Sampling the lipogenic hepatic acetyl-CoA pool in vivo in the rat. Comparison of xenobiotic probe to values predicted from isotopomeric distribution in circulating lipids and measurement of lipogenesis and acetyl-CoA dilution

Abstract

The acetylation of xenobiotics may provide a means for sampling hepatic cytosolic acetyl-CoA in vivo for isotopic studies of lipogenesis. Here, we test the accuracy of acetylated-sulfamethoxazole (SMX) in representing the true precursor pool for hepatic lipogenesis by comparison to a mathematical technique for estimating acetyl-CoA enrichment using the mass isotopomer distribution in circulating lipids. We then go on to measure hepatic fatty acid synthesis in intact rats using stable and radioisotopes. Specific activities and enrichments of SMX-acetate (the latter determined by high performance liquid chromatography-mass spectrometry) were monitored during fasting and refeeding. The dilution rate of hepatic acetyl-CoA relative to infused 13C- or 14C-acetates was 0.158-0.200 mmol/kg body weight/min during fasting, and did not increase significantly in rats refed with intravenous glucose at 25-30 mg/kg/min or refed ad libitum with chow, suggesting little additional input of acetate units. Plasma beta-hydroxybutyrate specific activity was much lower than SMX-acetate. The isotopomeric frequency distributions in circulating very low density lipoprotein (VLDL)-palmitate and VLDL-stearate were used to estimate the enrichment of the true precursor, hepatic acetyl-CoA, from a model based on the binomial distribution. The calculated acetyl-CoA values (7.28 +/- 0.49 molar percent excess (n = 16] based on isotopomeric frequencies were very close to measured SMX-acetate enrichments (7.44 +/- 0.41 molar percent excess (n = 21] and values within individual animals (n = 14) correlated very well (r2 = 0.90, p less than 0.0001). The contribution of VLDL-fatty acid by the de novo lipogenic pathway was similar using the stable isotope approach or radioisotopes (only 1-2% in fasted or intravenous glucose refed rats, 5% in chow refed). Combining fractional de novo lipogenesis values with absolute de novo lipogenesis rates allows estimation of total VLDL-triglyceride synthesis. In conclusion, the xenobiotic acetylation technique provides continuous access to the lipogenic hepatic acetyl-CoA pool in vivo and permits measurement of fatty acid synthesis. Isotopomer ratios in secreted lipids provide another method for estimating true precursor acetyl-CoA enrichments.