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Studies were conducted using cultured immortalized wild type (non-CF) and mutant (CF) DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) tracheal epithelial cells on the anti-inflammatory impact of agents that may alter ceramide and glutathione (GSH) metabolism. The CF cells demonstrated abnormally high levels of GSH and glutathione disulfide (GSSG), which could diminish intracellular production of ceramide, a key modulator of inflammation and apoptosis. Hence, additional cell culture studies were carried out with a known inducer of in situ ceramide synthesis, N-4(4-hydroxyphenyl) retinamide (fenretinide) on interleukin (IL)-8 release, intracellular ceramide content, and cellular proliferation in both the basal state and following the inflammatory stimuli of tumor necrosis factor (TNF) -alpha. Fenretinide treatment was associated with a dose-dependent increase in the cellular content of ceramide in both CF and non CF cells. Also, an inhibition of IL-8 release in the inflammatory condition of TNF-alpha treatment was observed following fenretinide treatment in the CF cells. As hyperbaric treatment of whey proteins was previously associated with improved survivability and higher GSH content in a Pseudomonas aeruginosa murine model of cystic fibrosis (CF), the anti-inflammatory role of low molecular weight peptides (< 1kDa) generated from enzymatic hydrolysis of native and pressurized whey protein isolates (WPI) was examined. Pressure treatment of WPI was associated with an enhanced protein digestibility and an altered peptide profile following in vitro digestion. The whey peptides were tested CF and non-CF lung epithelial cells to identify for their effects on GSH metabolism. The impact of the combined treatment of fenretinide and WPH was also tested in terms of apoptosis and cytokine release in cell culture. As opposed to non-CF cells, CF cells showed a strong downtrend in release of IL-8 following the combined fenretinide and whey peptide treatment. In addition, whey peptides protected wild type epithelial cells from the apoptotic effect of fenretinide. Our results suggest the usefulness of these agents as a pharmacological treatment in CF.

We developed a methodology to measure plasma hepatic glutathione (GSH) turnover and we tested it in rats treated with an inhibitor of GSH synthesis. Our goal was to determine whether protein intakes above NRC recommendations maximize hepatic GSH stores and turnover in vivo. We also wished to learn if plasma GSH, cysteine, or methionine concentrations or plasma GSH turnover could be used as noninvasive predictors of liver GSH status. Rats were fed purified diets containing 0, 5, 10, 20 or 40% casein for one week. The 0 and 5% casein diets were considered inadequate in protein, 10% marginal, 20% adequate and 40% excessive. Liver GSH content (mmol/liver) of rats fed 0 and 5% casein diets was 12.29 $ pm$ 1.11 and 16.43 $ pm$ 0.95, respectively, and increased to 23.62 $ pm$ 1.82 in the 10% group. Liver GSH content did not differ between the 20 and 40% groups. As dietary casein increased from 0-20%, free plasma GSH and cysteine concentrations and plasma GSH turnover increased, but did not increase further with the 40% diet. A sigmoidal relationship between plasma GSH turnover and hepatic GSH content was demonstrated. The best predictor of liver GSH content was not free plasma GSH concentration nor plasma GSH turnover, but the free plasma cysteine concentration.

Hydrostatic pressure has been demonstrated to induce major changes in secondary structure of whey proteins resulting in an increased digestibility in vitro, and possibly an improvement of the glutathione (GSH) inducing effect of whey proteins in vivo. Micro filtration and ion-exchange, two commonly used processing techniques in whey protein manufacture, generate whey proteins with different compositions. Two animal studies were designed to compare the digestibility and GSH inducing effects of whey protein isolates (WPIs) treated with three repeated pulse cycling of pressure (3-cycle) or single pulse of high pressure (1-cycle) and pressurized microfiltrated and ion-exchange WPIs. The results indicate that special hydrostatic pressure treatment on the proteins improves its growth stimulating effect, but does not enhance the GSH-inducing effect of WPI in the healthy growing rats. Difference among commercial whey protein products is also an important factor that affects the biological properties of the pressurized whey proteins. In conclusion, both proper pressure treatment and product composition should be considered in order to find the most bio-effective whey protein preparation.

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To examine the effects of fat composition and supplemental vitamin E (Vit E) and selenium (Se) on in vivo lipid peroxidation, diet-induced hypercholesterolemia, and glutathione (GSH) metabolism, male Syrian hamsters were fed for three weeks butter fat (BF-) or fish oil- (FO-)based diets supplemented with Vit E and/or Se. The effect of supplemental Vit E and Se on tissue lipid peroxidation (LPO), glutathione peroxidase (GSH-Px) activity and GSH concentrations differed between heart and liver and also was affected by dietary fat. The reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was more consistently associated with tissue lipid peroxidation than was tissue Vit E content. Plasma lipids were lowered with supplemental Se and Vit E. Se supplementation, however, exerted a more potent hypolipidemic effect than Vit E. A pro-oxidative action of Se in hearts of FO-fed hamsters was noted, which was inhibited by supplemental Vit E. Hence, the combination of Vit E and Se may offer the most benefit against diet-induced oxidative stress and hyperlipidemia.

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The hypothesis to be tested states that the pathology of Alzheimer's disease (AD) involves elevated levels of oxidative stress, resulting in elevated levels of cellular oxidative defense mechanisms. If the premise is true, than AD pathologically afflicted cells should have a higher demand for glutathione (GSH) as an innate oxidative defense mechanism hence; greater GSH concentrations, increased GSH resynthesis capabilities, and increased levels of cystathionine gamma-lyase (CNase). Alzheimer diseased and age matched control lymphoblast cells, obtained from OHSU's Oregon Brain Aging Study, were cultured, and GSH biochemistry was subsequently evaluated. GSH was depleted by exposing cells to the GSH depleting agent diethylmaleate (DEM) and the resulting GSH concentrations were measured. GSH resynthesis was measured after depleting GSH with DEM, to a level of approximately half base GSH concentration, then removing the depleting agent, resuspending the cells in fresh medium (DEM-free), and subsequently measuring GSH levels. GSH concentrations were measured by HPLC, and all data was normalized to cellular protein concentration. Cellular CNase specific activity levels were measured by adding cytasthionine, the CNase substrate, and then measuring the amount of cysteine produced by means of the DTNB assay. The AD cell lines showed no increase in base levels of GSH as compared to control cell lines. The AD cell lines showed a statistically significant increase in GSH resynthesis capabilities and cystathionine gamma-lyase specific activity levels. These findings add further weight to the AD oxidative stress hypothesis, which is based on the premise that the causative agent of AD pathogenesis is an increase in the level of cellular free radicals produced.