Use of mass isotopomer distributions in secreted lipids to sample lipogenic acetyl-CoA pool in vivo in humans.
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.
Department of Nutritional Sciences, University of California, Berkeley 94720.
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Hellerstein
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Kaempfer
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Shackleton
D000085:Acetates / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008055:Lipids / Q000096:biosynthesis; D008079:Lipoproteins, VLDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008954:Models, Biological; D013420:Sulfamethoxazole / Q000378:metabolism; D015262:Xenobiotics
D000085:Acetates; D002247:Carbon Isotopes; D005227:Fatty Acids; D008055:Lipids; D008079:Lipoproteins, VLDL; D015262:Xenobiotics; D000105:Acetyl Coenzyme A; D013420:Sulfamethoxazole