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1 Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA
2 Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco General Hospital, San Francisco, CA, USA
3 Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA; Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco General Hospital, San Francisco, CA, USA
* To whom correspondence should be addressed. E-mail: March{at}nature.berkeley.edu.
A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow turnover lipids. The glycerol moiety of TG is labeled from 2H2O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4% or 8% 2H2O in drinking water; body of 2H2O enrichments were stable at ca. 2.5 or 5.0%. TG-glycerol was isolated from adipose depots and liver during up-to 12-weeks of 2H2O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by gas chromatography/mass spectrometry. The combinatorial pattern (ratio of double- to single-labeled molecules) revealed the number of H-atoms in glycerol incorporating label from 2H2O(n) to be 3.8-4.0 of a possible 5 (or 80% of maximal incorporation) for adipose tissue and 4.6-4.8 for 10 week labeled liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4-4.6 x body 2H2O enrichment) indicating near-complete replacement of the liver TG pool, thereby confirming the combinatorial calculations and excluding dilution by unlabeled alpha-glycerol phosphate in newly synthesized TG. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster (60% newly synthesized at 7 d, k = 0.21 d-1) than other depots (k = 0.04-0.06 d-1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85-90% of calculated maximal values at 12 weeks (k = 0.05 d-1), excluding significant dilution by unlabeled alpha-glycerol phosphate. Turnover of plasma TG, modeled from 2H incorporation over 60 min, was 0.06 min-1 (half-life 11.5 min). In summary, use of of 2H2O labeling with MIDA of TG-glycerol allows measurement of new 
-glycerol-phosphate derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.
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