Formation of hyodeoxycholic acid from muricholic acid and hyocholic acid by an unidentified gram-positive rod termed HDCA-1 isolated from rat intestinal microflora.

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3alpha,6beta, 7beta-trihydroxy bile acid beta-muricholic acid into hyodeoxycholic acid (3alpha,6alpha-dihydroxy acid) by dehydroxylation of the 7beta-hydroxy group and epimerization of the 6beta-hydroxy group into a 6alpha-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3alpha, 6alpha,7alpha-trihydroxy acid), alpha-muricholic acid (3alpha,6beta, 7alpha-trihydroxy acid), and omega-muricholic acid (3alpha,6alpha, 7beta-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted beta-muricholic acid almost completely into hyodeoxycholic acid.

Stabilisation and partial purification of Triton X-100 solubilised trihydroxycoprostanoyl-CoA synthetase from rat liver.

The stability of rat hepatic trihydroxycoprostanoyl-CoA syntethase was studied in its native membrane environment and after solubilisation by Triton X-100, and compared to that of choloyl-CoA synthetase. The lability of both delipidated enzymes could be suppressed by high concentrations of polyols such as sucrose and glucose. Addition of phospholipids to the assay mixtures was necessary to restore the activity of the stabilized enzymes. For further chromatographic separations, the addition of the hydrotrope Triton H-66 to the glucose-stabilized Triton X-100 solubilised synthetases improved their recovery on different matrices. Gel filtration revealed a native molecular mass of the Triton X-100/Triton H-66/protein micelles of 212 and 207 kDa for choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase respectively.

Peroxisomal beta-oxidation. Purification of four novel 3-hydroxyacyl-CoA dehydrogenases from rat liver peroxisomes.

Peroxisomes are capable of beta-oxidizing a variety of substrates including the CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acids. The first reaction of peroxisomal beta-oxidation is catalyzed by an acyl-CoA oxidase. Rat liver peroxisomes contain three acyl-CoA oxidases: 1) palmitoyl-CoA oxidase, oxidizing straight chain acyl-CoAs; 2) pristanoyl-CoA oxidase, oxidizing 2-methyl-branched acyl-CoAs; and 3) trihydroxycoprostanoyl-CoA oxidase, oxidizing the CoA esters of the bile acid intermediates (Van Veldhoven, P.P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). We have now investigated whether the third step of peroxisomal beta-oxidation, catalyzed by a 3-hydroxyacyl-CoA dehydrogenase, is also catalyzed by multiple enzymes, using the 3-hydroxyacyl-CoA derivatives of palmitic acid, 2-methylpalmitic acid, and trihydroxycoprostanic acid as the substrates to monitor the dehydrogenase activities. In order to avoid contamination with mitochondrial 3-hydroxyacyl-CoA dehydrogenases, highly purified peroxisomes from untreated rats were employed as the enzyme source. Subfractionation of the peroxisomes revealed that the major portion of the dehydrogenase activities with all three substrates was present in the peripheral membrane protein fraction. Separation of this fraction on various chromatographic columns resulted in the purification of the well known multifunctional protein, a 78-kDa monomeric protein that displays 3-hydroxyacyl-CoA dehydrogenase plus hydratase activity, as well as of four additional novel dehydrogenases with different substrate specificities. Three of the enzymes are monomeric proteins of 35 kDa, 56 kDa, and 79 kDa, respectively. The latter enzyme also displays hydratase activity. The fourth enzyme is a dimer of 89 kDa, the subunits of which form a doublet at 40 kDa. The exact physiological role of each of the 3-hydroxyacyl-CoA dehydrogenases requires further investigation.

Comparison of fatty acid alpha-oxidation by rat hepatocytes and by liver microsomes fortified with NADPH, Fe3+ and phosphate.

Rat liver microsomes, when fortified with NADPH, Fe3+ and phosphate, can catalyze the oxidative decarboxylation (alpha-oxidation) of 3-methyl-substituted fatty acids (phytanic and 3-methylheptadecanoic acids) at rates that equal 60-70% of those observed in isolated hepatocytes (Huang, S., Van Veldhoven, P.P., Vanhoutte, F., Parmentier, G., Eyssen, H.J., and Mannaerts, G.P., 1992, Arch. Biochem. Biophys. 296, 214-223). In the present study we set out to identify and compare the products and possible intermediates of alpha-oxidation formed in rat hepatocytes and by rat liver microsomes. In the presence of NADPH, Fe3+ and phosphate, microsomes decarboxylated not only 3-methyl fatty acids but also 2-methyl fatty acids and even straight chain fatty acids. The decarboxylation products of 3-methylheptadecanoic and palmitic acids were purified by high-performance liquid chromatography and identified by gas chromatography/mass spectrometry as 2-methylhexadecanoic and pentadecanoic acids, respectively. Inclusion in the incubation mixtures of glutathione plus glutathione peroxidase inhibited decarboxylation by more than 90%, suggesting that a 2-hydroperoxy fatty acid is formed as a possible intermediate. However, we have not yet been able to unequivocally identify this intermediate. Instead, several possible rearrangement metabolites were identified. In isolated rat hepatocytes incubated with 3-methylheptadecanoic acid, the formation of the decarboxylation product, 2-methylhexadecanoic acid, was demonstrated, but no accumulation of putative intermediates or rearrangement products was observed. Our data do not allow us to draw conclusions on whether the reconstituted microsomal system is representative of the cellular alpha-oxidation system.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of 3-methyl-branched fatty acids in rat liver.

1. Subcellular fractionation of rat liver revealed that 3-methylmargaric acid, a monobranched phytanic acid analogue, can be activated by mitochondria, endoplasmic reticulum and peroxisomes. 2. Indirect data (effects of pyrophosphate and Triton X-100) suggested that the peroxisomal activation of 3-methylmargaric, 2-methylpalmitic and palmitic acid is catalyzed by different enzymes. 3. Despite many attempts, column chromatography of solubilized peroxisomal membrane proteins so far did not provide more conclusive data. On various matrices, lignoceroyl-CoA synthetase clearly eluted differently from the synthetases acting on 3-methylmargaric, 2-methylpalmitic and palmitic acid. The latter three however, tended to coelute together, although often not in an identical manner.

Synthesis and 29-14C-labeling of 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid. A bile acid occurring in peroxisomal diseases.

The synthesis and 14C-labeling of 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-carboxymethyl-5 beta-cholestan-26-oic acid by two different approaches is described. One of them involves chain elongation of cholic acid via Wittig-Horner condensation of its formylated 24-aldehyde with tetraethyl phosphonoglutarate. The resulting cholestenoate, on deprotection and hydrogenation, affords the unusual C29 bile acid in good yield. An alternative procedure consists in a malonic ester synthesis starting from the formylated 24-alcohol which, after conversion into a mesylate, is reacted with sodium salt of 2-carboethoxy-gamma-butyrolactone. Alkaline hydrolysis, decarboxylation, esterification with diazomethane and selective tosylation of the newly introduced primary hydroxyl function give a C28 precursor, which is easily chain-elongated into a labeled or unlabeled C29 bile acid by reaction with cyanide and hydrolysis. Due to the easy lactonization of some of the C28 intermediates, the latter method provides a better way for introducing a C-29 label than the sequence usually employed for carboxyl labeling of bile acids and consisting in a decarboxylative halogenation of the parent acid followed by substitution of the norhalogenide with [14C]cyanide and hydrolysis. The structure of the synthesized acid or its dimethyl ester is confirmed by 13C nuclear magnetic resonance spectroscopy and mass spectrometry, and is also shown by gas liquid chromatography to be identified with an authentic sample of biosynthetic C29 dioic bile acid extracted from body fluids of Zellweger patients.

The CoA esters of 2-methyl-branched chain fatty acids and of the bile acid intermediates di- and trihydroxycoprostanic acids are oxidized by one single peroxisomal branched chain acyl-CoA oxidase in human liver and kidney.

Rat liver peroxisomes contain three acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins; pristanoyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids (e.g. pristanic acid); and trihydroxycoprostanoyl-CoA oxidase, which oxidizes the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids (Van Veldhoven, P. P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). In the present report we demonstrate that human liver peroxisomes contain only two acyl-CoA oxidases: palmitoyl-CoA oxidase, which oxidizes the CoA esters of straight chain fatty acids and prostaglandins, and a novel branched chain acyl-CoA oxidase, which oxidizes the CoA esters of 2-methyl-branched fatty acids as well as those of the bile acid intermediates (which also possess a 2-methyl substitution in their side chains). The branched chain acyl-CoA oxidase was purified to near homogeneity by means of column chromatography. It appeared to be a 70-kDa monomeric protein that did not cross-react with antisera raised against rat palmitoyl-CoA oxidase and pristanoyl-CoA oxidase. No indication was found for the presence of a separate trihydroxycoprostanoyl-CoA oxidase in human liver. The branched chain acyl-CoA oxidase was present also in human kidney, suggesting that it is expressed in other extrahepatic tissues as well. Our results explain a number of clinical-chemical observations made in certain cases of peroxisomal beta-oxidation disorders.

Mitochondrial short-chain acyl-CoA dehydrogenase of human liver and kidney can function as an oxidase.

During an attempt to purify the peroxisomal acyl-CoA oxidases from human liver and kidney, we discovered a novel short-chain acyl-CoA oxidase, which was well separated from the known peroxisomal oxidases on various chromatographic columns. However, further experiments demonstrated that the novel oxidase is identical with the mitochondrial short-chain acyl-CoA dehydrogenase. (1) Subcellular fractionation revealed that the short-chain acyl-CoA oxidase is present in mitochondria and absent from peroxisomes. (2) The molecular mass (43 kDa) of the subunit of the purified oxidase was similar to that reported for the dehydrogenase. (3) The substrate spectrum of the oxidase was comparable with that described for the dehydrogenase. (4) On column chromatography, the oxidase and dehydrogenase activities co-eluted. Our results indicate that, in the absence of suitable electron acceptors, the short-chain acyl-CoA dehydrogenase is capable of transferring electrons directly to molecular oxygen, yielding potentially harmful H2O2. This raises the question as to whether the dehydrogenase might function as an oxidase in conditions in which the activity of the electron-transport chain is decreased, such as reperfusion after ischaemia.

The deficient degradation of synthetic 2- and 3-methyl-branched fatty acids in fibroblasts from patients with peroxisomal disorders.

The oxidation of pristanic and phytanic acids by human skin fibroblasts was compared to that of their synthetic analogues, 2-methylpalmitic and 3-methylmargaric acids. The synthetic compounds and natural substrates were degraded at comparable rates in control and X-linked adrenoleukodystrophy fibroblasts. The alpha-decarboxylation of 3-methylmargaric acid, similarly to that of phytanic acid, was affected in Refsum disease and Zellweger syndrome, but not in X-linked adrenoleukodystrophy. The beta-oxidation of 2-methylpalmitic acid, similarly to that of pristanic acid, was deficient in fibroblasts derived from patients suffering from Zellweger syndrome, confirming the importance of peroxisomes in the breakdown of 2-methyl-branched fatty acids. No deficiency was observed in fibroblasts from X-linked adrenoleukodystrophy patients. The 1-14C-labelled 2- and 3-methyl-branched fatty acids, which are easier to synthesize that the natural analogues, are therefore valuable tools for the diagnosis of human peroxisomal disorders.

Substrate specificities of rat liver peroxisomal acyl-CoA oxidases: palmitoyl-CoA oxidase (inducible acyl-CoA oxidase), pristanoyl-CoA oxidase (non-inducible acyl-CoA oxidase), and trihydroxycoprostanoyl-CoA oxidase.

Rat liver peroxisomes contain three acyl-CoA oxidases:palmitoyl-CoA oxidase, pristanoyl-CoA oxidase, and trihydroxycoprostanoyl-CoA oxidase. The three oxidases were separated by anion-exchange chromatography of a partially purified oxidase preparation, and the column eluate was analyzed for oxidase activity with different acyl-CoAs. Short chain mono (hexanoyl-) and dicarboxylyl (glutaryl-)-CoAs and prostaglandin E2-CoA were oxidized exclusively by palmitoyl-CoA oxidase. Long chain mono (palmitoyl-) and dicarboxylyl (hexadecanedioyl-)-CoAs were oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the former enzyme catalyzing approximately 70% of the total eluate activity. The very long chain lignoceroyl-CoA was also oxidized by palmitoyl-CoA oxidase and pristanoyl-CoA oxidase, the latter enzyme catalyzing approximately 65% of the total eluate activity. Long chain 2-methyl branched acyl-CoAs (2-methylpalmitoyl-CoA and pristanoyl-CoA) were oxidized for approximately 90% by pristanoyl-CoA oxidase, the remaining activity being catalyzed by trihydroxycoprostanoyl-CoA oxidase. The short chain 2-methylhexanoyl-CoA was oxidized by trihydroxycoprostanoyl-CoA oxidase and pristanoyl-CoA oxidase (approximately 60 and 40%, respectively, of the total eluate activity). Trihydroxycoprostanoyl-CoA was oxidized exclusively by trihydroxycoprostanoyl-CoA oxidase. No oxidase activity was found with isovaleryl-CoA and isobutyryl-CoA. Substrate dependences of palmitoyl-CoA oxidase and pristanoyl-CoA oxidase were very similar when assayed with the same (common) substrate. Since the two oxidases were purified to a similar extent and with a similar yield, the contribution of each enzyme to substrate oxidation in the column eluate probably reflects its contribution in the intact liver.

Alpha-oxidation of 3-methyl-substituted fatty acids in rat liver.

3-Methyl-substituted fatty acids are first oxidatively decarboxylated (alpha-oxidation) before they are degraded further via beta-oxidation. We synthesized [1-14C]phytanic and 3-[1-14C]methylmargaric acids in order to study their alpha-oxidation in isolated rat hepatocytes, rat liver homogenates and subcellular fractions. alpha-Oxidation was measured as the production of radioactive CO2. In isolated hepatocytes, maximal rates of alpha-oxidation amounted to 7 and 10 nmol/min x 10(8) cells with phytanic acid and 3-methylmargaric acid, respectively. At equimolar substrate concentrations, alpha-oxidation of branched fatty acids was approximately 10- to 15-fold slower than the beta-oxidation of the straight chain palmitate. In whole liver homogenates, rates of alpha-oxidation that equaled 60 to 70% of those observed in the hepatocytes were obtained. Optimum rates required O2, NADPH, Fe3+, and ATP. Fe3+ could be replaced by Fe2+ and ATP could be replaced by a number of other phosphorylated nucleosides and even inorganic phosphate without loss of activity. NADH could substitute for NADPH but not always with full restoration of activity. A variety of other cofactors and metal ions was either inhibitory or without effect. Scavengers of reactive oxygen species, known to be formed during the NADPH-dependent microsomal reduction of ferric-phosphate complexes, were without effect on alpha-oxidation. No evidence was found for the accumulation of NADPH-dependent or Fe(3+)-dependent reaction intermediates. Subcellular fractionation of liver homogenates demonstrated that alpha-oxidation was located predominantly, if not exclusively, in the endoplasmic reticulum. alpha-Oxidation, measured in microsomal fractions, was not inhibited by CO, cytochrome c, or ferricyanide, indicating that NADPH cytochrome P450 reductase and cytochrome P450 are not involved in alpha-oxidation. Our results indicate that, contrary to current belief, alpha-oxidation is catalyzed by the endoplasmic reticulum. The cofactor requirements suggest that alpha-oxidation involves the reduction of Fe3+ by electrons from NADPH and that it is stimulated by phosphate ions and nucleotides.

Mitochondrial and peroxisomal beta oxidation of the branched chain fatty acid 2-methylpalmitate in rat liver.

A number of isoprenoids (e.g. pristanic acid and the side chains of fat soluble-vitamins) is degraded or shortened via beta oxidation. We synthesized 2-methyl-palmitate and 2-methyl[1-14C] palmitate as a model substrate for the study of the beta oxidation of branched (isoprenoid) fatty acids in rat liver. 2-Methylpalmitate was well oxidized by isolated hepatocytes and its oxidation was stimulated after treatment of the animals with a peroxisome proliferator. Subcellular fractionation of rat liver demonstrated that 2-methylpalmitate is activated to its CoA ester in endoplasmic reticulum, mitochondria, and peroxisomes and that mitochondria and peroxisomes are capable of beta-oxidizing 2-methylpalmitate. At low unbound 2-methylpalmitate concentrations and in the presence of competing straight chain fatty acids, a condition encountered in vivo, peroxisomal 2-methyl-palmitate oxidation was 2- to 4-fold more active than mitochondrial oxidation. Treatment of rats with a peroxisome proliferator markedly stimulated mitochondrial but only slightly peroxisomal 2-methylpalmitate oxidation. The same treatment dramatically induced palmitoyl-CoA oxidase but did not change 2-methyl-palmitoyl-CoA oxidase activity. Our results indicate 1) that in untreated rats peroxisomes contribute for an important part to the oxidation of 2-methylpalmitate; 2) that treatment with a peroxisome proliferator stimulates mainly the mitochondrial component of 2-methylpalmitate oxidation; and 3) that palmitoyl-CoA and 2-methylpalmitoyl-CoA are oxidized by different peroxisomal oxidases.

Identification and purification of a peroxisomal branched chain fatty acyl-CoA oxidase.

Isoprenoid (branched) fatty acids such as pristanic acid can be degraded via beta-oxidation in peroxisomes. We synthesized 2-methylpalmitoyl-CoA as a model substrate in order to study the first step of the peroxisomal beta-oxidation of branched fatty acids, catalyzed by an acyl-CoA oxidase. 2-Methylpalmitoyl-CoA oxidase activity was found in rat liver homogenates. Subcellular fractionation demonstrated that the oxidase was confined to peroxisomes. 2-Methylpalmitoyl-CoA oxidase was also present in kidney and intestine. It was not induced in liver or in the extrahepatic tissues by treatment of rats with peroxisome proliferators or by feeding diets containing excess isoprenoids. The enzyme was partially purified together with palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase by heat treatment and ammonium sulfate fractionation of liver extracts. The partially purified preparation was chromatographed on various columns. 2-Methylpalmitoyl-CoA oxidase could be separated from the inducible (by peroxisome proliferators) palmitoyl-CoA oxidase and from trihydroxycoprostanoyl-CoA oxidase, but it always coeluted with the noninducible palmitoyl-CoA oxidase, recently described by us (Schepers, L., Van Veldhoven, P. P., Casteels, M., Eyssen, H. J., and Mannaerts, G. P. (1990) J. Biol. Chem. 265, 5242-5246). 2-Methylpalmitoyl-CoA oxidase was purified to near homogeneity in three chromatographic steps (anion exchange, hydroxylapatite, and gel filtration). Its apparent molecular mass is approximately 415 kDa, and it consists of identical subunits of approximately 70 kDa. The enzyme oxidized 2-methylpalmitoyl-CoA twice as rapidly as palmitoyl-CoA and pristanoyl-CoA as rapidly as palmitoyl-CoA, so that it can be considered as a branched fatty acyl-CoA oxidase. Since pristanoyl-CoA is one of its naturally occurring substrates we propose to name this enzyme pristanoyl-CoA oxidase.

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver.

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.

Presence of three acyl-CoA oxidases in rat liver peroxisomes. An inducible fatty acyl-CoA oxidase, a noninducible fatty acyl-CoA oxidase, and a noninducible trihydroxycoprostanoyl-CoA oxidase.

Mammalian liver peroxisomes are capable of beta-oxidizing a variety of substrates including very long chain fatty acids and the side chains of the bile acid intermediates di- and trihydroxycoprostanic acid. The first enzyme of peroxisomal beta-oxidation is acyl-CoA oxidase. It remains unknown whether peroxisomes possess one or several acyl-CoA oxidases. Peroxisomal oxidases from rat liver were partially purified by (NH4)2SO4 precipitation and heat treatment, and the preparation was subjected to chromatofocusing, chromatography on hydroxylapatite and dye affinity matrices, and gel filtration. The column eluates were assayed for palmitoyl-CoA and trihydroxycoprostanoyl-CoA oxidase activities and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results revealed the presence of three acyl-CoA oxidases: 1) a fatty acyl-CoA oxidase with a pI of 8.3 and an apparent molecular mass of 145 kDa. The enzyme consisted mainly of 52- and 22.5-kDa subunits and could be induced by clofibrate treatment; 2) a noninducible fatty acyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 427 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 71 kDa; and 3) a noninducile trihydroxycoprostanoyl-CoA oxidase with a pI of 7.1 and an apparent molecular mass of 139 kDa. It consisted mainly, if not exclusively, of one polypeptide component of 69 kDa. Our findings are probably related to the recent discovery of two species of acyl-CoA oxidase mRNA in rat liver (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furata, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137) and they probably also explain why in human peroxisomal beta-oxidation defects an accumulation of very long chain fatty acids is not always accompanied by an excretion of bile acid intermediates and vice versa.

Activation and peroxisomal beta-oxidation of fatty acids and bile acid intermediates in liver from Bombina orientalis and from the rat.

1. Bombina orientalis excretes mainly C27 bile acids: trihydroxycoprostanic and varanic acids. More than 90% of the trihydroxycoprostanic acid (THCA) present in the bile, was conjugated with taurine; varanic acid was present in the unconjugated form. 2. Trihydroxycoprostanoyl-CoA (THC-CoA) synthetase activity, required for the formation of the taurine conjugate, was present in the liver of Bombina orientalis. 3. Peroxisomal beta-oxidation, which catalyzes the oxidation of fatty acids as well as the conversion of C27 bile acids into C24 bile acids in rat and human liver, could be detected in liver of Bombina orientalis when palmitoyl-CoA was used as substrate, but not when trihydroxycoprostanoyl-CoA (THC-CoA) was used.

Deficient oxidation of trihydroxycoprostanic acid in liver homogenates from patients with peroxisomal diseases.

The activation of palmitate and trihydroxycoprostanic acid and the peroxisomal oxidation of palmitate, trihydroxycoprostanic acid and their CoA esters were measured in homogenates prepared from fresh liver tissue of patients undergoing hepatic surgery and from frozen postmortem liver specimens of controls, patients with Zellweger syndrome and a patient with pseudo-Zellweger syndrome, a deficiency of peroxisomal 3-oxoacyl-CoA thiolase. In contrast to the findings in control livers, peroxisomal beta-oxidation of palmitate and of palmitoyl-CoA was severely impaired, and oxidation of trihydroxycoprostanic acid and its CoA ester could not be detected in the livers of the patients affected by peroxisomal diseases. The finding in this paper, that the oxidation of trihydroxycoprostanoyl-CoA can be measured reliably in small amounts of human liver, will be of valuable help in the differential diagnosis and classification of peroxisomal disorders and will help to elucidate the exact nature of some of the defects present in these disorders.

Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver.

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.