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in the basis that large t. occur in this species, in why lymphatic trans it minimal in hamsters., sport of fibrous glass nph nodes is another of a dust that has little tenlial.10'18 This is ex it the concept that dust e into the lung interstirrtieles and that, theretransport is contingent an extracellular status, rich dust particles are te lymph nodes, tbere-lotoxicity. Since nonwould not be expected sintegration of tlie cells them, their extracelluheir penetration of the d subsequent lymphatdelayed. 2 RTP: Alveolar proleinoducUon in rodents. Arch Experimental endogenous J Path 28t211-221, 1952. erexperimentelle Untersuungsvermogen der Lunge. 1,1957. lignite and the derived he pulmonary response fo Health 14:450-400, 1967. 3 RTP:' Emphysema and rimental study on their nn'ron Health 18:340-349, liosis: An aspect from a 4:106, 1963. inbrodt HJ: Quantitative rebungen liber den Abin Lungen in die regionaHye Baht 149:367-384, >1: The recovery of quartz the lungs of rats. Arch 1957. : The requirements for Int Arch Gewerbepath 705 Lead Dosage and the Role of the Intranuclear Inclusion Body An Experimental Study Robert A. Goyer, MD; Daniel L. Leonard; John. F. Moore; Bonnie Rhyne, PhD; and Martin R. Krigman, MD, Chapel Hill, NC Biological parameters known lo be affected In lead poisoning were measured in rats following ingestion of graded dosages of lead. Intranuclear inclusion bodies are formed in renal tubular lin ing cells with smaller doses of lead than produce other changes. Decreased body weight is the next most sensitive abnormality. This is followed by in creased delta-aminolevulinic acid (ALA) excretion, retlcufocytosis, renal edema, and aminoaciduria. Anemia only occurs at the highest lead dosage. Over a wide range of lead ingestion, urinary lead excretion remains constant, although renal lead content Increases. Quantitative lead analyses of cell organelles show that lead is concentrated within the inclusion bodies. Relatively small amounts of lead are present in the cytoplasm and mitochondria. It is suggested that soft-tissue lead accumulates in the intranuclear inclusion body, thereby sparing toxic injury to cytoplasmic or ganelles. Th e METABOLIC effect of trace quanti ties of lead derived from environmental ex posure is not completely understood. It is not known whether a small amount of lead in body stores, that is, the body burden of lead, is in some way harmful or subclinically toxic.1-2 There is no doubt that lead is `po tentially lethal to humans and experimental animals, and the clinical manifestations of overt toxicity are well defined. However, when compared to the effects of other heavy metals such as cadmium and mercury, clini cal lead toxicity only occurs upon exposure Submitted for publication Nov 13, 1969; accepted Jan 7, 1970. From the Department of Pathology, University of North Carolina School of Medicine, Chapel Hill, NC. Reprint requests to Department of Pathology, University of North Carolina School of Medicine, Chapel Hill. NC 27514 (Dr. Goyer). to relatively large dosage. In addition, there is no clearly recognized biological require ment for lead. If a large amount of lead in body tissues is overtly harmful and none is required, accumulation of even trace amounts in body tissues may be undesirable or detri mental to optimal health. However, such a subelinica! effect is yet to be defined. More over, any subclinical effect is unlikely to be recognized until a better understanding of the metabolism of lead is obtained, particularly in terms of its effects on cellular organelles and metabob'sm. The need to attain this level of under standing is becoming increasingly urgent. Patterson estimates feat man, by mining and industrial usage, introduces approxi mately 100 times more lead into the envi ronment annually than would occur natural- Areh Environ Health--Vol 20, June 1970 N36908 DU P050312388 706 INTRANUCLEAR INCLUSION BODY--GOYER ET AL Table 1.--Renal Retention and Urinary Excretion ol Lead by Rats Given Varying Doses of Lead in Drinking Water tor Ten Weeks Animal Groups: Lead Content of Drinking Water (mg/ml) Lead Ingested (mg/Day/Rat)* Renat Lead (mg/gm Wet Weight) Urinary Lead Excretion (jag/Day/Rat)t % of Ingested Lead Excreted in Urine Control group 0.006 0.5 7.0 ...t 0.08 2.3 O.S 28.0 1.21 0.20 7.0 2.3 65.0 0.93 0.40 12 8.1 60.0 0.50 1.20 21 30.4 64.0 0.31 4.00 67 36.6 60.0 0.07 10.00 220 276 132.0 0.06 * Volume of.drinking water X lead content: average daily intake measured for one week lor each group of rats, t Twenty-four hour urine samples from all rats of each group were pooled before analysis. } See text for explanation (Results). Jy. This is reflected in body tissue levels. Recent surveys indicate that present day blood levels of the North American adult averages 0.25ju.g of lead per milliliter rather than an inferred natural level of 0.0026/xg of lead per milliliter. Blood lead levels of per sons with clinically recognizable lead poi soning are only slightly higher, 0.5/xg to 0.8fig of lead per milliliter,, The question exists, therefore, whether man has some threshold of tolerance to lead and, if so, what is the nature of such a mechanism and how can its limits be recognized.2 The present report is an experimental study of the sensitivity of various biological parameters in the rat to exposure to graded doses of lead. Particular interest is given to the kidney where the accumulation of lead is associated with the formation of intranu clear inclusion bodies and renal tubular dys function. Materials and Methods Experimental Design.--There are two as pects of the study. The first portion deals with the sensitivity of a number of biological param eters known to be abnormal in lead poisoning. Forty-two white, male Sprague-Dawley rats, weighing 150 gm to 175 gm, were divided into seven groups of six rats. Ail rats were caged in pairs and fed standard laboratory chow. Six groups were given tap water containing the following quantities of lead in the form of lead acetate: 0.08, 0.20, 0.40, 1.20, 2.00. and 10.0 mg of lead per milliliter. The control group received tap water without added lead. The animals were weighed weekly and 24-hour urine collections were made prior to killing at 10 weeks of age. The animals were anesthetized with pentobarbital (5 mg/100 gm body weight) and exsanguinated by cardiac aspiration into a heparinized syringe. Both kidneys were quickly excised, weighed., and a small portion of cortex was fixed in 2.5% glutaraldehyde with 0.1 M sodium cacodylate. The remainder of the kid neys was frozen at -20C until analyzed for lead and /1-glucuronidase. The second phase of the experiment concerns measurement of lead content of organs and organelles in young-adult, white, Sprague-Daw ley male rats given pulverized 1% lead as lead acetate mixed with powdered laboratory chow for about four months. Similar rats fed the same chow without added lead served as con trols. Both groups were given tap water for drinking. Analytical Methods.--Packed red-cell vol umes were determined by centrifugation of capillary tubes of heparinized blood using a microhematocrit centrifuge. Reticulocytes on blood smears were stained with 0.5% methylene blue. The dye was mixed with heparinized blood and the stain in a capillary tube. Blood cells and stain were al lowed to stand for ten minutes before making smears on glass slides. At least 2,000 red blood cells (RBC) were counted on slides from each rat. Urinary delta-aminolevulinic acid (ALA) was determined by the method of Davis em ploying prefilled, disposable exchange columns. Urinary c-amino nitrogen was determined by totaling the individual amino acids which had been separated and measured by ion-exchange column chromatography employing an auto matic amino-acid analyzer with 120 X 0.6-cm column containing cation exchange resin. Prior to analysis, proteins were precipitated from 0.025 of a 24-hour urine sample by adding an equal amount of 10% sulfosalicylie acid and centrifuging in the cold (4 C). Distortion of the Arch Environ Health--Vol 20, June 1970 f IXT elution patlern of ami of lead remaining in t] adding 0.2% edetic ac ing solution. Lead was measured and reaction with die method of Bessman ai of cells for isolation o inclusion bodies was ] centrifugation by met! from this laboratory. Beta-glucuronidase v od of Gianetto and de Re The relationship of renal retention and ui is shown in Table 1. urinary excretion of the amount ingested Environmental lead i gested by this group Fig 1.--Hematologic efi lead in drinking water fed PACKED RED BLOC AURINARY AMI ACID EXCR 100 ; 3 : >/ o .oa .2 mg Pb/ml OR DUP050312389 T AL id it Weeks esd % of Ingested )h Lead Excreted lat)t_________In Urine ...t J' 1.21 093 ______________ 0.50 031 ______________ 0.07 ______________ 0.06 eek for each group of rats, lysis. ;/100 gm body weight) trdiac aspiration into a h kidneys were quickly small portion of cortex iraldehyde with 0.1 M remainder of the kidOC until analyzed for >e experiment concerns ontent of organs and t, white, Sprague-Dawsrized 1% lead as lead dered laboratory chow Similar rats fed the :d lead served as coni given tap water for -Packed red-cell volby centrifugation of rinized blood using a =e. I smears were stained e. The dye was mixed and the stain in a 11s and stain were alinnutes before making t least 2,000 red blood d on slides from each ;vulimc acid (ABA) method of Davis em>!e exchange columns.3 ;en was determined by aiino acids which had mred by ion-exchange employing an auto:er with 120 X 0.6-cm exchange resin. Prior `re precipitated from sample by adding an ulfosalicylic add and * C). Distortion of the INTHAXUCLEAH 1XCLUSIOX HODY--COYER ET AL 707 elution pattern of amino acids by (lie presence of lead.remaining in the urine was corrected |>v addling 0.2% edetic acid (EDTA) to (he wash ing solution. bead was measured by wet digestion of tissue and reaction with dithizone according to the method of Bessman and Layne.' Fractionation of cells for isolation of organdies and nuclear inclusion bodies was performed by differential centrifugation by methods previously described from bills laboratory.5 Beta-glucuionidose was assayed by the meth od of Gianetto and de Duve.B Results The relationship of increasing ingestion to renal retention and urinary excretion of lead is shown in Table 1. For the control group, urinary excretion of lead slightly exceeds the amount ingested with drinking water. Environmental lead in laboratory chow in gested by this group must account for the relatively large urinary excretion. Balance studios on humans ingesting environmental quantities of lead have shown that only 5% to 10% of ingested lead is absorbed from the gastrointestinal tract.7 Animals given increased doses of lead ex crete a very much smaller fraction in their urine--the more lead ingested,, the smaller the percent appearing in the urine. How ever, the actual amount of lead appearing in the urine remains constant over a wide range of increased lead ingestion (0.20 mg to 4.00 mg of lead per milliliter of drinking water). This suggests some limit to the amount of lead excreted by the kidney. The amount absorbed, however, must increase with quantity ingested, since retention by the kidney increases with dosage. The result of increasing doses of lead on hematological parameters is shown In Fig 1. The- packed red-cell volume appears to in- Fig 1.--Hematologic effects of increasing doses of lead in drinking water fed to rats for ten weeks. Fig 2.--Renal effects and body weight resulting from Increasing doses of lead in drinking water fed to rats for ten weeks. * V*'*,,*'* * V ' 1 ,3 AM1N0ACI0UR1A j \300 200 S+2S0- 100- URINART A AMINOLEVULINIC ACI0 EXCRETION fes as X+2S.0. X 0 .08 .2 .4 1.2 4.0 10 mg Pb/ml DRINKING WATER RENAL D GLUCURONIDASE ifimm s ijE ^ 300 p KIDNEY LEAD CONTENT 200 - 100 - 30 4 35.6 50p 30 L r e n al e d e ma /j!8^Y.yO \ BODY WT J n soar w e ig h t X+2S.D. X 1.2 4.0 10 rag Pb/ml DRINKING WATER Arch Environ Health--Vol 20, June 1070 DUP050312390 708 INTRANUCLEAR INCLUSION BODY--COYER ET AL crease in rats receiving small doses of lead, intranuclear inclusions. These bodies, easily but declines at higher dosage levels. At 4.0 recognized on histological examination in the mg of lead per ml of drinking water, reticu kidneys of rats fed 0.40 mg of lead per milli locytes and ALA excretion are increased, liter of drinking water, have a characteristic whereas significant anemia only occurred at uhr.'istruclurnl appearance. An electron pho the highest level of lead ingestion. tomicrograph of an inclusion body is shown A number of other parameters which re in Fig 4. The morphological and histochemi- flect injury to the kidney and effect on body cal features have been well described in the weight are presented in Fig 2. Amino-acid past, and recent study in this laboratory has excretion was more variable in the lead-fed shown that most of the lead retained by the rats than in the control animals, but is not kidney in lead poisoning fa located within constantly elevated except in the rats receiv these inclusion bodies. ing the highest dosage of lead. Other soft tissues in the rat have only a A few rats receiving 1.2 and 4.0 mg of small fraction of the kidney lead concentra lead per ml of drinking water had elevated tion (Table 2). Analysis of lead content of renal jS-glucuronidase levels, and even the organelles obtained by differential centrifu rats in the groups receiving the highest dose gation shows that the concentration of lead showed some overlap with controls. Incre in control rats is highest in nuclei in the ments In lead content of kidneys and renal kidney (Table 3). Increase in organelle lead edema expressed as content, therefore, is not uniform. The mito- kidney weight body weight show a gradual progression Fig 3.--Correlation of lead content of kidney and different doses of lead fed to rats for ten weeks with expression of lead toxicity. from control values. Al though renal edema com mences with low lead dosage, it is not clearly discernible from control levels until lead ingestion reaches 4.0 mg of lead per milliliter of drinking water. Body weight is also quite variable among rats ingesting smaller doses of lead, but rats receiving 1.2 mg lead per milliliter of drinking water have consis tently lower than normal body weight. The expression of lead ef fects at different dosage levels fa related to lead con tent of kidneys (Fig 3). Apart from irregular incre ments at doses of 0.08 and 4.0 mg of lead per ml of drinking water, the rise in renal lead content is predict able, although increments at the highest dosage (10 mg of lead per milliliter of drinking water) are quite variable. The most sensitive param eter of lead poisoning in the rat is the presence of Arch Environ Health--Vul 20, June 1970 ! i j i >t ! t i i in t r a chondrial increment in only three or four ti whereas, the increment times. Lead per milligrt more concentrated in t! sion bodies, and sugges crease in nuclear conte: of lead poisoned rats is tion of the intranuclear Comm The most sensitive ingestion of lead, apart lead content, is the f dear inclusion bodies, ness of this parameU limited. It is possible, 1 fig 4.--Nt water contair contains den: DUP050312391 J4 s. These bodies, easily V ical examination in the tO mg of lead per milli- : ; r, have a characteristic ance. An electron phoiclusion body is shown; . ') logical and liistochemii well described in the 1 r in this laboratory has le lead retained by the ' , cling is located within j.5 n the rat have only a kidney lead concentra- '11 ysis of lead content of < y differential centrifu- , ! concentration of lead . ;hest in nuclei in the i ' :rease in organelle lead j.,,,; not uniform. The mito- (!, ( ' dney and different doses pression of lead toxicity. 1NTRANUCLEA/i IXCfMSlOX BODY--COYER ET AL chondrial increment in lead-poisoned rats is only three or four times control values, whereas, the increment in nuclei is about 30 times. Lead per milligram of protein is even more concentrated in the intranuclear inclu sion bodies, and suggests that the large in crease in nuclear content of lead in kidneys of lead poisoned rats is related to the forma tion of the intranuclear inclusion bodies. Comment The most sensitive index of increased ingestion of lead, apart from actual urinary lead content, is the formation of intranu clear inclusion bodies. The clinical useful ness of this parameter is obviously very limited. It is possible, however, to recognize these inclusions in urine sediment from per sons with clinical lead poisoning.8 Increased urinary ALA excretion and retlculocytosis occur at the same dosage of lead as does renal tubular dysfunction manifested by amino-aciduria and renal edema. Similar levels of sensitivity to lead by the hemato poietic and renal systems may be only coin cidental. However, both femur (containing bone marrow) and kidney contain higher concentrations of lead than other organs in the rat and, perhaps even more important, the biochemical manifestations in both or gans are related to a common organelle, the mitochondrion. Evidence for involvement of the mitochondrion in the anemia of lead poisoning comes from two sources. Deltaaminolevulinic acid, a precursor of porphyrin. rig 4.--Nucleus of proximal renal tubular-lining cell from kidney of rat Ingesting water containing 10.0 mg of lead per ml for 10 weeks. Inclusion body (arrow) contains dense staining central core surrounded by fibrillary outer core (x 14,000). ........... -- -- - `400 100 ^ WATER DUP050312392 710 INTRANUCLEAR INCLUSION BODY--GOVEli ET AC Table 2.--Lead Content* 6/ Various Tissues in Control and Lead-Poisoned Rats Tissue Control Rats Rats Fed \% Lead Acetate 515 to 17 Weeks Femur Kidney Liver Heart Brain Blood (/ig/ml) [2.7 \2.4 fO.8 ll.O J0.4 \0.4 h.7 \l,4 /0.4 \0.4 ?0.1 10.2 731 666 84 54 5.5 4.8 0.7 0.9 1.8 1.9 1.8 1.6 * Wet weight; pg of lead per gm of tissue. Table 3.--Lead Content* of Cellular Organelles From Rat Kidney Control Rats Fed X% Lead Acetate 15 to 17 Weeks Whole kidney homogenate Mitochondria Nuclei Isolated inclusion bodies 0.0016*0.016(8) 0.034-0.056(3) 0.30.6(2) ... 0.53-1.10(4) 0.13-0.16(2) 11.5-15.7(4) 49.2-68.5(4) * Content, jig of lead per mg of protein. is synthesized within the mitochondrion of the reticulocyte, and increased ALA excre tion is thought to reflect impairment by lead of ALA dehydrase.9 The function of the enzyme, as well as other steps in heme synthesis, is dependent on intact mito chondrial respiration.10 Also, respiration of reticulocytes from bone marrow is decreased in lead-poisoned rats.11 Renal tubular dys function in lead poisoning is also believed to be related to impairment of mitochondrial function.12'14 Decrease in rate of body-weight gain is another sensitive indicator of the metabolic effects of lead. Possible contributing factors include decreased intestinal absorption, un coupled oxidative phosphorylation, and ex cessive loss of urinary amino acids. The latter two defects are clearly shown to be operative in rats receiving larger doses of lead, but changes in these parameters are not descemible at the small doses at which decreased weight gain becomes apparent. The pathology of lead poisoning in rats and humans is comparable with respect to the hematopoietic and renal organ systems. In humans, however, the central nervous system (CNS) is also very sensitive to the toxic effects of lead, particularly in children in whom an acute encephalopathy and coma are likely to be manifestations of acute in toxication. The severity of the CNS disease in humans is likely to preclude the develop ment of chronic renal disease. However, it has been suggested that acute childhood lead poisoning may influence the develop ment of chronic renal disease later in life.16 This relationship is not confirmed in anoth er similar study.10 Correlations between biochemical and clinical manifestations of lead poisoning have been conducted in humans particularly for the purpose of improving early diagnosis of lead poisoning among lead industry work ers. Increased ALA excretion and urinary lead content are both useful indicators of early lead intoxications.17'18 Also, the occur rence of amino-aciduria in acute lead intoxi cation in children is well documented,19 and Clarkson and Kench have demonstrated varying degrees of amino-aciduria in a group of lead factory workers.20 However, the relationship of renal tubular dysfunction with a parameter of heme metabolism has not to our knowledge been studied in hu mans. The urinary excretion of lead remains relatively stable over a wide range of lead dosage. The amount retained by the kidney progressively increases over this dosage range. Also, the increased retention of lead appears to be related to formation of intra nuclear inclusion bodies. Recent studies have shown that renal excretion of lead occurs by two mechanisms, glomerular filtration and Iranstubular excretion.21 Lead entering tubular-lining cells from adjacent capillaries is excreted into tubular fluid. The stability of the lead content of urine suggests that the amount of lead actually excreted by these renal routes is limited although renal con tent increase's. Measurement of lead content in organelles shows that the concentration within the nucleus increases more rapidly than within whole kidney homogenate or a suUvllular mitochondrial fraction. Studies on the composition of the intranuclear inclu sion hodits have shown that they are comj x w <1 of a lead-protein complex, alt-bough tlie nature of the protein is at present Arch Environ Health---Vat -0. June 1970 ! " , * i INT, unknown.5 Nevertheli accumulating lead w minimizing the conce the cytoplasm. A corollary of this ochondria have a lin Mitochondria isolatec acetate for ten weeks impairment of respirt tive abilities.14 The pi preparations of mito< rats fed a similar di. contain 0.13pg to 0.1 gram of protein. This ated with mitochondri per limits compatible studies in this labors the adenosine diphosj ed respiration of mito 1. Scliroeder HA, Tipi burden of lead. Arc* E 1968. 2. Patterson CC: Conte environments of mane Ar 358,1905. 3. Davis JR, Andelman levnlinie acid (ALA) lev. modified method for th urinary delta-aminolevuli ion-exchange chromatogr? ran Health 15:53-59, 1967. 4. Bessman SP, Layne the. determination of Ieac presence of organic chel; Med 45:159-166,1955. 5. Goyer RA, May P, protein content of isola' bodies from kidneys of Invest, to be published. 6. Gianetto R, de Duve the binding of acid phi and cathepsin by rat li 59:433-483.1959. 7. Kehoe RA: Normal Environ Health 8:232-234, 8. Landing B, Nakai B of renal lead-inclusions a urinary sediment. Amer 1959. 9. Chisolm JJ: Distort of heme in lead intoxica 1964. 10. Rimington C: Po i-j thesis and its control. Ai 445):ll-24,1966. 11. Lessler MA, Cardc DUP050312393 T AL the central nervous . very sensitive to the jrticularly in children ,`phalopathy and coma 'estations of acute in;y of the CNS disease preclude tire developl disease. However, it that acute childhood nfiuence the developdisease later in life.15 >t confirmed in anoth- en biochemical and is of lead poisoning n humans particularly roving early diagnosis lg lead industry workaccretion and urinary 1 useful indicators of s.iT.18 Also, the occuria in acute lead intoxiell documented,19 and * have demonstrated amino-aciduria in a r workers.20 However, al tubular dysfunction heme metabolism has ; been studied in hu- tion of lead remains a wide range of lead etained by the kidney 3S over this dosage ased retention of lead to formation of intraes. Recent studies have stion of lead occurs by tnerular filtration and i.21 Lead entering tuadjacent capillaries is fluid. The stability of rine suggests that the dly excreted by these d although renal conrement of lead content .hat the concentration nereases more rapidly tlney homogenate or a irial fraction. Studies the intranuclear inclum that they are comein complex, although protein is at present INTRANUCLEAR INCLUSION BODY--GOYER ET AL 711 unknown.6 Nevertheless, it must function by accumulating lead within the nucleus, thus minimizing the concentration of lead within the cytoplasm. A corollary of this hypothesis is that mit ochondria have a limited tolerance to lead. Mitochondria isolated from rats fed 1% lead acetate for ten weeks or longer show partial impairment of respiratory and phosphorylative abilities.14 The present study shows that preparations of mitochondria isolated from rats fed a similar diet for a longer period contain 0.13/u.g to 0.16/tg of lead per milli gram of protein. This amount of lead associ ated with mitochondria must be near the up per limits compatible with function. Recent studies in this laboratory have shown that the adenosine diphosphate-(ADP) stimulat ed respiration of mitochondria isolated from kidneys of normal rats is completely inhibit ed when incubated in vitro for ten minutes with 0.15/ug to 0.20/ig of lead per milligram of protein. Intranuclear complexing of lead must therefore limit transport or transtubu lar flow of. diffusible lead. Toxic injury to cytoplasmic organelles is thereby spared. This mechanism may be important in man's ability to tolerate continued exposure to trace or environmental quantities of lead. This investigation was supported by Public Health Service contract PH-43-68-74 from the National in stitute of Environmental Health, and Public Health Service grant AM 21061 from the National Institute of Arthritis and Metabolic Diseases. Nonproprietary and Trade Names of Drug Pentobarbital--Nembutal. A1. Schroeder HA, Tipton IH; The human References body Effect of lead on reticulocyte respiratory activity. J burden of lead. Arch Environ Health 17:965-977, Cell Biol 39:171a, 1968. 12. Goyer RA: The renal tubule in lead poison- 12. Patterson CC: Contaminated and natural lead-ring: I. Mitochondrial swelling and aminoaciduria, environments of man. Arch Environ Health 11:344- 'Lab Invest 19:71-77,1968. 358, 1965. 13. Goyer RA, Krall AR, Kimball JP: The renal . Davis JR, Andelman SL: Urinary delta-amino- - tubule in lead poisoning: II. In vitro studies of levulinic acid (ALA) levels in lead poisoning: I. A >. mitochondria structure and function. Lab Invest modified method for the rapid determination of 19:78-83,1968. urinary delta-aminolevuiinic acid using disposable t 14. Goyer RA, Krall RC: Ultrastruetural transEor- ion-exchange chromatography columns. Arch Enyi-i-mation in mitochondria isolated from kidneys ofr.j-c' ron Health 15:53-59,1967. normal and lead poisoned rate. I Cell Biol 41:393- - , 4. Bessman SP, Layne EC: A rapid procedure for 400, 1969. JKv the determination, of lead in blood or urine in the 15. Henderson DA: A follow-up of cases of A ' presence of organic chelating agents. J Lab Clin -Tplumbism in children. A list Ann Med 3:219-224, Med 45:159-166, 1955. 1954. 5. Goyer RA, May P, Cates M, et al: Head and s 16, Tepper KB: Renal function subsequent to \x' protein content of isolated intranuclear inclusion childhood plumbism. Arch Environ Health 7:76-85, Si bodies from kidneys of lead poisoned rats. Lab 19655- Invest, to be published. 6. -Gianetto R, de Duve C: Comparative study of 37. Creraer K, Selander S: Control of lead work ers by determination of urinary delta-aminolevulinic the binding1 of acid phosphatase, ^-glucuronidase add. Brit J Industr Med 24:283-288, 1967. and cathopsin by rat liver particles. Biochem J 18. Kretser AJ, Waldron HA: Urinary delta-ami- 59:433-483, 1959. vnolevulinic acid and porphobilinogen in lead-ex- Kehoe RA: Normal metabolism of lead. Arch posed workers. Brit J Industr Med 20:35-40,1963. Environ Health 8:232-234, 1984. 19. Chisolm JJ Jr: Aminoaciduria as a manifes- 8. Landing B Nakai H: Histochemical properties tation of renal tubular injury in lead intoxication P _ of renal lead-inclusions mid their demonstration in ^pnd a comparison with patterns of aminoaciduria Jf urinary sediment Atner J Clin Path 21:499-503, seen in other diseases. J Pediat 60:1-11,1962. 1959. 9 20. Clarkson TW, Kench JE: Urinary excretion 9. Chisolm JJ: Disturbances in the biosynthesis ^of amino acids by men absorbing heavy metals . of heme in lead intoxication. 7 Pediat 64:174-187, ^ (uranium, cadmium and lead). Biochem J 62:361- 1964. 372, 1956. . 10. Rimington C: Porphyrin and haem biosyn- 21. Vostal J, Heller J: Renal excretory mecha- thesis and its control. Acta Med Scand 179(suppl y-nisms of heavy metals: I. Transtubular transport of " 445):ll-24,1966. heavy metal ions in the kidney. Environ Res 2:1-10, v 131. Lessler MA, Cardona E, Padilla F, et al: 1968. Arch Environ Health--Vol 20, June 1970 DU P050312394