ABSTRACT
In recent years, new stable isotopic methods have been developed that enhance our capabilities to assess metabolism (Brunengraber et al., 1997). These newer methods complement and, in some cases, exceed the capabilities of more traditional radioisotopic methods. The major advantage of the new methods is the improved ability to estimate the enrichment of a precursor during biosynthesis leading to more accurate estimates of metabolic rates. To put this new information in perspective, this chapter presents a tutorial describing the underlying basis of isotopic methods for the analysis of lipid metabolism. We show how the new stable isotopic methods differ from radioisotopic methods and explain the rationale for choosing among various types of isotopic tracers in speci$ c situations. The goal of this presentation is not limited to the fate of fatty acids in foods. We also include a discussion of methods for assessing the endogenous production of lipids in vivo. The literature citations offer examples of interesting applications but are not meant as an exhaustive
I. Introduction ............................................................................................................................ 67 A. Compartments and Pathways ........................................................................................... 68
II. Isotopic Tracers ...................................................................................................................... 69 A. General Properties ............................................................................................................ 69 B. Tracer Activity .................................................................................................................. 70 C. Radioisotopes ................................................................................................................... 71 D. Stable Isotopes ................................................................................................................. 72 E. Selecting the Tracer .......................................................................................................... 74
III. Practical Applications of Tracers in Lipid Metabolism .......................................................... 76 A. Flux Ratios Determined in Isotopic and Metabolic Steady State .................................... 76 B. Flux Rates Relative to Pool Size: Metabolic Steady State with Isotope Filling Pools ...... 76 C. Rate of Appearance .......................................................................................................... 77
IV. Estimating De Novo Biosynthesis of Lipids .......................................................................... 78 A. Deuterium Incorporation Approach ................................................................................. 78
V. Methods for Detecting 13C Atoms Rather than Molecules .................................................... 80 A. Gas Isotope Ratio Mass Spectrometry ............................................................................. 81 B. Gas Chromatography Combustion Isotope Ratio Mass Spectrometry ............................ 82
VI. Looking to the Future ............................................................................................................ 82 A. Mass Spectrometry as a Key to New Lipid Methodologies ............................................. 82 B. Isotopic Flux Studies as a Tool in the Genomics Era ....................................................... 83
References ..................................................................................................................................... 84
survey of all the noteworthy work in this $ eld. We bring to this discussion the viewpoint of metabolic physiologists who think in terms of pathways. To deal with the complexities of dietary lipid metabolism, the classic de$ nition of a pathway is expanded to include all processes from ingestion of food to oxidation. Thus, the major metabolic pathways of a dietary triglyceride involve many steps-oral ingestion, digestion, hydrolysis of the triglyceride, intestinal transport, chylomicron synthesis, export to plasma, > ux through various lipoproteins, uptake by cells, recirculation through the liver, and ultimately oxidation. To lay the groundwork for the tutorial, we begin with a review of the key concepts underlying the isotopic methodology used in metabolic studies.
