Blood glutathione status during exercise: effect of carbohydrate supplementation

Blood glutathione status and activities of antioxidant enzymes have been investigated during prolonged exercise with or without carbohydrate (CHO) supplementation. Eight subjects cycled at approximately 70% of maximal oxygen uptake to fatigue [134 +/- 19 (SE) min] on the first occasion (control, CON) and at the same work load and duration on the second occasion but with CHO ingestion during exercise. Blood reduced glutathione (GSH) concentration increased from 0.55 +/- 0.05 mM at rest to 0.77 +/- 0.09 mM after 120 min of exercise during CON (P < 0.01) but remained constant during CHO exercise. Blood glutathione disulfide (GSSG) levels were unchanged during CON and CHO exercise. Blood GSH + GSSG content and GSH/GSSG ratio were also significantly (P < 0.05) elevated during CON but not during CHO exercise. The increases in GSH and GSH + GSSG in CON were associated with decreases in plasma glucose and insulin levels. Activities of blood GSH peroxidase, GSSG reductase, and glucose-6-phosphate dehydrogenase were significantly increased during the CHO exercise, whereas only GSSG reductase activity was elevated during the CON ride. It is concluded that blood GSH increases during prolonged exercise and that CHO supplementation may prevent blood GSH increase possibly because of its inhibitory effects on hepatic hormonal releases, which stimulate GSH output.

Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity

Glutathione status and antioxidant enzymes in various types of rat skeletal muscle were studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus (SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscle GSH increased proportionally with Ex intensity and reached 1.8 +/- 0.08 mumol/g wet wt compared with 1.5 +/- 0.03 (P < 0.05) in resting controls (R). GSSG in DVL was increased from 0.10 +/- 0.01 mumol/g wet wt in R to 0.14 +/- 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereas GSH/GSSG ratio was unchanged. Activities of GSH peroxidase (GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutase activity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increases in GPX, GR, and CAT activities in DVL muscle. None of the antioxidant enzymes was affected by exercise in SO. It is concluded that rat DVL muscle is particularly vulnerable to exercise-induced free radical damage and that a disturbance of muscle GSH status is indicative of an oxidative stress.

Absorption and lymphatic transport of peroxidized lipids by rat small intestine in vivo: role of mucosal GSH

The absorption and lymphatic transport of peroxidized MaxEPA fish oil was studied using the lymph fistula rat to determine the role of mucosal glutathione (GSH) in intestinal metabolism of luminal lipid hydroperoxides. Decreasing intestinal GSH concentrations with buthionine sulfoximine (BSO, 1.15 +/- 0.20 nmol/g), diethyl maleate (DEM, 0.93 +/- 0.26 nmol/g), phorone (1.46 +/- 0.14 nmol/g), or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 1.54 +/- 0.18 nmol/g) compared with control (2.60 +/- 0.38 nmol/g) resulted in higher luminal recovery of the infused lipid hydroperoxide (% of infused dose): BSO (87.8 +/- 4.8%), DEM (86.1 +/- 1.3%), phorone (78.1 +/- 2.1%), and BCNU (71.7 +/- 4.8%) compared with control (52.8 +/- 4.3%). These results suggest that decreased elimination of luminal peroxidized lipids is associated with decreased tissue GSH. Treatment of rats with BSO, DEM, phorone, or BCNU resulted in dramatic increases in appearance of peroxidized lipids in lymph over 6-h lipid infusion (54.7 +/- 3.7, 57.7 +/- 4.6, 46.4 +/- 2.7, and 42.1 +/- 3.9 nmol, respectively) compared with control (20.5 +/- 3.4 nmol). The results are consistent with decreased intracellular metabolism of absorbed hydroperoxides and enhanced transport into lymph under GSH-deficient conditions. The current findings suggest that the function of the mucosal GSH peroxidase/oxidized glutathione (GSSG) reductase system may play an important role in intestinal handling of luminal lipid hydroperoxides. A compromised function of this detoxication mechanism in GSH-deficient states can significantly alter the metabolic fate of dietary peroxidized lipids.

The radiosensitivity of rat thymocytes

gamma-Irradiation in vitro apparently blocked a plasma-membrane associated, superoxide-producing, NADPH oxidase in rat thymocytes. Differential centrifugation of the mixed thymocytes indicated the smaller lymphocytes (approx. 6 microns diameter) to be the radiosensitive population. The oxidase system co-isolated in part with thymus nuclei and could be solubilized by detergent treatment [Bellavite, Jones, Cross, Papini & Rossi (1984) Biochem. J. 223, 639-648]. Endogenous NADPH was the rate-limiting component for superoxide formation in vitro. The level of NADPH was lowered by gamma-irradiation, an effect mimicked by GSSG in the presence of 50 microM-ZnCl2 to inhibit GSSG reductase. These findings are suggested as the metabolic basis for interphase death of small lymphocytes exposed to ionizing radiation.

Regulation of the pentose phosphate cycle

1. A search was made for mechanisms which may exert a `fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP+) show that at physiological concentrations of free NADP+ and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn2+ did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg2+ and NADP+ before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

Interrelationship of glutathione–cystine transhydrogenase and glutathione reductase in developing rat intestine

1. Glutathione reductase and glutathione–cystine transhydrogenase activity in supernatant fractions of whole homogenates and homogenates of mucosal and muscular layers were determined in developing rat intestine after determination of the optimum conditions for assay of the two enzymes. In jejunum from adult rat, the Km values for GSSG reductase and GSH–cystine transhydrogenase activities were 0.25mm-GSSG and 0.23mm-cystine respectively. 2. The two activities could be differentiated by stability studies since GSSG reductase was stable at 60°C for 10min and could be stored at 4°C for 24h without loss of activity. GSH–cystine transhydrogenase, on the other hand, was denatured at 60°C and completely inactive after 24h storage at 4°C. 3. Based on calculations of total activities, both enzymes increased from the eighteenth day until the animals were young adults. 4. Total GSSG reductase activity increased at a greater rate with age than total GSH–cystine transhydrogenase activity as evidenced by activity ratios for GSH–cystine transhydrogenase/GSSG reductase of 0.44 and 0.12 in ileum from suckling and adult rats respectively, and 0.31 and 0.24 in jejunum from suckling and adult rats respectively. 5. In mucosa from adult rats GSSG reductase was more active in the ileum than in the jejunum, whereas GSH–cystine transhydrogenase activity was higher in the jejunum. 6. GSH–cystine transhydrogenase was active only in the muscle cells of the ileum of 7-day-old rats but became localized primarily in the mucosal layer in the adult rat. However, GSSG reductase activity was distributed evenly between the two layers throughout the intestine.

The Regeneration of Reduced Glutathione in Normal and Glucose-6-Phosphate Dehydrogenase Deficient Human Red Blood Cells

Reduced glutathione (GSH) was rapidly regenerated in normal human red blood cells treated with the GSH oxidizing agent, methyl phenylazoformate. Erythrocytes of G-6-PD deficient males regenerated little, if any, GSH under the same conditions. The rate of regeneration of GSH in erythrocytes of G-6-PD deficient heterozygote females was similar to that of a mixture of normal red blood cells and erythrocytes of G-6-PD deficient males. It was compatible with the assumption of mosaicism of the erythrocytes in the heterozygote females. The rapid rate at which the normal erythrocyte can regenerate its GSH may render it capable of continuously absorbing free radicals derived from drugs without harmful consequences to the cell. Study of the rate of regeneration of GSH in erythrocytes treated with methyl phenylazoformate may be useful in the detection of deficiencies of G-6-PD, GSSG reductase, and hexokinase.