IMPROVED ESTIMATION OF ANAPLEROSIS IN HEART USING C-13 NMR

Anaplerotic enzymes, such as pyruvate carboxylase or malic enzyme, cat alyze reactions that fill up the pools of the citric acid cycle (CAC), thereby increasing the total mass of CAC intermediates. Relative anap lerosis (y) denotes the ratio of anaplerotic flux to the flux catalyze d by citrate synthase. We examine conventional methods [C. R. Malloy, A. D. Sherry, and F. M. H. Jeffrey J. Biol. Chem. 263:6964-6971, 1988; C. R. Malloy; A. D. Sherry, and F. M. H. Jeffrey, Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H987-H995, 1990] of measurement of y using C-13-labeled precursors and analysis of [C-13]glutamate labeling by n uclear magnetic resonance (NMR) spectroscopy. Through mathematical ana lysis and computer simulation, we show that isotropic enrichment of th e pool of pyruvate that is substrate for anaplerosis will severely dec rease the accuracy of estimates of y made with conventional methods no matter how small the mass of the pool of pyruvate. Suppose that the r ecycling parameter R denotes the fraction of molecules of pyruvate tha t contain carbons derived from intermediates of the CAC. Each means of estimation of relative anaplerosis in the peer-reviewed literature as sumes that R = 0, although this assumption has not been confirmed by e xperiment. We show that conventional formulas, using either fractional enrichments of carbons or isotopomer analysis, actually estimate at m ost y.(1 - R) instead of y during administration of [2-C-13]acetate an d unlabeled pyruvate. Using a new formula for estimation of y, we reca lculate values of y from the literature and find them similar to 50% t oo low. We assume that all anaplerosis is via pyruvate and that the di fference in isotopic enrichment between cytosolic and mitochondrial ma late is negligible.