Document Ndrej72Q6DeJ21rjMK6gpGbD

Thu Irioiogicnl idii'act-s of Iliad at Ley L - -. 1 -; I-lie there are current examples of db' ious le ad poison-- i-ip in improperly supervised .Industrial ope:qat ions, and i:i pediatric practice i/hare pica for old .paint is ohse rvc-:d, the luu, j. tu ai rn\it'.carte of lead cor.rotar.dt - ii% ami ient ur van at-- inccpharc" lit.s not been clearly defined. The lead c a?-cent rati in v;on-.industrial atmospheres: are very much lower than the cur vent industrial TLV (several puy/nv1 vs. 150 ysg/np} and evi- dencc of pluribisia is completely lacking, idWerthel ess it is reasonable to believe that at some point she `t of ppvious. lead y. loaning in the usual '~nia. body abo ption of lead ivay. >; o.telj a usag nlludw sufficient to cause d s cuvlmeoc of normaV physioLoy leal -nechanj . That thi point is not yet known- in related to that fact that trsditior si biochemical and clinicpl. approaches tc the detection 'of leap effects fit low levels have been relatively insensitive and incapable of detecting minimal alterations. The question of. the existancc and significance of ip.Lnii.ial alterations, bovhvor. Is central to cliscu.ssi.ons of the health significance ci the concentra- tier of alkyl, iced eeng;ounds in motcn: fuels. in exhaust, and OJ eor-soquqntly in ambient- atmospheres. KET 0017361 b2 a. This report contains a summary of discussions held in- .February, 1970, which attempted to: 1) identify biochemical and cl initial approaches to. the detection ..of. lead .effects, at lo,.' . 1 e:Vels; 2) establish the health signifi nance of whatever lead-related phenomena might be observed at these levels; and 3} suggest possible areas in which research ernphas i s might : be. rni'St fruitful in solving the presented prob ems. Participants included physicians with varying, .types c-f. e>;:peri enc e -with clinical l.ead toxicology, respected scientists in s syeral branches of clinical medicine and biochemistry, and 'invest!- g a. tors with primary interests and background a- in lead' metabolism; cti.-o. "XperiifEi! t ai toxicolcgy , -Several pa. it ie pciiCS , although eminent in their.respective fields, had no Aa jor interest in. .lead,. They were invited because of - their specializ ed clinical - shills and knowledge relevant to organ systems, khov^i to be affected-by lead. The material'presented"in' this report reflects the work of the' participants, their colleagues, and c thers in the re spec- tive "field's of investigation.- ;. -Participating Scientists IMS. I. Barry, M. D, The Associated'Octet Company, Ellesmere Port Cheshire-, England Ltd. Neals. Bric;k e r ; m, d . .'Washington University St:.' Louis , - Miss our 1' HE 0017^62  ,( - 2b - j. Julian Chisolm, Jr., M.D> John- Hopkins University Baltimore, Maryland Joseph It, Moyer, Pb.D University of eincinna a Cincinnati', Ohio Jerome F. Cole, Sc . D. Carl V. Moore, M. D. International Lead -Zinc Research- Washing t on- university Organ iration ' " St. Louis,. Missouri. Now. Ybrk, N. Y. v,- Kim Cramer, M.D. Universitetet I cbteborg Goteborg SV, Sweden' Jean M. Morganl M.D, University of Alabama Birmingham, Alabama . Robert E. Eckardt, M.D, Esso Research & Engineering Company Linden, New.Jersey Emil A. pfitzer, Sc.D university of ! tine-inn; ti Cincinnati, Ohio, Robert Feldman, M.D. Boston University Boston, Massachusetts George Roush,. Ty* JL t } \K L i. e Tulane Univers ity New Orleans, L ouisaan Abraham Goldberg,or.ivcrcity of Glasgow GI a s g oww. 1, Scotland D.Sc. , -P.'R-.C.P. George E. Schreiner, . Georgetown Tjr.l W a sh ing t on, D. C, M.D. Harold Goiz, M.D. American Petroleum Institute New York, N. Y. ., G. J. Stopps, M .B. , B S, ......... 1/ E. -I.; du Pont. 5e .Nemoik rs & Company Wilmington, De lav/are "Robert A.-coyer, M.D. " University of North Carolina; -Chapel Hill,''North Carolina Lloyd B. Tappet, M.D. University of Cincinnati Cincinnati, -Ohio Robert c, Griggs,. M.D. Western Reserve University Cleveland, Ohio ' ' - David. D. 'Ulmer, M.D. Harvard University. Bast on -, Mo ssachusetts Robert j. m. Horton, M.D. - Bert L. Valle M, D, National Air Pollution.Control Adrcinistration . Ha vard University Durham, Kor-th Carolina, . . Boston, Massac: ousetts Fa. 11 ace N, Jensen, M.D. George Washington University Washington, D. C. ?. 1 ;i V- * S Kl= 0017363 Summary of Reports oh' Environmental and Human Lead Levels The concentr3.ti.on of lead in soils, fqods, wat exp .and air is highly variable. Alimentary lead intake cov ers a range of approximately 0.12 to 0.35 mg/dsy.- The absorpti on of lead from the atmosphere varies 6 to 10-fold. in spite of this wide range of lead intake levels, the amouil : of lea d in man HE" 0017364 3 is: relatively constant, totalling about '1004(4 mg, . of which at least 90% is stored in the bone. Blood lead levels are even more constan]t, remarkably so in view of the wide range of exposures. When the'mean blood lead levels from'various.populations are examinedther seems to be an increase in the blood level a s one -goes from a rural to an urbanized area. This phenomenon no related to the density of motor vehicles in these respective communities, and the urban-rural gradient i; observ sd in areas of the world with very low number of cars, When one goes from society to society the differences in blood lead levels are n or i mpres-s ive, .he oiooa xeaa. .level or ru mans, tor example, approximates that for rural Nor th Americans Soft tissue lead levels and bone lead eve Is dp not cor- relate well in individuals. The lead in th bone seems to increase with age whereas the soft tissue-life nsi of .ead. does not rise after the end of the second decade In fe males the soft tissue lead levels tend to be slightly lower than in males, while in' bone the lead concentration in females .1. very much lower than in males. This lower Jt one lev il'of lead. h owev er, also rises with age as i n m.a 3.es. c hildren show very low levels in soft- tissue and bone, find no d liferon 0017365 the sexes is demonstrable. Whether the higher bone levels In older adults is due to accumulation with a ge or simply- due to the fact that these people were living art a. time v?hen there was more lead in the total environm.e nt is not known. Lead concentration in bones varies w b th the ions sale the lead content being higher in the more dense bon; s and highest in the petrous temple. Bone lead levels have been determined j n the sk eletons of: 40 Indians vino lived in Arizona during the period 700 to `i 1450 hiJi ) The specimens were from original, burial ites and did not include museum material. Soil samp 2 53 rrcm ,? ? -i-- ?v J- V* th body cavity and adjacent to the buried eletem C O n T* A j laps lead than the bone; therefore, the que tion of aontamin- at ion was excluded. Assay showed 6-8 ppm iri the b or e ash of mo r,'ib. Sacrificial skuj.3_s frorn Mexico, px cfcablv f roTu the tine of the Conquistadors, contained lead love _n the range of 2-4 ppm of the ash. Coprolites (mumroifi l fecal material) found with these skeletons gives evidence of the die; t on which fihh, the population lived. r. A small grains, grasses, f\~ - - The and food supply was pri the t'otal j -cal lee d c en centration was very low. .. . At some point in history, environmental and teral R 001J366 5 the-Civil War in this country. - contaiivbDead- in conce it rat ions which approximate those noted in modern autopsy mate cials, viz. 40 to 50 ppm. In view or the very great range of lead] intake between individuals,-jIa"n"d 'variability in lead absorption, the consistency of blood lead levels is extremely impressive;. One w onders if there is a biological basis for this consistency of blood lead levels, and whether' in fact the biological basi s of the consistency reflects a biological necessity jfor lead . We have no evidence for this, but we have no evidencecje that a t is not- the case. It is generally true, however, that when a metal W3./Wi-wl AW e,v\ (i -> c* rn ent merely on an incidental basis but rather as a si bstance i with specific biological importance. One cannot hei p but re- collect the early studies of so-called zinc roxicitx in which zinc was regarded solely as. a toxic racial responsibl e f or metal fume fever. Zinc was a typical trace metal -p: esent in amounts difficult to detect and. in association with only traces of information about its significance. When in 194] , it be- came apparent that zinc was- an essential constituent of the metalloenzyme carbonic ahhydrase it became apparent that zinc was.neither incidental or solely.toxic but in fact 1 ,.ad a spe- cific biological function. Thai: a simile:!r s . may apply 0017367 o in the case of lead has never been critically or systematicaII examined. In rnaking. inferences fram b 1 ood 1ead 1 ei>el dete[rmination., i it is important to remember that the analytical tedjuilcfues for lead assays at low levels do .not yield ikpressivfely con sistent results. In many situations the variation between replications is greater than the variation between i.ndividuais in a sampled population. In addition the precision of analyst; by a given laboratory may vary trom day to ahy. II. Summary of Reports on Lead and Sub-cellular physiol There are a number of metals jthe biological furiction of 1 *. | |4- 'T' *5 r\ l 1 metals in tissues and biological fluids is nteither an index of their importance or of their biological sfignificance, posi tive or negative. There is still active disfcussion as to whether selenium and chromium are essential trace medals or simplv incidental metals incorporated into tissues. bead is m a similar category. We do not know whether of; not lead has an essential biological function. Where metals have been known to interact with Essential biological systems., they either interact with prote. .ns ox* I with nucleic acids or with, integrated system^ such as organelles, KE 0017368 probably with the membranes. Among the proteins, those that have been examined extensively are the enzyjfnes. Eh cymes often contain a metal which is bound in such a specific it anner that it cannot be removed without loss of activity. Thi s phenoraenon is not related to activation or inhibition of an en zyme but rather to the intrinsic activity of the enzyme. A large numher of metals are bound to nucleic acids.. |The fun tion of these metal-containing nucleic acids is presently e ntireiy unknown. The relationship of metals to organelle f:unction is also not understood. The sites to which metals can attach inn biolog real materials are not infinite. Metals can attach to the eamino acid group of lysine, to the carboxyl groups of glutamic and aspartic acid, to the suifhydryl groups of cyst ene, p in other less common ways such as with the phe noxy gre up of tyro- b1 onds ar e stable. e v/ith a ny or all leva the t lead associates only with sulfhydryi.groups to f brm mere aptides. We do know that lead can be toxic unde t certai n circus-' stances, but so can any metal. Essential m etals pc t as metallic proteins.. When these metals are present in excess a nd a11 aclt e d to ligands which sure not the normal .active uetfil--bi V"; pr "i JV"jf f? f'-./TO RET 0017369' 3 of these metalloproteins, inhibition occurs,i[ i.e. , th is toxic, Whether a lead mefcaliopro'cein exijsts and has an essential bodv function is not.known, ! tork with car): oxypep t ida S 0 illustrat es s c. v sre aj. importa poin ts. C a rb oxypep tidase J_ & cl zinc -centa ini pg enzyri e which i has t W o functions: first as cl pept idase, se condly a S an es It .1 s DO s sib Is to r emove the normal zinc con stituant of the ensy me a nd replace it wit h va xi ous other met als. wh en the zinc is removed and rep la ced v?i th 1 C Ci.Ci j S'- ne inds th at the met a lloenzyme has 1 ost is pep t x dase prope rti es, but the est properties are increased; in fact, the lead harboxppleptida.se is a better esterase than the naturally occurring zxpc analog. The point here is that a lead raetal 1 oensyme pan have a par fectly legitimate biological purpose. Work with this enzyme is instructive in a second sense: it has been found that the metal of c arb oxypeSptIdas a is attached to three ligands: two imidazolium groups of jhistidihe and one carboxy group. .Sulpjhur-containing amino icids ape not in- volveu. T'his observation is contrary to many! of the e x oectaticns which have been expressed in the literature. Another metalloprocein, metal1othion ein, /as aiscovered first in the horse, then in other species anc:certair .y in man. K% 0017370 q Metallothionein is a e-admium-coutaining- protein with a molecular v/oight- of about - 6000 without the metal 6800 with the metal. The protein is composed of shme 52 arPL no acia groups, one-third of which are cystine. |According to I ticnal'thinking, this high concentration of sulfl y d ry 1 gr cc. .t p s should yield a material with, great avidity for le ad. This is not the case in. vivo, however, and lead i _> /"! Ot 3 f fic.ien.tly strongly bound to met a-i 1 oth i one in to disp lace cac niura or zinc, The balance between metals is essent ial in a .1 considers- .ons of metal biocheraistrv and toxicity. Meta interact and may be antagonistic, metal A protecting f OIYi t i 1 if feet of ii'CtTcij. J'J f ore metal A .exa^cresracx.o^ tno on o example is that of molybdenum intoxication which |.s observed in Somerset in England and in the western t of the United States, The manifestations of this diseat '.ay be enhanced e 1 i'i sly that lead, iron, and perhaps other' metals inter are relevant to this discussions but are n in v ays which well understood pends upon nurne ous factors, most of which are poorly understood Included tmong these factors are the amount and cha aeter o rctanao the penetration through membranes by motels,. .the :ani utv constant.for the several metal-ligand.bond; ;, and si or i.c lacr oi describing the special arrangement between rust a Is ? nd ligands. HE 0011371 lu It is relevant to examine systems Vhidh might possibly i be 'disturbed.by lead and hence represent.target syj t erns i or lead effect, one such .system is the pyruv:ate--carb< !xvrase system -whereby pyruvic acid is introduced into the Krebs cycle by acetyl co-enzyme A. This system is q conglomerate of sulf hydryl-ccntaiming enzymes- and co-enzymes, Including thiamine! i pyrophosphate, folic acid, and co-enzvme A dncl is ed sent ial in the energy metabolism of the brain. We know, for example, fha thiamine deficiency, as in Wernicke's syndrome or } ore Eik of f 1 s psychosis /,leads to gross functional abnorm; lities. A similar -syndrome has been observed in foxes fed on rails-wnich ps Jc~ 5-\ m v) --.. o r V; O' k\*n t?\ V f 1 < r n ^ 't~ thiamine- is destroyed, absent, or interfered -with by metal attachments to relevant sulfhydryl groups would be expected to yield the same clinical syndrome. That jt.he cent rai nervous system manifestations of lead poisoning map Qb ! P tea to ini;e foronce with pyruvic acid metabolism may well be p asible, The problem of mercury intoxication, as in me enn me -mercu- rialism of the Mad Hatter, the question.of arsenic toxicity md the therapeutic effect'of BAL, a sulfhy^ ry1 - C on|t a in ing material, are all relevant to the question ot metal bind!ng to sulfhydryl groups of the pyruvate-carbox^rase sv stem and the disturbance of this system by-extraneous metals KJS- 0017372 The peripheral nervous manifestations jo lead poisoning i' l: are not related to these phenomena, but it jis entir eiy possib] that lead may attach to sulfhydryl groups ojf the ns uroconduct;Lon i system peripherally, I - Another system where investigation may yield i rformation on the biochemistry of lead is that related) to oxid ative phos- i phorylation. This process depends upon the| integri ty of i mitochondrial membranes, which among other things, preserve | the respective levels of calcium and potassium i'nsi .e and outside the mitochrondria, Impairment of tljiis meirib ane by carbon tetrachloride is associated with a dramatic idnift of ! ,i_ i if `.'H H it * Pj r i i ri/ 1 f 1! 1 i > 1 * Kill r'\ V xidation and phosphorylation. The sulfhydryl ligands of the mit ocli onj drial membrane suggest that the attachment Of lead cj>r other metals to the membrane may interfere with it s intririi.sic mera- holism and its ability to protect the intra-jmitochor mai environment. ! Under such circumstances- the rrjstals mi yht be ... I expected to cause significant damage to the (energy rr e t abol i sru . i system, *: i There are undoubtedly ways in which the) effect of.lead on nucleic acids mav be measured. we `know that the binding of lead to .nucleic acids causes their hydrolysis. Research .reat i i r b . cl is currently less well devel or-e 3, "i'j owe v K6" 0017373 G u tomarv- of Rep or t s on Lend and the Kidney .. There is no doubt that lead in abnormally high amount: may cause acute or chronic renal injury, m childr :Ti acute lead intoxication may be associated with a irenal le Sion tvpical of the Panconi syndrome. In cases pf intox i cation without encephalopathy one nay see either atoino aci iur.ia or glycosuria or both. In more severe cases, such as pith. en- cephalopathy,the triad of the Faneoni syndrome may ` ic seen-. amino aciduria, rickets, and kyperpkosuria in the i resence of extreme hypophosphatemia in the range of 2 tog % (no. rial = 3.5 to 4,5 reg' %) , Fructose and glucose are excreted in the presen - \t f- ( t Tt'-r ~~t"I- * y, 1;y, sugars varies. Some children excrete closej to a gr am of ultra i in a day although the blood citrate .level ib norms! During the acute episode seme' of thes< chi Id. re n have shown the inappropriate secretion of ADH, I'nxs o.o ;rvat j on cannot be cittributed to treatment since' it has been observed before treatment is initiated. Treatment i V* reo .o it v/x th 2DTA has been shewn to cause a' transient depression in the s erura phos- phate levels, and a transient increase in aInino aci dnr j.a, With treatment, many of the elements df the sy ndrome are promptly corrected. The rceiituria is gone within a week; the aminoaciduria clears within a month (nemiennani it at i X E" 0017374 chromatography). Bone changes resolve within 7 t-o 8 moiiths. - EDTA mobilisation tests in which PTH was- used as well to mobilize lead stored in the bene have been attempted. In some cases sufficient PTH has been used to [raise the serum calcium level to more than 13 mg %. Althoujgh the a mount: or s\ calcium in the urine increased 3- to 4"fold,j there w as- no in- crease in the urinary excretion of lead abojve that , which >e was associated with EDTA alone. With respect to chronic renal injury tjhere is, of course, the Australian material which shows quite ejonvincin sly that under certain conditions of exposure lead ihtoxicat ion can i W /-. "-* V, -v- -i` w^v\"hv: 4*4 ci C'w' v-lii. y^si ixv.. J. t W L .U .1. 1..* C Cu'-nn U. '.A V-i t i --. 4 rv- /`I'." - ... have failed to demonstrate, however, an equivalent phen omen on in the United States, The EDTA mobilization tests in the Balti more group were normal and -it was not possible to d imonstrate chronic renal injury. Epidemiological factors must tlCC OUllt for the dissimilarity between Australian and Ame.ric rn exper- fence. In the United States the patients have chew d paint at some time between their first and third fear of ife. In Australia the children are in the 6-to 10-year age ranee and absorb lead through the ingestion of raindroos v/hiel i coh'caxn ite ad from decomposed paint on veranda rails and rooft ops This may represent a less "intense exposure 1 tr: a so? o prolonged 14 one. Many of the Australian- patients- have a jpes- cav is - aotormit ' and tophaceous gout. A number of cases of lead poisoning in z Quits h ive been bserved in Alabama in relation to the 'prolonged ex ssive con- sumption of .illegally-prepared whiskey Tnis disease occurs" as well in Georgia, the earolinas, and indeed may os 'expected anywhere that whiskey is illegally prepared i a solder ed vats and distilled, through old automobile radiator t' e>T*V | fOs v is evi~ dence that over 50 million-gallons of illegal ilcoaoll are distilled in 'this country every year and that over 50 Yo of the illegally-prepared alcohol contains more than ]_ rpyj q < lead/ "t j ? a t Mile"! oi~ Ltiiix3. c. oii"C3L3 riw even as high as 80 mg of lead per litei In Alabama experience the typical patient is a negro male i between the ages of 45 and 60. Perhaps some 25% of h ospital admissions in this group have been exposed to illegal alcohol. Those v?ho have experienced severe and prolong expc ur e. of 10 years or more are typically found to be anemic, to h a.ve a table renal insufficiency, and to show a nor: na i pye i ogram, although the kidneys, while symmetrical> are bceasionally somewhat smaller dj/xkveyg- than might be expect >d. The; e i s n o t'ctence of infection or of g 1 cnerulonepririti Tilt iro.ne seu- s nonspecific with very few cells, and .1. O iA R 0017376 protein/liter- is excreted in the urine, Thebe is ru glycosuria, Rena] biotsv shoves interstitial fibrosis without ini .amr-tation. Glomeruli are rather well preserved; however there .s evidence of deceneratinc tubules and a loss in the nu her or ; ubu1es, reb .eroris ox varying degrees us we 11. Patients in this group with long-'standi xq lead exposure and renal impairment may have symptomatic saturnine gout. This can be differentiated from primary or hereditary gou t by the fact that saturnine gout follows a pre-existing rena 3. disease and has no increased pool of urate but does have a or n >- m j * 't /" > ~ creased metabolic pool of urate, and renal i ijury ro .ows ratnor than proceeds the appearance of clinical gou t. One would not judge that any of these patients reflect sen sitivs r. espouses to lead insult inasmuch as they have been wi thout ex cep-cron severely dosed over long periods. From the morphological point of view tr e ciassi 1 hall marks of lead nephropathy are severe nephros eXerosis . aovane sc: tubular degeneration, interstitial fibrosis, ana im ranuciear inclusion bodies. The bodies ate acid-fast meiusior :S which d ` i v.' L. ntain iron, They are unlike inclusion bodies seen xn viral diseases which affect the kidneys, pa V: j CUirtti u.Lic uiseas HK 0011 1r ;ideraf ions of clinical. renal disease caused by lead particularly the relatively acute episode?; in-children, suegest possible physiological interpretation^ . The cute effec of lead upon the kidney is-`manifested by race of transephithelial transport.mechanisms' for a varic t of seem ingly -unrelated solutes: glucose, amino aejids, .phosphate, . and urate. In the normal kidney -these -soliites are res orbed in the renal tubule, against a coneentraticjh gradient, by an ,,, ... !- - energy-oependent, energy-generating transport meet i.n the lead syndrome the transport of each of these j tubule resorbed solutes is modified, giving rise co the - con- .v.-,.;; syndrome. It is known from studies of intestinal epithelium and isolated perfused nephron?; that the ttarisport oi glucose- i is in some way sodium-dependent. Studies of isolate *1 j. _ .,, t *t tl J..`civ_ 1, t_;,L d-oii. systems show that the transport of some amin Hii bo &oct iur dependent. There are also reasons to believe that phosphate transport is sodium-depen-dent. All these - presumably 'l; Osdnt. J1L U dependent transport'mechanisms then are impaired by lead. Simultaneously, uric acid is moved from the rlapiilari tS:i :-> m l- L> t>;c tubular fluid. The impairment of uric acj.i.d trap:-: sort; ov I lend results in a retention of uric acid and. urieemia There .'1 1"' ."i "C, r-. *. {" (l"' O' "1 a r- -j --* 4-T.^ -_ -J y-1 -i .... n-oxt re. 1 *-o o?d vm~dopendent. The mechanism of excretion of lead is not ful HE" 0*0173*\ **, understood but at least in the chicken it involve ne nass- age of lead from the capillary CiCJL Ohs ih> :ell to the tubular lumen. Lead is also filtered so that there imay conceivably be as well a reabsorptive mechanism for L In any case, ead crosses cells and in this transient'may alter the machinery for the transport of glucose, amino acids, jjhosphat s, and urates, the mechanisms for which appear * to lie I close]. y coupled to sodium transport. I Sodium transport is the principal energy consul :er in the kidney, accounting for at least 50% and perhaps 70 < r 80% of j 'cxygen and substrate consumption. The search for i common S suggests that one examine the sodium transport and the energ' t pr od u c t i orj mechanism in the kidney. A reasonable hypothesis is that lead .i.ri transit through the epithelial' cell modifies the structural and funcrtional in- tegrity of mitochondria. We know that'mitochondria are well II I, equipped for the transport of calcium, and it is quite possible that lead, is transported in the same way. iLead within tin Tn.i tochondria might well raodify the energy-pioduci ng i which drive the solute-transport.mechanisms .for glucose, phos phate, amino acids, and urate. In view of the evidence that those transport mechanisms are coupled to stxt-.um resorption. RE 0017379 it would be most surprising to find that a idefeet -i n sodium transport is not oresent as well v/hen these' lead-induced modifications are manifesi Definitive studies to examine- this hyp ornesrs have not you been conducted. Experiments have been conducted, h owever, which shed light on several features of the functional renal impairment due to lead. When rats are fed for two weeks ondiet containing 1% lead acetate, the total urinary amino acid nitrogen increases to approximat ely 2 to 4 times no: :rual, If the renal tubular- cells had been killed, as with ca- ;!ra ium, the excretion of alpha/amino nitrogen then would oe apprxirnai:e.Ly t.u. i;i experiments the renal tubular cells are fane CXQTlJjiq but at a less than optimal level... When - one does clearance studies, the cl i aroilc ( of glycine is not increased to the extent of that for he other amino acids; however the. amount of glycine is very much in ;reased in the urine. This reflects a marked increase in th a plasma content of glycine. an. overflow kind of amino acidur fa for glycine. One wonders whether rhis glycine comes fre n an in- paired conjugation of glycine and succinate m the s nthesis of delta amino levulinic acid, a question raised by IiaaerArcs neon years acre. The clearance of tyros in- arc! cil RE 0017380 19a is increased mors than that for the other amino acicli Since lead can form bonds with the phenoxy grou.pj of tvro sine and the iraidasol'iuin grouj) of histidine, one can specul ate that :he binding of lead to these two amino aci is re lated to tiicir clcarancs, I It has been suggested that the amino-|acidura may be related to changes in mitochondria. Isolated mi totrhondria have been studied and show swelling and a defect i n oxidative phosphorylation. This defect is deraonstra ted with a pyruvate substrate but not -with succinate. These, f indings indicate that lead interferes with early stages of electron transport, 1~ r-. ^ c? rones csisg incomplete aud not blfic'ciny eieciron transport entirely. Additional studies are required to localize the effect of lead on molecules in the "complex part of the electron transport system. Mitochondria from normal kidneys havej. been isolated and i- maintained in a medium with pyruvate-pliospliate buffer, no -. i EDTA, ana various concentrations of lead, | .At a leph concen- . t; tration of 1 x 10 J molar, there is a smal ef reel with partial uncoupling. At a concentration of 2.x 10" molar there is complete paralysis of respiration indicating a tox ic effect of the Lead upon kidney mitochondria. The concent ation of R 0017381 1 o a o effecting k.i< 3ney hnown to be toxic in Studies have bee tes of lead 1 oc a I i 2 a t i on in mere is more lead in the cell tocnhoornsa ria; how ever for those rats on 1% lead acetate diets there ls a small increment of lead in the nucleus, primarily in the . infra-- nuclear inclusion bodies. It is not possib I e or ap oroprlate to compare the concentrations of lead in ml :ochondr ia from ip vitro studies to in vivo studies. Mover iheless, the con- centrarions in both systems appear to be of the sam : order of magnitude. Concentration of lead-in the in - r anno 1 ; ar inclusi body can be demonstrated by autoradiographid tochn gue follow ing exposure to labelled lead. The morphological appearance of the in :ranucxe nr inc iu- sion body in rats is the same as that observed in hUmans acutely exposed to lead.. The electron mien oscopic Appearance of the body is characteristic. There is a dense ce, rfcral core surrounded by'a periphery of fibrillar mat irial co it a in ing ` some hind of material in the matrix. The nucleolus in the nucleus is normal under these conditions indicating that re has been no inhibition of protein synthesis. It is be- the formation of the intranuclc on no;, c-_ hi to _ dependent: of the x;ucloolus.j*^.d:t'--if{m-bea K E 0 017382 19c *. 'I Y' i-r , 1i i . 4^4Ne*.u-Iea-i? in-oiU'S'ioja- body is formed by accretion ..o c reaa- . complexed protein and f'kafe--4tee>-i^dy grows by' continu 3.1 addition of this material. The intranuclear inclusion bcdA?5 o :cur-. typi- cally only in the nuclei of the proximal-tubIplar cel Ls, The changes consisting of laminar figures representing sms' Ivosomal phenomenon commonly seen in the cells of thej distfal tubles / are not specific for lead poisoning. The intranuclear inclusion body may be (isolated by the . ' classical methods, for isolating nuclear matejrials ari|d nucleoli, A<? s-H- i-y'S. ! Subsequently ultrasonifieation de-s-t-r-i-es the jnucleoli v but.. does not damage the intranuclear inclusion body or a Iter their 5_r - . _K 7 - . sis ting mostly o.C int ranuc 1 eplar bodies can. [be obtal red from sixteen rat kidneys. When the-bodies are treated wi th RNA-- i ase or DlvA-ase there is little if any defectj, indie. ting the Yy"i '> y\ 1 V *1 t-,- \ I tA Vs``'T-i;. "S j presence of RNA or DMA or their complete absence. 'I reatnent A with proteolytic enzymes such as - tripsin.dods cause a change which apparently reflects the splitting off |oi: a protein fragment. The remaining materials presumably still contain I lead and some .protein. More positive identification has. not ~ 'i been possible with the quantity of "material [available, a! There is evidence that - lead enters the unns by both glo- merulnr filtration .and bv secret ion across- 1.011 -Leix. lininq . KE" 0017383 i col If. 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 shorn to be in jboth an inorganic and. an organic form. Tire organic form is 'a complc x with a small ligand which has not been positively identified. In cases of lead poisoning, it is apparently jthe organically A complex lead which increase s m tne urine. Intranuclear inclusion bodies may represent a cor age : of excess quantities of lead that enter the nucleu s of the proximal tubule cell. Bodies are noted id rats Wh ?.n their riri rA'I no *!-o'i*- ^ ravi h- t _ip S ^^ rr*Cf ]_0 ^ C* *p3vj I'll. , 2 T cvol of exposure v.hieh does not cause anemia or other sigr > of detri-- mental effect. Thus intranuclear inclusion bodies may be among the most sensitive indicators of chajnge. It has been noted, however, that effelcts can be observed at this and lower dose levels if rats- are blaced on a lew- calcium diet, i.e., 0.1 gram percent, which is jus surncien to prevent hypocalciumio. Purina Laboratory Chow includes calcium .in excess of that required by rats;. Gn th i c- low- calcium dicts lead at a concentration of 0. B rniiiig rams per i m i. II ill eter has to date les/d to abnormal it if s ^ inc lu 'ling elevat d-AL-A and blood lead levels. KE ,0017384 It should be noted that concentrations of lee in urxnKinq water to which rats show no significant ^response are still relatively high, and that the; rat is a: relativ elv res tant species to lead intoxication.' The rat jinay' serv e., however, as a model to study the various factors vhicjh inf] nee lead l .- intoxication such as low-calcium diets or pije-.existi ng renal I disease. ' It is possible to develop an hypothesis! which r elates - solute transport failure, disturbances of oxiidative. ph os ph ory 1 a-- Lien, and lead in the intranucleai; inclusion! bodies. Central to thi >thesis is evidence for the trans-tubular ss cretioa of O \s Vo O f blood lead is attached to the red blood celji, there is 3. .Ligand-- bound diffusable form of lead which passes ajeross th e renal ' tubular cell. Mitochondria are exposed to tjhis lead , which moves against a gradient into the nucleus and into! the.int ranuclear I inclusion bodies/ probably as a lead lipoproiein com.iplex. in V>.L", J | a sense, the inclusion body represents a protective mechanism tending to withdraw lead from the renal tubular cell If one is to examine the effect of lead \: on oxidative pho phorylation, it is useful to examine experiments whi :h have j neen acne in cases of experimental (h etrabarbenn chi or .de int ox-- ica exon, Wn-en mis on for j.naied nyo.r ocaxcon s ao:;an:-Sfered to 0017385 rats and oxidative phosphorylation is -examined with succi ! i glutamate, and oetinoate substrates-, one - -observes iphat-un coupling does not occur until some 10 to li hours ~i i. administration of the carhontetrachlorj.de. i There .3 then i rapid uncoupling which exist s for some 20 flours, ait ter which time the process returns to normal. It is [easy to uncoupls oxidative phosphorylation with lead in yiti o, in which cases the mitochondria show a high increase in le|ad conte nt. When in vivo studies are conducted, however, the| mitbehc idria, wliil showing a. very high concentration of lead;ajs compar ed to all ! other subcellular fractions, show no evident of is n r~l rs -f- ; t In some way which is not understood this intact ihirv.al manages to compensate and adjust for the lejid insul-p. One night suspect that in the intact organism Idad would have better opportunity to attach to active enzyrhe site than would be the case .in in vitro experiments. In vif ro experiments, in Which the effect of the order of addition id examin d, often * show that the addition of substrate and co-factors 1 influences the acessibilifcy of binding sites on enzymes] to adirunis-cerea metals. Presumably in the living animal in which cc o co-factor, substrate, and enzyme occur mdre activ 1 cud might have a bettor chance to interfere. to be the case, however. 0017386 Additional. clinical..and. experiment-al studies relevant to tine effect of lead upon renal function might be attempted; 1. A' group of patients with lead intoxication might be put on a salt--free diet. it is conceivable that- the distal tubule and the loop of Hehle might not be able to compensate for a defect in proximal tubular sodium resorption. A simple screening technique might test and demonstrate a defect in proximal tubular resorption of sodium. If the test is negative it is not meaningful; if it is positive, however, it is highly. !. Tubular micropuncturo techniques might be used to lit: v-ii V/iitf; j.r not imal tubular resorption of sodium. 3. In vitro techniques are available for studying sodium, transport in kidney tissue slices. 4. Uric acid is presumably transported by the same syste: that transports organic anions. The kidney uses as a primary 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- animohi pourat e from solution' in vitro and tne; influence of H 0017387 22 lead added to the medium defect in thS transport of PAI-I is induced, ................................................ " ..... - ----- 5. . Such slices may also be used in a similar .study of the uptake of amino acids. 6. One might also measure ATP levels in the kidney and levels of substrate and co-factors associated with g-lycolysis and oxidative phosphorylation. 7. The examination of oxidative phosphorylation in the mitochondria of lead poisoned, animals might be considered. 8. Amino acid infusions might be examined in lead poison ing to see if an aminoaciduria insues in any predictable manner, ."V Ta7 Oil i_ Id'S rev- C_ li.!_ _ ...;.1_1_ i.>G'l `v Ins t .... _ ** _r i . r- C <Tj\~i SC j \ 0 1 0. Studies of sodi urn transport in the r* 0 lated frog kid- ney . red blood cell. and other pilysi olog ica lly active tissues are 3 pprepriate. i i. The in vivo kin etics of glu c 03 s re sorption i n the prs se nee of lead can be examined by glue ose ti tration studies. Bicarbonate titration studies can be utilized to test this sodium-linked proximal tubular function. Summary _q Espprts pn Lead and the Kematopo.ietic system. Discussions on the hematopoietic effect's of lead focused to a major extent upon disturbances of heme synthesis. As heme synthesis occurs in virtually all cells and is essential MG' 0017388 23 for normal cellular function and- structural -integrity, however -the review associated with the hematopoietic system oroadly overlapped 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 read 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 hospitalised lead workers and children, however, the majorityhave anemia. In children the effects of lead are often com bined with 'iron deficiency which cause" an anemia to be even more widely noted. The anemia is typically microcytic and hypochromic; however it is sometimes r.ormccytic and normo chromic. The reticu1ocytes are usually elevated to a minor degree, perhaps 2 to 12%. There is an increased erythyrocyte index, meaning increased erthyropoietin- activity or at least increased stimulation for red cell production. There i.s no evidence that a g GP deficiency, such as is observed in some 10% of Alterican negroes, has any effect on anemia due to loud. Similarly, alcohol dees not appear to influence the tfE 0017389 M- In studies of red ceil survival in lead workers there . may be shortening in some-, In - hospitalized patients, how--'" ever, some half of the adults do show red cell survival decreases. Studies in children show that'the shortest sur vival time is associated with the shortest and most acute lead exposure. With intense, acute exposures an acute hemo lytic 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. Ferrokinetie studies show that iron is absorbed from . I. C/1 j h* ./ t" t . i~. o.i l,,l j.d Il-L lit >J-`l L'.c S._- J. 'w* U iron into the developing red cell does not seem to be impair JErp vitro studies, however, show that high concent rat ions of load seem to interfere with the passage of iron from trans ferrin to the developing red ceil. Long chronic exposure to lead appears to reduce the turnover of iron and the clearance of iron from the plasma. This is associated with a reduced utilization of iron for new red cell production. In acute exposures in children ferrokinstic studies are typical of what is seen in hemolytic anemias. There is a rapid uptake of Iron in marrow, a rapid clearance of iron from the plasma, ana a rapid utilization of iron for red coll production. UE 0017390  * r* * Stipplc^cells Have been classically recorded in lead .. poisoning. There is no correlation known between the num ber of stipple^cells and. the intensity and type of symptoms observed. Experiments in animals suggests that stipple^ cells are preferentially sequestered in the spleen. Splenectomy or administration of lead to spleneotomiaed animals results in the increase of number' of stipple^cells. The spleen reduces the number of stipple^, cells either by maturing, then and returning them to the systemic circulation or by destroy ing then;. osmotic fragility studies show that the lead -poisoiied red ce 1.1 is brittle and inelastic, chewing- decreased osmotic fragility or T^ri increased resistance to osmotic stress. This is undoubtedly a membrane.effect,, ATPase activity is decreased in the red cell membrane, and one notes increased permeability to cations, especially loss of potassium,through 'the membrane. It is likely that these observations are related to modi fications in osmotic fragility. A positive Coombs test, is observed in some patients with lead poisoning. Cells which are Coombs-positive, are those which are young, stippled, or . _ retxcu.LGcytp^TT This indicates that some pienibrane defects have picked up serum protein. Electron microscopic studies of normoblasts .rover: 1 a number of changes characterisf i.c of lead ini oxicafci.on: ,H* 0017391 a . V V'd' .^'y'b"p r- h -;-7 i-:b.by. k ' ; b 1 *"dV ,'V ' V. ' * V.V'.V.' V*v 'a'V,\: 1. Gaps in the membrane of the normablast nucleus are increased both, .in. number. and,,.in..size. .... ....................................................-............. 2. The protein-secreting Golgi apparatus is tremendousIv dilated, . 3. The mitochondria are enlarged. The crystal poles are pushed apart. 4. Myelin bodies are observed. These are electron dense bodies between the nuclear membrane and the mitochondria. 5. The mitochondria, are distributed in perinuclear fashion to form ring sideroblasts. Cells with this mitochondrial arrangement are termed ring sideroblasts because they are siderobi asf" , i.e. because the'" contain non-heme j rr>ri; and secondly, abnormal sideroblasts because the iron is located in mitochondria. In man . deposits of ferruginous micelles. or ferritin are not normally seen in the mitochondria. One cannot distinguish morphologically ring sideroblasts observed in lead poisoning from those observed ..in thalassemia or in alcoholism. In these materials derived from lead-poisonecl animals one may distinguish with elecfcronmicroscopic technique the molecule of ferritin, ' which, is characterized by a 4, S, 16 or 3 2-dot pattern. Special studies show the reticu.locyte to have a marbedly irregular outline and to contain numerous mit ochoncl r . a heavi 1 v K# 0017392 t .i letters v?ith noiHieme iron, the ferruginous micelles. Stippiinu - ,r in animals and patients is characteristic of lead poisoning,'* ^ Wi- The cause of the stippling is not understood, but the stipules themselves are composed primarily of ribosomal material. in she polychromatophiiic cells the "polychromasia is due to the retention of ribosomal material. In lead poisoning the in corporation of into phosphat iric 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 "plrariboma, ' the presence of which may be associated wju'ci> p.s nsinaoy ~os 5 > arid nypero'aHimHgi.d!bo.lAi.`eui.s.'ct. lor some time, it has been known that protoporphyrin, . coproporphyria,- 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 herr-e biosynthetic pathway. It is clear that lead affects multiple sites at con centrations of 10 to 10~3 molar. It has been shown that the main effects are early in the synthesis - stage at the formation or u-ALA,' and later at: the point at'which iron is inserted mb:- the protoporphyrin mol ecu 3. e by -heraesynthetase. These KE* 00173-93 processes take place within the mitochondria. More recently it has been shown that ALA dehydrase is inhibited as well. Ir is important to indicate that practically every living cell contains beiae, and chat these disturbances of heme forma tion are relevant not only to blood production but equally important to cytochrome formation as well. The kidney contain large amounts of ALA synthetase as does the liver. .Drugs which induce AL?* synthetase in the liver can produce Inonaous concentrations of this enzyme there, and the kidney as wall. Acute intermittent porphyria (A'lP) is in some respects 3 able model for the study of certain aspects of acute lead ixLuxicat3*.on.. This inborn error ,.of metabol ism tends to occur /V f' in families, most commonly in women of the 20 - ,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 Signs in clude tychardia, hypertension, decreased reflexes, and sensory abnormalities. D-ALA synthetase, d-ALA.- and PEG are increased in the blood. ALA -and PBG are elevated in the urine as well, asymptomatic relatives of persons with acute intermittent porphyria may show only the biochemical alteration disease, viz, , increased inary ALA and p e g . *e 0017394 i Other porphyrins which way be found in the urine on laborat cry examination are net excreted but rather are formed in the voided urine as degradation products.. Isolated and purified ALA and PBG show no evidence of pharraacolegicai 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 demyelin>i-2ation. The presence of macrophages containing lipid-staining material around the nerve gives evidence that these changes occur Emte-wortera. denvoii 7" ` n has hoar, rotor V iii- appropriate 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, andaxons. The pattern is strihingly 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 there is an association hot'-veer, the Ke 0017395 30 hepatic biochemical alterations ana the morphologic'a 1 train changes.'. Lead appears to-act directly on the train and nervou and hemaiopoetic systems. Thus, it would appear that All* and acute load 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 teen conducted on lead levels and heme pre cursors in children. When lead is moderately elevated in children into the 40-60 /jg/100g range one notes a relatively poor correlation between the blood lead level and Mil, corpor- pherin, and other heme precursors. This contrasts somewhat. r.n* hVt P j.h'b'at'i r~- v -? ve j'- H `u **" 2 ^ ^ Tv\`On''*^ ' v"_, p't Ti" ' ' > concentrations of these materials are almost invariably found at the 60yug/lOOg range. In studies of groups of normal children and those with mental retardation reflecting a variet 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 a l a dekydrase and intelligence. It has been found, however; that there is a significant correlation between blood .lead levels in the 40 /ag/100g range and AL7i dehydrsiss in the blood. The C1 i n g ^ 7^1 Ip 1 rfjh j_ 1" XC* P r; C F* O*'* C' Q~='Y \ '* "j_ C'T' ft 71 rv-f- C ci C i p"1i Ae-' HZ 0017396 There is experimental evidence -for a correlation between ALA dehydrase levels in liver, brain, and blood. Such evidenc is derived from studies on suckling rats who have received lead only through the milk of their mothers, who had been placed on a 5% lead acetate, diet after the litter had been delivered. Nearopatholcgical changes were observed in the suckling mice under these circunst&nces,, The clinical impli cations of this work are obviously not fully defined inasmuch as interrelations between lead, depressions of blood and brain ALA dehydrase, and anatomical changes of the central nervous svstein are not well understood'. i, hydrasc 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 enxym in the brain. {The presence of ALA dehydrase ;i n human brain may be assumed since positive enzyme activity was found in the brains; of two aborted fetuses 'which were at the 15th -and . i iu t d- *- .1. c> orxc.Ln.fion, exists between the blood lead Jove], in oLh'b sen ' KE: 0017397 and the ALA dehydrase activity in the brain. The potential significance of these-observations suggests that it is -ap propriate 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 docs were fed for 42 weeks on diets containing 0, 100, and 500 ppm dAfTlJilbi- 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 pphlbfhrbifeb;bon lead. F,'cv- . v t i . i. 1.[ e a - rioV .1.0 < " a a i - no- a i-c r o < n v i "t .1 > , . 5 t * i , ,r. i r,;a , -- was the same. No anitaals appeared to have been adversely affected by the ingestion of lead over this period. Although these animals did not appear to have suffered a detrimental physiological effect, there was the question of whether or not the dogs would respond differently to physiological stress. In these studies . the animals -were- .stressed through a reduction in blood volume of each dog to 50% of its original value through phlebotomy. The recuperation from this insult ap peared to be identical for the three groups of dogs,and the carves reflecting the blood paraxnaters could be essentially superimposed, one upon tl . other. The bleeding proceduro did not seam to influence' the blood load level cf the animals which returned to the prephlebotomy level some one week' aftor the loss, of blood. ' in the study of hemoglobin synthesis a convenient bio logical model is available in cultures of the photcsyntnetic microorganism, Rhodopseudoaor,as spheroldes. This organism grows very well, either as a facultative anaerobe, without oxygen, in. which ease it. produces baeteriochlorophyli, or in the presence of oxygen.,it will grow in the dark and manu facture primarily heme. Lead suppresses the growth of the organism very markedly when the culture is relatively low in iron, uhen supplies of .iron are sufficient the inhibit or'r affect 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 che organism is grown at -low concentrations of iron very little coproporphyrin is excreted. ' 'The"'addition of 0.03 ppm inane anew: to the culture medium, however, causes the excretion of largo ito-.ir.ts of coproporphyrin. In iron--supplemented cultures much cxgr.er quantities of manganese are tolerated before the- cxeesr. ''-'-proporphyrin appears. Lead potentiator the effect of man~: ve-'-e in causing increased .coproporpnyr xn excretion ana is ofv-fepon. If than still more iron is acicied :to the ireaius one overcomes the effect of manganese entirely and the effect of lead to a somewhat lesser extent, No amount of iron supplementation can completely -the reverse coproporphyria .production caused by lead. An examination of the other metabolites in the. tetrapyrrole synthetic pathway suggest that the en.cyme coproporphyrinogen oxidase or "corproginase" may be iron-dependent in that its activities are influenced by the balance between iron and other antagonistic metals, FerroCiieIats.se and ALA dehydrase may also be. affected by manganese and lead, but in the whole avrvira orcfsmsm me cerumen nase sir :a (=t t is r.o be the triesr important, _______ _on Lead and the Nervous System. Clinical man!testations 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~ t ensity of exposure.' The actual lead burden, hov/ever, must also include an unmeasured quantity of lead stored in the central and peripheral nervous systems as veil a?; in the skel eton and soft tissues. The persist i.na effects of encephaiconi:h such as mental retardation, epilepsy, d. behav-i ora] disorders may be the result of anatomical darnac ai.i.ng asm 0017400 acute intoxication or possibly .the result of chronic cellu lar damage resulting-from the slow release of stored lead in combination with continued exogenous exposure. Experience* observers have repeatedly emphasized the importance of con tinued environmental exposure to lead as an important factor v/hich increases the likelihood of vare permanent damage to the brain. It is reasonable to consider as wall the possibility that continued exposure may be caused by the slow release of stored amounts of lead which in themselves do not produce obvious symptoms but. nevertheless; may be capable of damaging certain . J. w V- .J. _L U* J. The concept of latent effects due to lead in persons who do not show obvious clinical plumbisra has been raised by Lane and otheres who believe that lead burdens insufficient to caus symptoms or to produce disability, have shortened life expect an ci.es because of premature development of 'nephritis or in some instances cerebral hemorrhage. The neurological manifestations of lead poisoning are variable and widespread. Lead workers may complain of lassitu irritability, depression, constipation, aiid abdominal diseomfo These symptoms are the same as those sometimes expressed by umortonnlly door eased individuals. KE 0017401 3 ob peripheral neuropathy is considered a common neurologic anifestation of lead poisoning, and involvement of the radial erve is frequently described. While the clinical picture may resent as a mononeuropafhy, neurological examination will fter*. demonstrate weakness in other muscles, and neuroelectrical tudies may show prolonged conduction times or decreases in he amplitudes of the action K 0017402 >V 36 potential. In some cases, spastic paraplegia and hyrerreflexia suggest upper arc ana lower' neuron damage. In such cases the differential diagnosis between lead nauromyelopathy and spinal cord degeneration such as in primary lateral sclerosis and. amyotrophic lateral sclerosis may be difficult. Lead encephOiopathy 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. Gpthalmo.1 ogica 1 disturbances are not common g_n systemic lead poisoning 7ilthoucfh p isad pigment at. ion of the retina has been desorrbed in adults. Ocular symptoms of lead poisoning have been more common in (.,_r 1 children in whom it is possible to recognize disturbances of the visual cortex, suprageniculate pathways, optic nerve, retina, lens, and intra- and extra-ocular muscles. 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 mere common. It is likely,however, that examples of peripheral neuropathy in children go : unrecognized in many eases, including those in whom .encephalopathy is dramatic and command;; clinical attention. Children with clinical ' " k'E 0017403 -v \ 37 evidence 'Off peripheral neuropathy. vi2,-senso.ry loas-and-- VJeakness ,sbow evidence, of denervation when tested electrically, M'Surachemic a1 studies of the brain in lead poisoning have been technically difficult and have most commoniv focused noon me errectti -f- f* o 4- ro-\ f alkyl lead compounds, the pharmacology for which is undoubtedly dissimilar to that for inorganic load, Although seizures encountered in acute lead cncephalo-" pathy may be due to neur ono~ excit ation related to local-edema or vascular lesions, these manifestations may be due to an interference with glutamic ac.id transport or metabolism re sulting in a reduction of ^-aminobutyric -acid (GABA) production sent moderator, and interference with .-GABA production might produce seizures by increasing neuronal excitability. Meta bolic steps which might be .susceptible to- the effects of lead include (1) glutamic acid decarboxylase (GAD) which carboxylate glut: a mate, to GABA; (2) glutamic acid dehydrogenase, (GHD) which reduces 2-keto .glutar-ate to glutamic acid; and (3). pyridcxal phosphate metabolized, from pyridoxine "which functions as the co-enzyme for transamination react.ions in the synthesis of GABA. Evaluation cf those systems and of the clinical efficacy of pyridoxine or glutamate in children -with seizures of acute lead poisoning might be wap ranted. RE 0017404 38 Recent electron microscopic, studies in,experimental lead neuropathy nave 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, have been demonsrrated microscopically. Two -mechanisms have been suggested -in explanation of the process of demyelination. and remyelination in lead nephropathy. One possibi lity is that a substance having porphobilinogen as a precursor is essential, for the maintenance of myelin. It is. known that in porphyria there is a metabolic block, which results, in the excretion of excess porphobilinogen and d-ALA ana concurrently a dev.ye.:,mar ; ,-,g jPVV*\v> t the .Specific. e'etu^e; vi wiiid: Lx rot known. The second possibility is that lead causes vasodHatton ana altered vascular permeability Which cause intraenaonearal edema and consequent damage to the Schwann cells by pressure or ischemia. Biochemical studies of lead nephropathy have concentrated on the motor endplate region of striated muscles, the point where the greatest proportion of synaptic acetylcholinesterase is localized. The introduction of lead into suitable neuro muscular preparations results in the precipitation of lead at the esteratie site of this enzyme,. The prime binding rite for load appears to be the post-synoptic sub-neural apparatus of the motor endplate. The b puling of the sub-nsural apparatus kE 0017405 . V. .f v : ' -PIWi-, t"- * ;:S 39 by.lead Is not influenced by the prior^admini&tra-ion diisopropyl fluorophosphate (DFp) or neostigmine. Hcv/ever, ttype of' treatment completely inhibits' cholinesterase a-ctivi Cholinesterase activity does appear to. be reduced in muscle in which the subneure.l apparatus has been bound by lead. s studies are part of an attempt to identify.some properties of the cholinergic receptor and the characteristics of load 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 vhfp'5 -r. a 5-T p>rS ?; <3 rIpprr-'O g r\y5 f n][l rv\v/-t r>0 v*'*7,0 cr rrn > 1 0 4`^ ^ ^ unship between the site of calcium releas o t \ the site of binding of divalent metal ions such as lead may explain the predilection for palsy in the more active if.us cl Lead and calcium may .also be interrelated through a moohunt which is reflected in the fact that a reduction in calcium concentration may produce neuromuscular blockade by decrees lug the presvnaptic release of acetylcholine. K 0017406 VI. Conference Summary - - ' This'conference reaffirmed a long known fact that' . poisonings due to moonshine liquor containing lead, pica for lead-containing material, and excessive occupational exposure to lead continue to occur. This fact represents a failxxre on the part c 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 lead manifestations of absorption. Such studies are obviously important to our understanding- .of;,the mechanisms and conse quences of lead intoxication, Gf even- greater importance, however, would be the development of sensitive and specific measures which could establish whether or not "normal" 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 studios is currently under exam.ina.tion. Ziminoacidur1a. Abnormal quantities of amino acids in urine occur only in people with overt lead -poisoning. Barely elevated levels of amino acids can be detected in urine of KE 0017407 41 workers with excess lead exposure, Aminqacid&ria,. therefore,, does not appear to be a -highly sensitive clinical study pro cedure for "nonttal" populations, - although- it ..is ^.certainly important for. research in cases of .lead intoxication. IncInsion Bodies. Nuclear inclusion bodies in kidney cell's- appear..tq. be relatively;spec!fic:lbut. 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 c:Duplexed with, mitochondria. intranuclear.inclusion- bodies should he. looked for in. cadaver studies , particularly in those v?ith hxgh body burdens of load,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. . ' Glomerular Filtration Rate. It was' suggested that the glomerular ...filtration .rate .might be a -useful screening measure.. However this measure'-was questioned as lacking .specif-ity for lead exposure relative' to renal disease in general. '`Sodium Pump" Inhibition, This phenomenon presents as a. hypothetical lead effect and requires research for verification Brochemlcal Functions. Two potentially sensitive indicators are available;- the measurement of the respiration of rniro- chondrxa of reticulocytes and ..the .measurement or.potassi .U'.'l 0017408 42 - loss across the red. blood,cel-1. membrane. - Bath, of these ' phenom'ena-xequi.'re--evaluation in 'animals-"and 'humans-: with,-lead------ -intQxication. :. Enzyme Changes. ' Enzymes which'.are considered to be most sensitive- to-;lead ."exposure ;are ' delta ! amino ievulir.ic acid' ' dehydr,ase:, . lipoamide dehydrogenase- and ATP-ase. changes- -.in enzymes 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 .b.e' competitive with ... ; these metals. However there 'are...no known example's of such i.nii.'.bit3.oi` .oy lead in in vivo systems. Cadaver scueies iiave shovm- that essential -trace: metals in-.soft-tissues are '-normal' despite.increased concentrations, of lead. - Herne., Synthcsis,, copropoyphyri'n and delta amino"leviilinic acid in urine are.sensitive and reasonably specific indicators of. lead exposure, .. These meas.urero.ents arc. 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 flow rather than to urine voliur.-, telm amino levulinic acid determinations in serum are supa-ruLed as ..a more refined measure of lead effect ..upon heme synthesis.. - HE 0017409 f' 1: (j. - V _ ., ' 43 The- behavior.. of-.ALA-dehydrase. under lead exposure situations requires exaraination ana '.interpretation from the point of.view " of health.- ...... . - .....nerve- Conduction- Time. -One of- the more practical-and refined 'tests : for Tneurplogxcal change, is the" measurement of : ; nerve -conduction time.- -However-; this precedure is considered: t-o be-more useful- fur following cases of lead intoxication than, for screening purposes. Analytical Variability.' ' For almost -every study, -. quest! about analytical-accuracy-and precision can be raised It i suggested that.' comparisons ~of data'.would' be facilitat'ed by col 1 aboration..through, a. central laboratory, ... This t.e.o` would avoid much controversy and 'expedite interpretat ms o.f: data. This conference reviewed the state of- current knew3.edge-, 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 lea.d to, improved measures of early response are aroat eot. ."It. 'has been reassuring-'to note that, .exhaustive *\ studies have not led to the. discovery of new lesi ons r'-lxnli -:: . ft-. q- ; lead, although it is also-clear that there- are major oif fe rences between species in response to' load absorption, Kf 0017410 v. x t 'M 44 ...... Several.heeds- for the immediate future-.-arerhighlighted* , 1, Prevention- of excessive 'and unusual exposures to lead-*- a broad problem for society-at-large 2. 'defined-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 v;ith_'respect to.fchanges in- renal, fundtioru-and,.heme synthesis. - 4. -Research .with. animals on .correlations between erizybe changes and adverse effects of lead. ... 5. Surveillance of'the environment and .populations fox- ` i.* i Cl X v rClO liC t? or iflci.il ' S load, .... ' 1i*ICJ ciiicl - C'UH CO* . 6. Surveillance for potentially.susceptible individuals and indications ' of synergistic . actions .or. .aggravation of exist ing disease. ; - - ........... . ._ - . * . 0017411