Hydroxy Dicarboxylic Acids Research Articles

Synthesis and Spectroscopic Characterization of Selected Water-Soluble Ligands Based on 1,10-Phenanthroline Core.

Water-soluble ligands based on a 1,10-phenanthroline core are relatively poorly studied compounds. Developing efficient and convenient syntheses of them would result in new interesting applications because of the importance of 1,10-phenanthrolines. In this manuscript, we describe novel and practical ways to introduce a carboxyl and, for the first time, a phenol and dithiocarboxyl group under mild reaction conditions. This strategy enables highly efficient and practical synthesis of suitable organosulfur compounds with high added value, high chemoselectivity, and a broad substrate range. We present the selective conversion of a hydroxydialdehyde in the form of 10-hydroxybenzo[h]quinoline-7,9-dicarbaldehyde into its derivative, unique hydroxydicarboxylic acid, by an oxidation procedure, giving 10-hydroxybenzo[h]quinoline-7,9-dicarboxylic acid. A similar procedure resulted in the formation of 9-methyl-1,10-phenanthroline-2-carboxylic acid by oxidation of commercially available neocuproine. An alternative method of obtaining 1,10-phenanthroline derivatives possessing carboxylic acid group can be based on the hydrolysis of ester or nitrile groups; however, this synthesis leads to unexpected products. Moreover, we apply Perkin condensation to synthesize a vinyl (or styryl) analog of 1,10-phenanthroline derivatives with phenol function. This reaction also demonstrates a new, simple, and efficient strategy for converting methyl derivatives of 1,10-phenanthroline. We anticipate that the new way of converting methyl will find wide application in chemical synthesis.

Seasonal variations of low molecular weight hydroxy-dicarboxylic acids and oxaloacetic acid in remote marine aerosols from Chichijima Island in the western North Pacific (December 2010–November 2011)

Concentrations of homologous hydroxy-dicarboxylic acids (diacids) (hC3-hC6) and keto-diacid (oxaloacetic acid) were measured in the atmospheric aerosols collected at Chichijima Island (27.04° N, 142.13° E) in the western North Pacific from December 2010 to November 2011. The monthly averaged concentrations of hydroxy-diacids and oxaloacetic acid were significantly higher in spring followed by winter and autumn. Molecular distributions of hydroxy-diacids demonstrated that malic acid was the most abundant species in all four seasons, followed by tartronic acid in winter and spring and 3- and 2-hydroxyglutaric acids in summer and autumn. Hydroxy-diacids and keto-diacid maximized in spring and winter when air masses originated from the Asian continent with westerly winds. The concentrations of total hydroxy-diacids and oxaloacetic acid ranged from 0.1 to 27.3 ng m−3 and <0.005 to 2 ng m−3, respectively. The enhanced concentrations of diacids and their intermediates in winter and spring are associated with a long-range atmospheric transport of pollutants from East Asia to remote Chichijima Island followed by photochemical processing of organic aerosols. Seasonal molecular distribution of hydroxy-diacids and oxaloacetic acid was found to be dependent on the source strengths and plausible photochemical processing to form smaller diacids. Moderate to strong correlations among hydroxy-diacids, oxaloacetic acid and low molecular weight (LMW) diacids suggest that hydroxy-diacids and oxaloacetic acid are the intermediates in the photochemical oxidation of LMW diacid. Hence, photochemical formation of the most abundant LMW diacids, i.e., oxalic acid, could be produced from hydroxy- and keto-diacid as intermediates.

Peripheral Neuropathy, Episodic Rhabdomyolysis, and Hypoparathyroidism in a Patient with Mitochondrial Trifunctional Protein Deficiency.

A combination of unexplained peripheral neuropathy, hypoparathyroidism, and the inability to cope with metabolic stress could point to a rare inborn error of metabolism, such as mitochondrial trifunctional protein (MTP) deficiency.Here, we describe a 20-year-old woman who was known since childhood with axonal motor sensory polyneuropathy of unknown origin. She presented with progressive dyspnoea, and increased muscle weakness, preceded by 6 days of fever, vomiting, and diarrhoea. Laboratory testing showed rhabdomyolysis, and hypocalcaemia with low parathyroid levels. The patient was intubated because of respiratory insufficiency and a viral and bacterial pneumonia was diagnosed. She was discharged after 16 days of admission. Metabolic screening, performed at the time of rhabdomyolysis, showed increased concentrations of long-chain 3-hydroxyacyl carnitine species, together with elevated urinary excretion of 3-hydroxy dicarboxylic acids. Decreased activity of long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-ketoacyl-CoA thiolase in peripheral lymphocytes and fibroblasts confirmed a MTP deficiency. Sequence analysis of the HADHB gene showed two heterozygous variants: c.209+1G>C (splicing defect) and c.980T>C (p.Leu327Leu). When the acylcarnitine profile was repeated after the episode of rhabdomyolysis had resolved it showed no abnormalities.Our case illustrates a cluster of peripheral neuropathy, episodic rhabdomyolysis, and hypoparathyroidism in a patient with MTP deficiency caused by mutations in the HADHB gene. It stresses the importance of performing metabolic screening when patients are most symptomatic, as normal results can be found at times when no metabolic stress is present. Screening is relatively easy and timely diagnosis has important implications for treatment.

Production of citramalate by metabolically engineered Escherichia coli.

Citramalic acid (citramalate) is a five carbon hydroxy-dicarboxylic acid and potential precursor for the production of methacrylic acid from renewable resources. We examined citramalate production in Escherichia coli expressing the citramalate synthase gene cimA. Although, knockouts in ldhA coding lactate dehydrogenase and glcB/aceB coding malate synthase did not benefit citramalate accumulation, knockouts in gltA coding citrate synthase, and ackA coding acetate kinase significantly increased citramalate accumulation compared to the control strain. A fed-batch process in a controlled fermenter using a glucose feed resulted in 46.5 g/L citramalate in 132 h with a yield of 0.63 g/g, over 75% of the theoretical maximum yield from glucose of 0.82 g/g. Biotechnol. Bioeng. 2016;113: 2670-2675. © 2016 Wiley Periodicals, Inc.

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.

Hydroxydicarboxylic Acids: Markers for Secondary Organic Aerosol from the Photooxidation of α-Pinene

Detailed organic analysis of fine (PM2.5) rural aerosol collected during summer at K-puszta, Hungary from a mixed deciduous/coniferous forest site shows the presence of polar oxygenated compounds that are also formed in laboratory irradiated alpha-pinene/NOx/air mixtures. In the present work, two major photooxidation products of alpha-pinene were characterized as the hydroxydicarboxylic acids, 3-hydroxyglutaric acid, and 2-hydroxy-4-isopropyladipic acid, based on chemical, chromatographic, and mass spectral data. Different types of volatile derivatives, including trimethylsilyl ester/ether, methyl ester trimethylsilyl ether, and ethyl ester trimethylsilyl ether derivatives were measured by gas chromatography/mass spectrometry (GC/MS), and their electron ionization (El) spectra were interpreted in detail. The proposed structures of the hydroxydicarboxylic acids were confirmed or supported with reference compounds. 2-Hydroxy-4-isopropyladipic acid formally corresponds to a further reaction product of pinic acid involving addition of a molecule of water and opening of the dimethylcyclobutane ring; this proposal is supported by a laboratory irradiation experiment with alpha-pinene/NOJ0 air. In addition, we report the presence of a structurally related minor alpha-pinene photooxidation product, which was tentatively identified as the C7 homolog of 3-hydroxyglutaric acid, 3-hydroxy-4,4-dimethylglutaric acid. The detection of 2-hydroxy-4-isopropyladipic acid in ambient aerosol provides an explanation for the relatively low atmospheric concentrations of pinic acid found during daytime in forest environments.

Chemical Compositions of Secondary Organic Aerosol from the Ozonolysis of Cyclohexene in the Absence of Seed Particles

Abstract The composition of the aerosol from the ozonolysis of cyclohexene in the absence of seed particles has been investigated by laboratory chamber experiments. The aerosol collected on filters was analyzed by a liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. Low-molecular weight products, i.e., dicarboxylic acids, oxocarboxylic acids, and hydroxydicarboxylic acids, as well as oligomers with molecular weights more than 200 were found in the aerosol.

Role of the charge in continuous beds in the chiral separation of hydroxy acids by ligand-exchange capillary electrochromatography.

This paper deals with the chiral separation of hydroxy acids using diallyl-dimethylammonium chloride as a positive charge-providing agent in the continuous bed. The chiral continuous bed was prepared by in situ copolymerization of monomers, including an L-4-hydroxyproline derivative as a chiral selector. This phase was applied to the chiral separation of hydroxy monocarboxylic acids and hydroxy dicarboxylic acids, respectively. The influence of both the selector concentration and the charge-providing agent on retention and separation was investigated.

Polar organic oxygenates in PM2.5 at a southeastern site in the United States

Abstract A field study was undertaken in Research Triangle Park, NC, USA during the summer of 2000 to identify classes of polar oxygenates in PM 2.5 containing carbonyl and/or hydroxyl functional groups and, to the extent possible, determine the individual particle-bound oxygenates that make up each class. The selected site was in a semi-rural environment with expected impacts from both biogenic and anthropogenic sources. PM 2.5 samples were collected and analyzed for gravimetric mass, inorganic composition by ion chromatography, polar oxygenated compounds by gas chromatography-mass spectroscopy after derivatization of the solvent extracts, organic and elemental carbon (EC) by a thermal–optical method, and chemical functional groups by infrared (IR) spectroscopy. The chemical analyses also included determining the polarity of the PM 2.5 by measuring the organic carbon (OC) concentrations in a series of extraction solvents of increasing polarity. The ambient PM 2.5 mass concentrations ranged between 9 and 30 μg m −3 , with an average mass composition of 28% sulfate, 6% nitrate, 12% ammonium, 39% OC compound, 2% EC, and 12% liquid water at a relative humidity of 43%. OC analyses of the organic extracts indicated approximately 89% of the extractable OC was more polar in nature than that extracted by n -hexane. IR analysis of the PM 2.5 was consistent with the presence of significant levels of polar carbonyl and hydroxyl functional groups. The polar nature of the organic fraction was further supported by the observation that the inorganic fraction accounted for only about 80% of measured liquid water concentrations in the PM 2.5 . The mass spectra data were consistent with the presence of the following classes of oxygenates: oxo monocarboxylic acids, trihydroxy monocarboxylic acids, dihydroxy dicarboxylic acids, hydroxy dicarboxylic acids, normal dicarboxylic acids, oxo dicarboxylic acids, methoxy dicarboxylic acids, tricarboxylic acids, triols, and photooxidation products of α -pinene and toluene. In particular, five secondary organic aerosol compounds observed in a smog chamber irradiation of an α -pinene/NO x /air mixture were detected in ambient PM 2.5 .

Clinical and Biochemical Findings of a Patient With Thanatophoric Dysplasia Type I: Additional Finding of Dicarboxylic Aciduria

Abstract Objective: A long-surviving thanatophoric dysplasia type I patient to age of 6 years is presented. Results and Conclusions: Molecular studies revealed a heterozygous point mutation, S249C in the fibroblast growth factor receptor 3 gene. Most of the clinical course was similar to previous reports, including hearing loss and acanthosis nigricans. Abnormal urinary excretion of dicarboxylic acids and 3-hydroxydicarboxylic acids was observed. We hypothesize that this was a consequence of the FGFR3 mutation.

Analysis of 3-hydroxydodecanedioic acid for studies of fatty acid metabolic disorders: preparation of stable isotope standards.

Current diagnostic tests to detect disorders of fatty acids metabolism, such as long-chain hydroxyacyl CoA dehydrogenase deficiency (LCHAD), are hampered by insensitivity or a long delay time required for results. Children with LCHAD deficiency are known to excrete 3-hydroxydicarboxylic acids with chain lengths of 10-16 carbons, but a quantitative method to measure excretion of these potentially diagnostically important compounds has not been reported. We report synthetic schemes for synthesis of 3-hydroxydodecanedioic acid and a di-deuterated analog, suitable for use in a stable-isotope dilution mass spectrometric analytical approach. Evaluation of several common derivatization protocols to produce a volatile derivative for gas chromatography determined that trimethylsyl derivatives produced the best efficiency and stability. Positive-ion chemical ionization mass spectrometry provided the greatest yield of characteristic ions. These results indicate the basic reagents needed to develop sensitive and accurate 3-hydroxydodecanedioic acid measurements for diagnosis of LCHAD deficiency and other fatty acid oxidation disorders.

Mitochondrial trifunctional protein deficiency associated with recurrent myoglobinuria in adolescence.

A 23-year-old man with recurrent myoglobinuria had low muscle-free carnitine levels and deficient fasting ketogenesis. Urinary organic acid analysis showed large amounts of C6-C14 3-hydroxydicarboxylic acids. Mitochondrial trifunctional protein (TP), harboring long-chain enoyl-coenzyme A (CoA) hydratase, long-chain 3-hydroxyacyl-CoA dehydrogenase, and long-chain 3-ketoacyl-CoA thiolase showed markedly decreased activity in fibroblasts. On immunoblot analysis, the TP content of his fibroblasts was less than 2% that of the control cells. TP deficiency can be a life-threatening disorder with early infantile onset, but it can also present in adolescence with recurrent myoglobinuria.

Inhibitory effect of 3-hydroxyacyl-CoAs and other long-chain fatty acid beta-oxidation intermediates on mitochondrial oxidative phosphorylation.

Among the abnormal biochemical findings, a mild to severe lactic acidaemia (Treem et al 1994) is frequently described in patients with a long-chain fatty acid β-oxidation disorder, together with a markedly increased plasma long-chain acylcarnitine fraction, abnormal excretion of C 6 -C 10 dicarboxylic acids and an accumulation of medium- and long-chain 3-hydroxydicarboxylic acids (Duran et al 1991). Our aim was to explain the lactic acidosis found in these patients and to associate this with the accumulation of long-chain fatty acid derivatives. Previously (Ventura et al 1995) we have reported that long-chain (C 14 -C 20 ) acyl-CoA esters, both saturated and unsaturated, inhibit the synthesis of ATP in digitonin-permeabilized normal human fibroblasts (Wanders et al 1993). Using the same system we investigated in this work the effects of palmitoyl-CoA, α,β-unsaturated palmitoyl-CoA, 3-hydroxy- and 3-ketopalmitoyl-CoA on the oxidative phosphorylation system.

Capillary electrophoretic determination of organic acids with indirect detection

Methods for the determination of several organic acids commonly found in foods and beverages, including oxalic, citric, acetic, tartaric, malic, succinic, lactic, carbonic, aspartic, glutamic, ascorbic and gluconic acids, by capillary electrophoresis (CE) with indirect absorbance detection were developed. Several absorbance providers, including chromate, p-hydroxybenzoate, phthalate, terephthalate, trimellitate and pyromellitate, were investigated for their suitability as background electrolytes (BGEs). CE was performed in the negative voltage (reverse polarity, detector towards anode) mode. The effects of pH and various additives on CE separations were evaluated. The BGE and pH each played a major role in affecting the selectivity and resolution of CE. All analytes except malate and succinate could be baseline resolved in one run by performing CE with 5 m M trimellitate (as the BGE)-1 m M tetradecyltrimethylammonium bromide at pH 9.0 in less than 10 min. On the other hand, the CE separation of the tri- and dicarboxylic acids and hydroxydicarboxylic acids (the first five) could best be obtained at pH 5.5 in 5 min. The precision of the method for most monoprotic analytes is typically less than 1% for the migration time and 1–4% for the peak area ( n = 6). The detection limit for most analytes is of the order of 2.0·10 −6 M. The new methods developed are rapid, sensitive and quantitative and can be readily applied to real food samples for quantitative analysis.

152 INCREASED URINARY EXCRETION OF DICARBOXYLIC ACIDS AND 4 HYDROXYPHENYLLACTIC ACID IN PATIENTS WITH ZELLWEGER SYNDROME

s-Oxidation of dicarboxylic acids has been shown to occur in mitochondria iind peroxisomes of rat liver in vitro. Patients with Zellweger syndrome (ZS) offer a unique possibility to study the importance of peroxisomes in the degradation of dicarboxylic acids in vivo. Methods: We quantified urinary organic acids in seven ZS patients with impaired peroxisomal s-oxidation but normal mitochondrial s-oxidation capacity by GCMS. Results: Increased amounts of dicarboxylic acids with an odd number of carbon atoms (e.g. azelaic (up to 23 vs <0.1 mmol/mol creatinine in controls), pimelic acid (up to 15 vs <0.1)) or an even number of carbon atoms (e.g. adipic (up to 180 vs <0.1 nmol/mol creatinine in controls), suberic (up to 140 vs <0.1), sebacic acid (up to 200 vs <0.1)) and 2-hydroxysebacic acid, a medium-chain hydroxydicarboxylic acid (up to 50 vs <0.1 mmol/mol creatinine in controls), were found in the urines of these patients. In addition, patients with ZS Slowed an highly elevated excretion of 4-hydroxyphenyllactic acid (up to 98 vs <0.1 mmol/mol creatinine in controls). Conclusion: These results prove the functional importance of peroxisomes in the s-oxidation of these compounds in vivo. The characteristic pattern of urinary organic acids in ZS should prompt for specific diagnostic procedures for peroxisomal disorders, when found in patients undergoing selective screening for inherited metabolic diseases.

Molecular and isotopic analyses of the hydroxy acids, dicarboxylic acids, and hydroxydicarboxylic acids of the Murchison meteorite

The hydroxymonocarboxylic acids, dicarboxylic acids, and hydroxydicarboxylic acids of the Murchison meteorite were analyzed as their tert-butyldimethylsilyl derivatives using combined gas chromatography-mass spectrometry. The hydroxydicarboxylic acids have not been found previously in meteorites. Each class of compounds is numerous with carbon chains up to C 8 or C 9 and many, if not all, chain and substitution position isomers represented at each carbon number. The α-hydroxycarboxylic acids and α-hydroxydicarboxylic acids correspond structurally to many of the known meteoritic α-aminocarboxylic acids and α-aminodicarboxylic acids, a fact that supports the proposal that a Strecker synthesis was involved in the formation of both classes of compounds. Isotopic analyses show these acids to be D-rich relative to terrestrial organic compounds as expected; however, the hydroxy acids appear to be isotopically lighter than the amino acids with respect to both carbon and hydrogen. The latter finding would not be expected if both classes of compounds came exclusively from common precursors as would have been the case for a Strecker synthesis.

3-Hydroxydicarboxylic and 3-ketodicarboxylic aciduria in three patients: evidence for a new defect in fatty acid oxidation at the level of 3-ketoacyl-CoA thiolase

Three patients presented with evidence of a fatty acid oxidation disorder. Analysis of urinary organic acids by gas chromatography/mass spectrometry demonstrated the presence of medium-chain (C6-C12) dicarboxylic, 3-hydroxydicarboxylic, and 3-ketodicarboxylic acids in all three urines. 3-Ketodicarboxylic aciduria is reported for the first time here, as are the mass spectra for 3-ketosuberic, 3-ketosebacic, and 3-ketododecanedioic acids and the oximated spectrum for 3-ketoadipic acid. The presence of 3-ketodicarboxylic acids suggests a defect at the level of a long-chain 3-ketoacyl-CoA thiolase, an enzyme for which a deficiency state has not previously been described. Our patients may represent the first cases of a long-chain thiolase defect.

14C-Labeling of a Succinimide Derivative SM-9018, a Novel Antipsychotic Drug.

cis-2- [4- [4- (1, 2-Benzisothiazol-3-yl) -1-piperazinyl] butyl] hexahydro-1H-isoindole-1, 3-dione hydrochloride (SM-9018) was labeled with carbon-14 individually at the imido carbonyl group and the benzisothiazole ring for use in metabolic studies. The synthesis of carbonyl-labeled SM-9018 was achieved according to the scheme shown in Fig. 3. 14C-Cyanation of the ketoester (3) with potassium [14C] cyanide (2) followed by acid hydrolysis of the resulting cyanohydrin (4) gave the hydroxydi-carboxylic acid (5) . Thermal dehydration of 5 produced the unsaturated anhydride (6) . Treatment of 6 with ammonia water and subsequent catalytic hydrogenation afforded the saturated imide (8) . Alkylation of 8 with 4-chloro-1-bromobutane followed by condensation of the resulting chloride (9a) with piperazinylbenzisothiazole (16a) gave free SM-9018 (10a), which was treated with hydrochloric acid to afford 1a. The overall yield of 1a from 2 was 34%.The synthesis of benzisothiazolyl-labeled SM-9018 was carried out according to the scheme shown in Fig. 4. Conversion of the di [14C] carboxyldisulfide (11) to the corresponding acid chloride (12) followed by treatment with ammonia water yielded the diamide (13) . Oxidative cyclization of 13 gave the benzisothiazolone (14) . Chlorination of 14 and subsequent replacement reaction of the resulting chloride (15) afforded the piperazinylbenzisothiazole (16b) . Condensation of 16b with the chloride (9b) gave free SM-9018 (lob), which was treated with hydrochloric acid to afford lb. The overall yield of 1b from 11 was 16%.

Gas chromatographic/mass spectrometric identification of 3-hydroxydicarboxylic acids in urine.

Organic acid profiles were routinely analysed for two patients suffering from a massive 3-hydroxydicarboxyluria and five patients suffering from ketoaciduria. The identification of the urinary, saturated and unsaturated 3-hydroxydicarboxylic acids as their trimethylsilyl derivatives was performed with electron impact and positive chemical ionization gas chromatography/mass spectrometry. Additionally, their methylene units have been determined on SE-52 and OV-1701 types of stationary phases.

Incomplete fatty acid oxidation. The production and epimerization of 3-hydroxy fatty acids.

3-Hydroxydicarboxylic acids are major urinary metabolites derived from fatty acid metabolism. These compounds are produced from the omega-oxidation of 3-hydroxy fatty acids. The production of the precursor 3-hydroxy fatty acids from incomplete beta-oxidation of fatty acids in rat liver mitochondria was investigated. Independent of the chain length or the concentration of fatty acid substrates, the accumulation of 3-hydroxyacyl intermediates was relatively constant at the concentration of 3-5 nmol/mg of mitochondrial protein. The extent of the incomplete oxidation was the same in Percoll gradient-purified mitochondria. Rotenone treatment increased the production of 3-hydroxy fatty acids. 3-Hydroxy fatty acids did not exist as pure L-enantiomer as expected from beta-oxidation. Instead, these metabolites were epimerized to a near racemic mixture of D- and L-isomers with a slightly dominant D-isomer (58 +/- 3%). By using deuterium-isotope labeling, the mechanism of epimerizartion was shown to be a rapid dehydration-rehydration through trans-2-enoyl-CoA. In addition, cis-3 and trans-3 fatty acids were produced; these metabolites were derived from the isomerization of trans-2-enoyl-CoA. Epimerase and isomerase were thought to be enzymes involved in the oxidation of unsaturated fatty acids. Current data have shown that the metabolism of these acids is actually through NADPH-dependent reduction pathways. The activities of epimerase and isomerase detected in rat liver mitochondria possibly function mainly in the metabolism of saturated fatty acids in a reverse role to the conventional concept.