STABLE-ISOTOPE TRACER AND GAS-CHROMATOGRAPHY COMBUSTION ISOTOPE RATIO MASS-SPECTROMETRY TO STUDY THE IN-VIVO COMPARTMENTAL METABOLISM OF DOCOSAHEXAENOIC ACID

A gas-chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) method using carbon 13 (C-13)-stable isotope to trace n-3 polyunsaturated fatty acids (PUFA) turnover in vivo is presented. Natural C-13 abundance of commercial n-3 PUFA was measured from 100 to 300 ng of fatty acids and was -27.58, -27.83, and -28.16 for 22:6n-3, 22:5n-3, and 20:5n-3, expressed as delta(13)C parts per thousand versus Pee Dee Belemnite (PDB), respectively. Precision of delta(13)C parts per thousand values was comparable for the three PUFA and gave relative standard deviations of 0.95-0.97%. Isotope enrichment of 0.0010 at.% could be detected. Triglycerides enriched in [C-13]22:6n-3 ([C-13]22:6-TG) were synthesized by growing a microalgae on [l-C-13]glucose. [C-13]22:6n-3 represented 36 wt.% of total triglyceride fatty acids and had an isotope enrichment of 2.0420 at.%, which was the double of natural abundance. The isotope enrichment of 22:6n-3 in lipids from rat lipoproteins and red cells could be followed as a function of time after ingestion of 3 mg [C-13]22:6-TG and showed specific patterns according to the lipid compartments. The retroconversion of [C-13]22:6n-3 was also detected in HDL phosphatidylcholine by the appearance of [C-13]22:5n-3 and [C-13]20:5n-3. On the other hand, 22:6n-3 natural C-13 abundance in human lipid classes of lipoproteins and blood cells has been measured using 10 ml plasma, even for the more limiting lipid compartments in terms of 22:6n-3 dose size. It is concluded that C-13-enriched n-3 PUFA along with GCC-IRMS can be used to study fatty acid fluxes in vivo, and that this method represents a convenient alternative to the utilization of radiotracer fatty acids in humans. (c) 1994 Academic Press, Inc.