NMR and isotope ratio mass spectrometry studies of in vivo uptake and metabolism of polyunsaturates by the developing rat brain

Abstract

This article describes the application of in vivo 13C-nuclear magnetic resonance (NMR) spectroscopy and gas chromatography (GC)-combustion-isotope ratio mass spectrometry to the study of brain uptake and metabolism of polyunsaturated fatty acids in the suckling rat model. NMR spectroscopy is uniquely suited to the non-invasive detection of nonradioactive metabolites in living animals. We applied this approach to the noninvasive detection of 13C-arachidonate in brain and liver of living suckling rats but found that technical limitations in our model, mainly poor signal-to-noise, largely prevent useful results at this time. However, in a tracer study using simultaneous doses of 13C-gamma-linolenate and 13C-arachidonate, 13C-NMR of tissue lipid extracts quantitatively demonstrated a 10-fold greater (liver) or 17-fold greater (brain) accumulation of pre-formed vs newly synthesized arachidonate. GC-combustion-isotope ratio mass spectrometry was used to trace the utilization of [U-13C]-alpha-linolenate into three products in the brain: docosahexaenoate, cholesterol, and palmitate. The rationale was that although alpha-linolenate is used in de novo lipogenesis, the quantitative importance of this pathway is unknown. Our results in the suckling rat show that 2-13% of carbon from [U-13C]-alpha-linolenate appearing in brain lipids is in docosahexaenoate while the rest is in brain lipids synthesized de novo. Overall, these results indicate that the suckling rat brain prefers pre-formed to newly synthesized arachidonate and that alpha-linolenate is readily utilized in brain lipid synthesis. These methods are suited to comparative studies of the metabolism of polyunsaturates and they support previous observations that the metabolism of some polyunsaturates such as alpha-linolenate extends well beyond the traditional desaturation-chain elongation pathway.