Measurement Of Acetyl-Coa Turnover (Approximate To Citric Acid Cycle Flux) In Perfused Rat Hearts By Isotope Dilution

We developed an isotope dilution technique to assess acetyl‐CoA turnover (≈ citric acid flux) in perfused rat hearts. In hearts, almost all acetate is used in citrate synthesis. Heart is perfused with buffer containing a low concentration of [ 2 H 3 ‐ 13 C 2 ] acetate (0.03 mM) which forms M5 and M4 acetyl‐CoA. The M4 acetyl‐CoA (greater than the small M4 component of M5 acetate) results probably from reversible enolization of acetyl‐CoA with partial 2 H loss. The buffer may also contain one labeled substrate which generates M2 acetyl‐CoA ([ 13 C 6 ] glucose or [1,2‐ 13 C 2 ] palmitate). Total acetyl‐CoA turnover is calculated from (i) the M5 acetate uptake, and (ii) the M5 acetyl‐CoA enrichment + the increase in M4 enrichment of acetyl‐CoA compared with the small M4 enrichment of [ 2 H 3 ‐ 13 C 2 ] acetate. The contribution of glucose or palmitate to acetyl‐CoA is calculated from the M2 enrichment of acetyl‐CoA. Loss of label between M5 acetate and citrate synthesis (acetylcarnitine, ketone bodies) is negligible. Rates of acetyl‐CoA turnover calculated by our method are similar to those calculated from O 2 uptake. The method was applied to measurements of acetyl‐CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). Supported by NIDDK RoadMap and the Case Mouse Metabolic Phenotyping Center.