CLINICAL AND BIOCHEMICAL APPROACHES TO THE STUDY OF LEAD
AT LOW LEVELS
L . B. Tepper, et al
Cincinnati University
Cincinnati, Ohio
February 1970
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.'/ Clinical aid Biochemical Approaches to the Study of Lead
at Low Levels
L. B. Tepper and E. A. Pfitzer
The Kettering Laboratory
Department of Environmental Health
College of Medicine
_ltoiversity of Cincinnati, Cincinnati, Ohio
'sponMt'lnj Agency«St ed *etHS» '
National Air Pollution Control Administration Technical Center
411 West Chapel Hill Street
Durham, North Carolina 27701
February 197Q
zctlon Cod«
B. Poforalflii Organization Rept. No.
10. Projsct/TcsVOorti Unit No.
CPA 70-14
11. Type ot Repot & Period Covered
rC«te
i. Abstract* Discussions were held in February, 1970, which attempted to: 1) identify bio-
chemical and clinical approaches to the detection of lead effects at Ion levels; 2) estab
lish the health significance of whatever lead-related phenomena might be observed at thes
levels; and 3) suggest possible areas in which research emphasis might be most fruitful i
solving the presented problems. Those discussions are summarized. Increasingly sophistica
ted methodology is being used to study the manifestations of excessive lead absorption.
The state of current knowledge about how lead interacts with nan and experimental living
systems was reviewed. The best Indicators of early response to lead are already in use.
H. Koy Bor* as) aeaxBM tadpii. M. OaatfUn
Air pollution
Lead Inorganic compounds '
Lead (metal)
Diseases
Health
Research
Diagnosis
17c. COSAT1 FfcHVBroo, 13/02. 06/06, 06/05, 06/20, 06/01
18. Distribution
Unlimited
B.Secwtty CIMJITM. Report)
UNCLASSIFIED
».J«comy Clan, mm Pegal
UBCLASflFIED
21. No. of P«g3»
30
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;':l> lifLEPAGE
ATTD-0617
«. Title and litjuiie • • . , .- , ,
Clinical and Biochemical Approaches to the Study of Lead
at Low Levels
' "'J3. Report Date ~~
February 1970
Pfi1
9. Performing Organization Name and Address
The fettering Laboratory
Department of Environmental Health
I College of Medicine
I Univejsitv of Cincinnati. Cincinnati, Ohio
12. Sponsoring Agency Nam and («3resl "
National Air Pollution Control Administration Technical Center
411 West Chapel Hill Street
Durham, North Carolina 27701
Perloralns Organization tooe
Performing Organization Hept. No.
JTT. dra
. CPA 70-14
ffi^pe of Report & Period Covered
111. Sponsoring Ajje'ncyWde
IS. Supplementary Hotel
IS. Abstracts Discussions were held in February, 1970, which attempted to: 1) identify bio-
chemical and clinical approaches to the detection of lead effects at low levels; 2) establ-
lish the health significance of whatever lead-related phenomena might be observed at these
levels; and 3) suggest possible areas in which research emphasis might be most fruitful in
solving the presented problems. Those discussions are summarized. Increasingly sophisticaj-
ted methodology is beiag used to study the manifestations of excessive lead absorption.. |
The state of current knowledge about how lead interacts with man and experimental living
systems was reviewed. The best indicators of early response to lead are already in use.
17. Key Holds and Doctseett Arutyilfc (a). Deiaiptcn
Air pollution
Lead inorganic compounds
Lead (metal)
Diseases
Health
Research
Diagnosis
176. I
»c.COSATl Field/Cm, '13/02. 06/06. 06/05. 06/20^06/01,
18. Dlsttiojllon Slfflenera
Unlimited
FORM HBS-eS7lt-70»
19. Security ClaulThis Rcoort)
UNCLASSIFIED
^.Security Class. (This Paje)
USCLASSIFIEO
21. No. 31 Pap«
30
'
_J
Clinical and Biochemical Approaches to
the Study of Lead at Low Levels
While there are current examples of obvious lead poison-1
ing in Improperly supervised industrial operations and in
pedlatric practice where pica for old paint is observed, the
health significance of lead compounds in ambient urban atmo-
spheres has not been clearly defined. The lead concentrations
in non-industrial atmospheres are very much lower than the
current industrial Threshold Limit Value (several jig/m3 vs
200 jug/m3), and evidence of a specific effect of lead is com-
pletely lacking. Nevertheless, it is reasonable to assume
that at some point short of clinical lead poisoning in the
usual sense, body absorption of lead will reach a magnitude
sufficient to cause significant changes in normal physiolog-
ical mechanisms. That this point is not yet known may be
related to the fact that traditional clinical approaches to
the detection of lead effects at low levels have been rela-
tively non-specific and incapable of detecting minimal
alterations. Nevertheless, it is the question of the exis-
tence and significance of minimal alterations or of non-specific
effects which is central to discussions of the health signi-
ficance of the industrial use of lead, its emission, and its
consequent appearance in ambient atmospheres, water,vand food.
This report contains a summary of discussions'^held. in
February, 1970, which attempted to: 1) identify biochemical
and clinical approaches to the detection of lead effects at
low levels; 2) establish the health significance of whatever
lead-related phenomena might be observed at these levels; and
3) suggest possible areas in which research emphasis might be
most fruitful in solving the presented problems. ^Participants
included physicians with varying types of experience with
clinical lead toxicology, scientists in several branches of
clinical medicine and biochemistry, and investigators with
primary interests and backgrounds in lead metabolism and ex-
perimental toxicology. Several participants, although eminent
in their respective fields, had no major interest in lead.
They were invited because of their specialized clinical skills
and knowledge relevant to organ systems known to be affected
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This report was furnished to the Air
Pollution Control Office by the
Kettering Laboratory in fulfillment
of Contract No. CPA-70-14.
Clinical and Biochemical Approaches to
the Study•of Lead at Low Levels
Report of a Symposium
February 1970
Summarized by:
L.B. Tepper
E.A. Pfitzer
The discussions summarized in this report were supported
under contracts with the National Air Pollution Control Ad-
ministration (CPA-70-14), International Lead Zinc Research
Organization, and the American Petroleum Institute and
through a grant of the National Institute of Environmental
-------
The material presented in this report reflects the work
of the participants, their colleagues, and others in the
respective fields of investigation.
Participating Scientists
P;S.I. Barry, M.D.
The Associated Octel
C ompany, Ltd.
Ellesmere Port
Cheshire, England
Neal S. Bricker, M.D.
Washington University
St. Louis, Missouri
J. Julian Chisolm, Jr., M.D.
Johns Hopkins University
Baltimore, Maryland
Jerome F. Cole, Sc.D.
International Lead Zinc
Research Organization, Inc.
New York, N. Y.
Kim Cramer, M.D.
Universitetet I Goteborg
G&'teborg SV, Sweden
Robert E. Eckardt, M.D.
Esso Research & Engineering
C ompany
Linden, New Jersey
Robert G. Feldman, M.D.
Boston University
Boston, Massachusetts
Abraham Goldberg, M.D.,
D.Sc., F.R.C.P.
University of Glasgow
Glasgow, W. 1, Scotland
Harold H. Golz, M.D.
American Petroleum Institute
New York, N. Y.
Robert A. Goyer, M.D.
University of North Carolina
Chapel Hill, North Carolina
Robert C. Griggs, M.D.
Western Reserve University
Cleveland, Ohio
Robert J. M. Horton, M.D.
National Air pollution Control
Administration
Durham, North Carolina
Wallace N. Jensen, M.D.
George Washington University
Washington, p. C.
Joseph H. Meyer, Ph.D.
University of Cincinnati
Cincinnati, Ohio
Carl V. Moore, M.D.
Washington University
St. Louis, Missouri
Jean M. Morgan, M.D.
University of Alabama
Birmingham, Alabama
Emil A. Pfitzer, Sc.D.
University of Cincinnati
Cincinnati, Ohio
George Roush, Jr., M.D.
Tulane University
New Orleans, Louisiana
David p. Ulmer, M.D.
Harvard University
Boston, Massachusetts
Bert L. Vallee, M.D.
Harvard University
Boston, Massachusetts
George E. Schreiner, M.D.
Georgetown University
Washington, D. C.
G. J. Stopps, M.B., B.S.
E. I. du Pont de Nemours t
Company
Wilmington, Delaware
Lloyd B. Tepper, M.D.
University of Cincinnati
Cincinnati, Ohio
I. Summary of Reports on Environmental and Human Lead Levels
The concentration of lead in soils, foods, water and air
is highly variable. The average alimentary lead intake covers
a range of at least 0.12 to 0.35 mg/day. There may be a 6-
to 10-fold variation between individuals in the average daily
exposure to lead from the atmosphere. In spite of the resul-
tant wide range of lead absorption levels, the amount of lead
in man is relatively constant, totalling about 100 to 400 mg,
of which at least 90% is stored in the bone.
Blood lead levels are even more constant, remarkably so
in view of the wide range of exposures. When the mean blood
lead levels from various populations are examined, there
seems to be a small increase in the blood level as one goes
from a rural to an urbanized area. This phenomenon is not
well related to the density of motor vehicles in these re-
spective communities, and the urban-rural gradient is observed
in areas of the world with a very low number of cars. In add-
ition there is evidence to show that processing of food
contaminates it with lead. When one goes from society to
society the differences in blood lead levels are not impres-
sive. The blood lead level of rural Peruvians, for example,
approximates that for rural North Americans.
Soft tissue lead levels and bone lead levels do not cor-
relate directly in individuals. The lead in the bone seems
to increase with age (at least through the fourth decade and
possibly beyond), whereas the soft tissue level of lead does
not rise appreciably after the end of the second decade. In
females the soft tissue lead levels tend to be slightly lower
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is very much lower than in males. This lower bone level of
lead, however, also rises with age as in males. Children
show very low levels in soft tissue and bone, and no differ-
ence between the sexes is demonstrable. Whether the higher
bone lead levels in older adults is due to accumulation with
age or simply due to the fact that these people were living at
a time when there was more lead in the total environment is
not known. Lead concentration in bones varies with the bone.
selected, the lead content being generally higher in the more
dense bones.
Bone lead levels have been determined in the skeletons
of 40 Indians who lived in Arizona during the period 700 to
1450 A.D. The specimens were from original burial sites and
did not include museum material. Soil samples from within
the body .cavity and adjacent to the buried skeletons con-
tained less lead than the bone; therefore, the question of
contamination was excluded. Assay showed 6 to 8 ppm in the
bone ash of the rib. Sacrificial skulls from Mexico, probably
from the time of the Conquistadors, contained lead levels in
the range of 2 to 4 ppm of the ash. Coprolites (mummified
fecal material) found with these skeletons give evidence of
the diet on which the population lived. The food supply was
primarily fish, small grains, and grasses; and the total
fecal lead concentration was very low.
At some point in history, environmental and skeletal
lead levels apparently rose. Bone samples from the time of
the Civil War in this country contain lead in concentrations
which approximate those noted in modern autopsy materials,
viz.,40 .to 50 ppm.
In view of the very great range of lead intake between
individuals and variability in lead absorption, the consis-
tency of blood lead levels is impressive, enough so to suggest
both regulation by a biological control mechanism and possi-
ble biological need. No evidence exists either to support
or to deny these possibilities. It is generally true, how-
ever, that when a metal is found with similar constancy, the
metal is not present merely on an incidental basis but rather
as a substance with specific biological importance. In the
early studies of zinc biology, for example, zinc was regarded
solely as a toxic metal since zinc fumes under appropriate
conditions were shown to be responsible for metal fume fever.
Zinc was a typical trace metal present in amounts difficult
to detect and in association with only traces of information
about its significance. When in 1941, zinc was shown to be
an essential constituent of the metalloenzyme carbonic an-
hydrase, it became apparent that zinc was neither incidental
nor solely toxic but in fact had a specific biological func-
tion. That a similar situation may apply in the case of
lead has never been critically or systematically examined.
In making inferences from blood lead level determina-
tions it is important to remember that the analytical
techniques for lead assays at low levels do not yield im-
pressively consistent results. In many situations the
variation between replications is greater than the variation
between individuals in a sampled population. In addition,
the precision of analysis by a given laboratory may vary day
to day.
II. Summary of Reports on Lead and Sub-cellular Physiology
The biological function of a number of metals is unknown.
The concentration and distribution of these metals in tissues
and biological fluids are neither an index of their importance
nor of their biological significance, positive or negative.
There is still active discussion as to the essentiality and
biological function of selenium and chromium; lead may be in
a similar category.
Where metals have been known to interact with essential
biological systems, they interact with proteins, with nucleic
acids, or with integrated systems such as organelles. The
proteins that have been examined most extensively are the
enzymes. Some enzymes, the so-called metalloenzymes, contain
a metal which is bound in such a specific manner that it can-
not be removed without loss of activity. A large number of
nietals are bound to nucleic acids. The function of these
metal-containing nucleic acids is presently entirely unknown.
The relationship of metals to organelle function is also not
understood.
The sites to which metals can attach in biological mate-
rials are not infinite. Metals can attach to the epsilon
aninoacid group of lysine, to the carboxyl groups of glutamic
and aspartic acid, to the sulfhydryl groups of cysteine, and
in other less common ways such as to the phenoxy group of Iyro-
sine. In the case of metals such as lead, bonds are stablo,
immobile, and polydentate. Lead can combine with any or all
of these groups; it would be misleading to expect that lea
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We do know that lead can be toxic under certain circum-
stances, but so can any metal. Essential metals often function
as components of metalloproteins. When these metals are pres-
ent in excess and attached to ligands which are not the normal
active metal-binding site of these metalloproteins, inhibition
occurs, i.e., the metal is toxic. Whether a lead metallo-
protein exists arid has an essential body function is not known.
Work with carboxypeptidase illustrates several important
points. Carboxypeptidase is a zinc-containing enzyme which
has two functions: first as a peptidase, secondly as an es-
terase. It is possible to remove the normal zinc constituent
of the enzyme and replace it with various other metals.' For
example, when the zinc is removed and replaced with cadmium,
one finds that the metalloenzyme has lost its peptidase prop-
erties, but the esterase properties are increased. Cadmium
carboxypeptidase, in fact, is a better esterase than the
naturally occurring zinc analog. Lead carboxypeptidase has
also been characterized and found to function as an active
esterase. The point here is that a lead metalloenzyme could
potentially have a perfectly legitimate biological purpose.
Work with this enzyme is instructive in a second sense:
It has been found that the metal of carboxypeptidase is at-
tached to three ligands: two imidazolium groups of histidine
and one carboxy group. Sulphur-containing aminoacids are
not involved. This observation is contrary to many of the
expectations which have been expressed in the literature with
respect to ligands which might bind lead in natural systems.
Another metalloprotein, metallothionein, was discovered
first in the horse, then in other species, and finally in man.
Metallothionein is a cadmium-containing protein with a mole-
cular weight of about 6000 without the metal, 6800 with the
metal. The protein is composed of some 52 aminoacids, one-
third of which are cysteine. According to traditional thinking,
this high concentration of sulfhydryl groups should yield a
material with great avidity for lead. indeed, this is easily
demonstrable to be so in vitro. However from lead poisoning
experiments in animals it has become evident that this is not
the case in vivo. In the intact animal lead is not sufficiently
strongly bound to metallothionein to displace either cadmium
or zinc.
A balance between metals is eaeential in all considera-
tions of metal biochemistry and toxicity. Metals interact
at common sites and thus may be antagonistic, metal A pro-
tecting from the effect of metal B, or metal A exaggerating
the effect of metal' B. An example is that of molybdenum
intoxication which is observed in Somerset in England and in
the western part of the United States. The manifestations
of this disease may be enhanced by the presence of excess
copper. It is quite likely that lead, iron, and perhaps
other metals interact in ways which are relevant to this dis-
cussion but are not well understood. The competition between
metals for ligands depends upon numerous factors, most of
which are poorly understood. Included among these factors
are the number and character of the ligands, the penetration
through membranes by metals, the stability constants for the
several metal-ligand bonds, and steric factors describing
the spacial arrangement between metals and ligands.
It is relevant to examine systems which might possibly
be disturbed by lead and hence represent target systems for
lead effects. One such system is the pyruvate-carboxylase
system whereby pyruvic acid is introduced into the Krebs cycle
by acetyl co-enzyme A. This system is a conglomerate of sulf-
hydryl-containing enzymes and co-enzymes, including thiamine-
pyrophosphate, folic acid, and co-enzyme A. It is essential
in the energy metabolism of the brain. We know, for example,
that thiamine deficiency, as in Wernicke's syndrome or Kor-
sakoff 's psychosis, leads- to gross functional abnormalities.
A similar syndrome has been observed in foxes fed on fish
entrails which contain thiaminase, a thiamine-destroying en-
zyme. The same clinical manifestations would be expected
whether thiamine is destroyed, absent, or its metabolic site
of action is otherwise interfered with by metal attachments
to relevant sulfhydryl groups. That the central nervous
system manifestations of lead poisoning may be related to
interference with pyruvic acid metabolism may well be possi-
ble. The problem of mercury intoxication, as in the chronic
mercurialism of the Mad Hatter, and the question of arsenic
toxicity and the therapeutic effect of diroercaptopropanol
(BAL), a sulfhydryl-containing compound, are all relevant to
the question of metal binding to sulfhydryl groups of the
pyruvate-carboxylase system and the disturbance of this system
by extraneous metals.
The peripheral nervous manifestations of lead poisoning
are not related to these phenomena, but it is entirely possi-
ble that lead may attach to sulfhydryl groups of the neurocon-
duction system peripherally.
Another system where investigation may yield information
on the biochemistry of lead is that related to oxidative phos-
phorylation. This process depends upon the integrity of
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.8
the.respective levels of calcium and potassium inside and
outside the mitochrondria. Impairment of this membrane by
carbon tetrachloride is associated with a dramatic shift of
calcium into the mitochondria -and an uncoupling of oxidation
and phosphorylation. The sulfhydryl ligands of the mitochon-
drial membrane suggest that the attachment of lead or other
metals to the membrane may interfere with its intrinsic meta-
bolism and its ability to protect the intra-mitochondrial
environment. Under such circumstances the metals might be
expected to cause significant damage to the energy metabolism
system.
There are undoubtedly ways in which the effects of lead
on nucleic acids may be measured. We know that the binding
of lead to nucleic acids causes their hydrolysis. Research
in this area is currently not well developed, however.
III. Summary of Reports on Lead and the Kidney
Lead in abnormally high amounts may cause acute or chronic
renal injury. In children-acute lead intoxication may be
associated with a renal lesion typical of the Fanconi syndrome.
In cases of intoxication without encephalopathy one may see
either aminoaciduria or glycosuria or both. In more severe
cases, such as with encephalopathy, the triad of the Fanconi
syndrome may be seen: aminoaciduria, rickets, and hyper-
phosphaturia in the presence of extreme hypophosphatemia in
the range of 2 mg/100 ml (normal .= 3.5 to 4.5 rog/100 ml).
Fructose and glucose are excreted in the presence of normal
blood levels; however, the ratio between these two sugars
varies. Some children excrete close to a gram of citrate in
a day although the blood citrate level is normal.
During the acute episode some of these children have
shown a picture suggestive of the syndrome of inappropriate
secretion of antidiuretic hormone (ADH). This observation
cannot be attributed to treatment since it has been observed
before treatment is initiated. Treatment itself with EDTA has
been shown to cause a transient depression in the serum phos-
phate levels, and a transient increase in aminoaciduria.
many of the elements of the syndrome are
The mellituria is gone within a week; the
aminoaciduria clears within a month {semiquantitative paper
chromatography). Bone changes resolve within 7 to 8 months.
EDTA mobilization tests in which parathyroid hormone is
used as well to mobilize lead stored in the bone have been
attempted. In some cases sufficient PTH has been used to
With treatment,
promptly corrected.
raise the serum calcium level to more than 13 mg/100 ml. Al-
though the amount of calcium in the urine increases 3- to 4-fold,
there is no increase in the urinary excretion of lead above
that which is associated with EDTA alone.
With respect to chronic renal injury there is, of course,
the Australian data which indicate that under certain condi-
tions of exposure lead intoxication can lead to chronic
nephritis. Studies in Boston and in Baltimore have failed to
demonstrate, however, an equivalent phenomenon in the United
States. The EDTA mobilization tests in the Baltimore group
were normal, and it was not possible to demonstrate chronic
renal injury. Epidemiological factors must account for the
dissimilarity between Australian and American experience.
In the United states the patients have chewed paint at some
time between their first and third year of life. In Australia
the children are in the 6 to 10 year age range and absorb lead
through the ingestion of raindrops which contain lead from
decomposed paint on veranda rails and rooftops. The exposure
in this case may be less intense but more prolonged. Many of
the Australian patients have a pes cavus deformity and to-
phaceous gout.
A number of cases of lead poisoning in adults have been
observed in Alabama in relation to the prolonged excessive
consumption of illegally-prepared whiskey. This disease oc-
curs as well in Georgia, the carolinas, and indeed may be
expected anywhere that whiskey is illegally prepared in sol-
dered vats and distilled through old automobile radiators.
There is evidence that over 50 million gallons of illegal al-
cohol are distilled in this country every year and that over
50% of the illegally-prepared alcohol contains lead at con-
centrations of more than 1 mg/1. Lead concentrations of 20
to 50 mg/1. and, rarely, even as high as 80 mg/1. have been
reported.
Renal disease has been observed in a number of these per-
sons exposed to lead-contaminated illegal alcohol. The extent
to which lead is the specific cause for the renal disease is
not established. In Alabama experience the typical patient
is a negro male between the ages of 45 and 60. Perhaps some
25% of hospital admissions in this group have been exposed
to illegal alcohol. Those who have experienced severe and
prolonged exposure of 10 years or more are typically found
to be anemic, to have a stable renal insufficiency, and to
show a normal pyelogram. The kidneys, while symmetrical, are
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10
is no evidence of infection or of glomerulonephritis. The
urine sediment is nonspecific with very few cells, and the
level of protein excretion less than 1 g/1. Glycosuria is
absent. Renal biopsy shows interstitial fibrosis without in-
flammation. Gloineruli are rather well preserved; however,
there is evidence of degenerating tubules and a loss in the
number of tubules. Most of the patients have nephrosclerosis
of varying degrees as well.
Patients in this group with long-standing lead exposure
and renal impairment may have symptomatic saturnine gout, so-
called because of its association with lead absorption. This
syndrome can be differentiated from primary or hereditary gout
by the fact that a) the onset of gout follows the onset of
renal disease, b) there is no increased pool of urate, c) the
clearance for urate and urea is reduced out of proportion to
decreasing creatinine clearance, and d) an anemia is invariably
present. In primary gout there is an increased metabolic
pool of urate, and renal injury often follows rather than
precedes the appearance of clinical gout. All patients thought
to have lead nephropathy have been without exception severely
dosed over long periods. That lead under different circum-
stances may either increase or decrease the excretion of urates
may reflect a variable disturbance in the renal handling of
urate and shifts in the balance between active resorption and
excretion of this solute.
The classical hallmarks of lead nephropathy are nephro-
sclerosis, advanced tubular degeneration, interstitial fibrosis,
and intranuclear inclusion bodies. The bodies are acid-fast
inclusions which do not contain iron. They are unlike inclu-
sion bodies seen in viral diseases which.affect the kidneys,
particularly cytomegalic disease.
Considerations of clinical renal disease caused by lead,
particularly the relatively acute episodes in children, sug-
gest possible physiological interpretations. The acute effect
of lead upon the kidney is manifested by a disturbance of
transepithelial transport mechanisms for a variety of seem-
ingly unrelated solutes: glucose, aminoacids, phosphate, and
urate. In the normal kidney these solutes are resorbed in the
renal tubule, against a concentration gradient, by an energy-
dependent, energy-generating transport mechanism. In the lead
syndrome the transport of each of these proximal tubule re-
sorbed solutes is modified, giving rise to the constellation
of abnormalities which constitute the Fanconi syndrome. It
is known from studies of intestinal epithelium and isolated
perfused nephrons that the transport of glucose is in some
11
way sodium-dependent. studies of isolated bacterial systems
show that the transport of some aminoacids can be sodium-
dependent. There are also reasons to believe that phosphate
transport is sodium-dependent. All these presumably sodium-
dependent transport mechanisms then are impaired by lead.
Simultaneously, uric acid is moved from the capillaries into
the tubular fluid. The impairment of uric acid transport by
lead results in a retention of uric acid and uricemia. There
are reasons for believing that uric acid transport may be
sodium-dependent. The mechanism of excretion of lead is not
fully understood, but at least in the chicken it involves the
passage of lead from the capillary across the cell to the
tubular lumen. Lead is also filtered so that there may con-
ceivably also be a reabsorptive mechanism for lead. In any
case, lead crosses cells and in this transit may alter the
machinery for the transport of glucose, aminoacids, phosphates,
and urates, the mechanisms for which appear to be closely
coupled to sodium transport.
Sodium transport is the principal energy consumer in the
kidney, accounting for at least 50% and perhaps 70 or 80% of
oxygen and substrate consumption. The search for a common
denominator for these various transport abnormalities suggests
that one examine the sodium transport and the energy production
and utilization mechanisms in the kidney.
A reasonable hypothesis is that lead in transit through
the epithelial cell modifies the structural and functional
integrity of mitochondria. We know that mitochondria are well
equipped for the transport of calcium, and it is quite possible
that lead is transported in the same way. Lead within the
mitochondria might well modify the energy-producing mechanisms
which drive the solute-transport mechanisms for glucose, phos-
phate, arainoacids, and urate. In view of the evidence that
these transport mechanisms are coupled to sodium resorption,
it would be most surprising to find that a defect in sodium
transport is not present as well when these lead-induced mod-
ifications are manifest.
Definitive studies to examine this hypothesis have not
yet been conducted. Experiments have been conducted, however,
which shed light on several features of the functional renal
impairment due to lead. When rats are fed for ten weeks on
a diet containing 1% lead acetate, the total urinary amlnoacid
nitrogen increases to approximately 2 to 4 times normal. If
the renal tubular cells had been killed, as with cadmium, the
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12
20 times normal. One can therefore infer that in the lead
experiments the renal tubular cells are functioning but at
a less than optimal level.
When one does clearance studies, the clearance of glycine
is not increased t.o the extent of that for the other amino-
acids; however the amount of glycine is very much increased
in the urine. This reflects a marked increase in the plasma
content of glycine, an overflow kind of aminoaciduria for
glycine. One wonders whether this glycine comes from an im-
paired conjugation of glycine and succinate in the synthesis
of d-amino levulinic acid, a question raised by Haeger-Aronsen
years ago. The clearance of tyrosine and histidine is increased
more than that for the other aminoacids. Since lead can form
bonds with the phenoxy group of tyrosine and the imidazolium
group of histidine, one can speculate that the binding of lead
to these two aminoacids is related to their clearance.
The aminoaciduria may be related to changes in mitochon-
dria. Isolated mitochondria have been studied and show
swelling and a defect in oxidative phosphorylatipn. This
defect is demonstrated with a pyruvate substrate but not with
succinate. These findings indicate that lead interferes with
early stages of electron transport, this interference being
incomplete and not blocking electron transport entirely. Ad-
ditional studies are required to localize the effect of lead,
but preliminary studies suggest that it occurs at "complex I"
or the NADHrcoenzyme Q reductase complex, the first part of
the electron transport system.
Mitochondria from normal kidneys have been isolated and
maintained in a medium with pyruvate-phosphate buffer, no
SDTA, and various concentrations of lead. At a lead concen-
tration of 1 x 10 M, there is a small effect with partial
uncoupling. At a concentration of 2 x 10~^ M there is com-
plete paralysis of respiration indicating a toxic effect of
the lead upon kidney mitochondria. The concentrations of lead
affecting kidney mitochondria are much lower than those known
tc be toxic in the whole kidney. It is not possible or apprc~
priate tc compare the effects on respiration of similar
concentrations of lead in mitochondria from in vitro studies
to in vivo studies. Nevertheless, similar concentrations in
both systems appear to cause respiratory impairment of the
same order of magnitude.
Studies have been conducted to identify the sites of
lead localization in organelles. In control rats more lead
is in the cell nucleus than in the mitochondria; however for
those rats on 1% lead acetate diets there is a large increment
13
of lead in the nucleus, primarily in the intranuclear inclu-
sion bodies. Concentration of lead in the intranuclear
inclusion body can be demonstrated by autoradiographic tech-
nique following exposure to labelled lead and by electron
microprobe analysis.
The morphological appearance of the intranuclear incJ vi-
sion body in rats is the same as that observed in humans
acutely exposed to lead. The electron microscopic appearance
of the body is characteristic. There is a dense central core
surrounded by a periphery of fibrillar material.containiny
some kind of material in the matrix. The nucleolus in the
nucleus is normal under these conditions indicating that
there has been no inhibition of protein synthesis. It is be-
lieved that the formation of the intranuclear inclusion body
is independent of the nucleolus and that the body is formed
by accretion of lead-complexed protein and grows by continual
addition of this material. Intranuclear inclusion bodies
occur typically only in the nuclei of the proximal tubular
cells. Pseudoinclusions consisting of laminar figures repre-
senting some lysosomal phenomenon are commonly seen in the
cells of the distal tubules in lead poisoning but are not
specific for this form of cellular injury.
The intranuclear inclusion body may be isolated by the
classical methods for isolating nuclei and nucleoli. Sub-
sequently ultrasonification destroys the nucleoli, but does
not damage the intranuclear inclusion bodies or alter their
morphology. Approximately 5 mg of material consisting mostly
of intranuclear bodies can be obtained from sixteen rat kid-
neys. When the bodies are treated with RNA-ase or DNA-ase
there is little if any effect, indicating the presence of
minimal amounts of RNA or DNA or their complete absence.
Treatment with proteolytic enzymes such as trypsin does cause
a change which apparently reflects the splitting off of a
protein fragment. The remaining materials presumably still
contain lead and some protein. More positive identification
has not been possible with the quantity of material available.
There is evidence that lead enters the urine by both
glomerular filtration and by secretion across tubular lining
cells. It is hypothesized that lead transported across the
tubular lining cell is in the form of a diffusable ligand
complex and that mitochondria are exposed to this complex.
Lead in the urine has been shown to be in both an inorganic
and an organic form. The organic form is a complex with a
-------
14
cases of lead poisoning, it is apparently the organically
complexed lead which increases in the urine.
Intranuclear inclusion bodies may represent a storage
of excess quantities of lead that enter the nucleus of the
proximal trubule cell. Bodies are noted in rats when their
drinking water contains lead at a concentration of 0.4 rag/ml
(400 ppm), a level of exposure in the adult rat which does not
cause anemia or other signs of detrimental effect.
It has been noted, however, that effects can be observed
at this and lower dose levels if rats are placed on a low-
calcium diet, i.e., 0.1 g/100 g, which is just sufficient to
prevent hypocalcemia. On the low-calcium diet, lead at a con-
centration of 0.2 mg/ml (200 ppm) has to date led to abnormal-
ities typical of clinical lead poisoning including elevated
d-ALA and blood lead levels.
Concentrations of lead in drinking water to which rats
show no significant response are still relatively high. This
suggests that the rat is a relatively resistant species to lead
intoxication. The rat may serve, however, as a model to study
the various factors which influence lead intoxication, such
as low-calcium diets or pre-existing renal disease.
It is possible to develop an hypothesis which relates
solute transport failure, disturbances of oxidative phosphoryla-
tion, and lead in the intranuclear inclusion bodies, central
to this hypothesis is evidence of the trans-tubular secretion
of lead across the proximal renal tubule. Although 95 to 99*
of blood lead is attached to the red blood cell, there is n
ligand-bound diffusible form of lead which passes across the
renal tubular cell. Mitochondria are exposed to this lead,
which moves against a gradient into the nucleus and into the
intranuclear inclusion bodies, probably as a lead-lipoprotcin
complex. In a sense, the inclusion body represents a protective
mechanism tending to withdraw lead from the cytoplasm of the
renal tubular cell.
If one is to examine the effect of lead on oxidative
phosphorylation, it is useful to examine experiments which
have been done in cases of experimental carbon tetrachlori
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16
energy substrate fatty acids, which are delivered primarily
by organic anions, e.g., paraaminohippurate. One can measure
how effectively rabbit kidney cortex slices concentrate para-
aminohippurate from solution in vitro and the influence on
the process of lead added to the medium.
5. Kidney cortex slices may also be used in a similar
study of the uptake of aminoacids.
6. ATP levels in the kidney and levels of substrate and
co-factors associated with glycolysis and oxidative phosphoryla-
tion can be examined.
7. The examination of oxidative phosphprylation in the
mitochondria of lead-poisoned animals might be considered.
8. Aminoacid infusions might be examined in lead poison-
ing to see if an aniinoaciduria ensues in any predictable manner.
9. Work on the renal transport of lead should be con-
tinued.
10. Studies of sodium transport in the isolated frog kid-
ney, red blood cell, and other physiologically active tissues
• are appropriate.
11. The in vivo kinetics of glucose resorption in the
presence of lead can be examined by glucose titration studies.
Bicarbonate titration studies can be utilized to test this
sodium-linked proximal tubular function.
IV. Summary of Reports on Lead and the Hematopoietic System
Discussions on the hematopoietic effects of lead focus
to a major exrent upon disturbances of heme synthesis. As
heme synthesis occurs-.in virtually all cells and is essential
for normal cellular function and structural integrity, a re-
view associated with the hematopoietic system broadly overlaps
with other areas, particularly those related to the nervous
system.
It has been known for years that lead can induce changes
in the mature red cell in .vivo, in vitro, and in studies where-
by red cells are exposed to lead in vitro and returned to the
living organism. Anemia due to lead has been described in
children and in adults. In lead workers on the job signif-
icant anemia is rather rare, perhaps noted in 2 to 3%. in
hospitalized lead workers and children, however, the majority
have anemia. In children the effects of lead are often com-
bined with iron deficiency. The anemia is typically microcytic
and hypochromic; however it is sometimes normocytic and normo—
chromic. The reticulocytes are usually elevated to a minor
degree, perhaps 2 to 12%. There is an increased erythro-
cyte index, meaning increased erthyropoietin activity or nt
least increased stimulation for red cell production. There
is no evidence that a G6P deficiency, such as is observed in
some 10% of American negroes, has any effect on anemia due to
lead, similarly, alcohol does not appear to influence the
prevalence or degree of anemia noted in lead workers.
Studies of lead workers show that red cell survival may
be shortened in some instances. In hospitalized patients,
however, the proportion with decreased erythrocyte life spans
increases to about 50%. In children the shortest survival
time is associated with the shortest and most acute lead ex-
posure. With intense, acute exposures an acute hemolytic
anemia may be seen. Some years ago patients with carcinoma,
who were treated with lead because of a presumed anti-neoplastic
effect of lead, were observed to have acute hemolytic anemias.
Ferrokinetic studies in lead poisoning have demonstrated
that iron is absorbed from the gut, transported, and bound
normally. The transfer of iron into the developing red cell
does not seem to be impaired. In vitro studies, however, show
that high concentrations of lead seem to interfere with the
passage of iron from transferrin to the developing red cell.
Long chronic exposure to lead appears to reduce the turnover
of iron, the clearance of iron from the plasma, and the utili-
zation of iron for new red cell production. In acute exposures
in children ferrokinetic studies are typical of what is seen
in hemolytic anemias. There is a rapid uptake of iron in mar-
row, a rapid clearance of iron from the plasma, and a rapid
utilization of iron for red cell production.
Stippled cells have been classically recorded in lead
poisoning. There is no correlation known between their num-
ber and the intensity and type of symptoms observed. Experi-
ments in animals suggest that stippled cells are preferentially
sequestered in the spleen. Splenectomy or administration of
lead to splenectomized animals results in an increase of num-
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18
stippled cells either by maturing them and returning them to
the systemic circulation or by destroying them.
The lead-poisoned red cell is brittle and inelastic.
Its decreased osmotic fragility or increased resistance to
osmotic stress is undoubtedly a membrane effect. ATPase
activity is decreased in the red cell membrane. Permeability
to cations is increased; potassium, especially, is lost through
the membrane. It is likely that these observations are re-
lated to modifications in osmotic fragility. The Coombs test
may be positive, particularly if blood is permitted to settle
and the test is done on the younger red cells at the top of
the column, presumably because transferrin and other globulins
may be retained on the cell.membrane of reticulocytes.
Electron microscopic studies of normoblasts reveal a
number of changes characteristic of lead intoxication:
1. Gaps in the membrane of the normoblast nucleus are
increased both in number and in size.
2. The protein-secreting Golgi apparatus is tremendously
dilated. .
3. The mitochondria are enlarged.
are pushed apart.
The crystal poles
4. Myelin bodies are observed. These are electron-dense
bodies between the nuclear membrane and the mitochondria.
5. In a variable proportion of nucleated red blood cells,
mitochrondria containing non-heme iron are arranged in a ring
about the nucleus (ringed sideroblast). The iron is present
as molecules of ferritin and as ferruginous micelles. The
chemical nature of the fine, dust-like ferruginous micelles
is unknown; they constitute an abnormal form of intracellular
iron. Similar ringed sideroblasts are found in primary sidero—
blastic anemia, in pyridoxine-responsive anemia, and in a
wide variety of secondary sideroblastic anemias. The patho-
genic mechanism common to all seems to be a defect in heme
synthesis.
Special studies show the reticulocyte to have a markedly
irregular outline and to contain numerous mitochondria heavily
laden with non-heme iron, the ferruginous micelles. Stippling
in animals and patients is characteristic of lead poisoning.
The cause of the stippling is not understood, but the stipples
19
themselves are composed of altered aggregated ribosomes. In
the polychromatophilic cells the polychromasia is due to the
retention of ribosomal material. In lead poisoning the in-
corporation of 32P into phosphatidic acid in mature red blood
cells is decreased.
When lead is administered subcutaneously over a period
of time in sufficient concentrations, a subcutaneous granuloma
forms, a "plumboma," the presence of which may be associated
with plasmacytosis and hypergammaglobulinemla.
For some time it has been known that protoporphyrin,
coproporphyrin, and d-ALA increase in the red cell in the
presence of lead intoxication. With improved isolation of
individual enzymes it has become possible to examine the ef-
fects of lead on the various stages of the heme biosynthetic
pathway. It is clear that lead affects multiple sites at con-
centrations of 10~5 to 10~3 M. The main effects are early in
heme synthesis at the formation of d-ALA, and later at the
point at which iron is inserted into the protoporphyrin mol-
ecule by heme synthetase. These processes take place within
the mitochondria. More recently it has been shown that ALA
dehydrase is inhibited as well.
Practically every living cell contains heme; these dis-
turbances of heme formation are relevant not only to blood
production but equally important to cytochrome formation as
well. The kidney contains large amounts of ALA synthetase as
does the liver. Drugs which Induce ALA synthetase in the liver
can produce enormous concentrations of this enzyme there, and in
the kidney as well.
Acute intermittent porphyria (AIP) is in some respects
a suitable model for the study of certain aspects of acute
lead intoxication. This inborn error of metabolism tends to
occur in families, most commonly in women of the 20 to 30 year
old age group. Presenting symptoms include acute unexplained
abdominal pain, vomiting, constipation, and neuropsychiatric
manifestations which encompass a broad range of phenomena
from motor weakness to psychiatric disturbances. Sighs in-
clude tachycardia, hypertension, decreased reflexes, and
sensory abnormalities. Concentrations of d-ALA synthetase,
d-ALA, and porphobilinogen (PEG) are increased in the blood.
ALA and PEG are elevated in the urine as well. Asymptomatic
relatives of persons with acute intermittent porphyria may
show only the biochemical alteration of this disease, viz.,
-------
Other porphyrins which may be found in the urine on lab-
oratory examination are not excreted but rather are formed in
the voided urine as degradation products.
Isolated and purified ALA and PEG show no evidence of
pharmacological activity; they are not pressor substances.
The clinical phenomena associated with AIP can be explained
on the basis of a disorder of the nervous system. Indeed it
has been shown that the peripheral nerves of patients with
this disease can undergo demyelination. The presence of macro-
phages containing lipld-staining material around the nerve
gives evidence that these changes occur ante-mortem. Brain
demyelination has been noted around vessels. The inappropri-
ate secretion of ADH has been noted in the presence of changes
in the thalamic region.
With lead a similar symptom pattern is observed: ab-
dominal pain, vomiting, constipation, and peripheral nerve
disturbances primarily of motor function. Anatomically, it
has been shown that lead has an effect on myelin sheaths,
Schwann cells, anterior horn cells, and axons. The pattern
is strikingly similar to that in AIP.
In AIP the main biological disorder is found in the liver,
which shows a high level of ALA, PBG, and ALA synthetase.
There is reason to believe that an association exists between
the hepatic biochemical alterations and the morphological
brain changes. Lead appears to act directly on the brain and
nervous and hematopoietic systems. Thus, it would appear that
AIP and acute lead poisoning have different basic causations
but show a final common pathway of clinical manifestation.
In the search for more subtle indices of lead effect,
examinations have been conducted on lead levels and heme pre-
cursors in children. When lead is moderately elevated in
children into the 40 to 60 /ig/100 g range one notes a relatively
poor correlation between the blood lead level and ALA, copropor-
phyrin, and other heme precursors. This contrasts somewhat
with the situation in industrial workers, for whom abnormal
concentrations of these materials are almost invariably found
at the 60/ig/100 g range. In studies of. groups of normal
children and those with mental retardation reflecting a variety
of etiologies, a difference in serum ALA between the two
groups has not been observed. Similarly, there has been no
difference in blood lead levels nor in the lack of correla-
tion between blood lead and intelligence, or between ALA
dehydrase and intelligence. A significant correlation has
been found, however, between blood lead levels in the 40/jg/100 g
21
range and ALA dehydrase in the blood. The clinical signif-
icance of this correlation is not established.
There is experimental evidence for a correlation between
ALA dehydrase levels in.liver, brain, and blood. Such evidence
is derived from studies on suckling rats who have received
lead only through the milk of their mothers, who had been fed
on a 5% lead acetate diet after the litter had been delivered.
Neuropathologioal changes were observed in the suckling mice
under these circumstances. The clinical implications of this
work are obviously not fully defined inasmuch as interrela-
tions between lead, depressions of blood and brain ALA dehydrase,
and anatomical changes of the central nervous system are not
well understood.
In summary, however, it has been shown that (1) ALA de-
hydrase activity is reduced in both the blood and brain of
lead-poisoned rats, (2) a significant correlation exists be-
tween blood and brain ALA dehydrase activity in normal and
lead-poisoned rats, and (3) a correlation exists between blood
lead and ALA dehydrase levels in children. It is possible
that diminution in ALA dehydrase activity in the peripheral
blood of children may reflect a similar reduction of the enzyme
in the brain. (The presence of ALA dehydrase in human brain
may be assumed since positive enzyme activity was found in
the brains of two aborted fetuses which were at the 15th and
17th week of. gestation.) Thus, it is likely that a negative
correlation exists between the blood lead level in children
and the ALA dehydrase activity in the brain. These observa-
tions suggest that it is appropriate to consider seriously
the possible effect on the developing brain of lead levels
previously regarded as normal.
Preliminary studies of lead-poisoned dogs have been con-
ducted to shed light on the physiological significance of
measurements which show a depression of ALA dehydrase. In
such studies dogs were fed for 42 weeks on diets containing
0, 100, and 500 ppm of added lead. At the end of the 42-week
period the blood ALA dehydrase level was virtually zero in
the group of dogs fed 500 ppm lead. However, the behavior
and appearance of health in all animals was the same. So an-
imals appeared to have been adversely affected by the ingestion
of lead over this period. Although these animals did not ap-
pear to have suffered a detrimental physiological effect, there
was the question of whether or not the dogs would respond dif-
ferently to physiological stress, in these studies the
animals were stressed through a reduction in blood volume
-------
The recuperation from this insult appeared to be identical
for the three groups of dogs, and the curves reflecting the
blood parameters could be essentially superimposed. The
bleeding procedure did not seem to influence the blood lead
level of the animals, which returned to the prephlebotomy
level some one week after the loss of blood.
In the study of hemoglobin synthesis a convenient .bio-
logical model is available in cultures of the photosynthetic
microorganism, RhodopseudomonaB spheroideo. This organism
grows very well, as a facultative anaerobe, without oxygen,
in which case it produces bacteriochlorophyll, or in the
presence of oxygen, it will grow in the dark and manufacture
primarily heme. Lead suppresses the growth of the organism
very markedly when the culture is relatively low in .iron.
When supplies of iron are sufficient,.the inhibitory effect
of lead on the growth of the organism is much less apparent.
The excretion level of coproporphyrin by this organism
clearly demonstrates the antagonism between iron on the one
hand and lead, manganese, and cobalt on the other. When the
organism is grown at low concentrations of iron and in the
absence of other metals very little coproporphyrin is excreted.
The addition of 0.03 ppm manganese to the culture medium,
however, causes the excretion of large amounts of copropor-
phyrin. In iron-supplemented cultures much higher quantities
of manganese are tolerated before the excess coproporphyrin
appears. Lead potentiates the effect of manganese in caus-
ing increased coproporphyrin excretion and is thus antagonistic
to the addition of iron. If then still more iron is added to
the medium one overcomes the effect of manganese entirely and
the effect of lead to a somewhat lesser extent. In all in-
stances the effect of a metal such as lead can only be stated
in terras of the relative concentration of other metals which
act at the same binding site.
An examination of the other metabolites in the tetra-
pyrrole synthetic pathway suggests that the enzyme coproporphy-
rinogen oxidase or coprcginase may be iron-dependent in
that its activities are influenced by the balance between iron
and other antagonistic metals including lead. Ferrochelatase
and ALA dehydrase may also be affected by other metals as well
as lead, in the whole growing organism the coproglnase site
seems to be a most important area for lead inhibition to be
manifest, and this may well provide a basis for the well-known
coproporphyrinuria of plumbism in man.
23
V. Summary of Reports on Lead and the Nervous system
' Clinical manifestations caused by excessive amounts of
systemic lead depend upon the particular structures which are
affected or damaged and the intensity and duration of exposure
Levels of lead in blood and urine may reflect the current in-
tensity of exposure. The actual lead burden, however, must
.also include an unmeasured quantity of lead stored in the
central and peripheral nervous systems as well as in the skel-
eton and soft tissues. The persisting effects of encephalopat
such as mental retardation, epilepsy, and behavioral disorders
may be the result of anatomical damage occurring during the
, acute intoxication or possibly the result of chronic cellular
damage resulting from the slow release of stored lead in combi
'atlon with continued exogenous exposure. Experienced observer
have repeatedly emphasized the importance of continued environ
mental exposure to lead as an important factor which increases
the likelihood of severe permanent damage to the brain.
An additional hypothesis is that continued exposure by
the slow release of stored amounts of lead, which in them-
selves do not produce obvious symptoms may, nevertheless, be
capable of damaging certain intracellular enzyme systems and
producing latent effects. The concept of latent effects due
to lead in persons Who do not show obvious clinical plumbism
has been raised by Lane and others who believe that lead bur-
dens insufficient to cause symptoms or to produce disability
may shorten life expectancies because of premature development
of nephritis or cerebral hemorrhage. The evidence for this is
not firm, however.
• The neurological manifestations of lead poisoning are
variable and widespread. Lead workers may complain of lassi-
tude, irritability, depression, constipation, and abdominal
discomfort. These symptoms are the same as those sometimes
expressed by emotionally depressed individuals.
Peripheral neuropathy is considered a common neurologic
manifestation of lead poisoning, and involvement of the radial
nerve is frequently described. While the clinical picture may
present as a mononeuropathy, neurological examination will
often demonstrate weakness in other muscles, and neuroelectri-
cal studies may show prolonged conduction times or decreases
in the amplitudes of the action potential. In some cases,
spastic paraplegia and hyperreflexia suggest upper arc and
lower neuron damage. In such cases the differential diagno-
sis between lead neuromyelopathy and spinal cord degeneration
such as in primary lateral sclerosis and amyotrophic lateral
-------
24
Lead encepholopathy has been considered more common in
children than in adults, in whom it may be misdiagnosed as a
brain tumor responsible for such symptoms as headache, vomit-
ing, diplopia, and periodic confusion, in conjunction with
evidence of increased intracranial pressure. Ophthalmological
disturbances are not common in systemic lead poisoning although
lead pigmentation of the retina has been described in adults.
Ocular symptoms of lead poisoning have been more common in
children in whom it is possible to recognize disturbances of
the visual cortex, suprageniculate pathways, optic nerve,
retina, lens, and intra- and extra-ocular muscle movement.
Of the neurologic manifestations of lead poisoning in
children, peripheral neuropathies have not been the most ap-
parent, and central nervous system manifestations have been
considered more common. It is possible, however, that exam-
ples of peripheral neuropathy in children go unrecognized in
many cases, including those in whom encephalopathy is dramatic
and commands clinical attention. Children with clinical evi-
dence of peripheral neuropathy, viz., sensory loss and weakness
show evidence of denervation when tested electrically.
Neurochemical studies of the brain in lead poisoning
have been technically difficult and have most commonly focused
upon the effect of alkyl lead compounds, the pharmacology
for which is undoubtedly dissimilar to that for inorganic lead.
Seizures encountered in acute lead encephalopathy may be due
to neuronal excitation related to local edema or vascular
lesions. These manifestations may be due to an interference
with glutamic acid transport or metabolism resulting in a
reduction of •Jf~aminobutyric acid (GABA) production. GABA has
been considered an inhibitory transmitter or depressant
moderator, and interference with GABA production results in
seizures by increasing neuronal excitability. Metabolic
steps which might be susceptible to the effects of lead in-
clude (1) glutamic acid decarboxylase (GAD) which carboxylates
glutamate to GABA; (2) glutamic acid dehydrogenase (GHD) which
reduces 2-Xeto glutarate to glutamic acid; and (3) pyridoxal
phosphate (B,P) metabolized from pyridoxine which functions
as the co-enzyme for transaminatlon reactions in the synthesis
of GABA. Further evaluation of these systems and of the clin-
ical efficacy of pyridoxine or glutamate in children with
seizures of acute lead poisoning is warranted.
Recent electron microscopic studies in experimental lead
neuropathy have shown that the primary damage is to Schwann
cells and myelin sheaths but that axons also show degenerative
and reactive changes. Steps in the remyelination process
25
have been demonstrated microscopically. Two mechanisms have
been suggested in the explanation of the process of demyelin-
ation and remyelination in lead neuropathy. One possibility
is that a substance having porphobilinogen as a precursor is
essential for the maintenance of myelin. It is Xnown that
in porphyria there is a metabolic block, which results in the
excretion of excess porphobilinogen and d-ALA and concurrently
a demyelinating process, the specific cause of which is not
known. The second possibility is that lead causes vasodila-
tion and altered vascular permeability which cause intra-
endoneural edema and consequent damage to the Schwann cells
by pressure or ischemia.
Biochemical studies of lead neuropathy have concentrated
on the motor endplate region of striated muscles, the point
where the greatest proportion of synaptic acetylcholinester-
ase is localized. The introduction of lead into suitable
neuromuscular preparations results in the precipitation of
lead at the esteratic site of this enzyme. The prime bind-
ing site for lead appears to be the post-synaptic subneural
apparatus of the motor endplate. The binding of the sub-
neural apparatus by lead is not influenced by the prior
administration of di-isppropyl fluorophosphate (DFP) or nee—
stigmine. However, this type of treatment completely inhibits
cholinesteraBe activity. Cholinesterase activity does appear
to be reduced in muscle in which the subneural apparatus has
been bound by lead, such studies are part of an attempt to
identify some properties of the cholinergic receptor and the
characteristics of lead binding at the motor endplate which
may be related to some aspects of the weakness found in lead
poisoning.
Other studies have focused upon calcium, which is nor-
mally present at the motor endplate in a bound form and is
released as the free ion following nerve stimulation. The
close relationship between the site of calcium release and
the site of binding of divalent metal ions such as lead may
explain the predilection for palsy in the more active muscles.
Lead and calcium may also be interrelated through a mechanism
which is reflected in the fact that a reduction in calcium
concentration may produce neuromuscular blockade by decreasing
the presynaptic release of acetycholine.
VI. Conference Summary
This conference reaffirmed a long known fact that poi-
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26
lead-containing material, and excessive occupational exposure -
t.o lead continue to occur. This fact represents a failure on
the part of health experts and other responsible elements of
society to prevent gross exposures to lead. • "
The presentations of this conference illustrate the'''in-
creasingly sophisticated methodology'."being used to study the
manifestations of excessive lead absorption. -^Such studies
arc obviously important to our understanding of the mechanisms
and consequences of lead intoxication. Of even greater im-
portance, however, would be the development of sensitive and
specific measures which could establish whether or not "nor-
mal" people are being harmed by current concentrations of
lead in the "normal" environment. No new, practical measures
are as yet available as screening procedures to detect very
early adverse responses to lead. The validity of new enzyme
studies is currently under examination.
Aminoaciduria. Abnormal quantities of aminoacids in
urine as a consequence of lead exposure occur only in people
with overt lead poisoning. Barely elevated levels of amino-
acids can be detected in urine of workers with excess lead
exposure. Aminoaciduria, therefore, does not appear to be a
highly sensitive clinical study procedure for "normal" popula-
tions, although it is certainly important for research in
cases of lead intoxication.
Inclusion Bodies. Nuclear inclusion bodies in kidney
cells appear to be relatively specific but to date have only
been found after relatively high exposures to lead. Their
only presently known function is as a storage site for excess
lead which has entered the nucleus of kidney cells and com-
plexed with mitochondria. Intranuclear inclusion bodies
should be looked for in cadaver studies, particularly in-
those with high body burdens of lead, and in cells in the
urinary sediment of humans with chronic lead exposure. In
addition various animal species should be used in laboratory
studies of nuclear inclusion bodies.
"Sodium Pump" Inhibition. This phenomenon presents as
a hypothetical lead effect and requires research for verifica-
tion.
27
Biochemical Functions. Two potentially useful indicators
of effect of lead are available: the measurement of the respira-
tion of mitochondria of reticulocytes and the measurement of
potassium loss across the red blood cell membrane. Both of
these phenomena require evaluation in animals and humans with
lead intoxication.
Enzyme Changes. Enzymes which are considered to be most
sensitive to lead exposure are d-aminolevulinic acid dehydrase,
lipoamide dehydrogenase and ATP-ase. Changes in enzyme
levels need to be correlated with possible adverse effects in
man and animals.
-- Essential Trace Metals. Many trace metals are essential
for normal body functions, and lead may be competitive with
these metals. However there are no known examples of such
inhibition by lead in in vivo systems. Cadaver studies have
shown that essential trace metals in soft tissues are normal
despite increased concentrations of lead.
Heme Synthesis. Coproporphyrin and delta amino levullnic
acid in urine are sensitive and reasonably specific indicators
of lead exposure. These measurements are of value in studies
with adults, but may be of less value in studies with children.
There are marked diurnal variations in these indicators, and
it is recommended that concentrations be expressed in units
relative to urine output per unit time rather than to urine
volume. Delta amino levulinic acid determinations in serum
are suggested as a more refined measure of lead effect upon
home synthesis. The behavior of ALA-dehydrase under lead ex-
posure situations requires examination and interpretation
from the point of view of health.
Nerve Conduction Time. One of the more practical tests
for sabacute and chronic neurological change is the measure-
ment of nerve conduction time. This procedure is considered
to be more useful for following cases of lead intoxication
than for screening purposes although lead poisoning should be
considered in the presence of unexplained delayed conduction
times.
Analytical Variability. For almost every study, questions
about analytical accuracy and precision can be raised. It is
suggested that comparisons of data would be facilitated by
collaboration through a central laboratory. This technique
would avoid much controversy and expedite interpretations of
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This conference reviewed the state of current knowledge
about how lead interacts with man and experimental living
systems,. It appears that the best indicators of early response
to lead are already in use.- Several areas of research which
may lead to improved measures of early response are apparent.
It has been reassuring to note that exhaustive animal
studies have not led to the discovery of new lesions due to
lead, although it is also clear that there are major dif-
ferences between species in response to lead absorption.
Several needs for the immediate future are highlighted.
1. Prevention of excessive and unusual exposures to
lead—a broad problem for society-at-large.
2. Refined studies of those cases of lead intoxication
which, unfortunately, continue to occur.
3. Refined studies on populations with elevated, but not
necessarily clearly harmful, exposures to lead, especially
with respect to changes in renal function and herae synthesis.
4. Research with animals on correlations between enzyme
changes and adverse effects of lead.
5. Surveillance of the environment and populations for
continuing evidence of man's existing and previous exposure
to lead.
6. Surveillance for potentially susceptible individuals
and indications of synergistic actions or aggravation of exist-
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