Pediat. Res. 11: 153-157 ( 1977) ATPasc lead poisoning glucose sodium intestinal absorption Experimental Lead Poisoning and Intestinal Transport 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 "pica" 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 enzyme 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 food intake was measured daily. tial amounts of a soluble lead salt. 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 nitrogen (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), calcium, 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 nervous system, 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 blood cell membrane 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 ); brain out with two I 0-ml portions of warm O. 15 M NaCl. The proxi­ metabolism (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 salts 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 phosphorus (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<OI scraped with a glass slide and immediately homogenized in a ••• P< 001 glass homogenizer with a Teflon pestle (Tri-R Instruments (67)) 150 with four volumes of 0.15 1\1 NaCl. The homogenate was centri­ fuged at 700 x g and frozen at -50°. A similar treatment was applied to kidney and liver specimens. The homogenates were thawed for the determination of total ATPase and (Na+-K+)­ ATl'ase (31 ). fructose-I .(1-diphosphatase (3Ci). succinic dehy­ 100 drogenase (8). pyruvate kinase ( l 0). tryptophan hydroxy lase (55). and protein (34). In other experiments, the integrity of the intestinal mucosa was assessed by perfusing the animals as indicated above with l''C]-PEG or with ul''Cj-aminoisobutyric acid (AIB) in tracer 50 levels added to the standard amounts of the marker or the amino acid in the buffer. After a 2 hr perfusion, blood specimens ,,ere taken from the suborbital sinus of the rats and deproteinized with an equal volume of 2 1\1 HClO.,. The supernatant was counted in a liquid scintillation spectrometer (5(1). The I' 'C]­ AIB uptake of the intestinal mucosa was determined by measur­ 2 4 10 20 40 80 mM ing the radioactivity taken up by that tissue and expressing it Fig.