Stable Isotope Tracer and Gas-Chromatography Combustion Isotope Ratio Mass Spectrometry to Study the in Vivo Compartmental Metabolism of Docosahexaenoic Acid

Abstract

A gas-chromatography combustion isotope ratio mass spectrometry (GCC-IRMS) method using carbon 13 ( 13C)-stable isotope to trace n-3 polyunsaturated fatty acids (PUFA) turnover in vivo is presented. Natural 13C 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 δ 13C‰ versus Pee Dee Belemnite (PDB), respectively. Precision of δ 13C‰ 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 [ 13C]22:6n-3 ([ 13C]22:6-TG) were synthesized by growing a microalgae on [l- 13C]glucose. [ 13C]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 [ 13C]22:6-TG and showed specific patterns according to the lipid compartments. The retroconversion of [ 13C]22:6n-3 was also detected in HDL phosphatidylcholine by the appearance of [ 13C]22:5n-3 and [ 13C]20:5n-3. On the other hand, 22:6n-3 natural 13C 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 13C-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.