Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans

M. K. Hellerstein, C. Kletke, S. Kaempfer, K. Wu and C. H. Shackleton Department of Nutritional Sciences, University of California, Berkeley 94720. Measurement of hepatic fatty acid (FA) and cholesterol synthesis has been limited by lack of access to the precursor pool, cytosolic acetyl-CoA. We present a method for inferring the enrichment of the true hepatic lipogenic precursor pool in humans using the frequency distribution of mass isotopomers within enriched circulating polymers of acetyl-CoA [very low-density lipoprotein (VLDL)-palmitate, VLDL-stearate]. Human subjects were infused intravenously (n = 16) with [1-13C]- or [2-13C]acetate. Oral sulfamethoxazole (SMX) was administered concurrently, and the acetylated conjugate (SMX acetate) was used to estimate independently the hepatic cytosolic acetyl-CoA enrichment. Isotopomer frequencies in VLDL-FA were determined by gas chromatography-mass spectrometry, whereas high-performance liquid chromatography-mass spectrometry was used to measure enrichments in SMX acetate. Based on the excess M2/excess M1 ratio in VLDL-FA, calculated acetyl-CoA enrichments were 5.59 +/- 0.33 molar percent excess (MPE), whereas SMX acetate enrichments were 5.38 +/- 0.31 MPE (the 2 methods were not significantly different). Mass isotopomer-calculated and SMX acetate-measured estimates of acetyl-CoA enrichments correlated very closely in individual subjects (r2 = 0.93; P less than 0.0001). De novo hepatic lipogenesis can be measured using isotopomer-calculated precursor enrichments compared with measured incorporation in specific isotopomers of VLDL-FA. In summary, excess isotopomer frequencies in secreted lipids provide a non-invasive technique for estimating hepatic cytosolic acetyl-CoA enrichments in humans in vivo and correlate closely with enrichments observed using the xenobiotic probe technique. Isotopomeric distributions represent a new strategy for accurate measurement of macromolecule synthesis that may be applicable to other classes of molecules besides lipids.