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Glutathione and Its Redox System in Diabetic Polymorphonuclear Leukocytes
Glutathione and Its Redox System in Diabetic Polymorphonuclear Leukocytes S.N. CHARI, N. NATH, PH.D., AND A.B. RATHI
Abstract: The level of reduced glutathione in diabetic polymorphonuclear leukocytes was found to be significantly decreased compared to normal. Although the activity of glutathione reductase remained unchanged, the activity of glutathione peroxidase was found to be decreased in the diabetic state. Ketosis was not found to additionally aggravate the glutathione system. Polymorphonuclear leukocytes obtained from insulin-treated patients showed significant restoration. KEY INDEXING TERMS: Polymorphonuclear leukocytes, Diabetes mellitus, Glutathione, Glutathione reductase, Glutathione peroxidase. [Am J Med Sci 1984; 287(3):14-15.]
Introduction
he presence of large amounts of reduced glutathione (GSH)I and T iis redox system 2. in polymorphonuclear leukocytes (PMNL), is 3
stated to be of vital importance in the protection of these cells against oxidative injury during phagocytosis. 4 Although numerous earlier studies were made on the contents of glutathione and its related enzymes in leukemic leukocytes,s.6 no information is available yet regarding the glutathione system in diabetic PMNL. Moreover, the content of membrane sialic acid stated to be dependent on the content of GSH7 has recently been reported to be decreased in the diabetic PMNL. It was therefore thought pertinent to examine the content of GSH and the activities of glutathione reductase and peroxidase in diabetic PMNL. Patients and Methods Patients. Heparinized fasting blood samples were drawn from normal subjects and diabetic patients in the age group of 40 to 50 years. The diabetic patients were divided into three groups: with or without ketosis and with treatment. All the diabetic patients had positivc glucose tolerance tests: hence they were truly diabetic and not merely hyperglycemic. Nonketotic and ketotic patients had postprandial blood sugar levels of 300 mg% to 450 mg% and 400 mg% to 500 mg% respectively. with 1.5 g% to 2 g% sugar in urine. Ketotic patients had a qualitative presence of ketone bodies in urine . Treated diabetics were receiving insulin from the past five to seven years and had a postprandial blood sugar level of 110 mg% to 170 mg% with no sugar in urine. None of the above groups had any n urine . None of the above groups had any infection at the time of this study.
Frolll thl' DI'Iumllll'/11 of Biochl'lIIistry, Na8pllr Vl/il'('I'sity. Na8PIlr. RI'(lrilll RI'qm'st,\',' N. N(/(h. Ph.D . . Re(/{/('/'. D('IJartllll.'/11 of Biochelllistry. M.G. Ro(/c/, LIT Call1(llls. N(/}I(llIr-440 010. INDIA .
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Isolation of PMNL. The method was essentially that of Soyum8 with slight modifications. Heparinized blood was allowed to stand for 45 minutes at 20°C to 25°C, and plasma was aspirated and centrifuged at 170 x g for ten minutes. Pellets were suspended in Hank's balanced salt solution (with glucose) and placed on top of a ficoll-paque mixture and centrifuged at 400 x g for 40 minutes. The remaining erythrocytes were disrupted by hypotonic lysis, and 97% to 99% PMNL were obtained by this procedure as viewed under phase contrast microscopy. The entire operation was carried out in a cold environment (O°C to 4°C). Reduced glutathione was determined by the method of Woodward and Fry.9 Enzyme assay, For the assay of glutathione reductase, cells were suspended in 70% glycerine as suggested by Strauss et al lo and enzyme activity was monitored by a decrease in optical density in Gilford spectrophotometer at 340nm using oxidized glutathione as substrate. II Glutathione peroxidase levels were determined on sonicated leukocytes by the method of Paglia and Valentine with H20 2 as substrate. 12 Proteins were estimated by the method of Lowry et al,13 using bovine serum albumin as standard. Results It is evident from Table I that the activity of glutathione perox-
idase and the content of GSH were significantly decreased in the diabetic PMNL as compared to normal (p
CHARI ET AL
TABLE EFFECT OF DIABETES MELLITUS ON THE ACTIVITIES OF GLUTATHIONE REDUCTASE AND PEROXIDASE AND THE CONTENT OF REDUCED GLUTATHIONE OF POLYMORPHONUCLEAR LEUKOCYTES Glutathione Reductase 1.1. moles NADPH oxidizedlmin/gm protein
Subjects Nonnal (7) Diabetic Ketotic (7) Diabetic Nonketotic (7) Diabetic Treated (7)
2.61 2.46 2.46 2.48
± ± ± ±
0.41 O.4I NS 0 .50 NS 0 .42NS
Glutathione Peroxidase nM NADPH oxidizedl min/mg. protein 70.66 48.08 46.80 65.76
± ± ± ±
4.42 4.04* 4.01* 4.15t
Reduced Glutathione mg/gm protein 15 .85 ± 2.14 9.11±2.41* 9.14 ± 2.21* 13.42 :!: 3.04t
*= t=
P<.OI as compared to lIormal. P<.OI as compared to diabetic ketotic. NS = NOIl-sigllificant. Number oj subjects are showlI ill parentheses. Values showlI are meall ± SD.
the oxidative burst during phagocytosis, leading to a defect in microbial killing, and that glutathione peroxidase-deficient patients are more susceptible to infection.4 In view of the observed derangement in the activity of glutathione peroxidase of diabetic PMNL, it becomes possible to account for the impaired leukocytic function of phagocytosis so often noticed in diabetes. 19 The significant decrease in the activity of glutathione peroxidase without any alteration in the activity of glutathione reductase also implies a higher accumulation of peroxides in the diabetic PMNL. In fact, this contention gains support from our preliminary findings, where we observed an increased concentration of thiobarbituric acid-reacting substance in diabetic PMNL. The reason for the reduction in the activity of glutathione peroxidase in diabetic PMNL is not understood. Whether the diabetic PMNL incur an insufficient supply of selenium and thus get a diminution in the activity of this selenoenzyme,20 is a matter for future investigation. The present results, however, clearly indicate a marked defect in glutathione metabolism in the diabetic PMNL. It is also evident that the changes observed are due to insulin insufficiency since PMNL obtained from insulin-treated diabetic patients showed considerable restoration. Acknowledgment The authors wish to acknowledge Professor C.H. Chakrabarti, Professor & Head, Department of Biochemistry, Nagpur University, Nagpur, for valuable advice, and Shri P.K. Mehta for technical assistance. References I. Burchill BR, Oliver JM. Pearsoh CB. et al: Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J Cell Bioi 1978; 76:439-447. 2. Reed PW: Glutathione and the hexose monophosphate shunt in phagocytizing and H 20 2 -treated rat leukocytes . J Bioi Chem 1969; 244:2459-2464. 3. Bracci R, Calabri G. Bellni F, et al: Glutathione peroxidase in human
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
leukocytes. Clill Chem Acta 1970; 29:345-348 . 4. Roos D. Weening RS. Voetman AA. et al: Prolection of phagocylic leukocytes by endogenous glutathione: Studies in a family with glutathione reductase deficiency. Blood 1979; 53:851-856. 5. Pisciotta AV, Daly M: The reduced glutathione content of leukocytes in various hematological diseases. Blood 1960; 15:421-422. 6. Harrop KR. Jackson RC: Biochemical aspects ofleukemias: Lcukocytic glutathione metabolism in chronic granulocytic Icukcmia. Ellr J Callcer 1969; 5:61-67. 7. Warren L, Felsenfeld H: The biosynthesis of sialic acid. J Bioi Chl'm 1962; 237:1421-1431. . 8. Boyum A: Isocilation of mononuclear cells and granulocytes from human blood. SC(lIId J Clill Lab Illvest (Suppl( 1968; 21 :77-'109. 9. Woodward GE, Fry EG: The determination of blood glutathione. J Bioi Chem 1932; 97:465-482. 10. Strauss RR, Paul BB. Jacobs AA, etal:The role of the phagocyte in host parasite interaction XLL: Leukocytic glutathione rcductase and its involvement in phagocytosis . Arch Biochelll Biophy.l' 1969: 135:265-271. II. Hom HD: Glutathione reductase, in Bergmeyer HV (ed): Mf!thml.l' ill Ellzymatic Allalysis. New York. Academic Press. 1963. pp 875-879. 12. Paglia DE. Valentine WN: Studies on the quantitative and qualitalive characterization of erythrocytic glutathione peroxidase. J L(/b Clill Mecl 1967; 70:158-169. 13. Lowry OH. Rosebrough NJ, Farr AL. et al: Protein measurcment with the folin phenol reagent. J Bio Chem 1951 ; 193:265-275 . 14. Gandhi CR. Roychowdhary D: Effect of diabetic mellitus on sialic acid and glutathione content of human erythrocytes of differcnt agcs . II/(I J E.tpt Bioi 1979; 17:585-587 . 15. Chari SN. Nath N: Sialic acid content and sialidase activity of polymorphonuclear leukocytes in diabetcs mcllitus. Am J Ml'cI Sci. in press. 16. Tsan MF, Mcintyre PA: Surfacc control of hexose monophosphatc shunt in human polymorphonuclcar leukocytcs. Blo1Jl/1974; 44:927. 17. Holmes-Gray B. Haseman J, Buron S. et al: The relationship or glutathione levels and metabolism of human leukocYles. Ff!d Pmc 1971; 30:693(a). 18. Bass DA. DeChatlet LR. Burk RI. et al: Polymorphonuclear leukocYlic bactericidal activity and oxidative metabolism during glutathione peroxidase deficiency.IIlJectlmlllllllity 1977; 18:78-'104. 19. 8agdade JD, Walters E: Impaired granulocytc adherence in mildly diabetic patients. Diabetes 1980; 29:309-311. 20. Flohe L. Gunzler WA. Shock HH : Glutathione pcmxidase a selenoenzyme. FEBS Letters 1973; 32: 132-134 .
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Abstract: The level of reduced glutathione in diabetic polymorphonuclear leukocytes was found to be significantly decreased compared to normal. Although the activity of glutathione reductase remained unchanged, the activity of glutathione peroxidase was found to be decreased in the diabetic state. Ketosis was not found to additionally aggravate the glutathione system. Polymorphonuclear leukocytes obtained from insulin-treated patients showed significant restoration. KEY INDEXING TERMS: Polymorphonuclear leukocytes, Diabetes mellitus, Glutathione, Glutathione reductase, Glutathione peroxidase. [Am J Med Sci 1984; 287(3):14-15.]
Introduction
he presence of large amounts of reduced glutathione (GSH)I and T iis redox system 2. in polymorphonuclear leukocytes (PMNL), is 3
stated to be of vital importance in the protection of these cells against oxidative injury during phagocytosis. 4 Although numerous earlier studies were made on the contents of glutathione and its related enzymes in leukemic leukocytes,s.6 no information is available yet regarding the glutathione system in diabetic PMNL. Moreover, the content of membrane sialic acid stated to be dependent on the content of GSH7 has recently been reported to be decreased in the diabetic PMNL. It was therefore thought pertinent to examine the content of GSH and the activities of glutathione reductase and peroxidase in diabetic PMNL. Patients and Methods Patients. Heparinized fasting blood samples were drawn from normal subjects and diabetic patients in the age group of 40 to 50 years. The diabetic patients were divided into three groups: with or without ketosis and with treatment. All the diabetic patients had positivc glucose tolerance tests: hence they were truly diabetic and not merely hyperglycemic. Nonketotic and ketotic patients had postprandial blood sugar levels of 300 mg% to 450 mg% and 400 mg% to 500 mg% respectively. with 1.5 g% to 2 g% sugar in urine. Ketotic patients had a qualitative presence of ketone bodies in urine . Treated diabetics were receiving insulin from the past five to seven years and had a postprandial blood sugar level of 110 mg% to 170 mg% with no sugar in urine. None of the above groups had any n urine . None of the above groups had any infection at the time of this study.
Frolll thl' DI'Iumllll'/11 of Biochl'lIIistry, Na8pllr Vl/il'('I'sity. Na8PIlr. RI'(lrilll RI'qm'st,\',' N. N(/(h. Ph.D . . Re(/{/('/'. D('IJartllll.'/11 of Biochelllistry. M.G. Ro(/c/, LIT Call1(llls. N(/}I(llIr-440 010. INDIA .
14
Isolation of PMNL. The method was essentially that of Soyum8 with slight modifications. Heparinized blood was allowed to stand for 45 minutes at 20°C to 25°C, and plasma was aspirated and centrifuged at 170 x g for ten minutes. Pellets were suspended in Hank's balanced salt solution (with glucose) and placed on top of a ficoll-paque mixture and centrifuged at 400 x g for 40 minutes. The remaining erythrocytes were disrupted by hypotonic lysis, and 97% to 99% PMNL were obtained by this procedure as viewed under phase contrast microscopy. The entire operation was carried out in a cold environment (O°C to 4°C). Reduced glutathione was determined by the method of Woodward and Fry.9 Enzyme assay, For the assay of glutathione reductase, cells were suspended in 70% glycerine as suggested by Strauss et al lo and enzyme activity was monitored by a decrease in optical density in Gilford spectrophotometer at 340nm using oxidized glutathione as substrate. II Glutathione peroxidase levels were determined on sonicated leukocytes by the method of Paglia and Valentine with H20 2 as substrate. 12 Proteins were estimated by the method of Lowry et al,13 using bovine serum albumin as standard. Results It is evident from Table I that the activity of glutathione perox-
idase and the content of GSH were significantly decreased in the diabetic PMNL as compared to normal (p
CHARI ET AL
TABLE EFFECT OF DIABETES MELLITUS ON THE ACTIVITIES OF GLUTATHIONE REDUCTASE AND PEROXIDASE AND THE CONTENT OF REDUCED GLUTATHIONE OF POLYMORPHONUCLEAR LEUKOCYTES Glutathione Reductase 1.1. moles NADPH oxidizedlmin/gm protein
Subjects Nonnal (7) Diabetic Ketotic (7) Diabetic Nonketotic (7) Diabetic Treated (7)
2.61 2.46 2.46 2.48
± ± ± ±
0.41 O.4I NS 0 .50 NS 0 .42NS
Glutathione Peroxidase nM NADPH oxidizedl min/mg. protein 70.66 48.08 46.80 65.76
± ± ± ±
4.42 4.04* 4.01* 4.15t
Reduced Glutathione mg/gm protein 15 .85 ± 2.14 9.11±2.41* 9.14 ± 2.21* 13.42 :!: 3.04t
*= t=
P<.OI as compared to lIormal. P<.OI as compared to diabetic ketotic. NS = NOIl-sigllificant. Number oj subjects are showlI ill parentheses. Values showlI are meall ± SD.
the oxidative burst during phagocytosis, leading to a defect in microbial killing, and that glutathione peroxidase-deficient patients are more susceptible to infection.4 In view of the observed derangement in the activity of glutathione peroxidase of diabetic PMNL, it becomes possible to account for the impaired leukocytic function of phagocytosis so often noticed in diabetes. 19 The significant decrease in the activity of glutathione peroxidase without any alteration in the activity of glutathione reductase also implies a higher accumulation of peroxides in the diabetic PMNL. In fact, this contention gains support from our preliminary findings, where we observed an increased concentration of thiobarbituric acid-reacting substance in diabetic PMNL. The reason for the reduction in the activity of glutathione peroxidase in diabetic PMNL is not understood. Whether the diabetic PMNL incur an insufficient supply of selenium and thus get a diminution in the activity of this selenoenzyme,20 is a matter for future investigation. The present results, however, clearly indicate a marked defect in glutathione metabolism in the diabetic PMNL. It is also evident that the changes observed are due to insulin insufficiency since PMNL obtained from insulin-treated diabetic patients showed considerable restoration. Acknowledgment The authors wish to acknowledge Professor C.H. Chakrabarti, Professor & Head, Department of Biochemistry, Nagpur University, Nagpur, for valuable advice, and Shri P.K. Mehta for technical assistance. References I. Burchill BR, Oliver JM. Pearsoh CB. et al: Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J Cell Bioi 1978; 76:439-447. 2. Reed PW: Glutathione and the hexose monophosphate shunt in phagocytizing and H 20 2 -treated rat leukocytes . J Bioi Chem 1969; 244:2459-2464. 3. Bracci R, Calabri G. Bellni F, et al: Glutathione peroxidase in human
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
leukocytes. Clill Chem Acta 1970; 29:345-348 . 4. Roos D. Weening RS. Voetman AA. et al: Prolection of phagocylic leukocytes by endogenous glutathione: Studies in a family with glutathione reductase deficiency. Blood 1979; 53:851-856. 5. Pisciotta AV, Daly M: The reduced glutathione content of leukocytes in various hematological diseases. Blood 1960; 15:421-422. 6. Harrop KR. Jackson RC: Biochemical aspects ofleukemias: Lcukocytic glutathione metabolism in chronic granulocytic Icukcmia. Ellr J Callcer 1969; 5:61-67. 7. Warren L, Felsenfeld H: The biosynthesis of sialic acid. J Bioi Chl'm 1962; 237:1421-1431. . 8. Boyum A: Isocilation of mononuclear cells and granulocytes from human blood. SC(lIId J Clill Lab Illvest (Suppl( 1968; 21 :77-'109. 9. Woodward GE, Fry EG: The determination of blood glutathione. J Bioi Chem 1932; 97:465-482. 10. Strauss RR, Paul BB. Jacobs AA, etal:The role of the phagocyte in host parasite interaction XLL: Leukocytic glutathione rcductase and its involvement in phagocytosis . Arch Biochelll Biophy.l' 1969: 135:265-271. II. Hom HD: Glutathione reductase, in Bergmeyer HV (ed): Mf!thml.l' ill Ellzymatic Allalysis. New York. Academic Press. 1963. pp 875-879. 12. Paglia DE. Valentine WN: Studies on the quantitative and qualitalive characterization of erythrocytic glutathione peroxidase. J L(/b Clill Mecl 1967; 70:158-169. 13. Lowry OH. Rosebrough NJ, Farr AL. et al: Protein measurcment with the folin phenol reagent. J Bio Chem 1951 ; 193:265-275 . 14. Gandhi CR. Roychowdhary D: Effect of diabetic mellitus on sialic acid and glutathione content of human erythrocytes of differcnt agcs . II/(I J E.tpt Bioi 1979; 17:585-587 . 15. Chari SN. Nath N: Sialic acid content and sialidase activity of polymorphonuclear leukocytes in diabetcs mcllitus. Am J Ml'cI Sci. in press. 16. Tsan MF, Mcintyre PA: Surfacc control of hexose monophosphatc shunt in human polymorphonuclcar leukocytcs. Blo1Jl/1974; 44:927. 17. Holmes-Gray B. Haseman J, Buron S. et al: The relationship or glutathione levels and metabolism of human leukocYles. Ff!d Pmc 1971; 30:693(a). 18. Bass DA. DeChatlet LR. Burk RI. et al: Polymorphonuclear leukocYlic bactericidal activity and oxidative metabolism during glutathione peroxidase deficiency.IIlJectlmlllllllity 1977; 18:78-'104. 19. 8agdade JD, Walters E: Impaired granulocytc adherence in mildly diabetic patients. Diabetes 1980; 29:309-311. 20. Flohe L. Gunzler WA. Shock HH : Glutathione pcmxidase a selenoenzyme. FEBS Letters 1973; 32: 132-134 .
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