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.

Long-term strategies for the treatment of Refsum's disease using therapeutic apheresis.

Refsum's disease is a rare autosomal recessive disorder of fatty acid metabolism. Poorly metabolized phytanic acid accumulates in fatty tissues, including myelin sheaths and internal organs, leading to retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, and renal, cardiac or liver impairment. Dietary restriction of phytanic acid in some cases is not sufficient to prevent acute attacks and stabilize the progressive course. Phytanic acid bound to large low density lipoproteins (LDL) and very low density lipoproteins (VLDL) molecules offers the possibility of extracorporeal elimination by lipid apheresis. We report on the long-term lipid apheresis treatment of four patients with severe Refsum's disease. Retinitis pigmentosa, peripheral polyneuropathy, cerebellar ataxia, anosmia, and sensorineural hearing loss were major symptoms exhibiting a progressive course. Lipid apheresis was performed for 5-13 years without severe complications. Maximum levels of phytanic acid before commencing chronic lipid apheresis were >300 mg/l. During steady state with lipid apheresis, mean phytanic acid before treatments was 87 mg/l and was reduced to 36 mg/l. Mean reduction rate was 59% per treatment. In all patients, abnormal motor nerve conduction velocity with signs of chronic denervation improved, morphological and functional stabilization of eye involvement was observed. Lipid apheresis prevented the extension of the disease to previously unaffected organs in three patients. Extracorporeal elimination of lipoprotein-phytanic acid complexes by lipid apheresis represents a pathophysiologically guided therapeutic approach, resulting in long-term improvement or stabilization of overall rehabilitation in patients with progressive Refsum's disease.

Biochemical and structural characterization of CYP124: a methyl-branched lipid omega-hydroxylase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) produces a variety of methyl-branched lipids that serve important functions, including modulating the immune response during pathogenesis and contributing to a robust cell wall that is impermeable to many chemical agents. Here, we report characterization of Mtb CYP124 (Rv2266) that includes demonstration of preferential oxidation of methyl-branched lipids. Spectrophotometric titrations and analysis of reaction products indicate that CYP124 tightly binds and hydroxylates these substrates at the chemically disfavored omega-position. We also report X-ray crystal structures of the ligand-free and phytanic acid-bound protein at a resolution of 1.5 A and 2.1 A, respectively, which provide structural insights into a cytochrome P450 with predominant omega-hydroxylase activity. The structures of ligand-free and substrate-bound CYP124 reveal several differences induced by substrate binding, including reorganization of the I helix and closure of the active site by elements of the F, G, and D helices that bind the substrate and exclude solvent from the hydrophobic active site cavity. The observed regiospecific catalytic activity suggests roles of CYP124 in the physiological oxidation of relevant Mtb methyl-branched lipids. The enzymatic specificity and structures reported here provide a scaffold for the design and testing of specific inhibitors of CYP124.

Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca2+ signaling through activation of free fatty acid receptor GPR40.

The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

phytanoyl-CoA dioxygenase domain-containing protein 1 plays an important role in egg shell formation of silkworm (Bombyx mori).

Yun7Ge is a giant egg mutant found in the silkworm variety Yun7. In comparison with the giant mutant Ge, the eggs of Yun7Ge are larger. The number of laid eggs and hatching rate of Yun7Ge are reduced, which is not conducive to reproduction. In this work, the target gene controlling giant egg trait is located on the Z chromosome and was determined through genetic analysis. Transcriptome results showed that phytanoyl-CoA dioxygenase domain-containing protein 1 (PHYHD1) on the Z chromosome was silenced, and the 25 chorion genes on chromosome 2 were remarkably downregulated. Sequence analysis showed that the 73.5 kb sequence including the PHYHD1 was replaced by a ~3.0 kb sequence. After knocking out the PHYHD1 by using CRISPR/Cas9, the chorion genes were significantly downregulated. Hence, the silencing of PHYHD1 leads to the downregulation of many chorion protein genes, thus directly causing giant eggs.

Lanthanide phytanates: liquid-crystalline phase behavior, colloidal particle dispersions, and potential as medical imaging agents.

Lanthanide salts of phytanic acid, an isoprenoid-type amphiphile, have been synthesized and characterized. Elemental analysis and FTIR spectroscopy were used to confirm the formed product and showed that three phytanate anions are complexed with one lanthanide cation. The physicochemical properties of the lanthanide phytanates were investigated using DSC, XRD, SAXS, and cross-polarized optical microscopy. Several of the hydrated salts form a liquid-crystalline hexagonal columnar mesophase at room temperature, and samarium(III) phytanate forms this phase even in the absence of water. Select lanthanide phytanates were dispersed in water, and cryo-TEM images indicate that some structure has been retained in the dispersed phase. NMR relaxivity measurements were conducted on these systems. It has been shown that a particulate dispersion of gadolinium(III) phytanate displays proton relaxivity values comparable to those of a commercial contrast agent for magnetic resonance imaging and a colloidal dispersion of europium(III) phytanate exhibits the characteristics of a fluorescence imaging agent.

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten-protein conjugate for antibody development.

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative-BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).

Alpha-oxidation.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched chain fatty acid, which is a constituent of the human diet. The presence of the 3-methyl group of phytanic acid prevents degradation by beta-oxidation. Instead, the terminal carboxyl group is first removed by alpha-oxidation. The mechanism of the alpha-oxidation pathway and the enzymes involved are described in this review.

Development of a Method for Measuring Phytanic Acid as a Lifestyle-related Disease Biomarker in Rat Serum Using Ultra-fast Liquid Chromatography-Ultraviolet Spectrophotometry Combined with a Modified 2-Nitrophenylhydrazine Derivatization Method.

A simple and rapid ultra-fast liquid chromatography-ultraviolet spectrophotometry (UFLC-UV) method combined with modified 2-nitrophenylhydrazine (2-NPH) derivatization was developed for determining phytanic acid (Phy) in rat serum. Serum Phy and heptadecanoic acid (the internal standard) were derivatized by 2-NPH at ambient temperature for 20 min and extracted in n-hexane. After extracting derivatized Phy (D-Phy) and derivatized IS from the reaction mixture, the extracts were separated with a YMC-Pack C8 column (150 × 3.0 mm i.d., S-3 µm) using an isocratic mobile phase comprised of acetonitrile:H2O (90:10; pH 4.4) at 0.5 mL/min. The detection wavelength was 228 nm. Linearity was observed over 1 - 20 µg/mL (r = 0.9997). The intra- and inter-day reproducibilities of D-Phy measurements were ≤13.0%. To our knowledge, this is the first report of the quantitative and qualitative measurement of serum Phy using 2-NPH derivatization and UFLC-UV. This method can be performed rapidly under mild conditions.

Brain pyruvate and 2-oxoglutarate dehydrogenase complexes are mitochondrial targets of the CoA ester of the Refsum disease marker phytanic acid.

Pyruvate and 2-oxoglutarate dehydrogenase complexes are strongly inhibited by phytanoyl-CoA (IC(50) approximately 10(-6)-10(-7) M). Palmitoyl-CoA is 10-fold less potent. Phytanic or palmitic acids have no inhibitory effect up to 0.3 mM. At the substrate saturation, the acyl-CoA's affect the first and second enzymatic components of the 2-oxoglutarate dehydrogenase complex, while the third component is inhibited only at a low saturation with its substrate dihydrolipoamide. Thus, key regulatory branch points of mitochondrial metabolism are targets of a cellular derivative of phytanic acid. Decreased activity of the complexes might therefore contribute to neurological symptoms upon accumulation of phytanic acid in Refsum disease.

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 beta-oxidation is impossible. Instead, phytanic acid is oxidized by alpha-oxidation, yielding pristanic acid. Despite many efforts to elucidate the alpha-oxidation pathway, it remained unknown for more than 30 years. In recent years, the mechanism of alpha-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 alpha-oxidation is pristanic acid. This fatty acid is degraded by peroxisomal beta-oxidation. After 3 steps of beta-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 heterogeneous, 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 involving phytanic acid and/or pristanic acid metabolism have been characterized on the molecular level.

Formation and characterization of planar lipid bilayer membranes from synthetic phytanyl-chained glycolipids.

The formability, current-voltage characteristics and stability of the planar lipid bilayer membranes from the synthetic phytanyl-chained glycolipids, 1, 3-di-O-phytanyl-2-O-(beta-glycosyl)glycerols (Glc(Phyt)(2), Mal(N)(Phyt)(2)) were studied. The single bilayer membranes were successfully formed from the glycolipid bearing a maltotriosyl group (Mal(3)(Phyt)(2)) by the folding method among the synthetic glycolipids examined. The membrane conductance of Mal(3)(Phyt)(2) bilayers in 100 mM KCl solution was significantly lower than that of natural phospholipid, soybean phospholipids (SBPL) bilayers, and comparable to that of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayers. From the permeation measurements of lipophilic ions through Mal(3)(Phyt)(2) and DPhPC bilayers, it could be presumed that the carbonyl groups in glycerol backbone of the lipid molecule are not necessarily required for the total dipole potential barrier against cations in Mal(3)(Phyt)(2) bilayer. The stability of Mal(3)(Phyt)(2) bilayers against long-term standing and external electric field change was rather high, compared with SBPL bilayers. Furthermore, a preliminary experiment over the functional incorporation of membrane proteins was demonstrated employing the channel proteins derived from octopus retina microvilli vesicles. The channel proteins were functionally incorporated into Mal(3)(Phyt)(2) bilayers in the presence of a negatively charged glycolipid. From these observations, synthetic phytanyl-chained glycolipid bilayers are promising materials for reconstitution and transport studies of membrane proteins.

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.

Utilization of sterol carrier protein-2 by phytanoyl-CoA 2-hydroxylase in the peroxisomal alpha oxidation of phytanic acid.

Since it possesses a 3-methyl group, phytanic acid is degraded by a peroxisomal alpha-oxidation pathway, the first step of which is catalyzed by phytanoyl-CoA 2-hydroxylase (PAHX). Mutations in human PAHX cause phytanic acid accumulations leading to Adult Refsum's Disease (ARD), which is also observed in a sterol carrier protein 2 (SCP-2)-deficient mouse model. Phytanoyl-CoA is efficiently 2-hydroxylated by PAHX in vitro in the presence of mature SCP-2. Other straight-chain fatty acyl-CoA esters were also 2-hydroxylated and the products isolated and characterized. Use of SCP-2 increases discrimination between straight-chain (e.g., hexadecanoyl-CoA) and branched-chain (e.g., phytanoyl-CoA) substrates by PAHX. The results explain the phytanic acid accumulation in the SCP-2-deficient mouse model and suggest that some of the common symptoms of ARD and other peroxisomal diseases may arise in part due to defects in SCP-2 function caused by increased phytanic acid levels.

The chemoenzymatic synthesis of the core trisaccharide of N-linked oligosaccharides using a recombinant beta-mannosyltransferase.

The chemical synthesis of the beta-mannosyl linkage of N-glycans has presented a great challenge to synthetic carbohydrate chemists. We have therefore investigated the application of beta-mannosyltransferases to the preparative synthesis N-linked core oligosaccharides. In this paper we report the chemoenzymatic synthesis of beta-D-mannopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose on a preparative scale using a phytanyl-linked acceptor in the presence of a recombinant beta-(1-->4)-mannosyltransferase.

[Refsum disease]. / La maladie de Refsum.

Refsum's disease, firstly described almost 50 years ago by the Norvegian neurologist Sigvald Refsum, is an autosomic recessive disease affecting mostly the Scandinavians and the populations originating from Northern Europe. The disease results from a specific enzyme deficiency of the first step of phytanic acid catabolism pathway. This deficiency leads to an accumulation of this C20 fatty acid in the serum and the tissues with a preference for adipose tissue, liver and kidneys. The clinical picture includes retinitis pigmentosa, peripheral neuropathy, ataxia and elevated cerebrospinal fluid protein concentration. Other less frequent manifestations include cranial nerves deficiency, myocardiopathy, renal tubular dysfunction and ichtyosis. The diagnosis relies on serum phytanic acid measurement. The treatment consists of a phytanic-acid free diet sometimes associated with plasmapheresis. This treatment is generally effective on neuropathy but not on cranial nerves dysfunction and retinitis pigmentosa.

Content and distribution of phytanic acid diastereomers in organic milk as affected by feed composition.

Phytanic acid (PA) is a bioactive compound found in milk that is derived from the phytol chain of chlorophyll, and the content of PA in milk fat depends on the availability of phytol from feed. In this study, the content of PA diastereomers was analyzed in milk sampled from five organic herds twice during the grazing season (May and September). The total content of PA was higher in September compared to May, but was not affected by breed (Danish Holstein or Danish Jersey). Total PA could not be directly related to intake of green feed items. The distribution between diastereomers was closely related to the amount of grazed clovers, where a higher intake resulted in a higher share of the RRR isomer.

Phytol/Phytanic acid and insulin resistance: potential role of phytanic acid proven by docking simulation and modulation of biochemical alterations.

Since activation of PPARγ is the main target for the antidiabetic effect of TZDs, especially when it heterodimerizes with RXR, we aimed to test the potential antidiabetic effect of phytol (250 mg/kg), the natural precursor of phytanic acid, a RXR ligand and/or pioglitazone (5 mg/kg) to diabetic insulin-resistant rats. Regarding the molecular docking simulation on PPARγ, phytanic acid, rather than phytol, showed a binding mode that mimics the crystal orientation of rosiglitazone and pioglitazone, forming H bonds with the same amino acids (S289, H 323, H 449 and Y 473), and the least energy level, which emphasizes their importance for PPARγ molecular recognition, activation, hence antidiabetic activity. In addition, docking on the RXRα/PPARγ heterodimer, revealed that phytanic acid has higher binding affinity and lesser energy score on RXRα, compared to the original ligand, retinoic acid. Phytanic acid binds by 3H bonds and shares retinoic acid in arginine (R 316). These results were further supported biochemically, where oral phytol and/or pioglitazone (5 mg/kg) improved significantly glucose homeostasis, lipid panel, raised serum adiponectin level and lowered TNF-α, reaching in most cases the effect of the 10 mg/kg pioglitazone. The study concluded that the insulin sensitizing/anti-diabetic effect of phytol is mediated by partly from activation of nuclear receptors and heterodimerization of RXR with PPARγ by phytanic acid.

Characterization of two Agrostis-Festuca alpine pastures and their influence on cheese composition.

Recently, there has been a renewed interest in mountain farming, and several studies have been carried out on milk and cheese obtained in the unique environmental conditions of the Alps, a 1300 km mountain chain, located in the north of Italy. In this paper, the influence, on some cheese constituents, of two very similar mountain grasslands, both dominated by Festuca - Agrostis , was investigated. The two pastures were located in the same area in the southeastern Italian alpine region and differed in sunshine orientation and exposure. Milk obtained from cows grazing on these pastures was used to produce a semi-hard traditional cheese. The differences observed between the cheeses of the two areas for both some hydrocarbons (1-phytene and 2-phytene) and trans-fatty acids can be explained by a different rumen environment created by the botanical composition of the two pastures. The multidisciplinary approach can be considered a successful strategy, suitable for studying markers of authenticity.

Morphology of 1-methyl-1-nitrosourea induced rat mammary tumours after treatment with precursor of phytanic acid or its combination with vitamin D analogue.

OBJECTIVE: The proposed therapeutical effect of phytol (PHY), a precursor of the phytanic acid (PHYA), on mammary tumours induced with 1-methyl-1-nitrosourea (MNU), was investigated in Sprague-Dawley rats in combination with vitamin D analogue, Seocalcitol (SEO). METHODS: Female Sprague-Dawley rats were administered intraperitoneally with MNU (50 mg/kg of body weight) at the 46th and 52th days of age. Controls and MNU animals received propyleneglycol appropriate to their body weight. PHY (MNU + PHY) (500 mg/kg) was administered after tumour detection (approximately in 100th day of the life) three times/week. Combination of PHY with SEO (7 µg/kg per week) was administered to rats after tumour detection (approximately in 100th day of the life) until the 181st day of age. Then the animals were sacrificed, the tumours removed, and fixed in 10% formalin. Haematoxylin and eosine stained sections were evaluated under microscope. RESULTS: Tumour invasiveness observed in all groups of animals was ranging from 80 to 90%. Treatment with PHY alone did not inhibit the progression of the MNU induced tumours in the rat breast but it decreased the tumour burden and volume in comparison with MNU treated controls. Decreased tumour burden and volume were induced by combined treatment of PHY with SEO. Malignity and invasivity of carcinomas were not affected. CONCLUSION: No redifferentiating effect on mammary tumour cells induced by NMU after treatment with PHY alone or in combination with SEO was observed in rats. SEO alone or in combination with PHY inhibited the progression of MNU induced mammary tumours and also inhibited the increase of tumour burden and volume in comparison with MNU treated control group. However, none of the compounds, either alone or in mutual combination, reduced the malignity or the number of invasive tumours in this experimental study.