Document 8Ry0B9YL2YmKpm6n72Q9dnpRy

M tb'fyf* 4 o f fruits. ,v : ? ^ R>ec) b h o ,h1 olog 1 c a 1 :2 f.is-1< of L e a d at L e v L evels ;ile there re current; examples of db virus le ad p o is on-- i-ip in i m properly s upervised Indus trial ope ations and in p o d i a l .ric practice- w h e r e pica for old pair;t is ohse ;:vc-;d, the I:^i L' . significance of lead c o n p u r d c in ani: lent u rtjan at neophe pc? lit.s not b e e n c l e a r l y defined. Th lead c one en.fruti in V;Oil industrial tmospheras: are v e r y much lower th a n the . curve;/!' induct rial TLV (several ,,uc/m3 vs .lid and evi- ChiVCO pf p l u m b i c m is c o mpletely l a c k i n g , lir/erfhcl ~s it is "Ofj'IO:.' Able to b e l i e v e that at some point s:tcr t o f .oty ior s read p. leaning in the usual nens-a', b o d y abo ptiOTl of lead H-Y. .-../ cf a usayiiiLudw sufficient tu cause d.!.sturbane 03 Of n o m a l p h y s i o l o g i c a l machanj sj.ic. T hat this point 1 not yet known- s related to that fact that traditior si bloc? ie.'ttical and cli.nica 1. approaches to the detection 'of lead effect; at low levels have been relatively insensitive and incapstk e of detect ing minimal alterations. The question of the existence and ;in niflounce of ipiniiial alterations, bov;haver, Is conerax to disc Uosrcus of tne health significance of one concen.tr tion of alxyj. lead compounds jn motc>t fuels OJ cnr-socprently in ao-bierv:. atmospheres. 2n cxn: Ctc.t.. a.no RE" 001 7361 2a Tills report contains a summary of discu s si on held in-.February, 1970, w h i c h attempted to: 1) id en tify bi ocheraical -. . and c l i nical approaches to. the d t e c t i o n .of. lead, e fiects. at lv? .levels; 2) est a b l i s h the h e a l t h .sighifi nance of w h a tever . lad-related p h e n omena might be observed at these levels; and 3} suggest p o s s i b l e areas in w h i c h r e s earch ernphas i s might :be. most fruitful in solving the p r e s e n t e d p rob em.3. Pa rticipants ' .included p h y s i c i a n s with varying, .types of.e xperd enc e -with - clinical l.ead toxicology, re s p e c t e d sc lent ie ts in s sveral . branches of clinical m e d i c i n e and bio c h e m i s try, and 'investi~ " gators w i t h p r i m a r y interests and bachgrourip s- in l ad' metabolism; h.r'.i exper j-iaeiiL&.!t oxi e ol og y Several par Li O.Lpcilf S , A.!_CiiOUCji"! eminent in t h e i r .respe c t i v e fields, h a d no rdaj or inter est in. .lead,. T h e y were invited b e c a u s e of -their sp e ciaiized c U n r e a l shills and.k n o w l e d g e relevant to organ syst ms. k n o w n to be affectera-by lead. -- Th e material' pre s e n t e d in this report r -fleet s the work of the participants', their colleagues, and others i: i the re spec-- tive ^field's of i n V e S t i g a t i o n r ""'"'-''7" - .' . . -Par t i c i p a t ing Scie n tists lbs. I. Barry, MID. ' The Associated'Octet . Ellesmere Port - " Cheshire-, England " Company, Ltd. . Neal'S. B r i b k e r M, D. W a s h i n g t o n Uni ye s i t_y st,' Louis,. Hisd ouri H E 00173 62 - 2b - ,( J. Julian Chisolm, Jr., M.D> John- Hopkins U n i v e r s i t y Baltimore, Maryland . ; Joseph It, Moyer, Pb.D . university of Cincinna ci .C in c i n n t i', Ohio Jerome F. Cole, Sc .D. , International L e a d -Zinc Res ear ch- Organi rat ion. .' " ' . . . Now. Ybrk, N. Y. ' Carl V. Moore, M. D. W a s h i n g t on- u n i v e r s itv St. L o u i s ,. Missouri. Kim Cramer, M.D. -. Universitetet I Goteborg : Goteborg SV, Sweden .. Jean M, M o r g a n U M.D. Univers ity of Alabama Birmingham, Alabama . Robert E. E c k a r d t , M.D, Erail'A. Pfitzer, Sc.D osso Research & Engineering Company university of Cincinn ti L i n d e n , N e w . erse Cincinnati, Ohio, Robert Feldman, M.D. . Boston University Boston, Massachusetts George Roush,. Jr. , M. T u lane Univers ity New Orleans, Louisian A b r a h a m Goldberg,- M . D f D . S c . , -F.R-.C.B. Ge o r g e E. ScTireini r, M.D.. university of Glasgow G e o r g e t o w n Tjr.1 /Vi'.\S1.tV Giasgow; w . 1 , Scotland W a s h ing t o n , D , C, Harold Goiy, M.D. .- Araerlean Petroleum Institute N e w York, S-]. Y . .. ' G . J . St o p p s , .B. , B S. ...... E . I . du P o n t .de '.Nemo'Jirs & Comp a n y Wilmington, Delaware "Robert A.--Gyer, M.D. " ` ' University of North Carolina! -Chapel Hill,''North Carolina L l o y d B. Teppi fr, M.D. U n i v e r s i t y of incinn;at i Cincinnati, -Ohio - Robert c . G r i g g s ,.M.D. : Western Reserve University Cleveland, Ohio '' - ' " * ' ' D a v i d .D . U l m e r , M.D. Harvard University. Bast on -, Mo ssachusetts Robert j. m . Horton, M.D. - Bert L. Va l l e e M-.D , National Air P o l l u t i o n .C o n t r o l A d m i n i s t r a t ion . H a r v a r d Un; v e r s t v Durham, North Carolina., Boston, Massac :husetts F o l i a c N, Jensen, M.D. George Washington University Washington, D. C. '' ' - ?. w?V-*S Kl= 0017363 .Il Summ a r y of Repo r t s on E n v i r o n m e ntal and Hurnn Lea d Levels T h e concent rat i on of lead in soils, fcjods, Wat ex', .and air is highly variable. Alimentary lead intake coders a range of a p p r o x i m a t e l y 0.12 to 0.35 mg/day.-: ,T h e absorpti on of lead from the atmosphere varies 6 to 10-fold. In spite of this w i d e range of lead intake levels, the amouit : Of lea d in man 0017364 3 is: r e l a t i v e l y constant, totalling about 'IGOrf^OO mg, .of w h i c h at least 90% is stored in the bone. \" B lood lead levels are e ven more constan]t, r e m a r k a b l y so in view of the wide range of exposures. When the'mean blood lead levels f r o m 'v a r i o u s .p o p u l a t i o n s are e x a m i n e d ,'ther seems to b e an increase in the b lood level a s one -goes from a rural to an urbanized area. This phenomenon no related to the dens i t y of motor vehicles in these respect ive communities, and the u r b a n -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 impres-s i v e , .he o i o o a xeaa. .lever or ru nans, tor example, approximates that for rural Nor th Americans Soft tissue lead levels and b o n e lead eve Is dp not c o r relate well in individuals. The lead in t h bona seems to increase w ith age whereas the soft tissue-life nsi of .ead does not rise after the end of the second decade In fe nales the soft tissue lead levels tend to be slightly lower than in males, w h i l e in' b o n e the lead c o n c e n t r a t i o n in females .1. very much lower than in males. This lower Jtone lev of lead, h owev er, also rises wit h age as in m.a3.e s . c hila r e n show v e r y lo w levels in soft- tissue and bone, find n o d iffercn 0017365 A the sexes is d e m o n s t r a b l e , wh ether the higher bone levels .in older adults is due t o a c c u m ulation w i t h a-ie or simply- due to the fact that these peo p l e w e r e living arc a. time w h e n there was more lead, in the total eavironme at is notknown. Lea d c o n c e n t r a t i o n in nones varies wp tii the bone sslec th lead content being higher in the more dense bone s and highest in the petrous temple. Bone lead levels h a v e b e e n d e t e r m i n e d .i i the sk o.1ep on s of.40 Indians w h o lived in A r i z o n a during th period /00 to m a i1450 ekS. The specimens w e r e from original 7 ites and did not include museum material. Soil samples from within thfc b o d y cavity and adjacent to the buried se Lc-cons lads lead than the bone; therefore, the q u e s t i o n of sont ami.n- at ion wa s excluded. A ssay showed 6-8 p p m in the b or.e ash ot the rib. Sacrificial skulls from Mexico, probably Srom the tine of the Conquistadors, c o n t a i n e d lead levels ir m o rar.ae of 2-4 p p m of the ash. C o p r o l i t e s (muiraoifio fecal material) found with these skeletons gives evidence of the diet on w hich the p o p u l a t i o n lived. T h e food supply. mPiV* i' 't\~FifSh',,pma l 1. o'Jrains- grasse-s,- and t he ..t'otai i cal lee ii c en c entration was very low. .' At some point in history, environmental fer ax 0013366 the-Civil W a r i n this country, -contaiivTDead- in conce ttrat ions w h i c h ap p r o x i m a t e those noted in m o d e r n autojisy mte trials, viz 40 to 50 ppm. - In v i e w of the v e r y great range of lead] intake between t1 individuals.jand v a r i a b i l i t y in lead absorpti);on , the ionsistency of blood lead levels is extremely impressive; One w onders if there is a biological basis for this consist) ncv of blood lead levels, and whether in fact the biological basi s of the consistency reflects a biological necessity or lead. We-have n o evidence for this, bu t we h a v e no evidencie that it is not- the case. It is generally true, h o w e v e r , that when a metal snivtr-l T.n.r. L U C I V tf --L V . U /-\v\ (i > < *" n i r*V, /v.m T-a/'-v-* *r , rns merer r > * .-a r- r> c.'-- * AS" JT* ent merely on an incidental basis but rather as a s batanee .| with specific biological importance. One cannot hel p but re collect the early studies of so-called zinc jroxioitv , in which zinc was r e g a r d e d solely as. a toxic metal r^sponsibl e for . metal fume fever. Zinc was a typical trace hiatal -p esent in amounts difficult to detect and in a ssociation -with only traces if i nformation about its significance. W h e n in 194] , it be- came apparent that zinc was- an essential constituent of the met:alloenzyme carbonic anhydrase it b e c a m e apparent T h a t zinc w a s . n e i t h e r incidental or solely .toxic but iln fact 1i.ad a s p e cific b i o l o g i c a l funcfieri. That a similar a ntna t i o n may apply 0017367 o in the case of lead has never been critically or systematical! examined. Xn rnaking. inferences fram b 1 ood 1ead 1ei/el dete[rmination., . ' I. it is important to r e m e m b e r that the analytical tecljuiicfues for lead assays at low levels do .not yield ikpressivfely c o n sistent results. in many situations the variation between repl i c a t i o n s is grea t e r tha n the v a r i a t i o n b e t w e e n i.ndividuals l_ in a sampled population. In addition the precision of analyst; b y a given laboratory may vary trom day to ahy, II. Summary of R eports o n L e a d and S u b - c ellular U h y s i o l T here are a number of metals jthe b i o l o g i c a l furiction of Ui'lKliO'.Vil. C*1*'j1ir*i I1 metals in tissues and b i o l o g i c a l 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 s e l e n i u m and c h r o m i u m are e s sential trace medals or simplv in- c i d ental metals i n c o r porated into tissues. bead is m a similar category. W e do not k n o w w h e t h e r of; not lead has an essential biological function. Where metals have been known to interact with Essential b i o l o g i c a l systems., they ei t h e r interact w ith prote. .ns ox* I with nucleic acids or with, integrated system^ such as o r g a n e l l e s , KE 0017368 p r o b a b l y w i t h the m e m b r a n e s . A m o n g the proteins, tnose 'chat: hav e b e e n e x a m i n e d e x t e n s i v e l y are the enzymes. Eh zyiaes often contain a metal which is bound in such a specific r armer that it cannot be remo v e d without loss of activity. Thi s p h e n o m e n o n is not related to activation or inhibition of an en zyme but rather to the intrinsic activity of the enzyme. A large num- h e r of me t a l s are b o u n d t o nucleic acids.. |The func tion of . 'l these metal-containing nucleic acids is presently nt ireiy unknown, T h e r e l a t i o n s h i p of metals t o organelle function is also not understood. The sites to which metals can attach in biolog rear mate rials are not infinite. Metals can attach t o the enc *i \o'n amino acid group of lysine, to the carboxyl groups or gruramic V and aspartic acid, to the suifhydryl groups of cyst ene, Rin other less c o mmon ways such as with the phelnoxy gre up of tyro- sine. in the case . of metals . such as lead, ' bonds 11 are stable, W I, ' immobile, and polyd^ntate.. Lead, can combin e w i t h any ox- all of these groups,and it is misleading to believe the t lead associates only with sulfhydryi..groups to form mere aptides . W e d o k n o w that lead ca n be toxic under certai n c ix-cunv- stances, but so can any metal. Essential metals ac proteins.. When these metals are present in sneers and attached o ligands w h i c h aire .not the normal .active letfiI-bi ppr"iJV"jf f? f-./rx RE" 0017369' s ol: these metalloproteins, inhibition o c c u r s ,1' i.e. i , the raeta] is toxic, Whether a lead mefcalioprotein exists and nas an essential b o d / function is not.known. Worlc w i t h c a r b o x y p e p t i d a s e illustrates several imporrant points. C a r b o x y p e p t i d a s e is a zinc-containiirig enzyrr.e w h i c h has t w o functions: first as a peptidase, secondly aft an esterase It is p o s s i b l e to remove the normal zinc constituent of the enzyme and replace it wi t h various other metals. Wh en the zinc is remo v e d and r e p laced w i t h lead, one finds th at the m e t a 11oenzyme has lost is peptidase properties, but the esteri i; properties are increased; in fact, the lead ciarboxyp eptidas is a b e t t e r esterase than the nat u r a l l y occuprinq zi nc a n a l o g . Th e poinc neie is tliat a lead m e t a l i o e n z y m e dan have fectly legitimate biological purpose. ; pa:, W o r k w i t h this enzyme is instructive in a sec on sense: j.c lias b e e n round that the metal of c arb oxypcajptIdas a is att a c h e d to three ligatas: two i m i d a z o i i u m groups cf (histidi ns cine one c a r b o x y group. .Sulphur-containing amino s.cids are not in- volved. Tills o bservation is c o n trary t o many! of the exp ectaticns which h a v e been expressed in the literature, i `I 7\nocher metalloprocein, metallothionein, ; was di!scoverei first in the horse, then in other species and|certs* ly in m a n . K 0017370 .. .. Met-allothi one-in is a e a d m i m u - c ontaining- p r o t e i n w it h a molecular Weight- of about -6000 without the meta] 6800 1 n the metal. Th e p r o t e i n is c o m posed of sme 52 ahlino acid groups, o n e -third of w h i c h .are cystine, Accordina to tra.dr' - iI t i c n a l 't h i n k i n g , this h i g h c o n c e n t r a t i o n jof s u l ft vclryi groups should y i e l d a m a t e r i a l with, great avidit ad. This is not the case in vivo, however, and lead is not ffielentiy strongly b o u n d to me t a-i1oth i one in to displace cad mium or z i n c , The b a l a n c e b e t w e e n metals is essent ial in a .1 considera tions of metal biochemistry and toxicity, Metal interact and may b e antagonistic, metal A protecting' f com tue exrece ox ii'C ' - a i . j j , o * i . a i 1. . . >if'1 J". -\- `erj example is that of. m o l y b d e n u m i n t o x i c a t i o n w h i c h is observed in Somerset in England and in the western part of the United States, The manifestations of this disea may b enhanced b y the p r e s e n c e of excess copper. It is cjuite lively that lead, iron, and p erh a p s other metals Interact in vj-ays which are relevant t o this discus s i o n s bu t are n ot well u n d e r s t o o d . T h e competition, b e t w e e n metals f o r .ligands! depends upon numer- ous factors, most of which are p oorly u n d e r s t c o d . Included. among these factors are the amount and character o ho j_ c: n a the p e n e t r a t i o n thro u g h m e m b r a n e s b y m e t a l s ,.-the .'stability on s tant .ror tne several m e t a l - l x c a u d .cono ieri b i n a the ep a c xal arrangement between HE 001-7371 ana s eric laccor tali? ;ind ligands, It is relevant to examine systems 'which might p o s s i b l y . i' be disturbed, by lead and hence represent target systems tor lead effect, One such .system is the pyru vate - c a r b o x y l a s e system vherebv pyruvic acid is introduced into the Krebs cycle b y acetyl co-enzyme A. T h i s sy s t e m is % conglomerate of sulf- hydry 1-ccntairsing enzymes- and co-enzymes, including thiaminepy rophosphate folic acid, and cc-enzyme A cnd is rentrai in the energy metabolism of the brain, we know, for example, tha t h i amine deficiency, as in W e r n i c k e 's synch orne or Korcavkof f 1s psychosis /leads to g"ross f u n c t i o n a l abnormc iliti.es. A similar syndrome has b e e n observed, in -foxes fed on f-rsh en rails -which m J(~ 1-g -I *. v n i r i . o - n r:0 <tv"0r?\f o r'hb r thiamine- is destroyed, absent, or interfered v/ith b y m etal attachments to relevant sulfliydryl groups would be expected to y i e l d the same c l i nical syndrome. That 'the eenijral nsrvou | s y s t e m manifes t a t i o n s of lead po i s o n i n g may be rela tea to lire feronce v;ith p".y'ruvic acid m e t a b o l i s m m*ay.'wejll b e pc s s i b l e . T h e p r o b l e m of m e r c u r y intoxication, as in the chrc m e mercu- r i a l i s m of the M a d Flatter, the q u e s t i o n .of arsenic :oxicity and the therapeutic effect of BAL, a sulfhydry1-c on t a m i g material, are all relevant to the question of metal bind!ng to s u Ifhydry1 groups c the pyruvate-carboxylase sy stem and the disturbance of this system bv-extraneous metals 0017372 Th e p e r i p h e r a l ner vous m a n i f e s t a t i o n s -of lead poi s o n i n g r ` I; are not r e l a t e d to these phenomena, but it |is entir eiv possiDle that lead may attach to sulfhydryl groups o|f the n =ur oc onduc 1 1on ` I system peripherally, 'i I A n o t h e r s y s t e m w here inves t i g a t i o n m a y yield i 'for mar x on on the b i o c h e m i s t r y of lead is that related to oxidtstive phos.i phorylation. This process depends upon thejintegr.il mitochondrial membranes, which among other things, preserve the r e s p e c t i v e levels of c a l c i u m and p o t a s s i u m insi de and outside the m i t o c h r o n d r i a , Impairment of tdjiis meirib] ane b y carbon t e t r a c h l o r i d e is a s sociated w i t h a dramatic Shift of i ' v . 1 s ' - i n 7 . `i 4 y , .""i i " n 'i 'V ! ih p , r 1 i i r i , * t i! i ; > 4 ' r u i . and phosphorylation. The sulfhydryl ligandi of the mitochon- | d r i a l m e m brane suggest that the attachment cj>f lead or other. metals to the m e m b r a n e ma y interfere w i t h itjs intrinsic met a Ii holism and its ability to protect the intra-jmitochondr iai environment. U n d e r such c i r c u m s t a n c e s - t h e rrjstals might be ' - ... I e x p e c t e d to cause significant damage to the energy rr.etabo3.isra . 'i svstem, * T h e r e are u n d o u b t e d l y ways in w h i c h thel effect o f .lead on nucleic acids mav b e measured. W e 'know that the binding of lead to nucleic acids causes their hydrolysis. Research in this area is cur r e n t l y less w ell devel oyse .2, h owe v *15.-. Sum m a rv- of Rep or t s on L e?xd and 'the K,id ney -- T h e r e is no doubt that lead in a b n o r m a l l y h i g h aracunr may cause acute or chronic renal injury, in children acute lead i n t o x ication may b e associated w i t h a irenal le $3.on typical of the panconi syndrome. In cases pf intox icat i o n without encephalopathy one nay see either akino aci furia or g l y c o s u r i a or both. In more severe cases, such as vith en- c e p h a l o p a t h y ,the triad of the Faneoni syndrome may ':;c seen-, amino aciduria, r i c k e t s , and k y p e r p k o s u r i a in the i.essence of extreme liypophosphdternia in the range of 2 fag % (no. mal = 3.5 to 4,5 m g '%), Fructose and glucose are excreted in the presen r- ........ . O '! -, - 1 ,i' Vi--'/~ -- -j--j-. ,, -- , -i i ... ; ; , sugars varies. Some c h i l d r e n excrete c l o sejto a gi am of cj.tra i in a day although the b l o o d citrate .level ib normal During the acute episode seme' of these; child.ro hav e shown the inappropriate secretion of ADH. This ohs ervatjon cannot b e cittributed to treatment s i n c e 'it has b e en observed b e f o r e ti trnent is initiat th EDTA has K - '/ lsn i .> 1-k i * > r . to canee a" trans erma ph os-- a , and a tr ansie: d u r i a, r e a trnent, many o ndrciit are treeted. The me ituria is gone Within a week; the Is c5;";rs vi th in KET 0017374 -.T - Arorntography). Bons changes resolve within ? to 8 months. E D T A m o b i l i s a t i o n tests in which. P T H wa-s* used as we to mobilize lead stored in the bene have been attempted, In sene ca ses sufficient; PTH ha s b e e n used to raise th Q s erum c a l c i u m level to more than 13 mg %. Althoujgh the aiaount ox ' 'r . i c a l c i u m in the urine i n c r e a s e d 3- t o 4-fold, there w a a n o in- crease in the u r i n a r y e x c r e t i o n of lead above that w h i c h wj associated with EDTA alone. W i t h respect to chronic renal injury there is, of course, the A u s t r a l i a n m a t e r i a l w h i c h shows quite ejonvincin jlv that under certain conditions of exposure lead ihtcxicat ion can i ,*> " r - i * i- / - \ e t V *y .X. C_ v~v C - 'w V - i i J u - v s - v- v V~- 4 * -i c it' t .U U ,1 . k.> c d i- n o t f jn O, '.4 V -t . } . w . J t vs .J.. .1 * *. * f I/*-. *--i -> v s x.J .> '4 U l A V . . I have failed to demonstrate, however, an equivalent Phenomenon in the United States, Th e E D T A m o b i l i s t iofc tests .n the; Balri- more g r o u p w e r e normal and -it was not p o s s i b l e to d m o n s u r a u e chronic renal injury. Epidemiological factors must tlC C OUil'C for the dissimilarity between Australian and American exper ience. In the U n ited States the patients have chewc d paint at some time b e t w e e n their first and third jj-ear o n e . In Australia the children are in the 6-to IG-y ar age ranee and absorb lead through the ingestion of raindrops which contain lead from decomposed paint on veranda rails and r o o f t o p s . Phis may represent a less `intense exposure I ire a mo? prolonged K 0017375 14 one. M a n y of the A u s t r a l i a n - p a t i e n t s 'have a jpes- cav s deformit and tophaceous gout A nu m b e r of cases of lead p o i s o n i n g in adults t ive been observed in Alabama in relation to the prolonged exea ssive con sumpt i o n of .illegally-prepared whiskey This di'seas- ce curs " as well in Georgia, the earolinas, and indeed m ay d o . expected anywhere that w h i s k e y is illegally p r e pared i n soi.oei ed vats and distilled, through old automobile radiators 'Til ,tii- e is evi- deuce that over 50 m i l l i o n gallons of illegal tie on oil are d i s t i l l e d i n 'this c o u n t r y every y ear and that over 50% of the illegally-prepared alcohol contains more than Si U C n o r U : i S ii!= ' .\r-.1" -ii -| m g .o lead/ ucr and even as high as 80 mg of lead per liter. In Alabama experience the typical patient is a n =gro male i b e t w e e n the ages of 45 and 60. Perhaps some 25 % of h aspital admissions in this group have been exposed to alcohol. T h o s e w h o h a v e e x p e r i e n c e d severe and prol o n g a expos ur s of iO y iars or m o r e are t y p i c a l l y found t o b e ant raic, t o h s.v e a s t a b l e renal insufficiency, and to s h o w a nor na i pye jlpgr a m , a l t h o u g h the kidneys, while s y m m e t r i c a l > are occasion 1J.V somewhat smaller dtidaaeyg- than might b e expect id. Tile re .is no evidence of infection or of glomeralonephriti inert is nonspecific wi t h very few cells, and Tne irj.ne seu.1. ofIi't HE" 0017376 p;cotein/liter is e x c r e t e d in the urine, T h e b e is n o glycosuria, Rena] b i o p s y shows interstitial fibrosis without inf .ammation. Glomeruli are rather well preserved; however, there s evidence of degenerating tubules and a loss in the number of :ubuies Host of the .patients have naphr.csc1er os s of varying aeqrcei as w e l l . Patients in this group with long-standihg lead exposa and renal impairment may have symptomatic saturnine gout. This can b e d i f f e r e n t i a t e d from p r i m a r y or h e r e d i t a r y goult b y the fact that saturnine gout follows a p r e - e x i s t i n g rena 3. aisease and has no increased pool of urate but does have a T.-, v u j > \ e.!. o r n >" m ' i > v-vr'x-. creased metabolic pool of urate, and renal ijury ro liows r a m e r than proceeds the appearance of clinical gou t. One would not esponsijudge that any of these patients reflect sen S i t i V S r to lead insult inasmuch as they ha v e b e e n w thout er ce p e ion severely dosed over long periods. - From the morphological point of view t e exassi 1 hall marks o f lead n e p h r o p a t h y are severe nephros c l e r c s is . aovancec: tubular degeneration, interstitial fibrosis, ina iru.ran u c l e a r inclusion bodies. The bodies are acid-fast i U C U U S l ! ns which d not contain iron, T h e y are unlike inclusion seen xn viral diseases which affect the Kidneys, vu cui ari maqaiic disease. ". , C o n s i d e r a t i o n s of c l i n i c a l .renal disej&se cans a by l e a d , i' particul-ar 3.y the r e l a t i v e l y acute episodes;! in-chxl Cren, suggest p o s s i b l e p h y s i o l o g i c a l interpretation^,. The aeute effe< of lead u p o n the k i d n e y is-'manifested b y a| disturb ance or t r a n s e p h i t h e l i a l transport.mechanisms' fox d. varxsr.v od ingly u n r e l a t e d solutes: glucose, a m i n o a d i d s p h o s p h a t e ,. and urate. In the no r m a l k i d n e y these solutes arein the r enal tubule, against a c o n e e n t r a 1 1 c}n g r a d e energy-dependent, energy-generating transp the lead syndrome the transport of each of these p tubule r esorbed solutes is modified, giving .1. CJ. _ . syndrome. It is k n o w n from studies of intestinal e pithelium " | " 1ancl isolated p e r f u s e d nephrons that the ttaisport of glucose is in some w a y s o d i u m - d e p e n d e n t . Studies ofj isolatetQ j etc "corexcii. systems sho w that the transpc of some arainjo ac id C-nil b &ac iurr ons t o b e l i e v L that P cospiriate transport is s o d i u m - d e p e n d e n t . A l l t h e s e p r e s u m a b l y thLt _LU1 do-pendent t r a nsport 'm e c h a n i s m s then are impaired b y |ead. , .I Simultaneously, -uric acid is m o v e d f rom the ciapiilari tie tubular fluid. The impairment of uric ac id trans sort: uy lead results in a retention of uric acid and jurieemia arc reasons for b e l i e v i n g that uri c - a c i d rani s vor-t ma There odi viTi-dooendent. T h e m e c h a n i s m of excretior of h S'5 is not fid u n d e r s t o o d but at least in the chicken it involves tjhe pas aqe of lead from the c a p i l l a r y across the" cell' 'to the tubular lumen. L ead is also filtered so that there may c o n c e i v a b l y b e as v?eil' a r e a b sorptive m e c h a n i s m for lead. In arty case, lead crosses cells and in this transient''ma\j alter the m a chinery . ior the transport of glucose, amino acids, jjfoosphat^s, and urates, the mechanisms for which appear to be closely coupled to sodium transport. Sod i u m transport is the p r i n c i p a l en e r q v consu: er m tne kidney, accounting at least 50% and perhaps 70 or 8 0 % of cxygen and substrate consumption. The search for common derouiinator for these vari o u s Lranspor L abuorwrid .1.L.:ios suygosta :hat one examine the so d i u m transport and the enex-gy? p r eemption mechanism in the kidney. A r e asonable h y p o t h e s i s is that lead .in transit through the epithelial cell modifies the structural and functional in tegr i t y of mitochondria. W e k n o w t h a t 'mito c h o n d r i a are well j e q u i p p e d for the transport of calcium, and :j.t is qu: that lead, is tran sporte d in the B3. Lfymi t o chondria migh t well raodd the v;hi ch drive the s oiuta- Irani?.por - it T.phai:e, amino ac id s , and "U cl!:e. In the ae transpo rt n echani `rm e coir lain thi mechanx sins ose, phoS" ;nce that, orption, -. K 00173 79 XO SU2?p r X s i.no* to f present as wall n sodium dueed h<-* modj.fications are manifest. 'v 'j I Definitive studies to examine this hypothesis have not been conducted. Experiments have been conducted, h owever, which shed light on several features of the functional renal impairment due to lead. whe n rats are fed for two iceks on a diet containing 1% lead acetate, the total urinary jauno acid n i t r o g e n increases to a p p r o x i m a t e l y 2 to 4 -times no rma.L. If the renal tubular cells h a d b e e n hilled, as with ca^-hiium, the excretion of aiph?/amino n i t r o g e n then w ould oe appr xixnate.ly -ii'.* \ _I uiO O I 1 0 r r i u 2 2. U ? ;G C u .ij 0 1 2 0 . 0 0 -b 02? *3 2 . c.<. e xperiments the renal tubular cells are func cj.onxnq but at less than optimal level, W h e n one does cl e a r a n c e s t u d i e s , the cl sarancs of glycine is net i n creased to the extent of that for the. other a m m o acids; h o w e v e r the amount of glyc i n e is very much .increased in the urine. Tills reflects a m a r k e d increa :e in cn e p l a s m a content of glycine, an. o v e r f l o w h i n d of amin 2) filO2O.Lij!ia. for glycine. One wonders v/hether rhis glycine comes fro:ti an xn~ p a i r e d c o n j u g a t i o n of glyc i n e and succinate m the s acnes is of delta a mino levulini c acid, a q u e stion raised b y IiaaerPror o o n v o cir ,o cHO'c, `\1ii-e clCh'it'cu'jCi? oi t'o'os trie snei ci'xot id ine K 0017380 19a is increased more than that for the other amino ac ids. Since lead can form bonds with the p h e n o x y group| of tvro sine and the imidazolxum group of histidine, one can specul ate that the binding of lead to these two amino aci is re lated to xf c learance, - It has been suggested that the amino- aciduria may b e related to changes in mitochondria. Isola ted mitebhondria have been studied and show swelling and defect in oxidative phosphorylation, This defect is dem o n s t r a ied with a p y r uvate substrate but not with succinate. These findings indicate that lead interferes with early stages of electron transport, m i s m t e r r o r o n c e being incomplete awl noli block.in g elec Iron transport entirely. A d ditional studies are requir ?d to localize the effect of lead on m o lecules in the "c ompiex o n e ' part or the electron transport system. M i t o c h o n d r i a from normal k i d n e y s havej b e e n is elated and main t a i n e d in a m e d i u m w i t h pyruvate-phospiiate b u f fer, no EDTA, an various concentrations of lead, At a lead cone en- .. ' . ...r? " ''' tration of 1 x 10 ~ molar, there is a small effect with partia uncoupling. At a c o n c e n t r a t i o n of 2.x 10~D molar h e r e is c o m plete paralysis of re s p i r a t i o n indicating a tox .c effect or the lead upon kidney mitochondria. The concent 'ation of ' H 0017381 leaa eff e c t i n g h i She; snewn to be toxic in Studies nave bo tes of lead 1 oc a Ii 2at i on in :iere is more lead in the -cel .tccnond: cxa; hcw- ever for those rats on 1% lead acetate diets there Is a small increment of lead in the nucleus, primarily in the Intra- nuclear inclusion bodies. It is not p o s s i b l e or ar h r o prlate :o compare the c o n centrations of lead in raifochondr ia rrom in v i t r o studies to in v i v o studies. Nevertheless, -lid- con- centrerions in b o t h systems appear to be of the sait ; croer or magnitude. Concentration of lead-in the intranucl ar inclus! b o d y can b e ' d e m o n s t r a t e d b y a utoradiographid tochni pue follow ing exposure to labelled lead. T h e m o r p h o l o g i c a l appearance of the intranucle ir inclu- sion b o d y in rats is the same as that observed in h Umans - acutely exposed to lead.. The e l e c t r o n mic; appearance of the body is characteristic. There is a dense ce itral core surrounded b y ' a p e r i p h e r y of fibrillar material co ita in ing ` some hind of material in the matrix. The nucleolus in the nucleus is normal under these conditions indicating that there; h a s b e e n n o i n h i b i t i o n of p r o t e i n synthesis, It is be- ieved that the f o rmation of the intranucIOio; j.ncrusi is ir.dependent o f the x;ucloolus.^~w.d:f'--d;G-daoIievrrl"'-'c-:iT: 19c -ittsAwei&'cwi- body is formed b y acc r e t i o n ..of lead- __ , comp]. exec p: grows by continual addition of th 1 s iicXtn ;ur-. typi~- cal ly only : j. The chang es con; oms lyosomal phenomenon i are n ot spei tubles / Th e in- class ical n\' i|d nucleoli, Suudb se q uent l1*y u l t r a s o n i f i c a t i o n de-s-t-r-i-es the nucleoli v b u t .. does not daa:mage the intranuclear inclusion -body or a Iter their t..tit.J"*"X*'i">-!-"1ology. .. 4 - -H T V - r cp _l W . O . J I < i . i . t~: ti sisti ng inos' ned from sixteen rat k i d n e y s . . W h e n the-bodies are treated wi th RNA'- - " i ase or BNA-ase there is little if any defect!, indie ting the Vv> y \ 1 v_>, t-- \ /. ' Y `/ s s ' <i v C : ! p r e sence of RNA or D M A or their compl e t e absence, 'Ireatment n " !' w i t h proteolyti c enzymes such as -t r i p s i n .dods cause a change which apparently reflects the splitting off |oi: a p r o t e i n fragment. The remaining materials presumably still contain '. '' '' j i lead and some protein. More positive identification has not Yfi.ai.'i. been possible with the quantity of "material javaiiabl e . - a! ' T h e r e is evidence that -lead enters the urine b y b o t h glo- ionitir lining . KX. 0017383 cell/. It is h y p o t h e s i z e d that lead t r a n s p o r t e d ac :oss the tubular lining ,cell is in t h e ..form of a diffusable liqandcomplex and that mito ehondria are expos ed to this complex. Lead in the urine has been shown i 0 be in both an inorganic and. an orga nic form. Th e organic form is 'a compie X wich a sma.ll ligand w h i c h has not b e e n p o s i t i v e l y identif l e d . In cases of lead poisoning, it is a p p a r e n t l y the orga nically A. complex lead which increases m tne urine. i I Intranuclear inclusion bodies may represent tor age ': of excess quantities of lead that enter the nucleu s of the . prxima], tubule cell. Bodies are noted inj rats w h e.n their n 'l 1! hd *r r ? p f * ^5 *j_v*i ^ 0 ^ /I m fr q 1 _ O l 3 t i * p 3 v `j I ' l l . , , 1ove 1 exposure which does not cause anemia or other sign : of detri-- mental effect. Thus intranuclear inclusion bodies may be among the most sensitive indicators of chajnge. It h a s b e e n oted, hov;ever, that effects can be observed . at this and lower dose levels if rats- re pl a c e d on a 1 cw-- c a l c i u m diet, i. e. , 0.1 g r a m percent, whicjh is jus t sufficxen to prevent hypocalc-iumio. Purina Laboratory chow includes c a l c i u m in excess of that required b y ratsj. Gn th is low- . ' c a l c i u m diet lead at a c o n e e n t r a t i o n of 0 .& millig rams p e r mi 11 i 1et er has to date les/id to abnormalities /inoli; ling elevat d-LA and blood lead levels. K E .0017384 It should be n o t e d that c o n centrations of lead .n arxnK- ing water to which rats show no significant ^response are still r e l a t i v e l y high, and that the; rat is a relativ elv r e s i s tant species to lead intoxication.' The rat jmay serv e, however, as a m o d e l to study the vari o u s factors vhicjh in flu* nee lead intoxication such as low-calcium diets or pxje-existl ng renal disease. .... ! ; It is p o s sible to develop an hypothesis! w hich xJelates solute, t r a n s p o r t failure, d i s t u r b a n c e s of oxjidative. p h o k p i n o r y 1 a TM Lien, and lead in the intranucleaj; inclusion! bodies, Central to this h y p o t h e s i s is evidence for the t r a n s-tubular ss l ^ i r" .i *-at xs-s. _il _ rx - u r M e' * ! .- I x ip v -' / `o t . ' u . b l o o d lead is a t t a c h e d to the red b l o o d celjl, there j . S 3. .L2-C.-1 rj, ! 1Q bound diffusable form of lead which passes abross th e renal ` tubular cell. Mitochondria are exposed to tbis lead which move against a gradient into the nucleus and into! t h e .int anuclear inclusion bodies, probably as a lead lipoprolein complex. In V>.L'\ \ a sense, the inclusion body represents a protective mechanism tending to v/itharaw lead from the renal tubular cell, If one is to exam i n e the effect of l e a d [on oxidative phos . i. phorylation, it is useful to examine experiments wh ich have ' , ' -j b een done in cases of e x p e r imental (hetrapa.i:bone chloride intox ication, W h e n this chi or inatod hydrocarbon, is admin rentered to E 0017385 rats and oxidative p h o s p h o r y l t ion is .examined vitri suecirx ' !' i glutamate, and act in pate substrates-, one -observes i::hat- un- i c o u p l i n g does not occur until some 10 to 'li hours er ^ 'I _ i. a d m i n i s t r a t i o n of the carbontetrachlorj.de. i T h e r e .1s then . 'i rapid u n c o u p l i n g w h i c h exists for some 20 flours, a r time the process returns to normal. It is easy to uno o u p 1s oxidative p h o s p h o r y l a t i o n wit h lead in. vitr"'j-jo, in vihl;ich cases I the m i t o c h o n d r i a show a h i g h increase in lelad content. whe n in v i v o studies are conducted, however, the mitbchc ilCl'/f d.Ci , T;V.ri Tl J- O ! showing a v e r y h i g h concent inti on of lead ^ajs compel so. to all other subcellular fractions, snow n o evidence o f 'in pairment of 'j /*%*' r i ^ 7o. p V c y l *-j -5~ a ! is n ot uneierstood ' / animal m d adjust ror m e . he int act organism b e t t e r o pportunity to attach to active enzyrhe si than would b e the case in- in v i t r o experiments. irg v i t r o e x p e r i m e n t s , in w h i c h the effect of the order of addit i o n id examine d,. often show that the addition of substrate and co-factors influences the accessibility of b i nding sites on enzymesj to administered metals. P r e s u m a b l y in the livinq animal in which cerribination: " " I of co-factor, substrate, and enzyme occur more actively, lead might h a v e a b e t t e r chance t o interfere. This .ape-jars not to to be the case, however. A d d i t i o n a l .clinical..and experintent-ai studies relevant - to tine effect of lead u p o n r enal function might b e attempted; 1. A' g r o u p of p a t ients with lead intoxieati.cn might be put on a salt-free diet. It is c o n c e ivable that- the distal tubule and the loop of Henle might not be able to compensate for a defect in proximal tubular sodium resorption. A simple screening technique r h t test and demo.nstrate a defect in p r o ximal tubular rest reion of sodium. If th.p test is xnegative, it is not meaningful; . it is posit acre , h o w e v e r , it is highly significant. . u n c t u r c techniqu es might .be used to imal tubular resorption of sodium. . 3. in v i t r o t echniques are available for studying sodium- transport in kidney tissue slices. 4. Uric acid is p r e s u m a b l y t r a n s p o r t e d b y the same system 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- arainohippurate from solution in vitro and the influence of u 22 l ead added t o the m e d i u m defect in th"!1ran sport of PAI-I is induced, ............... ..... ` ' .. -- 5. .Such slices m a y also b e use d in a similar study of the uptake of amino acids, . . 6. One might also measure A T P levels in the k i d n e y and levels of substrate and co-factors associated with g-lycolysis and oxidative phosphorylation. 7. The e x a m i n a t i o n of oxidative p h o s p h o r y l a t i o n in the mitochondria of lead poisoned, animals might be considered. 8. A m i n o acid infusions might b e e x a mined in lead p o i s o n ing to see if an aminoaciduria insues in any predictable manner. W o J-K. Oi l V i i*z: j l tr X i cl .L l a 1 1>y>O j T I ' O -L i . ... T. t } i"u .* *It i*v 1 0 . St udies of s o d i u m t.ransport in the relat ed fr og kid- ney . red bl ood cell, and oth er pi-lysiolog ically ac tive tissues are appr opr iete ,, 1 1 . Th e in vivo kinetic s of glucose resoroti on 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. J. Su m m a ry _af Espprts jan L e a d ar.d the Kematopo.ietic s ystem. G etrec of heme h e m e s y renesxs occ X. J, cell 23 for n ormal cel l u l a r fu net ion dA1*.1. s t n ' y - k J - p -, -{-* ' r \ 7 'the r e v i e w associated With rh e h emat < ove clapped w i t h other area.s , par ticu: -! y , -o n e r v o u s system. It has b e e n 'known for ye ar s the in the mature red cell in v-ivo, in y. by read cells are expo sed to lead inn_ v i t r o and returned to the living organism. Aneiri.3. due to in children and in adu I t s . nT .i. 1ead < ica nt a n e m i a is rather rai- r per haps h 0 : 3 pitalired lead verb ers and ch ildr ei i a . V anemia. In c h i l d ren l :jl c ? ef fect >rten ccrm- b ined wit h iron defici ency w n.ich c avi f. more widely noted, T he anemia is typically microcytic and h y p o c h r o m i c ; how ever it is sometimes norrnccyiio and n o r m o 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 s t i m u l a t i o n for red cell production. There i.s no evidence that a g GP deficiency, such as is observed in some 10% of American negroes, has any effect on anemia due to loud. Similarly, alcohol dees not appear to influence the r.-c - j. KE 0017389 24 ..... In studies of red ceil survival in lead workers there . may be shortening in- some-. In -h o s p i t a l i z e d patients, how--'" ever, some half of the adults do show red cell survival decreases. Studies in c h i l d r e n show t h a t *the shortest s u r v ival time, is associated with the shortest and most acute lead exposure. With intense, acute exposures an acute hemo lytic anemia ma y be seen,. Some y ears ag o p a t ients w ith carcinoma, who were treated with lead because of a presumed anti-neoplastic effect of lead, were observed to have acute hemolytic anemias. F erro k in o.t ic studies show "C1clt ir on is absorbe' l , "5 . r- ii.iu.I 1'Jd'J'J.liO*." normally. "1 ; r * -iJ . V. - J - Cx u iron into the dev-eloping red cel 1 d os:s not seeni to b In vitro studles, however, show that high concentrt load seem to interfere with the passage of iron from trans ferrin. to the d e v e l o p i n g red cell. Long chronic exposure to lead appeal's to reduce the turnover o f 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 who.t is seen in h e m olytic anemias. T h e r e is a rapid 'uptake of iron in narrow, a rapid clearance of iron from the plasma, and a r apid ut i l i s a t i o n of iron for red coll pre-duel loi'.. UE..0017390 i StipPle^cells 11ave b e n c l a s s i c a l l y recorded in lead .. poisoning.. T h e r e is n o co r r e l a t i o n k n o w n b e t w e e n the nura- . her of stipple^ cells and.the intensity and type of symptoms observed. Experiments in animals suggests that stipple^ceil; are preferentially sequestered in the spleen. Splenectomy or a d m i n i s t r a t i o n of lead .to splenec t o m i a e d 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 them. osmotic fragility studies snow that the lead-poisoned rp>ri j ct t"C JLr_02 clC't.i.Cf .i - i i-.u i e r e fragility or c^n increased r e sistance to osmotic stress. This --t. is u n d o u b t e d l y a m e m b r a n e .e f f e c t .* A T P a s e activity is decreased in the red cell membrane, and one notes increased permeability to cations, e s p e c i a l l y loss of p o t a s s i u m ,thro u g h 'the m e m b r a n e . It is likely that these observations are' related to modi fications in osmotic fragility. A p o s i t i v e Coombs test, is observed in some patients with lead poisoning. Ceils bitch are C combs-positive, are those .which are young, stippled, or .. re txcu.LccytpoTT T h u s indicates that some m e n b r a n e defects have picked up serum protein. E l e c t r o n micros c o p i c studies of normab 1r.s-ty reverb, a nu m b e r of changes character 1 s t i.c of -loud inf oxicati.cn: I. Gaps in the m e m b r a n e of the noriaablast nucleus are increased both, .in .n u m b e r .and^.in.size. .... ... ... ........... .. - ..... 2. The p r o t e i n - s e c r e t i n g Golgi apparatus is t r e m endously dilated, . 3. The m i t o c h o n d r i a are enlarged. The crystal poles are pushed apart. . 4. M y e l i n bo d i e s are observed. T hese are e l e c t r o n dense bodies between the nuclear membrane and the mitochondria. 5. The mitochondria, are di s t r i b u t e d in p erinuclear fashion to form ring sideroblasts. Cells with this mitochondrial arrangement are termed ring sideroblasts because they are siderrihl a s t " . i.e. `henenuo the'" c o n t a i n non-heme j rmi . and secondly, abnormal, sideroblasts because the iron is located in mitochondria. In man . deposits of ferruginous m i c e l l e s . 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-poisoned animals one may distinguish with elecfcronmicroscopic technique the m o l e c u l e of f e r r i t i n , 'w h i c h is chara c t e r i z e d b y a 4, S, 16 or 3 2-dot pattern. , Special studies show the retlcu.locvte to have a marbe&ly irregular outline and to cont a i n numerous m i t o c h o n d r i a he.avi 1v K # 0017392 / Uvten wit h non-heme iron, the ferruginous micelles. Stippiim- - ' ,p-ywiin animals and patients is c h a r a c t e r i s t i c of lead p o i s o n i n g ,k*'* * The cause of the stippling is not understood, but the stipples' themselves are c o m p o s e d p r imarily of ribosomal material. in she p o l y c h r o m a t o p h i l i c cells the `p o l y c h r o m a s i a is due to the retention of ribosomai material. In lead poisoning the in corporation of 32P into phosphatiiiic acid in mature red blood cells is decreased. . .. - : W h e n lead is administered sub cuta n e o u s l y over a period of time in sufficient concentrations, a subcutaneous granuloma forms, a "piumboma, ' the p r e s e n c e of w h i c h m a y be associated V - . n . i , p ' f. S " i ' S S Sp i * i Ji p } `a- iOJ l~' ;'-.':Cl`j l O S' 'S'-1 J.. ` : Cl . . for some time, it has b e e n k n o w n that protoporphyrin, coproporphyria,- and d - A L A 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 biosynthet ic pathway. It is c l ear that lead affects multiple sites at cen- ~f a '` . . . centrations of 10 t o 10 molar. It has b e e n shown that the main effects are early in the s y n t h e s i s .stage at the formation of d~ALA,' and later at: the point at'whi ch iron is inserted into the protoporphyrin molecule by -hemesynthetase. These Kf 0017393 processes take place within the mitochondria. More recently it has b e e n shown that A L A deh y d r a s e is inhibited as well. It is important to indicate that practically every living cell contains heme, and that these disturbances of heme forma tion are relevant not only to blood production but equally important to cytochrome formation as well. The k i d n e y contain A L A synthetase as does the liver. -Drugs synt h e t a s e in the liver W produc e sihormo' i' : thl s enzyme there, and the kidney as well A c u t e inte rmittent porp h v r la (AIP) j.S .1 v crrwr./r,i r o C*pe cts 3 able model for the study of cer tain asp ecr s of a:CUte 'jG3 C .ban.x j-v-a c3.Oil, This inb1yrn errer ..of met a b o I J.s m .x. ds <--) occur f in faim! lie s , moiSt COTOTUCinly in v;o m e n of the 20 - ,,30 -y cir- old age group. Pre senting symptoms inc lude acut e 1:lSX pi ai.r`3d abdo minal pain, vomitin.g, const Xpd Cio n , and neur sy ch iatrie man i.festat ions w h i c h en.compao s a br oad range ph en en a from motor weakness to psychiatric disturbances. Signs in clude tvehardia, hypertension, decreased reflexes, and sensory abnormalities. D-ALA synthetase, d~ALA, and PBG are increased in the blood. ALA -and PBG are elevated in the urine as well. Asyuptomatic relatives of persons with acute intermittent porphyria may show only the biochemical alteration of 11 1I S disease, viz, , increased X'rinary ALA and p u g . ne 0017394 Other porphyrins which may be found in the urine on , laborat cry examination are net excreted but rather are formed in the' v o i d e d u rine as d e g r a d a t i o n products., . Isolated and purified ALA and P3G show no evidence of . phar m a c o l e g i c a x activity; t hey are not p r e s s o r substances, ~ - The clinical phenomena associated with AIP can be explained on the b a s i s of a d i s o r d e r of the nervous system. Indeed it has been shown that the peripheral nerves of patients with . this disease can undergo derayellnyhation. The presence of macrophages containing lipid-staining material around the nerve gives evidence that these changes occur ante~-mortera. 1 Lilcj*), n. -i.. j. v* v--x i t ' d -- . _i_ . _ c - P. . . udu iM .>LC: ii f . i l u i III V i-.-,.-',,- . /f J-Ir- approprlate secretion of ADH has been noted in the presence of changes in the thalamic region. W i t h lead a similar symptom pattern is observed: ab dominal pain, vomiting, constipation, and peripheral nerve d i s t u rbances p r i m a r i l y of m otor f u n c t i o n / Anatomically, it has seen shown that lead, ha s an effect on myelin sheaths, Schwann cells, anterior h o r n cells, a n d a x o n s . T h e p a t t e r n 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 m a s o n to beli e v e that there is an a s s o c iation rwtvcon the Kf 0017395 30 hepatic l>iochemical alterations and the morphologtea 1 braii c h a n g e s L e a d appears to-set directly on the brain and m r ana hematopoeti.c systems. Thus, it vnvn la evopear tlm1v M F a acut e load poisoning have different basic causations but sh a f ivial common p a t h w a y of c j.xnic'.i. 'm a n i f e s t a t i o n . Xn tlie search for more : examin at icss h a v e b e e n condui cur sor S Xn chiIdre:n. W h e n L c h i 1dr en into the 40-60 /ag/l poor c orr elation b-etween the PiiOX XI"), C.nd other h e m e preen: T,71*-hj'V A-iits c**?*r*1A*tT*? concen ZTV.lions of these mate: at the 60 yug/lOOg r a n g e , In c h i l d r e n and those w i t h merit. ( of eti OxCj gies, a d ifference : gr oups ha s not bee n observed, - Similarly, there has bee no d i f f e r e n c e in b l o o d lead levels nor in the lack of corre tion'between' blood lead and intelligence, or b e t w e e n ALA' dehydrase and intelligence. It has b e e n found, h o w e v e r , th there is a significant co r r e l a t i o n b e t w e e n b]..ood lead level in the 40 /ag/100g range and AL7i d e h y d r a s e in the blood. Tit c i inicx* ipi.Q'j'jj_v,,jLc ^ncc Oir Jclix.x coxx'xl d '*xo~* i_s c m t 1i r^ldC' X 0017396 T h e r e is experimental, -evidence for a co r r e l a t i o n betw e e n ALA dehydrase levels in liver, brain, and blood. Such evidence is deri v e d from studies on su ckling rats w h o h a v e received lead only through the milk of their m o t h e r s , 'who had b e e n placed on a 5% lead acetate, diet after the litter had been delivered, Neur opathological changes were observed in the suckling nice under these cirounsfarces, The clinical impli- ' cations of this v/ork are obviously not fully defi n e d inasmuch as interrelations b e t w e e n lead, d epressions of b l o o d and b r a i n ALA dehydrase, and anatomical changes of the central nervous svs rein are not well understood', r-\ o r hydraso activity is reduced in b o t h the b l o o d and b r a i n of lead poisoned r a t s / (2) a significant c orr elation exists b e tween blood and brain ALA dehydrase .activity in normal, and lend p o i s o n e d rats, and (3) a correlation exists 'between b l o o d lead and A L A d e h ydrase levels in children. It is p o s s i b l e that d i m i n u t i o n in ALA deh y d r a s e activity in t h e 'peri p h e r a l blood of children may reflect a similar reduction of the enzya in the brain. (The p r e s e n c e of ALA d e h y d r a s e ;in h u m a n b r a i n may b e assumed since p o s i t i v e enzyme a c t i v i t y was found ire the brains' of two aborted fetuses -which were at the 15th a nd i; .i. 7 : X ! . - ' k i / j . V v. kX 1 3 :ori elation. exists b e t w e e n the b l o o d lead .!evel /> y g //.r: and the ALA dehydrase activity in the brain. The potential s i g n i ficance -of these - o b s e r v a t i o n s suggests tha.tt it is -aapp- p r o p r i a t e to c o n sider s e riously the p o s sible :ect on the d e v e l o p i n g b r a i n of lead levels p r e v i o u s l y regairrddeed as normmaall. Pr e l i m i n a r y studies of lead p o i s o n e d dogs have be een c o n- du c t e d to shed light on the p h y s i o l o g i c a l signi.ffiiccarniccee ojf . roeasurements which show a depression of ALA dehydrase. In such studies dogs were fed for 42 weeks on diets containing 0, 100, and 500 p p m of added lead. At the end of the 42-v/eek pe r i o d the b l o o d ALA deh y d r a s e level was v i r t u a l l y zero in the group of dogs fed 500 lead. Eow- . v.- 1 . 1b e nc ri.ciV .o s a c a *,-u w .:,,.cc- o i <*,->,i.... ,:5 <.i( ../1,t.a .s '.. as the same. N o an'itaals a p p e a r e d to h a v e b e e n adv e r s e l y 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 t h r o u g h a reduction in blood volume of each dog to 50% of its original value thro u g h phlebotomy. The re cu p e r a t i o n from, this insult ap~ peared to b e identical for the three groups o.f cicgsiand the carves reflecting the blood paramaters could be essentially super i m p o s e d , one upon th . ocher. The bleeding procedure did n o t .ssera t o influenc' the b l o o d load level of. the animals w hich returned to th e p r e p h i e b o t c m y level some one weak' nicer the loss, of blood.' In the study of hemoglobin synthesis a convenient bio logical model is available in cultures of the photosynthetic mi cr oorg&rsism, Rh od op seu d croon a s s p h r o d e s . This organism grows very well, either as a facultative anaerobe, without oxygen, in. w hi ch case it. produces b a c t e r i o c h i o r o p h y l l , or in the presence of oxygen^ it v;i3.1 g r o w in the dark and m anu facture primarily hems, Lead suppresses the growth of the organism very m a r k e d l y when the cult u r e is r e l a t i v e l y low ^ d 01^ * \ co c' '-j ' aV-, ; -i p. v? -P *1 ---ra -pji'-J -i*.` -v-T--,r\ - t-i'-. - - effect of lead on the g r o w t h of the o r g a n i s m is much less apparent. The exc r e t i o n level of coproporphyrin b y this- organism clearly- demonstrates the antagonism -between iron on the one hand and lead, manganese,..and cobalt on-the other, t h e n the organism is grown at -lo*w co n c e n t r a t i o n s of iron v e r y little cop-rop orphyr in is excreted. ^ T h e a d d i t i o n of 0.03 ppm manganc to the culture medium, however, causes the excretion of large amo-.ii'.ts of coprop orphyr in. In iron--supplemented cultures rmic uxer.ar quantities of man g a n e s e are tolerated before the- cxeos "cprop orphyr in appears. L e a d potent i a t e s the effect of :u a -' o in causing increase;; c opr op orphyr x 11 e x cretion a n a is thus antagonistic to the addition of v-fepon. If than still more iron is. ded ;tO 4t-i1-l,e medium one overcomes the ef f ec t ryf cinnS'0 tiresi y and t he ef f ec t of l eaa to a somewhat lesser extent, No amount of iron supplementat ion can completely -the reverse c o p r o p o r p h y r i a .production caused b y lead, .. An examination of the other metabolites in the tetrapyrrole a y"i3the t ic p atlv OK idase or "co: c)Ct ivit ies are n ci Qo-thh e r antag ooniiisti c metals. Ferrocheiat3.se and A L A deh y d r a s e mo y a.1s o be . 3.i O'**"owing erg on t eons to he the most imp o r t a:ot , Pu mrnary of Rep Cl iinicaall m]a n i f e s t a t i o n s caused b y e x c essive amount % sy 0 1.nni c le ad ! af fecte d or aai Le vels of 1ead i --sns it y of exp al so in clud e a: C t*ilcj_cJL and pe: K 0 ..L ct on c:id so ft O-Uoh as men tal ms-v b e t h e r e s u l t o f a n a t o m i c a l darnao e. 0017400 d behavioral axsoniors urn m g au r i n g m e \ acute intoxication or possibly the result of chronic cellu lar damage resulting 'from the s l o w release of stored lead in corahinatiora w i t h c o n tinued exogenous exposure. E x p e r i e n c e d observers have repeatedly emphasized the importance of con tinued environmental exposure ro Lead as an important factor v.hich increases the likelihood of severe permanent da m a g e to the brain. It is r e asonable to c o n sider as wall the p o s s i b i l i t y 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.u-j.'j . u .Ue o.'jverve eyCi-Cmu m e pi o c w o m y raven*1.-- o x.rw o s . 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 cause symptoms or to produce disability have shortened life expec-can dles b e c a u s e of p r e m a t u r e de v e l o p m e n t of '.nephritis or in some instances cerebral hemorrhage. neurol ogical inifestations of lead poisoning are e and wi o.0 sprs illtv, depress ymptoms are th %4~ *YU'1 :** ally dev roe sad yH o -o KE 3 -3b peripheral neuropathy is considered a common neurologic anitestation of lead poisoning, and involvement of the radial erve is frequently described. W h i l e the clinical pi c t u r e may resent as a mononeuropafhy, neurological examination will fren demonstraroo. r.rc ^ Ir in rao c* other muscles, and neuroelectrici tudies rnay sho w p r o l o n g e d cond u c t i o n times or d e c reases in he amplitudes of the action K 0017402 36 tic paraplegia and ron damage. In su lead' neuroxnyel opat cord degeneration such as in imary lateral sole amyotrophic lateral sclerosis may be difficult. Lead encephoiopathy has been considered more common in children than in adults, in w h o m it m a y b e misdiagnosed as a brain tumor responsible for such symptoms as h e a d a c h e , vomit ing , diplopia, and periodic confusion, in conjunction with evidence of increased i n t r acranial .pressure. Opthalmo.lcg.ica 1 disturbances are not common in systemic lead poisoning although t\ " " i 33.0. ulQrilSujCBtl'jiJ ui -le: jTG'CxiiB ii3.S u c SCl IOc:'..'. XTi yT0...t2 Ocular symptoms of lead poi s o n i n g h a v e b e e n more common in children in w h o m it is possible to recognize disturbances of the visual cortex, suprageniculate pathways, optic nerve, retina lens, and intra- and extra-ocular muscles. uf the n e u r o l o g i c ' m a n i f e s t a t i o n s of lead p o isoning in children, p e r i p h e r a l .neur o p a t h i e s h a v e not b e e n the most ap parent, and central nervous system manifestations have been considered mere common. It is likely, however, that examples of p e r i p h e r a l n e u r o p a t h y in c h i l d r e n go.: unre c o g n i z e d in many eases, i n cluding those in w h o m .encephalop CitUV -u tuva eorjicuvacir c.u.nicdi aiLCint i.< Cilx la x v: v:- cn c.1.x a i c.h J tfE. 001*7403 e v i dence of p h e r i p h e r a l neuropathy, viz:.-,- sen-soyy l o s s - a n d - -weakness ,ch-ow evidence of de n e r v a t i o n when tested electrically. Neurochemicai studies of the brain in lead poisoning have been technically difficult and have most commonly focused upon the effect of alkyl lead compounds, the pharmacolcgy for which is undoubtedly dissimilar to that for inorganic load. Although seizures encountered in acute lead ohe'ephalo- ' pa thy ma y be due to neur on o-~e x c i t a t i o n related to local -edema or vascular lesions, these manifestations may be due to an inierterence w ith glutaraic acrid transport or m e t a b o l i s m re-' suiting in a r e d u c t i o n of aminobutyric -acid (GABA) produ c t i o n t O ;aC _--i. G -V X eiXi xnr}. i c OJ.. y J- Cl' .!.o sent moderator, and interferen ce w i t h GAB A p r o d a c t i o n mightfc produce seizures by increasing neuronal excitability. Meta bolic- steps w h i c h might b e .susceptible to- the effects of lead include (1) glutaraic acid decar b o x y l a s e (GAD) which carboxylate glutamate, to GABA; (2) gluta m i c acid dehydrogenase, (GHD) -which reduces 2-ket o .glut ar-ate to glut auric acid; and (3). pyridoxal' p h o s p h a t e (B.gP) m e t a b olized from pyridox.tne which functions as the co-enzyme for transamination react.ions in the synthesis of GABA. Evaluation of those systems and of the clinical e f f icacy of p y r i domine or g l utamate in childr en -with seizures of acute lead poisoning might be warranted. Rts 0017404 w* 38 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 .remye i i n a t i o n process, have been demonsrrated microscopically. Two mechanisms have been suggested in explanation ox the process of derayelination. and remyeiination in lead nephropathy. One possibility is that a substance h a ving p o r p h o bilinogen as a precursor is essential for the mainte n a n c e of myelin. It is. k n o w n that . in porphyria there is a metabolic blech, which results, in the excretion of excess p o r p h o b i l i n o g e n and d-JILA ana c-oncurrentXy a dop.ye.1 xr.ar~eg puiveija the .specific. lV u s s >..'i wiii'j:.: not known. The second possibility is that lead causes vasodilation and altered vascular permeability which cause -intraenaensara 1 edema and consequent damage to the Bchwaim 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. Th e introduction of lead into suitable n e u r o m u s cular p r e p a rations results in the p r e c i p i t a t i o n of lead at . the coteratic site of this e n z y m e , . The prime binding site for load appears to b e the post- s y n a p t i c sub- nerve.:, apparatus of the motor endplate. The b puling of the sub-neural apparatus 39 by lead 'Is not influenced b y the pric&y administration. diiscpropyl fluorophosphate {DFF} or neostigmine. Hcwever, tV-v of tree traent c PTipIetely 'inhibits -ciholin-esterase activi ftv *w-< -1_ TM ne otera se. acti' rity does appe a r to. b e r e d u c e d in muscle in vd::1.Ch the sxibneu. :ai apparatus lias been, b o u n d by l e a d . s St a i are part of an attempt to identify, some properties o i th choli nergic receptor and the c h a r a c t eristics of lead ;ding at the sets of the Other S Xli < ,ly prese Tit <it the m otor e n d plate in a b o u n d form and is -rA5-Tpfl ?XS r"h'0-3 relai X OXi.i the: site of b i n1d i n g of divalent m etal ions such as lead may exp'llaaiinn the p`-xr-v*,e d i l e c t i o n for p a l s y in the m o r e active rr.usc.l Load and calcium may .also be interrelated through a meohuni which is r ef l e c t e d in the fact that a red.uct.ion in calc i u m concentration may produce neuromuscular blockade by decrees trig 'the p r e s y n a p t i c release of acetylcholine. K 0017406 V. C o n f erence Summary ' .. T h i s 'confrence r e a f f i r m e d a long k n o w n fact that' . poisonings due to moonshine liquor containing Dead, pica for lead-containing material, and excessive occupational exposure to lead continue to occur. This fact represents a failure on the part cf health experts and other responsible elements . of so ci e t y t o prevent gross exposures to -lead.. . The presentations of this conference illustrate tho in creasingly sophisticated methodology being used to study the lead manifestations of absorption. Such studies are obvicuslv f- . ` . .important to our understanding of the mechanisms and conse- . queneos of lead intoxication, Gf even-greater iimportance, however', would b e the development of sensitive and specific m e a s u r e s w h i c h could e s t a b l i s h w h e t h e r ' o r not "n o r m a l " people are being harmed by current concentrations of lead in the 'n o r m a l " environment. N o new, p r a c t i c a l measures are as yet .' a v a i l a b l e as screening p r o cedures to .detect v e r y e a r l y adverse res p o n s e s to lead. T h e v a l i d i t y of n e w enzyme studies is currently under examint ion. Aminoa cid u r l a . 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 HE 0017407 workers w i t h excess lead e x p o s u r e , Aminoaciduria,. .therefore^ d o e s not ap p e a r t o be a -highly sensitive clinical study p r o c e d u r e for "n o n s a l " populations, although it .is :certainly_ important for. r e s e a r c h in cases of .lead, intoxication, IncI n s i on Bodies. N u clear inclusion bodies in kidney cells appear., to.be r e l a t i v e l y ;specif i d b u t . .t o plate, h a v e only, b e e n f ound after r e l a t i v e l y hig h exposures `to lead. ...Their'. .' only presently, k n o w n function is as a storage site for excess lead 'which has ente r e d the nucleus., of k i d n e y cells and comp le with, mitochondria. Intranuclear inclpp ioru bodies should be . looked for in. c a d a v e r studies, p a r t i c u l a r l y in those v?ith h x g h b o d y burdens of load,and in cells in the urinary sediman of h u m a n s w i t h chronic, lead exposure. In addition various animal species should be used in laboratory studies of n u d e a inclusion bodies. . .. . . ' G l o m e rular F i l t r a t i o n Rate. It was' suggested that the g l o m e r u l a r ...filtration .rate .might b e a -useful screening me a s u r H o w e v e r this m e a s u r e ' w a s q u e s t i o n e d as lacking specif-ity .for lead exposure relative' to renal disease in general. '`S o d i u m P u m p " I n h i b i t i o n . This pheno m e n o n presents as a. h y p o t h e t i c a l lead effect and r equires .research for v e r i f i c a B rochemical Functions. Two potentially sensitive indiea are a v a i l a b l e r the .measurement of the respiration of rniro- c n o n d r x a ot r e t i culocytes and ..the .m e a s u r e m e n t of. p o t a s s i u m KE 0017408 42 loss across tlie re d b l o o d ,cel-1. meiTtoran. -Both' of 'these - 'phenomeiia-xpqui're.-evaluat-i-o'n in 'animal-s-- a n d humans-with.--lead- 'ihtpxication. :. ' . ' . Enzyrae C h a n g e s. Enzy m e s w h i c h .are c o nsidered to be most sen s i t i v e - t o -;l a d ."exposure ar e dai-ta :ainino l e v u l i n i c aci'd ' dehydr.ase:, .Tippamide. d e h y d r o g e n a s e -and ATP-ase. Changes- .in enzymes levels-need to be correlated.with possible adverse . e f f e c t s i n man -a n d .a n i m a l s . ' .....- - -- . - --- ---- -- - - E s s e ntial. Trace. Met a l a ..- M a n y .trace metals are essential for normal b o d y .functions,..: and .lead may .b.e' c o m p t i t i v e w i t h . these metals. H o w e v e r there 'are .no k n o w n examples of such inhibition by lead in in vivo systems Cadaver s tudies have shown. that essential-trace-metals in-.soft-tissues are normal' despite,.increased concentrations, of lead. . Heme.. S y n t h c s i s .. C o p r o p o r p h y r i n and delta amino"levlinic acid in u r i n e are.sensitive and r e a s o n a b l y specific indicators of. lead exposure, .. These .measurements' are'.'of value in studies w i t h adult s',""hut may.b of less' value' i n ':studies wit h children There are marked diurnal variations in these indicators, and it is r e c o m m e n d e d that- concent r a t i o n s b e - e x p r e s s e d in un.i-t.s- relative, to urine flow rather than to urine volume. t-eira avnino ievulinic acid determinations in serum are suggested as ..a more refined measure of lead effect .upon h eme synthesis.. KE 0017409 f 43 -.T h behavior.. of-.A L A-dehy.drase. u n d e r lead exposure situations 'requires examint ion a n d `i n t e r p r e t a t i o n from the point o f .v iew of health.- . - - ' : -- --nerve- Conduction- T i m a . One of- the more p r a c t i c a l -and refined "tests -.-for ```n e u r o L o g x c a b change, is th e - measurement of : ; nerve -conduction- time.- -However-; this p r e c e d u r e is c o n s i d e r e d : t-o b e - m o r e u s e f u l - f o r following' cases of lead into x i c a t i o n tha for screening purposes. *- * A n a l y t i c a l Variability.' For almost -every s t u d y , quest ion about- analyti cal acc u r a c y - a n d p r e c i s i o n can b e raised. It is suggested' that.' comparisons'-of data .would b e f acilitated b y - col lab oration, .through, a. c ent r a l laboratory, This vaohnigue . 'w o u l d avoid much c o n t r o v e r s y and'expedite interpretations of data. :' '' - T his c o n f e r e n c e r e v iewed th state of- current k n e w .ledge-, about, h o w lead interacts with man'and. experimental .living - systems. .It a p p e a r s .that the b e s t "indicators :of early respons -to-lead" are. already in use.. Several areas of research w hich may iead to, i m p roved measures of early response are a p p a t eut.. . It. "has b e e n r e a s s u r i n g - t o note that ..exhaustive animal. - studies h a v e not led to the, dis c o v e r y of ne w lesi ons due .to lead, a l t h o u g h it is also-clear that there- are major differer.-c b e t w e e n species in response to' load absorption, ' HE 0017410 44 - ...... -s.ev-erai-ineeds -for the immediate future-.-arerhighlighted* , " ' 1, Prevention- of exc e s s i v e 'and u n u s u a l exposures to lead a broad problem for society-at-large.- ' ' 2r liefined-studies of those- cases of .lead i n t o x i c a t i o n which, unfortunately. centinue to occur. ' . ' 3. Refi n e d studies on p o p u l a t i o n s w i t h elevated, but not necessarily clearly harmful, exposures to lead* especially . v.?ifh_ respect to.lchanges in- r e n a l funetioiwand. h e m e s y n t h e s i s . . . - 4. -Research .with.animals .on .correlations b e t w e e n enzyme changes and adverse effects of lead. ... '` . 5. Surv e i l l a n c e of- the environment and .populations fox- V d.*iClC? v j-ClCrliCt? Oi_ iflci.il'hi 01^X3 L aiid OU.K CC. load, " .. V" .' .- . 6. Surv e i l l a n c e for p o t e n t i a l l y . s u s c e p t i b l e individuals and indications 'of synergistic .actions .or..aggravation of exist ing disease. - --.. .. - - - --