275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3␣,7␣,12␣-trihydroxy-5␤-cholestanoic acid. In contrast, VLCS activated 3␣,7␣,12␣-trihydroxy-5␤-cholestanoate, but did not utilize any of the C 24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C 24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.

Peroxisomes play an essential role in a number of different metabolic pathways, including the ␤-oxidation of a distinct set of fatty acids and fatty acid derivatives. The importance of the peroxisomal ␤-oxidation system in humans is made apparent by the existence of a group of inherited diseases in which peroxisomal ␤-oxidation is impaired. This includes X-linked adrenoleukodystrophy and other disorders with a defined defect. On the other hand, many patients have been described with a defect in peroxisomal ␤-oxidation of unknown etiology. Resolution of the defects in these patients requires the elucidation of the enzymatic organization of the peroxisomal ␤-oxidation system. Importantly, a new peroxisomal ␤-oxidation enzyme was recently described called Dbifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity primarily reacting with ␣-methyl fatty acids like pristanic acid and di-and trihydroxycholestanoic acid. In this patient we describe the first case of D-bifunctional protein deficiency as resolved by enzyme activity measurements and mutation analysis. The mutation found (Gly 16 Ser) is in the dehydrogenase coding part of the gene in an important loop of the Rossman fold forming the NAD ؉ -binding site. The results show that the newly identified D-bifunctional protein plays an essential role in the peroxisomal ␤-oxidation pathway that cannot be compensated for by the L-specific bifunctional protein.

The mitochondrial metabolism of 5-enoylCoAs, which are formed during the (3-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of (3-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by mediumchain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cisoctadienoyl-CoA, which was isomerized to 3,5-octadienoylCoA either by mitochondrial A3,A2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-transoctadienoyl-CoA in the presence ofsoluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a A3',542'4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPHdependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoylCoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetylCoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the (3-oxidation of polyunsaturated fatty acids.The degradation of unsaturated fatty acids by 8-oxidation involves at least two auxiliary enzymes in addition to the enzymes required for the breakdown of saturated fatty acids (1). The auxiliary enzymes acting on double bonds are 2,4-dienoyl-CoA reductase or 4-enoyl-CoA reductase (EC 1.3.1.34) and A3,A2-enoyl-CoA isomerase (EC 5.3.3.8) (2).Chain shortening of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms leads to the formation of4-enoyl-CoAs, which are dehydrogenated by acyl-CoA dehydrogenase (EC 1.3.99.3) to 2,4-dienoyl-CoAs. An NADPH-dependent 2,4-dienoyl-CoA reductase, originally described by Kunau and Dommes (3), catalyzes the reduction of 2,4-dienoyl-CoAs to 3-enoyl-CoAs, which, after isomerization by A3,A2-enoyl-CoA isomerase to 2-enoylCoAs, can be completely degraded via the P-oxidation spiral.Unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms are, according to Stoffel and Caesar (4), chain-shortened to 3-enoyl-CoAs, which, after isomerization to 2-enoyl-CoAs by A3,A2-enoyl-CoA isomerase, reenter the p-oxidation spiral. If so, 5-enoyl-CoAs are intermediates that would pass once more through the ,8-oxidation spiral before being acted upon by A3,A2-enoylCoA isomerase. This prediction, however, is contradicted by a recent observation of Tserng and Jin (5) who reported that the mitochondrial -oxidation of 5-enoyl-CoAs is dependent on NADPH. Their analysis of metabolites by gas chromatography/mass spectrometry...