Klin. Wschr. 53, 879--880 (1975) - © by Springer-Verlag 1975
Kurze wissenschaftliche Mitteilungen The Activity of Erythrocyte Glutathione Peroxidase in Chronic Renal Failure E. Mfiller, A. Blumberg and H.R. Marti Medizinische Klinik, Kantonsspital Aarau, Switzerland Received March 3, 1975
Die Aktivitgit der Erythrocyten-Glutathion-Peroxidase bei chronischer Niereninsuffizienz. Zusammenfassung. Bei 18 Patienten mit terminaler chronischer Niereninsuffizienz unter zweimaliger w6chentlicher H~imodialysebehandlung war die vor Dialyse gemessene Aktivit~it der GlutathionPeroxidase der Erythrocyten nicht beeintrfichtigt sondern im Vergleich zu Kontrollpersonen leicht erh6ht (2,7 bzw. 2,1 Einheiten/ p < 0,01). Dies wurde auf die Verkfirzung des Durchschnittsalters der Zellen zurfickgefiihrt. Schliisselwgrter: Glutathion-Peroxidase, Erythrocyt, H/imolyse, Niereninsuffizienz, H/imodialyse. Summary. In 18 patients with terminal chronic renal failure on twice weekly maintenance hemodialysis, the predialysis activity of glutathione peroxidase was not impaired but rather slightly but significantly elevated when compared with healthy controls (2.7 versus 2.1 units/p < 0.01). This was attributed to the reduced mean cell age. Key words: Glutathione peroxidase, erythrocyte, hemolysis, renal failure, hemodialysis.
Erythrocyte glutathione peroxidase (GSH-Px) is of importance for the protection of red cells against oxidative damage (Cohen et al., 1964). Decreased activity of this enzyme can shorten red celt lifespan [2, 5 - t 1]. In chronic renal failure the mechanism leading to red cell injury is still largely unknown. Recently Hopkins et al., 1973, have reported on a patient with chronic pyelonephritis due to long-term phenacetin ingestion, in whom GSH-Px activity was persistently low over a period of 2 years. In 4 other cases with uremia the enzyme activity was within the normal range (Hopkins et al., 1973), although shortening oferythrocyte lifespan should have been accompanied by an increase in enzyme activity (Blumberg etal., 1972). A study by Yawata etal., 1973, indicates that a plasma factor inhibits the hexose monophosphate shunt; the activity of this metabolic pathway is of importance for the formation of reduced glutathione (GSH), the donor substrate of GSH-Px. In the present report the activity of GSH-Px is studied in 18 patients with terminal renal failure on maintenance hemodiaIysis.
GSH-Px activities were measured in washed and hemolysed red cells using the NADPH-coupled reaction according to the modification of Floh6 et aI., 1970. Determinations were performed at 37°C and 340 nm in a Beckman-DU spectrophotometer with an automatic recording device. The activity unit (u) is referred to 1 gram of hemoglobin and is defined as the quantity of enzyme which per ml and per minute will induce a change of 1 in the decadic logarithm of GSH concentration. Results
The normal range (2 + 2s) of GSH-Px activity as determined in 26 healthy control persons was 2,] + 0,7 u. The predialysis enzyme activities of the 18 patients with renal failure are represented in Fig. 1.4 of the measurements fell into the middle and 10 into the upper part of the normal range, whereas 4 exceeded it. The mean GSH-Px activity for all the patients was 2,7, significantly above normal (p < 0.01). The patients exhibited a normochromic anemia with hemoglobin concentrations of 6 - 1 1 gram/100 ml and with reticulocyte counts as corrected to 5 x 106 erythrocytes/mm 3 of 3 22°/00 . No correlation was found between corrected reticulocyte counts and GSH-Px activity (r=0.13, not significant); however mean GSH-Px activity was higher in patients with reticulocyte counts above 12°/o0 than in those below that level (3.0 versus 2.4). Predialysis serum creatinine was 10 - 16, blood urea nitrogen 50 120 rag/100 rot. U
5-
4-
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I
2-
m
2s
o Q
Patients and M e t h o d s
2s 9 men and 9 women aged 28 - 60 years were studied. The underlying disease was chronic interstitial nephritis due to phenacetin consumption in 7, polycystic kidney in 5, chronic glomerulonephritis in 4 and chronic interstitial nephritis of unknown etiology in 2. All were dialyzed twice weekly for 7 - 8 hours with coil type dialyzers (Ultraflo II or EX-0t). Dialysate was prepared with softened underground water not containing any disinfecting additive. With one exception the patients received no transfusion, Blood samples were taken immediately before dialysis.
Fig. t. Activity of GSH-Px in 18 patients with chronic renal failure on maintenance hemodialysis (black dots) in comparison with the normal range ( x + 2 s )
880
Kurze wissenschaftliche Mitteilungen Discussion
In these studies no indication for a decrease in GSH-Px activity was found. The slight elevation of the mean enzyme activity is in accordance with the moderately shortened erythrocyte lifespan of patients on twice weekly hemodialysis (Blumberg et all, 1972). Earlier studies have shown that the red cell concentration of reduced glutathione (GSH) is not diminished and that plasma pH can be normalized, whereas glutathione reductase activityis significantly elevated in these patients (Blumberg et al., 1972; Wernze et aL, 1965). Consequently, in chronic renal insufficiency no reduction of the GSH-dependent protection of the erythrocyte against oxidative injury has been demonstrated so far. Literature
1. Blumberg, A., Marti, H.R. : Red cell metabolism and haemolysis in patients on dialysis. Proc. Europ. Dialysis Transpl. Assoc. 9, 91 (1972) 2. Boivin, P., Galand, C., Hakim, J., Rog6, J., Gu&oult, N. : An6mie h+molytique avec d6ficit en gtutathion-peroxydase chez un adulte. Enzymol. biol. clin. 10, 68 (I969) 3. Cohen, G., Hochstein, P.: Generation of hydrogen peroxide in erythrocytes by hemolytic agents. Biochemistry 3, 895 (1964) 4. Flohb, L., Brand, I. : Some hints to avoid pitfalls in quantitative determination of glutathione peroxidase (EC 1~11.1.9). Z. klin. Chem. und klin. Biochem. 8, 156 (1970) 5. Glader, B.E., Conrad, M.E. : Decreased glutathione peroxidase in neonatal erythrocytes: Lack of Relation to hydrogen peroxide metabolism. Pediat. Res. 6, 900 (1972)
6. Hopkins, J., Tudhope, G.R. : Gtutathione peroxidase in human red cells in health and disease. Brit. J. Haematol. 25, 563 (t973) 7. Necheles, T.F., Boles, T.A., Allen, D.M. : Erythrocyte glutathione-peroxidase deficiency and hemolytic disease of the newborn infant. J. Pediatrics 72, 319 (1968) 8. Necheles, T.F., Maldonado, N., Barquet-Chediak, A., Allen, D.M.: Hemozygous erythrocyte glutathione-peroxidase deficiency: Clinical and biochemical studies. Blood 33, 164 (1969) 9. Necheles, T.F., Steinberg, M.H., Cameron, D. : Erythrocyte glutathione-peroxidase deficiency. Brit. J. Haematol. 19, 605 (1970) 10. Nechetes, F. : The clinical spectrum of glutathione-peroxidase deficiency. In: Glutathione, Proc. 16th Conf. German Soc. Biol. Chem. Floh+, L., Ben6hr, H.Ch., Sies, H., Waller, H.D., Wendel, A., Eds. Georg Thieme Publishers, Stuttgart 1974 11. Nishimura, Y., Chida, N., Hayashi, T., Arakawa, T. : Homozygous glutathione-peroxidase deficiency of erythrocytes and leukocytes. Tohoku J, exp. Med. 108, 207 (1972) 12. Wernze, H., Koch, W.: Ober den Glutathion-(GSH)-Gehalt der Erythrocyten bei chronisch renaler Insuffizienz. Klin. Wschr. 43, 451 (1965) 13. Yawata, Y., Howe, R., Jacob, H.S. : Abnormal red cell metaboIism causing hemolysis in uremia. Ann. Int. Med. 79, 362 (1973)
Prof. Dr. H.R. Marti Chefarzt Medizinische Klinik Kantonsspital CH-5000 Aarau Switzerland
Kurze wissenschaftliche Mitteilungen The Activity of Erythrocyte Glutathione Peroxidase in Chronic Renal Failure E. Mfiller, A. Blumberg and H.R. Marti Medizinische Klinik, Kantonsspital Aarau, Switzerland Received March 3, 1975
Die Aktivitgit der Erythrocyten-Glutathion-Peroxidase bei chronischer Niereninsuffizienz. Zusammenfassung. Bei 18 Patienten mit terminaler chronischer Niereninsuffizienz unter zweimaliger w6chentlicher H~imodialysebehandlung war die vor Dialyse gemessene Aktivit~it der GlutathionPeroxidase der Erythrocyten nicht beeintrfichtigt sondern im Vergleich zu Kontrollpersonen leicht erh6ht (2,7 bzw. 2,1 Einheiten/ p < 0,01). Dies wurde auf die Verkfirzung des Durchschnittsalters der Zellen zurfickgefiihrt. Schliisselwgrter: Glutathion-Peroxidase, Erythrocyt, H/imolyse, Niereninsuffizienz, H/imodialyse. Summary. In 18 patients with terminal chronic renal failure on twice weekly maintenance hemodialysis, the predialysis activity of glutathione peroxidase was not impaired but rather slightly but significantly elevated when compared with healthy controls (2.7 versus 2.1 units/p < 0.01). This was attributed to the reduced mean cell age. Key words: Glutathione peroxidase, erythrocyte, hemolysis, renal failure, hemodialysis.
Erythrocyte glutathione peroxidase (GSH-Px) is of importance for the protection of red cells against oxidative damage (Cohen et al., 1964). Decreased activity of this enzyme can shorten red celt lifespan [2, 5 - t 1]. In chronic renal failure the mechanism leading to red cell injury is still largely unknown. Recently Hopkins et al., 1973, have reported on a patient with chronic pyelonephritis due to long-term phenacetin ingestion, in whom GSH-Px activity was persistently low over a period of 2 years. In 4 other cases with uremia the enzyme activity was within the normal range (Hopkins et al., 1973), although shortening oferythrocyte lifespan should have been accompanied by an increase in enzyme activity (Blumberg etal., 1972). A study by Yawata etal., 1973, indicates that a plasma factor inhibits the hexose monophosphate shunt; the activity of this metabolic pathway is of importance for the formation of reduced glutathione (GSH), the donor substrate of GSH-Px. In the present report the activity of GSH-Px is studied in 18 patients with terminal renal failure on maintenance hemodiaIysis.
GSH-Px activities were measured in washed and hemolysed red cells using the NADPH-coupled reaction according to the modification of Floh6 et aI., 1970. Determinations were performed at 37°C and 340 nm in a Beckman-DU spectrophotometer with an automatic recording device. The activity unit (u) is referred to 1 gram of hemoglobin and is defined as the quantity of enzyme which per ml and per minute will induce a change of 1 in the decadic logarithm of GSH concentration. Results
The normal range (2 + 2s) of GSH-Px activity as determined in 26 healthy control persons was 2,] + 0,7 u. The predialysis enzyme activities of the 18 patients with renal failure are represented in Fig. 1.4 of the measurements fell into the middle and 10 into the upper part of the normal range, whereas 4 exceeded it. The mean GSH-Px activity for all the patients was 2,7, significantly above normal (p < 0.01). The patients exhibited a normochromic anemia with hemoglobin concentrations of 6 - 1 1 gram/100 ml and with reticulocyte counts as corrected to 5 x 106 erythrocytes/mm 3 of 3 22°/00 . No correlation was found between corrected reticulocyte counts and GSH-Px activity (r=0.13, not significant); however mean GSH-Px activity was higher in patients with reticulocyte counts above 12°/o0 than in those below that level (3.0 versus 2.4). Predialysis serum creatinine was 10 - 16, blood urea nitrogen 50 120 rag/100 rot. U
5-
4-
•
I
2-
m
2s
o Q
Patients and M e t h o d s
2s 9 men and 9 women aged 28 - 60 years were studied. The underlying disease was chronic interstitial nephritis due to phenacetin consumption in 7, polycystic kidney in 5, chronic glomerulonephritis in 4 and chronic interstitial nephritis of unknown etiology in 2. All were dialyzed twice weekly for 7 - 8 hours with coil type dialyzers (Ultraflo II or EX-0t). Dialysate was prepared with softened underground water not containing any disinfecting additive. With one exception the patients received no transfusion, Blood samples were taken immediately before dialysis.
Fig. t. Activity of GSH-Px in 18 patients with chronic renal failure on maintenance hemodialysis (black dots) in comparison with the normal range ( x + 2 s )
880
Kurze wissenschaftliche Mitteilungen Discussion
In these studies no indication for a decrease in GSH-Px activity was found. The slight elevation of the mean enzyme activity is in accordance with the moderately shortened erythrocyte lifespan of patients on twice weekly hemodialysis (Blumberg et all, 1972). Earlier studies have shown that the red cell concentration of reduced glutathione (GSH) is not diminished and that plasma pH can be normalized, whereas glutathione reductase activityis significantly elevated in these patients (Blumberg et al., 1972; Wernze et aL, 1965). Consequently, in chronic renal insufficiency no reduction of the GSH-dependent protection of the erythrocyte against oxidative injury has been demonstrated so far. Literature
1. Blumberg, A., Marti, H.R. : Red cell metabolism and haemolysis in patients on dialysis. Proc. Europ. Dialysis Transpl. Assoc. 9, 91 (1972) 2. Boivin, P., Galand, C., Hakim, J., Rog6, J., Gu&oult, N. : An6mie h+molytique avec d6ficit en gtutathion-peroxydase chez un adulte. Enzymol. biol. clin. 10, 68 (I969) 3. Cohen, G., Hochstein, P.: Generation of hydrogen peroxide in erythrocytes by hemolytic agents. Biochemistry 3, 895 (1964) 4. Flohb, L., Brand, I. : Some hints to avoid pitfalls in quantitative determination of glutathione peroxidase (EC 1~11.1.9). Z. klin. Chem. und klin. Biochem. 8, 156 (1970) 5. Glader, B.E., Conrad, M.E. : Decreased glutathione peroxidase in neonatal erythrocytes: Lack of Relation to hydrogen peroxide metabolism. Pediat. Res. 6, 900 (1972)
6. Hopkins, J., Tudhope, G.R. : Gtutathione peroxidase in human red cells in health and disease. Brit. J. Haematol. 25, 563 (t973) 7. Necheles, T.F., Boles, T.A., Allen, D.M. : Erythrocyte glutathione-peroxidase deficiency and hemolytic disease of the newborn infant. J. Pediatrics 72, 319 (1968) 8. Necheles, T.F., Maldonado, N., Barquet-Chediak, A., Allen, D.M.: Hemozygous erythrocyte glutathione-peroxidase deficiency: Clinical and biochemical studies. Blood 33, 164 (1969) 9. Necheles, T.F., Steinberg, M.H., Cameron, D. : Erythrocyte glutathione-peroxidase deficiency. Brit. J. Haematol. 19, 605 (1970) 10. Nechetes, F. : The clinical spectrum of glutathione-peroxidase deficiency. In: Glutathione, Proc. 16th Conf. German Soc. Biol. Chem. Floh+, L., Ben6hr, H.Ch., Sies, H., Waller, H.D., Wendel, A., Eds. Georg Thieme Publishers, Stuttgart 1974 11. Nishimura, Y., Chida, N., Hayashi, T., Arakawa, T. : Homozygous glutathione-peroxidase deficiency of erythrocytes and leukocytes. Tohoku J, exp. Med. 108, 207 (1972) 12. Wernze, H., Koch, W.: Ober den Glutathion-(GSH)-Gehalt der Erythrocyten bei chronisch renaler Insuffizienz. Klin. Wschr. 43, 451 (1965) 13. Yawata, Y., Howe, R., Jacob, H.S. : Abnormal red cell metaboIism causing hemolysis in uremia. Ann. Int. Med. 79, 362 (1973)
Prof. Dr. H.R. Marti Chefarzt Medizinische Klinik Kantonsspital CH-5000 Aarau Switzerland
