583
Minnesota, are comprised of minerals in this series.
The term "ironstone" includes oolitic iron oxyhydroxides (goethite
and hematite) and silicates (predominantly chamosite, another septa-
chlorite). Calcite and dolomite are the common constituents of ironstone
and its iron content ranges from 20-40%. Thicknesses and lateral extents
of ironstone deposits are generally an order of magnitude less than those
of iron formations. Thus, the reserves have been estimated as at least
428
100 times less. In certain locales, the near-surface ironstones
have been enriched by leaching of calcite. The Clinton Formation,
extending from Alabama to New York, is one of the world's most extensive
ironstone deposits. However, most of the high grade, secondarily enriched
ore has already been mined in the United States. Important deposits are
still being mined in northern Europe and coastal Newfoundland.
Other sedimentary iron ores include bog-iron deposits (e.g., accumu-
lations of iron oxyhydroxides in swampy areas and shallow lakes in
northern Europe and North America), and blackband and clayband deposits
14
-------
(e.g., the thin layers of siderite found in the coal sequences of
Appalachia and Great Britain). Neither of these are of much present
428
or future economic significance.
Iron deposits resulting from igneous activity or formed during
replacement by hydrothermal solutions occur primarily as magnetite and
hematite. The magmatic segregations of magnetite in Kiurna, Sweden, and
the hydrothermally enriched iron-formation in the Quadrilatero Ferrifero
of Brazil yield high-grade ores (60-70% iron). At present, both deposits
are of modest economic importance to the world and contain moderate
reserves. The contact metasomatic ores in the Cornwall area of
Pennsylvania, which contain about 40% iron, are moderately important
sources of iron now, but are potentially a large resource for the United
States. Other igneous- or hydrothermally-related iron ores in the
United States occur in Nevada, Utah, New York, and Missouri, but are
currently of minor economic importance and of small to moderate
428
potential.
The fourth class of iron ores was produced by surface or near-
surface enrichment of pre-existing low-grade deposits or by the weather-
ing of iron-containing rocks into laterites. The secondarily enriched
ores include the important "direct-shipping" and "wash ores" of the
Lake Superior region discussed above. Iron-rich laterites generally
result from the weathering of rocks high in iron, such as serpentine,
under tropical conditions with alternating wet and dry seasons. Such
laterites occur in Cuba and the Philippines and commonly contain 40-50%
iron, primarily as poorly crystallized goethite. Many laterites contain
15
-------
enough nickel to be mined for that element alone. Although presently
only of minor economic importance, the unmined quantities in the world
are large. The continental United States, however, has no major
428
lateritic deposits.
Iron in the Hydrosphere and in Solutions
The main features of the aqueous chemistry of iron and the
stability of iron compounds that most commonly participate in
chemical equilibria can be shown conveniently by Eh-pH diagrams. Eh
represents the redox potential in volts, calculated for equilibrium
conditions by means of the Nernst equation. Increasing positive
values of Eh represent increasing intensity of oxidation. Figure
1-1 is one form of such a diagram, which shows areas of dominance for
dissolved ferric and ferrous species, and for two hydroxy com-
plexes of each. The shading of Figure 1-2 shows the areas of stability
for ferric and ferrous oxyhydroxidesi;Fe(OH)3 and Fe(OH)2, siderite,
FeCO , pyrite, FeS., pyrrhotite, FeS, and- metallic iron, Fe*, and the
field of stability of liquid water at 25°C and 1 atm. Figure 1-2 also
illustrates the total solubility of iron as a function of Eh and pH
within the field of stability of liquid water, in the system specified
for the diagram. Techniques for preparing and interpreting Eh-pH diagrams
294 345 347
have been well documented. * * The system specified for Figures
l.-l and 1-2 includes water, ferric and ferrous ions, sulfur, and carbon, in
equilibrium at 25°C and 1 atm, with a total sulfur activity of 10~ ' mol/1
and a total activity for carbon dioxide species of 10 ' mol/1. These
measurements are equivalent to a sulfate concentration of 9.6 mg/1 and
16
-------
Eh,
volts .4
Water oxidized
0 2 4 6 8 10 12 14
FIGURE 1-1 Areas of dominance for dissolved ferric and ferrous
ions and hydroxide complexes in dilute solutions at
25 C and 1 atm. Drawing courtesy of J. D. Hem.
17
-------
volts
0
14
FIGURE 1-2
Fields of stability for solids and solubility of
iron between 10~3>0° and 10~9-00 mol/1 in a
system of iron, sulfur, carbon dioxide, and water
at 25°C and 1 atm. Total dissolved sulfur species
activity is fixed at 10~^-°° mol/1 (9.6 mg/1 as
sulfate ion) and t9ta-l dissolved carbon dioxide species
activity at 10 mol/1 (61 mg/1 as bicarbonate
anion). Drawing courtesy of J. D. Hem.
18
-------
a bicarbonate concentration of 61 mg/1, about the worldwide average
concentration of these compounds in river water. The stability
fields for solids shown in Figure 1-2 were extended into conditions
of low pH in which iron solubility would exceed the highest values
shown on the diagram (10~ * mol/1 or 56 mg/1). For siderite to
exist below pH 6, for example, a very high dissolved iron concentration
would be required.
Chemical thermodynamic data used in preparing the graphs were
777 443
selected from Wagman et al. and Langmuir. Their values yield
—38 49
a solubility product of Fe(OH)_ of 10 * . The solubility product
O ^ / /
for this material can range from about 10 to about 10 , depending
813
on the degree of crystallinity. The values chosen represent aged
but not dehydrated material. The minimum concentration of iron (10~ *
mol/1) shown in Figure 1-2 is 0.56 yg/1. A relatively low pH or redox
-3.00
potential, attainable in oxygen-depleted water, may stabilize 10
mol/1 or more of iron in solution. Iron sulfides generally have very
low solubilities, but they are unstable in the presence of oxidizing
agents. Metallic iron is unstable in water. The species greigite ^6384)
and mackinawite (FeS) may occur in natural systems, but they are less
stable than the forms considered in Figure 1-2.
Silicate rocks commonly contain iron and some iron silicate
minerals are important sources of dissolved iron. The iron from these
rocks is released during weathering.and subsequent control over the
solubility and mobility of iron is a function of processes involving
the secondary mineral species shown in Figure 1-2. The silicate minerals
may not behave reverslbly under earth-surface conditions. The thermodynamic
data for iron silicates are incomplete, but some of their effects on iron
258a
solubility have been examined.
19
-------
Complexes of ferric iron with fluoride, chloride, and sulfate are
strong enough to affect the chemical behavior of iron in solutions that
are enriched in these anions. Organic complexes of iron also may
influence iron solubility. In many systems in which organic ligands
are abundant, conditions will tend to be reducing, favoring the forma-
tion of ferrous rather than ferric complexes. The effects of reduction
as well as of complexing must be considered in such systems. Yet some
organic ligands are stable in the ferric species regions diagrammed in
Figure l-l. The tartrate ion, for example, forms a strong ferric complex.
Ferrous iron-tannic acid complexes and similar substances may be found in
734
swamps and other natural waters that contain organic coloring matter.
Some inorganic phosphate compounds of iron have low solubilities and
may affect the behavior of the element in environments such as eutrophic
lakes, agricultural soils, or sewage. Polynuclear complexes of iron
703
and their relation to biologic iron transport have been reviewed.
(The simplified system portrayed in Figures 1-1 and 1-2 only considers
hydroxide complexes.)
As a rule,the redox reactions of ferric hydroxide and ferrous and
ferric solutions are fast enough to reach equilibrium readily when the
pH approaches neutrality. The oxidation rate of aqueous ferrous iron
in aerated water has a second-order dependence on pH, and in acid
solutions it is very slow unless some catalytic effect is introduced.
Reactions involving sulfide species of iron generally require biologic
mediation to attain equilibrium, processes depending on an initial
oxidation or reduction of sulfur rather than iron.
20
-------
When the impositions of an external agent cause the Eh. or pH of a
solution containing equilibrated concentrations of dissolved iron to
change, the system's equilibrium will be upset (see Figure 1-2). Where
the necessary reactants are available to restore equilibrium, the
resulting chemical processes can serve as sources of energy. Microbiota
commonly utilize these sources to catalyze such reactions as the oxida-
tion of ferrous iron to precipitate ferric hydroxide. Where organic
material is present, organisms also can mediate reduction of ferric iron
or sulfate.
Electron transfers are a part of redox processes, and mechanisms
to facilitate transfers are established at active surfaces such as those
of ferric hydroxide precipitates or particles. Such processes can
explain coprecipitation of other metals with iron. The sorption of
various other dissolved metals by ferric hydroxides is Important in controlling con-
346
centrations of iron in soil moisture, river, and underground water.
Iron is involved in the synthesis of chlorophyll and its role as an
oxygen carrier in electron transfer is essential in the life processes
of plants and animals. Therefore, iron is a component of almost all
organic matter.
Iron in the Atmosphere
Iron raised from the soil by far exceeds iron residing in the atmosphere
from all other natural sources, including meteorites and volcanic eruptions.
The Natural Environmental Research Council of the United Kingdom began operating
several air sampling stations in sparsely populated areas in the Lake District
at Wraymires in April 1970. Most of the samples were analysed byy -ray
21
-------
spectroscopy. Indications that atmospheric iron at such sites far from
industrial activity came from the soil were substantiated in a later
extension of the study to other places. ' Data for about 30 trace and
more abundant elements were tabulated. "A remarkable uniformity in the
elemental composition of the general aerosol" at seven nonurban sites chosen
608
to represent rural and more industrial exposures was reported.
3
Iron concentrations ranged from 67-940 ng/kg air (about 0.09-1.2/ug/m )
during January-December 1972. At Lerwick in the Shetland Islands during
3
June 1972-May 1973, it was only 49 ng/kg air (about 0.06/ig/m ). By taking
ratios of concentration of iron to scandium in air, and normalizing these
ratios by the ironrscandium ratio in average soil (38,000 ppm:7 ppm), an
enrichment factor was arrived at to separate elements into those that were
"soil-derived" (that is, having enrichment factors near unity), and those of
"industrial" origin, that is, enriched relative to the soil by a few to many
608
times.
The concentration of iron in surface air layers at nonurban sites in
the United States during 1970-1974—represented by the average of intermittent
24-h measurements throughout the year--ranged from 0.049/ig/m at a site in
3
Hawaii County, Hawaii in 1972 to 1.091/ig/m in Park County, Indiana in 1970.
The median annual average for 80 complete site-years of record at 38 nonurban
3
sites during the same 5 yr was 0.255,/ug/m . Seventy-one percent of the annual
averages (57 out of 80) fell within the range 0.100-0.399/ig/m3. The above
statistics are based on analysis of EPA data summaries a by the Subcommittee
on Iron.
The highest 3-mo average of all nonurban site averages during the same
3
period was 2.80/ug/m at a site in Tom Green County, Texas during the first
quarter of 1971. The lowest 3-mo average fell below the lowest discrimination
22
-------
levels of the method of measurement (i.e., less than 0.006 to 0.101/ug/tn )
during one or more 3-mo periods at sites in Delaware, Hawaii, New Mexico,
South Dakota, and Wyoming.
Atmospheric iron concentrations for earlier years that are based on similar
193
measurements have been summarized. The concentration of iron and its per-
centage of the total of all species in suspended particulate matter at 10
3
remote nonurban U.S. stations for 1966-1967 was 0.15/ig/m and 0.717o, res-
cog
pectively. The latter figure compares with the lower end of the range of
the percentage of iron in soil. In the absence of more direct evidence, it
can serve as a rough indicator of the order of the iron fraction (one part
in 100 or 200) in soil-derived suspended particulates.
The gross global production rate of tropospheric particulates of all
n £-i
compositions and sizes is estimated by Hidy and Brock to be 10.7 million
metric tons/day, of which 94% or 10 million metric tons/day is attributed
to natural sources. Almost half of the daily production rate from natural
sources is attributed to sea spray (3 million metric tons), wind-blown dust
(20,000 to 1 million metric tons), forest fires (400,000 metric tons), and
volcanoes (10,000 metric tons). The remaining production of tropospheric
particulates from natural sources comes from the conversion to particulates
of gaseous emissions from vegetation, the nitrogen and sulfur cycles, and
volcanoes. Applying the ratio of iron to gross particulate from the
paragraph above to the production rate of wind-blown dust, one obtains a
rough idea of the rate of iron entry into the atmosphere from this source,
O £. I
viz., 100-10,000 metric tons/day. Hidy and Brock's estimates of global
tropospheric production rates of particulates await corroboration (cf=
Q1 A
Whelpdale and Munn ).
23
-------
MAN-MADE SOURCES OF IRON
At t;he Earth'8 Surface
Iron Ore. Most (95%) crude iron ore mined in the United States and in
the world (85%) in 1974 was extracted from open pits because most commercial
ore bodies lie close to the surface and have large lateral dimensions. Sites
of underground mining in the United States have declined from at least 30 mines
in 1951 to 6 underground mines in 1974. The present demand for iron ore of
higher iron content and greater uniformity of chemical composition and physical
structure requires that almost all crude ores mined be beneficiated before
shipment. High-grade ores are crushed and separated according to size fractions.
Lower-grade ores are processed more extensively to increase iron concentration
as well as to produce the correct physical characteristics for iron extraction
processes. After beneficiation, the ores with particles smaller than 0.625 cm
are agglomerated by sintering or pelleting. The beneficiated ore, known as
usable ore, had an average of 60.6% iron in 1974 in the United States. World
production of usable iron ore, iron ore concentrates and agglomerates, and their
total metal contents for 1974 are set forth in Table 1-3.
Usable iron ore production in the United States has been stable since
1964. Crude ore production, however, has slowly increased because of the rise
in the ratio of crude ore mined to usable ore produced. As a result of mining
lower-grade ores such as taconite, the ratio rose from 1.4:1 in 1954 to 2:1 and
2.6:1 in 1964 and 1974. Of all usable ore produced in 1974, 69% came from
Minnesota, 13% from Michigan, and the rest was divided among California, Utah,
Wyoming, Missouri, Pennsylvania, New York, Texas, and Wisconsin (Table 1-4).
The ore was produced by 35 companies operating 66 mines and 44 concentrating
429
plants. Figure 1-3 shows the locations of principal iron ore sources as
well as major U.S. ironmaking centers. Approximately one-third of the usable
24
-------
TABLE 1-3
Worldwide Production of Iron Ore, Iron Ore Concentrates,
and Iron Ore Agglomerates,
Gross Weight Iron Content
North America 141,273 86,447
South America 118,062 75,772
Western Europe 135,238 56,387
U.S.S.R. 224,989 132,743
Africa 63,560 38,717
Asia 111,423 59,639
Oceania 99,021 62,518
TOTAL 893,566 511,787
aDerived from Klinger.430
^In thousands of metric tons.
25
-------
TABLE 1-4
Crude Iron Ore (< 5% Manganese) Mined
in the United States By Region, 1974^
Region
Lake Superior
Southeast
Northeast
West
TOTAL
Number of Mines
Crude
Usable Ore
34
4
3
25
66
186,886
654
7,103
26,046
220,689
71,855
292
2,396
10,664
500°
85,705
Derived from Klinger.430
^In thousands of metric tons.
^Byproduct from processing of other ores.
26
-------
•ounces or IRON out MPOSITI OF COKING COAL,
AND IRONMAKINQ CIMTIM OF TNI UNITED STATES
FIGURE 1-3 Sources of iron ore, deposits of coking coal, and
ironmaking centers of the United States. Reproduced
from Klinger.429
27
-------
ore consumed in the United States each year is imported (Table 1-5). In 1974
Canada supplied 41%, Venezuela,32%, Brazil,14%, Liberia,6%, Peru,4%, and other
countries)3% of the imports. A relatively small amount of usable iron ore was
430
exported, mainly to Canada and Japan.
The iron and steel industry in the United States is concentrated in
about twenty metropolitan areas. Pennsylvania, Ohio, Indiana, Illinois,
Michigan, and New York produce about three-fourths of the nation's steel.
Iron ores produced in Minnesota and Michigan are usually shipped by railroad
and lake carriers to consuming centers. However, from December to April,
shipment of ores on the Great Lakes is impractical. Thus, ore must be stock-
piled, generally in the open, at mines or shipping ports during the winter, and
at consuming plants during the shipping season to maintain production during the
winter. Unprotected piles are possible sources of injections of particles high
in iron into the atmosphere. In other states, most ores are transported only
by rail. Pipelines are now being successfully employed to move iron ore concen-
trates as slurries. If pipeline use and winter shipment of ores by lake carriers
are increased, steel mills will no longer have to maintain as large inventories
429
of iron ore as before, which will reduce the size of unprotected piles.
Amounts of stockpiled ores in the United States are noted in Table 1-5.
Pollution problems related to mining iron ore are mainly associated with
large-scale operations. The average ratio tonnage of overburden and waste rock
429
to crude ore mined in the United States in 1974 was 1:1. Most of this waste,
.some of which is low-grade iron ore, is discharged onto large piles adjacent to
the open mine pits. Even larger amounts of wastes result from the beneficiation
of the crude ore into usable ore (approximately 130 million metric tons of
processing wastes were produced in 1974 in this country). Most of these wastes
are piled on the land, although some taconite wastes have been dumped into
28
-------
TABLE 1-5
U.S. Iron Ore Statistics
a
Usable Iron Ore
(< 5% manganese)
Production
Exports
Imports for consumption
Consumption
(Iron ore and agglomerates)
Stocks0
At mines
At consuming plants
At U.S. docks
Average for ,
19 70-19 74fc 1974^
84,934
3,194
ion 43,099
85,705
2,360
48,797
134,069
13,803
51,169
3,203
140,371
9,555
45,971
3,324
aDerived from Klinger.430
^In thousands of metric tons.
CAs of December 31, 1974.
29
-------
Lake Superior, Iron oxides, carbonates, or silicates in the waste piles would
either remain relatively insoluble or weather to become immobile ferric oxyhy-
droxides. Only in sulfide-containing rock would there be serious pollution
from acidic drainage from mines or ore and waste piles. Other environmental
problems are related mainly to the minerals associated with the iron ores, such
462
as the amphibole asbestiform fibers in taconite tailings.
Wastes from bauxite processing. Bauxite, the ore from which alumina and
eventually aluminum is extracted, contains from 1-25% ferric oxide. Consequently,
the residue resulting from alumina production is mainly iron oxide. In the Bayer
process, the waste is known as "red mud" and contains about equal amounts of
iron oxides and complex sodium-aluminum silicates. The red mud may be fed to a
subsequent lime-soda sinter step in the processing of bauxite containing more
than 8% silica to produce brown or sinter muds, which are primarily constituted
705
of iron oxides and calcium silicates. About 5.6 million metric tons of
these residues are generated annually at 8 alumina plants located along the
437
lower Mississippi River, the Gulf Coast, and in central Arkansas. The muds
821
from most of the plants are impounded in large adjacent lakes. Until several
years ago, as much as 1.7 million metric tons were discharged annually into the
Mississippi River by two alumina plants in Louisiana. Now, one of them has
developed a method to eliminate the river disposal. The red mud is first filtered
through sand and then stored as reserve landfill. Red mud waste in Arkansas is
705
occasionally used to neutralize acid waste generated at a nearby vanadium plant.
In another process, red mud is used to produce a substitute for fluorspar in
438
making steel.
Fertilizers. Iron-containing fertilizer supplements are applied to soils,
to foliage in aqueous sprays, or into trunks and limbs of fruit trees. Iron
30
-------
558
compounds have been applied experimentally in irrigation waters in Kansas.
The fertilizer substances used range from inorganic salts to organic compounds
and iron chelates, as listed in Table 1-6. Iron salts, such as ferrous sulfate,
have proved inefficient in enriching many soils because they oxidize and then
rapidly hydrolyze to insoluble ferric oxyhydroxides. Ferrous sulfate in solution,
however, has proved effective in sprays and is the most popular inorganic salt
for foliar application. Fritted iron is not suitable for fertilizing alkaline
748
or calcareous soils, but it can be effective in acidic soils. In addition to
ferrous sulfate, the iron compounds used most widely for correcting iron chlorosis
are the chelates. Although iron chelates have been applied to soils, they are
more effective as foliar sprays. The primary use of chelates is for correcting
iron deficiencies in citrus and other fruit trees, grain sorghum, and certain
vegetables. Recommended rates of application for aqueous solutions of ferrous
sulfate or iron chelates range from 12-24 g of iron/citrus tree (as a solution
558
of 0.6-1.0 g iron/1) to 0.5-1.7 kg iron/ha (as a solution of 6-8 g iron/1).
fit the Hydrosphere
Acid mine drainage. Acidic drainages high in dissolved iron are products of
the weathering of iron-containing sulfides in rocks exposed by mining to moist
air or oxygenated surface and ground waters. Most acid drainage results from
the decomposition of pyrite and marcasite in the coals and associated shales of
coal mines. During weathering, the sulfide oxidizes to sulfate, which decreases
the pH of the surface moisture or water present. The ferrous iron released
forms salts with the sulfate (in drier weather), dissolves in rain or ground
waters, or oxidizes. Acidic waters high in iron emerge as seeps from surface
mines or as small streams from underground mine portals. The dissolved ferrous
iron then oxidizes and hydrolyzes to ferric oxyhydroxides, a reaction that
31
-------
TABLE 1-6
Some Types of Iron Fertilizers
Source
Ferrous sulfate
Ferric sulfate
Ferrous oxide
Ferric oxide
Ferrous ammonium phosphate
Ferrous ammonium sulfate
Iron frits
Iron ammonium polyphosphate
Iron chelates
Iron polyflavonoids
Iron ligninsulfonates
Iron methoxyphenylpropane
Formula
FeS0
FeO
Fe203
Fe(NH4)P04.H20
(NH4)2S04-FeS04«6H20
Varies
Fe(NH4)HP20?
NaFeEDTA
NaFeHEDTA
NaFeEDDHA
NaFeDTPA
FeMPP
Approximate Percentage
of Iron
19
23
77
69
29
14
Varies
22
5-14
5-9
6
10
9-10
5-8
5
aFrom Murphy and Walsh.558
EDTA : ethylenediaminetetraacetic acid
HEDTA: hydroxyethylenediaminetetraacetic acid
EDDHA: ethylene diamine di (o^hydroxyphenylacetic acid)
DPTA : diethylenetriaminepentaacetic acid
32
-------
further acidifies the drainage. The oxidation is ordinarily very slow at a low
pH, but it is increased by iron-oxidizing bacteria. Dissolved ferrous iron
concentrations, which can be higher than 1,000 mg/1 in acidic waters draining
from underground coal mines, diminish downstream from the mines as waters
become neutralized by dilution or dissolved bicarbonate in non-acidic flows.
The precipitated ferric oxyhydroxldes coat the stream bed, accumulating as a
muddy orange layer that can be as deep as several centimeters near
mines.
516
About 17,600 km of streams have been affected by mine drainage, primarily
in the coal-mining regions of Appalachia. Abandoned mines contribute an estimated
60-70% of the drainage, with approximately 85% of that amount from exhausted
22
underground mines. Biesecker and George found that the pH values of many
streams containing acid drainage from mines in Appalachia were below 5; 35, 83,
•
and 22% of the 318 stream sites sampled had respective iron, manganese, and sul-
74
fate concentrations exceeding U.S. Public Health Service drinking water standards.
The streams sampled included several sites not affected by mine drainage in addi-
tion to those that were. Future acid mine-drainage problems will be greater in
the Appalachian than in the western coalfields, because the former contain coals
of generally higher sulfur content.
Prevention methods such as flooding or sealing mines have been investigated
in attempts to decrease the amount of acid drainage. Many acidic waters are
22
treated by neutralizing them with lime or limestone. One expense of the
treatment accrues from the disposal of the voluminous amounts of sludge, con-
821
sisting primarily of water and gelatinous, amorphous ferric oxyhydroxides.
Annual literature reviews on the treatment of coal mine drainage, as well as its
chemistry and occurrence, have been published by the Water Pollution Control
95
Federation for over 20 years; a recent one is by Boyer and Gleason.
Steel industry wastes. Iron exists in both dissolved and particulate form
in steel industry wastewaters. Water used to clean gases from blast and steel-
making furnaces contains very fine, suspended particles composed largely of
iron oxides. Wastewaters from continuous-casting and hot-rolling mill operations
33 '
-------
contain suspended iron oxide scale that is washed from the steel. Surface
oxides are also removed in hot rolling and other processes by acid pickling-
657,762,821
baths; the spent liquors have high dissolved iron concentrations.
More suspended solids and slightly less dissolved ferrous sulfate, generated
in wastes per ton of steel produced, are created with advanced processes for
steelmaking than with older technologies. (The newer techniques produce more
of certain types of pollutants because larger amounts of water are required
762
per day per ingot ton than for the older processes.) Annual literature
reviews of the treatment of wastes from the steel industry have been published
698
by the Water Pollution Control Federation for over 20 years. Wastewater-
producing processes, analyses of loads of raw wastes, and water-pollution
98a
control practices in the carbon and alloy steel industry have been described.
In the Atmosphere
The iron and steel industry. Man's most important metal is produced and..
recycled at prodigious and increasing rates. U.S. and world production of pig
iron and steel for 1974, and averaged for the 5-yr period 1970-1974, is set
645a
forth in Table 1-7. In 1974 the quantity of iron scrap and steel scrap
necessary to manufacture pig iron, steel ingots, steel castings, and foundry
and miscellaneous products in the U.S. was about 10% more than the total pig
iron consumed. Several descriptions of iron- and steelmaking technologies
and their potential for emission of pollutants are available. > » » jn
the making of iron and steel, vaporization, condensation, fracture, and chemical
reaction of raw materials and intermediate products result in the emission of
iron and iron-containing particulate matter to the atmosphere, directly or in
reduced quantity through pollution control systems. Figure 1-4 shows the
principal sources of particulates in iron- and steelmaking processes.
34
-------
TABLE 1-7
Iron and Steel Production in United States and the World, 1974-
Average for
Pig Iron 1970-1974- 1974-
United States 83,161 86,615
Worldwide 466,000 514,000
Raw Steel
United States-
Carbon
Stainless
All other alloys
Total
Worldwide—
109,129
1,478
13,082
123,688
641,000
114,857
1,950
15,388
132,195
707,000
-Derived from Reno.
—In thousands of metric tons.
£
-From data of the American Iron and Steel Institute. Includes
ingots, continuous-cast steel, and all other cast forms.
—Ingots and castings.
35
-------
FLUE GAS
(SINTER
OPERATION)
DUST, FINES,
AND COAL
SINTER
OPERATION
(P)
DUST
H
)N .
D
I
, 1
/CYCLON
SECONDARY
CLEANER
PRIMARY
CLEANER
IRON ORE
GAS
PURIFICATION
COAL
COKE
OPERATION
(P)
LIMESTONE
FINISHING
OPERATIONS
SCARFING
MACHINE
FIGURE 1-4 Flow chart of iron and steel processes, "(P)" denotes a
major source of particulate emissions if it is not con-
trolled. Reproduced with revisions from the U.S. Environ-
mental Protection Agency.764
36
-------
Emissions of iron to the atmosphere from U.S. iron and steel mills,
which can be related to production rates for pig iron and raw steel, are
localized, as shown in Figure 1-3 above and in Tables 1-8 and 1-9.
Table 1-10 lists raw steel production by furnace type for 1974 and averaged for the
5-yr period 1970-1974. Steel production by basic oxygen converter or electric
furnace is increasing while production by open»hearth furnace is declining.
Production data from cupola furnaces are not available, but in 1974 cupola
furnaces consumed 14,190,000 metric tons of scrap and 1,926,000 metric tons of
. . 227a
pig iron.
Air pollutant emission factors are ratios of estimates of the average rate
at which a pollutant is released as a result of some activity (e.g., industrial
production), to an index of the level of that activity (e.g., production rate
or capacity). Table 1-11, adapted from data collected for many iron- and steel-
764
making activities, lists emission factors expressed as weight of particulates
per unit weight of metal produced applicable to: iron and steel mills; ferro-
alloy production in electric smelting furnaces; gray-iron foundries; and steel
foundries. The efficacy of various pollution control systems in reducing
emissions to the atmosphere is indicated in Table 1-11 by reductions in emission
factor for a given operation.
That iron does enter the atmosphere from human activities will be shown
below. A recent survey by Steiner summarizes the relatively few measurements
available on the composition and particle-size distribution of emissions
(separately) from sinter plants, blast-furnace flues, open-hearth furnaces,
basic oxygen furnaces, electric furnaces, and foundry cupola furnaces. Particle
sizes smaller than 5 um may represent half or more of the weight of particulates
from open-hearth and electric furnaces, and also from cupola furnaces,
especially of the hot blast type. Typical reported iron fractions of partic-
ulate matter emitted in ferrous metallurgic processes are summarized in
Table 1-12.
37
-------
TABLE 1-8
U.S. Production of Pig Iron by State, 1974-
Production-
Number of
Blast Furnaces—
3,514
6,517
15,423
6,905
-
4,237
15,843
19,681
4,621
9
19
27
9
2
11
41
50
11 (4, 4, 3)
State
Alabama
Illinois
Indiana
Michigan
Minnesota
New York
Ohio
Pennsylvania
California, Colorado, Utah
Kentucky, Maryland, Texas
West Virginia
Total
-Derived from Reno.
—In thousands of metric tons.
-Data from the American Iron and Steel Institute. Total number of blast
furnaces as of January 1, 1975, 62 of which were out of blast.
The total as of January 1, 1974 was 204, 42 of which were out of
blast.
-Does not include 2 ferroalloy blast furnaces.
—Rounded
9,872
86,615^
18 (2, 10, 2, 4)
197-
38
-------
TABLE 1-9
U.S. Production of Raw Steel by State, 1974-
State Production—
California 3,895
Illinois 11,738
Indiana 20,945
Kentucky 2,452
Michigan 9,488
New York 4,985
Ohio 22,907
Pennsylvania 30,422
Rhode Island, Connecticut, New Jersey,
Delaware, Maryland 6,258
Minnesota, Missouri, Oklahoma, Texas,
Nebraska, Iowa 5,217
.Virginia, West Virginia, Georgia, Florida,
North Carolina, South Carolina, Louisiana 5,097
Arizona, Colorado, Utah, Washington, Oregon,
Hawaii 4,465
Alabama, Tennessee, Mississippi, Arkansas 4,325
TotalS. 132,195
-Derived from data of the American Iron and Steel Institute.
23
—In thousands of metric tons.
c
"founded
39
-------
TABLE 1-10
U.S. Steel Production by Type of Furnace^-
Average for
1970-1974-
verter 66,528
35,162
21,999
Percentage
53.8
28.4
17.8
1974^
73,983
32,204
26,008
Percentage
56.0
24.4
19.7
Furnace
Basic oxygei
Open hearth
Electric
Total- 123,688 100 132,195 100
a 645a
-Derived from Reno. Castings produced by foundries not covered in
American Iron and Steel Institute data are excluded.
—In thousands of metric tons.
—Rounded
40
-------
TABLE 1-11
Emission Factors-
Iron and Steel Mills—
Pig iron production
Blast furnaces
Ore charge, uncontrolled
Agglomerates charge, uncontrolled
Total, uncontrolled
Settling chamber or dry cyclone
Plus wet scrubber
Plus venturi or electrostatic precipitator
Sintering
Windbox, uncontrolled
Dry cyclone
Dry cyclone plus electrostatic precipitator
Dry cyclone plus wet scrubber
Discharge, uncontrolled
Dry cyclone
Dry cyclone plus electrostatic precipitator
Steel Production
Basic oxygen, uncontrolled
Venturi scrubber
Electrostatic precipitator
Spray chamber
Total Particulates-
55
20
75 (65-100, range)
30
7.5
0.75
10
1.0
0.5
0.02
11
1.1
0.055
25.5 (16-43, range)
0.255
0.255
7.65
41
-------
TABLE 1-11 (continued)
Iron and Steel Mills—
Open hearth
No oxygen lance, uncontrolled
Venturi scrubber
Electrostatic precipitator
Oxygen lance, uncontrolled
Venturi scrubber
Electrostatic precipitator
Electric arc—
No oxygen lance, uncontrolled
Venturi scrubber
Electrostatic precipitator
Baghouse
Oxygen lance, uncontrolled
Venturi scrubber
Electrostatic precipitator
Baghouse
Scarfing, uncontrolled
Electrostatic precipitator
Venturi scrubber
Ferroalloy Production—
Electric Smelting Furnace
Open furnace
50% FeSi -
75% FeSi
Total Particulates-
4.15 (2.9-6.0, range)
0.085 .
0.085
8.7 (4.65-11.0, range)
0.085
0.175
*»
4.6 (3.5-5.3, range)
0.09
(0.14-0.37, range)
0.045
5.5
0.11
(0.165-0.44, range)
0.055
£0.5
io.03
£0.01
100
157.5
42
-------
TABLE 1-11 (continued)
Ferroalloy Production^ Total Particulates-
90% FeSi 282.5
Silicon metal 312«5
Silicomanganese 97.5
Semi-covered furnace
Ferromanganese 22.5
Gray-Iron Foundries^
Cupola furnace, uncontrolled 8.5
Wet cap 4
Impingement scrubber 2.5
High-energy scrubber 0.4
Electrostatic precipitator 0.3
Baghouse 0.1
Reverberatory furnace 1
Electric induction furnace 0.75
Steel Foundries—
Melting
Electric arc 6.5 (2-20, range)
Open hearth 5.5 (1-10, range)
Open hearth, oxygen lanced 5 (4-5.5, range)
Electric induction 0.05
a_ 764
^Derived from U.S. Environmental Protection Agency data (published in 1973)
compiled by R. L. Duprey and R. Gerstle; data for iron and steel mills were
revised by W. M. Vatavuk and L. K. Felleisen.
-In kg of particles/metric ton of product. Emission factors are based on
field measurements of numerous sources„
43
-------
TABLE 1-11 (continued)
—In kg of particles/metric ton of metal produced, charged, or processed
(see notes b, e_, £, j*) .
-For carbon-type electric arc furnaces; for alloy-type furnaces, multiply
given value by 2.80.
p
—In kg of particles/metric ton of specified product. Emission factors are
based on few, if any, field measurements.
^Ferrosilicon with 50%, 75%, and 90% silicon, respectively.
%n kg of particles/metric ton of metal charged. Emission factors are
based on a limited number of field measurements.
—In kg of particles/metric ton of metal processed. Emission factors are
based on field measurements of numerous sources.
44
-------
-Derived from Steiner.
—Expressed in percentage by weight.
-Typical
d
-Total of Fe203, FeO, and Fe components: 5-26% by weight.
TABLE 1-12
a.b
Iron Fraction in Industrial Participates— —
Industrial Operation F£o°o FeO Fe
2. J "™"
Pig Iron Production
Blast furnaces
U.S. plants 36.5-50.3
European plants 5-40.0
Sintering 11.7-78 5(£
Steel Production
Basic oxygen
N.oncombusted gas 4.0 21.4 66.7
Combusted gas, A 90.0 1.5
B 80 56.0-57.7
C 11.5-16.4 65.1-68.8
Open hearth 61.3-96.5 55.9-68.0
Electric 19-60 4-11 5-36
Ferrous Foundries
Cupola d d d
45
-------
Emission factors for carbon monoxide have been estimated for uncontrolled
blast furnaces (875 kg/metric ton), sinter discharges (22 kg/metric ton),
basic oxygen furnaces (69.5 kg/metric ton), and electric arc furnaces (9 kg/
metric ton), as well as for uncontrolled cupola furnaces (72.5 kg/metric
764
ton). Carbon monoxide gas needs to be considered because of the potential
219a
for formation of the extremely toxic iron pentacarbonyl, Fe(CO),-. Measure-
ments of the presence or absence of such particulates in emissions from iron-
and steelmaking seem to be lacking in the literature; c.f. treatments of this
compound in the petroleum industry and elsewhere. ' Retention on
solid surfaces of low concentrations of iron carbonyl vapors has been discussed
.! 838a
recently.
Particulate matter is potentially a carrier for sorbed gases, as is
discussed in Chapter 8. It has been estimated that fine magnetite particles
can carry one monolayer of sulfur dioxide at 2 ppm sulfur dioxide and 75 mono-
layers at 66 ppm. Thus some proposed systems of sulfur dioxide control have
taken into account the concurrent particulate concentration.
Other sources of iron. Additional sources of iron and its compounds
found in the atmosphere as a result of human activities include: mining and
handling iron ore; rusting and weathering of exposed iron; weathering of iron
pigments in surface coatings; radioactive isotopes of iron; iron-containing
fertilizers; decay or burning of vegetation and refuse; and sea spray. None
of these sources compares with raising of soil by wind, or iron and steel
manufacturing, except possibly in highly special circumstances.
For a recent discussion of atmospheric corrosion of iron, including
differences between marine and industrial environments, see Spedding. The
artificially radioactive forms of iron are Fe-52, -53, -55, -59, -60, and
-61. Their radioactivity is induced in naturally occurring iron atoms
46
-------
directly, or in neighboring elements of the periodic table, such as cobalt
and manganese, which transmute to radioactive iron by decay. Physical data
on these forms of iron are available, and a body of literature exists on
radioactive elements present in the atmosphere during and after testing of
atomic and nuclear devices. Superphosphate fertilizers contain 2% iron,
some of which becomes airborne during or after application. The iron
fraction in true solution in sea water has been estimated at less than
723a
2 iig/1; the total iron present may be 10 times that quantity. Iron in
plankton may account for as much as 167o of the total iron. Thus sea spray
associated with surf and wind introduces iron into the atmosphere. There has
been some discussion of iron compounds as catalysts in certain chemical
reactions in the atmosphere, such as the formation of sulfuric acid mist from
sulfur dioxide and sulfurous acid, and the oxidation of nitrous oxide, '
47
-------
TRANSPORT
In Soils and Sediments
Soils. The weathering of rocks results in fragmented and/or chemically
altered materials that can then form a soil or be transported during erosion
as sediment or dissolved solids. Under aerobic conditions, the amount and
distribution of iron in most soil profiles are largely dependent on the inter-
actions of the following soil-forming processes:
1. Release of iron from the primary minerals by chemical weathering;
2. Movement of iron from the surface to the subsoil by percolation
of waters containing iron in solution or colloids;
3. Reaction of iron with dissolved and amorphous silica and alumina
to form clay minerals;
4. Precipitation or accumulation of iron as amorphous ferric oxyhy-
droxides in the subsoil, followed by slow crystallization to less
soluble oxyhydroxides;
5. Transport of iron from the subsoil to the surface soil by plant
46
absorption and eventual release through plant decomposition.
The rate of the first process generally increases with temperature and
moisture. The weathering rate is also dependent on the parent minerals:
ferromagnesium silicates and sulfides decompose relatively rapidly, whereas
magnetite and ilmenite weather so slowly that they are found as heavy minerals
in soils and sediments. Biochemical activity also increases weathering and
subsequent release of iron by attack of microorganisms or their metabolites.
Slightly acid waters produced by the dissolution of carbon dioxide from organic
matter decay also speed up weathering rates. Most iron released from the
minerals is ferric, although some organic compounds form chelates with ferrous
iron.
48
-------
The most aggressive and widespread microbial agents are bacterial slimes
and acids, phenols, and certain alkaline compounds produced during the decompo-
27
sition of organic residues or excreted by microorganisms into the soil solution.
In addition to mineral (nitric,hydrochloric, and sulfuric) acids, acidic organic
compounds are formed. The dissolving action of the latter acid group is sometimes
stronger because they often can complex with iron. Organic chelates found in
soil solutions are acids of low molecular weight (oxalic, citric, fumaric, and
others), fulvic and humic acids of higher molecular weight, and polyphenols.27»588
The weathering of ferrous sulfides in rocks or sediments, e.g., some
exposed coals and adjacent shales, is aided by the bacteria Thiobacillus ferro-
oxidans. The oxidation of ferrous iron is a source of energy to these micro-
organisms. They are most active in an acidic environment (optimal pH range of
27
1.7-3.5), become inactive above pH 6 and die at higher pH's. Bacterial
activity markedly increases the normally slow rate of inorganic ferrous iron
oxidation at pH values below 4.
The second process for distribution of iron in soils comes about more
rapidly at higher temperatures in highly organic, moist, acidic soils. Although
microgram quantities of trivalent iron can move through acidic soils in solution
as hydroxy-complexes, perhaps most dissolved iron is transported as organic
Fe(III) complexes. Divalent iron chelates also are mobilized and transported.
588
Although it has been claimed that some microorganisms reduce ferric compounds,
it appears that the organisms isolated are responsible for producing substances
27
(by destroying organic compounds) that then reduce the iron. In addition to
the low-molecular weight acids, polyphenols, and fulvic and humic acids mentioned
above, it is possible that water-soluble hydroxamates formed by soil organisms
778
may complex and transport iron. Iron is transported through soil in the form
of colloidal ferric oxyhydroxides and iron-containing clays that migrate downward
27 588
under the peptizing action of dissolved organic compounds. '
49
-------
The third process, iron reacting with silica and alumina to form iron-
containing clays, proceeds best at a near neutral pH. Most of the iron
reacting is ferric.
For the fourth process, the chemical or biologic environment of many sub-
soils must cause the iron migrating down into the subsoil to accumulate and
stabilize. Heterotrophic bacteria of the Siderocapsaceae family, for example,
produce morphologically distinct iron precipitates. The bacteria are widely
distributed in soils in which ferrous iron is highly unstable (with near-neutral
pH and higher oxygen pressure). These microorganisms utilize or oxidize the
organic portion of dissolved iron chelates, releasing the iron that then will
precipitate as ferric oxyhydroxides. Any released ferrous iron is rapidly
oxidized in such an environment. In very moist or flooded soils with lower
oxygen pressure and neutral to slightly acidic pH, bacteria such as Gallionella
can promote the rate of ferric oxyhydroxide precipitation by increasing the
27
oxidation rate of dissolved ferrous iron existing in ionic or chelated form.
Iron-manganese concretions and iron intercalations are formed by micro-
organisms at specific soil horizons. In rainy seasons, when soil capillaries
are nearly filled with water, iron-accumulating bacteria generally develop in *
thin zones where soil solutions come into contact with atmospheric oxygen. At
moderate humidities, when capillary spaces contain both air and water, iron
bacteria develop in dispersed, separate nidi, which become centers of accumulating
27
iron and manganese.
Ferric oxyhydroxides and clays accumulating in the subsoil can also increase
the deposition of iron by their absorption of iron-organic complexes. This
accretion could continue until the surfaces were saturated and then be followed
588
by microbial precipitation of iron. The negatively charged surfaces of clays
could also adsorb positively charged ferric oxyhydroxide soils that were migrating
downward.
50
-------
Ferric oxyhydroxides deposited inorganically or biogenically in soils
are generally amorphous or very poorly crystalline. With aging they can
slowly crystallize to more stable, less soluble oxyhydroxides. This has the
effect of stabilizing the accumulation of iron in the subsoil.
The rates of the five soil-forming reactions given change primarily with
climate and vegetation. In soils (commonly in moist, forested regions) where
the first through the fourth processes operate at greater rates than the fifth
iron accumulates in the B horizon. In northern Appalachian Spodosols, the
iron accumulated in the upper part of the B horizon is greater under coniferous
220
than deciduous forest. Where chemical weathering is intense and accumulation of
iron is more important than its transport, the very high iron contents of Oxisols
can result. These soils form in many tropical environments, especially during
dry seasons, when the ferric oxyhydroxides are dehydrated to hematite. Oxisols
can evolve over a long period of time in a nontropical climate, which has
228
occurred in southeastern Africa. In some moist, acidic soils with a high
organic content, the first and second processes may become much more important
to iron transport than the third and fourth ones, resulting in small iron con-
centrations in both upper and lower soil horizons. An oxygen deficiency in
these soils slows the decomposition of iron-organic compounds and thus the rate
of oxyhydroxide liberation.
When the rate of iron transport is low and the precipitation or stabiliza-
tion of iron as ferric oxyhydroxides predominates, iron distribution throughout
a soil can be relatively uniform. For example, in aerated, calcareous soils
in which carbonate equilibria buffer soil pH's near 8, ferric oxyhydroxides
severely limit the solubility of iron, and thus its availability to plants.
Many dry grassland soils (Mollisols) and arid soils (Aridisols) are calcareous
and cover large areas of the western half of the United States. High phosphate
51
-------
concentrations in a soil can also limit iron mobility. At acidic pH's (where
ferric oxyhydroxides are more soluble), ferric phosphates (such as strengite)
are more stable than calcium apatites (such as hydroxyapatite) and limit iron
solubility.
In poorly drained or flooded soils containing appreciable amounts of
organic matter, iron mobility is often much greater than in well-drained,
aerated soils. Organic compounds are oxidized, producing an anaerobic environ-
ment in which ferric iron is reduced. Concentrations of up to several milligrams
per liter of dissolved ferrous iron can exist in the soil solution. The concen-
trations of other dissolved constituents that could precipitate ferrous iron,
particularly carbonate, sulfide, and phosphate, limit the amount of dissolved iron
available. Major controls on the transport rate of dissolved iron in flooded
soils are concentration gradients for iron and the flow of water within the
soil.
Soil pH is one of the most important regulators of iron mobility and.by
extension, availability to plants. The optimal soil pH for iron supply is
about 6.0-6.8 for most plants. Within this range there is usually no iron
491
excess or deficiency. Other important factors, such as the presence of
organic chelates or excessive heavy metal micronutrients such as manganese,
copper, and zinc can increase or decrease the availability of iron to plants,
595
respectively.
In addition to controlling the solubility of iron in most soils, ferric
oxyhydroxides are important to reactions active at soil surfaces. Specific
surface areas of oxyhydroxides are especially large because they can coat parti-
cles of all size fractions and thus exert chemical activity much greater than
410
their concentrations would suggest. The isoelectric pH of hydrous ferric
52
-------
603
oxyhydroxides ranges from approximately 6.7-9, indicating a net positive surface
for many soils. This surface is shown by the association of anion adsorption
750
in soil clays with the presence of iron and aluminum compounds. Phosphates
are fixed by specific adsorption on oxyhydroxide surfaces. Certain trace metals
are coprecipitated in or adsorbed on the coatings. Indeed, the hydrous oxides
of iron and manganese are thought by Jenne to be the principal control on the
410
fixation of cobalt, nickel, copper, and zinc in soils and freshwater sediments.
_o
The adsorption of molybdenum (as MoO. ) is stronger on ferric oxyhydroxides than
on aluminum oxide and clays, and increases as soil pH decreases for positive
249
oxyhydroxide surfaces.
Sediments. The products of rock weathering can be transported by the erosional
agents/water (the most important), wind, and ice. Iron movement in surface running
water is principally as suspended matter: colloidal ferric oxyhydroxides, ferric
oxyhydroxide coatings on silts and clays, or iron-containing clays or micas.
Concentrations of suspended iron thus depend primarily on the total amount of sus-
pended matter and range from <0.1- >1,000 mg/1. A smaller amount of iron is
transported in the bedload of streams in heavy minerals, primarily magnetite and
ilmenite. The average concentration of iron in suspended sediments carried by
295
the world's streams to the ocean has been estimated to be 7%. However, when
the amount of annual iron flux is compared with the total dissolved and suspended
load of material carried by all erosional agents to the oceans, the average iron
concentration is 5.5%, about the same as' for the average continental crust. Thus,
it appears that for the world as a whole, weathering and erosion are not appre-
ciably altering the average iron concentration of the land. Relatively high
losses of iron to suspended sediments from land subject to greater erosion are
balanced by the accumulation of iron in Oxisols of the tropics.
The concentrations of iron in suspended sediments generally increase inversely
with particle size. Coarser sediments deposited near the ocean shore are poorer
53
-------
in iron than those in deeper sea areas, and the highest concentrations are found
in red clays far from shore. There are, however, no present-day examples of
sedimentary iron deposits approaching in composition and extent the ancient iron
407
formations or ironstones.
Because moisture and temperature control the movement of iron within aerated
sediments, transport tends to be similar to activity found in other soils in the
same climate. Where iron mobility is low and the leaching of silica by infil-
trating meteoric waters is important, iron formations and ironstones have become
enriched in iron, sometimes to depths as great as 400 m in tropical areas.
Most sedimentary deposits are ultimately subaqueous. Iron moves primarily in
solution in the pore waters, either by diffusion as a response to chemical gradients
or by flow of the water. In lake, estuary, sea basin, or lagoon-bottom sediments,
where silts and clays accumulate, often enough organic matter is decomposing
to produce reducing conditions. Then ferric oxyhydroxides are dissolved as the
iron is reduced to the ferrous state. The ferrous iron can then migrate in solution
and be reprecipitated, often as sulfides, but also as carbonates and phosphates.
Iron participates in a redox cycle at the sediment-water interface in lakes
that become stratified annually. The cycle is an important control on dissolved
phosphate concentrations just above the interface. In the summer, phosphate and
ferrous iron concentrations increase 'as adsorbed phosphate is released during the
reduction of ferric oxyhydroxides in the oxygen-depleted waters. During turnover
of the water in fall, the increase in dissolved oxygen causes ferrous iron to
be oxidized. Simultaneously, phosphate concentrations decrease by adsorption
807
freshly precipitated ferric oxyhydroxides.
Transport in the Hydrosphere
Data on concentrations of iron present in water from various sources are
59,480
abundant, but they contain certain inherent biases. Common analytic
54
-------
methods are insufficiently sensitive to detect iron concentrations much below
104
10 yg/1; hence.extremely low concentrations in fresh water are seldom
studied. Concentrations of iron in colloidal particulate form are influenced
347,419
by the method of pretreatment used in the sampling and analysis procedures.
For computing geochemical transport rates, the difference in chemical behavior
between the colloidal particulates and dissolved ions is not important, but
different forms of iron may behave dissimilarly in plant and animal metabolism.
Hence the form in which iron occurs in water requires consideration.
Reported concentrations of iron for the ocean range from a few tenths of
59 480
a microgram per liter up to about 3 yg/1. Livingstone reported a mean
of 670 yg/1 for "average" river water. Such numbers suggest a solubility
higher than that predicted in Figure 1-2. Because ocean and rivers generally
contain substantial amounts of dissolved oxygen, the stable forms of iron should
be ferric. Filters with pore diameters near 0.45 ym generally are used to
remove particulate matter before making analyses, and the dissolved state is
thus functionally defined as any material passable through openings of this
size. The iron reported in rivers, lakes, and oceans is generally a colloidal
419,813
suspension of ferric hydroxide particles. In some waters, notably the
drainage from swamps or bogs, iron may be present as a stable organic complex.
Such waters commonly are yellow or brown and may contain more than 1 mg/1 iron.
In some such waters, however, the organic matter carrying the iron is also
colloidal. Streams contaminated by acid mine drainage have a low enough pH to
retain more than 100 mg/1 of dissolved ferric and ferrous species, but chemical
reactions with sediment minerals and mixing of the water with alkaline inflows
eventually neutralize the acidity as the water moves downstream and the iron
is oxidized and precipitated as ferric oxhydroxide.
The range of iron concentrations in groundwaters is quite wide. In systems
where organic material is present, the oxygen content of the groundwater can be
55
-------
depleted and iron brought into solution in the ferrous form. Concentrations
as high as 50-100 mg/1 can be held in solution in these waters at pH 6.0 and
concentrations of 0.5-10 mg/1 are not unusual under many different kinds of
345a
geologic conditions. Groundwater with a measurable dissolved oxygen con-
tent is likely to contain no more than a few micrograms per liter of iron if
the pH is above 5. Pyritic material may be attacked by oxygenated water that
recharges the groundwater reservoir, and substantial amounts of divalent iron may
be brought into solution as the sulfur of the pyrite oxidizes to sulfate.
Once the oxygen is consumed, the ferrous iron can remain in solution.
In 1962, the U.S. Public Health Service set a drinking water standard of a
maximum of 0.3 mg/1 of iron, which the 1974 National Academy of Sciences recommen-
dations supported. Most public water supply systems in which iron in the raw
water exceeds this limit substantially lower the iron content by treating the
water before it is delivered to consumers. However, iron in pipes, tanks, and
other equipment in the distribution system exposed to water may be attacked by
corrosion. Thus iron occasionally may be present in tap water in greater
quantities than allowable. The 0.3 mg/1 limit was set for primarily aesthetic
purposes, as water excessively high in iron tends to stain plumbing fixtures
and laundry and may be turbid. Compounds such as sodium phosphate are sometimes
added in water treatment to complex dissolved iron and prevent or delay its
precipitation.
Mobilization, transfer, and fixation processes affecting iron in the hydro-
sphere and controlling its removal from and return to the lithosphere are cyclic.
For example, iron released by weathering and biochemical processes from soil and
rock minerals is carried to the ocean by rivers and the atmosphere. The average
301
residence time of the iron in ocean water has been estimated to be about 140 yr.
During this period it participates in chemical and biochemical processes that
ultimately precipitate it. The iron next becomes a part of accumulated sediment.
56
-------
The residence time of dissolved iron in the ocean is very brief compared to that
236
estimated for most other metals. Duce et^ al. reported that the iron content
of atmospheric particulates entering the ocean from direct fallout was only
slightly enriched above the average level for mineral matter at the earth's sur-
face. They concluded that most iron transported in this way came from natural
weathering processes.
The importance of biota in certain redox processes influencing aqueous
chemical behavior of iron has already been noted. Certain aspects of the topic,
however, are worthy of further consideration. Microbes and fungi present in
soil and subsurface environments may mobilize iron and bring it into solution
by mediating chemical reactions that may serve as energy sources. Pyrite, for
example, may be converted to ferrous and sulfate ions; usually oxygen from the
atmosphere is the oxidizing agent. The iron is not necessarily oxidized, but
it is made available for solution through decomposition of the pyrite. In
anoxic environments, ferric oxides or hydroxides may be reduced when certain
590
strains of microorganisms and an organic food source are present.
Once it has been brought into solution, aerobic species of bacteria common
in soils and surface waters may catalyze oxidation and precipitation of ferrous
345a
iron to ferric hydroxide. This reaction is thermodynamically favorable in
the presence of oxygen and thus can be an energy source. The ferric hydroxide
produced by the aid of "iron bacteria" such as Crenothrix or Leptothrix is
commonly encrusted on the cells' organic sheaths. Colonies of these microorganisms
constitute gelatinous masses of such material and these deposits occur in many
wells, pipelines or other places in which water that contains ferrous iron is
589
exposed to oxygen. The deposits are a nuisance in agricultural tile drains
277
in some parts of Florida. These iron-oxidizing bacteria have some
589
characteristics of fungi.
57
-------
Growth cycles of marine and fresh water algae and related aquatic
microorganisms substantially influence concentrations of iron in open water
bodies. Demand for iron by algae during an algal bloom can greatly reduce
concentrations in solution. This iron is released again upon death and decay
590
of the plants. Some systems may exist in which increased iron availability
can trigger an increase in algal growth. Possible correlations have been
advanced between "red tides" noted along the west coast of Florida and flooding
in coastal streams that was responsible for carrying organically complexed iron
422,515
into the seawater offshore (see also Chapter 3).
Transport in the Atmosphere
Direct transport from source to receptor. The atmospheric dispersal of
contaminant gases and particles of negligible settling speed is often treated
127 281 697
as simply the eddy diffusion of matter from point or line sources. ' ' '
715 754
' The diffusion model in predominant use applies the continuity equation
in a steady (mean) wind and permits the emergent plume to spread crosswind
vertically and horizontally while traveling downwind, so as to yield at any
given distance Gaussian profiles of concentrations of contaminant in those two
directions. Standard deviations of the two crosswind distributions of contam-
inant that have been widely accepted were derived indirectly from a number of
field experiments; they increase with distance from the source and vary with
atmospheric stability and with the duration of time over which the concentrations
are averaged. Values for these standard deviations ("diffusion parameters")
are chosen to obtain estimates of contaminant concentration, despite the absence
in most places of direct measurements of atmospheric stability, by schemes that
employ one or more of the meteorologic elements commonly measured or otherwise
known or estimated, viz., wind speed, solar irradiation, and cloud cover,
525
together with the time of day and sometimes the season. Another approach
58
-------
to these standard deviations of contaminant distribution makes use of the
measured range of fluctuations in wind direction at a suitable height above
the earth's surface when these data are available.
The empirical basis of the diffusion model for estimating contaminant con-
centrations at distances beyond a few tens of kilometers is weak, and other
901 fin*j Q-I n
methods must be considered. ' ' Other problems of the model include its
inapplicability in near calms or during precipitation, and the all-too-common
absence of a reliable climatologic lexicon of diffusion parameters for the
height, terrain geometry and surface cover, as well as for the geographic loca-
788
tion being considered. Some success has been realized in accounting for jet
and buoyancy effects on the downwind profiles of plume height versus distance,
an understanding important because of the major influence on ground-level con-
taminant concentrations that the effective height of emission exerts, especially
in the near field, i.e., within 20 or so chimney heights. *»715 Another
important effect on plume height arises from the distortion of airflow by
buildings and other structures in the vicinity of the place of emission, in an
extreme case even resulting in recirculation to the origin. Approaches to
estimating the effect of architecture and construction on the performance of
relatively short stacks and chimneys have been proposed. ' '' * Mean-
while, allowance for or even recognition of this effect, e.g., in the fitting
of pollution models to measurements, is spotty.
As particles increase above 10-30 urn in aerodynamic diameter, deposition on
the earth's surface and thus loss from the diffusing plume must be taken into
account. This phenomenon is commonly treated by regarding the particulate
plume to be similar to a gaseous plume that is tilted downward at an angle
whose tangent is the ratio of the settling speed of the particle to the wind
speed, the angle of tilt changing with particle size.
59
-------
Thus the estimation of contaminant concentrations resulting from specified
emission rates of, for example, iron-containing particles, is common practice,
but one whose results should be accepted with reserve,, The consequences of
plausible alternatives to the model inputs advanced from meteorologic and other
perspectives might be useful to contemplate, and if even fragmentary measure-
ments of contaminant concentrations are available, patterns of correspondence
and dissimilarity should be noted for the information they provide on the dis-
persal process.
Reentrainment of deposited dust and raising of soil by the wind. Contam-
inants not only enter the atmosphere from stationary and mobile sources of
emission, but enter or reenter when winds are strong enough to raise the soil,
sand, or previously deposited dust. ' Little air movement is required when
the particles are fine and dry. The presence in the eastern U.S. of dust from
the Great Plains in periods of drought and the sighting of Sahara dust far out
to sea are not rare. As a rule, as the wind speed increases, the concentration
of airborne dust near the surface will rise— a result of increasing flux of
particles from the surface—but then it will fall as dilatation begins to
dominate production. With huge reservoirs of dry dust available, the high dust
concentration and low visibilities characteristic of dust and sandstorms are
reached. This mobility of iron-containing soil and deposited dust is neces-
sarily reflected in the measurements of airborne iron in remote areas discussed
above. On local scales, particulate loss from ore piles may be controlled by
use of moisture, covers, and wind breaks. The process of reentrainment has
/TOO
been studied and described for an important case by Sehmel and Lloyd.
60
-------
Observed ambient concentrations. Measurements of iron concentrations in
surface air from the National Air Surveillance Network between 1970-1974 were
summarized earlier for nonurban sites. Similar data for 210 urban sites com-
prising 604 complete site-years of record show annual average values ranging
3 3
from 0.23^ug/m at a site in St. Petersburg, Florida in 1972 to 12.12 ug/m at
2
a site in Steubenville, Ohio in 1971. The median annual average was 1.295/ig/m
or about 5 times the median annual average for iron at nonurban sites. (The
statistics above are based on analysis of EPA data summaries by the Subcom-
mittee on Iron.)
Figure 1-5 shows the cumulative frequency distribution of the annual
average iron concentrations over the 604 site-years of record at urban sites
from 1970-1974, and also corresponding data for the 80 site-years of record at
nonurban sites previously noted. The highest 18 (3%) concentrations for urban
sites came from 14 places in Ohio, Indiana, Pennsylvania, and Kentucky (cf.
Figure 1-3). The lowest 18 came from 13 sites all located in coastal states
including Hawaii, with the single exception of one site-year at Lincoln,
„ , , 765a
Nebraska„
3
The highest 3-mo average was 16.0jug/m at the Steubenville site in the
3
second quarter of 1971; the second highest, 14,6 ug/m , was measured at a site
in East Chicago, Indiana during the same period. Both sites are near major
iron and steel mills. The tendency observed in the data toward maximum con-
centrations of iron in air during the first half of the year is consistent with
the expectation that strong winds affect the contents of surface air.
The primary 24-h and annual mean standards for total suspended particulate
o
matter are 260 and 75 ug/m , respectively. Based on the EPA's composite annual
average data from 1,096 sites monitored between 1970-1974, ambient concentrations
61
-------
210 Urban Sites
600
c
-------
of total suspended particulates declined from 80 to 66^ig/m during the 5 years
studied. The percentage of air monitors reporting values exceeding the
primary standard decreased from 16 to 8% for the 24-h average, and from 50 to
23% for the annual average.
Of 236 air-quality control regions (one or more monitors) reporting minimum
acceptable data in 1974, 99 exceeded the 24-h primary standard and 111 exceeded
the annual primary standard. "Nonpoint sources"--defined as sources emitting
fewer than 90 metric tons/yr of total suspended particulates—also contributed
to the excessively high amounts.
It is interesting to note that attainment and maintenance of these standards
is adversely affected by two kinds of fugitive emissions: those generated from
industrial operations and released to the atmosphere through plant apertures
other than the primary exhaust system; and those generated by the force of the
wind or human activity on the land, including wind-raised particulates from
croplands, unpaved roads, and exposed areas at construction sites, as well as
£ Q f\
surface material made airborne by vehicles and machines from streets, crop-
lands, etc. Fugitive dust is a major problem in the arid West, but it is not
confined there. Nationwide estimates of pollutant emissions have declined
steadily in tonnage of particulates between 1970-1974. One should expect the
"soil-derived" fraction of iron in the ambient air to increase or decrease in
quantity independently of the decline in emissions, according to cultivation
practices and the frequency of dry, windy conditions.
63
-------
CHAPTER 2
MICROORGANISMS AND IRON
Iron is thought to be a universal requirement for microbial cells whether
572
they be prokaryotes or eukaryotes, i.e., bacterial or fungal species. The
only possible exception is the lactic acid bacteria. These organisms flourish
in environments, such as dairy products, which are notoriously low in absolute
and/or available iron concentrations, and they do not contain any cytochrome or
148
hydroperoxidase enzymes. The absence of catalase affords a quick presumptive
test for colonies of lactic acid bacteria growing on agar surfaces. In lactic
acid bacteria, the normal iron-containing ribotide reductase is replaced by a
functionally equivalent vitamin B enzyme system. Hence, in these species,
iron is not even required for synthesis of DNA. Although it is a generally
nutritious element for microbes, certain hazardous ecologic and environmental
factors are associated with microorganisms and iron that need to be evaluated.
METABOLISM
Assimilation
All known aerobic and facultative anaerobic organisms possess multiple
systems for acquiring this crucial nutrient. These systems fall into
two general classes: low affinity, or nonspecific, and high affinity, or
575
specific. In the first system, almost any inorganic or organic iron compound
will suffice to support growth, provided it is furnished in substantial amounts.
The biochemical mechanism of operation of this pathway is not well understood,
because no convenient way exists to probe the biochemical genetics of the system.
In contrast, the high-affinity system depends on the elaboration of specific
carrier molecules, called siderophores (formerly, siderochromes), and the cognate
surface receptors for the iron-laden form of the carrier. Formation of sidero-
phores and their specific receptors may be induced by growth of the organism in
environments either low in absolute or available iron concentrations.
64
-------
Physiologically Active Iron Compounds in Microbes
As a rule, microorganisms contain most of the iron compounds found in the
cells of higher plants and animals. Ferredoxins, nonheme iron-containing compounds
that serve as electron transfer agents, are abundant in all species requiring a
carrier of low-potential electrons, such as the Clostridia, and in all organisms
488
performing photosynthesis or nitrogen fixation. It is probably physiologically
counterproductive for microbes to synthesize oxygen carriers or possibly even iron
storage compounds. However, hemoglobin-like pigments and ferritin have been found
213
in certain species. As expected, siderophores have not been detected in strict
anaerobes or lactic acid bacteria. Table 2-1 lists iron compounds reported to
exist in microbial species.
Dissimilation
Biologic degradation of microbial iron compounds should be similar to that
of higher species, although the subject does not seem to have been investigated
systematically. An organism, Pseudomonas FC-1, has been isolated and shown to
be capable of growing on ferrichrome A, a fungal siderophore that was its sole
793
source of carbon and nitrogen. Intracellular microbial iron compounds usually
are biologic catalysts and are present in small amounts within the organism.
No special problems with pollution or other ecologic disturbances are anticipated
in the biodegradation of these substances and no toxic substances should be pro-
duced.
DEFICIENCY
General Effects of Iron Limitation
Among the aerobic and facultative aerobic microorganisms, the general
effect of iron deprivation will be the switch of the metabolism to a more anaer-
obic character. Glycolysis proceeds without utilizing iron catalysts; the latter
begin to influence energy metabolism at the level of the tricarboxylic acid cycle
65
-------
TABLE 2-1
Iron Proteins and Enzymes in Microorganisms
Substance
Oxygen-binding proteins
Hemoglobinlike proteins
Leghemoglobin
Iron storage
Ferrltin
Phosphoproteins
Hydroperoxidases
Catalase
Peroxidase
SuperozLde dismutase
Electron-transfer proteins
Berne proteins
Cytochromes
Nonheme proteins
Iron-sulfur proteins
Nitrogenase
Glutamate synthase
Hydrogenase
Ribotide reductase 82
Iron flavoproteins
Dlhydroorotate dehydrogenase
Succinic dehydrogenase
Nitrate reductase
Xanthine oxidase
HADH° dehydrogenase
Malate vitamin K reductase
Adenylylsulfate reductase
NABPIT sulfite reductase
formate dehydrogenase
Oxygenases
Beme type
Tryptophan dioxygenase
Honheme type
Diverse substrates
Iron-activated enzymes
Aconitase
D-Altronic acid dehydrase
L(+)-Tartrate dehydrase
Unclassified iron enzymes
Enzymes acting on amines,
•mine acids, and other
substrates
Source
Restricted distribution
Rhizobia
Phycomyces blakesleeanus
Unknown
Widely distributed in aerobes
Restricted distribution
Widely distributed in aexobes
Widely distributed
Widely distributed
Kitrogen-fixing species
Escherichia coli
Restricted distribution
Escherichia coli
Zymobacteriun oroticiua
Widely distributed
Micrococcus denitrificans
Micrococcus lactilyticus
Azotobacter vinelandii
Mycobacteriug phlel
Desulfovibrio vulgaris
Enteric bacteria
Pseudomonas oxalaticus
Widely distributed
Widely distributed
Coincident with tricarboxylic
•cid cycle
Escherichia coli
Pseudomonas
sp.
Phosphodiesterase
Widely distributed
? Coincident with cyclic AMP
aFrom Neilands572 uniess otherwise noted.
^Zinc, copper, and manganese, as well as iron, have been
detected in superoxide dismutases from different sources.
^Nicotinamide adenine dinucleotide, reduced.
Nicotinamide adenine dinucleotide phosphate, reduced.
317a
66
-------
(aconitase, succinic dehydrogenase) and the respiratory chain (heme and nonheme
iron). Thus, a facultative anaerobe such as Escherichia coli has a substantially
higher iron requirement when offered succinate rather than glucose as an energy
source. Microorganisms seem to be infinitely adaptable to the hardships imposed
537
by living in media low in iron. Certain species of Clostridia, when grown
without iron, produce a low-molecular weight flavoprotein (flavodoxin) with the
same redox potential and biologic function as ferredoxin. The metabolism
475
possible at low levels of iron has been characterized.
The production of numerous commercial pharmaceutical products depends
on microbes grown at low levels of iron. Formation of citric acid by Aspergillus
niger and rlboflavin by Eremothecium ashbyii are examples of commercial fermenta-
537
tions based on growth under iron-poor conditions. Iron represses the synthesis
10-12
of a number of bacterial toxins. The mechanism for inhibiting synthesis of
557
the Corynebacterium diphtheriae toxin has been investigated.
Siderophores
Chemical nature, mechanism of action, and distribution among species. Sidero-
phores are the low-molecular weight (about 500-1,000 daltons), high-affinity
carriers found in practically all aerobic and facultative anaerobic microorganisms
229,417,571-576
in which they have been sought. These compounds are virtually
30
ferric-specific, have large formation constants for trivalent iron (about 10 or
573
higher), and the liganding atoms are generally all oxygens. The complexes
are in d5 orbitals and exhibit rapid exchange kinetics. Chemically the sidero-
phore ligands are usually classified as hydroxamates or catechols. The former
are common to higher microorganisms, such as fungi, whereas the latter are
usually encountered in the prokaryotes. Ferrichrome, a cyclic hexapeptide ferric
trihydroxamate, is the prototype of the hydroxamate class of siderophores. Ferri-
chrome and ferrichrome A, and the related compounds ferrichrysin, ferricrocin,
ferrirubin, and ferrirhodin are growth-promoting iron chelates. Hydroxamate
67
-------
siderophores are found in Neurospora, Ustilago and other basidiomycetes, and
in species of the mold genera Aspergillus and Penicillium. Enterobactin, also
called enterochelin, is the prime example of the catechol class and it is pro-
duced by the enteric bacteria. The latter, however, may also produce a hydroxa-
mate, such as aerobactin.
Except for the mycobactins, siderophores tend to be exceptionally water-
soluble. They can usually be extracted into either benzyl alcohol or phenol-
chloroform (1:1, vol/vol) from water or aqueous salt solution at neutral or
acidic pH. Diluting the organic phase with diethylether and back extraction
with water returns the siderophore to the water. If a siderophore does not have
a hexadentate coordination center (e.g., rhodotorulic acid), it may form poly-
nuclear complexes with iron that are practically impossible to extract into an
organic solvent. When this happens, it is best to isolate the substance in the
absence of iron0 Similar considerations apply to crystallization of the siderophore
or its metal complex. In general, the ligands and chelates may be crystallized from
the lower alcohols. The hydroxamates are much more stable than the catechols,
but ferric hydroxamates decompose upon excessive dry or steam heat. Mineral
acid hydrolysis of the ferric hydroxamate will lead to disproportionation of
the hydroxylamino group and extensive degradation. A variety of techniques are
available for removing iron: extraction with 8-hydroxyquinoline, treatment with
dilute alkali in the cold, or continuous extraction with ether.in acidic media.
The hydroxamic acid group is weakly acidic and has a pKa_ of around 9. The
hydroxylamino function is easily oxidized at neutral or alkaline pH; it has a
pK.a close to 5. The paramagnetic ferric ion causes line broadening in the nmr
spectra, but the resonances of the I aluminum or gallium complexes are sharp and
481,482
well resolved.
The hydroxamate siderophores give the Czaky test for bound hydroxylamines;
719
respond (inexplicably) to the Folin-Ciocalteu phenol reagent; are oxidized
68
-------
by periodate to the acyl moiety and a cis-nitrosoalkane dimer, and by performic
acid to two carboxylic acid fragments; are reduced (if not too hindered) by
Raney nickel and hydrogen gas at 22.5 kg pressure; are hydrolytically reduced
by hydriodic acid; and are cleaved by nonreducing mineral acid to the carboxylic
acid and hydroxylamine components. The hydroxylamine moiety will reduce alkaline
tetrazolium without heat, thus affording a sensitive spray for chromatograms.
The catechol class of siderophores give the Arnow test and those containing
the dihydroxybenzoyl nucleus have a characteristic blue fluorescence and an
electronic absorbancy band near 310 nm in the ultraviolet region.
The hydroxamate siderophores are well-detected by chromatography on silica
375
gel, either in columns or on thin layer plates. Enterobactin, however, is
more difficult to discern by chromatography; 57o ammonium formate (wt/vol) in 0.5%
formic acid (vol/vol) will move it slightly on cellulose thin layers or paper
and in 6% acetic acid it has an Rf of 0.29. The iron complex is retarded
chromatographically on cellulose weak anion exchangers or on Sephadex" LH-20.
In both catechol and hydroxamate siderophores, the ferric ion is attached
to an asymmetric, bidentate five-membered ring. The metal-binding center may
be chiral, provided there is optic activity in the ligand; geometric isomers
642
are also formed. Ferrichrome A, ferrichrysin, and ferric mycobactin have
been examined by X-ray diffraction and shown to have the A-cis configuration,
whereas ferric enterobactin is probably A-cis. Isostructural Cr(III) analogs
are employed for assignment of configuration and in biologic transport experi-
ments for elucidation of the preferred optic isomer.
The electronic absorption spectra of the ferric hydroxamate siderophores
573
display hypsochromic and bathochromic shifts that are dependent on pH. The
red-orange to tea-colored 3:1 complexes have a maximum absorbancy at 425-440 nm
with molar extinctions of approximately 3,000. Upon acidifcation, tris-ferric
Specific products and trade names are cited solely for illustrative purposes.
Mention does not imply an endorsement from the National Academy of Sciences or
the National Research Council.
69
-------
acethydroxamate will decompose through the 2:1 to the 1:1 complex. The 1:1
complex is purple, with a maximum absorbancy near 500 run and an extinction of
about 1,000. In contrast, a trihydroxamate such as the ligand of ferrichrome,
will retain the 3:1 structure even at pH 2.
Ferric enterobactin is a wine-colored, water-soluble, trivalent anion with
a molar absorptivity of 5,600 at 495 nm. The ferric ion stabilizes the ligand,
although the molecule can suffer oxidation of the phenolic hydroxyls and hydro-
lysis of the ester bonds in the inner ring.
Both enterobactin and deferriferrichrome undergo a drastic conformation
483
change upon metal complexation, which enables the membrane receptors to
recognize only the metal-laden form.
Although a very large number of molecular species transport iron in ferrous-
ferric microbial systems, predictably, the tris-catechol and tris-hydroxamate
ligands are the most efficient carriers of the ferric ion. A microbial product
qualifies as a siderophore if:
30
* it has a high formation constant (about 10 or greater) for Fe(III),
high relative avidity for Fe(III) over Fe(II), and kinetic lability;
• biosynthesis can be induced when iron in the microorganism's
environment is low; and
• it is capable of transporting iron in microbes naturally or artificially
40
lacking high-affinity iron transport systems.
Substances with classic siderophoric chemical structures can be assigned
to one of seven families. Of these families, one is constituted of catechol
and the other six are hydroxamic acids. The following figures and captions
supply formulas and diagrams of the prototypes and a few other members of each
of the seven families. A more detailed list of structures has been published
571
elsewhere.
70
-------
The prototype of the catechol family is enterobactin, the cyclic trimer
of 2,3-dihydroxy-N-benzoyl-L-serine, shown in Figure 2-1. Ferric enterobactin
has been isolated from all enteric bacteria examined for its presence. A com-
pound of related structure, providing two catechols and one salicylic acid
728
moiety bound to spermidine, has been isolated from Micrococcus denitrificans.
The ferrichromes, set forth in Figure 2-2, are cyclic hexapeptides comprised
of a tripeptide of ferric S-N-acyl-S-N^hydroxy-L-ornithine and a tripeptide of
small, neutral amino acids such as glycine, serine, or alanine. To provide the
"hairpin turn" and the cross-g structure in the cyclohexapeptide, one residue
must always be glycine (R = H in Figure 2-2) . The acyl group furnishing the
R in Figure 2-2 may be acetate or some small carbon piece derived therefrom,
such as trans-g-methylglutaconic acid, as in ferrichrome A, or anhydromevalonic
acid, as in ferrirubin or ferrirhodin. In ferrichrome itself, R is methyl and
123
R = R = R = H. Albomycin (grisein) has a close structural relation to ferri-
chrome; it too displays broad-spectrum antibiotic activity.
The rhodotorulic acid family have in common the diketopiperazine of
N-acyl-6-N-hydroxy-L-ornithine. Members of the rhodotorulic acid family—
rhodotorulic acid, dimerumic acid, and ferric coprogen—are diagrammed in
Figures 2-3, 2-4, and 2-5.
The citrate-hydroxamate family (Figure 2-6) of siderophores has at least
six members, three of which have been isolated. The remaining three theoretical
members are awaiting discovery, hence the trivial name "awaitin."
The mycobactin family (Figure 2-7), produced by mycobacteria, has many
individual members because of the different substitutions in the R groups, as
marked in Figure 2-7. The molecule has six potentially optically active
carbon atoms, noted as a, b, c, d, e, and f in Figure 2-7.
The structure of the fusarinine family is shown in Figure 2-8. Fusarinines
(also called fusigens) are obtained from species of fusaria and other fungi.
71
-------
387a
FIGURE 2-1 Ferric enterobactin. Reproduced from Isied et al.
72
-------
R
©
R
FIGURE 2-2 Basic structure of the ferrichrome cyclohexapeptide ferric
trihydroxamates. In ferrichrome itself, which has a tri-
glycine sequence. R1 = R2 = R3 = H and R = CH-. Reproduced
from Neilands.571
73
-------
0 OH
ii i
H3C-C-N
N-C-CH:
I II
HO 0
571
FIGURE 2-3 Rhodotorulic acid. Reproduced from Neilands.
0 OH
CH3 C-N
HOCH2
CH20R
571
FIGURE 2-4 Dimerumic acid. Reproduced from Neilands.
74
-------
CH3
571
FIGURE 2-5 Ferric coprogen. Reproduced from Neilands.
75
-------
0 R
u I
CH
HN ^ (CH2)n
CH2
\
HO—C —C02H
N— C— CH3
\
(CH2)n
C
8
FIGURE 2-6 The basic structure of the citrate-hydroxamate family.
For the three known members, schizokinen, aerobactin
and arthrobactin, R = H or COOH and n = 2 or 4. Simi-
lar structural perturbations at R and n would afford
three additional hypothetical members: awaitins A, B,
and C. Reproduced from Neilands. •*-
76
-------
FIGURE 2-7 The basic structure of the mycobactin family. Depending
on the source organism, different substituents occur at
R! - R^. The ligand has six centers of potential optical
activity (a-f). Reproduced from Neilands.
77
-------
NH2 HO 0
I I II
HO-f-C-C-(CH2)3-N-C CH2-CH2-0--H
A >=<
H CH3
FIGURE 2-8 The fusarinine family. In the structure shown, n may mean
a 1-, 2- or 3-linear arrangement, or a 3-cyclic arrangement.
A corresponding series is acetylated on the amino group.
Reproduced from Neilands. '
78
-------
The ferrioxamines, represented in Figure 2-9, are linear or cyclic
molecules containing repeating units of l-amino-u>-N-hydroxyaminoalkane and
succinic or acetic acids. The antibiotic ferrimycin is a further elaboration
of ferrioxamine B.
Citrate can be considered as a primitive siderophore in that it tends
703
to form polynuclear complexes at ligand/ferric ion ratios <20:1.
The most noteworthy feature of the siderophores is that iron regulates
292
their biosynthesis. No matter what the level of available iron is, it is
likely that there can be a constitutive level of siderophore synthesis. How-
ever, at less than about 1 >ug-atom iron/1, the substances are leaked to the
medium and, in the case of rhodotorulic acid, the siderophore of the red yeast
OQ
Rhodotorula pilimanae, production reaches almost 10 g/1. In addition to the
siderophore itself, the appropriately specific membrane receptor proteins are
also overproduced by the culture when environmental iron concentrations are low.
Such activity may not be true for the ferrichrome receptor in enteric bacteria;
such organisms synthesize only enterobactin and not ferrichrome.527
Biochemical genetic experiments with Salmonella typhimurium prove conclu-
sively that the biologic functions of the siderophores are to solubilize and
619
transport iron. Thus/mutants that are unable to biosynthesize enterobactin
can only be grown in citrate medium after addition of excess iron or an organic
agent that can decompose ferric citrate, a complex which Salmonella cannot trans-
575
port. Several mechanisms exist to deliver siderophore iron in enteric bacteria.
It may be donated to the cell surface or, alternatively, the intact chelate may
penetrate the organism. In both instances the iron is liberated by reduction,
regardless of whether the ligand is hydrolyzed. The requirement for reduction
may obviate transport of nonnutritious metal ions such as Al(HI) • The presence
of the ferric complex in the cytosol would provide the cell with a specific
small molecule that could act as an effector to signal the biologic compatibility
of the external environment. Various ferrichromes and coprogen can act as
375
germination factors in Neurospora crassa.
79
-------
H-N' CONH CONH
\ / \ / \ I
(CH2)5 (CH2)2 (CH2)5 (CH2)2 (CH2)n (CH2)2
N-C N-C N-C
ii
ii
-CKC
ii
-.0 0
R
I
H-N
CONH CONH
\ / \ / \
(CH2)5 (CH2)2 (CH2)5 (CH2)2 (CH2)n R|
\ / \ / \ I
N-C N-C N-C
i ii
— 0^ 0
-0
II
0
-0
II
.0
FIGURE 2-9 Cyclic and linear forms of the ferrioxamine family.
In the ^.inear structure, various substituents occur at
R and
> lines
R1; n
may be 4 or 5. Reproduced from Neilands.-
80
-------
254
Working with Ustilago sphaerogena, . Emery proved that ferrichrome
could shuttle iron into the microorganism. The Cr(III) complex is also incorporated
by this organism, showing that the A-cis configuration is one of the actively
470
transported species.
Table 2-2 summarizes the known distribution of siderophores among species;
the list is growing rapidly.
Phage-bacteriocin interaction. Siderophores have been shown to protect
sensitive cells of enteric bacteria against certain phages and bacteriocins
797
("protein antibiotics" or "killer proteins"). Thus ferrichrome, although
it is not made by Escherichia coli, protects that organism from phages T^, Tr,
and 80, from colicin M, and from albomycin, a structurally related antibiotic,
by a mechanism known to be one of adsorption competition for a common outer
492
membrane receptor. Enterobactin protects the organism against colicin B
796
by the same mechanism of competition for the enterobactin receptor. In
Salmonella typhimurium, phage ES 18 and ferrichrome similarly compete for a sur-
face receptor. These protections are quite specific because the various agents
are binding to a similar or closely related locus in the receptor complex.
Siderophores, synthetic ferric chelates, and inorganic iron compounds
796
generally protect Escherichia coli against colicins B, V, and la. This
process is not totally understood, except that it is not one of adsorption
competition for the outer membrane receptor. The mechanism may amount to
repression of the surface receptor by iron, or the metal may somehow interfere
with how the colicin works. However, siderophores provide extremely potent pro-
tection, which is likely to be of biologic importance.
Guinea pigs kept on a synthetic diet high in iron fare poorly. The
gastrointestinal tract becomes distended and it represents a large fraction of
the carcass weight (G. Briggs, personal communication). The addition of as little
424
as 5 yQ/mi enterobactin to the diet enhances weight gain and restores the
81
-------
TABLE 2-2
Known Sources of Siderophores
a
Compound
A. Phenolates
Enterobactin (enterochelin);
Cyclo-tri-2,3-dihydroxy-N-
benzoyl-L-serine
2-N,6-N-di-(2,3-dihydroxy-
benzoyl)-L-lysine
2,3-Dihydroxy-N-benzoyl-L-
serine
2,3-Dihydroxybenzoylglycine
(itoic acid)
2,3-Dihydroxybenzoylthreonine
N1N8-bi£- (2,3-dihydroxybenzoyl)
spermldine
2-Hydroxybenzoyl-N-L-threonyl-
N4- [N^-bi^- (2,3-dihydroxy-
benzoyl) ] spermidine
Tri-2,3-dihydroxybenzoyl serine,
methyl ester
B. Hydroxamates
1.
Ferrichrome family
Ferrichrome
Ferrichrome A
Ferrichrome C
Ferrichrysin
Ferricrocin
Ferrirubin
Sources
Aerobacter aerogenes
Escherichia coli
Salmonella typhimurium
Azotobacter vinelandii
Aerobacter aerogenes
Escherichia coli
Salmonella typhimurium
Bacillus subtilis
Klebsiella oxytoca
Micrococcus denitrificans
Micrococcus denitrificans
Escherichia coli
Ascomycetes
Basidiomycetes
Fungi imperfecti
including Aspergillus,
Penicillium, and probably
Verticillium
Ustilago maydis and sphaerogena
Cryptococcus melibiosum
Aspergillus melleus, terreus,
and oryzae
Aspergillus fumigatus, humicola,
nidulans, and versicolor
Paecilomyces varioti, Penicillium
variable, and Spicaria
^Compiled from Diekmann,22^ Murphy et^ al. ,559 and Neilands.572"575 The organisnl
listed are typical sources of specific siderophores; however, they may be synthe-j
sized by other species. Thus enterobactin (MB) is generally produced by the
enteric bacteria.
-------
TABLE 2-2 (Continued)
Compound
Sources
Ferrlrhodin
Albomycins
Form 69
Form e
Form <$i
Grisein
Ferribactin
Sake colorant A
Verticillins
Rhodotorulic acid family
Rhodotorulic acid
Aspergillus nidulans and versicolor
Actinomyces griseus
Streptomyces subtropicus
Streptomyces griseus
Pseudomonas fluorescens
Aspergillus oryzae
Verticillium dahliae
Leucosporidium scottii (allotype,
mating type a)
Rhodosporidium toruloides (mating
type)
Rhodotorula glutinis
var, dairenensis (type)
var. glutinis graminis (type)
rubra (type)
rubra (type)^
Sporidiobolus
johnsonii (type)
ruinenii (type)
Sporobolomyces
albo-rubescens (type),
hispanicus, pararoseus (type),
and roseus
Dimerumic acid (dimerum acid) Fusarium dimerum
Coprogen (compound XFe)
Deacetyl coprogen
3. Citrate-hydroxamate family
Schizokinen
Terregens factor (arthro-
bactin)
Aerobactin
4. Mycobactins
5. Fusarinines (fusigens)
Neurospora crassa, Penicillium
camemberti, chrysogenum, notatum,
and urticae
Bacillus megaterium
Anabaena (blue-green alga) sp.
Arthrobacter pascens
Aerobacter aerogenes 62-1
Mycobacteria
Fusarium roseum; other species of
Fusaria, Aspergillus, Gibberella
and Penicillium
Originally known as Rhodotorula mucilaginosa.
83
-------
TABLE 2-2 (Continued)
Compound
Source
6. Ferrioxamines
Linear ferrioxamines:
Ferrioxamine B
Ferrioxamine DI
Ferrioxamine G
Ferrioxamine A..
Ferrimycin A
Cyclic ferrioxamines
Ferrioxamine E (ferric
nocardamine)
Ferrioxamine D£
Miscellaneous ferrioxamines
Metabolite C
Ferrioxamine Ao
C. Other hydroxamates
Aspergillic acids
Aspergillie, neoasper-
gillic, muta-aspergillic,
hydroxyaspergillic, and
neohydroxyaspergillic
acids
Mycelianamide
Pulcherriminic acid (2,5-
diisobutyl-3,6-dihydroxy-
pyrazine-1,4-dioxide)
Hadacidin
Actinonin
Micromonospora
Nocardia
Streptomyces pilosus and other
Streptomyces species
Nocardia
Streptomyces pilosus
Streptosporangium roseum
Chainia
Chromobacterium violaceum
Aspergillus flavus
Aspergillus sclerotiorum
Penicillium griseofulvum
Candida pulcherrima and related
yeasts
Fabospora ashbyi, dobzhanskii,
and lactis
Pencillium aurantio-violaceum
F4070b and frequentans
Streptomyces sp.
84
-------
TABLE 2-2 (Continued)
Compound Sources
2,4-Dihydroxy-7-methoxyl- Certain tissues of higher plants,
l,4-bezoxazin-3-one such as corn seedlings and
(DIMBOA) seedlings of higher grasses;
lettuce; leaves of tomato, cauli-
flower, leek and cabbage; and
extracts of carrot roots.
Mycelium of Streptomyces sp.
Viridomycin A Streptomyces viridaris
Thioformin (fluopsin) Pseudomonas fluorescens
D. Ferrous ion-binding compounds
Pyrimine Pseudomonas GH
Ferroverdin Streptomyces sp.
85
-------
gastrointestinal tract to its normal fraction of the carcass weight. It is
highly likely that the enterobactin alters the balance of the microbial flora
of the gut or the metabolism of the organisms therein. Enterobactin has been
shown to display pacifarin activity (nutritional immunity) in mice challenged
795
with virulent strains of Salmonella typhimurium.
The siderophore-phage-colicin interaction may determine aspects of
how sewage systems work, but basic research on this problem has yet to be
done.
Transport of actinide elements. It is estimated that 90 metric tons of
Plutonium exist in the world, manufactured in the past quarter century by fission
reactors. Siderophores represent one of the few natural ligand systems that
could coordinate plutonium effectively and initiate its insertion into the food
132a
chain. Plants have been shown to concentrate plutonium from the soil.
Animals kept on a low-iron diet have been found to take up plutonium more
efficiently; once in the bloodstream, the metal is carried by transferrin.
It may be excreted as the citrate chelate. It is a possibility that
this man-made element can be moved by natural ligand transport systems
. ^ , . , . 132a
intended for iron.
Distribution in nature. Assays with Salmonella typhimurium enb-7 have
found substances with the biologic properties of siderophores in mold-ripened
cheese, such as the camembert and blue varieties (S. Ong, personal communication),
and in soil and dung. The presence of these substances in excrement
357
accounts for the natural growth of the coprophylic fungus, Pilobolus. Several
members of the ferrichrome family of siderophores have been detected in the
Japanese fermented beverage, sake, which is prepared with the aid of Aspergillus
oryzae. The specific siderophore schizokinen occurs in the blue-green microalgal
693
species Anabaena and the capacity to synthesize hydroxamate chelates may enable
86
-------
such organisms to monopolize the limited iron supply in lake waters and so
559
to dominate other algal forms.
Pharmacology. Aromatic hydroxamic acids are potent carcinogens, but
this property has not been ascribed to the aliphatic members of the series.
If the hydroxamic acid bond were hydrolyzed, an alkyl-substituted hydroxylamine
would result. Hydroxylamines are well known to be mutagenic and therefore
may be carcinogenic. Little is known about the pharmacology of this
class of microbial products, although rhodotorulic acid has been touted as
306
a possible drug for treating persons suffering from chronic iron overload.
, the mesylate salt of deferriferrioxamine B, is the drug of choice
25
for treating acute episodes of accidental iron poisoning in small children.
Neither the hydroxamate ligands nor their iron complexes appear to be very
well absorbed by humans. Hence, the substances must be injected to treat
chronic iron overload. To control acute iron poisoning, the agents need to
be administered orally as well as injected.
TOXICITY
Iron- and Sulfur-Oxidizing Bacteria
On the prebiotic earth, the elements were in their reduced states. They
still retain this form in those segments of the lithosphere not contacted by
94
the oxygen gas derived from photosynthesis. Exposure of inorganic sulfur
compounds such as iron pyrite to oxygen will result in the oxidation of the
sulfur and the generation of acid. The latter, in turn, will stabilize the
ferrous ion and
87
-------
maintain it in solution as a substrate for the "iron bacteria." These reactions
lead to acid mine water and such low pH values that few forms of life other than
the Thiobacilli survive. The reactions bring about the precipitation of iron,
as exemplified by the gelatinous, golden-yellow ferric oxyhydroxide .
Acid mine water is the product of a complex series of transformations of
iron pyrite and related sulfide minerals. Some of these transformations are
336,495,839
chemical and some are catalyzed by specialized microbes . Pyrite and
marcasite (both FeS2) are oxidized in air to a solution of ferrous sulfate and
sulfuric acid:
FeS2 + H20 + 3-1/2 0^ =^ FeS04 + H^
The autotrophic bacterium, Thiobacillus f errooxidans , utilizes the ferrous ion
as an energy substrate:
+2 j. T. f errooxidans . o
?e + 1/4 02 -I- H+ , - ' _.._ N Fe+3 + 1/2 HO
Pyrite reduces the ferric ion, precipitating elemental sulfur (S°) :
2Fe+3 + FeS2 ___ i 3Fe+2 + 2S°
The elemental sulfur may be oxidized by the ferric ion or by the sulfur -oxidizing
autotroph, Thiobacillus thiooxidans ;
2S° + 12Fe+3 + 8H,0 12Fe+2 + 2S07 + 16H+
z. • i±
o T. thiooxidans
S + 1-1/2 02 + H20 - --- 1- 2H+ + S04
Although the rate-limiting step in this series needs to be defined, the
process can be retarded by covering the exposed minerals or eliminating standing
water.
Iron and Infection
Because pathogenic microbes require iron and because available iron in host
tissues may be limiting, organisms with a well developed system for acquiring
this metal may have special virulence. This subject has been reviewed by
799
Weinberg, who believes that the capacity to synthesize siderophores is a prime
determinant of virulence. This thesis is supported by his observations that:
elevated temperatures in the host (fever) inhibit synthesis of specific
88
-------
291
siderophores (e.g., enterobactin)} ' susceptibility to infections is enhanced
in hyperferremic animals and in hypotransferremic individuals; and devious
mechanisms are employed by the host to deny iron to invading pathogens. Among
the infections claimed to be dependent on iron supply are gram-negative septi-
cemia and meningitis, malaria, coliform pyelonephritis, gas gangrene, listeriosis
605
and systemic candidiasis. Payne and Finkelstein have demonstrated a role of
iron in the virulence of Neisseria gonorrhoeae.
As noted, factors imparting nutritional immunity have been termed pacifarins,
one of which, in the case of salmonellosis in the mouse, has been characterized
795
as enterobactin.
132
In surveying the role of iron in infection and immunity^ Bullen et al.
concluded that, taken as a whole, there was "ample evidence" that iron is of
crucial importance "in the mechanism of resistance to a variety of bacterial
342
infections." Yet Hegenauer and Saltman have argued that in many instances
the observed growth-promoting effect of highly-saturated iron transferrin may
be attributed to release of metal ion bound adventitiously to nonspecific sites
722
on the protein. Sussman reviewed the relevant data and concluded that
despite the evidence from experimental animals, hyperferremia does not contribute
534
to the course of human infections. Miles and Khimji found no correlation
between pathogenicity and capacity of enteric bacteria to synthesize chelates.
836
Yancy et al. showed that a strain of Salmonella typhimurium rendered
defective in the biosynthesis of enterobactin displayed greatly reduced viru-
lence in the mouse. However, the debate still continues. Ultimately, the
concept of "optimum" iron nutrition, as noted by Chandra,1-'3a may prove to be
correct.
Bacterial Toxins
Although the formation of a number of bacterial exotoxins is known to be
regulated by iron, diphtheria toxin is of special interest in view of the
559
advances in knowledge of its biochemistry and molecular biology. The toxin
89
-------
acts catalytically to effect the adenosinediphosphorylribosylation of the
EF-2 protein required for polypeptide chain elongation in the translocation
process. Toxin production is programmed by a 3-DNA phage that infects
Corynebacterium diphtherias. In the presence of bacterial extracts, iron
binds the 3-phage of DNA to nitrocellulose filters, suggested that iron is a
557
corepressor of the tox gene.
Mechanisms triggering dinoflagellate blooms, those "red tides" that cause
mass mortality of fish and other aquatic life, are as yet incompletely under-
422
stood. One factor triggering their activity may be the availability of iron
(cf. Chapter I).
MICROBIAL CORROSION
389
Microbial corrosion of iron has been reviewed thoroughly by Iverson.
Economic losses attributed to corrosion of iron and steel in the United States
has been estimated to be from $500 million-$2 billion annually. Corrosion,
defined as "the destructive attack of a metal by chemical or electrochemical
reactions within its environment," includes rusting, tarnishing, patina forma-
tion, pitting, selective leaching, stress cracking, and intergranular corrosion.
Both fungi and bacteria are involved in corrosion processes. The most active
bacteria are Thiobacillus, Desulfovibrio» and Desulfotomaculum, all of which
transform sulfur compounds. By virtue of the elaboration of oxygen gas, algae
are also involved. The chemical agents involved in biologic corrosion are
varied, and include inorganic and organic acids, hydrogen sulfide, elemental
sulfur, mercaptans, and other substances. Microorganisms growing on the surface
or vicinity of metals may produce substances that establish electrochemical
cells leading to corrosion. Biologic corrosion can be combated by altering
the environment in such ways as using microbial inhibitors, protective coatings,
or cathodic protection.
90
-------
CHAPTER 3
IRON AND PLANTS
Iron is essential to the growth processes of all plants. If a plant is
green, it usually has adequate iron. Green plants require a continuous supply
108,116
of iron as they grow, because iron does not move from older to newer leaves.
114,116
Iron absorption and transport are genetically controlled by the rootstock.
For example, when the iron-efficient Hawkeye soybean top (Glycine max L., Merr.)
is grafted to the iron-inefficient PI-54619-5-1 (T203) soybean rootstock, iron
chlorosis, shown in Figure 3-1, develops in the youngest leaf because the root-
stock cannot make iron available from the soil.
434
Soils do not usually lack iron per se, but in most calcareous soils
592
availability of ferric ions may be insufficient for plant growth. For each
pH unit increase above 4.0, the solubility of Fe(III) decreases by a factor of
448,782
1,000. When the amount of iron available to plants does not meet their
minimum needs, plants develop chlorosis, a mineral deficiency disease that will
manifest itself in the yellowing or blanching of normally green parts, such as
leaf tops, and they may die.
Chlorosis is more common in plants grown in alkaline soils. However, some
plants grow well on alkaline soils because they are endowed with a biochemical
mechanism that makes iron available to them from the soil, that is, they are
iron-efficient. The plant and the soil must be compatible for maximum efficiency
and the factors involved in such a relationship are discussed in this chapter for
representative species.
In the United States, iron deficiency is most likely to occur west of 100°
487
longitude (roughly the western half of the country). Lock found approximately
258 species or varieties of plants in western states exhibiting naturally occurring
741
iron chlorosis. Thorne and Peterson estimated that 55% of the world land area
782
receives fewer than 51 cm of rain annually, and Wallace and Lunt indicated
that 25-30% of the world's land surface is calcareous. Hence,iron deficiency in
plants is most likely to be found in these areas and is considered a world problem.
91
-------
FIGURE 3-1 Rootstocks of T203 and Hawkeye soybeans affect the
development of iron chlorosis and absorption and
translocation of radioiron from Quinlan soil (pH 7.5).
Top (left to right) photograph and autoradiograph are
of T203 top on Hawkeye rootstock; bottom photograph
(left to right) shows Hawkeye top on T203 rootstock.
The new leaves of T203 are green on the Hawkeye root-
stock, whereas the new leaf of the Hawkeye soybean is
chlorotic on T203 rootstock. No radioiron moved from
the old leaf to the new one (see also Figure 3-3).
Reproduced from Brown et a
92
-------
IRON SUPPLY
Inorganic and Stored Iron
The most common sources of iron for plants are the seed itself, the growth
medium, and sprays. Germinating seeds usually contain sufficient iron to meet
108 383
nutrient requirements of seedlings. Hyde et_ al. suggested that phyto-
ferritin in cotyledons of pea was the form of iron stored by young seedlings.
Chemical factors such as high pH and phosphate that interfere with uptake of iron
from the growth medium do not interfere with the plant's use of iron from cotyle-
19
dons. Most iron added as a spray remains in the plant tissue where it is
applied (see Figure 3-2), making it necessary for iron to be applied continually
to the new growth. Iron nutrition in plants depends largely on chemical factors
in roots, e.g., release of hydrogen ions and reduction of ferric iron, that
affect absorption and translocation of iron from the growth medium. It is not
uncommon to see two varieties of the same species growing in the same alkaline
soil, with one variety iron-deficient (chlorotic) and the other iron-sufficient
(green), as shown in Figure 3-3. The soybean cultivars of Figure 3-3 differ in
112
their ability to respond to iron stress. The chemical reactions that release
hydrogen ions and reduce ferric iron are induced by iron stress within the plant
where the roots and soil meet and make iron available to the plant. Plants are
classed iron-efficient if they respond to iron stress and iron-inefficient if
they do not. Iron-inefficient plants become chlorotic and will die on many
alkaline soils unless iron is made soluble by chelation, as illustrated in Figure
3-4.
Chelated Iron
Iron chlorosis can be corrected by adding the appropriate iron chelate to
153,341,373,405,469,783
the soil or nutrient solution. The primary role of the
chelating agent is to make the iron water-soluble and more accessible to the
93
-------
FIGURE 3-2 When the chelating agent FeDTPA was sprayed on chlorotic
leaves, it corrected the chlorosis only in spots where
the residue accumulated (right specimen). Note that the
new leaf (left specimen) that developed after the plants
were sprayed is completely chlorotic. Reproduced from
Holmes and Brown.
94
-------
FIGURE 3-3 Plant species (bottom photograph) and varieties within
species (top photograph) differ in their susceptibility
to iron deficiency. Top (left to right) T203 (iron-
inefficient) and Hawkeye (iron-efficient) soybeans grown
on Quinlan soil (pH 7.5). The iron-inefficient plants
serve as indicators. Photographs courtesy of J. C. Brown.
95
-------
FIGURE 3-4 Iron-inefficient T203 soybeans grown on 16 calcareous
soils (left 4 plants) responded to 150 kg FeDTPA/ha
added to their soils (right 4 plants). Photograph
courtesy of J. C. Brown.
96
-------
747
plant root, because roots extract iron from synthetic chelates, and ferric
chelates must be reduced to ferrous ones before the ferrous ion can be absorbed
154 746
by the plants. For a more detailed discussion of the subject, see Tiffin.
Plant species differ in ability to absorb iron from iron chelates, and
124
chelating agents themselves can compete with roots for iron. The ability of
EDDHA [ethylene diamine di (oj-hydroxyphenylacetic acid)] to chelate iron was
determined in nutrient solutions containing ethylenediaminetetraacetic acid
(EDTA), diethylenetriaminepentaacetic acid (DTPA), and cyclohexanediaminetetra-
124
acetic acid (CDTA), as competing chelating agents. The apparent stability
82
constants for FeEDTA, FeCDTA, FeDTPA, and FeEDDHA are 24.8, 29.3, 27.9, and
283
greater than 30, respectively. When chelating agents and iron are in equal
molar concentration, EDDHA competes successfully with EDTA, DTPA, and CDTA for
124
iron. However, when the concentrations of chelating agents increase, they
124
compete with EDDHA for iron in the following order: EDTA < DTPA < CDTA, a
relationship diagrammed in Figure 3-5.
Plant roots differ, as do chelating agents, in their ability to compete for
124,125
iron. Wheatland milo (Sorghum bicolor L., Moench) was unable to use iron
from FeEDDHA unless the iron concentration greatly exceeded the EDDHA concentra-
tion. For example, at 2 x 10 M iron and 0.16 x 10 M EDDHA, the milo took up
120 yg iron. At the same iron concentration (2 x 10~5M), but with the EDDHA
_5 108
concentration elevated to 1 x 10 M, the sorghum took up only 13 yg iron.
As can be seen in Figure 3-6, okra (Hibiscus esculentus L.) and wheat (Triticum
aestivum L.) developed iron-deficiency chlorosis when the molar concentration of
108
DTPA exceed the molar concentration of iron. Plant species may be able to
alter the activity of a metal ion by increasing or decreasing the concentration
of a specific chelating agent inside the plant or in the root exudate. In this
way, the type and concentration of a chelating agent, coupled with iron availa-
bility, determine the uptake of iron and other nutrient elements by plants.
97
-------
I
Q
UJ
u.
,f
g
2.0
1.5
1.0
0.5
O O
A = CHELATING AGENT *
B =
• - •- r .
1==:==:^^^.
^"^^!\
V
\\\ EDTA
\\ '
\\
\ \ DTPA
\^-
\
\ CDTA
A=0 .16 .5 1 2 4 6
6=4444444
C = 2 2 2 2 2 2 2
Fe
EDDHA
^-\
N
M
12
4
2
x I0'5 M
x ID'5 M
xlO'5 M
•
• — -__^
-
— • — ^^
18 36
4 4
2 2
FIGURE 3-5 The capacity of EDDHA to form FeEDDHA was affected by
the concentration of the competing chelating agents
in solution at 4 x 10~5M iron. No competition for
iron existed until the molar concentration of chelating
agents exceeded the molar concentration of iron. Repro-
duced from Brown et a
98
-------
FIGURE 3-6 When the molar concentration of DTPA exceed the molar
concentration of iron, DTPA competed with the roots of
okra and wheat (top and bottom photographs, respectively)
for iron. The nutrient solution contained 1 x 10 M iron
and (both genotypes, left to right) 0.16, 1, 2,_6, and
18 x 10~-*M DTPA. Reproduced from Brown et^ al
99
-------
Both the growth medium and the plant variety itself contribute to making a
continuous supply of iron available.
CONDITIONS AFFECTING IRON UPTAKE AND TRANSPORT
Soil and Environment
The hydrogen ion concentration of a soil is a crucial factor governing
the distribution of plants in nature. Some plants grow well only on acid soils;
others grow better on alkaline soils. For example, bentgrass (Deschampsia
flexuosa) develops iron-deficiency symptoms in a growth medium with a pH above
594
6, whereas mustard (Sinapis alba L.) grows well under similar conditions.
782
Wallace and Lunt listed the following as principal causes of iron chloro-
sis: poor iron supply; excessive calcium carbonate; bicarbonate in soil or irri-
gation water; overly irrigated or high-water conditions; high phosphate; high
concentrations of heavy metals such as manganese, copper, and zinc; low or high
temperatures; high light intensities; high concentrations of nitrate nitrogen;
unbalanced cation ratios; poor aeration; certain organic additions to the soil;
viruses; and root damage by nematodes or other organisms. All these factors are
more effective in a natural alkaline soil, and several may be operating at the
same time. • For example, the role of bicarbonate in precipitating iron depends
in part, on the interrelationship between bicarbonate, phosphate, calcium and
ferric ions in the soil. A moist calcareous soil containing decomposing
organic matter provides a condition for maximum bicarbonate-ion accumulation that
122
may increase phosphate availability and decrease the iron available to plants.
Bicarbonate per se does not appear to be a direct cause of iron-deficiency
107
chlorosis. Instances of iron chlorosis have been associated more with phosphate
107
concentration in solution than bicarbonate anion concentration. Phosphate is
514 312
considered a ligand that competes with the plant for iron. Greenwald and
596
Olsen et al. observed that bicarbonate increased the solubility of phosphorus
100
-------
in solution. Phosphate strongly influences iron absorption and translocation
72 108,553,742,782
in plants. This phenomenon has been well characterized.
112
More recent research has indicated that use of iron by plants is dependent
on the plant species or variety grown. Plant nutrition is entering an era in
which equal emphasis is rightly being placed on the plant as well as on the
soil for improving the efficiency of crop production.
Plant Response to Iron Stress
The term "iron stress" implies that a plant is deficient in iron. Plants
are classed as iron-efficient if they respond to iron stress by inducing bio-
chemical reactions that make iron available in a useful form, and iron-inefficient
if they do not. An iron-efficient plant may respond to iron stress without any
visual iron-deficiency symptoms, whereas an iron-inefficient plant develops
chlorosis. When plants respond to iron stress, the following products or bio-
chemical reactions are more likely to occur in iron-efficient than in iron-
inefficient plants:
• release of hydrogen ions from the roots;
• release of reducing agents from the roots;
• reduction of ferric iron at the roots; and
• increases in organic acids (particularly citrate) in roots.
Response to iron stress is adaptive and is known to be determined genetically
54,786,805
in several plant species.
425
Hydrogen ions released from roots. Kirkby and Mengel, working with
tomatoes (Lycopersicon esculentum Mill.), reported an elevated pH in nutrient
solution with nitrate, and a reduced pH with ammonium nutrition. With nitrate
as the source of nitrogen, the pH increased to pH 7.2 for T3238fer (iron-
inefficient) but decreased to pH 4.3 for T3238FER (iron-efficient) tomatoes
120
when subjected to iron stress. When supplemental iron was added to the
101
-------
iron-sufficient T3238FER tomatoes, hydrogen ions were not released from their
roots and the pH increased to 7.2, the same as for T3238fer. During an 8-h
absorption period, iron-stressed T3238FER plants absorbed and translocated
approximately 20 times more iron-59 to their tops than iron-sufficient T3238FER,
120
and 78 times more iron-59 than iron-stressed T3238fer tomatoes. The iron-
inefficient T3238fer did not respond to iron stress under any of the experimental
conditions.
When hydrogen ions are released from roots and the pH is lowered to make
the environment of the root more acidic, iron is made more available for plant
594
uptake. Although beneficial, this change is not always the ultimate solution
to iron chlorosis. For example, iron-stressed Wheatland and "B-line" (iron-
inefficient) sorghum and Pioneer 846 and KS5 (iron-efficient) sorghum release
about the same quantity of hydrogen ions into the growth medium, but they differ
118,121
in their absorption and transport of iron. This difference is associated
121
with greater reduction of Fe(IH) to Fe(II) at the root by the iron-efficient
than the iron-inefficient sorghum lines. Each factor has its own effect on
how plants use iron and may differ with variety or plant species and their growth
environment.
Reducing agents released from roots. In addition to releasing hydrogen
117 114
ions, iron-efficient soybeans and tomatoes release "reductants" from
their roots in response to iron stress. Reductants is a word coined to desig-
nate compounds released by roots that reduce Fe(III) to Fe(II). Chelating agents
interfere with the activities of reducing agents, but such interference may be
111
eliminated by increasing the concentration of the reductant in solution.
Iron-stressed Hawkeye (iron-efficient) soybeans released more reductant
into solution than the iron-stressed T203 (iron-inefficient) variety, but
102
-------
iron uptake was not increased when the T203 plants were placed in the Hawkeye
111
solutions. This may mean that reductants in the external solution indicate
a leaky root resulting from the release of hydrogen ions into the nutrient
121
solution, although reductants have been found in nutrient solutions at pH 7.
More important may be the adaptive production of reductants inside the root or
20,110
at the root surface that keeps iron in the more available ferrous form.
Over the past 20 yr, reducing agents associated with iron transport have
been identified in microorganisms. The excretion of metal-binding phenolic
acids by iron-stressed Bacillus subtilis might be a mechanism to correct iron
388
deficiency. Phenolic acids accumulated by iron-stressed Bacillus subtilis
do not seem to be involved in iron uptake, but serve to solubilize the metal in
613
the growth medium. The addition of iron to growth cultures will inhibit
136
phenolic acid excretion by Bacillus subtilis. Although not identified,
20 110
the reductants released by iron-efficient soybeans and tomato respond
similarly to the phenolic acids released by Bacillus subtilis.
Ferric iron reduced at root. Reduction of Fe(III) to Fe(II) is another factor
induced by iron stress and it occurs principally in the young lateral roots of
20,111 110
iron-efficient soybeans and tomato. Sites of reduction were determined
by transferring the iron-stressed plants to nutrient solutions containing FeHEDTA
110
and potassium ferricyanide, K~Fe(CN),. A blue precipitate (referred to here
as Prussian blue) formed in the epidermal areas of the root where one form of
the ferric ions was reduced by the root, shown in Figure 3-7. Reduction of
Fe(III) occurred in areas that were outside the root and accessible to bathophenan-
110
throlinedisulfonate (BPDS). Reducing conditions were established by adding
BPDS, 10% in excess of Fe(HI), to the nutrient solutions. As the ferric ion was
reduced, Fe(II) was trapped in solution as ferrous BPDSo. Hence most of the iron
was not transported to the plant top. '
103
-------
FIGURE 3-7 Left photograph shows sites of reduction of Fe(III) to Fe(II)
(dark areas are Prussian blue formations) on lateral
roots, and on the area of elongation and maturation of
the primary root of T3238FER (iron-efficient) tomatoes.
No reduction can be found on the T3238fer roots. The
right photograph is a cross section of a lateral root of
T3238FER. Prussian blue crystals have formed on the per-
iphery of the root. The three dark spots inside the root
reveal contamination occurring while the specimen was being
photographed. Reproduced from Brown and Ambler.
104
-------
If the iron-efficient soybean roots were given iron as FeHEDTA for 20 h
and then rinsed free of FeHEDTA and placed in nutrient solutions containing potas-
sium ferricyanide, Prussian blue (indicative of ferrous iron) appeared throughout
the protoxylem of the young lateral roots, as can be discerned in Figure 3-8,
and throughout the regions where the root elongates and matures in the primary
20,109
root, (see also Figure 3-7, left). According to this test, the metaxylem
of the iron-efficient plants contained no ferrous ions.
Elevation of organic acids (particularly citrate) in roots. Iron-deficient
222
plants usually contain more citric and malic acids than normal green plants. '
TO c O Q £ fj. £
' ' Citric acids chelate iron and keep it soluble in an external solu-
/: co
tion, and they may function similarly inside the plant. A striking relation-
ship was observed to exist between iron and citrate transported in the xylem
113 123
exudate, ' which is plotted in Figure 3-9. When iron increased, the citrate
increased; a decrease in iron was paralleled by a decrease in citrate. This
relationship held if iron stress were induced in the plant by limiting the iron
113 123
supply or if zinc, azides, or arsenate ' were used to induce iron stress.
743-745
Tiffin, using electrophoresis to follow the migration of the chelated
metal, identified ferric citrate in the xylem exudate of several plant species.
Enough citrate was always present to chelate the metal, and citrate in excess of
that needed for iron chelation migrated as an iron-free fraction behind the iron-
citrate band.
169
Clark et^ al. showed that malic, acetic and trans-aconitic acids were
ineffective in moving iron-59 electrophoretically in acetate, citrate, isocitrate,
trans-aconitate, and malate buffers. Citric acid moved iron to the annode whenever
present on the electrophoretogram, and competed successfully with the other acids
169
for iron. Clark e_t al. also determined that iron-efficient WF9 corn (Zea mays
L.) absorbed and transported more iron than iron-inefficient ys corn, but the
105
-------
FIGURE 3-8 Protoxylem of iron-stressed lateral roots of the iron-
efficient Hawkeye soybean contained Prussian blue (dark
areas) indicating Fe(II) continuous in protoxylem until
union with metaxylem of the larger root, enlarged 20
times. The iron-stress-response mechanism reduced Fe(III)
to (Fell) and kept the ferrous ions available in the pro-
toxylem. Reproduced from Ambler et^ al.
106
-------
120
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xlO" M FeEDDHA in Absorption Nutrient
FIGURE 3-9 When the iron concentration in the stem exudate was
increased by increasing the iron concentration in the
nutrient solution, the citrate concentration in the
exudate also increased in Hawkeye (HA) soybeans, but
not in the iron-inefficient T203 variety. Both culti-
vars were subjected to iron stress before being trans-
ferred to the nutrient solutions containing different
concentrations of the metal. Reproduced from Brown and
Tiffin.123
107
-------
latter contained sufficient citric acid in the xylem exudate to transport iron
in vitro. These findings indicate that the iron-inefficlent corn roots do not
respond to iron stress and the metal is not made available for transport in the
xylem exudate. The translocation of iron in the plant involves more than
citrate chelation of iron in the root per se.
ROLE OF THE IRON-STRESS-RESPONSE MECHANISM
Internal Root Control
The term "iron-stress-response mechanism" is used to denote a positive
response to iron deficiency that induces biochemical or physiologic reactions
within the plant that makes iron available for plant use. This mechanism operates
in green plants that grow well on calcareous alkaline soils. Chlorotic plants
found in such land areas are iron-deficient because the iron-stress-response
168
mechanism is not functioning. Clark and Brown found that when WF9 (iron-
efficient) and ys (iron-inefficient) corn genotypes were grown together with
ys1:WF9 ratios of 4:0, 3:1, 2:2, 1:3, and 0:4 plants per container, WF9 in the
presence of ys-^ released more hydrogen ions, reduced more Fe(III) at the root sur-
face, and took up more iron than the ys, genotype. All ys1 plants, regardless
1 168 1
of the ratio to WF9, developed iron chlorosis. In a similar study using Hawkeye
and T203 soybeans with T203:Hawkeye ratios of 28:0, 24:4, 16:12, 4:24, and
18
0*.24, Ambler and Brown found that regardless of ratio, Hawkeyes took up 80%
111
more iron than T203's. In another study, iron uptake by T203 soybeans was
not increased when they were placed in solutions in which iron-stressed Hawkeye
soybeans had released hydrogen ions and reducing agents and had absorbed and
transported 80% of the iron available. It was concluded that iron absorption
and transport are controlled inside the roots and iron uptake is greatest when
the response mechanism is functioning.
108
-------
Both iron-efficient and iron-inefficient roots can have several hundred
mlcrograms of iron per gram of root, but the iron-inefficient plant may die
from lack of iron in its tops. In contrast, iron is made available to tops by
118
the iron-efficient roots in response to iron stress. In a similar way, iron
114
may remain in the nutrient solution as a ferric chelate or a ferric phos-
109
phate, and not be taken into the plant until it is made available for
absorption and transport through chemical reactions induced by iron stress.
Iron usually is used in plant tops once it is made available for transport by
the roots.
Mechanism of Iron Uptake
Iron absorption and transport, induced in response to iron stress, involves
the release of hydrogen ions by the root, which lowers the pH at the root zone. This
favors Fe(III) solubility and reduction of Fe(III) to Fe(II). Iron-efficient roots
also release reductants in response to iron stress. These agents, along with re-
duction at the root surface, reduce Fe(III) to Fe(II), which enters the root pri-
marily through the young lateral roots. It is likely that the Fe(II) is kept
reduced in the roots by the reductant. Ferrous irons are present throughout the
protoplasm and may or may not have entered the root by a carrier mechanism. The
root-absorbed Fe(II) is oxidized to Fe(III) near the metaxylem, chelated by
citrate, and transported in the metaxylem to the top of the plant.
Other Chemical Reactions Affected
The chemical reactions induced by iron deficiency may affect a plant's nitrate
reductase activity, use of iron from ferric phosphate and from FeEDDHA, and
tolerance to heavy metals.
Nitrate reductase activity. The products of iron stress concomitantly
increase nitrate reductase activity in roots, shown in Figure 3-10 for tomatoes.
109
-------
10
8
6
4i
2
DAY 2
DAY 6
DAY 8
O-(J OO O-1 O-5
Fe, mg/l
8
£3
T3238fer
_ _ J3238FER
O-O O-1 O-5
Fe, mg/l
*Statistically significant at 1% level.
**Some iron removed from roots with EDDHA treatment.
FIGURE 3-10 When subjected to different iron concentrations in nutrient
solutions (NO-j-N), nitrate reductase activity increased in
T3238FER roots, and the pH decreased in the nutrient solution
in response to iron stress. Neither the iron-sufficient
T3238FER and T3238fer'tomato showed these responses. The
plants were 21 days old when transferred to these treatments.
Drawings courtesy of J. C. Brown and W. E. Jones.
110
-------
In both induced nitrate reductase activity and induced "reductants" activated in
response to iron-stress, a substrate is reduced: i.e., nitrate to nitrite ions
708 110
by nitrate reductase, and Fe(III) to Fe(II) by the reductant. The induced
nitrate reductase activity decreased when iron was made available to the
plants.
153
Use of iron from ferric phosphate. Phosphate, the plant, and the
chelating agent, ferrous ferrozine, Fe(II)3-(2-pyridyl)-5,6-bis(4-phenylsulfonic
acid)-l,2,4,triazine, compete for the iron in nutrient solution. Hawkeye and
T203 soybeans were used as test plants. Within 2 days after adding 16 mg phos-
phorus as phosphate to the nutrient solution, only about 10% of the ferrous
ferrozine was colorimetrically detected in solution. The iron was removed from
ferrous ferrozine, and it appeared as a suspension of iron phosphate. Phosphate
was dominating the system for iron. Four days later, all plants developed some
chlorosis in the new leaves. The iron-efficient soybeans responded to this stress
by releasing more hydrogen into solution and reducing more ferric to ferrous ions
than the iron-inefficient variety. During this process, ferrous ferrozine re-
appeared in the solutions containing the Hawkeye plants, but not the T203 speci-
mens. The iron-efficient plants now dominated the competition for iron. On
day 17 (final harvest), the green iron-efficient soybeans contained 86 yg
iron/g of tops, whereas the chlorotic iron-inefficient soybeans contained only
36 vig iron/g of tops. In the latter, phosphate still dominated the system for
iron.
114
Use of iron from FeEDDHA. Iron-inefficient T3238fer tomato plants
developed iron chlorosis because they could not absorb iron from FeEDDHA, as in
Figure 3-11. In contrast, the iron was available to iron-efficient T3238FER tomato
plants because they could reduce Fe(III) to Fe(II). For plants to use Fe(III)
111
-------
FIGURE 3-11
T3238fer (left) and T3238FER (right) tomatoes as they
appeared when grown in nutrient solution containing
10 yM iron supplied as FeEDDHA. The iron-inefficient
T3238fer tomato could not use iron from FeEDDHA.
Reproduced from Brown et al. ^
112
-------
154
from several ferric chelates, they first must reduce ferric to ferrous chelates,
which generally have a much lower stability constant than the ferric compound.
119
Tolerance to heavy metals. Heavy metals added to soils in pesticides,
fertilizers, manures, sewage sludge, and mine wastes can cause various degrees
(depending on genotype) of iron deficiency to develop in plants. An iron-efficient
plant is more tolerant to heavy metals than an iron-inefficient one. For example,
copper added as a fungal spray to citrus trees and zinc added as the oxysulfate
to control bacterial spot in peach trees accumulated in the soils in concentrations
119
toxic to plant growth. When iron is made available, it counteracts the effect
702
of the heavy metals.
GENOTYPIC DIFFERENCES AND THEIR RELATION TO IRON STRESS
Iron Deficiency and Excess
A recessive gene controls the uptake of iron in iron-inefficient T203 soy-
805 47,54 786
beans, ys^/ys^ corn, and T3238fer tomato. These plants usually develop
iron chlorosis on alkaline soils, and to a much lesser extent on some acid soils.
In contrast, the most iron-efficient plants are usually green on alkaline soils,
and may even develop symptoms of iron poisoning on some acid soils. For example,
594
Olsen found that bentgrass developed iron-deficiency symptoms in nutrient
solutions (pH above 6), but mustard grew well under these conditions with ferric
sulfate as the source of the metal. When the pH of these solutions decreased
from 6 co 4, symptoms of iron toxicity developed in the mustard, but not the bent-
594
grass. These same relationships were found in soils. Iron-efficient Bragg
soybean tops were reported to contain 45, 353 and 1,320 yg iron/g and iron-
inefficient Forrest soybean tops had 22, 54, and 112 pg iron/g when grown on
alkaline Quinlan soil (pH 7.5), acid Bladen soil (pH 4.3), and in nutrient
solution containing 2 mg iron/1 as FeHEDTA, respectively (unpublished observations,
J.C. Brown and W. E. Jones). Bragg soybeans showed signs of iron overload
113
-------
in the nutrient solution and Forrest soybeans developed symptoms of iron
deficiency on the Quinlan soil. These relationships are illustrated by Figure
3-12. Iron toxicity is only about 10% as prevalent as iron deficiency. How-
ever, the increased uptake of the metal by iron-efficient plants grown on acid
soils may be injuring plant growth, although the condition has not been recog-
nized or documented.
Iron and its Relationship to Phosphorus-efficient and Phosphorus-inefficient
Plants
533
Mikesell et al. concluded that B-line sorghum was less iron-efficient
than KS5 sorghum because the B-line took up more phosphorus than the KS5 line and
121
that phosphorus interfered with the availability of iron. Brown and Jones
confirmed these findings and showed further that KS5 responded more to iron
stress than B-line. They concluded that the accumulation of phosphorus and
insufficient iron-stress response both contributed to iron deficiency in B-line
sorghum.
Other sorghum lines grown on alkaline soil developed iron chlorosis on
the phosphorus-efficient but not on the phosphorus-inefficient genotypes
(unpublished observations, J. C. Brown and W. E. Jones). When the sorghum
lines were under phosphorus stress (deficient in phosphorus), only the phos-
phorus-efficient genotypes survived, as can be seen in Figure 3-13. The
phosphorus-efficient genotypes took up 15 times more phosphorus-32 than the
phosphorus-inefficient plants.
Zinc Stress as an Inducer of Iron Uptake
For some unexplained reason, zinc deficiency induces iron uptake in some
17,391,792
plant species or varieties. Zinc-deficient corn plants accumulate
excessive iron in their tops; evidently the excessive iron concentration
114
-------
FIGURE 3-12 Bragg (a) and Forrest (b) soybeans grown in nutrient
solutions containing 2 mg iron/1 and grown (c and d)
in Quinlan soil (pH 7.5). Note the symptoms of exces-
sive iron on Bragg (a), but not on Forrest (b) and the
iron-deficiency symptoms on Forrest (d), but not on
Bragg (c) . Photographs courtesy of J. C. Brown and
W. E. Jones.
115
-------
FIGURE 3-13 When SC369-3-1JB, PI-405107 and NK212 (top left to
right) sorghum were grown on Quinlan soil (pH 7.5),
SC369-3-1JB developed iron chlorosis, but NK212 did
not. In the bottom series of photographs, NK212
specimens grown on Bladen soil (pH 4.3) developed
phosphorus-deficiency symptoms, but SC369-3-1JB did
not. The PI-405107 genotype was intermediate in its
response. Photographs courtesy of J. C. Brown, R. B.
Clark, and W. E. Jones.
116
-------
was associated with zinc stress in the plant and not with the level of iron
391,792 17
in the soil solution. Ambler and Brown, working with Sanilac (zinc-
inefficient) and Saginaw (zinc-efficient) navy beans (Phaseolus vulgaris L.),
showed that when Sanilac plants developed zinc-deficiency symptoms, as in
.Figure 3-14, they contained twice as much iron (665 yg iron/g) and nearly twice
as much phosphorus (1.7%) in their tops as Saginaw. Sanilac tops contained
18 ug zinc/g compared to 23 ug zinc/g in Saginaw tops. Zinc-deficient cotton
(Gossypium hirsutum L.) contained 412 pg iron/g compared to 94 pg iron/g top
in the zinc-adequate plants (unpublished observations, J. C. Brown and W. E.
Jones).
Interference of Molybdenum Stress with Iron Uptake
360
Molybdenum is required by flavoproteins that transfer electrons and by
52,226,259,580
nitrate reductase, the enzyme that breaks down nitrate.
62
Barry and Reisenauer determined that molybdenum affected the reductive
capacity of Marglobe tomato roots. Iron uptake was depressed when molybdenum
concentrations were both low (less than adequate) and high (more than adequate)
in the nutrient solution. The plants receiving adequate molybdenum were the
most effective in raising the redox potential, and those that were not supple-
mented were the least effective. The increased redox potential corresponded
to the production of ferrocyanide, i.e., the reduction of Fe(III) to Fe(II),
and an increased uptake of iron by the plant. Iron accumulation was maximal
at marginally adequate levels of molybdenum nutrition.
Role for Plant Breeding
The plant breeder must select or develop iron-efficient cultivars to be
used where the mineral deficiency is a problem in crop production. In past
years, and with very little economic success, attempts have been made to
117
-------
FIGURE 3-14
Zinc-deficiency symptoms appear on Sanilac leaves
(left) but not on Saginaw plant leaves (right), when
both are grown in a split medium of Shano soil (top)
and nutrient solution (bottom). Reproduced from
Ambler and Brown.^
118
-------
correct problems of iron nutrition by changing the soil to fit the plant.
Plant breeding now offers the possibility of controlling iron chlorosis by
tailoring the plant to fit a problem soil. In some cases, varieties, lines,
or hybrids already developed or selected will overcome the problem. In others,
however, more iron-efficient lines will need to be developed.
834 787
Citrus and grape growers in Texas and Utah, respectively, controlled
genetically iron chlorosis by grafting iron-efficient rootstocks onto desirable
704
scions. Sprague believes that genetic knowledge is adequate to provide the
necessary support for a productive cooperative effort between physiologists and
geneticists to develop iron-efficient plants. Heslop-Harrison suggests that
"the most effective way to obtain a growth pattern efficient in a given environ-
355
ment for a particular purpose is to breed a genotype for the job." It is
necessary to know the nutrient requirements of crop plants before fitting them
4
to a particular soil. For example, when the Hawkeye soybean was replaced by
new,iron-Inefficient soybean varieties in central and north-central Iowa (cal-
226
careous soil), iron chlorosis developed in the new soybean varieties. These
plants were probably not tested for iron efficiency before they were released ...
to the field.
FORMS AND FUNCTIONS OF IRON IN PLANTS
If iron absorption and transport depend on the induction of the iron-stress-
response mechanism, then these induced biochemical reactions should enhance the
use of the element throughout the plant. Iron is an essential component of
260,631,632
many heme and nonheme enzymes and proteins. Nucleic acids contain
286
iron, and a ribosomal chromoprotein has been identified in rat liver that
532
contains 20% iron. The iron concentration of this protein varied with the
physiologic state of the organism. Storage forms of iron in plants include
119
-------
383,578,679
phytoferritin and ferric phosphate. Iron accumulation in different
plant parts generally follows this order: roots > old leaves > young leaves
755
> stems.
Some of the iron in plants is chelated. When a cell develops iron stress,
i.e., deficiency, iron chelates with low stabilities will fail to form and the
631
metal will be distributed among other ligands. For example, in catalase,
the iron exchange is rapid, and in cytochrome a_, it is slow. Catalase activity
115,802
has been reduced in several plant species as a result of iron deficiency.
631
Price indicates that data are insufficient to decide whether or not iron
compounds in physiologic systems behave as simple chelates. He suggests that
because chlorosis is one of the earliest symptoms of iron deficiency, the com-
pound responsible for the condition is one of the least stable among the physio-
logically essential forms of iron. Phosphate may act as a competing ligand
for iron in such a system.
This chapter has emphasized iron absorption and transport rather than its
221,260,358,359,563,631,63
forms and functions, which have been thoroughly reviewed.
RESEARCH NEEDS
Plant species and varieties within species differ in their iron require-
ments and tolerance to high concentrations of mineral elements, which complicates
determining the nutrient requirements of plants so that the plant and the soil
can be made compatible. The plant breeder will be challenged to develop plants
that are nutritionally adapted to problem soils. The agronomist, plant physiolo-
gist, biochemist, and horticulturist will need to supply the geneticists with
techniques for identifying a desirable factor or trait for a plant. For example,
iron-inefficient plants develop less iron-stress response than iron-efficient
120
-------
plants, and this range in response is the basis of a technique for screening
118
plants for iron-efficiency. A limited supply of iron and some control of
pH in the growth medium are required in this technique. Degree of iron chlorosis
is all that is needed to give the plants an efficiency rating. When the geneti-
cally controlled reaction is identified in the plant that responds to iron stress,
a more specific technique may be developed and used by the plant breeder to
identify iron efficiency in that particular genotype.
Regional laboratories for testing soils and plant tissues could be estab-
lished to meet the needs of a specific area. Soils and crops could be characterized
for what cultivars would be most suitable to the area and what supplements should
or should not be added to the soil. The genetic control of nutrition is well
established and a genetic program is suggested to fit plants nutritionally to
problem soils, i.e., iron-deficient, saline, and manganese- and aluminum-toxic.
Recognition of the role of plants in controlling mineral absorption and transport
signals a new direction for plant nutrition. Related programs, based on the
pooling of resources and expertise, would conserve our soils, improve the effi-
ciency of fertilizers and increase crop production.
121
-------
CHAPTER 4
IRON METABOLISM IN HUMANS AND OTHER MAMMALS
TOTAL BODY IRON
The essential concentration of iron in the vertebrate organism increased
by two orders of magnitude over that of lower living forms when hemoglobin
evolved as an oxygen transport vehicle. Total body iron concentrations of
various animal species, estimated on the basis of the sum of individual iron
fractions, varies between 25-75 mg/kg body weight. The adult human male has
approximately 49 mg iron/kg and the adult female has approximately 38 mg/kg,
equivalent to a total body iron content for the 80 kg male of about 4 g and
89
about 2.5 g for the 65 kg female. Miscible iron has been determined by
on o
isotopic dilution studies to be 42 mg/kg in the male; this somewhat lower
estimate is assumed to result from incomplete mixing with the pool of storage
iron. Variations in iron content within individuals of one species and among
other vertebrate species are explained by differences in circulating hemoglobin
mass and/or by differences in storage iron. The general proportions of
erythron iron, other essential tissue iron, and storage iron in humans are
summarized in Table 4-1.
Essential body iron in humans is generally proportionate to lean body mass
at about 35 mg/kg, and a variable amount of storage iron must be added to this
amount. At birth, total body iron concentration is increased by the elevated
476
red cell mass and the presence of appreciable storage iron in the liver.
During the first month of life, stores increase still further because of the
decrease in circulating red cell mass. Thereafter, rapid growth and greater
red cell requirements result in a transfer in the opposite direction so that
iron stores become virtually exhausted between the sixth and twenty-fourth
683,701
months. Little direct evidence exists of the size of iron stores in
later childhood, but as can be seen in Table 4-2, serum ferritin values indi-
184,648
cate a limited iron reserve, perhaps 5 mg/kg until after the age of 15.
122
-------
TABLE 4-1
Body"Iron Contents of Adult Humans
Myoglobin
Cell enzymes
Age
6 mo
Male Female
marrow
ig erythrocytes
les
md hemosiderin
' IRON
TABLE
Mean Values for
yg/kg mg/80.kg yg/kg
2 160 2
28 2,240 26
*4 320 =3
=2 160 -2
13 1,040 5
49 4,000 38
4-2
Serum Ferritin
mg/65 kg
130
1,690
195
130
325
2,470
as a Function of Age
rborn
10
.0 yr
18 yr
•45 male
45 female
5 male
5 female
Ferritin, yg/1 .
110
5
21
22
94
25
124
89
123
-------
Iron reserves of some 1,000 mg in the male are created between the ages of 15
and 30; adult females maintain low iron stores of about 300 mg, but after meno-
pause they increase to the level found in males.
Although various chemical measurements of the iron content of body tissues
758
have been made in animals and humans, such analyses are of limited utility.
The difficulty lies in the lack of attention paid to separating hemoglobin iron
from other iron, because of the very large concentrations of iron in circulating
red cells that contaminate those tissues. Hemoglobin, ferritin-hemosiderin, and
myoglobin are the only quantitatively important body-iron compounds that have been
found: Therefore, any appreciable concentration of iron in the tissues is assumed
to represent one of those components.
Body iron contents of animals vary with age, depending on the iron endow-
ment at birth, the duration of breast-feeding, the rate of growth, and repro-
432,758
ductive requirements. With the exception of the pig, whose exceedingly
285
rapid growth frequently outstrips iron supply, mammals easily acquire and
maintain essential body iron, and iron deficiency is exceedingly rare.
THE NATURE OF BODY IRON
Iron Compounds of the Blood
Red cell hemoglobin constitutes the largest fraction of body iron. Hemo-
globin is composed of four polypeptides each weighing about 16,000 daltons, and
each possessing a heme moiety within which an iron molecule is located. The two
different subunits of the protein together form a tetrameric molecule, (aS)^.
Each chain is coiled into an a-helix folded around the heme group in such a way
as to provide a nonpolar environment for its heme. This folding is essential in
611
permitting the iron to combine with oxygen without being oxidized. In
unoxygenated hemoglobin, the iron atoms are five-coordinated, and are forced
124
-------
556
out of the plane of the heme ring. Upon oxygenation, the sixth coordinate
site is occupied by oxygen, causing the three electrons of the iron atom to
rearrange. This alteration reduces the radius for the iron atom, enabling it
to move back into the heme plane. These changes in structure are essential
for the normal functioning of hemoglobin as an oxygen carrier.
Hemoglobin accounts for approximately 85% of essential iron in the human
female and 60% of the total in the male, a distinction largely related to the
difference between the size of iron stores in the two sexes. Hemoglobin con-
centration in normal humans depends on ambient oxygen tension, hemoglobin
3
affinity for oxygen, and circulating testosterone levels. In childhood, affinity
of hemoglobin for oxygen is somewhat decreased, perhaps related to an increased
organic phosphate concentration. The rise in phosphate is thought to be respon-
sible for a slightly lower hemoglobin concentration 'because it makes more oxygen
146
available to tissues. The sex difference in adult hemoglobin concentration
is undoubtedly linked to the effect of testosterone on erythropoietin stimulation
of the marrow. The effect of altitude on hemoglobin is well established. It
is proportional to decreases in arterial oxygen saturation, resulting from
380
changes in ambient oxygen tension.
The physiologic norm for an individual's hemoglobin concentration varies
considerably, and it is extremely difficult in mild anemia to separate physiologic
variations from deviations produced by pathologic states. It has also been compli-
cated to define mean normal values of hemoglobin concentration for normal popula-
tions. Surveys are problematic in that they embrace a considerable number of
iron-deficient individuals, some of whom are anemic. One study of a Swedish
population showed a hemoglobin response by 17% of the "normal" adult population
290
to iron administration.
Statistical approaches have been developed for surveys to exclude individuals
180
with iron deficiency. Mean hemoglobin concentrations from residents of differ-
ent parts of the world have not demonstrated differences in hemoglobin concentration
125
-------
based on racial derivation, although the existence of such variances often has
been suggested. For example, mean hemoglobin concentrations from individuals
in ten states, set forth in Table 4-3, suggest racial differences, but dissimilar
dietary customs also may have been responsible. The values in Table 4-3 are
certainly influenced by a population with iron deficiency. In subjects sampled
from the Pacific Northwest, about 4% of the adult males and 20% of the menstruating
184
females were iron-deficient, and half of that number had demonstrable anemia.
Normal hemoglobin values for other mammals are even less well defined, but
reports do show characteristic species variations that influence body iron
432,758
content. Thus, some sheep have a hemoglobin content approximately half
600
that of a human, apparently explained by a marked increase in oxygen dissociation.
In animals such as dogs and goats, hemoglobin concentration may fluctuate markedly
because their spleens can temporarily hold as much as one-third of the red cell
mass, although the total intravascular hemoglobin in these species is quite similar
to that of humans.
Transferrin is the other iron compound of the blood with an important physio-
548
logic function. This plasma component is a glycoprotein with a molecular
weight of about 80,000; it has a prolated ellipsoid shape with an axial ratio of
1:3 and is composed of a single polypeptide chain with 2 identical carbohydrate
6
side chains. Although this protein is pleomorphic—with some 18 variants
296
described in humans—these differences are not known to affect iron transport.
Transferrin is predominantly synthesized in the liver. Its production and losses
296
parallel those of albumin. The normal concentration of the protein in humans
180
is about 2.3 g/1 of plasma, equivalent to an iron-binding capacity of 3.3 mg/1.
Transferrin concentration changes inversely in relation to changes in body iron
801
stores. In individuals with adequate body iron, the iron-binding capacity of
transferrin is only 20-45% saturated; the remainder constitutes a latent capacity.
126
-------
TABLE 4-3
Median Hemoglobin in Populations of Ten States, 1968-1970a
Age
< 2
2-5
6-12
White
11.1
12.1
12.6
Black
10.7
11.3
12.0
Spanish-
Surnamed
12.3
12.5
12.9
Male
Female
13-16
17-44
45-59
> 59
White
14.1
14.9
14.9
14.6
Black
13.0
14.2
13.9
13.5
Spanish-
Surnamed
13.8
15.0
14.6
14.2
White
13.1
13.3
13.4
13.6
Black
12.3
12.8
12.8
12.6
Spanish-
Surnamed
13.1
12.9
13.4
13.4
Derived from data of the U.S. Department of Health, Education, and Welfare.
761
127
-------
Mean plasma iron concentration and total iron binding capacity in many animal
89,758
species are quite similar to humans. A higher plasma iron in some
animals appears to be a reflection of a much greater amount of iron absorbed—
185
with substitution of a low-iron diet, plasma iron falls to about 100 ug/dl.
Essential Tissue Iron
Three types of iron-containing compounds in the body perform essential meta-
207
bolic functions. The first includes all other heme iron compounds. Of these,
13
myoglobin is the largest fraction, amounting to about 15 mg iron/kg muscle.
Its structure is closely related to the monomeric unit of hemoglobin: it contains
one polypeptide chain attached to a heme group with a single iron atom. It
functions as a link in the oxygen transport chain, accepting oxygen from the
blood and storing it for utilization during muscle contraction. Cytochromes a_,
b_, and c^ and PASO represent another set of tissue heme proteins located in the
582
mitochondria and other cellular membranes, and which aid in electron transport.
Of these, cytochrome c_, a pink protein with a molecular weight of 13,000, is the
best characterized. Catalase and peroxidase are other heme iron enzymes. A
second category consists of metabolically active compounds with an enzymatic
318
function, but in which iron is not in the form of heme. In mitochondria,
for example, nonheme compounds account for far more iron than do the cytochromes.
A large portion of this iron is in a group of proteins designated as metallo-
flavoproteins. Metalloflavoproteins are involved in oxidative metabolism and include
reduced nicotinamlde-adenine dinucleotide, succinate, and cx-glycerophosphate
dehydrogenases. Other enzymes of this group, such as monoamine oxidase,are not
yet purified, but are presumed to contain iron. The final category includes
enzymes such as aconitase and microsomal lipid peroxidase, which do not contain
iron yet require it as a cofactor. Iron in a loosely bound form is required for
hydroxylation of proline and lysine in protocollagen, steps essential in the
128
-------
synthesis of collagen. This catalog is far from complete, and it is likely that
additional compounds with important metabolic activities will be identified in
the future.
Storage Iron ,
Iron within the body is stored as ferritin and hemosiderin, which together
represent the second largest fraction of iron after hemoglobin. Ferritin is a
334
specialized tissue protein designed for iron storage. It has a molecular
weight of about 450,000 and is composed of some 24 subunits. Within the central
cavity of ferritin, masses of iron are deposited principally as ferric hydroxide,
but they contain some phosphate as well. Hemosiderin is considered to be,an
aggregate form of ferritin and showsup as golden brown granules when seen by
light microscopy. More detailed examination by the electron microscope suggests
that these masses are composed of closely packed ferritin molecules. Because a
variety of organic constituents are also included in the aggregate, it is postu-
lated that these masses actually are disintegrated, ferritin-loaded, cellular
organelles. These iron storage compounds are widely distributed in nature
197,383
among different animals, plants, and fungi.
With a positive iron balance, the inflow of iron induces the synthesis of
apoferritin, and, when it becomes loaded with iron, the ferritin molecule is
233
protected against degradation as well. Storage ferritin is synthesized by
free polyribosomes, whereas serum ferritin may be produced by the endoplasmic
636
reticulum of the reticuloendothelial cells and hepatocytes. The ratio between
ferritin and hemosiderin differs according to the total amount of iron stored
within the cell. At lower concentrations of tissue iron, ferritin predominates;
at higher concentrations, most of the iron found is hemosiderin. The
exact mechanism whereby iron loads into the ferritin molecule or is released
129
-------
is largely unknown, although it may involve a Fe(III) ->• Fe(II) ->• Fe(III) cycle.
In mammalian species, diverse molecular forms of ferritin have been identified;
in humans, different profiles are observed, according to whether the ferritin
originates from heart, liver, or other tissues. The proposal has been made
that these different species of ferritin represent various combinations of
233
dissimilar subunits.
IRON BALANCE IN HUMANS
Iron balance represents the relationship between iron absorbed and iron lost
by the individual. The notion that iron exchange was very small originally came
524
from careful chemical balance studies. A better understanding was achieved
through isotopic measurements of iron losses, more accurate quantitative estimates
of iron requirements for growth and pregnancy, and measurements of iron absorption
from food.
Physiologic Iron Losses
Losses in the male. Because of the small amounts of iron that men lose
daily and the difficulties involved in distinguishing true losses from the
presence of contaminating iron not actually excreted, it became evident that
chemical measurements of excreted iron were unsatisfactory. Direct determination
of radioactivity in urine, feces, and sweat after the intravenous injection
of iron-59 permitted a distinction between excreted iron and contaminating
234
fractions but presented considerable technical difficulties. Such studies
led to an estimate of about 1 mg/day of iron loss in the adult male, a figure
that has been subsequently confirmed. Similar figures were later obtained by
140,620
counting total body iron, but the relatively short half-life of iron-59
and the extremely limited daily loss of the isotope from the body made precise
quantitation difficult. Long-term studies of body iron turnover employing
130
-------
iron-55, in which the specific activity of the circulating red cell mass was
268
followed over several years, have provided the most accurate data. A trans-
lation of these isotopic measurements into absolute amounts of iron lost,
however, involved an assumption of complete body mixing and a definition of pool
size. Analysis of the red cell activity curve showed an initial mixing component
of about one year's duration and thereafter an exponential decrease at a rate of
about 11%/yr in the adult American male; this derivation was estimated to be
308
equivalent to an iron loss of about 12 yg/kg/day or 1 mg for an 80 kg man.
Losses for other normal subjects in other countries studied by the same technique
308
were about 14 yg/kg/day.
308
The source of these basal iron losses has been categorized. The largest
single fraction, amounting to about 0.4 mg/day, consists of red cells entering
the gut lumen. Additional losses of smaller magnitude, listed in Table 4-4,
are derived from the iron content of the bile and exfoliated intestinal cells.
These three sources of gastrointestinal iron are the basis for two-thirds to
three-fourths of the total body loss. Urinary iron loss is of little consequence.
Losses from the skin could not be accurately measured by chemical techniques
because of surface contamination with iron. Radioactive measurements permit
determination of the uptake by epithelial cells from transferrin, totaling about
2.5 yg/kg/day; this amount should represent the maximal iron available for loss
through cell exfoliation, although some individual variation may occur. Perhaps
the greatest fluctuations occur in blood loss from the intestinal tract, which may
be increased by local irritants such as aspirin, alcohol, and other drugs. Other
losses (skin, gastrointestinal mucosa, and urine) are affected by the level of
serum iron, and therefore decrease during states of iron deficiency and increase
in iron overload. However, the extent of iron losses probably is not reduced to
131
-------
TABLE 4-4
Estimated Major Iron Lossesa
80 kg male
Source yg/kg/day mg/day
Gastrointestinal region
blood 5.0 0.4
mucosa 1.0 0.1
bile 2.5 0.2
Urine 1.0 0.1
Skin 2.5 0.2
TOTAL 12.0 1.0
Adapted from Green et^ al.
132
-------
less than 50% in iron deficiency and not elevated more than twice normal in
iron overload.
Special iron losses in females. Special requirements for iron face the
adult female. Menstrual blood losses have been shown to average about 0.6
172,321
mg/day if distributed over the month. Therefore, the mean total iron
loss in the menstruating female is about 20 yg/kg/day or 1.4 mg for the 65 kg
female. This figure is consistent with radioactive measurements showing an
268
overall loss of red-cell specific activity of 20%/yr. The distribution curve
for menstrual blood loss is skewed: 11% of women lose over 80 ml blood/mo,
321
representing an iron loss of more than 1 mg/day. This phenomenon is plotted
in Figure 4-1. Thus, at least 10% of menstruating women have iron requirements
more than twice that of the male population.
The other unique loss for the female is associated with pregnancy. Two
sets of requirements may be drawn up for pregnancy, summarized in Table 4-5.
One represents iron needed during pregnancy, and the other, the actual iron
49,195
lost with pregnancy and delivery. When distributed over 9 months,
the requirement becomes 3.5 mg/day and the cost, 2.5 mg/day. The increased
requirements are imposed largely during the last 6 months of pregnancy,
making the daily requirement during that period even greater.
Requirements during infancy and childhood. In infancy and childhood,
attention is directed to the iron required for growth. The newborn is endowed
with sufficient excess iron by the polycythemia present at birth and the hepatic
iron stores to meet requirements for doubling body size during the first four
months. In the premature infant, the reserve is greatly reduced—moreover,
816
growth requirements are increased. Through infancy, childhood, and adoles-
cence, requirements of growth amount to 30 mg iron/kg increase in body weight.
133
-------
100-
50-
0
50
100 150 200 240
MENSTRUAL BLOOD LOSS, ml
460 540
FIGURE 4-1 Distribution of menstrual blood loss in a population sample from
women in Goteborg, Sweden. Reproduced from Hallberg et al.321
-------
TABLE 4-5
Total Iron Requirements for Pregnancy
External iron loss
Expansion of red-blood cell mass
Fetal iron
Iron in placenta and cord
Blood loss at delivery
Total requirement12
Cost of pregnancy
Average ,
mg
170
450
270
90
150
980
680
Range,
mg
150-200
200-600
200-370
30-170
90-310
580-1,340
440-1,050
Blood loss at delivery not included.
Expansion of red cell mass not included.
135
-------
701
Information is limited concerning iron losses in infancy; they are assumed
to be at least equivalent to those of the adult on a kilogram basis. The fre-
quent finding of guaiac-positive stools in infants and demonstrations of increased
losses of radioiron in the stool suggest that infants lose more iron that do
367
adults. Much of this early bleeding may result from an immunologic reaction
to cow's milk. A composite of average iron requirements throughout life is shown
89
in Figure 4-2.
Iron Intake and Absorption
Iron absorption is the product of the amount of iron in the diet, its
availability, the influence of various luminal factors, and the behavior of the
intestinal mucosa. Much of the work on absorption has been carried out in
animals, and although this research has been useful in developing certain general
criteria, its relevance to humans is questionable because of species differences.
Mucosal regulation of absorption. That the intestinal mucosa regulates
316
iron absorption has been known since 1945. Most iron is absorbed in the duo-
denum and upper jejunum, although the mucosa throughout the intestine is capable
of absorbing iron because of the favorable intraluminal conditions in that portion
of the gut.
Oral administration of iron salts has permitted a more direct evaluation of
the intestinal mucosa because highly available iron could be presented to the
mucosal cell without the modifying effects of food. Body iron stores appear to
91,186,616a
be the most important influence on the absorptive capacity of the mucosa.
In subjects with marginally low iron stores whose hemoglobin or plasma iron con-
186,435
centrations were not changed, absorption was increased. Conversely,
excess iron stores resulted in diminished absorption.
An increased role of erythropoiesis following hemorrhage, hemolysis, or
exposure to decreased ambient oxygen tension also has been shown to increase iron
136
-------
15-
I
5
FIGURE 4-2 Iron requirements in humans. The daily iron requirement
through life is indicated by the continuous black line.
For those over the age of 12, the line divides into the
requirements of the female (upper line) and the male (lower
line). The dotted line indicates available iron in the
normal diet. The shaded area during the first year of life
indicates a period of negative iron balance when the infant
utilizes iron stores. The black arrows indicate the two
critical periods when intake and loss are of similar magni-
tude. Reproduced from Bothwell and Finch.
137
-------
91,177,340,530,710
absorption. A return to more normal conditions will tend to
91,530,803
decrease erythropoiesis and iron absorption. Whereas it has been
proved in animals that hemolytic anemia increases iron absorption, data for
humans are less convincing. Only with disorders in hemoglobin synthesis, i.e.,
thalassemia and sideroblastic anemia, does iron absorption appreciably increase.
531 671
Anemia per se has been described as increasing iron absorption. Schiffer
found that markedly less iron was absorbed in patients with aregenerative anemia
when their hemoglobin was restored by transfusion. Still another condition
affecting absorption is inflammation, during which iron transport across the
mucosa is reduced, accompanied by a. similar decrease in iron release by hepa-
tocytes and reticuloendothelial cells.
Although these factors and possibly others are known to modify iron absorp-
tion, the means by which it is accomplished is not known. Considerable effort
has gone into the search for a humoral factor in the blood, yet convincing
752
results have not been obtained. Iron absorption may be regulated by iron
178,179,804
within the intestinal cells received during their formation. How-
ever, others have observed that the iron content of mucosal cells in man is not
16,42a
appreciably changed during iron deficiency or overload. Even if this
suggestion is correct, it remains unexplained as to how the iron content of
these cells is modified, inasmuch as plasma iron does not diminish with depleted
iron stores, yet absorption increases.
The nature of the absorptive process is as yet unclear, although interesting
377
observations have been made. Hubers et^ .aJU found that iron-deficient rats
have twice as much iron in their brush border as those of iron-adequate rats,
suggesting that the brush border might be one point of control. Within the cell,
several iron-binding substances have been found in the particle-free fraction of
138
-------
363,606,622,831
mucosal cell homogenates. A transferrin-like protein has been
demonstrated that is found in greatest concentration in the upper part of the
small intestine of rats. It is increased by iron deficiency and by pretreatment
of iron with phenobarbital. The administration of phenobarbital also results
in iron absorption, but it is not demonstrated in the intestinal mucosa of mice
378
with sex-linked anemia who have impaired iron absorption. This protein is
not precipitated by antiferritin, antitransferrin, or antilactoferrin antisera.
622
A similar protein has been described by Pollack. However, proof adequate
to establish any given protein as an important transport mechanism for iron has
not yet been provided. Notable amounts of iron are held as ferritin within
the mucosal cell in inverse relationship to the amount absorbed. This binding,
however, is not thought to regulate iron absorption, but merely to hold iron
not designed to enter the body, since the metal is lost when the mucosal cell
is sloughed.
Lmnlnal factors. The amount of dietary iron absorbed depends not only on
the absorptive behavior of the intestinal mucosa, but on the effect of various
luminal secretions and gastrointestinal motility. It has been demonstrated
both in humans and experimental animals that absorption is usually reduced when
183,512,561
gastric acid secretion is reduced or eliminated. Similarly; the
administration of hydrochloric acid to individuals with achlorhydria increases
183,403 811
absorption in humans and animals. Because inorganic iron compounds
are more readily solubilized in acidic than in neutral or alkaline media, it
is not surprising that the pH of the stomach is important to absorption.
Gastric juices may contain other substances facilitating or inhibiting iron
absorption, but on the basis of present data, it is impossible to identify any-
thing other than a nonspecific absorption of iron to mucoproteins secreted
139
-------
550
within the stomach. Pancreatic secretions have also been thought to modify
iron absorption, but the most recent animal and clinical studies do not support
this idea. Thus it is inappropriate to assume any effect other than alkaliniza-
41,225,415,560
tion of the pH in the upper bowel.
Alkalinization precipitates iron and makes it unavailable for absorption.
At the same time, the digestion of food in the upper duodenum by pancreatic
enzymes might well be expected to elaborate chelates such as cysteine and histi-
dine, which might improve availability of iron for absorption. Studies of the
physical form of iron in the stomach and duodenum indicate that iron is bound
predominantly to macromolecules in the stomach, but micromolecular iron is found
400
in the duodenum. Ascorbic acid secreted into the stomach of some animals and
into the duodenum from the bile has also been proposed as an aid to iron absorp-
175
tion, but the acid's quantitative importance is unknown. The role of the
digestive process and of possible chelates secreted into the lumen is unclear.
Iron absorption also is affected by the motility of the gastrointestinal
tract. Retarded gastrointestinal motility is accompanied by increased absorption,
whereas operative procedures on the stomach that shorten emptying time are asso-
141,511
ciated with decreased absorption. Such observations indicate that the
"hopper" function of the stomach is essential for normal iron absorption.
IRON IN THE DIET
Humans ingest less iron than other animal species. Average dietary iron
intake in developed countries is about 6 mg/1,000 cal; in developing countries,
49,761
the content is more variable, averaging about 10 mg/1,000 cal. The higher
values in developing countries may be a function of extrinsic iron that is con-
taminating food during its procurement and preparation. The iron content of
various foods is listed in Table 4-6.
140
-------
TABLE 4-6
Iron Content of Foods
a
I. Low Iron Content (<0.7 mg iron/100 kcal)
mg iron/
Food 100 kcal
Apples, raw 0.6
Avocado, raw 0.4
Beer trace
Bologna, frankfurters 0.6
Bread, white, unenriched 0.3
Butter 0
Cheese, cheddar 0.3
Cheese, cottage, creamed 0.3
Chocolate, semisweet 0.5
Codfish, broiled 0.6
Corn, fresh, cooked 0.7
Egg whites, chicken 0.2
Honey 0.2
Ice cream trace
Lamb, loin chop, broiled 0.4
Margarine 0
Milk trace
Oil, salad or cooking 0
Orange juice 0.4
Peaches, canned in syrup 0.4
Peanut butter 0.3
Peanuts, roasted 0.4
Pears, raw 0.5
Potato chips 0.3
Rice, brown, cooked 0.4
Rice, white, unenriched, cooked 0.2
Salad dressing, mayonnaise 0.1
Salad dressing, mayonnaise-like 0.1
Sugar, white, granulated O.I
Sweet potatoes, baked 0.6
Wheat flour, white, unenriched 0.2
mg iron/100 g
edible portion
of food
0.3
0.6
trace
1.8
0.7
0
1.0
0.3
2.6
1.0
0.6
0,
0,
0.1
1.3
0
trace
0
0.2
0.3
2.0
2.2
0.3
1.8
0.
0.
0.5
,2
,1
0.9
0.8
II. Medium Iron Content (0.7-1.9 mg iron/100 kcal)
Almonds, dried
Apricots, raw
Beef, ground, cooked
Beef, round, broiled
Beef, T-bone steak, fried
Bread, white, enriched
Bread, whole wheat
Carrots, raw
0.8
1.0
1.1
1.9
0.6
0.9
0.9
1.7
aPrepared by Elaine Monsen.
4.7
0.5
3.2
3.7
2.7
2.5
2.3
0.7
141
-------
TABLE 4-6
(Continued)
rag iron/
Food 100 kcal
Cereals, prepared breakfast
enriched at 2-7% USRDA per oz. 0.4-1.0
Chicken, dark, cooked 1.0
Chicken, white, cooked 0.8
Cranberries, raw 1.1
Eggs, chicken, whole 1.4
Eggs, chicken, yolk 1.6
Gingerbread, made with enriched flour 0.7
Liverwurst 1.8
Molasses, light (first extraction) 1.7
Melon, cantaloupe 1.3
Oatmeal, cooked 1.1
Onions, mature, cooked 1.4
Oranges, raw 0.8
Peaches, raw 1.3
Pork, medium-fat, roasted 0.8
Potatoes, white, baked 0.8
Prunes, dehydrated, uncooked 1.3
Raisins, uncooked 1.2
Rice, white, enriched, cooked 0.8
Sardines, canned in oil, drained 1.4
Soup, canned, vegetable beef, ready
to serve 0.9
Squash, winter, baked 1.3
Sugar, brown 0.9
Tuna, canned in oil, drained 1.0
Turnips, cooked 1.7
Walnuts, black 1.0
Wheat flour, white, enriched 0.8
Wheat flour, whole grain 1.0
III. High Iron Content (>2.0 mg iron/100 kcal)
Apricots, dried 2.1
Artichokes, cooked 4.2
Asparagus, cooked 3.0
Baby cereals, enriched >13.5
Beans, green, cooked 2.4
Beans, kidney, cooked 2.0
Broccoli, cooked 3.1
Caviar, granular 4.5
Cereals, prepared breakfast
enriched at 25% U.S. RDA per oz. 3.8
Clams, hard shell 9.4
Coffee, instant, dried 4.3
Fish flour, from fish fillets 2.0
Fish flour, from whale fish 12.2
Giblets, chicken, fried 2.6
mg iron/100 g
edible portion
of food
1.4-4.0
1.7
1.3
0.5
2.3
5.5
2.3
5.4
4.3
0.4
0.6
0.4
0.4
0.5
2.9
0.7
4.4
3.5
0.9
2.9
0.3
0.8
3.4
1.9
0.4
6.0
2.9
3.3
5.5
1.1
0.6
>50.0
0.6
2.4
0.8
11.8
15.0
7.5
5.6
8.0
41.0
6.5
142
-------
TABLE 4-6
(Continued)
Food
Heart, beef, braised
Kidneys, beef, braised
Lettuce, iceberg
Lettuce, romaine
Liver, calves, fried
Molasses, blackstrap (third extraction)
Mung bean sprouted seeds, cooked
Oysters, fried
Peas, frozen, cooked
Soybeans, mature, cooked
Spinach, fresh, cooked
Squash, summer, cooked
Tomatoes
Turnip greens, cooked
Wheat bran, commercially milled
Wheat germ, commercially milled
mg iron/
100 kcal
3.1
5.2
3.8
7.8
5.4
7.6 .
3.2
3.4
2.8
2.1
9.6
2.9
2.3
.3
.0
5,
7.
2.6
mg iron/100 g
edible portion
of food
5.9
13.1
0.5
1.4
14.2
16.1
0.9
8.1
1.9
2.7
2.2
0.4
0.5
1.0
14.9
9.4
143
-------
Employing previously discussed Estimates of the amount of iron required
to maintain balance, adult males in developed countries should be absorbing
about 6% of dietary iron, and adult females should require about 12%. In
developing countries, 4% and 8% absorption, respectively, would seem adequate.
These requirements do not appear difficult to achieve when compared to absorp-
tion data obtained by the use of iron salts in fasting subjects. However,
they are large when the limited availability of dietary iron is considered.
In the past, the total iron content of the diet has been emphasized, and there-
fore on the iron contents of individual foods. However, it is not practical to
modify the iron content of a diet greatly by changing its composition except
through fortification, and food iron content per se: does not correlate well with
the amount of iron absorbed.
Availability of Dietary Iron
Until recently, direct attempts to measure dietary iron availability have
had limited success. Chemical balance studies have been technically difficult
and imprecise. To understand the availability of food iron in humans, it was
necessary to develop a methodology capable of overcoming certain problems.
186
One complicating factor was the variation in iron absorption among individuals,
which included differences in iron balance affecting the mucosal setting for
iron absorption and meal-to-meal fluctuations. The latter presumably was
related to physiologic differences in secretions and in motility of the gastro-
intestinal tract. Differences caused by variations in iron stores were controlled
by carrying out comparisons in the same individual, employing two isotopes of
iron. Sporadic variations were dealt with by studying about a dozen individuals
of the same age and sex, or by administering repeated doses of isotope so as to
103
determine absorption from the number of meals.
Employing an adequate experimental design, the absorption of iron from
single foods was evaluated. When compared to other forms of iron, hemoglobin
144
-------
was found to be particularly well absorbed by humans, and more important,
was found to be uninfluenced by chelates that could block absorption of nonheme
176
iron. Heme is taken intact into the mucosal cell, where it is catabolized
808
by heme oxygenase with the release of its iron.
Many foodstuffs have been biosynthetically labeled and their absorption
measured, as charted in Figure 4-3. Vegetal iron was poorly absorbed in normal
452
subjects (1-10%), whereas meat iron was better absorbed (5-20%). When these
studies were extended to evaluate mixtures of tagged foods, it became apparent
that individual foods interacted, affecting the availability of all the different
forms of nonheme iron present. Indeed, virtually all nonheme iron in a single
meal had the same availability. When added to a complex meal, a tracer dose of
radioiron salt was absorbed in the same level as nonheme iron in individual
187
food articles. Similarly, a tracer dose of radioactive heme was found to be
320,454
absorbed to the same degree as heme iron in food. These two extrinsic
tags provided the first accurate means of determining iron absorption from the
normal diet.
The two-pool, intrinsic tag method (heme and nonheme) has yielded estimates
of food iron absorption that closely agree with estimates based on normal body
iron losses. For example, meals composed of aliquots of all food consumed in a
typical 6-wk diet and doubly tagged with radioactive heme and nonheme iron were
77
administered to 32 young men. The total daily intake of iron in these men was
17.4 mg, of which only 1 mg was in the form of heme iron. Total absorption
averaged 1.25 mg/day. Absorption of nonheme iron averaged 5.3% or represented
0.88 mg/day, whereas absorption of heme iron averaged 37% or 0.37 mg of iron.
, 454
Similar data were obtained by Layrisse and Martinez-Torres, who fed their sub-
jects a meal of meat, black baansv maize, and rice containing a total of 4.5 mg
iron. In their normal subjects, absorption from 1.5 mg of heme iron was 27%
(0.34 mg), compared to an absorption of 3 mg of nonheme iron of 6% (0.12 mg) .
145
-------
Dose of
food Fe
N° coses
20-
15-
-^ 10-
-------
Total absorption from the meal was 0.46 rag iron,, These studies underline the
important contribution made by the heme iron to the diet.
If individual components affecting the availability of nonheme iron in a meal
could be identified, absorption from any type of diet should be predictable. A
number of inhibitors and enhancers of food iron absorption have been identified.
Phytic acid is often considered a potent inhibitor of iron absorption; however, the
effects of phytate on iron utilization are difficult to interpret because of several
intervening factors. For example, the number of iron atoms sequestered by the
phytate molecule have an effect. Iron from certain iron-phytate complexes is less
available for hemoglobin formation than that from more soluble iron salts. ' a>
' However, Morris and Ellis recently reported that iron from monoferric
phytate is readily available to rats. The research indicates that much of the iron
in wheat is monoferric phytate. Moreover, wheat iron is readily available to
rats and humans. Evidently different iron-phytate complexes have differing
availabilities, which must be taken into account when considering the effect of
phytate on iron availability.
Another complication is that inorganic phosphates also depress iron utiliza-
343
tion. Thus, in experiments in which the total phosphorus content of the diet
is not constant, it is not possible to separate the effects of phytate from those
resulting from increased phosphate content of the diet. a Phytate in vegetal food
sources (such as legumes) is often complexed to protein or other components of the
plant and may be less reactive than soluble phytate salts. The addition of soluble
salts of phytic acid to diets has reduced iron availability, but naturally-occurring
f Q oa
phytates may not act the same way. For instance, Sharpe e£ al. found that adding
sodium phytate to milk reduced the iron absorption of human subjects. However, when
the phytate was supplied by oats, no correlation was observed between the phytate
content of the oats and iron availability. Similarly, Welch and Van Campen a
found that the phytate concentration of soybeans was not correlated with the avail-
147
-------
ability of the soybean iron to rats. Thus it is not at all certain if naturally-
occurring phytates have the same influence on iron utilization as soluble phytate
salts.
Finally, recent reports of the effect of fiber on iron utilization have fur-
ther complicated analysis. Several investigators have reported that substitution
of whole-wheat bread for white bread or the addition of increasing quantities of
, „, _ _, ,. _ .,, , . ..... _. 75,2803,4093,6883,8153
wheat bran to the diet will depress iron utilization,, ' '
This phenomenon had been attributed to the phytate content of the whole mesl or
643 a
bran products. Yet phytin-free fiber'can reduce iron utilization in humans and
rats. Since wheat bran contains:both fiber and phytate, it is hard to discern
which is the critical factor in reduc'lhg iron utilization. At present it is
difficult, if not impossible, to assess the individual effects of fiber, phytate,
and phosphate on iron utilization.
Undoubtedly, a considerable number of substances may interfere with iron
absorption. Calcium and phosphate salts and ethylenediaminetetraacetic acid (EDTA),
which are added to American diets as food preservatives, have been shown to reduce
192 543
iron avsilability. ' At present, two food components have been shown to
enhance absorption. The availability of nonheme iron increases when the meal con-
tains animal tissues. ' For example, the substitution of 100 g beef for an
equivalent amount of egg albumin in a test meal increased absorption more than
191
5 times. Thus meat is important not only as a source of heme iron, but as an
enhancer of absorption of nonheme iron. Muscle from animals and fish and liver
have this property, whereas milk and cheese do note Ascorbic acid is another
potent enhancer of iron absorption because it can reduce iron and form a chelate
with ferric iron at low pH, effects that maintain solubility at the higher pH of
the duodenum. Recent studies have shown an enhancing effect on nonheme iron
absorption by relatively small amounts of ascorbic acid that were either contained
in or added to food during its preparation. For example, 60 mg
148
-------
ascorbic acid added to a meal of rice more than tripled absorption of iron,
and 150 g of papaya containing 66 mg ascorbic acid increased iron absorption
456
more than fivefold when taken with a meal of maize. Such studies of
dietary iron availability employing the extrinsic tag have clarified information
on iron absorption and loss and pointed out that availability may be even more
important than content in determing the amount of iron absorbed from food.
Some limitations in the extrinsic tag technique as applied to nonheme
iron absorption have become evident. These shortcomings do not invalidate
its use as described, but need to be considered in special situations.
Certain iron salts used in fortification, such as pyro- and orthophosphates
are incompletely miscible with the nonheme dietary iron pool.190 Certain forms
of food iron—such as ferritin and hemosiderin with their masses of ferric
hydroxide are also incompletely miscible—and it is presumed that soil iron
457
would be even more so.
Balance and Stores
From the information available on iron requirements and available iron in
the diet, humans appear to be uniquely restricted in external iron exchange.
Basal exchange in the adult male of 12 yg/kg/day may diminish to as little as
6 yg/kg/day with iron deficiency, and the adult menstruating female, with an
average requirement of about 20 yg/kg/day may decrease her daily requirement to
about 15 yg/kg. An iron-deficient American male should be able to absorb a
maximum of about 50 yg of iron/kg/day from his average diet, whereas the iron-
deficient menstruating female might be expected to absorb about 35 yg/kg/day.
These availability estimates for either sex may be halved, if the diet does
not contain enough meat or ascorbic acid. It is apparent that the male has a
considerable margin of safety, whereas a major portion of the female population
must be at risk. A prevalence of a 4% iron deficiency in the adult male popula-
tion (most of which is related to pathologic bleeding), compared to a 20% iron
184
deficiency in the adult female population, reflects this distinction. The
inability of most pregnant women to meet iron requirements is acknowledged by
149
-------
the routine therapeutic administration of iron salts. A high prevalence of
iron deficiency is also found in infancy, in which requirements of growth out-
195
strip available dietary iron. There is no question that iron balance in
menstruating and pregnant women and in infants is perilous.
One of the best indicators of an individual's iron balance is the extent
of ferritin and hemosiderin stores. They are normally found equally divided
749
among the hepatocytes, reticuloendothelial cells, and striated muscle.
These stores have been evaluated in various ways. The most direct but least
practical method is phlebotomy, which mobilizes iron stores; approximately 1 g of
storage iron has been measured in the adult male and one-third that much in the
42a,597,634
female. Another approximation of storage iron is provided by
707
examining a marrow aspirate for hemosiderin. In healthy individuals, a
negative correlation has been shown between the hemosiderin of the marrow and
800
the total iron binding capacity of the plasma. However, the effect of other
factors, particularly protein malnutrition, on the iron binding capacity, vitiate
the usefulness of this measurement in evaluating stores. One of the more in-
formative procedures has been the assay of a postmortem liver specimen for
nonheme iron. The results of some 4,000 determinations in 18 different coun-
160
tries have been reported by Charlton et al., and mean values for their male
subjects are summarized in Table 4-7. Assuming that the liver iron of such
individuals represented about one-third of storage iron, estimates of the size
of body iron stores could be made as well as comparisons among different popu-
lations .
401,535
Immunologic methods have been developed to measure serum ferritin.
The values obtained in several of experimental situations showed a close rela-
tionship to iron stores. That is, the geometric mean of about 90 in males as
compared to 30 in females was proportionate to the difference between iron
stores in the two sexes. A relationship has been shown between serum ferritin
188
and iron absorption and between serum ferritin and iron stores as determined
785
by phlebotomy. Only in hepatic disease and inflammation does the ferritin
478
level appear to deviate from this relationship.
-------
TABLE 4-7
Hepatic Storage Iron Concentrations
a
Males
Females
Countries
Great Britian
Sweden
Czechoslovakia
United States
South Africa
Caucasian
Bantu
No. of
Subjects
182
422
187
232
97
318
Mean, mg/kg
158
159
212
188
258
818
No. of
Subjects
128
300
187
121
42
133
Mean, mg/kg
156
129
182
144
161
270
Rhodesia
Bantu
Nigeria
142
681
76
228
Bantu
India
New Guinea
Venezuela
Brazil
Mexico
87
203
110
204
74
151
189 96
112 67
106
203
187
198
187
79
Adapted from Charlton et al.
151
-------
INTERNAL IRON METABOLISM
Internal iron exchange in mammals is dominated by iron requirements for
hemoglobin synthesis. The iron content of the erythron is about 1 g/kg tissue,
some 2 orders of magnitude greater than the essential iron content of other
body tissues. In the design of internal iron exchange, therefore, some means
had to be created whereby the disparate needs of individual tissue could be met
and excess iron could be stored, accomplished through the function of a plasma
548
protein, transferrin. The phylogenic appearance of this plasma protein co-
incided with the appearance of hemoglobin in red cells. When, as a genetic
oddity, transferrin is absent in a human, the unique finding of iron deficiency
anemia despite iron overload of other body tissues is observed. This phenomenon
304
is clear evidence of the protein's essential function in iron exchange.
The first steps in binding appear to be the interaction of transferrin with
iron, and the release of three protons; the second step involves the inclusion
of bicarbonate in the iron transferrin complex and isf associated with the
development of a salmon-pink color. The reverse reaction occurs with iron
release. Iron binding above pH 7.2 is maximal, with a binding constant in plasma
24 -1 9
of approximately 10 M ; as the pH is reduced to < 6.5, iron starts to disso-
721
ciate, and it is nearly completed at pH 4.5. In vivo transferrin shuttles iron
back and forth between body tissues without being used up itself. The behavior
of the two iron-binding sites has been the subject of some controversy: it is
undecided if they participate independently and equally in the binding and
release of iron, or if differences in affinity exist between them and certain binding
8
tissues. Recent evidence favors the homogeneous behavior of transferrin iron,
625
with specific tissue receptors decisively determining the exchange. The ability
7,730,828
of transferrin to bind a variety of trace metals is noteworthy. However,
152
-------
the patterns in which these metals are released are quite different from that
of iron and they are not assimilated by immature erythrocytes in any appreciable
amount.
Current methods for measuring plasma iron largely exclude hemoglobin and
386a
are therefore assumed to represent isolated transferrin iron. However, they
will measure part of parenterally injected preparations such as iron dextran.
The normal level of human plasma iron is about 100 yg/dl and saturation of trans-
89
ferrin with iron is about 33%. Whereas transferrin concentration is relatively
stable from hour to hour and day to day, serum iron in the normal individual may
vary over 24 h between 50-200 yg/dl. Diurnal variations are normal, with morn-
ing values at least 50% higher than evening values. The higher levels of plasma
iron commonly found in animals are related to a much greater iron absorption,
185
and fasting will reduce the level to about 100 yg/dl. Decreases in plasma
iron are associated with exhaustion of iron stores, inflammation, increased
89
erythropoiesis, and reduction in transferrin concentrations. Elevations in
plasma iron are caused by increased absorption, hepatic damage, increased blood
destruction (in particular, ineffective erythropoiesis), and the parenteral
administration of iron. Figure 4-4 sets forth relationships between plasma iron
and transferrin concentration observed in various diseases.
The erythron contains 90% of essential body iron in most mammalian systems
and is therefore the primary user of transferrin iron. Iron is taken up by the
immature red cell, incorporated into its hemoglobin, and remains within the
cell for its life-span. Uptake initially involves the adherence of the trans-
ferrin iron complex to membrane receptors. Then the complex is internalized
in microtubles and iron is removed, followed by the return of transferrin to
348,413
the surrounding plasma. Within the erythroid cell, more than 80% of the
153
-------
TRANSFERRIN
Unbound
\ 3
1
1
Hemolyfic anemia
(ineffective erythropoiesis) °
Idiopathic J
hemochromatosis
Reticuloendothelial \[
iron overload
Hypoproteinemia [ 4
Bound
Normal
g Iron deficiency
ra Infection
^^^^^ Hemolytic anemia
iililllll
>0^^^^^^^
56^^^^^^
^
Saturation (%)
FIGURE 4-«4 Relationships between transferrin and plasma iron in
disease. Reproduced from Bothwell and Finch.
89
154
-------
586
iron is delivered to the mitochondria, where it is converted into hemoglobin.
Excess iron is deposited in the cytoplasm in the form of ferritin aggregates.
Although a coordination exists between heme and globin synthesis that keeps
their production balanced, the ratio of production of these moieties has little
effect on the uptake of iron by the developing red cell. If hemoglobin synthesis
is reduced, excess iron will accumulate in ferritin stores with aberrant globin
synthesis or in mitochondria with abnormal heme synthesis.
Usually, transferrin iron above 50 yg/dl provides the erythroid marrow with
the amount of iron it needs for hemoglobin synthesis. However, the adequacy of
iron supply is influenced by changes in requirement by the erythroid marrow as well
as by fluctuations in plasma iron concentration. As production increases, the
minimal level of plasma iron required to support erythropoiesis increases propor-
404
tionately. The adequacy of iron supply versus erythron needs may be monitored
by red cell protoporphyrin, which increases as the ratio of plasma iron supply to
441
erythron needs falls.
Quantitative aspects of iron exchange between transferrin and the erythron
270
have been studied in detail with iron isotopes. The basal turnover of this
fraction depends on the life-span of the red cell and varies from about 3%/day
in mice to 0.8% in humans. However, with the wastage iron of erythropoiesis,
iron turnover within the erythron is greater than that calculated from red cell
life-span. Each day in the adult human, some 25 mg of iron is used by the
270
erythron. The anatomic location of the erythroid marrow may be visualized by
770
the positron camera after injection of iron-52, and the uptake and release of
iron may be measured by surface probes placed over marrow-rich areas such as the
271
sacrum.
The other component of the erythron iron circuit is the reticuloendothelial
cell, which processes the iron of senescent or defective red cells and returns it
155
-------
to plasma transferrin. The wastage iron of erythropoiesis is released to the
reticuloendothelial cell at the time of red cell maturation. Within the reticulo-
endothelial cell, red cell hemoglobin is catabolized. Its iron is either directed
to the cell membrane to be taken up by transferrin, an exchange in which cerulo-
637
plasmin participates, or it is deposited within the cell as ferritin. In
266
dogs, about one-half of catabolized red cell iron goes by each pathway; in
267
humans, only one-third is held in storage. The partition between these two
pathways is inconstant, and the variation is thought to be largely responsible
for the diurnal variation in plasma iron. During inflammation, the greater
deposition of ferritin iron within the reticuloendothelial cell results in lowered
266,353
iron concentrations in plasma.
An alternate pathway for transferrin iron uptake involves the liver and
270
other parenchymal tissues. The hepatocyte accepts and stores iron when uptake
by the erythron decreases, as illustrated by in vivo profiles obtained in patients
270
with aplastic anemia. Studies in the rat show some localization in muscles,
skin, and subcutaneous tissues, but the largest amount resides in the intestinal
162
mucosa, reflecting the ability of that species to excrete iron through the gut.
The only other well-delineated pathway in humans is the exchange of plasma iron
549
with the extravascular transferrin pool. The total format of internal iron
exchange is shown in Figure 4-5. The diagram does not illustrate the uptake by
hepatocytes of the hemoglobin iron released through intravascular hemolysis of
351
red cells.
Isotopic studies of plasma iron kinetics have provided a better appreciation
150
of these various pathways and their functional interrelationships. More
detailed data on kinetics is available for humans than for other mammals.
Iron turnover through the plasma is considerably greater than was expected for
the needs of the circulating red cell mass, an excess largely explainable by a
156
-------
INTERNAL IRON EXCHANGE
Circulating
Erythrocytes
Gut
Extrovascular
exchange
FIGURE 4-5 Internal iron exchange. Reproduced from Hillman and
Finch.
365
157
-------
624
reflux of radioiron from tissues. Part of this reflux represents the wastage
iron of erythropoiesis, amounting to about 24% of plasma iron turnover in humans
189
with a half-time of about 7 days. The second portion of this reflux, 8% of
plasma iron turnover with a half-time of about 7 h, relates to extravascular
exchange of transferrin iron. In humans, absorbed iron comprises only about 3%
of plasma iron turnover and has little effect on internal exchange. By contrast,
185
as much as 50% of plasma iron turnover in the rat may be derived from absorption.
Detailed ferrokinetic analyses of humans have shown that the nonerythron turnover
fraction, including extravascular flux and parenchymal uptake, is proportionate
189
to the amount of plasma iron. Thus, by an empirical calculation of this frac-
tion and its subtraction from the plasma iron turnover, the amount of erythron
iron turnover can be ascertained accurately. The erythron in a variety of dis-
270
orders has shown a large range of iron uptake: from 0-4 mg/kg (normal, 0.4 mg/kg).
Parenchymal uptake is more limited and reaches maximal values of about 0.6 mg/kg,
and only when plasma iron is high and transferrin is saturated. It is not possible
to measure iron stores by short-term kinetic measurements, because the early
mixing of radioiron with stores is limited. As erythron iron cycles through the
reticuloendothelial cell, it will mix with ferritin, but approximately a year
308
is required for stores to reach equilibrium. Hemosiderin stores may not
exchange iron. Kinetic measurements revealed the difference between reticulo-
270
endothelial and parenchymal iron storage. Reticuloendothelial iron stores
are the products of red cell catabolism, whereas parenchymal iron stores are
created when elevated plasma iron leads to an increased hepatocyte uptake. Hepa-
tocyte loading may be further augmented in hemolytic states when the hemoglobin
and ferritin are transported to them.
158
-------
IRON BALANCE IN ANIMALS
Information on iron balance in mammals is more fragmentary than data for
humans, but marked differences are known to exist that must be taken into
account when interspecies inferences are drawn. For example, the rat has an
active excretory mechanism whereby plasma iron is taken up by the gut mucosa
162,178
and lost from the body when mucosal cells are exfoliated. Under basal
conditions, 10-15% of the plasma iron turnover follows this pathway, and the
amount may be increased when an animal is given iron supplements. A rat's
dietary iron intake is greater than 100 times that of a human's when expressed
185
on a per kilo basis. Absorption of nonheme iron is more efficient than in
humans, whereas heme iron is less well absorbed. As much as 50% of the iron
entering a growing rat's plasma may be derived from absorption, and the plasma
iron is elevated to 2-3 times that of humans as a consequence. Growth require-
ments are much greater, and erythropoiesis in the newborn rat is 2-4 times
greater than that of the fully grown animal when expressed per kilogram body
weight because of the requirements imposed by expansion of blood volume. These
differences make comparison between rats and humans difficult. The use of the
rat to evaluate iron availability of foods is equally suspect. From the stand-
point of iron balance, rats are far better off than humans. Not only is iron
deficiency virtually nonexistent, but it has been impossible to produce a pattern
of tissue damage similar to that seen in humans with iron overload.
These differences observed in the rat apply to a greater or lesser degree
758
to other animal species. Differences are to be expected in the adequacy
of iron balance at different ages, the rate of iron exchange, and the ability
to absorb heme versus nonheme iron. Some animals, such as the pig, are born
with very low iron stores and others, such as the rabbit, with very adequate
reserves. Animals generally seem to have little problem in maintaining iron
159
-------
balance unless removed from their natural environment and placed on an arti-
ficial diet.
Although information concerning iron metabolism of most farm animals is
limited, enough concern has arisen about their nutritional needs to define dietary
iron requirements. Swine appear to have the greatest problem in maintaining iron
758
balance, because of their very rapid growth and low iron stores at birth.
Estimates of food iron required for baby pigs range from 60-125 mg iron/kg dry
521
diet. Matrone e_t al. reported that shoats fed a milk substitute needed
98
125 mg/kg of food iron to maintain normal growth and hemoglobin. Braude et al.
stated their estimates in another way: baby pigs must retain 21 mg of iron/kg
live weight increase to maintain normal iron status. All these estimates of
iron requirements are complicated by the variable utilization of dietary iron.
521
Matrone et al. found that 30% of the iron in swine diets was utilized for
756
hemoglobin synthesis, whereas Ullrey et al. found that 82, 61, and 50% of
the food iron was utilized when the diets contained 25, 35, and 125 mg iron/kg
98
diet, respectively. Braude et^ al. reported from 30-60% retention of orally
administered iron, according to the method of dosing. As a result of these and
other studies, the National Academy of Sciences has recommended that diets for
569
growing pigs contain at least 80 mg iron/kg dry matter. Requirements for
adult swine are not known; however, because parasitism is not a problem,
natural feed ingredients evidently provide adequate iron intake.
The iron requirements of poultry for growth have been studied. Hill and
364
Matrone suggested on the basis of blood data that a diet containing 50 mg
216
iron/kg would meet the chick's requirement. Subsequently, Davis et al.
reported that 4-wk-old chicks required between 65 and 105 mg iron/kg diet to
i
maintain normal weight gains and hemoglobin levels. They later observed that
chicks fed a soybean protein or a casein-gelatin based diet required 75-80 mg
160
-------
217
iron/kg of diet. Thus, it appears that diets for growing chickens should
contain a minimum of 50 mg iron/kg and that 80-100 mg of the element are re-
quired to provide an adequate safety margin. Reliable estimates for mature
chickens are not available; however, the demand for iron by laying hens is
very high. An average hen's egg contains about 1 mg of iron—thus laying hens
require some 200 mg iron/yr for egg production. Although hemoglobin levels
will decline slightly with the onset of laying, no striking reduction of body iron
638
stores has been measured in laying hens. Because iron deficiency is not con-
sidered a practical problem in poultry, it should be safe to assume that diets com-
posed of natural feedstuffs supply enough iron for growing and laying chickens.
Veal calves fed unsupplemented whole-milk diets rapidly developed anemia.
520
Matrone et al. fed calves a diet of whole milk that provided 30 or 60 mg
iron/day depending on whether or not iron supplements were added. Both diets were
adequate in maintaining normal growth and hemoglobin levels from birth to
40 wk of age. In this case, 30 mg iron/day was sufficient for calves in the
age group studied; however, the dose is considerably smaller than the value of
79 99
100 mg/day suggested by Blaxter et al. Bremner and Dalgarno reported that
for calves on a fat-supplemented skim milk diet, 40 yg iron/g diet prevented
all but a very mild anemia when fed from 17 days to 11 wk of age. The hemo-
globin levels of these calves were increasing at the end of the experiment, and
the authors speculated that they would have been normal had the dietary regime
been continued to the calves' normal slaughter weights. They found that similar
results were obtained when iron was supplied as ferrous sulfate, ferric citrate,
or ferric EDTA; however, iron phytate was not utilized as well as the more solu-
ble iron compounds. Requirements for mature cattle are not known; however, it
has been estimated that milking cows require 50-60 mg iron/day and that pregnant
432
cows need 60-80 mg/day. These levels should be met by virtually any diet
normally consumed by these animals.
161
-------
450
Data on iron requirements of sheep are limited. Lawlor et al. experi-
mented with sheep fed purified diets containing levels of iron ranging from
10-280 ppm. They found that 10 ppm was clearly inadequate for growing lambs,
25 ppm did not support normal growth, and 40 ppm appeared to be adequate. They
concluded that the minimal requirement for iron in growing lambs was in excess
of 25 ppm but no more than 40 ppm. No experimentally derived estimates of iron
requirements for mature sheep are available; however, mature sheep need about
432
10-15 mg of iron/day. This requirement is met when the animal's feed averages
10-15 ppm iron. As forages and feed grains uniformly contain considerably more
than 15 ppm iron, healthy sheep fed normal diets should have no trouble in
meeting their iron requirements.
There are several extensive compilations of feed analyses that include data
536,567-569 757
on iron content. Underwood summarized the iron content of common
animal feeds: grasses, 100-250 mg/kg; legumes, 200-300 mg/kg; cereal grains,
30-60 mg/kg; and oilseed meals, 100-200 mg/kg. Except for milk and milk by-
products, feed byproducts of animal origin contain high levels of iron. The
following figures are representative of the iron content of some of these
materials: bloodmeal, 3,108 mg/kg; fishmeal, 381 (range,210-800); meatmeal,
439 (range, 180-920); dried skim milk, 52 (range,5-104) mg/kg dry material.
Additionally, many inorganic materials fed to animals as sources of calcium and
phosphorus—i.e., ground limestone, rock phosphate, ground oyster shells, and
757
dicalcium phosphate—contain high levels of iron, frequently more than 500 mg/kg.
The high levels of iron in common animal feeds explain why iron deficiency is not
generally a practical problem in farm animals, except in young animals consuming
only milk or milk-product diets. Specific information, however, on the availa-
bility of these different iron salts is lacking.
162
-------
CHAPTER 5
IRON DEFICIENCY
PREVALENCE
Worldwide and in the United States, iron deficiency is the most
common nutritional problem and cause for anemia. However, estimates of
prevalence are difficult to determine with precision because of problems
in defining what constitutes iron deficiency.
Three stages of iron deficiency can be defined. The mildest form,
depletion of iron stores, sometimes termed pre-latent iron deficiency, is
characterized by decreased or absent storage iron. No other abnormalities
are identifiable. To detect pre-latent iron deficiency, iron stores must
be measured in one of three ways: by phlebotomy to the point of iron-deficient
339,785
erythropoiesis, which yields a quantitative assessment of iron stores
(the amount of iron in the hemoglobin removed); by a semiquantitative esti-
mate derived from the amount of stainable hemosiderin in bone marrow
641
aspirates or biopsies; or by measuring an increase in intestinal iron
344,830
absorption, which is correlated with decreasing iron stores. Because
none of these methods has been practical for large population studies, con-
siderable interest has arisen in the recently introduced serum ferritin
measurements. These sensitive imraunoradiometric assays appear to reflect
691,718,760,761,785
iron stores. Although few reports have been published to
date on the efficacy of serum ferritin concentrations to detect iron deficiency
134,184,829
in large population studies, the approach seems a promising one.
The second stage, latent iron deficiency, ensues after iron stores are
depleted. It is characterized by reduced serum iron concentration, elevated
transferrin concentration, and a resultant drop in transferrin iron saturation
35
to less than 15%. Concomitantly, hemoglobin synthesis is impaired by iron
163
-------
lack,causing erythrocyte protoporphyrin concentration to rise to more than
441
70 yg/dl. However, in this stage of iron deficiency, circulating hemo-
globin concentrations remain within the normal range, although treatment with
290
iron results in slight hemoglobin increases.
Overt iron deficiency anemia, the third stage, is triggered when the
restricted hemoglobin synthesis contributes to a measurable decrease in the
concentration of circulating hemoglobin or in the volume of packed erythro-
cytes (hematocrit). Tests of large populations have been able to discern
only overt or latent iron deficiency. Studies that add serum ferritin
measurements to transferrin saturations and erythrocyte protoporphyrin
determinations may detect the earliest stage of iron deficiency, which will
provide increased precision in defining iron deficiency in population sur-
134,184,401,402,478
veys.
Another difficulty in estimating the prevalence of iron deficiency in
population studies is the uncertainty about what constitutes an exact range
of normal hematologic values. Anemia is difficult to define with precision
for different age groups. Most "normal" values are arbitrary and represent
the best judgments of committees of experts; indeed, sharp demarcation of
49
continuous variables may be impossible. Another problem stems from
sampling biases: populations with varying susceptibilities to iron
deficiency must be included to obtain a valid distribution rather than
projecting population data from easily sampled groups. Older studies often
suffered from the loose attribution of iron deficiency as the cause for most
of the anemias detected. Multifactorial causes of anemia and the difficulty
of separating serum iron changes associated with infection or chronic
disease from those of iron deficiency contribute to the problems of inter-
49
preting prevalence data. These complexities are discussed by Beaton, in
a thoughtful review of the epidemiology of iron deficiency.
164
-------
When values of transferrin iron saturation of less than 15% are used
35
as a criterion of iron deficiency, the prevalence of iron deficiency in
830
selected populations of various countries is shown in Table 5-1. These
results—from relatively small numbers of subjects in each group (48-220)—
emphasize a population particularly at risk (pregnant women), but they also
show the incidence of iron deficiency in men and nonpregnant women. The
study also demonstrates the discrepancy between iron deficiency as defined
by low transferrin saturation, and anemia as defined by hemoglobin levels
below arbitrary standards. In addition, other or coincidental causes of
anemia, such as those based on deficiencies of vitamin 8,2 or folic acid,
are identified. By the criterion of low transferrin saturation, 40-99% of
the pregnant women sampled from the 6 countries studied were iron-deficient.
Nonpregnant women showed 11.4-42.5% iron deficiency, and men showed a.range
of 0-8.8% iron deficiency.
Other large population studies have confirmed the prevalence of iron
829
deficiency in many countries of the world. In the United States, infor-
mation about the prevalence of iron deficiency has been derived largely
195
from patients seen by physicians, but two large populations have been
760,761
surveyed. The first was designed to obtain information about nutri-
761
tional deficiencies in a low-income population of the United States.
The second study, the "HANES" preliminary report, provides a more balanced
760
appraisal of nutrition in the United States. Again, discrepancies are
apparent between different criteria of anemia (such as hemoglobin and hema-
tocrit values) and raise questions concerning the standardization of methods,
the criteria of iron deficiency, and differences among populations studied.
Iron deficiency, estimated from a serum transferrin saturation of less than
760
15%, was observed in about 10% of children aged 1-5 yr. Projecting this
percentage to the total population of children in the United States, the
165
-------
TABLE 5-1
Prevalence of Anemia a
and of Iron, Vitamin Bi->, and Folate Deficiencies*
Country
Israel
Poland
India
(Delhi)
India
(Vellore)
Mexico
Venezuela
Subjects
Women
pregnant
nonpregnant
Men
Women, pregnant
Women
pregnant
nonpregnant
Women
pregnant
nonpregnant
Men
Women
pregnant
nonpregnant
Men
Women
pregnant
nonpregnant
Men
Propor-
tion
of cases
with
anemia, 7.
47.0
29.0
13.6
21.8
80.0
64.3
56.0
35.0
6.0
26.6
11.7
0.9
37.0
14.9
1.9
Proportion (2) of cases with the fol-
lowing deficienies
Serum
iron,
< 50 uR/dl
36.0
15.0
12.1
31.4
10.0
9.5
99.0
51.0
7.1
30.5
22.1
6.2
56.8
14.0
7.5
Trans -
ferrin
satur-
ation,
< 157.
46.3
11.4
8.8
40.0
51.7
25.8
99.0
42.5
5.1
61.2
28.1
3.6
59.7
18.9
0
Serum
vitamin
B12>
< 80 pg/ml
2.0
0
0
0.5
49.0
26.7
0
3.0
0
7.1
0
1.1
23.0
1.0
2.0
Serum
folatej
< 3 ng/ml
6.3
5.1
1.6
1.4
9.0
0
2.0
6.5
6.0
3.5
15.1
9.5
18.8
The following hemoglobin concentrations per deciliter of blood are
considered to indicate the presence of anemia:
Children, 6 mo-6 yr < 11 g/dl
Children, 6-14 yr < 12 g/dl
Adult males < 13 g/di
Adult females, nonpregnant < 12 g/dl
Adult females, pregnant < 11 g/dl
^Derived from the World Health Organization.
830
166
-------
authors estimated that a total of 383,000 children met this criterion of
iron deficiency. Adolescent girls and women during their reproductive
years were far more likely to be iron-*deficient than men, consistent with
physiologic iron losses of menses and pregnancy (see Chapter 4). Consider-
able additional information is included from separate analyses of subgroups
of different races and economic status. However, some information of
interest to epidemiologists is hidden by analyses that define low trans-
ferrin saturation differently (
-------
CAUSES OF IRON DEFICIENCY
Most cases of iron deficiency in humans are caused by insufficient
dietary iron, iron losses, or malabsorption of iron. Although each of
these causes has important health implications, the problem of insufficient
iron assimilation is probably the problem most amenable to solution.
Insufficient Dietary Iron
People fail to assimilate enough iron from the food that they eat
because of absence of iron in the food itself; insufficient quantities of
iron-containing foods; ingestion of cleaner food to which no extraneous
iron has been added during processing; interference with absorption because
of natural or artificial inhibitors of dietary iron absorption; and reduced
intake of substances that promote iron absorption. Often a combination of
these factors will play a role in reducing the assimilation of iron from
food (see Chapter 4).
As can be seen from Table 4-6, many foods are poor in iron. Prominent in
this list are milk and milk products, many fruits and vegetables, sugar,
and salad and cooking oils. There is little wonder that infants and small
children, whose major caloric intake is from cow's milk, are at risk of
iron deficiency. Intake of mother's milk instead of cow's milk improves
the absorption of iron. Selecting foods low in iron may
be a function of habit, economic necessity, or of religious and cultural
reasons, but the result is the same: an iron intake insufficient to balance
losses and eventual development of iron deficiency.
The quantity of food consumed by most people is based on their caloric
49
requirements. Accordingly, men and boys eat more food than women and
girls. Although iron content and availability vary considerably in indi-
vidual foods, the mixtures of foods people eat provide a fairly constant
49
iron concentration in relationship to caloric intake. Beaton has
168
-------
tabulated this iron:kilocalorie ratio from calculated and measured patterns
of dietary iron consumption in many countries. In the United States, the
iron concentration in milligrams per kilocalorie ranged from 4.9-6.3 in
49 760
one study and was about 6.0 in another. Iron intake calculated in re-
lation to energy requirements for men expending 3,000 kcal yields about
18 mg dietary iron from the average American diet. Women whose energy
expenditure is approximately 2,000 kcal would be expected to consume about
12 mg dietary iron. This latter value is close to that actually measured in
50
a group of 97 Canadian women and exceeded the mean value (9.9 mg) recorded
544
for 13 young American women. Caloric intakes of children are related to
760
age, but vary within the range of 1,500-2,000 kcal, which would provide
9-12 mg iron from diets similar to those consumed by adult members of a
family. Thus, lower caloric intakes in women and children place them at
risk of insufficient dietary ingestion of iron.
The possibility that iron intake is somewhat dependent on the addition
of extraneous iron during food preparation was emphasized by the major dis-
crepancies found in calculated and chemically analyzed iron concentrations
of diets sampled primarily from institutional dietary kitchens in a variety
49
of foreign countries. Contamination of acid foods from iron cooking pots
545
has been demonstrated by Moore and the addition of iron to Kaffir beer
83
consumed by the Bantu is well documented. The converse, reduced iron con-
tamination of foods from the use of stainless steel and glass cooking vessels,
seems to be more widespread in developed countries where modern food processing
prevents marked contamination. Several studies have showed close corre-
50,544,832
lation when iron content of diets from actual chemical measurements
794
is compared to calculated iron content from food tables.
169
-------
Availability of iron in foods depends considerably on absorption
from single food sources as well as the interaction of foods on the ab-
sorption potential of the iron compounds they contain. The graph of foods
labeled with radioactive iron shown in Figure 4-3 indicates a wide range
451
of variation in absorption of food iron. As a rule, iron in cereals
and vegetables is poorly absorbed, whereas iron from animal sources,
especially heme-containing compounds, is absorbed much better. Furthermore,
absorption of vegetable food iron is almost doubled when it is ingested
458,517,519a
with heme from meat, liver or fish or when ascorbic acid is
77,139,435,667
added. Iron absorption reflects that of the least well
absorbed vegetable when two vegetables are mixed, and is the same as the
vegetable iron when up to 60 mg of a ferric or ferrous iron salt is mixed
78,187,454,455
with a vegetable. Addition of a chelating agent (desferrioxa-
187,435
mine) diminishes the absorption of vegetable iron. Heme iron compounds
from meat, liver and fish are not affected by desferrioxamine or ascorbic
187,454
acid.
These results have been interpreted as evidence of two major iron
pools, i.e., one a heme and one nonheme pool with the various iron compounds
in each pool having the same percentage of absorption. Each pool is affected
by substances present in food that inhibit or enhance the metal's absorption.
Furthermore, iron compounds used for food fortification are absorbed to the
187,455
same extent as foods in the nonheme pool.
The distinction of dietary iron according to iron pools has permitted
measurements of iron absorption from complex dietary mixtures in which the
interactions of iron from many sources were once difficult to discern. A
tracer dose of iron salt such as iron-59 chloride to label the nonheme pool
170
-------
mixed with iron-55-labeled hemoglobin provides separate measurements of
the nonheme and heme pools of a dietary mixture and an accurate estimate
of percentage and total absorption from the two forms of iron. This
extrinsic method of labeling also makes it possible to measure absorption
from iron compounds used for food fortification as well as from the naturally
occurring iron of foods. Diets with an adequate total amount of iron but
containing little heme, which enhances absorption of the rest of the iron
intake, may not be adequate to meet nutritional requirements. Thus, forti-
fication of foods with iron will be dependent not only on the amount of iron
added, but also on the overall availability of iron in the diet.
Natural dietary inhibitors that decrease iron absorption include phy-
343
tates, phosphates, oxalates, and carbonates, which form insoluble pre-
760 231 76,455,518
cipitates. Substances in tea (tannins), corn, milk and
76,139,546
eggs also reduce absorption. Artificial substances that may
interfere with iron absorption include ethylenediaminetetraacetic acid
192,278
(EDTA), which is added to many foods as a preservative.
The antacids given with various types of therapeutic iron salts reduce
247,339,667
absorption of the mineral. No information is available about
the effects of antacids on food iron absorption per se, although decreased
absorption of the nonheme component would be likely. Some types of clay
538
and dirt consumed accidentally or intentionally by subjects with pica
will reduce iron absorption. Pancreatic secretions (probably because of
56
the bicarbonate component) also reduce iron absorption.
Iron Losses
Iron balance in the human subject can be upset by iron losses of two
sorts: common physiologic losses that exceed iron intake and pathologic
iron losses. Iron deficiency may be a consequence of either.
171
-------
The obligatory physiologic losses in the urine, feces, sweat, and
desquamation of cells from epithelial surfaces are discussed in Chapter 4,
as is normal menstrual blood loss. Unrecognized menorrhagia is one of the
most frequent causes of iron deficiency in women during their reproductive
years. Using analyses of subtle changes in hemoglobin concentrations, mean
corpuscular hemoglobin concentrations, and serum iron levels, Scandinavian
investigators concluded that the upper range of normal menstrual losses was
321
about 60-80 ml. Above this level, bleeding was labeled abnormal or
659
menorrhagic. Factors increasing menstrual flow include genetic factors,
658 838
multiple childbirth, and the use of intrauterine devices for contra-
171,584
ception. Oral contraceptives tend to reduce menstrual losses.
As mentioned, pregnancy and delivery contribute to physiologic iron
losses. Lactation depletes the maternal iron stores by approximately
195
0.5-1.0 mg/day, which may be partially counterbalanced by the absence of
menses during lactation.
When pathologic causes of iron loss are examined, most of them
involve blood loss. In sheer numbers, the menorrhagia suffered by 10-25%
of women is the most common form of abnormal blood loss throughout the world.
Other obvious forms of blood loss are nosebleeds, wound bleeding, hematuria,
and bleeding hemorrhoids. Less overt are the many sources of gastrointestinal
bleeding (shown in Table 5-2) that cannot be detected solely by clinical means.
In men and postmenopausal women, occult blood loss into the gastrointestinal
tract is the most common cause of iron deficiency. The frequency of lesions
69
responsible for this bleeding was estimated by Beveridge. He found that
hemorrhoids were responsible in 10% of the cases, salicylate ingestion in
8%, peptic ulceration and hiatus hernia in 7% each, diverticulosis in 4%,
neoplasms in 2%, and undefined causes in 16%.
172
-------
TABLE 5-2
Sources of Blood Loss in the Alimentary Tracta
Esophagus
Varices
Hiatus hernia
Stomach
Varices
Ulcer
Carcinoma
Gastritis
Le iomyoma
Small bowel
Ulcer
Aberrant pancreas
Meckel's diverticulum
Telangiectasia
Polyp
Carcinoma
Regional enteritis
Helminthiasis
Vascular occlusion
Intussusception
Volvulus
Le iomyoma
Biliary tract
Trauma
Cholelithiasis
Neoplasm
Aberrant pancreas
Ruptured ancurysm
Intrahepatic bleeding
Colon
Ulcerative colitis
Amebiasis
Carcinoma
Telangiectasia
Diverticulum
Rectum
Hemorrhoids
Ulceration
Carcinoma
aFrom Fairbanks et al.
264
173
-------
Infestation with hookworm is a common cause of blood loss and iron
459
deficiency in tropical countries. It is estimated that a worm load
sufficient to produce 1,000 eggs/g of feces will result in the loss of
2.1 ml blood (0.8 mg iron)/day when Necator americanus is the parasite
652
and 4.5 ml blood (1.8 mg iron)/day when Ancylostoma duodenale is the agent.
Further studies set an upper limit on the degree of parasitism -~ 2,000 eggs/g
feces in women, and 4,000-5,000 eggs/g in men--that would not undermine the hemo-
460
globin concentrations of a population. Parasitism of greater degrees pro-
duced an anemia directly correlated with egg counts because blood and iron
losses exceeded the compensatory iron absorption mechanisms.
Other less common forms of abnormal iron loss include blood donation
and disorders that produce intravascular hemolysis followed by iron loss as
hemoglobin or hemosiderin in the urine. Blood donation results in the loss
of about 200 mg iron per unit removed (assuming a 450-ml unit of blood with an
average hemoglobin concentration of 14 g/dl). Repeated blood donations at
intervals of about two months can be tolerated by many male donors whose body '
597
iron stores are depleted, but who avoid anemia by increasing their absorp-
323
tion through a generous intake of dietary iron. Women donors, at risk
because of menstrual losses as well as the lesser intake of dietary iron,
282
frequently develop iron deficiency unless they receive iron supplementation.
Iron can be lost through hemosiderinuria resulting from intravascular
hemolysis. In intravascular hemolysis, depletion of haptoglobin leads to
filtration of hemoglobin dimers through the glomerulus and iron deposition
in tubular cells. These cells, laden with hemosiderin, are later excreted
into the urine. Although uncommon in the general population, this process
may induce profound iron deficiency in patients with paroxysmal nocturnal
174
-------
hemoglobinuria, prosthetic heart valves, atrial myxoma, or malaria. Urinary
262
iron losses from intravascular hemolysis may be as high as 34 mg/day.
An even rarer cause of iron deficiency is pulmonary siderosis, brought
on by hemorrhage into pulmonary alveoli. Iron, deposited as hemosiderin,
remains in the alveoli and is not resorbed into the body to any major degree.
Thus, large deposits of iron may be found in the lung although the individual
is iron-deficient.
Malabsorption
Compared to blood loss, impaired gastrointestinal absorption of iron is
a rare cause of iron-deficiency anemia. Two major types of impaired iron
absorption are recognized. One results from gastrointestinal surgery and
the other from various disorders commonly lumped together under the term
"malabsorption syndrome."
2
Iron-deficiency anemia occurs in most patients after total gastrectomy
and in 10-50% of patients who have a partial gastrectomy, especially those
36,324,368
who undergo the Billroth II surgery that bypasses the duodenum.
Typically, the anemia develops progressively during a 10-year period after
the operation and, predictably, it is more frequent and severe in menstru-
36
ating women. Studies with iron radioisotopes indicate that food iron
324,368,616
absorption is impaired; the subjects' absorption of medicinal
iron was normal but did not increase appropriately in response to the
38,324,700
anemia.
In malabsorptive syndromes such as celiac disease caused by gluten entero-
pathy, epithelial cells are damaged and villi in the proximal small intestine
31,32
atrophy. Consequently, iron and various other nutrients are not well absorbed.
Clinically detectable improvement following treatment with a gluten-free diet
31,138
results in improved iron absorption . in most patients.
175
-------
DELETERIOUS EFFECTS OF IRON DEFICIENCY
Several tissue changes and metabolic abnormalities have been observed
in association with iron deficiency in human beings and in experimental
animals. In some cases the causal relationship of iron deficiency is clear,
but in many instances the association is merely inferential. In the follow-
ing discussion, an effort will be made to distinguish between the two.
Specific Tissue Changes
Iron deficiency, despite its wide prevalence and its associated morbidity,
rarely causes death. Consequently, information from autopsies is scarce
723,823
about pathologic tissue changes. More information is available
for tissues that can be studied readily through biopsy during life, such as
peripheral blood lymphocytes, bone marrow, the oral cavity, and the gastro-
394
intestinal tract. These tissues contain rapidly proliferating cells that
are more likely to show a lack of iron earlier than cells with a slow turn-
207
over.
Bone marrow. The most obvious change in the bone marrow of an iron-
deficient patient is the absence of sideroblasts and of stainable iron
granules in reticuloendothelial cells as storage iron deposits become
288,331,641
mobilized. The erythroid precursors in a hyperplastic marrow
203,822
increase, but hypoplasia may also be observed, accompanied by in-
67,126
creased numbers of reticulum and plasma cells. Poor correlation
often exists between the degree of severity of the anemia and the degree of
67,784
erythroid hyperplasia. Normoblasts with ragged edges and reduced
822
cytoplasm are characteristic of iron-deficient marrow. However, most of
these changes are too general or variable to be pathognomonic.
Gastrointestinal tract. Lesions connected with iron deficiency have
been described in all portions of the gastrointestinal tract. The predomi-
nant lesion is mucosal cell atrophy, which sometimes includes a component
176
-------
395,396
of mononuclear cell infiltration. In cells of the oral mucosa, Jacobs
found abnormally thin epithelia, increased mitoses in the prickle cell layer,
decreased melanin, greater numbers of inflammatory cells in the subepithe-
lia, and keratinization. Similar changes have been described in biopsies
37
of tongues with atrophied filiform papillae. Both lesions are reversed
37
with iron therapy, although the tongue changes require 7-14 days.
The clinical manifestations of these lesions in the oral cavity include
angular cheilitis, a sore red tongue, pallor, and often soreness of the
buccal mucosa. These changes are not specific, however, because similar
abnormalities are exhibited in disorders such as deficiencies of folic acid
and vitamin B .
Additional lesions in the hypopharynx associated with iron deficiency
are mucosal atrophy and web formation in the post-cricoid area of the
pharynx and upper esophagus. This syndrome, characterized by difficulty in
swallowing, is termed sideropenic dysphagia. When the clinical features of
gastric achlorhydria and gastric atrophy are involved, especially in middle-
aged women, this constellation is termed the Paterson-Kelly or Plummer-Vinson
418,542,604,723,774
syndrome. In some patients (5-15%), the disorder has
5,163,382,392,397,835
progressed to carcinoma. However, many investigators
believe that the causal connection of iron deficiency with the various mani-
164,165,252,
festations of the Plummer-Vinson syndrome is far from explicit.
392,397,398,835
Especially troublesome to clearcut explanation is the geo-
graphic distribution of the syndrome, which predominates in Northern Europe
and the United States. Almost no reports of it exist from Africa, South
America, or the Far East, areas where iron deficiency is far more common.
Moreover, similar lesions have been described in the absence of any current
252
or past evidence of iron deficiency. Whether factors other than iron
177
-------
deficiency predispose to the post-cricoid webs and gastric atrophy is a
moot question, particularly because the incidence of Plummer-Vinson syndrome
has decreased in recent years.
Gastritis, gastric atrophy, and diminished acid and intrinsic factor
33,
secretions are additional abnormalities related to iron deficiency anemia.
214,397,399,467,823
Histamine-fast achlorhydria has been found in about 50%
399,467,713
of patients with iron-deficiency anemia, and an even higher inci-
33,214,412,466
dence of gastritis has been reported. Whether the gastritis
and gastric atrophy are a cause or a consequence of the iron deficiency is
33,214,466
debatable; either explanation is possible for individual cases.
Iron therapy has improved the appearance of the gastric
33,214,399,467,713
lesions and returned acid secretion to normal in some patients,
126,468
especially young people, chronic cases, and patients whose gastric
atrophy is not severe. However, recovery is uncommon in patients older than
399,467,713
30. Antibodies circulate to gastric parietal cells in about a
third of patients with iron-deficiency anemia and histamine-fast achlorhy-
203a,513
dria. Again, the causal relationship of antibodies to iron deficiency
is obscure.
Atrophy of small intestinal mucosal cells in children with iron-
314,562
deficiency anemia has been described. Treatment with iron restored
39
these mucosal lesions to normal. However, Baker criticized the adequacy
562
of the biopsy specimens in these studies because he could detect no marked
morphologic abnormalities in 20 patients with severe iron-deficiency anemia.
423
The observations of Kimber and Weintraub may be of greater importance.
They found intestinal malabsorption of radioiron by severely iron-deficient
children and puppies, a condition reversible after iron repletion. They
attributed the phenomenon of malabsorption to decreased iron-containing
intestinal enzymes.
178
-------
Integument. The progressive changes in fingernails from brittleness,
thinning, flattening to eventual concavity (koilonychia or spoon nails) are
372
associated with iron deficiency and respond completely to iron therapy.
Decreased cystine content of the nails has been reported in individuals with
406
koilonychia. It is unknown whether iron deficiency interferes with cystine
metabolism or if another defect alters its intake or metabolism. Koilonychia
is rare in the United States and is no longer a common disorder in other
332
countries. Hair loss has been associated with iron deficiency in women,
but such a symptom is not very specific.
Neurologic changes. Various neurologic symptoms have been attributed
to iron deficiency, including numbness, paresthesia, and pain, yet all with-
out objective clinical findings. However, papilledema and increased intra-
cranial pressure not due to other causes and which disappear with iron
145,431
therapy are well documented.
The high concentrations of nonheme iron found in certain parts of the
brain (substantia nigra, globus pallidus, and the red nucleus) are of the
325
same order as those in the liver. Some of this nonheme iron is ferritin;
the rest is not clearly characterized by either type or function. In the
development of iron deficiency, nonheme iron concentrations in the brain do not
325
drop, as do the nonheme hepatic iron stores. Yet a brief period of
severe iron deficiency in the young rat results in a deficit of brain iron
that persists in the adult animal, despite a subsequent adequate intake of
212
iron.
Nasal mucosal atrophy (ozena). Ozena, a disorder that occurs with iron
57,58
deficiency in eastern European countries, is practically unknown in the
United States and the rest of the world. The association of atrophic rhinitis
with iron deficiency may be fortuitous, as others have reported failure of
554.
iron therapy to correct the condition.
179
-------
Metabolic Defects
No exact measurement of organ or tissue dysfunction exists that
clearly divides the effects of iron deficiency from those of anemia. The
question of whether or not patients without anemia but with definite deple-
tion of stores and possible depletion of some key tissue iron component
68
have symptoms related to their iron depletion is unresolvable. Clinicians
have observed two discordant patterns. Some patients without overt anemia
but with depleted iron stores and borderline lowering of serum iron concen-
tration and transferrin saturation exhibit symptoms of profound fatigue and
apathy that are alleviated after iron therapy. Yet other people with
moderately severe anemia and clearcut iron deficiency may be vigorous,
asymptomatic, and completely unaware of their anemia. Faced with these
inconsistencies and uncertain about which key iron-containing biochemical
systems to study, investigators have been relatively slow to probe the
metabolic defects of iron deficiency with the same enthusiasm accorded to
the more easily and successfully measured aspects of the anemia.
Biochemical Compounds and Reactions Altered by Iron Deficiency
Heme proteins. Heme proteins, which function in the process of oxi-
dative metabolism, include hemoglobin, myoglobin, the cytochromes, catalase,
and peroxidase. Because iron is central to the function of these proteins,
its insufficiency would be expected to affect all cells profoundly. In the
absence of adequate iron, hemoglobin and myoglobin synthesis is decreased,
and the function of the heme proteins is changed quantitatively rather than
qualitatively. Much evidence confirms the decrease in hemoglobin in iron-
deficient subjects; little is known about the effect of iron deficiency on
human myoglobin. Myoglobin concentrations in various muscles of experimental
animals respond differently to iron deficiency. In rat skeletal muscle,
180
-------
210
myoglobin concentration markedly decreases, but the concentration of myo-
globin in the animal's cardiac muscle is unchanged, and absolute amounts of
210
myoglobin appear to increase during iron deficiency. This phenomenon may
be related to the workload of the organ, because myoglobin is similarly spared
in the diaphragm.
Cytochromes function in the electron transport system. Cytochromes
a. (cytochrome oxidase), b_, and £ are located in the cristae of mitochondria
and are responsible for transforming cellular energy into adenosine tri-
phosphate. Cytochrome P450 is located in microsomal membranes of the liver
and aids in the oxidative degradation of drugs and various metabolites.
Cytochrome b^ is found in many membranes; it functions in a variety of
metabolic activities unique to its cell of residence. Levels of cytochrome
activity in iron deficiency vary in different organs. Biochemical and
histochemical measurements have shown that cytochrome oxidase is reduced
204,212a,393
in the buccal and intestinal mucosa of iron-deficient patients.
In iron-deficient rats, the enzyme is markedly decreased in intestinal
mucosa and skeletal muscle, virtually unaffected in brain and heart
207
muscle, and only slightly below normal in kidney and liver. The micro-
209
somal cytochromes P450 and ^ are hardly affected by severe iron deficiency.
Catalase and peroxidase are involved in the reduction of endogenously
produced hydrogen peroxide. These enzymes are widely distributed in many
cells, and they are usually contained in small organelles such as peroxi-
somes of liver cells or granules of leukocytes. Congenital absence or
marked deficiency of catalase is remarkably asymptomatic in many people
1
with acatalasia, although others are afflicted with necrotic mouth lesions.
42,509,661
Human red blood cell catalase is lowered in iron deficiency anemia;
measurements in other tissues have been made in experimental animals. Rat
65
liver catalase remains normal in iron deficiency.
181
-------
Metalloflavoproteins. Metalloflavoproteins include succinate dehydro-
genase, a-glycerophosphate dehydrogenase, and NADH-dehydrogenase of mito-
chondria, and monoamine oxidase, xanthine oxidase, and aldehyde dehydro-
genase in the cell sap. These enzymes all contain nonheme iron and are
present in many tissues of the body. Knowledge of their activities in iron
deficiency is virtually nil in human studies and meager in experimental
30,66,725
animals.
Enzymes with iron as a cofactor. This class of enzymes includes
aconitase (citrate or isocitrate hydrolase). They are deficient in whole
724
blood and leukocytes of iron-deficient patients, and are normal or
64
decreased in various tissues of the iron-deficient rat. Hydroxylation
of proline and lysine in collagen synthesis is dependent upon a poorly
635
identified iron-requiring enzyme system. The ferrous iron chelating
agent a, a -dipyridyl interferes with collagen synthesis when given
812
parenterally to normal rats and iron-deficient rats show a similar deteriora-
167
tion. Impaired collagen formation has been proposed as a possible cause
724
for poor wound healing that accompanies iron deficiency. However, it
510
is more likely that the defect is a nonspecific consequence of the disorder.
Other enzyme systems. Enzymatic activities in which iron is neither a
51
component nor a cofactor may be diminished in iron-deficient rats. Becking
showed a 50% reduction of the activity of glucose-6-phosphate reductase,
phosphogluconate dehydrogenase, and malate dehydrogenase in iron-deficient
rats compared to normal control animals. Iron deficiency led to reduced
disaccharidase activity in the brush border of dog intestinal epithelial
369 354
cells. DNA synthesis in marrow is inhibited in human iron deficiency,
650
and a similar impairment has been observed in HeLa cells treated with
the chelating agent desferrioxamine, perhaps because of interference with
370
ribonucleotide reductase.
182
-------
Mitochondrial Damage
Injury to mitochondria in several tissues of iron-deficient experimental
animals and humans has been demonstrated by electron microscopy and other
207-209,302,409,647
analytic techniques. The mitochondria are uniformly
enlarged and radiolucent. In human bone marrow they also appear to be in-
creased. Similar findings in the heart muscle of iron-deficient
rats have suggested that greater numbers of enlarged mitochondria may account
for the cardiac hypertrophy in excess of that caused by the increased work-
load imposed by the anemia. Mitochondria from the small intestine of iron-
647
deficient rats are abnormally fragile. Swelling, vacuplation, and breakdown
of cristae in mitochondria from lymphocytes of patients with iron-deficiency
409 209
anemia and in the hepatocytes of severely iron-deficient rats attest to
the diffuse abnormalities produced in these important subcellular organelles.
However, translation of these morphologic abnormalities into specific func-
tional defects has not been accomplished.
Functional Abnormalities
Although many symptoms of iron deficiency remain unexplained, measur-
able functional abnormalities associated with iron deficiency have been
documented in humans and experimental animals. Most of these abnormalities
involve complex interactions between a number of organs and biochemical
mechanisms. Iron deficiency impinges upon growth, physical and mental per-
formance, reproduction, and susceptibility to infection. Descriptions of
these and other conditions are supplied below.
Growth. The effect of iron deficiency on growth rate appears to have
been studied much more carefully in laboratory animals than in humans,
perhaps because of the difficulty in separating nutritional iron deficiency
206,211,523
from other dietary insufficiencies in children. Studies in rats
indicate that depressed growth is a late consequence of iron deficiency
183
-------
that appears long after the onset of severe anemia. Two studies found no
48,135
association between hemoglobin concentrations and weight in children,
although addition of iron to the diet of male infants produced an increase
412a
in weight gain not seen in female infants. Judisch e_t^ ad. observed two
patterns of infant growth. The first was a rapid weight gain that slowed
when iron deficiency developed; the second was a normal weight gain that
accelerated when iron was supplied. They concluded that iron administration
was associated with increased body weight in infancy, but they were uncertain
if such a weight gain were desirable.
Physical performance. Although lassitude and tendency to be fatigued
68
easily are common in patients with iron deficiency even without anemia,
documentation of decreased physical performance from iron deficiency alone
is rare. It has been difficult to define for humans effective endpoints
539
of tests of endurance or maximal response to workloads.
215
In one study, reduced heart rate following exercise was observttd after
194
correction of iron-deficiency anemia with iron therapy. In another,
pulmonary efficiency was depressed during fairly vigorous exercise in
patients with iron-deficiency anemia. Respiratory function was improved by
775 293
iron therapy but not by placebo. Viteri and Torun and Gardner et al.
showed that the capacity for physical work was impaired even with small
(1-2 g/dl) decreases of hemoglobin concentration within the normal range.
However, Hallberg and his colleagues produced iron deficiency by phlebotomy
in otherwise normal student volunteers and were unable to detect any appre-
ciable differences in the performance of various standardized physical tasks
between the phlebotomized subjects and controls (personal communication,
L. Hallberg).
184
-------
In rats running a treadmill, forced and voluntary running time declined
with iron-deficiency anemia 25-30% below that of normal controls. Perform-
ance improved 3 days after iron therapy and was restored to normal 7 days
244 272
after treatment. In another study, the treadmill running of iron-
deficient rats was markedly impaired as compared to normals. The effects
of anemia were eliminated by equalizing the hemoglobin concentrations in
both groups of animals. Attempts to identify a biochemical defect in
skeletal muscle disclosed that the rate of mitochondrial oxidative phos-
phorylation with a-glycerophosphate as substrate was uniquely responsive
to iron replacement and paralleled recovery of treadmill performance in
iron-deficient rats treated with iron. Reduced concentrations of cytochromes
and myoglobin and low rates of oxidative phosphorylation with other sub-
strates were observed in these iron-deficient rats, but none of the functions
improved rapidly after iron administration.
Spontaneous basal activity diminished in rats in the presence of iron-
297
deficiency anemia. This reduction was independent of the severity of
anemia, and it was rapidly reversed after treatment with iron. In school
289
children studied by Gandra and Bradfield, daily energy expenditure was
not dependent upon the degree of anemia. A similar observation was made in
39
studies of iron-deficient and normal children in an orphanage.
Mental performance. Efforts to show quantitative reduction in tests
of scholastic achievement or other mental performance in relationship to
623
iron deficiency have been notoriously difficult to assess. Significantly
lower test scores were reported in a group of iron-deficient adolescent boys
798
and girls compared with matched normal control subjects. Another study,
376
reported only in summary form, compared 2 groups of black children aged
3-5 from low-income areas of Philadelphia. One group was anemic (9-10 g
185
-------
hemoglobin/dl blood), and the other was injected with enough iron to raise
the hemoglobin concentration to 12 g/dl. Comparisons of the two groups by
a battery of tests indicated that iron-deficient children exhibited strikingly
decreased attentiveness, more aimless manipulation, narrower attention span,
and less complex and purposeful activity when compared to their normal counter-
parts. However, no significant differences were revealed by their scores on
720
the Stanford-Binet and Goodenough intelligence tests. Sulzer reported
251,253
similar conclusions from a different group of psychologic tests. Others
have been unable to detect any differences in the performance of psychologic
tests in iron-deficient and normal patients even when patients served as
251
their own controls.
An unusual behavioral disturbance of unknown etiology seen in iron
deficiency is pica, or the habitual desire for unnatural articles of food.
A long list of specific cravings has been recorded, including earth or clay,
264
laundry starch, ice, olives, writing chalk, and cigarette ashes. Pica in
adults usually disappears soon after treatment with iron and may recur repeat-
edly with the return of iron-deficiency anemia. How iron deficiency is related
to this craving is completely unknown. A similar behavioral disturbance,
.374
breath-holding compulsions, has been attributed to iron deficiency, but no
direct cause and effect relationship has been proved.
Observations of decreased monoamine oxidase activity in iron-deficient
725,726 137,837
rats and humans raise the possibility that this enzyme may
be responsible for some of the behavior disorders of iron deficiency.
Lack of monoamine oxidase in the brain of an iron-deficient patient might
simulate the effect of drugs given to inhibit monoamine oxidase and produce
side effects of restlessness and irritability. To date, these relationships
are completely conjectural.
186
-------
Reproduction. Because iron-deficiency anemia is especially common in
pregnant women, it is surprising that little information is available about
the effects of iron deficiency on fetuses or on a mother's ability to carry
the pregnancy to term successfully. No available evidence exists that iron
deficiency in a population has a major effect on the ability to conceive
633
or on early fetal wastage. The suggestion that iron deficiency in the
mother leads to iron deficiency in the infant resulting from depleted
714 224,284,445,717,827
iron stores at birth has been refuted.
The fetus apparently has first option on the maternal plasma iron.
Placeatas from severely iron-deficient mothers weigh more than those from
53
women who received adequate iron during pregnancy. Such hypertrophy
might result in greater blood flow to the fetal placenta, but this propo-
sition has not been proved.
Susceptibility to infection. The relationship between human iron
status and susceptibility to infection is controversial and has been
722
thoughtfully reviewed by Sussman. Almost all pathogenic microorganisms
have an absolute requirement for iron and their interaction with a human
host may be affected by the host's iron status. Evidence can be presented
that iron deficiency both increases the risk of infection and that it protects
722
against it; the case for neither view is compelling. Considerable addi-
tional information is required about the effects of iron on bacterial growth
and virulence, host defense, and the interaction between iron and its phy-
siologic binding proteins, transferrin and lactoferrin, before the connection
between iron and susceptibility to infection is clarified.*
Potential for absorbing other metals. Iron deficiency in the rat
enhances absorption of a variety of metals. Manganese absorption is
230,529,621,736
increased in iron deficiency, and neither metal affects the
*See also Chapter 2.
187
-------
736
rat's absorption of the other. However, when manganese was perfused into
the duodenums of human subjects with varying iron stores, its absorption
increased in those with iron deficiency. Conversely, absorption was inhibited in
736
individuals with adequate iron stores. Retention of a tracer dose of
manganese-54 10 days after absorption by an iron-deficient patient was no
736
greater than by a patient with normal iron stores.
621,738,740,768
Absorption of cobalt by rats and humans is increased in
the presence of iron-deficiency anemia. Both metals are absorbed predomi-
737
nantly in the proximal small intestine of the rat. In both species,
increasing the concentrations of either metal inhibits the absorption of
668,669,739
the other, suggesting that iron and cobalt share at least part
of a common absorptive pathway. These features have been used in a cobalt
769
test for human iron-deficiency anemia.
Absorption of cadmium by iron-deficient mice was four- to sixfold
328
greater than by iron-replete mice. Moreover, cadmium toxicosis produces
133,624a
iron-deficiency anemia in the mouse and rat, which can be corrected
by additional iron. Whether or not human beings with iron deficiency absorb
greater amounts of cadmium is not known.
Absorption of lead increases in rats fed iron-deficient diets, as does
426,695
the toxic quality of the metal. It is unknown if a similar increase
in lead absorption occurs in iron-deficiency anemia of children. The fre-
quent association of iron deficiency and lead poisoning in children aged 1-3
166,727
yr in low-income groups raises the possibility of increased lead
absorption as a secondary effect of iron deficiency. However, pica asso-
ciated with iron deficiency often leads to ingestion of lead from paint and
plaster of older buildings in which these children live and clouds the ques-
tion of whether increased lead absorption or increased availability is at
issue.
188
-------
Absorption of cesium, magnesium, mercury, calcium, and copper was not
621
increased in iron-deficient rats.
189
-------
CHAPTER 6
ACUTE TOXICITY OF INGESTED IRON
Acute toxicity of ingested iron appears to be related strictly to
exposure to therapeutic iron preparations. It is not associated with inges-
tion of naturally occurring or other commercially produced substances. In
the United States, an estimated 2,000 cases of accidental iron poisoning
201,806
occur annually, with approximately 12 deaths. During an 18-mo-period, in whicH
1,645 children who had ingested toxic agents were seen, ferrous sulfate was
the agent in 6.2% of the incidents.
In 1947 Forbes pointed out that small children were accidentally poisoning
276
themselves by swallowing oral iron preparations prescribed for adults.
Before that report, only sporadic comments were published on acute, potentially
lethal poisoning from ingested iron. Numerous case reports and reviews of acute
14,152,257,263,274,279,449,814,815
iron toxicity have been published since.
EPIDEMIOLOGY
Acute iron toxicity is found most commonly in children aged less than
14,201
5 yr, predominately in the 1-2 yr age group. Poisoning results when a
child encounters the parent's iron medication and, attracted by the superficial
resemblance to candy and the taste of the sugar-containing coat, it ingests many
tablets before discovery. In adults, cases of acute iron toxicity occur almost
152,257,279,449
exclusively because of suicidal intent.
Acute iron toxicity caused by sources other than medicinal iron prepara-
tions does not happen because of the large quantities of iron that must be
ingested to produce major poisoning. The average human lethal dose is about
201
200-250 mg iron/kg body weight. Thus the average adult male must swallow
14 g of elemental iron (234 ferrous sulfate tablets). For the average 2-yr-old
child, 3 g of elemental iron (50 ferrous sulfate tablets) is the average lethal
dose.
190
-------
However, fatalities have been reported from much smaller doses. The
ingestion of 1.2-2.4 g ferrous sulfate led to death in a 17-mo-old child;
but the death was precipitated by peritonitis contracted following surgery
656
to relieve ferrous sulfate-induced pyloric stenosis. Yet a 17-mo-old
143
child recovered after swallowing a dose as large as 4.2 g iron. In one
review, the range of fatal amounts of ingested iron was 0.96-3.6 g elemental
14
iron, whereas for nonfatal cases it was 0.3-3 g. Although a few adults will
develop mild symptoms of gastrointestinal irritation following ingestion of
60-180 mg of medicinal iron, this quantity, the usual therapeutic dose, usually
produces no symptoms of overload or acute adverse effects.
CLINICAL STAGES
14,806
The features of acute iron poisoning have been well described and
may be divided into four phases.
• Approximately 30 min after ingestion of the iron, vomiting—which
may be bloody—begins in 80% of the patients. More than half the
patients become drowsy or lethargic. Diarrhea, often bloody,
develops in over 40% of the victims. The pallor, tachycardia,
and rapid respirations that often develop may indicate impending
shock. A patient may die during this phase, which extends over
6-12 h.
• During the second phase, improvement is noted, stupor clears,
and the vomiting and diarrhea subside. This phase lasts 10-12 h
and may be followed by complete recovery or sudden relapse.
• Many patients will relapse after the phase of improvement ends.
Fever, pneumonitis, shock, coma, convulsions, and death may
follow. This phase occurs approximately 20 h after ingesting
the iron.
191
-------
• In those surviving for 3-4 days after iron ingestion, recovery
is rapid. In a small number of patients, gastric obstruction
related to pyloric stenosis and stricture formation will develop.
Laboratory findings during the first three phases may include marked
leukocytosis, evidence of metabolic acidosis, hyperbilirubinemia, and deranged
263,814
blood coagulation. Serum iron levels vary, yet do have prognostic value.
In one report, shock or coma occurred in only 9 of 112 patients (8%) with an
initial serum iron value less than 500 ug/100 ml, but in 37% of those with an
806
initial value in excess of 500 ug/100 ml. The onset of coma or shock indi-
806,815
cated a serious prognosis. Without treatment, all patients with these
signs died. If neither coma nor shock were present, all patients survived.
806,819
With current modes of treatment, the mortality rate is 11-17%.
PATHOLOGY
651
In addition to the removal of tissues during surgery, several autopsies have
' 14,152,279
been reported in children and adults who died from acute iron poisoning.
In the stomachs examined, hemorrhagic necrosis and sloughing of areas of
mucosa with extension into the submucosa were common. The necrotic surfaces
were covered with iron-positive pigment. The mucosa was diffusely congested and
infiltrated with polymorphonuclear leukocytes and mononuclear cells. Basement
membranes of lymphatics, capillaries and venules of submucosa and serosa stained
iron-positive. Platelet thrombi were numerous in the submucosal capillaries and
veins. Changes in the small intestine were the same as described for the stomach
but were less severe. However, the most striking changes were in the proximal small
651
intestine. Infarctions, particularly of the ileum, were found to be associated
with thrombosis of the mesenteric veins. Autopsies revealed hemorrhagic necrosis of
502
the periportal portion of the liver lobules. Iron stains of these areas showed
192
-------
finely dispersed iron deposits in portal vein endothelium, Kupffer cells, and
periportal reticulum. Other organs exhibited only general conditions of edema,
cloudy swelling, and areas of hemorrhage.
PATHOPHYSIOLOGY
Local Events
Hemorrhagic necrosis of the gastrointestinal tract is a direct effect of
the interaction of iron and gastric hydrochloric acid. This strongly acid
solution containing ferrous and ferric ions, chloride, and usually sulfate,
produces coagulation of protein and extensive corrosion. Although the injury
to the intestinal tract may be severe enough to lead to scarring, the notion
that loss of blood and fluid from the gastrointestinal tract is a major cause
of shock and death has been disproved.
Systemic Events
Most of the systemic effects of poisoning are associated with absorption
of excessive amounts of iron, resulting in the appearance of plasma iron not
bound to transferrin. In experimental situations employing intravenous injec-
tions of ionic iron, exceeding the iron-binding capacity has produced such
striking symptoms and events in humans as sneezing, nausea, cutaneous flushing,
tachycardia, and hypotension. ' In dogs, excessive iron is rapidly
absorbed across intact intestine, where it is responsible for profound meta-
644 645
bolic consequences leading to death. ' Thus, intestinal mucosal damage
need not be invoked to explain the excessive absorption of iron or the follow-
ing systemic events.
Coagulation defects. No systematic study of clotting and iron poisoning
has been reported in humans. Studies in animals suggest that alterations
in coagulation are secondary to a direct effect of iron on the various proteins
193
-------
involved in blood coagulation. These reactions can be simulated by a direct
addition of ferrous sulfate to human plasma.
Metabolic acidosis. Acidosis is a prominent and consistent feature of
experimental iron poisoning in dogs and a common development in iron poison-
ing in children. Three factors are involved with the onset of acidosis.
Hydrogen ions are released from the conversion of ferrous iron to the ferric
form in the circulatory system, i.e.,
1/4 02 + Fe"*"*" + 5/2H20 > Fe(OH)3 + 2 H+.
Organic acids such as citric and lactic acids are accumulated prior to circu-
644
latory failure. Electron microscopic studies of ferrous sulfate-induced
liver damage show mitochondrial injury that constitutes an anatomic basis for
824
the organic acid accumulation. In addition to the reasons just discussed,
lactic and citric acids accumulate as a result of the anaerobic metabolism
associated with shock.
Shock. Shock can be an early feature even of severe nonfatal iron poison-
ing. Therefore, it is more than a terminal event. The peripheral circulatory
644
failure is induced by absorbed iron. Although how the process works has not
been characterized fully, it appears that iron directly affects the peripheral
vasculature. In dogs given fatal amounts of iron, six alterations were con-
sistently found; reduced cardiac output; increased total peripheral resistance;
decreased plasma volume; hemoconcentration; decreased total blood volume; and
644
lowered blood pressure. Using this and other information, Whitten and
Brough presented a hypothetical sequence of the pathophysiologic events of acute
iron poisoning, but the data on which they based their sequence were insufficient
to substantiate the hypothesis.
194
-------
TREATMENT AND PREVENTION
Details of treatment are beyond the scope of this report. It is based
on efforts to rid the victim of unabsorbed iron by using gastric lavage and
cathartics. In patients suspected of having ingested near-lethal amounts
of iron and in those with serum-iron concentrations in excess of the binding
capacity, the physician attempts to promote urinary excretion of the extra
plasma iron by binding it with a chelating agent, deferoxamine mesylate.
143 274 705a
Early treatment is most important. ' ' The shorter the interval
between ingestion of the iron compound and the initiation of therapy, the
better the prognosis.
Prevention is the key to this public health problem. Millions of pre-
scriptions are written each year in the United States for iron-containing
medications. Moreover, iron compounds are available for purchase without pre-
scription. Because many of the users are women with young children, consider-
able opportunity exists for iron ingest ion and poisoning to occur in that
susceptible group. Public education and the necessity for physicians and
pharmacists to make their patients aware of the hazard are therefore of the
utmost importance.
In summary, no known natural or dietary sources such as water, food, or
any beverage are likely to result in acute iron toxicity. Acute poisoning
from ingested iron comes from accidental ingestion of medicinal iron, mainly
by children one to two years of age. Iron poisoning may be fatal and early
treatment is crucial. Prevention of exposure is the most important aspect
of the problem.
195
-------
CHAPTER 7
CHRONIC IRON TOXICITY
In relation to the total body iron content, the amount of exchange that
occurs between humans and their environment is small. Disturbances of iron
balance are common, but they are almost invariably such that a reduction in
the total body iron content results, as discussed in Chapter 5. A major pur-
pose of this chapter is to explore if iron overload does, indeed, result in
toxicity.
As the quantities of storage iron (soluble ferritin and insoluble hemosi-
derin) in the body rise, the ratio of hemosiderin to ferritin increases. Thus
an increase in storage iron is called hemosiderosis or siderosis, and it may be
relative or absolute. A relative siderosis is a part of any anemia not caused
by blood loss, and merely represents an internal redistribution of the body's
iron, with less in the red cell mass and more in stores. Siderosis may also be
localized to certain organs; in idiopathic pulmonary siderosis, it is the lungs
and in paroxysmal nocturnal hemoglobinuria, the kidneys. Such conditions are
examples of focal siderosis. When the total iron content of the body is increased,
the extra iron is laid down in storage compounds, and it is called absolute sidero-
sis or iron overload. The term hemochromatosis is applied when the organs con-
taining grossly excessive amounts of storage iron show pathologic evidence of
damage, usually fibrosis.
IRON OVERLOAD
Pathogenesis
When the total body iron content is increased, the extra iron enters either
through the gastrointestinal tract or by the parenteral route. In some circum-
stances, both mechanisms may be operative.
Oral Iron Overload
Excessive dietary iron intake. Although the absorptive mechanism normally
adapts itself to the body's need for iron, nevertheless the actual quantities
196
-------
89
absorbed increase progressively as the intake of absorbable iron rises. Sub-
stantial iron overload theoretically can be a function of large amounts of absorb-
able iron, but the condition is rare except in southern Africa, where alcoholic
159
beverages are prepared in iron drums and pots; The mean iron content of such
brews has been found to vary between 40-80 mg/1 and the pH of the beverages is
low. In Western countries, an analog sometimes is encountered in wine-drinking
506
alcoholics. However, the iron content of wines is considerably lower than
that of the beer consumed by South African blacks; red wines usually contain
26,506,610
between 5-6 mg/1 and white wines lesser quantities. Finally, there
have been occasional reports of patients who have misguidedly continued to consume
157
iron tonics over extended periods.
There is good reason to question the traditional assumption that dietary
iron overload is simply a function of the increased quantities of the metal in-
gested. Only when the dietary iron is available for absorption is the overload
likely to occur. The importance of availability is well illustrated by the situ-
ation prevailing among South African blacks. On the basis of stool analyses, the
92,779
daily intake of many men was found to be between 100-200 mg. Most of this
contaminating iron is consumed in maize porridge cooked in iron pots, or in beer
made from maize and sorghum fermented in iron drums. Only very small quantities
of the iron in the porridge are absorbed. In contrast, the absorption of iron in
the beer is good, presumably as a result of the formation of soluble, easily
87
absorbed complexes during the fermentation process. Teff, a grain consumed in
Ethiopia, is heavily contaminated with iron but iron overload is not a problem,
371
since the extra iron is present in an unabsorbable form or forms.
Excessive absorption from diets of normal iron content. Oral iron overload
is possible even when the dietary iron content is not inordinate. In individuals
with idiopathic hemochromatosis, amounts of iron in excess of body requirements
197
-------
89
are regularly absorbed, and iron slowly builds up in the body over many years.
The metabolic defect responsible for the inappropriate absorption is unknown.
Indeed it seems likely that what is called "idiopathic hemochromatosis" may
include several separate diseases affecting at different sites the processes by
199
which iron absorption is regulated.
Regardless of the nature of the defect(s) responsible for increased iron
absorption, there is good evidence that idiopathic hemochromatosis is inherited.
Between 25-50% of first-degree relatives exhibit iron overload of varying degree,
157
and siblings are the most commonly affected. The majority show only a modest
increase in body iron stores, but some are heavily siderotic. It is rare to find
severe iron overload in successive generations, but families exist in which it
303
has been noted. Because different patterns of expression of the disorder have
670
been described, debate on the mode of inheritance has been spirited. However,
such questions become irrelevant if it is accepted that the condition is not a
homogeneous one, and that any of several defects may be associated with excessive
absorption of iron from the gut. The most common form of inheritance appears to
be an intermediate one in which the heterozygote manifests minor derangements of
iron metabolism, such as a raised plasma iron concentration and a moderate
increase in iron stores, and the homozygous state eventually leads to the accumu-
670,818
lation of massive amounts of iron. In contrast, in those families in
which successive generations have been affected with fully developed hemochroma-
43,88,219
tosis, the mode of inheritance appears to be dominant. In addition,
there are reports of patients with the disease who were the offspring of con-
sanguineous marriages, in which neither parent showed any abnormality; presumably
218,219,587
the genetic defect in these circumstances was recessive.
Resolution of the uncertainty is further compounded by the fact that the
ultimate expression of the gene in an individual can also be influenced by
198
-------
157
external factors, such as the amount of iron in the diet and physiologic
and pathologic blood losses. Severe iron overload thus would be expected
to occur less frequently in affected females and to be uncommon in countries
such as India, in which the quantities of available iron in the diet are small.
Conversely, hepatic dysfunction in the iron-loaded liver might be more widespread
273
and severer in individuals exposed to a hepatotoxin, such as alcohol. Whereas
a small proportion of patients with alcoholic cirrhosis certainly do show severe
305 499,501
siderosis, iron stores are usually normal in alcohol abusers. Iron
overload is rare enough in alcoholics outside of southern Africa to suspect
that consumption of alcoholic beverages is not the only factor in such subjects.
It seems reasonable to suppose that some of them might be heterozygous for a
hemochromatosis gene and, had they not been exposed to alcohol, would never have
developed a recognizable clinical syndrome.
Parenteral iron overload. In several refractory anemias, life is sustainable
81,84,89,144,427,506,593,676,694
over long periods through repeated blood transfusions.
Because each 500-ml blood transfusion contains more than 200 trig iron, very large
quantities can be introduced via this route. In the absence of bleeding, the body's
capacity to excrete the extra iron is extremely limited—a few milligrams daily at
308
the most (see also Chapter 4). As a result, massive quantities of storage iron
accumulate. Although iron overload is a serious problem in some patients with
acquired aplastic anemias, the majority succumb to other complications of bone
marrow failure before the stage of massive overload has been reached. Patho-
logic changes are more common in thalassemia major and other conditions asso-
ciated with defects in hemoglobin synthesis, including refractory sideroblastic
anemia, pyridoxine-responsive anemia, and refractory normoblastic anemia, and they
are occasional manifestations of a variety of chronic hemolytic states. There are
199
-------
two reasons for parenteral iron overload. First, the realization that the
quality of life in affected individuals is improved by maintaining the hemo-
globin at levels closer to normal has encouraged the regular use of transfusion
540
therapy from an early age. Secondly, in anemias such as thalassemia major in
which erythroid activity is markedly increased, yet ineffective in delivering
44,
viable red cells into the circulation, iron absorption from the gut is increased.
97,258,688
The iron overload that develops is thus not simply the result of
repeated transfusions, but also includes an oral component. Hence the useful
term "iron-loading anemias" coined by Crosby.
Although parenteral overload also could theoretically result from repeated
treatment with injectable iron compounds, no published evidence shows that it
represents a practical problem.
Prevalence
The prevalence of iron overload in different populations varies not only
with the frequency with which certain genes occur in different populations, but
with the composition of the average diet. It is not only the dietary iron con-
tent that is important, but also its availability for absorption. Meat is a
good source of absorable iron, whereas cereals and other vegetables are poor
518,752
sources. Therefore, it is not surprising that no reports of clinically
manifest oral iron overload have come from India. Differences in prevalence
among certain Western countries may represent a subtle interplay between the
frequency of genes associated with a tendency to absorb too much iron and the
content of easily absorbable iron in the diet. For example, the greater pre-
valence of clinical hemochromatosis in Australia as compared to the United
Kingdom may relate to the greater per capita consumption of meat in Australia.
506
In France, Germany and Italy, the consumption of red wines could be responsible.
200
-------
A real difficulty in assessing the prevalence of iron overload in different
countries is that quantitative methods for determining the amount of storage iron
in tissues have come into general use only recently. The quantitative approach
has required either the chemical estimation of the iron concentrations in
affected tissues or the measurement of the urinary excretion of iron after admin-
istering specific chelating agents. Such a distinction has considerable relevance.
506
Histologically visible iron often is noted in organs such as the liver, and if
cirrhosis is present the condition can be labelled hemochromatosis when the quan-
tities of iron are merely moderate. With these reservations, current U.S. statis-
tics indicate a prevalence of 1 in 20,000 hospital admissions and 1 in 7,000
273 264
hospital deaths in one study, and 4 in 100,000 necropsies in another. Much
higher, although misleading, figures would be obtained if statistics were collected
in diabetic or liver clinics, because the presenting clinical features are so com-
monly related to diabetes or hepatic dysfunction. Perhaps the most objective figures
available are those from a study in which nearly 4,000 liver specimens, the
majority obtained from hospital necropsies in 18 different countries, were ana-
160
lyzed chemically for iron. In only 3 samples were the concentrations more
than 5 times normal and none was anywhere near the figure of 20-50 times normal
found in hemochromatosis. Therefore, severe iron overload, at least as judged
by necropsy figures, is a relatively uncommon phenomenon in most countries.
As noted, the exceptions are confined to southern Africa, where many popu-
lations consume home-brewed drinks high in iron. There have been two attempts
129,159
to define the prevalence in this region. In one study carried out in
Johannesburg, 70% of black adult males and 25% of black adult females dying in
90
hospital were found to have excessive quantities of iron in the liver. Both
prevalence and severity increased with age. Although the degree of siderosis
was mild or moderate in most subjects, 20% of middle-aged males had concentrations
201
-------
above 2% dry weight, comparable to amounts described in Western countries for
subjects with idiopathic hemochromatosis. As exposure to inordinate quanti-
ties of iron in South African blacks is largely confined to adults, iron stores
are not abnormally high in infants and children; indeed, iron deficiency is not
158
uncommon in these groups.
The prevalence of those anemias most commonly associated with severe iron
overload is not known with any certainty. Aplastic anemia is a rare condition,
and has a median survival of only about two years; but thalassemia major and
several other chronic refractory anemias are compatible with survival for a number
of years. These individuals are attracting the attention of investigators in-
540
terested in the possible long-term effects of severe iron overload. Thalassemia
major reaches its greatest prevalence (1 in 400) in Mediterranean peoples, yet most
of the current knowledge concerning the effects of the accompanying iron overload
has been obtained from studies of small numbers of affected individuals whose
parents migrated to the United States, the United Kingdom, or Australia. Such
patients have usually been studied more systematically and they tend to have been
treated with more intensive regimens of blood transfusions. The prevalence in
these countries varies, but it is very low. In the United Kingdom just over 200
patients are thalassemics, most of them inmigrants from Cyprus and the
540
northwest part of India.
Rate of Iron Accumulation
Most patients with idiopathic hemochromatosis develop clinical symptoms in
273,689
middle age, when body iron content ranges from 20-40 g. This deposit can
result from an average positive iron balance of about 2-3 mg daily, but it is
doubtful if the accretion is uniformly spread out over the preceding years.
The increased stores tend to depress iron absorption, so that by the time clinical
817
manifestations appear the absorption rate is often within the normal range,
which is, of course, inappropriate to the iron-overloaded state. After stores
202
-------
have been removed by phlebotomy, the absorption rate rises to between 5-7 mg
daily, suggesting that it is initially high and then gradually declines as the
71,817
stores accumulate. That the clinical manifestations are 10 times
more common in men than in women is probably caused by lower iron intakes and
273
greater losses in females.
In dietary siderosis as it occurs in black South Africans, the body iron con-
tent builds up from late adolescence until the fifth decade. The quantities of
iron in the body of the severely affected individuals are similar to those found
387
in idiopathic hemochromatosis. Limited isotopic studies suggest a positive
daily balance of up to 3 mg between ages 20 and 50 yr. However, the rate of
92
accumulation may not be uniform.
Transfusional hemochromatosis is of concern in aplastic and hyperplastic
89
anemias. With aplastic anemia, the rate of iron deposition is directly related
to the frequency and the number of blood transfusions, although the quantity of
iron in the body may be much less than was present in the transfused blood, as
thrombocytopenia with resultant blood loss is a frequent complication in such
patients. In hyperplastic anemias such as thalassemia major, good correlation
exists between the amount of iron administered in donor blood and the size of the
iron stores, but there is also an appreciable contribution from the gut, especially
when the subjects are very anemic. As a result, the rate of iron accumulation is
greater than in the aplastic group, although the additional load from the excessive
540
absorption varies according to the level at which the hemoglobin is maintained.
Distribution of Iron
In idiopathic hemochromatosis the highest storage iron concentrations are
689
found in the liver and pancreas, where they are between 50-100 times normal.
In the thyroid the concentration is usually about 25 times normal*and in the heart,
203
-------
pituitary, and adrenals, between 10-15 times normal. Lower concentrations of
about 5 times normal are found in the skin, spleen, kidney, and stomach.
The striking histologic feature of the disorder is the widespread presence
of the golden-yellow iron storage compound, hemosiderin, in the parenchymal cells
235,689
of many organs. Deposition of hemosiderin is heavy in hepatocytes and
bile duct epithelium, with lesser amounts in Kupffer cells. In the pancreas,
hemosiderin deposits are most prominent in the exocrine cells, but are also
found in the islets of Langerhans. Epithelial cells of the thyroid, the parathy-
roid, and the anterior pituitary glands are all affected, whereas in the adrenals
the iron deposits usually are confined to the zona glomerulosa. Hemosiderin
granules generally are scarce in the testes. Cardiac involvement is a cardinal
feature, and deposits are heaviest in the perinuclear region of the muscle fibers
131,689
of the ventricles. Smaller amounts are present in the atria and conducting
system. Considerable attention has been devoted to investigating the deposition
240,
of iron in the joints. The synovial lining may be laden heavily with hemosiderin.
329,689
Electron microscropy has shown that type B cells are involved rather
675
than the macrophage-like type A cells.
Unlike the massive iron deposits in parenchymal cells throughout the body,
reticuloendothelial involvement is not prominent; splenic concentrations of iron
156,689
are only modestly raised, and iron in the reticuloendothelial cells of the
102
bone marrow is often within the normal range.
The distribution of iron in the dietary iron overload of South African blacks
has been studied at all stages of its development, since the degree of siderosis
in autopsied subjects ranges from mild to massive. The pattern is very different
from that observed in idiopathic hemochromatosis. In the earliest stages, an
increase in hemosiderin granules is detectable in the hepatic parenchymal and
86
*•
Kupffer cells. With hepatic concentrations of 5-10 times normal, the deposits
204
-------
in these cells get denser, and the portal tract macrophages become involved. When
iron concentrations reach 20 times normal, heavy deposits are observable in all
three sites. Hemosiderin is visible in the spleen from an early stage, and with
more severe degrees of iron overload, deposits may even exceed those in the
86
liver. Reticuloendothelial involvement is also apparent in the bone marrow,
288
which may contain as much as 10 g storage iron. There is moderately close
102
correlation between the amounts of storage iron in the marrow and the liver.
Iron deposits elsewhere in the body are relatively scanty in most subjects.
Thus siderosis is largely confined to the hepatocytes and the reticuloendothelial
362,387
cells.
This pathologic picture is the usual one in black South Africans with
iron overload, but in one set of circumstances a different pattern occurs.
About 19% of severely siderotic individuals also exhibit micronodular
96 387
cirrhosis. ' In such subjects notable deposits are also found in the
parenchymal cells of a number of their organs, including the pancreas, thyroid,
adrenals, pituitary and myocardium. The concentrations of iron are comparable
to those described in idiopathic hemochromatosis.
The distribution of iron in subjects with parenteral iron overload is
influenced by the nature of the underlying anemia and the length of time over
which blood transfusions are administered. When the marrow is hypoplastic, the
major impact is on the reticuloendothelial cells of the spleen, liver, and bone
89
marrow. This occurs because donor blood is eventually broken down in these
sites, and since the plasma iron turnover is reduced, there is little tendency
for it to leave. If, however, the condition persists over many years, the iron
is slowly redistributed and marked parenchymal loading in tissues may result.
In the second group of anemias, the marrow is hyperplastic rather than hypo-
plastic, which means that external and internal iron kinetics are different from
205
-------
those prevailing in the hypoplastic anemias. This variation affects the distri-
bution of the excess iron. A large proportion of the red cells produced by the
overactive erythroid marrow are so defective that they do not even enter the cir-
culation, and those that do have a shortened life span. As a result, erythropoietic
activity is great, and the plasma iron turnover markedly increases. Much of the
iron released from the catabolized hemoglobin of both these cells and the transfused
blood is rapidly returned to the plasma. In addition, iron absorption in such anemias
is typically increased despite the developing iron overload. A major contribution
from the gut is particularly likely in those conditions in which hemoglobin synthe-
sis is defective, since the red cells superficially resemble those found in iron
deficiency anemia. As a result, oral iron therapy may mistakenly be given over
extended periods, as illustrated by reports of severe iron overload in subjects
with refractory anemias and hypercellular marrows who have received few or no
89
transfusions.
Although the ultimate iron distribution in subjects with refractory anemias and
hypercellular bone marrows may be influenced by numerous extrinsic factors, wide-
spread parenchymal deposits of iron generally are inherent in the disorders themselves.
Presumably deposition is a function of the rapid iron turnover through the plasma,
with subsequent unloading of iron onto the parenchymal cells of various organs.
Unloading is almost certainly facilitated by the iron-saturated transferrin
bathing the tissues; at least for the liver, iron uptake by hepatocytes is greatly
increased under such circumstances. Why the concentrations of iron are greater in
some organs than others is not known, but the rate of blood flow through the organ
and the relative number of transferrin receptors could well be responsible.
Reticuloendothelial deposits are also prominent in refractory anemias with hyper-
649
cellular marrows. Such a feature is not surprising, since iron turnover through
these cells is several times normal. That splenectomy in thalassemia major is
206
-------
followed by some redistribution of iron in the liver and an increased amount in
61,649,826
hepatocytes is a subject of some dispute.
It is worth considering iron distribution in the liver in more
detail, because the one central denominator of all forms of iron overload is
involvement of the hepatocytes. Hepatocyte uptake of plasma iron is facilitated
182,408
by high- saturation of transferrin, a factor found in all forms
of iron overload, except for the dietary form occurring in black South Africans.
Another factor in those conditions associated with increased absorption from the
gut (i.e., idiopathic hemochromatosis, dietary iron overload, and refractory
anemias with increased but ineffective erythroid marrow activity) is that some
of the absorbed iron may be deposited directly in hepatocytes from the portal
venous blood. Normally such iron is transferrin-bound, but if the percentage
saturation of the metal is high and a large amount is absorbed, a good proportion
809
may not be. Any unbound iron is deposited immediately in the hepatic parenchy-
mal cells. In addition, in any anemia characterized by an appreciable degree of
intravascular hemolysis, hemoglobin in the plasma, whether bound to haptoglobin
351
or hemopexin, is taken up by hepatocytes; a proportion of such iron might be
expected to remain there. Finally, it is possible that some iron deposited in
hepatocytes may be derived from the small amount of iron circulating in the plasma
as ferritin or bound to a protein other than transferrin.
.Associated Pathologic Findings
When reviewing the pathologic findings associated with severe iron overload,
it is customary to consider idiopathic hemochromatosis as the prototype. The
major feature of the disorder is the presence of fibrosis in heavily siderotic
organs, particularly the liver. A fine monolobular cirrhosis is almost invari-
627,689
ably found, the lobules typically separated by wide bands of fibrous tissue.
420
However, sometimes a multinodular, postnecrotic type of cirrhosis may develop.
207
-------
In young subjects the fibrosis can be less severe. Carcinoma of the liver is
273
an important late complication, occurring in about 15% of patients. It is
235
usually a hepatoma, but cholangiomas also have been reported. In the pancreas
the number of islets is often reduced, and fibrosis and degenerative changes
689
develop in acinar cells. Some fibrosis may also occur in the pituitary,
adrenals and thyroid, but it is not prominent. Testicular atrophy is manifested
in the majority of subjects. Dilation and hypertrophy appear in the myocardium;
131
occasionally degeneration of myocardial fibers and fibrosis are reported.
The large joints may show calcium pyrophosphate deposition in hyaline and fibro-
240
cartilage. Villous hyperplasia and fibrous thickening of the synovium have
780
been noted, as have degenerative changes in the cartilage. These pathologic
changes are seen once the clinical features have become manifest; little is known
of what occurs during that long latent period, extending over half a lifetime,
during which the iron content of the body is slowly increasing. It is clear
from studies of affected siblings that hepatic siderosis precedes the onset of
89
fibrosis.
In the dietary iron overload that occurs so commonly in South African blacks
a clearer idea of the pathogenesis of iron overload has been obtained, particularly
in regard to hepatic changes. The results of several studies indicate that a defi-
nite association exists between severe iron overload and significant portal fibrosis
86,90
or cirrhosis. This association cannot be explained by the prominence of
severe siderosis in older subjects, because marked portal fibrosis or micronodular
cirrhosis occurs in only 13% of black adults over age 40 in whom the siderosis
is minimal, compared to 71% in severely siderotic individuals (iron concentra-
90
tion, > 2.0% dry weight). Iron loading in the parenchymal cells of other
organs is uncommon. However, if deposition occurs, it is usually not accompanied
by fibrosis. The only other common pathologic finding in these subjects is
208
-------
osteoporosis, which is especially marked in the spine and may lead to collapse
681
of vertebrae, particularly in the lumbar region. A link between this form
of bone disease and severe iron overload has been demonstrated, and chemical
analysis of necropsy specimens has shown an inverse correlation between hepatic
503
iron concentration and the mineral density of iliac crest bone.
Associated pathologic changes in subjects with transfusional hemochromatosis
are influenced by the nature of the underlying anemia and the age at which it
occurs. In an analysis of 20 patients with hypoplastic anemia who had received
more than 47 1 of blood (equivalent to 25 g iron), 12 showed increased portal
89
fibrosis, but only 1 person had fully developed cirrhosis. In contrast,
cirrhosis was much more prevalent in 31 patients with refractory anemias asso-
ciated with hypercellular erythroid marrows. Cirrhosis was diagnosed in 26 and
increased fibrosis was noted in another 4. Although these data suggest that iron
i
overload involving the reticuloendothelial system is better tolerated when
parenchymal deposits are not present from an early stage, different investigators
have used different pathologic criteria, so that any judgments can be only tenta-
tive.
More concrete information relating to thalassemia major has become available.
Iron concentrations in the organs of five subjects were observed to be in the range
826
described in idiopathic hemochromatosis. All were cirrhotic and three showed
frank pancreatic fibrosis. Cardiac hypertrophy and hyaline degeneration
of myocardial fibers were found, but very little fibrosis. In a study of 41
patients, 39 of whom had thalassemia major, congestive cardiac failure developed
255
in 26 and pericarditis in 19. Necropsies were performed on 11 subjects. Iron
deposition was widespread, as was tissue fibrosis, especially in the liver, pan-
creas, gonads, thyroid, pituitary, and adrenal glands. The heart was dilated and
hypertrophied and microscopy revealed large amounts of iron in muscle cells and
209
-------
histiocytes. Myocardial fibers showed focal degeneration and widespread fibro-
sis. In a study in which 32 liver biopsies were performed on children with
thalassemia major, the severity of the fibrosis correlated closely with both
649
the patient's age and hepatic iron concentration. The rate of fibrotic pro-
gression was a function of the liver iron concentration; when the concentration
was less than 3% dry weight, progression was relatively slow, whereas it accelerated
at higher concentrations. Although the severity of the fibrosis correlated with
the degree of parenchymal siderosis, its relationship to the reticuloendothelial
involvement was closer. An analysis of 207 patients showed that most of the 37
who had died had succumbed at an early age because of inadequate transfusion
540
therapy and/or hypersplenism. The 8 older subjects had received an average
of 130.2 1 of blood (range, 68.2-188.9), and death was due to cardiac failure in
all of them. Despite its presence in the liver and exocrine pancreas, there
was no fibrosis in the heart. A child who had received 65.8 1 of blood for con-
genital aplastic anemia died of myocardial insufficiency, and her heart was
665
examined by electron microscopy. In addition to intracytoplasmic deposits,
some iron was consistently present within the nuclei and mitochondria of the
myocardium. The mitochondria were swollen and disrupted, and the myofibrils
were reduced.
Clinical Features
The clinical presentation in patients with idiopathic hemochromatosis has
89,264,273,689
been fully described. Therefore, this discussion will examine
the relationship between the iron deposits and the manifestations of the disease.
Attention will be paid first to the functional disorders that affect those organs
in which the highest concentrations of iron occur. Then the possible connection
between excessive quantities of iron in the body and certain secondary and in-
direct effects on body function will be considered.
210
-------
Direct associations between iron overload and clinical manifestations in
idiopathic hemochromatosis. The major manifestations are skin pigmentation,
hepatomegaly, diabetes, cardiac abnormalities, endocrine changes and arthropathy.
Bronzed skin pigmentation is common in most subjects. Excessive melanin is an
invariable finding, but hemosiderin deposits are seen in about 50% of patients,
as well; in the latter group, the skin is a peculiar slate gray.
Symptoms and signs associated with involvement of the liver are nonspecific, and
include cachexia, weight loss, and weakness. The liver is almost always enlarged
and firm, and signs of hepatic dysfunction, such as palmar erythema, spider angio-
mata, loss of body hair, and testicular atrophy, may appear. About 50% of patients
have splenomegaly, but ascites is rare. The liver disease usually runs
629
a protracted and benign course, except when alcohol abuse complicates matters.
Hepatoma eventually supervenes in about 15% of patients, its development suggested
273
by unexplained weight loss, fever, nodular enlargement of the liver, and ascites.
Symptoms related to the onset of diabetes are observed in more than half the
239,712
subjects at the time of diagnosis, and administration of insulin is usually
necessary. It was once thought that the diabetes was ascribable to islet cell
failure resulting from local iron deposits, but recent studies have indicated that
its etiology is more complex: at least three factors may be involved. Insulin
deficiency is undoubtedly one of them, but insulin resistance brought on by the
630
cirrhosis is another. Carbohydrate intolerance is more severe in hemochroma-
totics than it is in subjects with alcoholic cirrhosis, and this difference is
712
associated with a notably smaller insulin response. In addition, the preva-
lence of diabetes mellitus unassociated with marked siderosis is high in relatives
of subjects with idiopathic hemochromatosis, suggesting that inheritance of
40,239
traits for diabetes is independent of the inheritance of hemochromatosis.
Cardiac complications are the presenting feature in about 15% of subjects,
but as the disease progresses the percentage rises, and they represent perhaps
211
-------
the commonest cause of death. Supraventricular and ventricular arrhythmias of
various types have been described, and both left- and right-sided heart failure
may develop. The manifestations are usually those of a congestive cardiomyopathy.
In subjects under age 40, cardiac manifestations are more frequently the pre-
senting feature and almost always the cause of death, which usually follows
within a year of diagnosis unless specific venesection therapy is instituted.
Endocrine changes are common in idiopathic hemochromatosis. The most
striking are loss of libido, impotence, amenorrhea, and other evidence of hypo-
100,273
gonadism. For example, 70% of 115 patients studied exhibited loss of
781
body hair and testicular atrophy. Although it has been customary to ascribe
these changes to hepatic dysfunction, recent hormonal assays indicate that often
692
both the anterior pituitary and target organ fails Luteinizing hormone
levels were found to be low in most hemochromatotic subjects with testicular
atrophy, but normal in individuals with other types of cirrhosis and a similar
711
degree of gonadal atrophy. It has been established that the low levels of
luteinizing and follicle-stimulating hormones were the result of pituitary
70
rather than hypothalamic disturbance. Testosterone concentrations have also
been found to be below normal, and they do not rise after specific trophic
stimulation. These results are compatible with a primary disturbance of pitui-
tary gonadotrophic function followed by secondary and irreversible failure of
gonadal function. Disturbances of other pituitary trophic hormones, however,
are uncommon.
Although arthropathy was recognized only recently as a complication of
674
idiopathic hemochromatosis, its symptoms are often present. In half of those
240
individuals with such symptoms, chondrocalcinosis has been noted. Acute
attacks of inflammatory synovitis are characteristic of the condition, but other
227
clinical syndromes have been recognized.
212
-------
Iron overload from fermented beverages. In studies of this condition in black
South
/Africans, attention has been directed to features characteristic of idiopathic
hemochromatosis. Yet it must be emphasized that the degree of siderosis found
in the majority of subjects is not as great as in the idiopathic disease, and
that even in heavily siderotic individuals the iron is normally confined to the
liver and the reticuloendothelial system. Marked hepatic siderosis occurs in a
96,387
very high proportion of those subjects who develop micronodular cirrhosis.
The prognosis in such individuals is poor. The majority succumb within a year
to the effects of liver failure and/or portal hypertension. As noted, the
iron distribution in many siderotic individuals with micronodular cirrhosis is
different from that found in the absence of cirrhosis. In cirrhotic subjects,
iron is heavily deposited in the pancreas and other organs; one set of autop-
sies revealed that as many as 20% had suffered from diabetes mellitus during
387
life. In another study, 7% of black males attending a diabetic clinic had
680
severe siderosis and micronodular cirrhosis. The clinical presentation in
such individuals was characteristic: affected patients were usually male,
middle-aged, and underweight. Their livers were firm and enlarged, and insulin
was needed to control the diabetes. Other features characteristic of idiopathic
hemochromatosis have not been reported in this variety of dietary iron overload.
Specifically, neither cardiac failure nor an endocrinopathy have been demon-
strated to be associated with the siderosis. However, certain other manifesta-
tions, including ascorbic acid deficiency and osteoporosis, which appear to be
indirect consequences of the increased body iron stores, do occur (see below).
Transfusional siderosis. Clinical manifestations similar to those of idio-
pathic hemochromatosis have been noted, especially when the affected individuals
have survived for extended periods. As mentioned, cirrhosis is rare in patients
with aplastic anemia, but it develops almost invariably in those with
213
-------
refractory anemias and hypercellular bone marrows who live long enough. Dia-
betes has been noted in both groups. In 20 patients with aplastic anemia who
had received more than 47 1 of blood, 20% had overt diabetes and in another 25%,
89
glucose tolerance was impaired. Overt diabetes mellitus also is frequent
among older patients with thalassemia major; and in one study, insulin response
446
to glucose was delayed and/or diminished in 6 out of 8 thalassemic subjects.
Two of these had insulin-dependent diabetes, as did 3 out of 8 patients studied
540
elsewhere.
Other manifestations of disturbed endocrine function in patients with
thalassemia major are particularly prominent in those patients who survive
45 411
into the teens. Growth is almost uniformly retarded, but not from
growth-hormone deficiency, as growth-hormone responses to a number of stimuli
remain normal. It is probable that chronic anemia is the major factor,
because growth rates have improved in subjects who have been transfused more
414 570
intensively. ' Although clinical evidence of hypogonadism is usual,
its cause is uncertain. Levels of gonadotrophic hormones have been reported
as being appropriate to the ages of the patients. ' ' In contrast,
levels of luteinizing hormone were low in 4 of 5 adults tested despite low
447
estrogen and testosterone levels. Thyroid and adrenal function are usually
normal. Hypoparathyroidism is thought to be a preterminal complication of the
,. 540
disease.
Possible indirect sequelae of iron overload. Certain disorders of a
more general biochemical nature than those just discussed may occur in iron
overload. Observations were initially made in black South Africans, but such
malfunctions may also occur in other varieties.
The first condition that has been noted is ascorbic acid deficiency,
which appears in almost all South African blacks with substantial iron over-
681
load. Frank scurvy will develop in some. Ascorbic acid deficiency is
214
-------
most prominent in dietary iron overload, but it has also been noted in some
subjects with idiopathic hemochromatosis and transfusional siderosis, particu-
541,591,790
larly in patients with thalassemia major. Moreover, when guinea
pigs were made siderotic by repeated injections of iron dextran, they became
scorbutic even when their diet contained enough of the vitamin to be adequate
791
for the controls.
South African blacks with massive iron overload are not only ascorbic-
cno AQ1
acid depleted, but frequently osteoporotic. ' Some conception of the
frequency and strength of the association may be gained from the results of
a radiologic survey of 110 asymptomatic middle-aged manual laborers.
Seventeen were found to be osteoporotic, as evidenced by vertebral body
503
deformity and other signs of decreased bone density. In another combined
clinical and histologic investigation, the association between the bone
disease and severe iron overload was found to be highly significant, a con-
clusion further underlined by the finding in a necropsy study of a negative
503
correlation between hepatic iron concentration and mineral bone density.
Statistical analysis suggested that age alone was not responsible for the
correlation. In addition, a striking correlation exists between clinical scurvy
681
and osteoporosis in this population. Tissue stores of ascorbic acid have
been shown to be extremely low in osteoporotic individuals, even when they
£ Q-l
do not exhibit frank scurvy.
The incidence of osteoporosis in other forms of iron overload has not been
accurately documented, but it has been observed in individuals with idiopathic
223,237
hemochromatosis. Of special interest is a report that beef cattle in an
area of New Zealand where the iron content of the water is very high develop a
form of siderosis very similar to that seen in black South Africans. In many
severely affected animals, osteoporosis of the vertebrae, sternum, and ribs is
335
prominent.
215
-------
Relationship to Tissue Damage
The particular toxic role of iron in inducing specific manifestations
of organ failure associated with severe iron overload will be analyzed in
this section,
Iron overload and the liver. Hepatic fibrosis is common to all forms
of iron overload, and the evidence suggests that the relationship is a causal
one. Almost all subjects with idiopathic hemochromatosis have cirrhosis
?73 689
by the time the condition becomes clinically manifest, ' ' and in affected
88 818
but preclinical siblings iron overload precedes the onset of cirrhosis. '
In dietary iron overload the facts are even more persuasive, because the incidence
of serious portal fibrosis or cirrhosis correlates directly with hepatic iron
90
concentrations. This correlation is illustrated in Figure 7-1. Similarly,
in patients with transfusional hemochromatosis, especially those with chronic
refractory anemias associated with increased but ineffective erythroid
activity, the fibrous response appears to accelerate once iron concentration
649
in the liver reaches 3% dry weight. The relative rarity of cirrhosis in
subjects with aplastic anemias who have been transfused repeatedly may be a
89
function of two factors„ The iron derived from transfused blood primarily
goes to the reticuloendothelial system. Because of the reduced marrow activity,
relocation of this iron to other sites is slow. Evidently reticuloendothelial
iron, even when it is within the liver, is less likely to produce cirrhosis
than iron in hepatocytes. The second potentially important factor is time.
Most patients with aplastic anemia succumb within a relatively short time,
whereas subjects with other forms of refractory anemia may survive for decades.
The evidence that iron is a fibrogenic agent and can cause hepatic
cirrhosis appears overwhelming. Yet several questions still remain unresolved
concerning the influence of extraneous factors on the genesis of hepatic
fibrosis in siderotic subjects. In transfusional siderosis, they may include
216
-------
g>
1 0-0.19
£ 0.2-0.49
g 0.5-1.99
DC
Z
111
o
I
o
DC
2.0+
0
20
40
60
80
PREVALENCE OF MODERATELY SEVERE PORTAL FIBROSIS
AND PORTAL CIRRHOSIS, %
FIGURE 7-1 Correlation between increasing concentrations of
hepatic storage iron and portal fibrosis in black
subjects with dietary iron overload. Replotted
from data of Bothwell and Isaacson.
217
-------
serum hepatitis and possibly even chronic anoxia. In the other forms of iron
overload the most important factor is alcohol: at least 25% of the cases of
0*7*^
idiopathic hemochromatosis have a history of excessive alcohol consumption. '
In the dietary iron overload of South African blacks, alcohol is of even
greater importance because the source of the excess iron is a fermented
92 779
alcoholic beverage. ' Subjects with the greatest concentrations of
liver iron thus tend to be the heaviest drinkers, and the ones whose diets
are the least nutritionally adequate. Analysis is further bedevilled by the
development of secondary iron overload in a small number of subjects with
O "I f\ £OQ QOfl
alcoholic cirrhosis who absorb excessive amounts of iron. ' '
/
Despite these complications, the ubiquity of hepatic fibresis in iron
overload regardless of pathogenesis supports the notion that iron is central
in its production. However, iron probably is only a low-grade fibrogenic
agent that requires high local concentrations and protracted exposure to exert
its effects. Such an interpretation .would be compatible with a necropsy
study of dietary iron overload in which 30% of the bodies with hepatic iron
90
concentrations above 2% dry weight showed no substantive portal fibrosis.
It would also fit with the experimental observation that the siderotic liver
298
is particularly vulnerable to nutritional, metabolic, and toxic insults.
Iron overload and the pancreas. Diabetes is one of the commonest clinical
manifestations of idiopathic hemochromatosis: about 80% of idiopathic hemochroma
tosis patients have diabetes. Its occurrence in cases of dietary iron overload
and transfusional hemochromatosis strengthens the concept that iron overload
plays a part in its pathogenesis. Evidently heavy iron deposits in the islet
cells of the pancreas lead to reduced insulin output and hence diabetes. How-
ever, such a conclusion is not as straightforward as it would first appear.
Not only are the iron deposits much less marked in the endocrine than in the
218
-------
235,689
exocrine cells, but insulin levels are only diminished in some diabetics
239,712
with idiopathic hemochromatosis. In addition, familial glucose intoler-
40,239
ance and cirrhosis also contribute to the pathogenesis of the diabetes.
Insulin levels have not been measured in South Africans with dietary iron over-
load, but they have been found to be reduced in patients with thalassemia. In
summary, insulin lack, secondary to damage of islet cells by iron, may be one of
the factors responsible for diabetes in some patients with iron overload.
Iron overload and the endocrine disorders. Hypogonadism is common in
89 711
subjects with idiopathic hemochromatosis. ' Iron deposits are prominent
in the anterior pituitary, and hormonal studies have demonstrated low levels
of luteinizing and follicle-stimulating hormones unresponsive to hypothalamic-
releasing hormones. Testosterone levels are reduced and do not rise after
administration of trophic hormones, although testicular iron deposits are
scanty. ' Hypogonadism is found in siderotic thalassemics. Its pattern
is similar to that in idiopathic hemochromatosis, although it is unknown if
540
the primary defect is at the pituitary or the hypothalamic level. The few
published results suggest that iron deposits in the pituitary may have a
selective effect on function in some patients. The sex trophic hormones are
447
the most susceptible, because the levels of other trophic hormones are
usually normal. ' Interpretation of results is complicated by the sparse-
540
ness of normal standards for childhood and puberty. The testicular failure
should be at least partly ascribable to lack of trophic stimulation.
Iron overload and the heart. The most direct evidence of iron toxicity
273
has been obtained for disorders of cardiac function. Cardiac failure is
suggested as the commonest cause of death in idiopathic hemochromatosis, as is
true for thalassendc subjects whose lives have been prolonged by maintaining
255 540
an adequate hemoglobin level with repeated blood transfusions. ' In
219
-------
idiopathic hemochromatosis the cardiopathy is more marked when the clinical
89
disease materializes in youth. Why this should be so is not known, but the
phenomenon may relate to the rate of iron accumulation. Fatal cardiopathies
in young adults and adolescent subjects with thalassemla who have been
heavily transfused are particularly noteworthy, because the age factor tends
to discredit the role of variables such as excessive alcohol intake and
anemia in the genesis of the cardiopathy. Cardiac complications have not been
159
described in dietary iron overload, and two possible explantations exist
for this apparent anomaly. Few subjects accumulate appreciable amounts of
iron in the myocardium even when they are heavily overloaded with iron, and
those who do, succumb rapidly to hepatic failure.
Iron overload and arthropathy. The frequent occurrence of chondrocal-
cinosis in idiopathic hemochromatosis has led to speculation as to how the
iron deposits might contribute to its genesis. It has been suggested that
329
the excessive iron in the joint tissues may inhibit pyrophosphatase. Since
this enzyme normally hydrolyzes pyrophosphate to the more soluble ortho-
phosphate, its inhibition promotes pyrophosphate deposition. Complications
in joints have not been described in other forms of iron overload.
Iron overload, ascorbic acid deficiency, and osteoporosis. The diet of
black South Africans with iron overload certainly does not contain optimal
amounts of ascorbic acid, but that alone will not explain the severe tissue
fifti
depletion of ascorbic acid they exhibit. Ascorbic acid nutrition is adequate
in nonsiderotic subjects whose diet is similar except for the beer, and much
504
of the available ascorbic acid is rapidly catabolized in siderotic individuals.
When large doses of ascorbic acid are given to such subjects, only small
quantities of the vitamin appear in the urine. Instead, more of an end oxida-
tion product of ascorbic acid, i.e., oxalic acid, is excreted. Presumably
220
-------
the rise results from the large deposits of ferric iron, as a similar phenomenon
790
has been observed in other forms of iron overload. Abnormal ascorbic acid
metabolism also has been demonstrated in dietary iron overload by tracing the
330
radioactively labeled vitamin. Under such circumstances, the ascorbic
acid is also rapidly catabolized, although not to oxalic acid; a large fraction
of the carbon-14 label is excreted as carbon dioxide.
The ascorbic acid deficiency accompanying dietary iron overload has
several consequences, one of which is interference with the release of iron
477
from reticuloendothelial cells. Such inhibition provides at least a partial
explanation for the prominent reticuloendothelial deposits in those subjects.
It undoubtedly accounts for their frequently normal plasma iron concentrations,
because administering ascorbic acid produces a sharp rise to more appropriate
789
levels. The relatively low degree of transferrin saturation may well be the
reason why the unloading of iron onto parenchymal tissues is so rare in this
form of iron overload.
That the ascorbic acid deficiency of dietary iron overload is a manifesta-
tion of deranged vitamin metabolism and not merely the result of a poor dietary
intake has been confirmed by the finding of reduced tissue ascorbic acid con-
centrations in well-nourished subjects with other forms of iron overload,
790
especially thalassemia major. In addition, administration of large doses of
ascorbic acid to patients with an adequate diet is also followed by elevated
oxalic acid excretion. Finally, it has been possible to reproduce a similar
condition in animals. Guinea pigs made siderotic by injecting iron dextran
develop severe ascorbic acid depletion, even when their diets contain adequate
791
amounts of the vitamin.
There is little doubt that iron overload is connected with ascorbic acid
deficiency, but the relationship between iron overload and osteoporosis is
221
-------
not so well defined,, At least one of the links between the two conditions is
ascorbic acid deficiency. Scurvy is known to be associated with osteoporosis
in children and experimental animals, which has been ascribed to the necessity
of ascorbic acid for ostepgenesis, including collagen synthesis, osteoblast
772
maturation, and osteoid formation. Bone resorption may be increased in
ascorbic acid deficiency; semiquantitative microradiography of the osteo-
porotic bones of ascorbic acid-depleted guinea pigs has revealed not only the
expected diminution in the bone formation surface, but an increase in the bone
791
resorption surface. A similar enlargement of the bone resorption surface
has been observed in osteoporotic black South Africans with severe dietary iron
791
overload. In addition, the results of experiments with radioactive calcium
suggest that decreased bone formation and increased bone resorption may be
present simultaneously. Finally, repletion with ascorbic acid may lead to
decreased urinary calcium excretion in such subjects.
Although all this evidence is compatible with the thesis that ascorbic
acid deficiency induced by iron overload is responsible for the osteoporosis,
especially when coupled with the occurrence of osteoporosis in other forms of
iron overload, it is not conclusive. Other factors—such as alcoholism, malnu-
trition, and associated liver disease—may all play a part. However, their
specific roles remain to be elucidated.
Iron overload and porphyria cutanea tarda. Porphyria cutanea tarda is
440
common in siderotic South African blacks. Other sources indicate that mild
to moderate iron overload is frequent. in porphyria cutanea tarda patients
in other countries. ' The majority of such patients have a history of
alcohol abuse, and hepatic fibrosis or cirrhosis are often found.
Possible reasons for the association include the large iron content of
some alcoholic drinks, the enhancement of iron absorption that alcohol
itself may cause, and the increased iron absorption sometimes observed in
222
-------
820
alcohol-induced liver disease. Indeed, greater than normal rates of iron
753
absorption have been found in subjects with porphyria cutanea tarda.
However, the association could be causal in the opposite direction, that
is, increased iron deposits may affect the pathogenesis of the porphyria. For
example, rats given large doses of iron dextran become porphyric when given
729
hexachlorobenzene more readily than do control animals. Of possible
relevance, too, is the finding that the rate of in vitro uroporphyrin synthesis
439
increases when ferritin and cysteine are added to the system. Although
increased amounts of iron may potentiate the metabolic defect in porphyria
cutanea tarda, it is not an essential trigger, as the condition has been
observed in subjects with normal storage iron concentrations,,
Effects of therapy in iron overload. If increased iron deposits are
capable of damaging tissues and impairing their function, then removal of the
iron would be expected to reverse or at least arrest those changes. In the
belief that iron is_ noxious, the treatment of idiopathic hemochromatosis by
269
venesection therapy was started almost 30 years ago. The rationale was a
simple one. It was argued that the deficit created (+ 400 mg iron/1 blood)
in Lhe red cell hemoglobin would have to be made good from the iron stores.
It was soon confirmed that patients with idiopathic hemochromatosis were
able to tolerate the removal of large amounts of blood for protracted periods,
so that it was possible to return the body iron content to normal within a
89
year or two. Subsequent studies assessed the effects of such treatment
on the function of the most severely affected organs and investigated
whether or not survival was prolonged. It has not been possible
to institute venesection treatment in siderotic black South Africans, who are
typically among the less sophisticated members of the community and unwilling
223
-------
to see large amounts of blood removed. No judgment can thus be made on its
therapeutic effects in dietary iron overload. For obvious reasons, venesection
has not been practical in subjects with transfusional hemochromatosis.
However, attempts have been made to reduce body iron content in such subjects
by using specific iron chelating agents such as desferrioxamine and diethylene-
25
triaminepentaacetic acid (DTPA).
±t should be stressed that no controlled clinical trial has been per-
formed in which two carefully matched groups of patients with idiopathic hemo-
chromatosis have been monitored, with one being venesected and the other not.
Despite this drawback, the uniform consensus nevertheless has been that remov-
ing iron is of definite value to the patient. In general, treated patients
live longer, and the longer survival is often accompanied by weight gain,
80 819
reduced skin pigmentation and hepatomegaly, and improved cardiac function. '
Gross disturbances in hepatic function are unusual at the time of
diagnosis, and therapy usually corrects minor abnormalities. isolated reports
exist of 6 patients in whom hepatic fibrosis or cirrhosis was judged on the
305
basis of liver biopsy findings to have reverted to normal. In a more
systematic study of 85 patients, 5 who were initially cirrhotic were judged to
QQ
exhibit only hepatic fibrosis 3-9 yr later. However, difficulties in inter-
preting liver histology on the basis of needle biopsy are well known. There-
fore, no final judgment is possible on the degree to which cirrhosis in
idiopathic hemochromatosis is reversible by iron removal. There is no evidence
80
that portal hypertension is benefited by venesection therapy. The incidence
of hepatoma does not appear to be reduced by treatment. In fact, the disorder
may even be greater in treated subjects, although the incidence could be a
function of their longer survival rate. Of 45 patients dying from the disease
on
in one study, 13 (29%) succumbed to hepatoma. Ten of the 13 completed a
224
-------
course of venesection therapy, while 3 died within 6 mo of commencing therapy.
Three patients died within 3 yr and the remaining 7 died 4-15 yr after the
initial venesections.
The degree to which glucose intolerance can be modified by therapy has
been carefully documented by Williams and his coworkers, who studied 85 hemo-
chromatotic patients, about two-thirds of whom were diabetic at the time of
80
presentation. The effects of venesection therapy were modest at best.
Improved glucose tolerance, designated as a reduction in insulin requirements
of at least 12 units/day, or a conversion to oral hypoglycemic therapy, occurred
in only 28% of the patients. Fifty-eight percent showed no change in require-
ments for antidiabetic agents, and 14% needed more insulin after venesection
therapy.
Cardiac function often improves after removing excess iron, and venesection
treatment undoubtedly has reduced the number of deaths ascribable to the
OAO OA1 ^ 1 *^ S99 ftQA
specific cardiopathy. ' ' ' ' Many patients have not required any
maintenance cardiac therapy after the iron has been removed, which further
validates the efficacy of such therapy. Timely treatment may also prevent onset
819
of clinical manifestations of cardiac disease in affected individuals.
No relief of hypogonadism or arthropathy has been recorded after
80
venesection treatment.
The most complete statistics on the effects of therapy on survival are
80
those of Bomford and coworkers. They have updated and extended their earlier
study, in which they found that the 5-yr mortality in 40 phlebotomized patients
819
was only 11%, compared to 67% in a retrospective group of 18 patients.
The newer figures are based on 85 phlebotomized patients and a control group
of 26 subjects made up of the original 18 patients plus another 8 who had
refused therapy, died soon after diagnosis, or in whom diagnoses were made at
225
-------
necropsy. The survival at 5 yr was 64% in the treated group and 15% in the
untreated one. Patients who had died from both groups were matched by age and
by the presence of complications, including diabetes, cirrhosis, hepatic
failure, and hepatoma. The analysis again yielded a highly significant
difference, shown in Figure 7-2. The mean log survival in 45 dead treated
patients was 4.3 yr, compared to 2.9 yr in the untreated ones. Twenty-two
percent of the treated group succumbed to neoplasms other than hepatoma,
whereas none did in the untreated group. In another study, retrospective
analysis also revealed an improved survival rate in venesected patients, with
3 deaths in 19 treated individuals as compared to 4 deaths in the untreated
group of 7 subjects.
Data on the effects of chelation therapy in thalassemia major are limited
but encouraging. Two groups of children were followed from 5-8 yr. The
treated group was given 0.5 g desferrioxamine intramuscularly 6 days a wk
45 649
as well as 2 g DTPA/unit of transfused blood, ' In both groups, hemoglobin
concentrations were maintained between 8-15 g/100 ml, and at the start of the
trial, the 9 children in each group were matched according to age, amount of
blood transfusedj and hepatic iron concentrations. At the end of 6 yr,
significant differences were measured in the concentrations of iron in the
liver; in the group given chelating agents, mean values were 2.6% dry weight,
as compared to 4.27o in the control group. The extent of hepatic fibrosis was
also carefully assessed, using an index obtained from camera lucida drawings
of liver biopsy specimens. Initially the fibrosis indices overlapped widely,
but by the end of the study a highly significant difference had emerged; the
fibrosis index had become more marked in the untreated group, yet had remained
the same in the chelated group.
226
-------
YEARS AFTER DIAGNOSIS
FIGURE 7-2 Cumulative survival after diagnosis in 45 treated
and 18 untreated patients with idiopathic hemo-
chromatosis. The vertical lines at each interval
represent +1.standard error. Reproduced from
Bomford et~al.80
227
-------
These differences might have been more dramatic had more effective chela-
tion therapy been available. Desferrioxamine is effective only after a certain
degree of siderosis has been reached, equivalent to 37.6-47 1 of transfused
540
blood. Therefore, it only is possible to contain the iron overload within
limits well within the range found in untreated idiopathic hemochromatosis.
Despite the limitations, the arrest of the hepatic fibrosis by such therapy .
provides strong indirect confirmation of the fibrogenic potential of heavy
iron deposits in the liver. Other noteworthy clinical observations also came
out of this investigation. Puberty was delayed in 4 out of 5 control children,
but in only 1 out of 4 treated patients, and a growth spurt had occurred only
in those subjects in whom puberty was not delayed. The incidence of diabetes
and its clinical manifestations were similar in each group. In another study
in which a larger dose of desferrioxamine was given, some improvement in
clinical symptoms and signs was noted—the electrocardiogram and liver function
684
tests showed a return toward normal.
Mild to moderate siderosis commonly accompanies porphyria cutanea tardaa
Clinical manifestations subside and porphyrin synthesis decreases when iron
639
stores are depleted by venesection therapy. It is uncertain if only the
removal of iron is important for improvement. The subject who embarks on a
venesection program may well also stop drinking, which may contribute to the
improvement. Iron nevertheless appears to play some key role, since urinary
9 Sfi
porphyrin excretion has diminished during venesection therapy even in
497
patients who have continued to drink. Biochemical relapse has been induced
498
by replenishing the iron stores.
Animal models of iron overload. The discussion thus far is based on
analyses of human disease and its treatment. For a closer examination of the
chronic toxicity induced by the metal, an acceptable animal model is neces-
sary. Unfortunately, attempts to create such a model have been unsuccessful.
228
-------
Much work and many ingenious approaches have not produced hemochromatosis
by dietary manipulation. Various species have been fed diets supplemented
with iron, and a number of methods have been used to try and increase iron
absorption. They have included reducing the phosphate content of the diet,
cyclic starvation and feeding, ligation of the pancreatic duct, splenectomy
and administration of such substances as DL-e£hionine, polysorbate 20 and
89
D-sorbitol. Despite such maneuvers, body iron concentrations usually have
stayed much lower than those found in idiopathic hemochromatosis. In most
instances, the iron has been confined to the liver and reticuloendothelial
system, although minimal deposits have sometimes been noted in the pancreas,
thyroid, and myocardium,, Tissue damage such as hepatic cirrhosis or pancreatic
fibrosis has never been produced by simple dietary iron overload. Success
was, however, claimed by workers who gave a choline-deficient, high-fat diet
CQQ
and excessive iron to rats for more than a year. The animals developed
fatty livers and progressively increasing cirrhosis; the changes could be
prevented by folic acid. However, the hepatic iron concentrations were
relatively modest (only 6 times normal), so that their relevance to hemo-
chromatosis is questionable. Similar doubt must be cast on studies in which
cirrhosis has been produced in rats by feeding iron supplements plus carbon
421
tetrachloride, although these experiments do support the contention of Golberg
298
and Smith that iron overload makes the liver more susceptible to other
noxious agents. Their view has not gone unchallenged, however, as other
workers have been unable to demonstrate such difference when they used
OOQ onc
ethionine as the second supplement. ' The discrepancies may be explain-
able by the finding that iron loading does not potentiate the early "hepatitic"
598
stage of ethionine injury, but rather the second "cirrhotogenic" stage.
This second stage involves interaction between excess iron and the cell membrane,
421 824
but the exact mechanism has not been defined. '
229
-------
Iron has also been administered parenterally to animals as colloidal iron,
saccharated iron oxide, iron ascorbate gelatin, iron dextran, and red blood
cells. It is usually injected intravenously, but the intraperitoneal and
intramuscular routes also have been used. The final body iron content has
varied between 0.1-3.3 g/kg body weight, and animals were tested anytime
89
from 4 wk to 7 yr later. When animals are given very large doses of iron,
certain pathologic changes are produced, but they have been unlike those found
in human hemochromatosis. In guinea pigs, for example, subcutaneous injections
of inorganic iron to a total dosage of 1 g/kg body weight caused hemorrhagic
phenomena in the lungs and adrenals and patchy parenchymal damage of the
585
liver. However, neither cirrhosis nor pancreatic fibrosis was induced.
The only morphologic changes in rats subjected to huge doses of parenteral iron
were proliferation and hypertrophy of the hepatocytes, ascribed to the induc-
232
tion of protein synthesis. In another experiment, massive iron overload
(2.5-3.'i g/kg body weight) was produced in dogs. It caused anorexia, apathy,
and weight loss with death after 5-10 mo, but autopsies revealed little morpho-
106 479
logic evidence of liver injury. Greater success was achieved by Lisboa,
who produced cirrhosis in dogs after 4 yr by injecting iron intravenously as
iron dextran to an accumulated dosage of 3.5-5.8 g/kg body weight. It
should be noted that the tissue iron concentrations in these studies were
several times' greater than those encountered in idiopathic hemochromatosis,
and that no pathologic changes appeared even after extended periods during
another study in which the iron levels were comparable. Tests for hepatic
and cardiac function and for glucose tolerance in these dogs were normal.
Serum proteins, liver function as measured by sulfobromophthalin sodium, and
glucose tolerance have also been found to be normal in iron-loaded rats and
rabbits. Animal experiments in which siderosis has been accompanied by
alcohol have also been negative. When rhesus monkeys were given iron dextran
intravenously (0.5 g/kg) and were then exposed to alcohol, or alcohol plus a
230
-------
low-protein diet, or carbon tetrachloride, no hepatic fibrosis was apparent
564
in any of the groups even after 110 wk.
The reasons for the almost uniform failure of parenteral iron-loading
experiments are several. The first is undoubtedly the predominant reticulo-
endothelial localization of the injected iron complexes. Although some iron
may be redistributed eventually, the amounts taken up by parenchymal cells
are usually small. In one study in dogs, for example, almost no iron moved
out of the reticuloendothelial cells even after the passage of 7 yr.
Therefore, the pattern is totally dissimilar to that found in hemochromatosis.
If these experiments prove anything, it is the capacity of the reticulo-
endothelial system to tolerate huge amounts of iron. Another drawback is the
necessarily short duration of most such studies, compared to the length of
exposure in the human diseases. Finally, species may well differ in their
susceptibility to the noxious effects of excessive tissue iron.
Mechanisms involved in iron toxicity . Despite the largely negative
results of attempts to produce experimental hemochromatosis in animals, the
cumulative experience in human subjects suffering from iron overload of
diverse etiologies strongly suggests that iron i£ noxious to tissues. For it
to exert these effects, two criteria must be satisfied: the metal must be
present in sufficient concentrations for long periods of time, and it must be
present in parenchymal calls. In contrast, the reticuloendothelial cells
appear do be admirably equipped to store excessive quantities of the metal.
(In the dietary overload encountered in South African blacks, the massive
quantities of iron in the spleen and reticuloendothelial cells of the bone
marrow do not appear to exert any direct pathologic effects.) Why this strik-
ing difference in cellular susceptibility exists is not known. Knowledge of
the mechanisms by which iron does damage tissues is itself largely speculative.
231
-------
Iron has been shown to accumulate in liver cell lysosomes of iron-loaded
rats, and liver biopsies from humans with hemochromatosis have also revealed
dense deposits of ferritin and hemosiderin in structures tentatively diagnosed
/"O oi Q
as lysosomes. ' The activity of hepatic lysosomal enzymes has been shown
to be markedly increased in patients with various types of iron overload and their
61 9
lysosomes were strikingly more fragile than normal. Perhaps iron accumula-
tion damages the lysosomal membrane, which then releases acid hydrolases into
the cytoplasm and initiates cell damage. These abnormalities return to
normal after removing, excessive iron from the liver, which suggests that the iron
612
causes them. However, the mechanism by which it injures the lysosomes is not
known. The iron deposits may catalyze the formation of free radicals and
they, in turn, may damage lysosomes and other subcellular organelles through
/••t r\
lipid peroxidation.
The other metabolic abnormality induced by iron overload is depletion of
159
ascorbic acid levels in tissues (see above). Whether chronic subclinical
depletion of the vitamin has effects other than scurvy is not known.
Lack of its nonspecific antioxidant action may damage susceptible tissues.
Serum concentrations of vitamin E (also an antioxidant) have been found to be
384
decreased in thalassemia major.
CARClNOGENESIS, MUTAGKNESIS. AND TERATOGENESIS
Carcinogenesis needs to be considered as a complication of iron deficiency,
of iron overload states, and of exposure of specific tissues to iron. In connec-
tion with epithelial changes in the esophagus of patients with chronic iron
deficiency, a few reports have appeared in the older literature of carcinoma of
382
the mid-esophagus. Althoughthis condition has been assumed to be related to
iron deficiency, the causal relationship is poorly established. A more definite
association has been found between carcinoma of the liver and parenchymal iron
232
-------
overload, described in approximately 15% of patients with idiopathic hemochrom-
273
atosis. The malignancy may become manifest years after the removal of excess
819
iron; presumably the changes have a long gestation period not reversible by
iron removal. A remote possibility exists that the malignancy is caused by
some other substance that is also absorbed in excessive amounts by hemochroma-
totics and which is not removed by phlebotomy. Neoplasms of other organs also
819
are more frequent in idiopathic hemochromatosis. There is no suspicion of
local malignancy accountable to gastrointestinal ingestion of inordinate amounts
of iron.
Intramuscular injections of iron dextran into animals have produced sar-
298,496
coma. This finding has implications for the therapeutic use of this
substance in treating iron-deficient patients who cannot respond to oral iron.
Isolated case reports of local malignant change in such patients give some sub-
309
stance to this. However, iron dextran is now given largely intravenously,
which should circumvent the possibility of risk.
Iron has not been reported to be mutagenic or teratogenic.
233
-------
CHAPTER 8
INHALATION OF IRON
SOURCES OF EXPOSURE
For Animals
The exposure of animals to inhaled iron is almost exclusively a
laboratory phenomenon. Syrian golden hamsters, guinea pigs, mice, and rats
97a
are the most commonly used animals in such experiments. Laboratory
exposure usually is through natural inhalation or through intratracheal injec-
tions of iron oxide particles of 5 urn or less and in single doses as high as
7 mg/m3 or a 2% solution.21'3483
Although some reports ' suggest that pulmonary fibrosis can
occur after inhalation of iron oxide, others studying tumor formation ' '
failed to note fibrosis in the lungs when iron oxide served as a vehicle for
benze/faj^pyrene and other polycyclic hydrocarbons found in coal tars. '
The lungs of animals exposed to ambient iron oxide near steel mills
and mines have not been examined for the effects of such exposure per se .
Cattle exposed to airborne dust
particularly high in fluorides near steel plants have been autopsied. Their
lungs showed anthracosis, along with slight to moderate involvement of the
regional lymph nodes with carbonaceous material. No evidence of any fibro-
sis or deposition of iron oxide was found. These reactions decreased as the
distance from the plant increased.
FOT Humans
Human exposure to inhaled iron generally comes from an occupational
source. The following occupations present risks of inhalation of dust and
234
-------
fumes of iron and its alloys and compounds: iron-ore mining, arc welding,
379
metal (iron) grinding, iron and silver polishing, metal working, sintering,
scarfing (personal communication, M. Bundy), and pigment manufacturing and
rubber manufacturing.
173
The Industrial Hygiene Association issued an ambient air quality guide
for iron oxide, Fe 0~. It stated that no potential acute or chronic hazards
were observed in exposed humans and animals even at the highest air concentra-
833
tions reportedo Iron oxide alone is "biologically inert" in that it
produces no direct tissue injury in experimental animals or humans as a result
of exposures in the work place.
EFFECT AND FATE OF INHALED IRON
In Animals
Pulmonary clearance. When inhaled, iron oxide particles penetrate into
O (if.
the lung parenchyma of mice and may be retained for more than 100 days. The
particles are observed inside macrophages collected around terminal bronchioles
275
and lymphoid tissue. Fisher, using Freund's complete adjuvant (FCA) to
study clearance of iron-59 oxide in the rat, found that clearance was increased
when iron oxide was administered 1-3 days after the use of FCA, because then the
response of free macrophages was the greatest. The amount of iron cleared in
the first 24 h as well as the rate of clearance during the later phases were
increased compared to control animals 40 days after exposure. These findings
suggest that phagocytic action is important in both early and late clearance
phases.
Saffiotti et al. reported that a small portion of the iron-oxide dust
introduced into the lungs of Syrian golden hamsters remained within the lungs
and the tracheobronchial lymph nodes throughout the lifespan of the animals.
235
-------
Macrophages engulf the iron particles that reach the alveoli, penetrate
the alveolar wall with this burden, and ultimately transport the particles
through this means. Some of the particles are carried to the trachio-
bronchial lymph nodes. This process of deposition, retention, and clearance
is not unique to iron oxide—it is common for all particles Syum or smaller
that enter the alveolar spaces. Alveolar macrophages appeared to affect
461
the clearance of radioactive iron dust from cats, rats, and monkeys. The
bronchi of all species were almost completely cleared in less than 2 days,
whereas the alveoli took 16-28 days in the rata and cats and 280-300 days in
the monkey.
626
Pulmonary toxicity. Using a scanning electron microscope, Port et al.
found acute changes in the surface morphology of hamster tracheobronchial
epithelium following thrice-weekly intratracheal administration of ferric
oxide in 10 doses of 5 mg each. These multiple infusions of ferric oxide
particles smaller than 5 yim suspended in an 0.9% salt solution produced
a loss of ciliated cells and broad areas of abnormal, enlarged, unciliated
cells with roughened or wrinkled surfaces. The abnormal cells were thought
to be areas of epithelial hyperplasia but their significance remains unknown.
This is the first and so far the only report of epithelial changes induced
from administration of relatively large doses of ferric oxide. Whether lower
doses given over a longer exposure time would produce the same response or if
changes would be found in the lower airways of the bronchial tree is not known.
The scanning microscope seems to be a useful tool for exploring reactions to
what was previously considered an "inert" dust particle.
Iron as a vehicle. Iron oxide can serve as a vehicle to transport
21
pollutants into the body via the air passages. Amdur and Underbill exposed
guinea pigs to open-hearth dust (90% iron oxide) alone or in combination with
236
-------
several concentrations of sulfur dioxide. Open hearth dust alone produced
no detectable respiratory response even when given at a concentration as high
o
as 7 mg/m . Neither did it affect the response to any of the sulfur dioxide,
which was administered in doses of 1.6-2.6 ^ig/ml. The findings were similar
21
for an aerosol of iron oxide. Soluble iron salts, however, do potentiate
21
responses to sulfur dioxide.
In Humans
Pulmonary clearance. The fate of iron particles that enter the human
lung through occupational exposure has been studied. Lung biopsies of seven
welders with siderosis have shown that some iron remains free in the alveoli
and bronchioles, but most of it is taken up in macrophages and transported
into the lymphatic channels. Much of the incoming dust is collected on the
mucus blanket and is expectorated; the dust is recognized easily by the worker
327
because of its rust color.
47? ASA A«S 64^
Injury to the lung. Investigators ''' who studied the
mortality patterns of 59,000 steelworkers for 13 yr (1953-1966) were unable
to find any toxic effect that might be attributable to the inhalation of iron
oxide.
•k
In their study of 4,588 crane operators in steel plants where exposure
472
to ambient iron oxide occurs, Lerer et al. reported that the total mortality
experience, as reflected in a relative risk of 1.00, was the same as the
mortality experience of workers who had never operated cranes. When analyzed
by specific causes, the Caucasian craneman had a 10% deficit mortality risk
for lung cancer. That is, 51.3 deaths were predicted and 47 were observed.
ic
Crane operators were selected because of reports in the literature that they
stood an excess risk of dying from lung cancer.
237
-------
The relative risk from other respiratory diseases was 0.81 (24 deaths
reported versus 28.8 expected), a decrease when compared to other occupations
in the same plant. In a long-term study of 9,655 open hearth workers,
mortality from lung cancer was usually less than 1.00 and- mortality from non-
malignant respiratory diseases was similar to that of other steelworkers in
643
the same plant, a relative risk of 1.00.
Caucasian crane operators showed an overall excess risk of dying from
472
cardiovascular and renal diseases, with a relative risk of 1.09. The excess
risk seemed to be related to particulate exposure,with the greater risk in
areas of greater particulate exposure. The mortality sample of non-white crane
operators was too small to determine relative risks for cardiovascular disease.
Open-hearth workers showed a deficit in mortality from cardiovascular diseases
with a relative risk of 0.90, which is statistically significant at the 1%
level. If particulates, i.e« iron oxide, are related to this phenomena, as
proposed in Lerer and coworkers* discussion of crane operators, then it is
exceedingly difficult to explain the deficit in cardiovascular disease mortality
643
in the open hearth workers studied by Redmond et al.
The open-hearth workers had an excess mortality risk (17.6 deaths pre-
dicted versus 31 found)- from diseases of the digestive system, particularly
ulcers. The relative risk was 1.22, which was statistically significant at the
5% level. Heat stress and physical exertion, rather than particulates, may
have been at the root of the disorder.
489
Lowe did not note the influence of iron oxide in inducing chronic
bronchitis and emphysema in steelworkers he examined in regard to exposure to
atmospheric dusts in the workplace. The disorders were related only to the
smoking habits of the steelworkers.
238
-------
732
Teculescu and Albu performed several lung-function studies on
14 workers with a mean age of 43 and who had been exposed to pure iron oxide
dust for an average of 10 yr. They did not find abnormalities compatible
with pulmonary fibrosis, although chest radiographs showed nodular opacities.
These opacities are symptomatic of pulmonary siderosis.
Siderosis, the accumulation of iron oxide in the lungs, is a form of
pneumoconiosis. Detailed information on the X-ray appearance, pathologic
327 552
and physiologic responses of the disorder has been published elsewhere. '
If exposure to pure iron oxide ceases, the dramatic nodulations on the chest
X-ray disappear. ' They would not subside if fibrosis were a feature of
the pneumoconiotic nodule of siderosis.
Although the term pulmonary siderosis is reserved for the accumulation of
pure iron oxide, it is probable that inhaling pure iron oxide never leads to
fibrotic pulmonary changes, whereas inhaling iron oxide plus certain other
substances most certainly does. Reports of pulmonary fibrosis in
hematite miners ' indicate that prolonged exposure, usually over 20-30 yr,
is required, although some signs have been noted after 10-yr exposures. It
should be noted that the ore to which the miners were exposed contained
approximately 10% free silica, a known fibrogenic agent. When pulmonary fibrosis
seems to be associated with occupational exposure to iron oxide, careful
investigation usually will uncover a simultaneous exposure to free silica.
Thus many exposures are to a mixture of materials and these additional particu-
lates (silica) may indeed produce fibrotic changes in the lungs. The resulting
disease is called silicosis.
*
In this chapter, siderosis only refers to pneumoconiosis resulting from the
inhalation of iron particles, and not to the cases of excess iron in the
blood or tissues discussed in Chapter 7.
239
-------
Some iron compounds are injurious when inhaled. However, the danger-
ous properties of an iron compound are a function of the ligand with which the
iron is coupled. Thus ferric arsenate and ferric arsenite possess the poison-
ous property of the arsenical ligand. Similarly, iron pentacarbonyl, Fe(CO),.,
known as iron carbonyl, is one of the most dangerous of the metal carbonyls,
having toxic and flammable properties.
This metal carbonyl is a yellow-brown liquid at ambient conditions and is
lOOa
highly flammable. Its presence may be suspected whenever high partial
pressures of carbon monoxide come in contact with iron or steel vessels. Iron
carbonyl is formed in gas-manufacturing processes, and it (along with nickel
carbonyl) must be removed to eliminate soot formation at the ultimate burning
point. Iron carbonyl may be formed in illuminating gas that has passed through
iron pipes, in water gas, in coal gas stored underground, and in gases coritain.-
ing carbon monoxide held under pressure in steel cylinders. Traces of this
carbonyl have been found in gases produced by gasification of oil or refinery
gas with steam over nickel catalysts at atmospheric pressure. Liquid iron
carbonyl is used as an antiknock agent in some gasolines.
The signs of iron carbonyl poisoning in animals are similar to those of
other metal carbonyls and include respiratory distress, cyanosis, tremors, and
paralysis of the extremities. Death may be immediate or delayed for a few
days. Human exposures similarly produce signs and symptoms similar for the
common metal carbonyls. Immediate exposure produces giddiness and headache,
and they are occasionally accompanied by dyspnea and vomiting. The signs and
symptoms may be relieved if the individual is removed to fresh air; however,
dyspnea returns 12-36 h later, with the addition of cyanosis, fever, and cough.
240
-------
If the exposure has been to lethal concentrations, death follows in 4-11 days.
Pathologic changes include pulmonary hepatization, vascular changes, and
degeneration of structure in the central nervous system.
CARCINOGENESIS*
94a
Boyd et al. studied the mortality of Cumberland iron-ore miners through
death certificates of 5,811 men from 2 mining communities and who died between
1948 and 1967. Comparison of the iron miners' experience with that of other
local men and the relevant national experience led to suspicions of an occupa-
tional hazard of lung cancer associated with hematite mining. Earlier,
Faulds and Stewart a had reported an increased risk in Cumberland iron-ore
miners after studying post-mortem findings. During the 20-yr period studied by
Boyd, among all iron miners (underground and surface workers) the 42 lung
cancer deaths observed were 50% higher than the number expected (28), an
increment statistically significant at the 5% level. However, the number of
deaths from other cancers (74) was very close to what was expected (71). Com-
paring underground and surface workers, the excess of lung cancer was confined
to the underground group (observed 36, expected 20.58); there were 6 deaths
among the surface workers, and 7.13 had been expected. The higher incidence
of lung cancer mortality in the underground miners persisted when tested by
the same method against the proportionate mortality determined from the
national standards. Post-mortem examinations revealed a high proportion (37%)
of oat-cell tumors. This percentage was similar to the figure (43%) found by
Saccomanno et al. for neoplasms among uranium miners in Colorado. Data
on radon concentrations in the Cumberland mines were available for 1968, the
See Cole and Goldman's review of occupational carcinogenesis for a com-
parison of risks from iron and other metallic dusts and fumes.170a
241
-------
year after the completion of the Boyd et^ al. study. In 3 of the 4 mines
tested, none of the levels—ranging from 30 pCi/1 to 300 pCi/l--were below
30 pCi/1, the maximum permissible level recommended by the International
Commission on Radiologic Protection. The lung cancer risk in the British
miners was much less than that estimated for radium mines in Schneeberg,
Germany, Jacymov, Newfoundland, and Colorado. The radon concentration in
these latter mines was many times that found in the Cumberland mines. The
average was 100 pCi/1 at Cumberland compared to 2,900 pCi/1 in Schneeberg
(even after considerable improvements were made); the highest radon concentra-
tion measured at the Cumberland mines (320 pCi/1) was far below the respective
measurements of 25,000 pCi/1 and 59,000 pCi/1 for the Newfoundland and
Colorado mines. It may be, therefore, that the limit of a twofold increase
in lung cancer estimated for the West Cumberland iron miners is compatible
with their lesser exposure to radon in comparison with that of the other groups
of miners, for whom risk estimates extend upwards of 10 times normal for the
Colorado miners. The exposed rock in the Cumberland has not revealed unusual
radioactivity, and it is speculated that the radioactivity is carried by the
mine water, which is believed to be the source of it in the Newfoundland
94a
fluorspar mines. Others have thought radon to be the probable causative
552
agent in the development of lung cancer in hematite underground miners.
*5 OT"U
Ishinishi e± al. repeatedly instilled iron dusts collected from an
open-hearth furnace as well as dusts containing benzo^a/pyrene into the
tracheas of albino rats. The suspensions injected were 7.5 mg iron dust,
7.5 mg iron dust plus 1 mg benzo^a/pyrene, and 1 mg benzo/a/pyrene suspended
in 0.2 ml distilled water. The iron dusts and benzo^a/pyrene particles were
no larger than 5 ^um. The iron dusts contained 52% iron and less than 1% nickel,
242
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chromium, and arsenic. No difference in the death rate occurred between the
experimental groups (8 out of 90) , and the control group (4 out of 30) . Both
groups had been followed throughout their entire life. After 15 weekly instal-
lations spread over about 4 months, one male rat out of 14 had a malignant
tumor and 3 female rats out of 15 had tumors in the iron-dust group. In the
iron dust plus benzo/a.7pyrene group, 8 out of 17 males and 3 out of 9 females
had tumor So In the benzo/q/pyrene group, 3 males out of 13 and 3 females out
of 14 had tumors. There were no lung tumors in the control group comprised of
12 males and 14 females. Several sarcomas of organs or tissues were detected,
but they were also found in the controls. The lung tumor rates were about
twice as high in the iron dust plus benzo/aj^pyrene group as in either alone.
Because the dust from the iron furnace contained a small amount of nickel,
chromium, and arsenic (known carcinogens) , it may not be possible to conclude
with certainty that iron itself has a carcinogenic potency. (This work also
reviewed Saffiotti's experiments, which showed that ferric oxide alone did not
produce respiratory tumors.)
A link with carcinoma of the lung has not been reported in surface mining
o o£ Ofl"7 A 1 A
operations. Although questions have been raised, ' ' it has been
generally concluded that neither iron ore dust (hematite) nor ferric hydroxide
predispose to malignancy in humans.
Ferric oxide given to hamsters, mice, and guinea pigs by inhalation or by
intratracheal route has not been found to be carcinogenic. Ferric oxide
particles can serve as a vehicle for transporting known carcinogenic agents
(e.g., benzo/q7pyrene) into the lungs of animals and can produce squamous
metaplasia as well as lung cancer under such circumstances. ' ' ' a>
683a,705b _. £J1 „ •,*,•,, , 626
The use of scanning electron microscope as described by Port et al.
may prove to be extremely useful in determining if neoplastic changes in
bronchial epithelium occur with exposure to iron.
243
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CHAPTER 9
SUMMARY
IRON IN THE ENVIRONMENT
Iron comprises about 5.4% of the continental crust. Its concentration
in soils ranges from 0.7-4.2%, where it exists primarily as ferric oxyhydroxides
and in clay minerals. Crude ores have from 20-69% iron, usually in the form
of iron oxide minerals. Iron mobility in the regolith generally increases at
lower oxidation potentials and pH's in flooded soils and subaqueous sediments
where ferrous iron can exist in solution, at lower pH's and highly moist
aerated soils, in the presence of complexing organic compounds dissolved in
soil solutions, and with decreasing concentrations of iron-precipitating anions
such as sulfide, carbonate, and phosphate. Bacteria also influence the trans-
port of iron by increasing the rate of iron oxidation, producing complexing
organic substances or inorganic acids through their metabolism, or oxidizing
or utilizing organic parts of chelates to release iron that can then accumulate
as ferrie oxyhydroxides.
At chemical equilibrium in a well-aerated river, the dominant forms of
iron usually are ferric,. Unless organic or inorganic complexing agents are
present, the solubility of ferric iron cannot be more than a few micrograras
per liter at pH's greater than 4, and the solubility at neutral pH is much lower.
River water generally contains some particulate ferric oxyhydroxide, however,
which may be in the colloidal size range. Iron concentrations in sea water
range from a few tenths of a microgram per liter up to about 3 jug/1. In mildly
reducing environments, dissolved ferrous species are dominant and iron
solubility at equilibrium may exceed 50 mg/1 at pH 6. Ground waters in many
244
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areas of the United States contain appreciable amounts of ferrous iron. Con-
centrations between 0.5-10 mg/1 are common in such areas and larger concentra-
tions are occasionally measured. When these waters are aerated, the iron is
oxidized and precipitated as ferric oxyhydroxide. In strongly reduced systems,
iron may be retained by ferrous sulfides, which are highly insoluble until the
sulfur is oxidized. Metallic iron is chemically unstable in water and many
water supplies contain iron that has been derived from corrosion of pipe and
other metal surfaces. Organic complexes of ferrous and ferric iron are found
in surface and ground waters, and may increase the solubility of iron or
influence its reactivity in other ways. Redox and precipitation reactions of
iron generally are fast at near neutral pH levels, and chemical equilibrium is
attainable. Many of the processes are mediated by microorganisms.
Iron in the atmosphere in remote areas of the United States and United Kingdom
o
is present in year-average concentrations as low as 0.05-0.09 Aig/m ,whereas in a U. So
3
iron and steel center, it has reached a 1-yr maximum average of 12>ig/m and a 3-mo
3
maximum average concentration of 16/ig/m . Atmospheric iron in remote areas is large-
ly soil-derived. The 5-yr (1970-1974) median concentration at remote sites in
3 3
the United States was found to be 0.26/ag/m ; at urban sites, 1.3yug/m , or
5 times as high. Maximum concentrations tend to occur in the windy season,
i.e., in late winter and early spring. Lowest urban iron concentrations are
found in coastal states.. The iron fraction of soil-derived particulates is
roughly 1 part in 100 or 200; the global rate of entry of iron into the atmos-
phere is of the order of 1,000 metric tons/day.
World production of pig iron and raw steel within the last 5 years has
reached 500 million and 700 million metric tons and continues to increase. The
U. S. contribution, more than half of which comes from Pennsylvania, Ohio, and
Indiana, amounts to 17-19% of the world total. Total particulate emission
245
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factors for individual manufacturing operations are known to be affected
markedly by the type and degree of pollution control employed. Estimation
of atmospheric contaminant concentrations resulting from given emission rates
is common practice. Estimation of soil quantities raised by the wind is less
common, but practicable, and some measure of control through cultivation
methods is available. The overall annual average concentration of total air-
3
borne particles in the United States decreased from 80 to 66/ig/m between 1970
and 1974.
The mining and processing of iron or certain materials containing iron
minerals affect the environment in several ways. Waste accumulated from iron
ore mining presents a problem only when sulfides are present to produce acidic
drainage. Extraction of aluminum from bauxite results in wastes containing
1-25% ferric oxide, and these residues amount to about 5.6 million metric tons
annually. Most of the wastes are deposited in adjacent lakes. If uncontrolled,
emissions from steel mills can contribute appreciable quantities of iron oxides
to the air but the only problem demonstrated to date has been soiling. The
carrier properties of these particles have not been adequately investigated,
and may be of concern. Wastewaters high in iron produced by the steel industry
generally are sufficiently treated. A major environmental problem relating to
iron-containing minerals involves the decomposition of pyrite and marcasite in
coal mines, which can result in acid drainages with high dissolved iron concen-
trations seeping into streams. Some 17,600 km of streams have been affected by
drainage pollution from both active and exhausted underground and surface mines.
An additional problem lies in disposing of the voluminous amounts of sludge,
primarily ferric oxyhydroxides, that result from the treatment of acidic
drainage.
246
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MICROORGANISMS AND IRON
Iron-containing enzymes are ubiquitous in microorganisms (bacteria, yeast,
molds, and the microalgae), where they catalyze essential cellular reactions
and promote the grand chemical cycles of the biosphere. Aerobic and facultative
anaerobic microorganisms are equipped with diverse mechanisms for acquiring the
ferric ion. The high-affinity uptake pathway, which involves organic carriers
(siderophores) and their corresponding membrane receptors, is presently the best
understood. Both siderophore and cognate receptor are under powerful repression
by iron. Certain enteric bacteria, viz., Escherichia coli and Salmonella
typhimurium, form receptors for exogenous siderophores, such as the ferrichromes,
which they do not themselves synthesize. Citrate may be viewed as a rudimentary
siderophore: E_. coli has an inducible uptake system for ferric citrate,
whereas 55. typhimurium lacks this permease.
The formation of siderophores and matching receptors in pathogenic species
may endow the microorganism with a competitive advantage in iron-poor environ-
ments, for example, in the tissues of the host. Similarly, in a mixed culture,
those species capable of elaborating siderophores may sequester the available
iron and thus achieve superiority over organisms lacking this ability—especially
if the latter also are unable to transport the siderophore made by its
competitor. Metabolically available iron will repress synthesis of surface
receptors for siderophores which, in turn, will render the organism resistant
to certain bacteriocins and bacteriophages.
Little is known about the possible role of siderophores in conditioning the
soil and making iron available to plants. The same is true of the pharmacology
of siderophores and their affect upon mammalian iron transport. Introducing
increased amounts of dietary iron may change the microflora of the human large
intestine.
247
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Rhodotorulic acid and 2,3-dihydroxybenzoic acid are two microbial products
that are being evaluated clinically as agents for removing excess body deposits
of iron. Desferal"', the trade name of desferrioxamine B, a siderophore from
Streptomyces pilosus, is the drug of choice for treating acute, accidental iron
poisoning in young children. Its utility to patients with chronic iron over-
load is being investigated.
IRON AND PLANTS
Plants require a continuous supply of iron for growth because it is an
essential component of many heme and nonheme enzymes and carriers. Iron con-
centration is not usually deficient in soils. However, 25-30% of the world's
land is calcareous in the surface horizon, and availability of trivalent iron in
aqueous solutions of most calcareous soils is inadequate for plant growth. Iron
poisoning may also be observed in plants growing on acid soil. For more than
50 years agronomists have tried to change the soil to fit the plant, but no
economical way has been found to supply iron to plants on problem soils. It has
been appreciated that some plants are able to use iron in calcareous soil (iron-
efficient plants) and some cannot (iron-inefficient plants). Iron-efficient
plants use nitrate, iron from ferric phosphate, and ferric chelates, and they
tolerate other heavy metals better than iron-inefficient plants. This affinity
is genetically controlled and it is possible to avoid iron chlorosis by
genetically tailoring the plant to fit a problem soil»
IRON METABOLISM IN HUMANS AND ANIMALS
Iron in the human body is largely in the form of hemoglobin within
circulating erythrocytes, which serve to transport oxygen. In the plasma, iron
is bound to transferrin, which distributes the iron among body tissues accord-
ing to their needs. Myoglobin, cytochromes, and various other iron-dependent
248
-------
tissue enzymes perform vital functions in all body cells, yet constitute a
small fraction of body iron. Surplus iron is deposited as ferritin and hemo-
siderin, predominantly in reticuloendothelial cells, liver parenchyma, and
muscles.
A unique feature of human iron metabolism is the extremely limited external
iron exchange. Recent studies indicate a marked limitation in the availability
of food iron for absorption. Available iron varies from < 1 to about 5 mg/day,
according to the composition of the food ingested. Heme iron is well absorbed,
whereas the absorption of nonheme iron is highly variable, depending on the
presence and amount of blocking and enhancing substances in the diet. The
intestinal mucosa also, modifies absorption according to individual needs, and
in men this regulation is effective. However, the amount of dietary iron avail-
able to women and infants is so low as to threaten iron balance even when
absorption is maximal. The iron requirements of pregnancy are so great as to
exceed the amount of iron that can be absorbed from an unsupplemented diet.
Despite the many improvements in modern nutrition, iron balance in females
and infants remains precarious. Reduction in caloric intake and diminished
iron content of food because of decreased contamination during procurement or
preparation undoubtedly contribute to the problem. Perhaps the most important
cause, however, is that most dietary iron is now derived from vegetable and
other nonanimal sources and thus is of very low availability.
Iron nutrition in animals presents few problems because of their higher
intake and more efficient absorption. However, the pig is an exception; its
rapid growth makes fortification or supplementation necessary.
IRON DEFICIENCY
Iron deficiency in the United States and worldwide is a major health
problem. In sheer numbers of people affected, it is one of the most prominent
249
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nutritional deficiencies. Iron deficiency is most common in children, in women
during their reproductive years, and particularly in the pregnant woman. In
adult men, nutritional iron deficiency is rare, and bleeding is the more important
cause of the deficiency. The early stages of iron deficiency before the develop-
ment of overt anemia were once difficult to measure. Accurate tests, including
serum ferritin concentration, transferrin saturation, and red cell proto-
porphyrin content are now available for evaluating the iron status of populations.
Because iron is essential to oxidative metabolism of all body cells as well
as a key element in many enzymatic functions, it is not surprising that iron
deficiency produces many structural and functional abnormalities. The abnormal-
ities in red blood cells, gastrointestinal tract, and integument are rather
easily demonstrated. Defects within the cell itself (except for altered mito-
chbndrial morphology) are more subtle. The effects of iron deficiency on
behavior, mental and physical performance, growth, reproduction, and suscepti-
bility to infection are poorly defined and difficult to separate from the
consequences of anemia and other factors. Animal studies indicate that iron
deficiency may predispose to increased absorption of other heavy metals and
potentiate their toxicity.
ACUTE TOXICITY OF INGESTED IRON
Acute poisoning from ingested iron is unlikely to be encountered from any
source other than medicinal iron. No known natural or dietary sources--such as
water, food, or any beverage--are likely to cause acute iron toxicity.
CHRONIC IRON TOXICITY
Dietary iron overload rarely, if ever, occurs, but overload may be produced
by large volumes of alcoholic beverage high in iron, as happens in black South
Africans. Otherwise, iron overload is caused by abnormal mucosal regulation
that permits excessive absorption from a diet of normal iron content, or by
250
-------
the parenteral administration of iron, usually in the form of transfused red
cells. Conspicuous differences exist in iron distribution in different types
of overload; in idiopathic hemochromatosis, deposits are almost exclusively
in the parenchymal tissues, particularly in the liver, whereas with transfusions
the iron is deposited predominantly in the reticuloendothelial cell. These
differences are important, because it is the parenchymal location of iron which
is harmful.
The two genetic disorders most commonly associated with iron overload are
thalassemia and idiopathic hemochromatosis. Thalassemia major is relatively rare
in the United States, affecting only about 5,000 persons. Current U. S. figures
suggest that the prevalence of clinically manifest idiopathic hemochromatosis is
of similar magnitude (1 in 20,000 hospital admissions and 1 in 25,000 hospital
necropsies). Asymptomatic siblings with increased iron are considerably more
frequent; it remains speculative whether such individuals would accumulate
sufficient iron to produce full-blown clinical hemochromatosis if exposed to a
high dietary intake of iron or excess alcohol.
Chronic parenchymal iron overload produces characteristic clinical mani-
festations, including hepatomegaly and liver dysfunction, diabetes, cardiac
failure, endocrine abnormalities, arthritis, and abnormal skin pigmentation.
These symptoms can be arrested or reversed in the individual with idiopathic
hemochromatosis through the removal of excess iron by phlebotomy. In the iron
overload of thalassemia, chelates have been used to remove excess iron, and
evidence has been obtained that this treatment can arrest tissue damage.
Carcinoma of the liver is a complication of idiopathic hemochromatosis and
a causal relationship between iron and this malignancy is thought to exist.
The intramuscular injection of iron dextran in animals has produced sarcoma, and
the association has been reported in a few instances in humans.
251
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INHALATION OF IRON
Ambient air contaminated with iron oxide has not been responsible for a
discernible fibrotic response in humans or animals. Laboratory exposure of
animals (mice, rats, guinea pigs, and hamsters) to iron oxide produces only an
accumulation of iron oxide in the pulmonary parenchyma without any evidence of
fibrosis. Eipthelial hyperplasia in the tracheobronchial tree of the hamster
has been the only finding noted.
Occupational exposure to iron oxide in the work place has been found to
produce pulmonary siderosis; however, it is not accompanied by fibrosis. No
other documented response has been noted. Pulmonary siderosis itself is a
benign disorder.
Suspicions of an occupational hazard of lung cancer associated with hematite
mining have not been conclusively proved. Whenever an increased risk has been
suspected, another more logical cause can be proposed. The probable cause of
the increased risk is the presence of radon in the work environment.
252
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CHAPTER 10
RECOMMENDATIONS
1. Additional research on the aqueous chemistry of iron and precipitated
iron compounds is needed to improve the treatment of high-iron waters
such as the products of acid mine drainage.
The chemical mechanisms and thermodynamics or the precipitation
and aging of ferric oxyhydroxides should be studied, as well as
the effects of other elements and organic ligands.
20 Because coal mining can be expected to increase, we should continue to
seek the most practical means of preventing or diminishing the forma-
tion of acid mine drainage.
3. More information is needed on airborne iron concentrations in relation
to the iron content of regional soils and dusts deposited in the vicinity
of industrial centers.
As a contribution to background data on environmental exposure to
chemical species, more information is needed on airborne iron
concentrations as they relate to the iron content of regional
soils, and also to iron deposited on the earth's surface in the
vicinity of industrial centers.
4. Basic research should be encouraged on the adsorption and catalytic
properties of particulate iron compounds in the atmosphere to determine
their importance in carrying substances or promoting reactions that
affect biologic systems.
Emphasis should be placed on the surface properties of ferric
oxides, as well as the effect of their mode of formation and
history on these properties.
253
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5. Studies of the relative solubilities of iron compounds in soils should
be pursued to aid in determining the influence of different soils on iron
availability to plants.
In particular, the solubilities of iron incorporated in different
clays, metal oxides, organic chelates, and the effect of incorporat-
ing other elements on the solubility of ferric oxyhydroxides in
soils should both be investigated,,
6. Basic biochemical research on iron should be promoted.
In order to achieve the objectives of Recommendations 6-8, research
on molecular and cellular aspects of iron metabolism must be con-
t inued.
7. More information is needed on the basic mechanism and regulation of iron
transport in microorganisms.
• Induction and derepression mechanisms and microbiologic iron-
transport systems need to be examined in relation to microbial
virulence and resistance to infection.
• It must be determined if siderophores supply or deny iron to
higher organisms, and the pharmacologic impact of these sub-
stances on animals needs to be ascertained.
• Siderophore ligands, although virtually ferric-specific, may form
complexes with toxic metal ions such as plutonium and move such
substances through the food chain. The possibility of this problem
needs to be recognized and studied.
254
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8. Clarification of the molecular processes of absorption and transport
and their regulation in plants is needed.
• The manner in which an iron-deficient environment induces
metabolic changes that enhance iron uptake should be determined„
The subjects to be investigated should include the source of
hydrogen ions and the source and nature of reductants released
by roots.
• The movement of ferrous iron into roots and its oxidation should
be studied. The internal pathway of iron, including its oxida-
tion to ferric citrate and ultimate utilization in plant tops,
also should be scrutinized.
• How other metals and chemical compounds affect the iron metabolism
of plants should be pursued in greater detail.
9. The matching of plant species with local soil conditions should be
pursued.
Regional soil and plant tissue testing laboratories could be
! established for this purpose.
10. Iron nutrition should be investigated further.
• Methods have been devised to measure iron availability in food,
but studies of enhancing and inhibiting factors are required to
understand the intricacies of availability. Investigating the
availability of iron in water and its effect on the iron balance
of populations consuming water high in dissolved iron also would
be helpful.
• The effect of iron deficiency on absorption of other metals,
particularly lead, should be evaluated.
255
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11. The prevalence of iron deficiency in the United States should be better
defined.
• Large-scale population surveys employing a battery of sensitive
screening tests for iron deficiency in statistically valid
samples of U. S. subjects are needed.
• The relation between iron deficiency and diet should be examined,
12. The effects of iron deficiency other than anemia should be defined.
• Particular attention should be given to the importance of the
depletion of tissue-dependent tissue enzymes.
• More discriminating tests of functional abnormalities produced by
iron deficiency should be designed. Such tests should include
observations of behavior as well as examinations of subcellular and
organ functions,
• The interaction between iron and its physiologic binding proteins,
transferrin and lactoferrin, needs to be elucidated before the
connection between iron and susceptibility to infection is clarified.
13. Improved diagnostic and therapeutic approaches to chronic iron toxicity
should be found.
• The mechanism by which excess iron produces tissue damage should
be determined and its carcinogenicity defined. This task would be
simplified by the development of a suitable animal model.
• A better means of detecting individuals with excessive iron absorp-
tion should be developed.
• More efficient chelating agents and methods of chelate administra-
tion should be sought to improve the treatment of patients with
iron overload who cannot be phlebotomized.
256
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APPENDIX
ANALYSIS OF IRON IN ENVIRONMENTAL AND BIOLOGIC SAMPLES
Many .analytic techniques have been developed to measure iron in a
variety of materials. More than 1,200 methods exist for the microdeter-
mination of iron in air, water, waste, and biologic samples. Listed in
descending order of popularity, the methods were reported as follows:
spectrophotometry, 42%; electrochemical, 17%; atomic absorption spectro-
photometry, 11%; emission spectrography, 10%; complexometric or volumetric,
10%; radiochemical, 4%; fluorescence, 2%; chromatography, 1.6%; catalytic
or kinetic, 1.1%; mass spectrography, 0.8%; microprobe, 0.5%; and chemi-
luminescence, 0.2%.
ENVIRONMENTAL SAMPLES
Although atomic absorption spectrophotometry (AAS) is a relatively
recent development, it has become the method of choice for a large portion
of published research during the past 20 years. Many of these less popular
methods were created for specific problems and as such are only applicable
to such situations. At present, AAS appears to be the preferred technique
for determining iron in air, water, and waste samples because of its
specificity, speed, and absence of interferences.
Many colorimetric reagents have been proposed for•the spectrophoto-
metric determination of iron, but the most common reagents remain phenan-
throline and its derivatives and bipyridyl and tripyridyl. Most
Environmental Protection Agency (EPA) and Association of Official Agricul-
tural Chemists (AOAC) official procedures employ one of these reagents.
257
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Optical emission and X-ray spectrography have been used to determine
iron in environmental samples, but few laboratories have elected to use
these techniques routinely. Both techniques are less accurate than the
433
atomic absorption and spectrophototnetric procedures. Kopp has reported
a coefficient of variation of 7% at a concentration of 6A rig iron/ml
for his optical emission technique. An interesting application of X-ray
spectrography was reported in which water was filtered through a resin-
coated filter paper to retain the metals that were then analyzed by energy-
699
dispersive X-ray fluorescence.
Because of their .wide usage at this time, only the atomic absorption
and coloritnetric procedures will be given a general review. Detailed in-
24,34,
structions may be found by consulting the references for this section.
104,245,419,433,566,609,663,699,731,733,735,765,771,766
Reliable results
only can be obtained from good representative samples properly prepared for
analysis. Again, detailed instructions for sample preparation are beyond
the scope of this report, but some special procedures will be discussed to
provide guidance in selecting a sample preparation technique.
Sample Preparation
Water samples. Ferrous and ferric iron are found in water as dissolved
solids, colloidal solids, and suspended solids. Under reducing conditions,
iron is ferrous, but upon exposure to air or oxidizing materials it will con-
vert to ferric iron. Ferric iron may hydrolize and form insoluble, hydrated
ferric oxide. The ferric oxide may adhere to the container wall unless the
pH of the sample is low. In general, the word "iron" is used to denote a
total of both the ferrous and ferric states.
258
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If ferrous and ferric iron are to be determined separately, a portion
of the water sample must be filtered and acidified immediately after col-
lection. Extreme care must be exercised to prevent the oxidation of the
ferrous iron and hydrolysis of the ferric ion during filtration.
Colloidal iron tends to adhere to the walls of plastic containers. If
suspended iron is to be measured separately, the sample should be shaken
vigorously to ensure homogeneity just before removing a portion for analysis.
Samples should be analyzed as soon after they are collected as possible to
avoid changes in the sample from bacterial growth or oxidation.
If only dissolved iron is to be determined, the water can be filtered
731,766
through a membrane filter with a pore size of 0.45^im. The filtrate
is used to determine the dissolved iron. However, the pore size of the
419
filter will affect the amount of soluble iron found. Kennedy et al.
have demonstrated the variations in the amount of soluble metals in water
samples according to the pore size of the filter being used. The suspended
material retained on the filter can be measured for suspended iron, or the
total iron can be measured on the sample as received and the suspended iron
calculated by difference.
Two books may be consulted for specific steps in collecting and pre-
104,731
paring water samples.
Particulate air samples. It is standard to collect ambient air samples
on 20 x 25 cm fiberglass filters and follow the general procedure of the
National Air Sampling Network. Environmental samples are collected on a mem-
brane or a fiberglass filter. The collected particulate can be leached from
the fiberglass filters by repeated treatments of a hot, 1:1 hydrochloric
acid;nitric acid mixture. The leaching solutions are combined and taken to
259
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dryness before solubilizing for analysis. The blank filter should always
be checked for iron contamination.
Most membrane filters can be destroyed in the hot acid mixture. How-
ever, ester-type filters may require repeated ashing with nitric acid to
eliminate all the carbon residue of the filter. One type of polyvinyl
chloride membrane filter on the market must be given an additional treat-
ment with perchloric acid to complete the destruction of Che filter.
Organic membrane filters can be employed for most sampling except when
hot metal sparks may strike the filter and burn holes in it. If this is
a possibility, a fiberglass filter should be used.
Because of the rigorous acid treatment necessary, it is not possibly
to differentiate between ferrous and ferric iron in air samples.
Waste material samples. It is very difficult to collect a representa-
tive sample of waste materials because they generally are a mixture of solids
and liquids. Rigorous ashing with nitric acid is usually required to destroy
all organic materials and solubilize the iron,, Sometimes an extra ashing with
hydrogen peroxide or perchloric acid is necessary. Perchloric acid is safe to
use if a small amount of sulfuric acid is present and if most of the organic
material already has been destroyed with nitric acid.
BIOLOGIC SAMPLES
Feeds and Fertilizers
Problems unique to analysis of feeds and fertilizers are usually those
of collection and preparation of representative samples. The actual analysis
by atomic absorption and/or the various spectrophotometric procedures is
straightforward and differs little from procedures used for other materials.
Animal byproducts used as feeds are analyzable by techniques used for the
*
Biologic samples are discussed below.
260
-------
appropriate animal tissues; accordingly, this discussion of feed analysis
will concentrate on plant materials. Much of the material presented here
27a 312a
has been excerpted from methods reported in two sources. '
Sampling. The AOAC method recommended for sampling bagged fertilizers
is to use a slotted tube with a solid cone tip at one end to remove a core
of fertilizer diagonally from end to end. For lots of 10 or more bags, at
least 10 bags should be sampled. For lots of less than 10 bags, at least
10 samples should be taken, at least one from each bag. For bulk fertilizers,
at least 10 cores should be taken from different regions. The samples should
be made composite, thoroughly mixed, and reduced to required size by repeated
quartering.
Preparation of samples. The AOAC method for preparing inorganic fertil-
izers for mineral analysis is as follows. Dissolve 1 g of well-ground sample
in 10 ml hydrochloric acid in a 150-ml beaker. Boil and evaporate solution
to near dryness on a hotplate; do not bake,, Redissolve residue in 20 ml of
0.5 N hydrochloric acid, boiling gently if necessary. Filter through fast
paper into a 100-ml volumetric flask, washing paper thorougly with distilled
water. Dilue to volume with distilled water. If further dilutions are neces-
sary, they can be made with 0.1 N hydrochloric acid.
The various organic materials that comprise fertilizers can be prepared
for analysis by employing the appropriate dry ashing or wet digestion
techniques used for plant samples. With both inorganic and organic fertil-
izers, precautions should be taken to obtain representative samples and to
minimize contamination during all phases of preparation.
Plants
Collection. Plant samples for iron analysis can be collected in paper
or polyethylene bags if they have not been treated with talc or other
261
-------
materials used to prevent the bags from sticking together. Aluminum containers
are generally not satisfactory for fresh material because they may be corroded
by plant exudates. Selecting representative plant samples in the field is
critical, especially if mixed forages are being collected. One way of achiev-
ing this is to mark off small areas randomly, collect all plant material in
each area, and make them into one or more composite samples. Sampling of feed
grains is best accomplished in sampling tubes; however, tubes made of noncon-
taminating materials should be used.
Cleaning and drying. Samples should be cleaned and dried as soon as
possible after collection. Contamination of plant samples with soil can be
a serious problem and all adherent materials should be removed. Plants can
be cleaned by rinsing thoroughly with 0.2 N hydrochloric acid or by washing
them in detergent solution followed by distilled water rinse. If the same
sample is to be used for multiple mineral analyses, care should be taken to
avoid leaching losses of the more soluble elements. After thorough cleaning,
samples are usually air-dried. The most common procedure is to spread out
the plant material in a thin layer and dry it overnight in a forced air oven
at 70 C. Drying at higher temperatures may result in decomposition and loss
of dry matter.
Grinding and subsampling. Grinding can be a serious source of iron con-
tamination for plant materials. Grinding sometimes can be avoided by analyz-
ing an entire plant; however, if this is not possible, special precautions
must be taken to avoid contamination. Using a mortar and pestle or a ball mill
made of agate are satisfactory methods. Fragile materials such as dried leaves
can often be crushed by hand in a plastic bag. The use of metal-containing
mills is often a source of serious but inconsistent iron contamination. Porce-
lain, flint, or mullite instruments also may contribute to iron contamination.
262
-------
Ground samples should be dried overnight prior to analysis. After drying,
they can be stored in glass or plastic containers with plastic lids. The
sample containers should not be more than half-full to allow for thorough mixing
before subsamples are taken. Even finely ground materials may segregate
because of differences in particle size and density; i.e., stems and leaves
often grind differently and thorough mixing is required to assure representa-
tive samples.
Dry ashing. Most dry ashing procedures in use have been modified to meet
312a
the needs of the individual users. The following methods are typical.
Samples (usually 1 or 2 g) are weighed into appropriate containers and
placed in a cool muffle furnace. The temperature is raised to 500 + 50° C
for 4-5 h or overnight. Any unashed organic materials can be removed by wetting
the sample with 2 ml of 5 N nitric acid, slowly evaporating to dryness, then
returning the sample to a muffle furnace and heating to 400° C for 15 min. The
muffle furnace should be cool « 200 C) when the acid-treated samples are
introduced, or violent decomposition may occur. Nitric acid treatment generally
is not required. After the sample is thoroughly ashed, it is moistened with
a small amount of distilled water and 2 ml of concentrated hydrochloric acid
is added. The liquid is evaporated to dryness on a steam plate and allowed
to bake 1 h to dehydrate the silica. The residue is then dissolved in 2.5 ml
of 2 N nitric acid and transferred to a volumetric flask. The container should
be rinsed several times with hot distilled water; add washings to the volumetric
flask. After the sample has cooled and been diluted to volume, allow any
silica to settle out or remove it by centrifugation before analysis.
If plant materials that contain large amounts of silica are being ashed,
the foregoing procedure sometimes is modified to eliminate potential adsorp-
tion of minerals to the silica. Some disagreement exists as to whether or not
263
-------
this problem is serious and the modified procedure may be necessary only when
there are large amounts of silica and analysis of the solution for minerals
that are present in very small concentrations is anticipated. in this modified
method, the samples are treated in the manner previously outlined, including
the step of ashing in a muffle furnace. After ashing, the samples are moistened
with distilled water, and 5 ml of 48% hydrofluoric acid and 5 drops of con-
centrated sulfuric acid are added. The samples are then heated gently on a
hotplate until the hydrofluoric acid evaporates. The residue is dissolved in
5 ml of 2 N nitric acid and transferred to a volumetric flask with the aid of
a stream of hot water.
Wet digetion. In many laboratories, wet digestion of samples with nitric
acid and perchloric acids is preferred to dry ashing. Both methods appear to
be satisfactory for determining iron. In a typical procedure, a 2-g sample of
plant material is weighed into a 250-ml beaker, allowed to stand overnight in
20-31 ml of concentrated nitric acid, heated gently until the initial vigorous
reactions have subsided, and slowly evaporated to near dryness. After the
sample has cooled, 10 ml concentrated nitric acid, 2 ml concentrated sulfuric
acid, and 10 ml of 72% perchloric acid are added„ The sample is then returned
to the hotplate, covered with a watchglass and heated until all organic matter
is dissolved and the solution is colorless. The coverglass is then removed and
the sample is evaporated to near dryness at just below the boiling point. The
residue is dissolved in 5 ml of 2 N nitric acid and transferred to a volumetric
flasko Silica generally presents few problems when wet digestion is employed.
The use of perchloric acid to digest samples necessitates a number of
precautions. The removal of easily oxidized material by pretreatment with
nitric acid is essential, as is the addition of sulfuric acid to prevent exces-
sive heating during the evaporation step. Samples should not be allowed to
char when evaporating the nitric acid; if charring occurs, more nitric acid
264
-------
should be added and the sample heated until all charred material dissolves.
If charred material appears in the sample after perchloric acid has been added,
the sample must be cooled and more nitric and perchloric acids added. Heat-
ing charred samples in the presence of perchlorates will almost inevitably
result in an explosion. Additionally, perchloric acid fumes should only be
discharged in hoods designed for perchloric acid use. Alternatively, samples
can be digested in Kjeldahl flasks and the acid fumes exhausted via a glass
manifold connected to a water aspirator. The problem of perchlorate can be
605a
avoided by digestion with sulfuric acid and hydrogen peroxide.
METHOD OF ANALYSIS
Atomic Absorption Spectrophotometry
Atomic absorption Spectrophotometry offers the fastest analytic method
for determining iron in environmental and biologic samples. (Most plant
materials contain enough iron so that no special techniques are required.)
It is specific, with generally little or no interference from the other metals
in the sample matrix. Four lines in the iron spectrum may be used to analyze
solutions, depending upon the iron concentration in the solution to be analyzed.
The wavelengths and sensitivities of the four lines are listed in Table A-l.
Although the detection limit (defined as the concentration at which the
signals from the analyte is twice the background signal) will vary from
instrument to instrument, it is approximately O.OOS^ug/ml at 248.3 nm when
an air-acetylene flame is used. Citric acid has been reported to suppress
the absorbance by up to 50% at a concentration of ZOO^ug/ml. The nitrous
oxide-acetylene flame is supposed to eliminate most interferences.
265
-------
Table A-l
Wavelengths and Sensitivities of the Iron Spectra Used in AAS—
Wavelength,
run
248.3
372.0
386,0
392.0
Band Pass,
run
0.2
0.2
0.2
0.2
Optimal
Range,
2.5
25
50
800
Working Typical Sensitivity
jig/ml
10
- 100
- 200
- 3200
ug/ml
0.062
0.55
0.90
17.0
-From Varian Techtron.
—Sensitivity is the concentration in an aqueous solution expressed in ug/ml
that absorbs 1% of the radiation passing through a cloud of atoms being
determined.
266
-------
With the development of the graphite furnace, carbon rod, and tantalum
ribbon accessories for atomic absorption spectrophotometers, the detection
limit for iron has been reduced by several orders of magnitude. However, it
will still vary depending upon the equipment, sample matrix, and specific
operating conditions. For instance, Ediger et_ al.., using the HGA-2000 graphite
furnace, reported the detection limit for iron in saline water as
0.00002 mg/ml, whereas Bagliano £t al., using the HGA-70 graphite furnace,
O /
reported a detection limit of 0.001 mg/ml for iron in uranium oxide.
The samples to be analyzed must be free of all suspended particulate to
prevent the aspirator system from clogging. The matrix of the calibration
standards should be similar to that of the samples to be analyzed. If the
sample matrix is very complex, analysis can be accomplished by the method of
addition, which will reduce the matrix effects between standards and samples.
The iron in the sample can be concentrated and matrix effects reduced by
chelating and extracting the iron with ammonium pyrrolidine dithiocarbamate
and methyl isobutyl ketone.
Operating procedures are described in the manufacturer's methods manuals
and will vary slightly from instrument to instrument. Specific procedures
for AAS analysis have been well characterized.24'104'566'609'731'735'765'771
Spectrophotometric Procedures
Review of the literature indicates that many colorimetric procedures
have been proposed. However, at this time, either thed,aL'- or ?: > ?)'-
bipyridyls and the phenanthrolines appear to be the reagents of choice. ' '
731 733
' In general, both of these colorimetric reagents have good sensitivity
and color stability, obey Beer's law, and can be used over a wide range. The
procedures are relatively simple and direct. Both systems react with ferrous
iron so that it is possible to determine the ferrous iron directly and the
267
-------
total iron after reduction to the ferrous states,, The ferric iron can be
calculated by difference.
For accurate results, the iron in the sample must be, available readily
and not bound up in stable complexes such as those that form with pyro-
phosphates. Since the reagents react in acidic solutions, the precipitation
of hydroxides and phosphates can be prevented.
Conclusions
Colorimetric and atomic absorption spectrophotometry offer satisfactory
procedures for measuring iron in a variety of environmental and biologic
samples. The specific choice of method must be chosen by the analyst, based
on the type of sample, analysis desired, equipment available for performing
the analysis, and any special requirements of the federal agency having juris-
diction. If total iron is to be determined, atomic absorption spectrophoto-
metry is probably the best method. If ferrous and ferric-iron need to be
• : . . • •< . : • . . "
measured separately, a colorimetric procedure must be used.
HUMAN AMD ANIMAL TISSUES
Assays of interest include measurement of blood iron compounds, primarily
used to identify iron deficiency; measurements of iron stores, of utility in
evaluating iron deficiency and iron overload; and isotopic measurements, for
determining absorption, loss, and internal iron exchange.
The blood of humans and other vertebrates have three important iron
compounds. Hemoglobin constitutes most of blood iron and usually is measured
colorimetrically by the cyanmethemoglobin method. Plasma transferrin
iron, is the compound of especial interest because it represents transport iron
and reflects the amount of the metal available to body tissues. As a rule,
474
plasma iron is measured colorimetrically. It is not influenced by
the presence of hemoglobin, whereas free iron, large amounts of ferritin, and
268
-------
parenteral iron do affect plasma iron. Measuring transferrin-binding capacity
is important because the availability of transferrin iron is influenced by the
degree of saturation of the iron-binding protein. Colorimetric and radio-
180
active methods have been described. Serum ferritin may be assayed radio-
535
immunometrically; the protein is useful in detecting iron deficiency and
overload. Although not an iron compound itself, red cell protoporphyrin
increases when the iron supply to the marrow becomes inadequate and therefore
441
serves as an additional gauge of iron deficiency. However, erythrocyte
protoporphyrin also rises with lead poisoning and certain unusual intrinsic
disorders of porphyrin metabolism.
Reticuloendothelial iron stores can be determined by examining a marrow
35
aspirate for particulate hemosiderin within the reticuloendothelial cells or
by determining the amount of;iron in marrow particles. Hepatic iron stores
may be evaluated by the intramuscular injection of desferrioxamine and sub-
sequent analysis of the urine excreted during the next 24 h. This technique is
333
particularly effective when parenchymal iron overload is suspected. Serum
ferritin is useful in evaluating reticuloendothelial and parenchymal iron stores
402
since its concentration usually parallels the size of tissue iron deposits.
Nonheme hepatic iron may be analyzed directly in tissue obtained by biopsy and
160
necropsy.
Much of the information concerning iron turnover has been made possible
through the use of iron isotopes. Iron-59, with a half-life of 44 days, is the
most convenient and its /^-radiation is detectable by crystal counting in vitro,
89
by surface counting, and by total body counting,, Iron-55 has a half-life of
4 yr; its X-rays are of such low energy that samples must be processed by wet
ashing for liquid scintillation counting. Iron-55 and -59 can be counted
242
differentially in the same sample with minimal cross-counting. A crude
269
-------
method for evaluating absorption is determination of the degree of elevation in
247
plasma iron after orally administering iron to a fasting subject. More
accurate determinations can be made by measuring red cell or total-body activity
89
2 wk after ingestion of radioiron. Absorption of nonheme and heme iron from
complex meals is ascertainable through extrinsic radioiron tags added to the
meal.77'187
270
-------
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359
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-78-017
4. TITLE AND SUBTITLE
IRON
7. AUTHOR(S)
Subcommittee on Iron
2.
3. RECIPIENT'S ACCESSIOt*NO.
5. REPORT DATE
February 1978
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Committee on Medical and Biologic Effects of
Environmental Pollutants
National Academy of Sciences
Washington, D.C. 20418
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory ..RTF,
Off ice 'of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
15. SUPPLEMENTARY NOTES
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
68-02-1226
13. TYPE OF REPORT AND PERIOD COVERED
NC
•'"••- - 14. SPONSORING AGENCY CODE
EPA-600/11
• • • .
16. ABSTRACT
This document surveys the effects of organic and inorganic iron that are relevant
to huma.ns and their environment. The biology and chemistry of iron are complex and
only pajtially understood. Iron participates in oxidation reduction processes that
not only affect Its geochemical mobility, but also its entrance into biologic .systems.
Hydrated ferric oxide surfaces have adsorbent properties and may act as reaction sites
and catalysts. In biologic systems, the iron atom is incorporated into several proteir
enzymes that participate in many oxygen and electron transport reactions.
The report addresses the quantity and form of iron in the environment, its move-
ment and the interaction between inorganic and organic forms of the metal. Some plants
have capabilities of retrieving iron from the soil; vertebrates in general appear to
he able to achieve satisfactory iron balance. Humans are the outstanding exception -
hundreds of millions of the world's peoples are iron-deficient because of inadequate
amounts of available iron the diet; deficiency may thus be the major iron-related
environmental health problem faced by humans. The presence of large deposits of ferri-
tin and hemosiderin in parenchymal tissues has been shown to result in damage to
several vital organs. Acute iron toxicity has been reported, but only with the ingest-
ion of large amounts of iron salts. Pulmonary inhalation of iron compounds from
industrial exposure has not been shown to be a hazard.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
iron
air pollution
toxicity
health
ecology
chemical reactions
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.lDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page)
UNCLASSIFIED
c. COSATI Field/Group
06 F, H, T
21. NO. OF PAGES
366
22. PRICE
EPA Form 2220-1 (9-73)
360
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