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.     

Rapid stable isotope dilution analysis of very-long-chain fatty acids, pristanic acid and phytanic acid using gas chromatography–electron impact mass spectrometry

A common feature of most peroxisomal disorders is the accumulation of very-long-chain fatty acids (VLCFAs) and/or pristanic and phytanic acid in plasma. Previously described methods utilizing either gas chromatography alone or gas chromatography–mass spectrometry are, in general, time-consuming and unable to analyze VLCFAs, pristanic and phytanic acid within a single analysis. We describe a simple, reproducible and rapid method using gas chromatography/mass spectrometry with deuterated internal standards. The method was evaluated by analysing 30 control samples and samples from 35 patients with defined peroxisomal disorders and showed good discrimination between controls and patients. This method is suitable for routine screening for peroxisomal disorders.     

Stereochemistry of the peroxisomal branched-chain fatty acid alpha- and beta-oxidation systems in patients suffering from different peroxisomal disorders

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid derived from dietary sources and broken down in the peroxisome to pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) via alpha-oxidation. Pristanic acid then undergoes beta-oxidation in peroxisomes. Phytanic acid naturally occurs as a mixture of (3S,7R,11R)- and (3R,7R,11R)-diastereomers. In contrast to the alpha-oxidation system, peroxisomal beta-oxidation is stereospecific and only accepts (2S)-isomers. Therefore, a racemase called alpha-methylacyl-CoA racemase is required to convert (2R)-pristanic acid into its (2S)-isomer. To further investigate the stereochemistry of the peroxisomal oxidation systems and their substrates, we have developed a method using gas-liquid chromatography-mass spectrometry to analyze the isomers of phytanic, pristanic, and trimethylundecanoic acid in plasma from patients with various peroxisomal fatty acid oxidation defects. In this study, we show that in plasma of patients with a peroxisomal beta-oxidation deficiency, the relative amounts of the two diastereomers of pristanic acid are almost equal, whereas in patients with a defect of alpha-methylacyl-CoA racemase, (2R)-pristanic acid is the predominant isomer. Furthermore, we show that in alpha-methylacyl-CoA racemase deficiency, not only pristanic acid accumulates, but also one of the metabolites of pristanic acid, 2610-trimethylundecanoic acid, providing direct in vivo evidence for the requirement of this racemase for the complete degradation of pristanic acid.     

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARalpha-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.     

Measurement of plasma pristanic, phytanic and very long chain fatty acids by liquid chromatography-electrospray tandem mass spectrometry for the diagnosis of peroxisomal disorders

High pressure liquid chromatography with a narrow bore C8 column has been used to separate pristanic, phytanic and very long chain fatty acids, important in the diagnosis of peroxisomal disorders, for their accurate isotope dilution quantification by tandem mass spectrometry. The fatty acids, isolated from plasma, were analysed as trimethylaminoethyl ester (quaternary ammonium) derivatives. Analysis time was 2.5 h and sample requirement was 10 microl of plasma. Good agreement with GC-MS methods for the levels of pristanic and phytanic acids, C26:0/C22:0 and C24:0/C22:0 ratios were obtained for 12 plasma samples from peroxisomal disorder patients and a set of controls.     

Human metabolism of phytanic acid and pristanic acid

Phytanic acid is a methyl-branched fatty acid present in the human diet. Due to its structure, degradation by β-oxidation is impossible. Instead, phytanic acid is oxidized by -oxidation, yielding pristanic acid. Despite many efforts to elucidate the -oxidation pathway, it remained unknown for more than 30 years. In recent years, the mechanism of -oxidation as well as the enzymes involved in the process have been elucidated. The process was found to involve activation, followed by hydroxylase, lyase and dehydrogenase reactions. Part, if not all of the reactions were found to take place in peroxisomes. The final product of phytanic acid -oxidation is pristanic acid. This fatty acid is degraded by peroxisomal β-oxidation. After 3 steps of β-oxidation in the peroxisome, the product is esterified to carnitine and shuttled to the mitochondrion for further oxidation. Several inborn errors with one or more deficiencies in the phytanic acid and pristanic degradation have been described. The clinical expressions of these disorders are heterogeneus, and vary between severe neonatal and often fatal symptoms and milder syndromes with late onset. Biochemically, these disorders are characterized by accumulation of phytanic and/or pristanic acid in tissues and body fluids. Several of the inborn errors involoving phytanic acid and/or pristanic acid metabolism have been characterized on the molecular level.     

Peroxisomes contain a specific phytanoyl-CoA/pristanoyl-CoA thioesterase acting as a novel auxiliary enzyme in alpha- and beta-oxidation of methyl-branched fatty acids in mouse

Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo beta-oxidation directly but instead must first undergo alpha-oxidation to pristanic acid, which then enters beta-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor alpha (PPARalpha) and is up-regulated in mouse liver in a PPARalpha-dependent manner.     

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.     

Pristanic acid and phytanic acid: naturally occurring ligands for the nuclear receptor peroxisome proliferator-activated receptor alpha

Phytanic acid and pristanic acid are branched-chain fatty acids, present at micromolar concentrations in the plasma of healthy individuals. Here we show that both phytanic acid and pristanic acid activate the peroxisome proliferator-activated receptor alpha (PPARalpha) in a concentration-dependent manner. Activation is observed via the ligand-binding domain of PPARalpha as well as via a PPAR response element (PPRE). Via the PPRE significant induction is found with both phytanic acid and pristanic acid at concentrations of 3 and 1 microM, respectively. The trans-activation of PPARdelta and PPARgamma by these two ligands is negligible. Besides PPARalpha, phytanic acid also trans-activates all three retinoic X receptor subtypes in a concentration-dependent manner. In primary human fibroblasts, deficient in phytanic acid alpha-oxidation, trans-activation through PPARalpha by phytanic acid is observed. This clearly demonstrates that phytanic acid itself, and not only its metabolite, pristanic acid, is a true physiological ligand for PPARalpha. Because induction of PPARalpha occurs at ligand concentrations comparable to the levels found for phytanic acid and pristanic acid in human plasma, these fatty acids should be seen as naturally occurring ligands for PPARalpha.These results demonstrate that both pristanic acid and phytanic acid are naturally occurring ligands for PPARalpha, which are present at physiological concentrations.     

Inactivation of the peroxisomal multifunctional protein-2 in mice impedes the degradation of not only 2-methyl-branched fatty acids and bile acid intermediates but also of very long chain fatty acids

According to current views, peroxisomal beta-oxidation is organized as two parallel pathways: the classical pathway that is responsible for the degradation of straight chain fatty acids and a more recently identified pathway that degrades branched chain fatty acids and bile acid intermediates. Multifunctional protein-2 (MFP-2), also called d-bifunctional protein, catalyzes the second (hydration) and third (dehydrogenation) reactions of the latter pathway. In order to further clarify the physiological role of this enzyme in the degradation of fatty carboxylates, MFP-2 knockout mice were generated. MFP-2 deficiency caused a severe growth retardation during the first weeks of life, resulting in the premature death of one-third of the MFP-2(-/-) mice. Furthermore, MFP-2-deficient mice accumulated VLCFA in brain and liver phospholipids, immature C(27) bile acids in bile, and, after supplementation with phytol, pristanic and phytanic acid in liver triacylglycerols. These changes correlated with a severe impairment of peroxisomal beta-oxidation of very long straight chain fatty acids (C(24)), 2-methyl-branched chain fatty acids, and the bile acid intermediate trihydroxycoprostanic acid in fibroblast cultures or liver homogenates derived from the MFP-2 knockout mice. In contrast, peroxisomal beta-oxidation of long straight chain fatty acids (C(16)) was enhanced in liver tissue from MFP-2(-/-) mice, due to the up-regulation of the enzymes of the classical peroxisomal beta-oxidation pathway. The present data indicate that MFP-2 is not only essential for the degradation of 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acid but also for the breakdown of very long chain fatty acids.     

Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency

We identified a new peroxisomal disorder caused by a deficiency of the enzyme alpha-methylacyl-coenzyme A (CoA) racemase. Patients with this disorder show elevated plasma levels of pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid (DHCA and THCA), which are all substrates for the peroxisomal beta-oxidation system. alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (2S)-isomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. In this study we used liquid chromatography/tandem mass spectrometry (LC-MS/MS) to analyze the bile acid intermediates that accumulate in plasma from patients with a deficiency of alpha-methylacyl-CoA racemase and, for comparison, in plasma from patients with Zellweger syndrome and patients with cholestatic liver disease.We found that racemase-deficient patients accumulate exclusively the (R)-isomer of free and taurine-conjugated DHCA and THCA, whereas in plasma of patients with Zellweger syndrome and patients with cholestatic liver disease both isomers were present. On the basis of these results we describe an easy and reliable method for the diagnosis of alpha-methylacyl-CoA racemase-deficient patients by plasma analysis. Our results also show that alpha-methylacyl-CoA racemase plays a unique role in bile acid formation. - Ferdinandusse, S., H. Overmars, S. Denis, H. R. Waterham, R. J. A. Wanders, and P. Vreken. Plasma analysis of di- and trihydroxycholestanoic acid diastereoisomers in peroxisomal alpha-methylacyl-CoA racemase deficiency. J. Lipid Res. 2001. 42: 137;-141.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

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.     

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.     

Subcellular localization and physiological role of alpha-methylacyl-CoA racemase

alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (S)-stereoisomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. Patients with a deficiency of alpha-methylacyl-CoA racemase accumulate in their plasma pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid, which are all substrates of the peroxisomal beta-oxidation system. Subcellular fractionation experiments, however, revealed that both in humans and rats alpha-methylacyl-CoA racemase is bimodally distributed to both the peroxisome and the mitochondrion. Our findings show that the peroxisomal and mitochondrial enzymes are produced from the same gene and that, as a consequence, the bimodal distribution pattern must be the result of differential targeting of the same gene product. In addition, we investigated the physiological role of the enzyme in the mitochondrion. Both in vitro studies with purified heterologously expressed protein and in vivo studies in fibroblasts of patients with an alpha-methylacyl-CoA racemase deficiency revealed that the mitochondrial enzyme plays a crucial role in the mitochondrial beta-oxidation of the breakdown products of pristanic acid byconverting (2R,6)-dimethylheptanoyl-CoA to its (S)-stereoisomer.     

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+.     

Separation of phytanic and pristanic acid by high-pressure liquid chromatography: application of the method

The synthesis of pristanic acid from phytanic acid, and a simple reversed-phase high-pressure liquid chromatographic (HPLC) method for the separation and purification of these acids, is described. A base-line separation of [U-3H]phytanic and [U-3H]pristanic acid is achieved with a graphitized carbon column. The isoprenoid metabolites formed after incubation of cultured fibroblasts with phytanic or pristanic acids are extracted with a Sep-Pak C18 cartridge and separated from the substrates by the same reversed-phase HPLC used for substrate purification. The methods are suitable for studies on the mechanisms for degradation of phytanic acid. Recently, different inborn errors with accumulation of phytanic acid have been defined. The present method will be a useful tool in our efforts to define these metabolic defects and their subcellular localization.     

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.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.