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Pediat. Res. 11: 153-157 ( 1977) ATPasc glucose sodium intestinal absorption

Experimental Lead Poisoning and Intestinal of Glucose, Amino Acids, and Sodiu1n

RAUL A. WAPNIR,"" RAMON A. EXENI, MELINDA MCVICAR, AND FIMA LIFSHITZ

North Shor<' U11i1·crsit_1· Hospital, [)<'part111e11t of l'<'iliatrics, Manlwss<'t, and Cornell Unii·ersity Medical Coll<'/;<', lJ<'part111<·11t of Pcdiatrics, Ne11· York, Nc11· York, USA

Summar)' ing on the dietary status of the animals (5, 19, 22, 50, 51 ). This model, however, provides the closest similarity to the conse­ J1ncnilc rats fed a diet containing I% lead acetate for 7 quences of "" in children (26, 42, 47, 52). The n:lationship weeks, in addition to an impaired growth rate and renal function between adequacy of diet and the extent of lead poisoning derangements, suffered malahsorption of glucose and certain damage has been investigated in experimental studies (5, 22, 50) amino acids, as assessed by an in 1·i1·0 perfusion technique. The and in nutritional surveys of children (6, 38). reduction in glucose absorption ranged between 10% and 31 % The purpose of this study was to determine the effects of a when the carbohplrate was pumped in concentrations of 2-80 chronic intake of lead on the intestinal transport of glucose. 1111\I. This alteration was compatible with a noncompctith·c t:nic amino acids, and sodium, and the possibility of cellular and/or of transport inhibition. The intestinal absorption of glycine, physiologic damage to the intestinal mucosa, in addition to the lysine, and phcn_ylalaninc were, rcspccth·cly, decreased 22, 18, previously described impaired growth rate and multiple renal and 15% when these amino acids were present at 1 1111\l levcls. function derangements. Sodium transport was scnrely reduced (57.6 ± 17.9 (SEl\l) vs. 124.2 ± 17.4 µE(J/min·cm) and intestinal mucosa (Na+-K+). ATPasc was concomitantly lower in the lead-intoxicated rats I\IATERIALS AND METHODS 086.4 ± 19.0 vs 268.4 ± 29.8 1111101 P/min·mg protein). llow­ Male Wistar rats, weighing (10-80 g, in groups of four to eight, c,·cr, this was not altered in liver and kidney. Further­ were given ad libi111111 a complete diet (56), containing I% lead more, intestinal mucosa fructosc-1,6-diphosphatasc, succinic dc­ acetate plus free access to water, for a period of 7 weeks. The hydrogcnasc, p)Tll\'atc kinase, and tryptophan hydroxylasc were effective lead intake was estimated to be 600 µg/kg/day. During not different in experimental and control animals. These studies the last week of the experiments. urine collections were carried substantiate the presence of functional and biochemical abnor­ out over several 24-hr periods. The experimental animals were malities in the intestinal mucosa of )'Oung rats when fed substan­ housed in metabolic cages and intake was measured daily. tial amounts of a soluble lead . It is, therefore, reasonable to Urinary glucose was determined by a glucose oxidase method accept the 11ossibility that physiologic damage occurs in tissues (58). Amino acid (23), inorganic phosphate ( I 8). and directly subjected to high and persistent lc\'els of a toxic agent, creatinine nitrogen (9) were assayed by colorimetric procedures. as it occurs in other organs, underscoring the parallelism be­ Atomic absorption techniques (Perkin-Elmer, model 305A tween transport mechanisms at the renal and intestinal levels. (59)) were applied for lead (32), , and magnesium ddcr­ minations (3) and flame photometry for sodium and potassium Speculation assays ( 60). The gastrointestinal s:pnptoms often associated with chronic The intestinal absorption of glucose, amino acids, and electro­ lead intoxication may be related to ph_ysiologic damage of the lytes was studied in the rats using an in l'il'O procedure (55, 5(1). intestinal mucosa transport mechanisms. Therefore, intestinal Glucose, in concentrations of 2 to 80 1111\1, labeled with ''C in mucosa could be considered another primary target organ for tracer amounts, plus I mM single L-amino acids, including "H lead poisoning, as the kidney, the crythropoictic system, and tracer, in both cases at a level about 50,000 dpm/ml. were added ncnous tissue arc known to be. to Krebs-1 lcnseleit bicarbonate buffers. Labeled metabolites were purchased from New England Nuclear (61). The tonicity was maintained by adjusting the concentration of NaCl. All Lead intoxication in humans has been shown to affect the chemicals were obtained from Fisher Scientific Co. ( 62). Poly­ central , the hematopoietic system, and the kid­ ethylene glycol (PEG), 11101 wt 3 ,000-3, 700, Carbowax 4000 (J. neys ( 12. 13, 16, 25). Susceptibility of red T. 13aker Co. ((13)), 6.0 g/liter, served as a nonabsorbable (Na +_K+)-ATl'ase to lead poisoning has been shown in workers marker and the ratio of its concentration (35) in the buffer to suffering the consequences of excessive exposure to this agent that of the perfusates was used as a correction factor for the (27). The lead burden in children is the highest among those calculation of absorption rates. The perfusing liquids were bub­

living in urban ghettos, particularly if they chronically ingest bled with 0 2 :C02 , 95:5 (Liquid Carbonic (64)), and maintained small amounts of wall peelings covered with lead-containing at 37° during the experiments. 111 brief. the animals were anes­ paint (6). This has been called "a preventable childhood disease thetized with I .2 g/kg urethane given intraperitoneally. The of the slums" (42). abdominal cavity was opened by a midline incision and the small Numerous investigators have used rats as an animal model of intestine cannulated below the ligament of Treitz. A 20 cm-long lead intoxication, duplicating alterations in kidney tubules (24); jejuna! segment was utilized and the intestinal contents washed changes in growth, behavior, and catecholamines (21 ); out with two I 0-ml portions of warm O. 15 M NaCl. The proxi­ (43, 48); and damage to the hematopoictic mecha­ mal junction was attached to a peristaltic pump (Harvard Appa­ nisms (37, 39). The oral administration of lead has been ratus (65)) and perfused at a rate of 0.20-0.23 ml/min. The chosen in most studies, in spite of variable rates of absorption, concentration of the glucose or amino acids was determined by estimated between 2% and 10% of the ingested dose, depend- isotope dilution computation after counting aliquots in a liquid 153 154 \VAl'NIR /:'T AL

Table l. Urinan· c.rcrction of \'/lrious 111ctalwlitcs in /cad-poisoned rats ( l'h) 1 ------Expcri111ental groups

Pb Control I' ------Urine volu111e (1111/24 hr) I 0.5 ::': 1.3 (34) 4.7 ± 0.8 (20) <0.001 Lead (µg/24 hr) I h5.8 ± 3J.h(55) '!.3 ::': 1.2 (2J) <0.001 Glucose (111g/24 hr) 10.67 ± I .'J7 (2J) 3.38 ± 0.78 (1.1) <0.01 AAN/creatinine N 0.86 ± 0.0') (22) 0.53 ± 0.04 (14) <0.01 Ca/creatinine N I .hi ::': 0.24 (22) O.M, ± 0.15 ( 13) <0.01 1\lg/creatininc N 2:U ::': 10.J (21) h4. I ::': 13.8 ( 14) <0.05 l'/creatinine N l .lh ::': tU8 (21) 2.4!i ± 0.70 (14) NS Sodiu111 (µEq/24 hr) (,')4 ± 7') (38) 1,255 ± I !i(, ( I h) <(l.01 l'otassiu111 (µEq/24 hr) l ,O!i5 ± I <,3 (35) 2.0')5 ± 209 ( 11) <0.001 ------1 Means ± SEM. Nu111ber of assays in parenthesis. Glucose oxidase was used for the dcter111ination of glucose (58); colori111etric 111cthods for the assay of a111ino acid nitrogen (AAN) (23), inorganic (18), and creatinine N ('!); ato111ic absorption spectrophoto111etry for kad (32), calcium, and magnesiu111 (3); and fla111e photo111ctry for sodiu111 and potassiu111. The significance of the difference of 111eans was deter111ined by a nonpain:d Student's /-test ( 17). scintillation counter (Beckman, model LS-203 ((1(1)) with a pre­ cision better than 0.7'/r and an efficiency above 50':r, for ''C. or m,n cm MEANS± SE M 20';,, for "11. 200 CJ CO.Tl!OLS At the end of the perfusion. a 30 cm-segment of the small CJ L[A0-TRlAT£0 intestine. distal to the cannulated portion. was excised, washed • P< 05 twice with ice-cold 0.15 1\1 NaCl. and the intestinal mucosa •• P

RESULTS Glucose intestinal absorption ,,as significantly reduced in Pb rats over the 1, hole range of concentrations of glucose values Rats fed lead acetate (Pb) failed to grow at the same rate as (Fig. I). The kinetics of this phenomenon was compatible with a controls (C) fed the same diet without lead. At the end of the noncompetitive type of inhibition (Fig. 2). The representation of test period the experimental animals weighed 128.0 ± 4.2 g vs. V vs. V/S and S/V vs. S have been shown to be better graphic 177 .8 ± 8.3 g for the C group (means± SEM. 32 d.f .. 1 = 5.35. transformations than the Lineweaver-Burk plot ( 15). I'< 0.00 l ). Comparable weight differences were observed from At the same time. the intestinal transport of lysine, glycine, the first ,,eek of the experiments. This weight discrepancy was and phenylalanine was inhibited. but other neutral amino acids not due to a reduced food or fluid intake. There ,,as no observ­ such as AlB, tyrosine. and tryptophan were not affected (Table able . 1lowcver. the food efficiency in the Pb rats was 2). A simultallL'ous severe reduction of sodium intestinal absorp­ far below that exhibited by the C animals (expressed as grams of tion was observed in the Pb rats. hut potassium influx across the weight gained by 100 g of food; Pb: 8.02 ± 0.57, C: 12.8'J ± small intestinal mucosa was not impaired. This finding was l .(15. l O d .f .. 1 = 2. 7'). I' < 0 .02). As described earlier by Goyer concomitant with a decrease in the levels of small intestinal (24). Pb rats had aminoaciduria and glycosuria associated with mucosa (Na 1 -K 1 )-ATPasc measured in homogenates obtained polyuria. 1\lon:over. we found that the rats treated with Pb had from the jcjunum inunL·diately contiguous to the perfused seg­ proportionally higher losses of calcium. In contrast, the renal ment. In contrast. total and (Na+ K +)-ATPasc activity of kidney of sodium. potassium. and magnesium was reduced in and liver homogcnatcs was the same for Ph and C animals (Fig. the experimental animals (Table I). The results in this study 3). Thc alteration in the enzymatic capability of the intestinal were expressed in several instances in terms of creatinine nitro­ mucosa appeared to be narrowly restricted. since four other gen to compensate for weight differences. although the validity present in that tissue and representative of key meta­ of data expressed in this fashion has been questioned in human bolic pathways in carbohydratc and amino acid metabolism were clinical chemistry ( 44). not altered in the Pb-poisoned rats (Fig. 4). EXPERII\IENTAL LEAD POISONING 155

It was considered possibli.: that those alterations in intestinal n moles P transport could be caused by nonspecific damage of the intestinal mi"nrngproi mucosa by Pb. Such damage could result in leakage of the 3LILI MEANS±SEM marker, ''C-labcled PEG, into the circulation. However, radio­ p activity in the blood of both Pb and C rats was indistinguishable 20 0 CO ~ THO, S from background (Table 3). In addition . the blood-to-lumen p passage of glucose, measured by the appearance of glucose in ~£ AD • P< 05 collected perfusates when no sugar was added to the Krebs­ 100 Henscleit buffer, was the same in Pb and C animals. and negligi­ ble when compared with lumen-to-blood absorption rates . On No K the other hand. the lumc· n-to-bloml passage of l''C-jAil3 was TO TAL higher in the Pb-poisoned rats . Furthermore, a higher amount of radioactivity was present in the intestinal mucosa, suggesting a KIDN EY LI VER n moles P certain extent of increased permeability to solutes of small mo­ min mg pro! lecular weight. However. no ultrastructural alterations could be observed in electron microscopy preparations of the intestinal IUO mucosa of Pb rats aft.:r 7 weeks of dietary treatment (53) . ~o

- , TOTAL ATPase rju

., Fi g . 3. (Na•-K +)-ATPase and total ATPase in the intestinal n111cosa . 7 kidney. and liver of Ph-poisoned rats. after? weeks on the diet . and their 1 TPase 4 UC -. controls. determined as detailed in Rcfen:nce 31. The (Na -K +)-A in the kidney and liver of hoh groups of animals. not sl1tl\1·n here. was indistinguishable. Eight rats used in each group.

i pMEA NS±S EM 1' I CJ ~-lJ(_i / . ,-~' ~; ·• ~. • I .: :·, , UO n moles P _ u_ _ u_ n mo!es i min mq pr mg pr mg pr mg pr -. hr ', \. ,x \ 08 4 47 15 '7 . :·- -~. 8 y_ ·--- 2 4 6 20 40 60 80 S(mM) s .J 3 3 06 I 10 I hg. 2. Kinc·tics of the intestinal transpllrt of glucllse in Ph-pllisllned ; 04 2 n 2 and control rats determined hy the i11 1-ii'o technique detailed under Marcrials and M<'lhods. By mathen1atical extrapllbtion. the statistically 5 02 identical K,,, values correspomling to the Pb-pllisllned and control ani­ mals were .'i(, . I and .'i'JA 111:\1. rc·spectivelv. The· corresponding. signifi­ cantly different. V,,, 1·:tlue, wc·re 2(,7 and -D.'i nmlll/ min·cm . respc·c­ FD Pase SDH PK TRPHYD tivdy. Thc·se data indicated a noncompetitive inhibition of glucose trans­ port in Ph-poisoned rats. Fllr c·ad1 point, six Ill eight animals were used Fig . 4_ Intestinal mucosa enzymatic activity in Ph-pllisllnc·d and con­ in the perfusion experiments. trol rats. FDPasc: fructose-1,6-diphosphatase: SDH: succinic dehydro­ genase: PK: pyruvate kinase: TRl'.IIYD.: tryptllphan hydrnxylase. 111 11011e of these cases there was a significant difference bc·twc·c· n the two and numher of rats. as in Figure J. Table 2. /111c.\'li11al tra11s1)(1rt of 11111i110 acids //1/(I clcctrolrtc·s in grllups. Duration of treatment lewl-poi.1·011cd mis (Ph)' ---- Experimental groups DISCUSSION l'h Cllntrol I' - - ---· Transp1>rt of solutes across membranes is a compkx phenome­ Aminll acids non \\'hich associates the passage of an clectrulyte. sodium . with Lysine J.21 :± 0 . 17 (40) J.lJ4 :t 0 .22 (20) < 0.02 that of glurnse and amino acids (2. 49 , 52). In this context. Glycine 2.7h ':: 11 .20 (4J) J . .'i4 :t ll.2h (21l) < ll .ll_'i (Na ' -K • )-;\Tl'asc has bc·cn shown to play a kcy role in sodium < 0.1)) l'he11ylalani11c· J.J.'i ± 0.llJ (44) J.

Tabk 3. ,\s.H·sslll('llt of i11tcsti11al 11111co.rn integrity' ------Experiment a I groups

Test Pb Control I' t\. Lumen-to-blood [''C]PEG leakage Background Background NS 13. 13lood-to-lumen glucose inllux (nmol/min·cm) 0.352 :!: 0.175 0.425 :!: 0.227 NS C. Lumen-to-blood ['·'C[AIB passage (mean% of buffer activ­ 0.2lJ :!: 0.02 0.15 :!: 0.02 < 0.001 ity attained in blood) D. ["C[t\lB uptake by intestinal mucosa (µCi /g protein) 0.045(, :!: 0.0045 0.0263 :!: 0.0063 < 0.05

1 Four animals in each group were used for every test. Results arc expressed as the means :!: SET\I. Test ,\ was carried out with a Krehs-1 knsekit 1 1 buffer containing (1.0 g/litcr polyethylene glycol (PEG) and 50,000 dpm/ml of [ I ,2- · CjPEG. Test B was performed with a glucose-free Krebs­ I fcnsekit buffer. perfused for 2 hr under the conditions described in l\faterial., and Methods. Tests C and I) were conducted with I mf\l a­ aminoisobutyric acid (t\lB) and tracer amounts of ["C]t\lll. n:kasc of ADI I (2lJ). Corrclalion of mcmbranc Iransporl dc­ I() timcs that of hu111ans (-1). Evcn in thc abscncc of lcad­ fcl'!s in thc gut and thc kidncy has bccn dcmonstratcd in thc containing paint, a child in an urban cnvironmcnt may inhalc human discascs, cystinuria. Lowc's syndromc. and llartnup·s and ingest amounts of that heavy mctal far in cxccss of thc (5-1). It is. Ihcrcforc, no! unrcasonablc that a toxic agcnl such as "maximum daily pcrmissiblc" !cad intake (33). Thc conditions !cad may havc simultancous cffccts on transport mcchanisms in in this study offcr a reasonablc parallcl to many children two of thc organs which rcgulatc cxchangcs of thc inncr milicu arc exposed to. 1 louschold dust can contain over I % of !cad with thc cnvironmcnt. At any ratc. othcr factors nccd to bc (33) and bc thc main vcctor in cases of !cad poisoning (33. -1(,) . takcn into considcration. namcly. compctition bctwccn ions for Such an cxposurc can bc aggravatcd by a rate of !cad absorption activc sites in the proximal tuhulc . as high as 50% in childrcn (I) and ncwhorn rats (30). which In our cxpcrimcnts. thc biol'11cmical and physiologic damag~· cxcccds by an ordcr of magnitude the proportion of kad rc­ of thc intcstinal nrncnsa could hc dctected bdorc gross altcration taincd by adult mammals (I'). 50. 51). It is. thcn. r1:asonablc Ill of small intcstinal cclls obscrvable by clcctr,rn microscopy. How­ acccpt thc possibility that physiologic and biochcmical damagc cvcr. biochcmically. thc small intcstinal mucosa appcarcd to be occurs in tissucs dircctly subjectcd to high and pcrsistcnt lcvcls scnsitivc to !cad at an carlicr stagc than thc kidncy. Goycr (2-1) of a toxic agcnt. as it occurs in othcr organs. As littlc as I mg has shown that IO wccks ullllcr comparablc conditions werc a !cad/day has bccn considcrcd cnough to cause !cad poisoning in minimum rcquircd to inducc markcd mitochondrial swclling in childrcn ovcr a (,-month period (25). This amount would corrc­ thc proximal tubulc of thc kidncy . spond to 70-80 µg/kg/day for a 12-1-1-kg child. In conscquencc, Thc observation that thc irnpairmcnt of glucosc transport was thc 6()() µg/kg/day ingestcd by thc rats is a conscrvativc scaling compatiblc with a noncompctitivc typc of inhibition is ulllkr­ down of dosagcs. standablc undcr thc assumption that the hcavy mctal prmluccs a Thc pervasivcness of thc biologic injurics which !cad can causc nonspecific altcration in thc availability of activc sitcs for the has bcen rcccntly extcmkd to possiblc intrautcrinc and nconatal translocation of glucosc and ccrtain amino acids. rather than a brain damagc duc to containing high conccntra­ compctitivc binding of thc inhibitor. prcsumably !cad. to specific tions of the heavy mctal. A study conductcd in Scotland associ­ activc sites for those suhstann:s . The circumstances that not all atcd this paramcter in the watcr supply availablc lo mothcrs neutral amino acid absorption ratcs wcrc equally rcduccd can he during g1:station. and th1:ir infants during th1: first y1:ar of lifc, interprcted in thc context of thcsc mctabolitcs sharing common with mental rctardation in youngsters. In a remarkable rcsponsc transport mcchanisms and si111ultancously posscssing spccific to a public . thc city of Glasgow adoptcd spccial mcmbranc carricrs which arc distinct from thc for111cr and cx­ water trcatment prccautions in an cffort to dccrcasc lcad con­ hibit diffcrcnt functional propcrtics. This possibility has heen tamination (7). discusscd in prcvious studi1:s considcring co111pctition among It is conccivablc, thercforc. that manifestations amino acids at thc intcstinal murnsa lcvcl (55. 57). and biochcmical changcs in thc intcstinal mucosa obscrved in Pb Thc failurc of thc Pb rats to grow al thc sa111c ratc as thc C rats may providc a cluc to the naturc of somc of thc physiologic animals was not duc to a decrcased food intake. In fact. rats abnormalitics pr1:sent in humans chronically poisoncd with !cad. acccptcd rcadily thc !cad acctatc ration . The swcet tastc of this particularly carly in lifc . salt . long ago known as " of !cad" (-15) contributcd to undiminishcd intakc in thc 1:xpcrimcntal group. Thcrdor1:. thcir l;1gging 11Tight gain could hc rclatcd cithcr to a dcficient rcnal C ONCLUSION function . to a malabsorption phcnomc·non. or to intcrfcrcncc by A dict containing I % !cad acctatc fcd for 7 wecks to young lc,1d of othcr k1:y mctaholic mcchanisms (12. I 6. 25. 27). Natu­ rats produccd an impair1:d intcstinal absorption of glucosc and rally. a combination of thesc possibilitics should hc a primary ccrtain aminl> acids, with a concomitant rcduction of sodium considcration. Thc dcficicnt dcvdopmcnt llf thcsc animals car­ transport. Intestinal mucosa (Na+-K+)-ATPasc was lowcr in rics a strong rL·scmhlance to the human condition of juvenilc lcad-intoxicatcd animals. although this cnzymc was not altcrcd !cad poisoning (6. 13. 25). as wcll as to other forms of kidncy in kidncy and livcr. suggcsting the intestinal mucosa as a pri111ary discasc oftcn associatcd with failurc to grow. such as renal targct organ in !cad poisoning. tubular acidosis (-11) and chronic urcmia (28). In humans. gas­ trointcstinal symptoms haw b1:cn dcscrihed in the majority of a group of 58 children suffcring an increased !cad burden (-17) . REFERENCES AND NOTES The alterations of sodium transport, expcrimentally dcmon­ stratcd in th1:se studics. mav have induc1:d water flux ahnormali­ 1. Akxandc r, F. \V.: The uptake of kad hy rhildTL'll in difkrcnt cnviro nmcnls. tics which could be responsihlc for somc of thc intcstinal disturb­ Environ. Health Pcrspcct. 7: 155 (1'174). .., Alvarado, F., Torrcs-Pinl'do. R .. MatL'U , L., and Rohinson, J. \V .: Counh:r am:cs associatnl with kad ing1:stion. Th1:re have h1:cn also oh­ transport hctwccn and amino acids in rahhit ikum. Fed. Eur. Bin­ scrvations of radiologic cvidcrn:c of duodcnal ulcerations in chcm. Soc. Lett., 8: 153 (1'170). children known to havc ing1:stcd kad-containing. nonfood sub­ 3. Analytii.:al r--.kthods for Atomk Absorption Spectrophotometry (Pcrkin-Elm~r stanccs (1-1). Co., Norwalk, Conn.). 4. Arena, J. ~I.: Poisoning. hi. 3. p. ~I (Charles C Thomas, Springfield, Ill. In r1:gard to thc dos1: r1:lationship in animal mndcls of kad I 'J74). poisoning. it should bc notcd that thc tolcrance of rats is at !cast 5. llaernskin. H. D .. and Grand. J . A .: The relation of protein intake to lead EXPERil\1ENTAL LEAD POISONING 157

poisoning in rats. J. l'har111acol. Exp. Ther.. 7-1 : 18 (19-12). Kaplan: l\klhods in E nzy mology. Vol. 2. p. 5-13. (Arademic Press. New 6. llarltrop. 0 .. and Killala. N. J . P .: ractors innueneing exposure of children to York, 1955). kad. Arch. Dis. Childhnod . .J.J: -17<, (I'/<,­ 12. Chisolm. J. J .: Aminoaciduria as a manifestation of renal tubular injury in lead li sm of c~c) glucose hy thl.' of suckling rats intoxicaled with inorganic intoxil'atinn and a comparison with patterns of aminoaciduria St.'.'L'll in other lead. J. Neurochem .. 22: 581 (197-l). diseases. J. Pe,lial., Ml: I ( I 9(,2). 4.t. Paterson. N .: Relative constancy of 24-hour urine volume and 2-l-huur cn:ati­ 13. Chisolm. J . J .. and Kaplan. E.: Lead poisoning in childhood: Comprehensive nine output. Clin. Chim. Acta. /8: 57 (1%7). manag,·menl and prevention. J. Pedial.. 73: 9-12 (1968). 45. Parkes, G. D., and l\lellor. J. W.: l\1odern . p. 7111 I-+. Dl.' Rosa. S.: Pl.'rsonal comnnmil:ation. (Thl.'Sl.' ohsL'rvations wl.'rl.' condtH,.'h..'d at (l.ongmans. Green and Co .. London. 1946). the Hospital de Ni1ios, Oucnos Aires, Argentina .) -16. Perkins. K. C.. and Oski. F. A.: Elevated hlood lead in a 6-month-old breast­ IS . Dowd. J. E. , and Riggs. D .S.: A comparison of L"stimatcs of ~lk·hal:lis­ fed infant: The role of newsprint logs. l'ediatries, 57: -12<, (1'17(1). r-..kntL"n l..inl'lic constants from varilHIS lim.:ar transformations. J. Bil,!. -17. Pucschel, S. 1\1., Kopito, L., and Schwachman, II.: Children with an increased Chem .. 2.JO: 8/,3 (1%5). lead hurden -A screening and follow up study. J. Amer. ~led. Ass., 222: l/1. Erenherg. G .. Rinsler, S.S .. and Fish. 13. G.: Lead neuropalhy and siekle cell 462 (1972). disease. P,·diatrics.5.J: -138 (J

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