AJP - Endocrinology and Metabolism, Vol 261, Issue 4 E479-E486, Copyright © 1991 by American Physiological Society
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.
