Glutathione S-transferase in mucus of rat small intestine.

Glutathione S-transferases in the small intestine function in detoxification of electrophilic compounds ingested in foods, dietary supplements, and orally administered drug preparations. Although the required substrate glutathione (GSH) is synthesized in the intestinal enterocytes, the rate of synthesis is slow compared to both the maximal GST activity and the rate of uptake of luminal GSH. GSH is supplied to the intestinal lumen in the bile, and normal luminal concentrations in the rat are about 250 microM. The present study was designed to test the hypothesis that exogenous GSH is used for intestinal conjugation by glutathione S-transferase. The results show that 250 microM of extracellular GSH stimulated conjugation of 1-chloro-2,4-dinitrobenzene by approximately 300% in rat intestinal enterocyte preparations. However, an unexpected finding was that most of this stimulated activity did not depend upon uptake of GSH by the enterocytes but was due to glutathione S-transferase associated with mucus. Immunohistochemistry of glutathione S-transferase in the intact small intestine confirmed that a portion of the GST is present in the mucus layer. The presence of this detoxication enzyme in the extracellular mucus layer provides a novel mechanism for preventing direct contact of potentially toxic dietary electrophiles with the intestinal enterocytes.

Evaluation of methylated DNA binding protein-1 in mouse liver.

DNA methylation (DNA-5-methylcytosine [MeC]) plays a key role in regulation of gene expression. Highly methylated genes tend to be silenced whereas hypomethylated genes have an increased potential for expression. One way in which methylation may lead to inhibition of transcription is by permitting the binding of methylated DNA-binding proteins, which then block access of transcription factors to DNA. A particular methylated DNA-binding protein, MDBP-1, has been identified in nuclear extracts prepared from various human and rat tissues, and binds in a sequence-specific manner to DNA containing methylated cytosines (P. C. Supakar et al., 1988, Nucleic Acids Res. 16, 8029-8044). In the current study, an electrophoretic mobility shift assay (EMSA) was employed to assess the presence of MDBP-1 in protein isolated from mouse liver and to examine characteristics of its binding to DNA. The ligand used in our EMSA was a 32P-end-labeled, double-stranded, symmetrically methylated oligonucleotide containing the protein's binding site, 5'-CTAGATMGT-CAMGGMGAT-3' (M denotes 5-methyl-cytosine). Utilizing protein extracted from B6C3F1 mouse liver, we observed a complex that is competed by non-labeled, double-stranded, fully methylated ligand but not by an excess of a 26-mer, double-stranded oligonucleotide containing the binding site for AP-1. No complex was formed when a nonmethylated, double-stranded ligand, or our single-stranded ligands (methylated or unmethylated) were used as EMSA ligands. Complex formation was observed utilizing double-stranded, hemimethylated ligands; however, the affinity of MDBP-1 for these was one-tenth the affinity of MDBP-1 for fully methylated, double-stranded ligand. Additionally, protein isolated from C57BL/6 and C3H/He mouse liver was found to bind specifically to fully methylated ligand and hemimethylated ligands for MDBP-1, with similar characteristics as the binding of B6C3F1 mouse liver protein. These results indicate that MDBP-1 is present in mouse liver, and that the degree of methylation determines the strength of binding of MDBP-1 to DNA.

Glutathione measurement in human plasma. Evaluation of sample collection, storage and derivatization conditions for analysis of dansyl derivatives by HPLC.

Literature values for human plasma GSH vary over 10-fold despite the use of apparently valid analytical procedures for GSH measurement. The purpose of this study was to develop a procedure to minimize error in sample collection, processing and storage that could contribute to such differences. HPLC with fluorescence detection of dansyl derivatives was used for quantification. The results show that collection of blood with a butterfly needle and syringe reduces overestimation due to limited hemolysis and that use of a preservation solution designed to inhibit autooxidation and enzymatic degradation allows quantitative recovery of both GSH and GSSG. Stability tests showed that non-derivatized samples were stable for at least 2 months at - 80 degrees while dansyl derivatives were stable in the dark at 0-4 degrees for 12 months. Results from 59 healthy individuals (20-43 years) provided a mean (+/-1 SD) GSH value of 2.09+/-1.14 micromolar.

Glutathione in human plasma: decline in association with aging, age-related macular degeneration, and diabetes.

Blood samples were analyzed for GSH and GSH redox state in 40 age-related macular degeneration (ARMD) patients (> 60 y), 33 non-ARMD diabetic patients (> 60 years), 27 similarly aged non-ARMD and nondiabetic individuals (> 60 years), and 19 younger individuals (< 60 years) without ARMD or diabetes. Results showed a significantly lower plasma GSH in older individuals (ARMD, diabetes, and controls) than in younger individuals (p < .01). Total GSH (GSHt) obtained following treatment with dithiothreitol was significantly lower only in diabetic cases (p < .05) but also approached significance for ARMD cases (p = .089). Estimation of redox potential indicated that the plasma GSH pool is considerably more oxidized in all of the older groups. Analyses of whole blood GSH showed that GSH was significantly lower in diabetic cases compared to the other groups, but did not reveal any difference associated with age or ARMD. In contrast, GSSG in whole blood was significantly higher in the older groups compared to the younger controls. The results suggest that in studies of age-related pathologies, oxidation of GSH may be a more important parameter than a decline in pool size, while in specific pathologies such as diabetes, both oxidation and a decline in pool size may be important.