Calvin A. Lang

The Effect of Aging on Glutathione and Cysteine Levels in Different Regions of the Mouse Brain

Abstract A general glutathione (GSH) deficiency occurs in many tissues of the aging mouse. However, there is no information on GSH in the aging brain even though it has been involved in a number of neurobiologic reactions. To this end, C57BL/6 mice, 3–31 months old, representing the growth, maturation, and aging periods of the life-span were studied. Brain cortex, hippocampus, and stem samples were dissected, processed, and analyzed specifically for reduced and oxidized glutathione (GSH, GSSG) and cyst(e)ine using high performance liquid chromatography with dual electrochemical detection. The GSH content of each brain region varied in the order brain cortex > brain hippocampus > brainstem. However, the GSH profiles of all regions were the same through the life-span, namely, high values during growth dropping to a maturation plateau and then decreasing 30% during aging. In contrast to GSH, the order of cysteine levels was brain cortex < brain hippocampus < brainstem and no life-span changes occurred in any region. In addition, the brain GSSG and cystine contents of all regions were very low and did not change during the life-span. Thus, the GSH loss was not accountable by oxidation to GSSG or degradation to cyst(e)ine. Altogether these results demonstrated a GSH deficiency in brain tissues of aging mice like that found previously in other tissues. These findings suggest an increased susceptibility of the aging brain to oxidative damage.

Differential distribution of free and bound glutathione and cyst(e)ine in human blood

The redox status of free and bound glutathione (GSH) and cyst(e)ine (Cys) is altered by oxidative stress, drugs, and disease. Most studies measure only their free forms and not the bound forms, which may have a crucial protective role. For this reason, we determined free and bound, reduced and oxidized GSH and Cys in whole blood, red cells, and plasma of human blood from healthy adults. Distinct compartments of GSH and Cys were found. In whole blood, > 99% GSH was in red cells, of which 16% was bound. GSH values were the same for red cells in whole blood or in cells isolated from the same samples. Only 0.5% of GSH was in plasma, all of which was bound. In contrast, 97% of Cys was in plasma and only 3% in red cells. This was a remarkable separation of these closely related metabolites in the same tissue. In plasma, 60% of Cys was bound. Also, strong correlations were shown of bound vs free Cys and also vs free plus bound Cys. The bound Cys was more constant and suggested that it is a metabolic reserve. Our findings demonstrate the occurrence of significant bound forms of GSH and Cys and have implications for future studies in disease and toxicology.

Glutathione and morbidity in a community-based sample of elderly

This study examined the association of blood glutathione level, a potential marker of physiological/functional aging, with a number of biomedical/psychological traits in a subgroup (N = 33) of a representative sample of community-based elderly. Higher glutathione levels were associated with fewer number of illnesses (p < 0.05), higher levels of self-rated health (p < 0.01), lower cholesterol (p < 0.05), lower body mass index, and lower blood pressures. Subjects with diagnoses of arthritis, diabetes, or heart disease (as assessed by physicians) had at least marginally significant lower glutathione levels than those who were disease free. Glutathione, together with age and a measure of suppressed anger, accounted for 39% of the variance of an index of morbidity. Glutathione, by itself, accounted for 24% of the variance. To our knowledge, this is the first evidence of an association of higher glutathione levels with higher levels of physical health in a sample of community-based elderly. Further studies in large samples are needed to investigate glutathione as a potential overall health risk factor for morbidity among the elderly.

Glutathione peroxidase and reductase activities in the aging mouse

Previous results from this laboratory demonstrated that glutathione concentrations decrease in aging mouse tissues. In this investigation glutathione (GSH) peroxidase and glutathione (GSSG) reductase activities were measured in tissues of standardized aging mice. Methods were validated for the quantitative determination of both enzymes in liver, kidney and heart tissues. GSH peroxidase activities were 27-53% lower in liver, kidney and heart of very old (36 months) mice compared to mature (10 months) mice (P less than 0.01). The same aging decreases were found with either hydrogen peroxide or cumene hydroperoxide as substrate. In a similar way GSSG reductase activities in liver and kidney were 25-28% lower in the old versus mature mice (P less than 0.01), but heart levels were unchanged. Further the lower GSSG reductase levels were unaffected by FAD supplementation in vitro. The changes in specific activity for both enzymes were not due to changes in organ weights and total protein contents, which were constant from 10 to 36 months of age. These decreases in GSH peroxidase and GSSG reductase do not account for the lower GSH levels in aging. Of special importance, however, is that these decreases indicate that detoxification via glutathione peroxidase and glutathione reductase could be impaired in senescence.

Sample processing alters glutathione and cysteine values in blood

The accurate assessment of glutathione status of blood is essential for its use as an index of health and aging. A major variable in glutathione analysis is sample processing, and identification of optimal standard conditions is needed. Thus our objective was to evaluate several methods to determine which one yields maximal levels of free and bound glutathione and cyst(e)ine in blood. Reduced glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), and cystine were analyzed specifically by an HPLC-dual electrochemical method. The highest GSH levels were found in ultrafiltrates of hemolysates, which were 58% greater than those in acid extracts of whole blood, and accounted for 96% of the free and bound GSH in borohydride-reduced samples; GSSG was undetected. The next highest values were in acid extracts of hemolysates which were 13% greater than in extracts of whole blood; both extracts contained GSH and GSSG. Their GSSG contents expressed in GSH equivalents comprised 7-9% of GSH + GSSG. Cys levels were highest in ultrafiltrates which were 11-fold greater than in acid extracts of whole blood, accounting for 62% of the total cyst(e)ine pool. In summary, the results indicate that ultrafiltration of hemolysates is the blood processing method of choice to obtain maximal values of free and bound GSH and cyst(e)ine.

Protein glutathiolation in human blood.

Glutathione (GSH) exists in both free and protein-bound (glutathiolated) forms (GSSP). Protein glutathiolation may represent an important post-translational regulatory mechanism for proteins. However, there are little data regarding the regulation of glutathiolation in blood. Our objectives were to examine GSSP levels of human blood by determining the distribution and variability of blood GSSP, as well as its relationship to free GSH and hemoglobin in healthy adults. To this end, we used a newly modified method allowing for rapid analysis of both GSH and GSSP in blood. GSSP was found in red cells with levels ranging from 4 to 27% of total (free+bound) GSH (mean+/-SD: 12.1+/-4.5%) with a concentration of 0.13+/-0.05 microEq GSH/mL (mean+/-SD). No correlations were observed between GSSP and either GSH (r=-0.085) or hemoglobin (r=0.086). Together these results suggest that the extent of protein glutathiolation in blood is substantial ( approximately 0.1 mmol/L). While the interindividual variation in GSSP is large (34%), its levels are apparently not regulated by GSH content.

Magnesium Deficiency Inhibits Biosynthesis of Blood Glutathione and Tumor Growth in the Rat

Abstract Previously we found that blood glutathione (GSH) levels increase in response to tumor growth in the rat and that this increase is not prevented with zinc deficiency. We also found that zinc deficiency which inhibited tumor growth did not prevent this increase in blood GSH. Therefore, the objectives of this study were to determine the effects of another nutritional modification, namely magnesium deficiency, on blood GSH status and on tumor growth. Magnesium was selected because it is an obligatory cofactor in GSH synthesis and in all biosynthetic reactions involving ATP. To this end, magnesium- and zinc-deficient rats with and without tumors were compared to pair-fed control rats with and without tumors. After 32 days of depletion, the rats were killed, and blood samples were analyzed for nonprotein sulfhydryls (SH) and specifically for GSH. The key finding was that in magnesium-deficient rats with or without tumors, blood GSH levels were low and SH levels were normal indicating a decrease in GSH biosynthesis. In contrast, zinc deficiency affected SH and GSH in parallel. Thus, these two deficiencies must act by different mechanisms. The zinc data verified our earlier results obtained with a different tumor type and rat strain, for blood GSH levels increased in tumor-bearing rats fed control diets, and zinc deficiency did not prevent this increase. Depletion of magnesium or zinc was equally effective in inhibiting tumor growth. These results provide in vivo evidence of a magnesium requirement for GSH biosynthesis in rat erythrocytes. Further, the results suggest that magnesium deficiency may inhibit tumor growth by limiting GSH synthesis from SH precursors.

Correction of a glutathione deficiency in the aging mosquito increases its longevity.

The decrease of tissue glutathione (GSH) concentrations in different senescent organisms gave rise to our hypothesis that a glutathione deficiency is a biochemical cause of the aging process. A rigorous test of this notion would be the correction of the deficiency and concomitant increase in life span. To this end, adult mosquitoes were fed magnesium thiazolidine-4-carboxylic acid, and their GSH levels and life spans were determined. The GSH levels increased 50–100% (P < 0.005) regardless of the age when feeding was initiated or whether the feeding period extended over 2 days or the entire life span. Also the median life spans increased 30–38% over control values (P < 0.005). The responses were specific for the thiazolidine carboxylate moiety, because MgCl2 had no effect. These findings confirm the GSH deficiency hypothesis and demonstrate a specific biochemical mechanism of aging that can be nutritionally modified.

Acetaminophen-induced depletion of glutathione and cysteine in the aging mouse kidney

Glutathione (GSH) plays an essential role in the detoxification of acetaminophen (APAP) and the prevention of APAP-induced toxicity in the kidney. Our previous results demonstrated that a GSH deficiency is a general property of aging tissues, including the kidney, suggesting a hypothesis that senescent organisms are at greater risk to APAP-induced renal damage. To test this, C57BL/6NIA mice of different ages through the life span were injected with various doses of APAP, and the extent of GSH and cysteine (Cys) depletion and recovery were determined. At time intervals up to 24 hr, kidney cortex samples were obtained, processed and analyzed for glutathione status, namely GSH, glutathione disulfide (GSSG), Cys and cystine, using an HPLC method with dual electrochemical detection. In the uninjected controls, GSH and Cys concentrations decreased about 30% in the aging mouse, but the GSSG and cystine levels were unchanged during the life span. APAP administration depleted the kidney GSH and Cys contents in a dose- and time-dependent manner. Four hours after APAP administration, GSH levels of the young, growing (3- to 6-month) and the mature (12-month) mice decreased 34 and 58%, respectively, and recovered to near control values by 24 hr (95 and 98%). In contrast, the extent of depletion in old (31-month) mice was greater (64%) and the 24-hr recovery was less, returning only to 56%. Likewise, Cys levels of the young and mature mice decreased 49 and 65%, respectively, 4 hr following APAP, and increased to 99 and 85% by 24 hr. In contrast, in old mice, there was a 78% depletion after 4 hr followed by a recovery of only 65% by 24 hr. These results demonstrated clearly that in the aging mouse kidney, a GSH and Cys deficiency occurs that is accompanied by an impaired APAP detoxification capacity.

Glutathione monoethyl ester protects against glutathione deficiencies due to aging and acetaminophen in mice

Our previous results indicated that glutathione (GSH) and/or cysteine (Cys) deficiency occurs in many aging tissues and also after acetaminophen (APAP) administration. The aim of this study was to investigate whether GSH monoethyl ester (GSH-OEt) can correct these deficiencies. Mice of different ages (3-31 months) through the life span were sacrificed 2 h after i.p. injection of GSH-OEt (10 mmol/kg). In separate experiments, old mice (30-31 months) received the same dose of ester 30 min before the administration of APAP (375 mg/kg) or buthionine sulfoximine (BSO, 4 mmol/kg), an inhibitor of GSH synthesis. Liver and kidney samples were analyzed for GSH and Cys by HPLC. The hepatic GSH and renal cortical GSH and Cys concentrations were about 30% lower in old mice (30-31 months) compared to mature mice (12 months). GSH-OEt corrected these aging-related decreases. APAP decreased both hepatic and renal cortical GSH and Cys concentrations in old mice, but GSH-OEt prevented these decreases. GSH-OEt also prevented the BSO-induced decreases in hepatic and renal GSH concentrations. The results demonstrated that GSH-OEt protected against GSH deficiency due to biological aging as well as APAP-induced decreases in old mice.