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...

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Phenylbutyrate is used in humans for treating inborn errors of ureagenesis, certain forms of cancer, cystic fibrosis and thalassemia. The known metabolism of phenylbutyrate leads to phenylacetylglutamine, which is excreted in urine. We have identified phenylbutyrylglutamine as a new metabolite of phenylbutyrate in human plasma and urine. We describe the synthesis of phenylbutyrylglutamine and its assay by gas chromatography/mass spectrometry as a tert-butyldimethylsilyl or methyl derivative, using standards of [(2)H(5)]phenylbutyrylglutamine and phenylpropionylglutamine. After administration of phenylbutyrate to normal humans, the cumulative urinary excretion of phenylacetate, phenylbutyrate, phenylacetylglutamine and phenylbutyrylglutamine amounts to about half of the dose of phenylbutyrate. Thus, additional metabolites of phenylbutyrate are yet to be identified.

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