Competition of pyruvate with physiological substrates for oxidation by the heart: implications for studies with hyperpolarized [1-13C]pyruvate

Abstract

Carbon 13 nuclear magnetic resonance (NMR) isotopomer analysis was used to measure the rates of oxidation of long-chain fatty acids, ketones, and pyruvate to determine the minimum pyruvate concentration ([pyruvate]) needed to suppress oxidation of these alternative substrates. Substrate mixtures were chosen to represent either the fed or fasted state. At physiological [pyruvate], fatty acids and ketones supplied the overwhelming majority of acetyl-CoA. Under conditions mimicking the fed state, 3 mM pyruvate provided approximately 80% of acetyl-CoA, but under fasting conditions 6 mM pyruvate contributed only 33% of acetyl-CoA. Higher [pyruvate], 10-25 mM, was associated with transient reduced cardiac output, but overall hemodynamic performance was unchanged after equilibration. These observations suggested that 3-6 mM pyruvate in the coronary arteries would be an appropriate target for studies with hyperpolarized [1-(13)C]pyruvate. However, the metabolic products of 3 mM hyperpolarized [1-(13)C]pyruvate could not be detected in the isolated heart during perfusion with a physiological mixture of substrates including 3% albumin. In the presence of albumin even at high concentrations of pyruvate, 20 mM, hyperpolarized H(13)CO(3)(-) could be detected only in the absence of competing substrates. Highly purified albumin (but not albumin from plasma) substantially reduced the longitudinal relaxation time of [1-(13)C]pyruvate. In conclusion, studies of cardiac metabolism using hyperpolarized [1-(13)C]pyruvate are sensitive to the effects of competing substrates on pyruvate oxidation.

Fig. 1.

¹H-decoupled ¹³C nuclear magnetic resonance (NMR) spectrum of a heart extract. The working heart was supplied with a mixture of ¹³C-enriched long-chain fatty acids, ketones, lactate, pyruvate, and glucose in concentrations typical of a fed, rested animal. The multiplets of glutamate carbons 1–5, shown as insets, are the result of ¹³C-¹³C spin-spin coupling and reflect the relative rates of oxidation of these substrates. Ala, alanine; βHB, β-hydroxybutyrate; Lac, lactate; Ac, acetate; Suc, succinate; Asp, aspartate; Mal, malate; Cit, citrate; D, doublet; Q, quartet; S, singlet; T, triplet.

Fig. 2.

Influence of pyruvate concentration ([pyruvate]; [Pyr]) on the carbon-4 resonance of glutamate. These ¹³C spectra were acquired from hearts exposed to the “fed” mixture of substrates at graded [pyruvate], 0.12 mM (bottom) or 1.0, 3.0, and 6 mM (top). The fraction of acetyl-CoA derived from pyruvate relative to the fraction from long-chain fatty acids is indicated by the ratio (S + D34)/(Q + D45). At 3 mM pyruvate, the oxidation of long-chain fatty acids is inhibited. F, resonances arising from oxidation of ¹³C-enriched long-chain fatty acids; P, resonances arising from oxidation of ¹³C-enriched pyruvate, Q, quartet due to J₄₅ and J₃₄ coupling; D34, doublet due to J₃₄ coupling; D45, doublet due to J₄₅; S, singlet due to glutamate with ¹³C in position 4 but not position 3 or 5. J is standard notation for the spin-spin coupling constant.

Fig. 3.

Influence of [pyruvate] on sources of acetyl-CoA and flux through pyruvate dehydrogenase (PDH) using a fed buffer. A: relative rates of oxidation of ketones (dotted line), long-chain fatty acids (broken line), or pyruvate (solid line) are shown. At 3 mM pyruvate, oxidation of competing substrates is largely suppressed. B: flux through PDH. Flux through PDH is maximal at 3–6 mM pyruvate. Data are means ± SD. Analysis of variance revealed statistically significant responses to increased [pyruvate] for pyruvate (P < 0.0001), fatty acid (P = 0.004), ketone body oxidation (P = 0.006), and PDH flux (P = 0.0004). *P < 0.05 [pyruvate] vs. [pyruvate] = 0.12 mM. $P < 0.05 for fatty acids and ketone bodies vs. respective 0.12 mM data point.

Fig. 4.

Effect of 6 mM pyruvate on the contribution of ketones (KB), long-chain fatty acids (FA), and pyruvate (Pyr) to acetyl-CoA. Hearts exposed to the fed mixture derived ∼80% of acetyl-CoA from 6 mM pyruvate (open bar); the contribution of ketones (gray bar) or fatty acids (solid bar) was negligible. However, hearts exposed to the “fasted” mixture preferentially oxidized ketones. Pyruvate contributed only 33% of the acetyl-CoA. Data are means ± SD. *P < 0.05 vs. respective Pyr.

Fig. 5.

Influence of [pyruvate] on hemodynamic performance of the isolated working heart. “Pre” and “Post” refer to measurements immediately before and 5 min after switching to 6, 10, 15, or 25 mM pyruvate, respectively. Within the 1st min of switchover, there was a variable decrease in developed pressure that recovered quickly. Heart rate, developed pressure, and O₂ consumption were not altered substantially by exposure to graded [pyruvate]. Data are means ± SD. All pre vs. post data sets were not significantly different from each other for heart rate and myocardial O₂ consumption (MV̇o₂). Developed pressure decreased significantly with all concentrations included in the repeated-measures model (pre vs. post difference 13.3 mmHg, 95% confidence interval, 8.8–17.9 mmHg, P < 0.0001). However, no significant pre vs. post differences were observed within any concentration, analyzed with Bonferroni-Holm adjusted multiple comparisons.

Fig. 6.

¹³C Spectra of isolated hearts exposed to HP [1-¹³C]pyruvate. A: 3 mM HP [1-¹³C]pyruvate injection in heart in the presence of 3% BSA and no fatty acids. B: 3 mM HP [1-¹³C]pyruvate injection in heart in the presence of 3% BSA and 0.4 mM fatty acids. C: 20 mM HP [1-¹³C]pyruvate injection in heart in the presence of 3% BSA and no fatty acids. D: 20 mM HP [1-¹³C]pyruvate injection in heart in the presence of 3% BSA and 0.4 mM fatty acids.

Fig. 7.

Effect of albumin on the spin-lattice relaxation time (T₁) of 2.5 mM [1-¹³C]pyruvate. ♦, Fatty acids present; ■, no fatty acids present. When fatty acids were not present, T₁ times were not able to be determined at [albumin] >2%. The two data sets were significantly different from each other at [albumin] ≥0.5%.