Betty J. Mills

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.

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.

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.

Dietary nordihydroguaiaretic acid increases the life span of the mosquito.

Abstract Our previous findings indicated that a major characteristic of aging organisms is a decrease in reducing capacity. Our objectives were to correct this impairment by administration of nordihydroguaiaretic acid (NDGA), a potent reducing agent, and to determine the effect on adult longevity of the mosquito. NDGA supplements were included in the axenic larval medium or adult diet of mosquitoes of different ages. The mean adult life spans of both sexes increased 42-64% over controls (P < 0.025), and the most effective doses were 0.001% for females and 0.005% for males. This NDGA effect was dependent on the age when feeding was initiated, since only biosynthetically active larvae and young adults were responsive. Also the effect was not due to dietary restriction. These results confirm the life span-enhancement effect of NDGA using defined conditions and establish the importance of redox status in the aging process.

The Verification of a Mammalian Toxicant Classification Using a Mosquito Screening Method

Routine toxicity screening of substances using rodents can be time consuming and expensive. For these reasons various alternative methods have been developed, but many lack standardization and acceptance. It was hypothesized that a satisfactory classification of mammalian toxicants could be obtained using a simple mosquito (Aedes aegypti) screening method. To this end, median lethal concentration (LC50) values were determined for a wide variety of mammalian toxicants representing a range of toxicity classes. Each substance was assayed in aqueous solution by incubation for different times, up to 24 hr, using at least 30 individual larvae and six different concentrations, for valid statistical analysis. With few exceptions, the resultant toxicity classes were the same as those determined using mammalian models. This classification was validated further by the very high correlation (r = 0.97) determined between the mosquito LC50 values and the rat LD50 values. These results suggest that this mosquito method can be used to rapidly screen compounds of potential mammalian toxicity.