USE OF STABLE ISOTOPICALLY LABELED TRACERS FOR STUDIES OF METABOLIC KINETICS

利用稳定同位素研究底物代谢动力学

Studies of substrate metabolic kinetics using stable isotope labeled tracers require accurate and precise isotopic enrichment measurements. The gas chromatograph/mass spectrometer (GC/MS) is a versatile instrument for analysis of metabolic tracers but is less precise or accurate than isotope ratio mass spectrometers (IRMS). It may be inconvenient to convert target metabolites to gases (2H2, 13CO2, 15N2) analyzed by IRMS, and GC/MS permits measurements of enrichment of single components within biological mixtures. The limited accuracy and precision of GC/MS places restrictions on tracer infusion protocols, and large doses of stable isotope tracers must be infused over long periods to achieve detectable enrichment. Typically, tracer to tracee ratios (TTR) of 3-5% must be achieved. Stable isotope tracer studies could benefit from improved accuracy and precision of GC/MS measurements. We find it is possible, under carefully selected conditions, to measure very low isoto pic enrich ments (TTR 0.001-0.1%) by GC/MS. Obesity is a major US public health problem because of its increasing prevalence and causal relationship with many medical illnesses. Metabolic diseases associated with obesity and aging, such as type II diabetes mellitus, dyslipidemia, and coronary artery disease, are related to abnormalities in glucose and lipid metabolism. Body fat mass and abnormalities in glucose and lipid metabolism also increase with aging. We have evaluated independent effects of obesity and aging on fat and carbohydrate metabolism in humans in vivo using stable isotope tracers and GC/MS. By combining isotope tracer, microdialysis, and abdominal vein catheterization techniques, we developed a four-compartment model of adipose tissue glycerol kinetics. We find that fat oxidation is decreased and carbohydrate oxidation increased during moderate intensity exercise in elderly compared to young adult subjects, even though lipolysis rates were similar. These data suggest an ag ing-induced impairment in skeletal muscle fat oxidation. Exercise training normalized fat oxidation in elderly subjects. In patients with severe upper-body obesity, we find that diet-induced weight loss decreases whole body lipolytic rates. During short-term fasting, obese women exhibit blunted lipolytic and plasma insulin responses and reduced norepinephrine spillover, an index of sympathetic nervous system activity.