MEASUREMENT OF FRACTIONAL LIPID-SYNTHESIS USING DEUTERATED WATER ((H2O)-H-2) AND MASS ISOTOPOMER ANALYSIS

Fractional biosynthesis of palmitate, stearate, and cholesterol was de termined with deuterated water ((H2O)-H-2) using mass isotopomer analy sis in Hep G2 and MCA sarcoma cells in culture. The method employed di ffers from previous ones in that the number of deuterium atoms from (H 2O)-H-2 incorporated into newly synthesized molecules was determined a nd not assumed. After correction for background natural abundances, th e isotopomer distribution due to deuterium incorporation in fatty acid s and cholesterol was shown to follow a simple binomial distribution d epending on the deuterium enrichment in water (p) and the maximum numb er of deuterium atoms incorporated per molecule (N). Under a wide rang e of (H2O)-H-2 enrichments, N could be determined to be 17 for palmita te, 20 for stearate, and 20 for cholesterol by regression analysis or from a series of consecutive mass isotopomer ratios. The fraction deri ved from de novo synthesis was given by the ratio of the observed to t he theoretical deuterium enrichment, which is the product (N x p). The new synthesized fraction of palmitate and stearate by Hep G2 cells fo r the length of the experiment was found to be 77 and 65%, respectivel y. These values were confirmed by experiments with [U-C-13]glucose as the precursor. In MCA sarcoma cells grown in lipid-poor medium, the av erage values for fractional synthesis of palmitate, stearate, and chol esterol were 70, 35, and 70%, respectively. This approach should be ge nerally applicable to the simultaneous determination of fractional syn thesis of a number of compounds with either deuterium or C-13 tracers. Its application is only limited by the accuracy of mass spectrometric analysis.