ABSTRACT
A new LC/MS method has been developed for the simultaneous measurement, in water and wastewater samples, of all species contained in commercial samples of linear type of alcohol ethoxylate (AE) surfactants including fatty alcohols. The method requires derivatization of the terminal hydroxyl of each surfactant species with 2-fluoro-N-methylpyridinium p-toluenesulfonate, which imparts a permanent cationic charge, allowing all species including the fatty alcohols and those with only one ethoxylate to be effectively detected by electrospray MS. Detection limits of typically <10 ppt for each individual species were attained in treated wastewater, in which total AE concentrations (combination of up to 114 individual species) are not expected to exceed 10 ppb. The method was validated for clean water as well as sewage influent and effluent samples.
Subject(s)
Alcohols/analysis , Environmental Monitoring/methods , Fatty Alcohols/analysis , Surface-Active Agents/analysis , Water Pollutants, Chemical/analysis , Chromatography, Liquid , Mass Spectrometry , Sensitivity and Specificity , Sewage/chemistry , Waste Disposal, FluidABSTRACT
AIMS/HYPOTHESIS: Substantial evidence suggests an important role for the expression of GLUT4 in adipocytes, in the pathogenesis of insulin resistance and Type II (non-insulin-dependent) diabetes mellitus. We investigated whether oxidative stress decreases GLUT4 expression by impairing DNA binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter. METHODS: 3T3-L1 adipocytes were exposed to micromolar H2O2 concentrations and GLUT4 expression and binding of nuclear proteins to defined DNA sequences were assessed. RESULTS: GLUT4 mRNA was decreased after at least 4 h exposure to H2O2, without a major change in the stability of GLUT4 transcripts. Nuclear protein extracts prepared from oxidized cells showed decreased binding to the insulin responsive element of the GLUT4 promoter but not to other DNA sequences. The direct effect of oxidation on the binding to the insulin response element was shown by the observation that in vitro oxidation of nuclear extracts with H2O2, n-ethylmaleimide or diamide decreased protein-DNA complex formation. This, and decreased binding capacity observed in nuclear extracts from oxidized cells, were partly reversible by subsequent treatment with a reducing agent. Protein binding to a consensus DNA sequence for nuclear factor 1 transcription factors was decreased 16 % by oxidation, whereas no change was observed in the protein content of several isoforms of these proteins. CONCLUSION/INTERPRETATION: Oxidative stress causes decreased GLUT4 expression, associated with impaired binding of nuclear proteins to the insulin responsive element in the GLUT4 promoter.
Subject(s)
DNA/metabolism , Insulin/physiology , Monosaccharide Transport Proteins/genetics , Muscle Proteins , Nuclear Proteins/metabolism , Oxidative Stress/physiology , Promoter Regions, Genetic/genetics , Response Elements/genetics , 3T3 Cells/drug effects , Animals , Consensus Sequence , DNA/genetics , Diamide/pharmacology , Ethylmaleimide/pharmacology , Glucose Transporter Type 4 , Hydrogen Peroxide/pharmacology , Mice , Monosaccharide Transport Proteins/metabolism , Oxidants/pharmacology , RNA, Messenger/metabolismABSTRACT
Data suggesting the involvement of increased oxidative stress in the pathophysiology of diabetes has raised interest in the potential therapeutic benefit of antioxidants. Although beneficial metabolic effects of antioxidant supplementation have been suggested, an antioxidant mode of action, particularly in skeletal muscle, has not been documented. In the present study, we evaluate the metabolic effects of a gamma-linolenic acid-alpha-lipoic acid conjugate (GLA-LA) in streptozotocin-induced diabetic rats, and assess its potential mode of action by comparing its effects with equimolar administration of LA and GLA alone. Ten days of oral supplementation of 20 mg/kg body weight GLA-LA, but not LA or GLA alone, caused a mild reduction in fasting blood glucose concentration as compared with vehicle-treated diabetic rats (375 +/- 11 vs. 416 +/- 16 mg/dl, p = 0.03), with no change in fasting plasma insulin levels. A peripheral insulin-sensitizing effect could be observed with GLA-LA, LA, and GLA treatments, as demonstrated by a significant (p < 0.04) 23%, 13%, and 10% reduction, respectively, in the area under the glucose curve following an intravenous insulin tolerance test. This effect was associated with a 67% and 50% increase in GLUT4 protein content in the membranes of gastrocnemius muscle of GLA-LA and LA-treated animals, respectively; however, no change was observed with GLA treatment alone. Interestingly, both GLA-LA and LA treatments corrected a diabetes-related decrease in the gastrocnemius muscle low-molecular-weight reduced thiols content. These data demonstrate insulin-sensitizing properties of the GLA-LA conjugate by distinct mechanisms attributable to each of its components, which are associated with antioxidant effects.
