Effect of Zinc Deficiency on Blood Glutathione Levels

Transcription

1 Effect f Zinc Deficiency n Bld Glutathine Levels BETTY J. MILLS, ROBERT D. LINDEMAN AND CALVIN A. LANG Luisville Veterans Administratin Medical Center and the Departments f Bichemistry and Medicine, University f Luisville Schl f Medicine, Luisville, KY ABSTRACT The specific effect f zinc deficiency n bld glutathine (GSH) cncentratins has nt been clearly demnstrated, fr earlier results culd have been due t differences in dietary intake. T clarify this, we determined bld GSH cncentratins ver a 17-day perid in yung, mature rats fed a zinc-deficient diet and cmpared them with pair-fed cntrls. Glutathine cncentratins based n hemglbin cntent increased in bth grups but at different rates. After 17 days, the GSH levels increased 20% in deficient rats and 29% in cntrl rats (P < 0.005). The GSH levels in deficient rats were 11% lwer than in cntrls by 10 days (P < 0.025) and remained significantly lwer thereafter. These results indicate that the decrease in bld GSH levels was a specific result f zinc deficiency. J. Nutr. Ill: , INDEXING KEY WORDS bld glutathine â zinc deficiency Changes in glutathine cncentratins have been bserved in tissues and in bld with age (1,2), diet (3,4), xenbitic administratin (5, 6) and disease states (7-9). Thus, the bld glutathine level may be a sensitive indicatr f alteratins in nrmal metablism. In a study t deter mine the effect f zinc deficiency n the rate f tumr grwth, we fund that bld glutathine (GSH) was decreased in rats that were bth tumr-bearing and zincdeficient cmpared t nrmal cntrls.1 The effect f zinc-deficiency alne was nt investigated. The nly ther study relating bld GSH levels and zinc status shwed that erythrcyte GSH was significantly lwer in zinc-deficient, weanling rats cmpared t cntrl rats fed ad libitum (10). Hw ever, the results were equivcal, fr the 2-fld greater fd intake by the cntrls culd accunt fr the GSH decrease. Our bjective was t determine the specific effect and time curse f zinc deficiency n bld GSH levels by the use f pair-fed animals. Yung, mature rats were used t minimize pssible interferences due t the rapid grwth f weanlings. METHODS Animals. Male rats f the Sprague- Dawley strain (Labratry Supply C., Indianaplis, IN) were hused individu ally in suspended stainless steel cages with screen bttms, with a waste-flushing munted system. n a rack Fr the zinc-deficient grups, cages, bttles, sip pers and feeders were rinsed with 10 mm EDTA slutin after they were washed. The rats were cnditined t the purified weighed cntrl diet fr 7 days and then and distributed int weightmatched grups that were fed ne f the diets described belw. Their age was 66 days and weight [mean ±SEM (number Received lr publicatin 25 Nvember Presented in abstract: Fed. Prc , I

2 BLOOD GLUTATHIONE IN ZINC DEFICIENCY 1099 f animals)] was 256 ±2.56 g (41) when the experimental diets were initiated. Rats fed either the cntrl r zincdeficient diet were killed at intervals thrughut a 17-day perid. They were anesthetized with 35 mg/kg ketamine hydrchlride (Ketaset, Bristl-Meyers C., Syracuse, NY), and bld was b tained by intracardiac puncture using dis psable plastic syringes with stainless steel needles cntaining heparin. The rats were then killed by cervical dis lcatin. Dietary treatment. The cntents f the basal, purified diet (Ralstn Purina C., Richmnd, IN) are listed in table 1. Tw grups f rats were pair-fed the fllwing diets: 1) a zinc-sufficient cntrl diet which cntained 26 ppm zinc and tap water cntaining less than 1 ppm zinc; r 2) a zinc-deficient diet which cntained less than 1 ppm zinc and deinized water. Strict pair-feeding measures ensured that cntrl rats received the same amunts f fd as their weight-matched cunter parts fed the zinc-deficient diet. Analysis f bld glutathine. Cncen tratins f glutathine (GSH) and hem glbin were determined using the methd f Beutler et al. (11). Acid-citrate-dextrse slutin (ACD) was added t freshlydrawn bld in the rati f 150 //,1/ml bld and thrughly mixed by gentle inversin f the tube. A 9.1% (v/v) hemlysate was prepared by adding 2.0 ml f water t 0.20 ml f the ACD sus pensin. T determine the hemglbin cncentratin, 0.10 ml f the hemlysate was added t 5.0 ml f a ferricyanidecyanide slutin, and the absrbance at 540 nm was measured. Three milliliters f metaphsphric acid (Mathesn, Cleman and Bell, Nrwd, OH) were added t the remaining 2.1 ml f hemlysate, mixed well and allwed t stand at rm temperature fr 15 minutes. The mixture was then filtered thrugh Whatman N. 1 paper (Whatman Bichemicals Ltd., Clif tn, NJ). T 0.30 ml f filtrate were added 0.70 ml f 0.5 M-Na2 HPO4 buffer, ph 9.0, and 0.10 ml f 5-5'dithibis-(2-nitrbenzic acid) and its absrbance at 412 nm was read within 10 minutes. Fr Cmpsitin TABLE 1 IngredientsSucrseEgg whiteslka FleRP mix'rp vitamin zinc)2dl-methininechline mineral mix (± chlridecrn illarddextrinbitin%by f the basal diet 1Prvides the fllwing amunts f ingredients (mg/kg diet): thiamin hydrchlride, 20; ribflavin, 20; niacin, 90; pyridxine hydrchlride, 20; D-calcium pantthenate, 60; flie acid, 4; D-bitin, 0.4; my-insitl, 200; vitamin B-12, 20 /xg; menadine sdium bisulfite, 20; vitamin A acetate, 22 lu/g; chlecalciferl, 2.2 lu/g; and DL-alpha tcpherl acetate, 50 lu/kg. 2 Five percent f the RP mineral mix prvides the fllwing mineral nutrients: calcium, 0.75%; phsphrus, 0.45%; ptassium, 0.46%; sdium, 0.29%; chlride, 0.29%; magnesium, 0.065%; manganese, 65 mg/kg; irn, 60 mg/kg; zinc, 20 mg/kg; cpper, 15 mg/kg; fluride, 5 mg/kg; cbalt, 3.2 mg/kg; chrmium, 3 mg/kg; idine, 0.6 mg/kg; mlybdenum, 0.8 mg/kg; and selenium, 0.2 mg/kg. each set f assays a standard curve using GSH (Sigma Chemical C., St. Luis, MO) in diluted meta-phsphric acid was prepared. Zinc analysis. Plasma btained by centrifugatin f heparinized bld at 500 x g fr 20 minutes was diluted 1/5 with 5% (v/v) glycerl and then analyzed fr zinc cntent by atmic absrptin spectrscpy (12). A standard f pled human serum was analyzed rutinely t ensure reprducibility f the prcedure. Statistical analysis. Cmparisns be tween Student's grups f-test were (13). perfrmed with the RESULTS AND DISCUSSION The results indicate that a decrease in bld GSH levels ccurred as a specific result f zinc deficiency. This decrease was nt due t general malnutritin r t differences in fd intake between the weight

3 1100 MILLS, LINDEMAN AND LANG 110 UJ 105 LU > 100 UJ cc 95 DAYS 10 ON DIETS Fig. l Relative weight changes f rats pair-fed cntrl and zinc-deficient diets. The 100% value was 256 ±2.56 g (41 rats). Each pint represents the mean ±SEM f fur t six rats. â â,cntrls; O O, zinc deficient. zinc-deficient and cntrl grups, fr their bdy weights were the same thrughut the experiment (fig. 1), and bth grups cnsumed the same amut f fd (17.8 g/day). Their kidney and heart weights als were the same, but the livers f the zinc-deficient rats were 20-30% heavier than the cntrls frm the 3rd t 13th day 15 (table 2). Since these livers appeared nrmal n grss examinatin, this dif ference is unexplained but may reflect a cmpensatry respnse t the zinc de ficiency. The bld GSH cncentratins f bth grups 17-day increased significantly experimental perid during the (P < 0.005) TABLE 2 Effect f zinc depletin n rgan weights f grupcntrl1zinc-deficientdays Dietary ndiet n. samples liver8.73 weightkidneyg ± 1All cntrl values were pled. 2 Mean ±SEM. 1P < cmpared t the cntrl grup a11.2 ± ±0.452" a11.5 ± 0.398"9.16 ± ±0.343Organ ± ± ± ± ± ± ±0.073Heart a P < 0.05 cmpared t the cntrl grup.

4 BLOOD GLUTATHIONE IN ZINC DEFICIENCY 1101 and prbably are related t maturatin f the animals (fig. 2). By the 10th day the GSH levels in zinc-deficient rats were 11% lwer than the cntrls, and this dif ference cntinued thrugh the 17th day (P < 0.05). In these experiments GSH cncentra tin was based n hemglbin cntent. Hwever, the changes in GSH cncentra tin were nt due t variatins in hem glbin levels, fr in bth grups the hemglbin cncentratins were nrmal and cnstant thrughut the experiment. The hemglbin cncentratins [mean ±SEM(number f rats)] were: cntrls, 13.5 ±0.348 g/dl (11); zinc-deficient, 13.6 ±0.198g/dl(27). Plasma zinc cncentratins were de creased t 65% f cntrl values by 3 days (P < ) and plateaued at 50% f the cntrl level after 5 days (fig. 2). Plasma zinc levels are knwn t decrease rapidly in respnse t dietary zinc de ficiency (14). There are several findings that relate glutathine cntent with zinc status. The decrease in GSH may be related t the bservatin that in zinc-deficient rats, there is a 7-fld increase in the urinary excretin f glutamic acid (15). Thus, there may be a decreased availability f this precursr f GSH. Als, zinc may have an imprtant rle in the maintenance f reduced glutathine. Evidence fr this is that zinc ins inhibited NADPH xidase activity in liver micrsmes (16) and this culd maintain NADPH levels necessary fr GSSG reductase activity and the re generatin f GSH. Finally, bld GSH and zinc have been implicated in the maintenance f erythrcyte membrane integrity. Zinc is an inte gral cmpnent f the membrane (17) and may play a rle in its stabilizatin, pssibly reacting with sulfhydryl grups t frm stable mercaptides (18). Further, GSH has been implicated as a membrane stabilizer since spntaneus hemlysis was related t glutathine xidatin (19). Fr these reasns, the prtective effect f zinc may be lst with zinc deficiency and result in a significant decrease in GSH. Further investigatin will be necessary S 80  u BLOOD GSH O \  >- CONTROLS ZN-DEFICIENT BLOOD GSH \ PLASMA ZINC 5 10 DAYS ON DIETS Fig. 2 Bld glutathine and plasma zinc cn centratins during zinc depletin. Upper graph, bld GSH zinc-deficient, cncentratins. Cntrls, â â ; O O. Lwer graph, relative cncentratins zinc-deficient f bld GSH and plasma zinc in rats. The plasma zinc cncentratin [mean ±SEM(number f rats)] fr the cntrl grup was 146 ±3.93  ig/dl(12). Bld GSH, O plasma zinc, Oâ â â O. Each pint represents O, the mean ±SEMf three t six rats. t determine whether the GSH decrease in zinc deficiency is due t its cnversin t xidized glutathine, r t a lss f ttal glutathine. ACKNOWLEDGMENT The authrs wish t acknwledge Pat rick J. Higgins fr his expert technical assistance. LITERATURE CITED 1. Abraham, E. C., Taylr, J. F. & Lang, C. A. (1978) Influence f muse age and erythrcyte age n glutathine metablism. Bichem. J. 774, Lambert, G. H. & Thrgeirssn, S. S. (1976) O 15

5 1102 MILLS, LINDEMAN AND LANG Glutathine in the develping muse liver. I. Develpmental curve and depletin after acet aminphen treatment. Bichem. Pharmacl. 25, Hsu, P. J. J. M., (1968) Anthny, W. Incrpratin L. & Buchanan, f glycine-l-14c int liver glutathine in zinc-deficient rats. Prc. Sc. Exp. Bil. Med. 127, Tateishi, N. & Higashi, T. (1978) Turnver f glutathine in rat liver. In: Functins f Glutathine in Liver and Kidney (Sies, H. & Wendel, A., eds.), pp. 3-7, Springer-Verlag, New Yrk. 5. Neish, W. J. P. & Rylett, A. (1963) Az dyes and rat liver glutathine. cl. 12, Bichem. Pharma 6. Byland, E. & Chasseaud, L. F. (1970) The effect f sme carbnyl cmpunds n rat liver glutathine levels. Bichem. Pharmacl. 19, Theil, G. B., Brdine, C. E. & Dlan, P. D. (1961) Red cell glutathine cntent and stabil ity in renal insufficiency. J. Lab. Clin. Med. 58, Gswitz, F., Lee, G. R., Cartwright, G. E. & Wintrbe, M.M. (1966) Erythrcyte reduced glutathine, glucse-6-phsphate dehydrgenase, and 6-phsphglucnic dehydrgenase in patients with myelfibrsis. J. Lab. Clin. Med. 67, Macdugall, L. G. (1968) Red cell lism in irn-deficiency anemia. J. metab Pediatr. 72, Hsu, J. M. (1976) Zinc as related t cystine metablism. In: Trace Elements in Human Health and Disease, vl. l (Prasad, A. S., ed.), pp , Academic Press, New Yrk. 11. Beutler, E., Durn, O. & Kelly, B. M. (1963) Imprved methd fr the determinatin f bld glutathine. J. Lab. Clin. Med. 61, Analytical Methds fr Atmic Absrptin Spectrphtmetry (1976) Perkins-Elmer Crp., Nrwalk, CT. 13. Snedecr, G. W. (1946) Statistical Methds, 4th à d.,pp , The Iwa State Cllege Press, Ames. 14. Mills, C. F., Quarterman, J., Chesters, J. K., Williams, R. B. & Dalgarn, A. C. (1969) Metablic rle f zinc. Am. J. Clin. Nutr. 22, Hsu, J. M. (1977) Zinc deficiency and alter atins f free amin acid levels in plasma, urine and skin extract. In: Zinc Metablism: Current Aspects in Health and Disease (Brewer, G. J. & Prasad, A. S., eds.), pp. 73â 83,Alan R. Liss Inc., New Yrk. 16. Chvapil, M., Ludwig, J. C., Sipes, I. G. & Misirwski, R. L. (1976) Inhibitin f NADPH xidatin and related drug xidatin in liver micrsmes by zinc. Bichem. Pharmacl. 25, Chvapil, M., Mntgmery, D., Ludwig, J. C. & Zukski, C. F. (1979) Zinc in erythrcyte ghsts. Prc. Sc. Exp. Bil. Med. 162, Chvapil, M. (1973) New aspects in the bi lgical rle f zinc: a stabilizer f macrmlecules and bilgical membranes. Life Sci. J3, Fegler, G. (1952) Relatinship between re duced glutathine cntent and spntaneus haemlysis in shed bld. Nature 170,