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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                                                                                     APTD-0617
                                                         4. TltKi ena S
                                                         .'/  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

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     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

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                              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.,

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     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

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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

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                              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
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     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.
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     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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                              28
     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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