Assay of the Concentration and 13C-Isotopic Enrichment of Malonyl–Coenzyme A by Gas Chromatography–Mass Spectrometry

Journal of Biological Chemistry

Thomas

et al. 2004

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The goal of this study was to test the relationship between malonyl-CoA concentration and its turnover measured in isolated rat hearts perfused with NaH 13 CO 3 . This turnover is a direct measurement of the flux of acetyl-CoA carboxylation in the intact heart. It also reflects the rate of malonyl-CoA decarboxylation, i.e. the only known fate of malonyl-CoA in the heart. Conditions were selected to result in stable malonyl-CoA concentrations ranging from 1.5 to 5 nmol⅐g wet weight؊ 1 . The malonyl-CoA concentration was directly correlated with the turnover of malonyl-CoA, ranging from 0.7 to 4.2 nmol⅐min ؊1 ⅐g wet weight ؊1 (slope ‫؍‬ 0.98, r 2 ‫؍‬ 0.94). The V max activities of acetyl-CoA carboxylase and of malonylCoA decarboxylase exceeded the rate of malonyl-CoA turnover by 2 orders of magnitude and did not correlate with either concentration or turnover of malonyl-CoA. However, conditions of perfusion that increased acetylCoA supply resulted in higher turnover and concentration, demonstrating that malonyl-CoA turnover is regulated by the supply of acetyl-CoA. The only condition where the activity of malonyl-CoA decarboxylase regulated malonyl-CoA kinetics was when the enzyme was pharmacologically inhibited, resulting in increased malonyl-CoA concentration and decreased turnover. Our data show that, in the absence of enzyme inhibitors, the rate of acetyl-CoA carboxylation is the main determinant of the malonyl-CoA concentration in the heart.Malonyl-CoA is a key intermediate of fatty acid synthesis in lipogenic organs. It is also an inhibitor of carnitine palmitoyltransferase I, a regulator of fatty acid oxidation in most tissues (1-3). A number of studies have documented the modulation by malonyl-CoA of fatty acid oxidation in the heart under physiological and pathological conditions, such as maturation (4), diabetes (5), increased cardiac work (6), and postischemic reperfusion (7). In the heart, malonyl-CoA formed by cytosolic acetyl-CoA carboxylase (ACC) 1 is, as far as is known, disposed only via decarboxylation catalyzed by malonyl-CoA decarboxylase (MCD). Thus, cytosolic acetyl-CoA and malonyl-CoA are the two components of a substrate cycle that regulates carnitine palmitoyltransferase I activity and the rate of mitochondrial fatty acid oxidation in the heart.No information is available on the relationship between the concentration of malonyl-CoA and its turnover, i.e. with the flux of ACC in the intact heart. The rate of turnover of malonyl-CoA is likely to be regulated by the cytosolic acetyl-CoA concentration and by the activities of ACC and MCD. The concentrations of cytosolic acetyl-CoA and malonyl-CoA in the heart are much lower than the K m of the two enzymes for their respective substrates (6, 8). Thus, it is unlikely that the modulation of ACC and MCD activities exerts a tight control on malonyl-CoA turnover. It was previously shown that the myocardial content of malonylCoA is elevated when acetyl-CoA levels are increased by either activation of pyruvate dehydrogenase (8, 9) or by perfusing the ...

Section: Methodsmentioning

confidence: 99%

“…We recently developed a mass spectrometric technique for measuring the malonyl-CoA turnover in isolated organs, based on the time course of its labeling from NaH 13 CO 3 (12). The malonyl-CoA concentration can also be assayed in the same samples by isotope dilution using an internal standard of [U-13 C 3 ]malonyl-CoA.…”

mentioning

confidence: 99%

Regulation of Malonyl-CoA Concentration and Turnover in the Normal Heart

Reszko¹,

Journal of Biological Chemistry

Thomas

et al. 2004

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“…The 13 C-labelling of the C1 + 2 fragment of BHB (another probe of mitochondrial acetyl-CoA [7,8]) was calculated as the difference between the labelling of the whole molecule and that of the C3 + 4 fragment [15]. The 13 C-labelling of liver malonyl-CoA [12] and of acetate in the effluent perfusate [16] were assayed as described previously. The labelling of acetylcarnitine was measured by LC-MS (liquid chromatography-MS) [17].…”

Section: Analytical Proceduresmentioning

confidence: 99%

Probing peroxisomal β-oxidation and the labelling of acetyl-CoA proxies with [1-13C]octanoate and [3-13C]octanoate in the perfused rat liver

Adams

Bian

et al. 2005

Biochemical Journal

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We reported previously that a substantial fraction of the acetyl groups used to synthesize malonyl-CoA in rat heart is derived from peroxisomal β-oxidation of long-chain and very-long-chain fatty acids. This conclusion was based on the interpretation of the 13 Clabelling ratio (malonyl-CoA)/(acetyl moiety of citrate) measured in the presence of substrates that label acetyl-CoA in mitochondria only (ratio < 1.0) or in both mitochondria and peroxisomes (ratio > 1.0). The goals of the present study were to test, in rat livers perfused with [1-13 C]octanoate or [3-13 C]octanoate, (i) whether peroxisomal β-oxidation contributes acetyl groups for malonylCoA synthesis, and (ii) the degree of labelling homogeneity of acetyl-CoA proxies (acetyl moiety of citrate, acetate, β-hydroxybutyrate, malonyl-CoA and acetylcarnitine). Our data show that (i) octanoate undergoes two cycles of peroxisomal β-oxidation in liver, (ii) acetyl groups formed in peroxisomes contribute to malonyl-CoA synthesis, (iii) the labelling of acetyl-CoA proxies is markedly heterogeneous, and (iv) the labelling of C1 + 2 of β-hydroxybutyrate does not reflect the labelling of acetyl-CoA used in the citric acid cycle.

“…At t ϭ 15 min, unlabeled bicarbonate was replaced by 40% enriched NaH 13 CO 3 (28), and the heart was quick-frozen at up to 35 min. The enrichment of HCO 3 Ϫ /CO 2 was measured in samples of effluent perfusate taken just before clamping the heart.…”

Section: Organ Perfusion Experimentsmentioning

confidence: 99%

“…The acetonitrile/water fraction is evaporated in a Savant vacuum centrifuge at room temperature. The residue is dissolved in 1 ml of water which is divided into two aliquots for the assays of the mass isotopomer distributions of (i) propionyl-CoA via propionylsarcosine pentafluorobenzyl ester and (ii) methylmalonyl-CoA 3 and succinyl-CoA via the tert-butyldimethylsilyl derivatives of the corresponding acids (27,28). The measured mass isotopomer distributions were corrected for the contributions of naturally occurring heavy isotopes (31).…”

Section: Sample Preparationmentioning

confidence: 99%

Assessing the Reversibility of the Anaplerotic Reactions of the Propionyl-CoA Pathway in Heart and Liver

Reszko

Journal of Biological Chemistry

Pierce

et al. 2003

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While a number of studies underline the importance of anaplerotic pathways for hepatic biosynthetic functions and cardiac contractile activity, much remains to be learned about the sites and regulation of anaplerosis in these tissues. As part of a study on the regulation of anaplerosis from propionyl-CoA precursors in rat livers and hearts, we investigated the degree of reversibility of the reactions of the propionyl-CoA pathway. Label was introduced into the pathway via NaH 13 CO 3 , [U- 13 Adequate energy production via the citric acid cycle (CAC) 1 requires not only a constant supply of acetyl-CoA, but also a fairly constant pool of the catalytic intermediates which carry the acetyl groups as they are oxidized. Although the process of anaplerosis in micro-organisms was discovered in the 1960s (1), its importance for the homeostasis of mammalian cells (particularly cardiomyocytes) was recognized only in the 1980s (2, 3) (for a recent review, see Ref. 4). While the crucial role of anaplerosis for hepatic gluconeogenesis is self-evident, investigations in the heart suggested that stimulating anaplerosis from exogenous substrates could become part of the treatment of myocardial reperfusion injury and other cardiomyopathies (5-10).In a recent clinical study (11), the hypoglycemia as well as the mechanical performance of the heart and muscle of patients suffering from long chain fatty acid oxidation defects was greatly improved by replacing the fat component of their therapeutic diet, i.e. trioctanoin (a medium-even-chain triglyceride) by triheptanoin (a medium-odd-chain triglyceride). The only difference between the metabolisms of octanoate and heptanoate is the production of propionyl-CoA from the latter. In a follow-up investigation in pig hearts, propionate infusion was found to be a very effective anaplerotic substrate. Anaplerosis from 0.25 mM [U-13 C 3 ]propionate amounted to 9% of the rate of the CAC (12, 13). This led us to investigate the regulation of the propionyl-CoA pathway in livers and hearts of animals.Propionyl-CoA is formed from the activation of propionate and from the catabolism of isoleucine, valine, threonine, methionine, cholesterol, odd-chain fatty acids, and C 5 -ketone bodies (14). In mammalian tissues, the metabolism of propionyl-CoA (Fig. 1) involves (i) the formation of (S)-methylmalonyl-CoA by biotin-dependent propionyl-CoA carboxylase (15), (ii) the conversion of (S)-to (R)-methylmalonyl-CoA by methylmalonyl-CoA racemase (16), and (iii) the isomerization of (R)-methylmalonyl-CoA to succinylCoA by cobalamine-dependent methylmalonyl-CoA mutase (17-19) (for a recent review, see Ref. 20).Early in vitro work with purified enzymes revealed different degrees of reversibility of the reactions catalyzed by propionylCoA carboxylase (21, 22), methylmalonyl-CoA racemase (23), and methylmalonyl-CoA mutase (24). In the presence of a mixture of purified propionyl-CoA carboxylase, methylmalonylCoA racemase, and methylmalonyl-CoA mutase, only a few percent of the radioactivity of [ 14 C]succinyl-CoA...