Phytanic acid metabolism in health and disease

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which cannot be beta-oxidized due to the presence of the first methyl group at the 3-position. Instead, phytanic acid undergoes alpha-oxidation to produce pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) plus CO(2). Pristanic acid is a 2-methyl branched-chain fatty acid which can undergo beta-oxidation via sequential cycles of beta-oxidation in peroxisomes and mitochondria. The mechanism of alpha-oxidation has been resolved in recent years as reviewed in this paper, although some of the individual enzymatic steps remain to be identified. Furthermore, much has been learned in recent years about the permeability properties of the peroxisomal membrane with important consequences for the alpha-oxidation process. Finally, we present new data on the omega-oxidation of phytanic acid making use of a recently generated mouse model for Refsum disease in which the gene encoding phytanoyl-CoA 2-hydroxylase has been disrupted.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo beta-oxidation but are first subjected to fatty acid alpha-oxidation in which the terminal carboxyl-group is released as CO(2). The mechanism of alpha-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid alpha-oxidation, and the complete alpha-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid alpha-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

Stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids in rat liver

The stereochemistry of the alpha-oxidation of 3-methyl-branched fatty acids was studied in rat liver. R- and S-3-methylhexadecanoic acid were equally well alpha-oxidized in intact hepatocytes and homogenates. Subcellular fractionation studies showed that alpha-oxidation of both isomers is confined to peroxisomes. Dehydrogenation of 2-methylpentadecanal, the end-product of the peroxisomal alpha-oxidation of 3-methylhexadecanoic acid, to 2-methylpentadecanoic acid, followed by derivatization with R-1-phenylethylamine and subsequent separation of the stereoisomers by gas chromatography, revealed that the configuration of the methyl-branch is preserved throughout the whole alpha-oxidation process. Metabolism and formation of the 2-hydroxy-3-methylhexadecanoyl-CoA intermediate were also investigated. Separation of the methyl esters of the four isomers of 2-hydroxy-3-methylhexadecanoic acid was achieved by gas chromatography after derivatization of the hydroxy group with R-2-methoxy-2-trifluoromethylphenylacetic acid chloride and the absolute configuration of the four isomers was determined. Although purified peroxisomes are capable of metabolizing all four isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, they can only form the (2S,3R) and the (2R,3S) isomers. Our experiments exclude the racemization of the 3-methyl branch during the alpha-oxidation process. The configuration of the 3-methyl branch does not influence the rate of alpha-oxidation, but determines the side of the 2-hydroxylation, hence the configuration of the 2-hydroxy-3-methylacyl-CoA intermediates formed during the process.     

Phytanic acid alpha-oxidation: identification of 2-hydroxyphytanoyl-CoA lyase in rat liver and its localisation in peroxisomes.

Phytanic acid is broken down by alpha-oxidation in three steps finally yielding pristanic acid. The first step occurs in peroxisomes and is catalysed by phytanoyl-CoA hydroxylase. We have studied the second step in the alpha-oxidation pathway, which involves conversion of 2-hydroxyphytanoyl-CoA to pristanal catalysed by 2-hydroxyphytanoyl-CoA lyase. To this end, we have developed a stable isotope dilution gas chromatography-mass spectrometry assay allowing activity measurements in rat liver homogenates. Cell fractionation studies demonstrate that in rat liver 2-hydroxyphytanoyl-CoA lyase is localised in peroxisomes. This finding may have important implications for inherited diseases in man characterised by impaired phytanic acid alpha-oxidation.     

The chemical biology of branched-chain lipid metabolism

Mammalian metabolism of some lipids including 3-methyl and 2-methyl branched-chain fatty acids occurs within peroxisomes. Such lipids, including phytanic and pristanic acids, are commonly found within the human diet and may be derived from chlorophyll in plant extracts. Due to the presence of a methyl group at its beta-carbon, the well-characterised beta-oxidation pathway cannot degrade phytanic acid. Instead its alpha-methylene group is oxidatively excised to give pristanic acid, which can be metabolised by the beta-oxidation pathway. Many defects in the alpha-oxidation pathway result in an accumulation of phytanic acid, leading to neurological distress, deterioration of vision, deafness, loss of coordination and eventual death. Details of the alpha-oxidation pathway have only recently been elucidated, and considerable progress has been made in understanding the detailed enzymology of one of the oxidative steps within this pathway. This review summarises these recent advances and considers the roles and likely mechanisms of the enzymes within the alpha-oxidation pathway.     

The subcellular localization of phytanic acid oxidase in rat liver

Peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia) show a series of enzymatic defects related to peroxisomal dysfunctions. Accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) has been found in several of these patients, caused by a defect in the alpha-oxidation mechanism of this acid. The fact that the alpha-oxidation of phytanic acid is defective in the peroxisomal disorders as well as in classical Refsum's disease makes it likely that this oxidation normally takes place in the peroxisomes. A series of experiments preformed to localize the phytanic acid oxidase in subcellular fractions of rat liver show, however, that the alpha-oxidation of phytanic acid is a mitochondrial process. Free phytanic acid is the substrate, and the only cofactors necessary are ATP and Mg2+.     

Identification of pristanal dehydrogenase activity in peroxisomes: conclusive evidence that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid which, due to the methyl-group at the 3-position, can not undergo beta-oxidation unless the terminal carboxyl-group is removed by alpha-oxidation. The structure of the phytanic acid alpha-oxidation machinery in terms of the reactions involved, has been resolved in recent years and includes a series of four reactions: (1) activation of phytanic acid to phytanoyl-CoA, (2) hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, (3) cleavage of 2-hydroxyphytanoyl-CoA to pristanal and formyl-CoA, and (4) oxidation of pristanal to pristanic acid. The subcellular localization of the enzymes involved has remained enigmatic, with the exception of phytanoyl-CoA hydroxylase and 2-hydroxyphytanoyl-CoA lyase which are both localized in peroxisomes. The oxidation of pristanal to pristanic acid has been claimed to be catalysed by the microsomal aldehyde dehydrogenase FALDH encoded by the ALDH10-gene. Making use of mutant fibroblasts deficient in FALDH activity, we show that phytanic acid alpha-oxidation is completely normal in these cells. Furthermore, we show that pristanal dehydrogenase activity is not fully deficient in FALDH-deficient cells, implying the existence of one or more additional aldehyde dehydrogenases reacting with pristanal. Using subcellular localization studies, we now show that peroxisomes contain pristanal dehydrogenase activity which leads us to conclude that the complete phytanic acid alpha-oxidation pathway is localized in peroxisomes.     

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.     

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.     

Omega-hydroxylation of phytanic acid in rat liver microsomes: implications for Refsum disease

The 3-methyl-branched fatty acid phytanic acid is degraded by the peroxisomal alpha-oxidation route because the 3-methyl group blocks beta-oxidation. In adult Refsum disease (ARD), peroxisomal alpha-oxidation is defective, which is caused by mutations in the gene coding for phytanoyl-CoA hydroxylase in the majority of ARD patients. As a consequence, phytanic acid accumulates in tissues and body fluids. This study focuses on an alternative route of phytanic acid degradation, omega-oxidation. The first step in omega-oxidation is hydroxylation at the omega-end of the fatty acid, catalyzed by a member of the cytochrome P450 multienzyme family. To study this first step, the formation of hydroxylated intermediates was studied in rat liver microsomes incubated with phytanic acid and NADPH. Two hydroxylated metabolites of phytanic acid were formed, omega- and (omega-1)-hydroxyphytanic acid (ratio of formation, 5:1). The formation of omega-hydroxyphytanic acid was NADPH dependent and inhibited by imidazole derivatives. These results indicate that phytanic acid undergoes omega-hydroxylation in rat liver microsomes and that an isoform of cytochrome P450 catalyzes the first step of phytanic acid omega-oxidation.     

Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.     

A dual function alpha-dioxygenase-peroxidase and NAD(+) oxidoreductase active enzyme from germinating pea rationalizing alpha-oxidation of fatty acids in plants

An enzyme with fatty acid alpha-oxidation activity (49 nkat mg(-1); substrate: lauric acid) was purified from germinating pea (Pisum sativum) by a five-step procedure to apparent homogeneity. The purified protein was found to be a 230-kD oligomer with two dominant subunits, i.e. a 50-kD subunit with NAD(+) oxidoreductase activity and a 70-kD subunit, homolog to a pathogen-induced oxygenase, which in turn shows significant homology to animal cyclooxygenase. On-line liquid chromatography-electrospray ionization-tandem mass spectrometry revealed rapid alpha-oxidation of palmitic acid incubated at 0 degrees C with the purified alpha-oxidation enzyme, leading to (R)-2-hydroperoxypalmitic acid as the major product together with (R)-2-hydroxypalmitic acid, 1-pentadecanal, and pentadecanoic acid. Inherent peroxidase activity of the 70-kD fraction decreased the amount of the (R)-2-hydroperoxy product rapidly and increased the level of (R)-2-hydroxypalmitic acid. Incubations at room temperature accelerated the decline toward the chain-shortened aldehyde. With the identification of the dual function alpha-dioxygenase-peroxidase (70-kD unit) and the related NAD(+) oxidoreductase (50-kD unit) we provided novel data to rationalize all steps of the classical scheme of alpha-oxidation in plants.     

Phytanic acid alpha-oxidation in human cultured skin fibroblasts.

We studied the oxidation of [1-14C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and 14CO2 in human skin fibroblasts. The specific activity for alpha-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid alpha-oxidation enzyme system in peroxisomes.     

The enzymic formation of long chain aldehydes and alcohols by alpha-oxidation of fatty acids in extracts of cucumber fruit (Cucumis sativus).

1. An enzyme system that catalyses the alpha-oxidation of fatty acids to shorter chain products is present in acetone powders of cucumber fruits. 2. In the absence of NAD+, the predominant product from palmitic acid is pentadecanal. Addition of NAD+ gives rise to a homologous series of n-alkanals, the concentrations of which are in the same order as that reported in the volatile products formed on homogenization of cucumbers, i.e. C15 greater than C14 greater than C13 greater than C12. 3. Pentadecan-1-ol is also formed from palmitic acid in the absence of added NAD+; C15, C14 and C13 n-alkanols are produced in the presence of NAD+. 4. The substrate specificity for saturated fatty acids is in the order C12 less than C14 greater than C16 greater than C18. Unsaturated C18 acids are oxidized more readily than stearic acid. 5. The alpha-oxidation system is inhibited by dithiothreitol, cysteine, imidazole and certain metal ligands (CN-, N3-, diphenylthiocarbazone) but not by EDTA. 6. Differences between the alpha-oxidation system in cucumber and those previously reported in other plants are discussed.     

Alpha-oxidation of 3-methyl-substituted fatty acids and its thiamine dependence.

3-Methyl-branched fatty acids, as phytanic acid, undergo peroxisomal alpha-oxidation in which they are shortened by 1 carbon atom. This process includes four steps: activation, 2-hydroxylation, thiamine pyrophosphate dependent cleavage and aldehyde dehydrogenation. The thiamine pyrophosphate dependence of the third step is unique in peroxisomal mammalian enzymology. Human pathology due to a deficient alpha-oxidation is mostly linked to mutations in the gene coding for the second enzyme of the sequence, phytanoyl-CoA hydroxylase.     

alpha-oxidation of fatty acids in higher plants. Identification of a pathogen-inducible oxygenase (piox) as an alpha-dioxygenase and biosynthesis of 2-hydroperoxylinolenic acid.

A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsis were recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523-1537). Linolenic acid incubated at 23 degrees C with preparations containing the recombinant enzymes underwent alpha-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z), 14(Z)-heptadecatrienal (83%), an alpha-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 degrees C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z), 12(Z),15(Z)-octadecatrienoic acid in alpha-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same alpha-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z), 15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 degrees C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 degrees C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for alpha-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic alpha-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into alpha-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both alpha-dioxygenases, and that members of this new class of enzymes catalyze the first step of alpha-oxidation in plant tissue.     

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.     

Phytanic acid alpha-oxidation: accumulation of 2-hydroxyphytanic acid and absence of 2-oxophytanic acid in plasma from patients with peroxisomal disorders.

A stable isotope dilution method was developed for the measurement of 2-hydroxyphytanic acid and 2-oxophytanic acid in plasma. In plasma from healthy individuals and from patients with Refsum's disease, 2-hydroxyphytanic acid was found at levels less than 0.2 mumol/l, whereas the acid accumulated in plasma from patients with rhizomelic chondrodysplasia punctata, generalized peroxisomal dysfunction, and a single peroxisomal beta-oxidation enzyme deficiency. In plasma from both healthy controls and patients with peroxisomal disorders, 2-oxophytanic acid was undetectable. Four different groups of diseases were characterized with a defective phytanic acid alpha-oxidation and/or pristanic acid beta-oxidation: 1) Refsum's disease, with a defect at phytanic acid alpha-hydroxylation; 2) rhizomelic chondrodysplasia punctata, with a defect at 2-hydroxyphytanic acid decarboxylation; 3) generalized peroxisomal disorders, with defects at 2-hydroxyphytanic acid decarboxylation and at pristanic acid beta-oxidation; 4) single peroxisomal beta-oxidation enzyme deficiencies, with a defect at pristanic acid beta-oxidation, resulting in an impaired phytanic acid alpha-oxidation by inhibition. The results indicate that 2-hydroxyphytanic acid decarboxylation and pristanic acid beta-oxidation take place in peroxisomes.     

Odd-and even-numbered fatty acids. Their contrasting behavior in normal and fowlpox virus-infected epithelium.

Upon infection with fowlpox virus, the amount of odd-numbered fatty acids in chick scalp epithelium shows a significant decrease compared with control values. This effect begins quite early and progresses throughout the period of infection. Individual members of the odd-numbered family (C15--C27 inclusive) were quantitatively related to the group as a whole during most of the infection. Experiments involving the administration of labeled acetate in vivo demonstrated an increase in the synthesis of even-numbered fatty acids and a decrease in the synthesis of odd-numbered fatty acids in infected epithelium. The reduced synthesis of odd-numbered fatty acids in infected epithelium could also be demonstrated with labeled propionate. The influence of the alpha-oxidation pathway was assayed in chick scalp epithelium in vivo by the administration of [1-14C,9,10-3H] stearic acid. The C17 acids formed had a 3H/14C ratio similar to that of the C16 acids, indicating that most label incorporation into C17 was due to beta-oxidation to acetate followed by resynthesis into fatty acids. C17 fatty acids from control and infected epithelium had similar 3H/14C ratios, indicating that the alpha-oxidation pathway probably does not contribute to the differences in odd-numbered fatty acid content observed. In assays for fatty acid synthetase activty, both [14C] acetyl-CoA and [14C]-propionyl-CoA were used as initial acceptors. The specific activities of preparations from infected scalp were similar to those of control preparations with both substrates. These results suggest that there is no decline in the ability to utilize propionate for fatty acid synthesis in infected epithelium.