Disruption of mitochondrial bioenergetics and calcium homeostasis by phytanic acid in the heart: Potential relevance for the cardiomyopathy in Refsum disease.

Refsum disease is an inherited peroxisomal disorder caused by severe deficiency of phytanoyl-CoA hydroxylase activity. Affected patients develop severe cardiomyopathy of poorly known pathogenesis that may lead to a fatal outcome. Since phytanic acid (Phyt) concentrations are highly increased in tissues of individuals with this disease, it is conceivable that this branched-chain fatty acid is cardiotoxic. The present study investigated whether Phyt (10-30 µM) could disturb important mitochondrial functions in rat heart mitochondria. We also determined the influence of Phyt (50-100 µM) on cell viability (MTT reduction) in cardiac cells (H9C2). Phyt markedly increased mitochondrial state 4 (resting) and decreased state 3 (ADP-stimulated) and uncoupled (CCCP-stimulated) respirations, besides reducing the respiratory control ratio, ATP synthesis and the activities of the respiratory chain complexes I-III, II, and II-III. This fatty acid also reduced mitochondrial membrane potential and induced swelling in mitochondria supplemented by exogenous Ca2+, which were prevented by cyclosporin A alone or combined with ADP, suggesting the involvement of the mitochondrial permeability transition (MPT) pore opening. Mitochondrial NAD(P)H content and Ca2+ retention capacity were also decreased by Phyt in the presence of Ca2+. Finally, Phyt significantly reduced cellular viability (MTT reduction) in cultured cardiomyocytes. The present data indicate that Phyt, at concentrations found in the plasma of patients with Refsum disease, disrupts by multiple mechanisms mitochondrial bioenergetics and Ca2+ homeostasis, which could presumably be involved in the cardiomyopathy of this disease.

Dietary phytanic acid-induced changes in tissue fatty acid profiles in mice.

The aims of this research communication were to investigate the in vivo tissue accumulation of phytanic acid (PA) and any changes in the tissue fatty acid profiles in mice. Previous in vitro studies have demonstrated that PA is a milk component with the potential to cause both beneficial effects on lipid and glucose metabolism and detrimental effects on neuronal cells. However, there is limited information about its in vivo actions. In this study, mice were fed diets containing either 0.00 or 0.05% 3RS, 7R, 11R-PA, which is the isomer found in milk and the human body. After 4 weeks, adipose tissue, liver and brain were harvested and their fatty acid profiles were determined by gas chromatographic analysis. The results showed that PA and its metabolite pristanic acid accumulated in the adipose tissue of PA-fed mice, and that dietary PA decreased the hepatic compositions of several saturated fatty acids such as palmitic acid while increasing the compositions of polyunsaturated fatty acids including linoleic acid and docosahexaenoic acid. However, dietary PA neither accumulated nor had a high impact on the fatty acid profile in the brain. These results suggested that dietary PA could exert its biological activities in adipose tissue and liver, although the brain is relatively less affected by dietary PA. These data provide a basis for understanding the in vivo physiological actions of PA.

Phytanic acid activates PPARα to promote beige adipogenic differentiation of preadipocytes.

A better understanding of the mechanisms of beige and brown adipogenesis is needed for developing strategies to prevent and treat obesity and associated metabolic disorders. Phytanic acid (PA) exists in a wide range of foods, especially in milk fat and marine foods, but its effects on obesity and beige adipogenesis remain poorly defined. The objective is to investigate the effects and regulatory mechanisms of PA in the beige adipogenesis. In 3T3-L1 preadipocytes, PA elevated the expression of brown adipogenic markers, suggesting that PA promotes beige adipogenic differentiation in committed adipogenic cells. In uncommitted C3H10T1/2 cells, while PA increased PGC1α expression, it did not increase brown adipogenic regulators PRDM16 or UCP1 expression, suggesting that PA had no significant effects on brown adipocyte commitment. PA also enhanced mitochondrial biogenesis and oxygen consumption. Promotion of both mitochondriogenesis and beige adipogenic differentiation were blocked by using PPARα antagonist or with Pparα knockdown, showing that PA-mediated beige/brown adipogenic differentiation is dependent on PPARα. Additionally, the PA-regulated effect is independent on ß3-adrenergic receptor. Taken together, PA promotes beige adipogenic differentiation, but not the commitment of progenitor cells to the brown adipocyte lineage. PPARα is a key mediator during PA-induced beige/brown adipogenic differentiation.

Low Phytanic Acid-Concentrated DHA Prevents Cognitive Deficit and Regulates Alzheimer Disease Mediators in an ApoE-/- Mice Experimental Model.

Alzheimer's disease (AD) is the main cause of dementia and cognitive impairment. It has been associated with a significant diminution of omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) levels in the brain. Clinical trials with DHA as a treatment in neurological diseases have shown inconsistent results. Previously, we reported that the presence of phytanic acid (PhA) in standard DHA compositions could be blunting DHA's beneficial effects. Therefore, we aimed to analyze the effects of a low PhA-concentrated DHA and a standard PhA-concentrated DHA in Apolipoprotein E knockout (ApoE-/-) mice. Behavioral tests and protein expression of pro-inflammatory, pro-oxidant, antioxidant factors, and AD-related mediators were evaluated. Low PhA-concentrated DHA decreased Aß, ß-amyloid precursor protein (APP), p-tau, Ca2+/calmodulin-dependent protein kinase II (CAMKII), caspase 3, and catalase, and increased brain derived neurotrophic factor (BDNF) when compared to standard PhA-concentrated DHA. Low PhA-concentrated DHA decreased interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) protein expression in ApoE-/- mice when compared to standard PhA-concentrated DHA. No significant differences were found in p22phox, inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx), superoxide dismutase 1 (SOD-1), and tau protein expression. The positive actions of a low PhA-concentrated DHA were functionally reflected by improving the cognitive deficit in the AD experimental model. Therefore, reduction of PhA content in DHA compositions could highlight a novel pathway for the neurodegeneration processes related to AD.

Naturally occurring 3RS, 7R, 11R-phytanic acid suppresses in vitro T-cell production of interferon-gamma.

BACKGROUND: Among the eight stereoisomers of phytanic acid (PA), the 3RS, 7R, 11R-isomer is naturally occurring and is present in foods and the human body. PA is considered to have possible health benefits in the immune system. However, it remains undetermined whether these effects are elicited by the 3RS, 7R, 11R-PA isomer, because previous studies used a commercially available PA whose isomer configuration is unknown. In this study, we synthesized a preparation of 3RS, 7R, 11R-PA, and investigated its in vitro immunomodulatory effects, especially the T-cell production of interferon (IFN)-γ, which is associated with various autoimmune diseases. This study also investigated the effects of 3RS, 7R, 11R-PA on NF-κB activity in order to address the mechanism of its immunomodulatory effects. METHODS: Mouse splenocytes and purified T-cells were stimulated with T-cell mitogens and incubated with 3RS, 7R, 11R-PA, followed by evaluation of IFN-γ production. The effect of 3RS, 7R, 11R-PA on NF-κB activity was also investigated using an A549 cell line with stable expression of an NF-κB-dependent luciferase reporter gene. RESULTS: 3RS, 7R, 11R-PA significantly reduced in vitro IFN-γ production at both the protein and mRNA levels, and was accompanied by decreased expression of T-bet, a key regulator of Th1 cell differentiation. The results indicated that NF-κB-mediated transcriptional activity was significantly decreased by 3RS, 7R, 11R-PA and that GW6471, an antagonist of peroxisome proliferator activated receptor α (PPARα), abrogated the inhibitory effect of 3RS, 7R, 11R-PA on NF-κB activity. CONCLUSIONS: The present study suggests that 3RS, 7R, 11R-PA is a functional and bioactive fatty acid, and has a potentially beneficial effect for amelioration of T-cell mediated autoimmune diseases. This study also indicates that interference in the NF-κB pathway via PPARα activation is a potential mechanism of the immunomodulatory effects of 3RS, 7R, 11R-PA.

Effects of Low Phytanic Acid-Concentrated DHA on Activated Microglial Cells: Comparison with a Standard Phytanic Acid-Concentrated DHA.

Docosahexaenoic acid (DHA, 22:6 n-3) is an essential omega-3 (ω-3) long chain polyunsaturated fatty acid of neuronal membranes involved in normal growth, development, and function. DHA has been proposed to reduce deleterious effects in neurodegenerative processes. Even though, some inconsistencies in findings from clinical and pre-clinical studies with DHA could be attributed to the presence of phytanic acid (PhA) in standard DHA treatments. Thus, the aim of our study was to analyze and compare the effects of a low PhA-concentrated DHA with a standard PhA-concentrated DHA under different neurotoxic conditions in BV-2 activated microglial cells. To this end, mouse microglial BV-2 cells were stimulated with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) and co-incubated with DHA 50 ppm of PhA (DHA (PhA:50)) or DHA 500 ppm of PhA (DHA (PhA:500)). Cell viability, superoxide anion (O2-) production, Interleukin 6 (L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GtPx), glutathione reductase (GtRd), Caspase-3, and the brain-derived neurotrophic factor (BDNF) protein expression were explored. Low PhA-concentrated DHA protected against LPS or H2O2-induced cell viability reduction in BV-2 activated cells and O2- production reduction compared to DHA (PhA:500). Low PhA-concentrated DHA also decreased COX-2, IL-6, iNOS, GtPx, GtRd, and SOD-1 protein expression when compared to DHA (PhA:500). Furthermore, low PhA-concentrated DHA increased BDNF protein expression in comparison to DHA (PhA:500). The study provides data supporting the beneficial effect of low PhA-concentrated DHA in neurotoxic injury when compared to a standard PhA-concentrated DHA in activated microglia.

A Phytol-Enriched Diet Activates PPAR-α in the Liver and Brown Adipose Tissue to Ameliorate Obesity-Induced Metabolic Abnormalities.

SCOPE: Peroxisome proliferator-activated receptor alpha (PPAR-α) is a ligand-activated transcription factor that regulates lipid and carbohydrate metabolism. We investigate the effects of naturally occurring PPAR-α agonists, phytol, and its metabolite phytanic acid, on obesity-induced metabolic disorders using a mouse model. METHODS AND RESULTS: A luciferase reporter assay shows that phytanic acid potently activates PPAR-α among PPAR subtypes. In high-fat-diet-induced, severely obese mice, a phytol-enriched diet increases phytanic acid levels in the liver and adipose tissue, where PPAR-α is abundantly expressed. A phytol-enriched diet ameliorates severe obesity and the related metabolic abnormalities of white adipose tissue. Moreover, the expression of PPAR-α target genes in the liver and brown adipose tissue is enhanced by a phytol-enriched diet, suggesting that phytol and phytanic acid activate PPAR-α in these organs. We confirm that phytanic acid treatment induced PPAR-α target gene expression in both primary hepatocytes and brown adipocytes from wild-type mice, but not in these cells from PPAR-α-deficient mice. CONCLUSION: A phytol-enriched diet may increase phytanic acid levels in the liver and brown adipocytes, thereby activating PPAR-α in these organs and ameliorating obesity-induced metabolic diseases.

Phytol-induced pathology in 2-hydroxyacyl-CoA lyase (HACL1) deficient mice. Evidence for a second non-HACL1-related lyase.

2-Hydroxyacyl-CoA lyase (HACL1) is a key enzyme of the peroxisomal α-oxidation of phytanic acid. To better understand its role in health and disease, a mouse model lacking HACL1 was investigated. Under normal conditions, these mice did not display a particular phenotype. However, upon dietary administration of phytol, phytanic acid accumulated in tissues, mainly in liver and serum of KO mice. As a consequence of phytanic acid (or a metabolite) toxicity, KO mice displayed a significant weight loss, absence of abdominal white adipose tissue, enlarged and mottled liver and reduced hepatic glycogen and triglycerides. In addition, hepatic PPARα was activated. The central nervous system of the phytol-treated mice was apparently not affected. In addition, 2OH-FA did not accumulate in the central nervous system of HACL1 deficient mice, likely due to the presence in the endoplasmic reticulum of an alternate HACL1-unrelated lyase. The latter may serve as a backup system in certain tissues and account for the formation of pristanic acid in the phytol-fed KO mice. As the degradation of pristanic acid is also impaired, both phytanoyl- and pristanoyl-CoA levels are increased in liver, and the ω-oxidized metabolites are excreted in urine. In conclusion, HACL1 deficiency is not associated with a severe phenotype, but in combination with phytanic acid intake, the normal situation in man, it might present with phytanic acid elevation and resemble a Refsum like disorder.

Phytanic acid attenuates insulin-like growth factor-1 activity via nitric oxide-mediated γ-secretase activation in rat aortic smooth muscle cells: possible implications for pathogenesis of infantile Refsum disease.

BACKGROUND: Infantile Refsum disease (IRD), a peroxisomal disease with defective phytanic acid oxidation, causes neurological impairment and development delay. Insulin-like growth factor-1 (IGF-1) regulates child development and to understand molecular mechanism(s) of IRD, we examined the effect of phytanic acid (PA) on IGF-1 activity. METHODS: Bromodeoxyuridine (BrdU) incorporation was measured in rat aortic smooth muscle cell (SMC) cultures following treatment with fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) or IGF-1 in the absence or presence of PA. Gene expression and protein contents of IGF-1 receptor (IGF-1R) and PDGF receptor (PDGFR) were examined using quantitative PCR and western blotting. RESULTS: PA inhibited mitogenic activities of FBS, PDGF and IGF-1 with more pronounced effect on IGF-1-induced bromodeoxyuridine (BrdU) incorporation. Palmitic acid or lignoceric acids did not inhibit IGF-1 activity. PA had no effect on PDGFR mRNA/protein levels but markedly increased IGF-1R mRNA levels. PA and nitric oxide (NO) markedly decreased IGF-1R protein. L-NAME, a NO synthase inhibitor and DAPT, a γ-secretase inhibitor, alleviated PA-induced decrease in IGF-1R protein. Both PA and NO donor increased γ-secretase activity which was alleviated by L-NAME. CONCLUSION: This study demonstrates that PA attenuates IGF-1 activity possibly through IGF-1R impairment and NO-mediated modulation of γ-secretase activity.

Phytanic acid activates NADPH oxidase through transactivation of epidermal growth factor receptor in vascular smooth muscle cells.

BACKGROUND: Phytanic acid (PA) has been implicated in development of cancer and its defective metabolism is known to cause life-threatening conditions, such as Refsum disease, in children. To explore molecular mechanisms of phytanic acid-induced cellular pathology, we investigated its effect on NADPH oxidase (NOX) and epidermal growth factor receptor (EGFR) in rat aortic smooth muscle cells (RASMC). METHODS: Smooth muscle cells were isolated from rat aortae using enzymic digestion with collagenase and elastase. Cultured RASMC were treated with varying concentrations (0.5-10 µg/ml) of phytanic acid in the presence/absence of fetal bovine serum (FBS) and/or EGFR inhibitor, AG1478. Following treatment with experimental agents, NOX activity was assayed in RASMC cultures by luminescence method. Gene expression of NOX-1 and p47phox was assessed using RT-PCR. NOX-1, p47phox and, total EGFR protein and its phosphorylated form were measured by Western blotting. RESULTS: Treatment of RASMC with supraphysiological concentrations (>2.5 µg/ml) of PA significantly (p < 0.01) increased the NOX activity. PA also significantly increased gene/protein expression of NOX-1 and p47phox whereas p22phox and p67phox remained unaffected. Interestingly, PA (2.5-10 µg/ml) markedly (2-3 folds) increased the total and phosphorylated EGFR. Treatment of cells with EGFR inhibitor, AG1478, significantly blocked the PA-induced enhancement of NOX activity. CONCLUSIONS: Our findings that PA transactivates EGFR and induces NOX activity in vascular smooth muscle cells provide new insights into molecular mechanisms of PA's role in cancer and Refsum disease.

The human liver fatty acid binding protein T94A variant alters the structure, stability, and interaction with fibrates.

Although the human liver fatty acid binding protein (L-FABP) T94A variant arises from the most commonly occurring single-nucleotide polymorphism in the entire FABP family, there is a complete lack of understanding regarding the role of this polymorphism in human disease. It has been hypothesized that the T94A substitution results in the complete loss of ligand binding ability and function analogous to that seen with L-FABP gene ablation. This possibility was addressed using the recombinant human wild-type (WT) T94T and T94A variant L-FABP and cultured primary human hepatocytes. Nonconservative replacement of the medium-sized, polar, uncharged T residue with a smaller, nonpolar, aliphatic A residue at position 94 of the human L-FABP significantly increased the L-FABP α-helical structure content at the expense of ß-sheet content and concomitantly decreased the thermal stability. T94A did not alter the binding affinities for peroxisome proliferator-activated receptor α (PPARα) agonist ligands (phytanic acid, fenofibrate, and fenofibric acid). While T94A did not alter the impact of phytanic acid and only slightly altered that of fenofibrate on the human L-FABP secondary structure, the active metabolite fenofibric acid altered the T94A secondary structure much more than that of the WT T94T L-FABP. Finally, in cultured primary human hepatocytes, the T94A variant exhibited a significantly reduced extent of fibrate-mediated induction of PPARα-regulated proteins such as L-FABP, FATP5, and PPARα itself. Thus, while the T94A substitution did not alter the affinity of the human L-FABP for PPARα agonist ligands, it significantly altered the human L-FABP structure, stability, and conformational and functional response to fibrate.

Effects of cis-9,trans-11 and trans-10,cis-12 conjugated linoleic acid, linoleic acid, phytanic acid and the combination of various fatty acids on proliferation and cytokine expression of bovine peripheral blood mononuclear cells.

Fatty acids may have an impact on immune functions, which is important in times of increased mobilization of body fat, e.g., around parturition. The aim of the present study was to investigate the effects of the CLA isomers cis-9,trans-11 and trans-10,cis-12, phytanic acid (PA), linoleic acid (LA) and a fatty acid (FA) mixture (containing 29.8% palmitic acid, 6.7% palmitoleic acid, 17.4% stearic acid and 46.1% oleic acid) on the proliferation of bovine blood mononuclear cells (PBMC) in vitro using alamar blue (AB) and 5-bromo-2'-deoxyuridine (BrdU) assay. Quantitative real time polymerase chain reaction analyses were performed to evaluate the expression of interleukin (IL)-4, IL-10, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and peroxisome proliferator-activated receptor (PPAR)-γ in response to cis-9,trans-11 and LA. The IC50 values did not differ between the investigated FA, but there were differences within the proliferation in the response of these FA in a concentration range between 20 and 148 µM (e.g., increased proliferation after treatment with lower concentrations of LA). No differences occurred when different FA combinations were tested. ConA stimulation increased the expression of TNF-α and IFN-γ, whereas IL-10 decreased. In general, neither the baseline expression nor the ConA-stimulated mRNA expression of cytokines and PPAR-γ were affected by the FA. In conclusion, all FA inhibit the proliferation of PBMC dose dependently without significantly altering the induced cytokine spectrum of activated bovine PBMC.

Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes.

BACKGROUND: Phytanic acid (PA) is a chlorophyll metabolite with potentials in regulating glucose metabolism, as it is a natural ligand of the peroxisome proliferator-activated receptor (PPAR) that is known to regulate hepatic glucose homeostasis. This study aimed to establish primary porcine myotubes as a model for measuring glucose uptake and glycogen synthesis, and to examine the impact of physiological amounts of PA on glucose uptake and glycogen synthesis either alone or in combination with insulin. METHODS: Porcine satellite cells were cultured into differentiated myotubes and tritiated 2-deoxyglucose (2-DOG) was used to measure glucose uptake, in relation to PA and 2-DOG exposure times and also in relation to PA and insulin concentrations. The MIXED procedure model of SAS was used for statistical analysis of data. RESULTS: PA increased glucose uptake by approximately 35%, and the presence of insulin further increased the uptake, but this further increase in uptake was non- additive and less pronounced at high insulin concentrations. There was no effect of PA alone on glycogen synthesis, while the insulin stimulation of glycogen was increased by 20% in the presence of PA. PA neither stimulated glucose uptake nor glycogen synthesis in insulin-resistant myotubes generated by excess glucose exposure. CONCLUSIONS: Primary porcine myotubes were established as a model of skeletal muscles for measuring glucose uptake and glycogen synthesis, and we showed that PA can play a role in stimulating glucose uptake at no or inadequate insulin concentrations.

Disruption of mitochondrial homeostasis by phytanic acid in cerebellum of young rats.

Phytanic acid (Phyt) brain concentrations are highly increased in Refsum disease, a peroxisomal disorder clinically characterized by neurological features, cardiac abnormalities, and retinitis pigmentosa. Considering that the pathogenesis of cerebellar ataxia, a common finding in this disease, is still unknown, in the present work we investigated the in vitro effects of Phyt at concentrations similar to those found in affected patients on important parameters of mitochondrial homeostasis in cerebellum from young rats. The respiratory parameters states 3 and 4 and respiratory control ratio (RCR) determined by oxygen consumption, membrane potential (∆Ψm), NAD(P)H pool content, and swelling were evaluated in mitochondrial preparations from this cerebral structure. Phyt markedly increased state 4 respiration, whereas state 3 respiration, the RCR, the mitochondrial matrix NAD(P)H content, and ∆Ψm were decreased by this fatty acid, being the latter effect partially prevented by N-acetylcysteine. These data indicate that Phyt behaves as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor disrupting mitochondrial homeostasis in cerebellum. It is proposed that these pathomechanisms may contribute at least in part to the cerebellar alterations found in Refsum disease.

Marked inhibition of Na+, K(+)- ATPase activity and the respiratory chain by phytanic acid in cerebellum from young rats: possible underlying mechanisms of cerebellar ataxia in Refsum disease.

Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.

Phytanic acid disturbs mitochondrial homeostasis in heart of young rats: a possible pathomechanism of cardiomyopathy in Refsum disease.

Phytanic acid (Phyt) accumulates in tissues and biological fluids of patients affected by Refsum disease. Although cardiomyopathy is an important clinical manifestation of this disorder, the mechanisms of heart damage are poorly known. In the present study, we investigated the in vitro effects of Phyt on important parameters of oxidative stress in heart of young rats. Phyt significantly increased thiobarbituric acid-reactive substances levels (P < 0.001) and carbonyl formation (P < 0.01), indicating that this fatty acid induces lipid and protein oxidative damage, respectively. In contrast, Phyt did not alter sulfhydryl oxidation. Phyt also decreased glutathione (GSH) concentrations (P < 0.05), an important non-enzymatic antioxidant defense. Moreover, Phyt increased 2',7'-dichlorofluorescin oxidation (DCFH) (P < 0.01), reflecting increased reactive species generation. We also found that the induced lipid and protein oxidative damage, as well as the decreased GSH levels and increased DCFH oxidation provoked by this fatty acid were prevented or attenuated by the reactive oxygen species scavengers melatonin, trolox, and GSH, but not by the nitric oxide inhibitor N: (ω)-nitro-L: -arginine methyl ester, suggesting that reactive oxygen species were involved in these effects. Next, we verified that Phyt strongly inhibited NADH-cytochrome c oxidoreductase (complex I-III) activity (P < 0.001) in heart supernatants, and decreased membrane potential and the NAD(P)H pool in heart mitochondria, indicating that Phyt acts as a metabolic inhibitor and as an uncoupler of the electron transport chain. Therefore, it can be presumed that disturbance of cellular energy and redox homeostasis induced by Phyt may possibly contribute to the cardiomyopathy found in patients affected by Refsum disease.

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.

In vitro evidence that phytanic acid compromises Na(+),K(+)-ATPase activity and the electron flow through the respiratory chain in brain cortex from young rats.

Phytanic acid (Phyt) tissue concentrations are increased in Refsum disease and other peroxisomal disorders characterized by neurologic damage and brain abnormalities. The present work investigated the in vitro effects of Phyt, at concentrations found in these peroxisomal disorders, on important parameters of energy metabolism in brain cortex of young rats. The parameters analyzed were CO(2) production from labeled acetate and glucose, the activities of the citric acid cycle enzymes citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase, as well as of the respiratory chain complexes I-IV, creatine kinase and Na(+),K(+)-ATPase. Our results show that Phyt did not alter citric acid cycle enzyme activities, or CO(2) production from acetate, reflecting no impairment of the functionality of the citric acid cycle. In contrast, respiratory chain activities were reduced at complexes I, II, I-III, II-III and IV. Membrane synaptical Na(+),K(+)-ATPase activity was also reduced by Phyt, with no alteration of creatine kinase activity. Considering the importance of the electron flow through the respiratory chain for brain energy metabolism (oxidative phosphorylation) and of Na(+),K(+)-ATPase activity for maintaining membrane potential necessary for neurotransmission, the data indicate that Phyt impairs brain bioenergetics at the level of energy formation, as well as neurotransmission. It is presumed that Phyt-induced impairment of these important systems may be involved at least in part in the neurological damage found in patients affected by disorders in which brain Phyt concentrations are increased.

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).

The influence of the branched-chain fatty acids pristanic acid and Refsum disease-associated phytanic acid on mitochondrial functions and calcium regulation of hippocampal neurons, astrocytes, and oligodendrocytes.

Pristanic acid and phytanic acid are branched-chain fatty acids, which play an important role in diseases with peroxisomal impairment, like Refsum disease (MIM 266500), Zellwegers syndrome and alpha-methylacyl-CoA racemase deficiency (MIM 604489). Several studies revealed that the toxic activity of phytanic acid is mediated by multiple mitochondrial dysfunctions. However, the action of pristanic acid on brain cells is still completely unknown. Here, we exposed astrocytes, oligodendrocytes and neurons in mixed culture to pristanic acid and phytanic acid to analyse cellular consequences. Pristanic acid exerts a strong cytotoxic activity on brain cells, displayed by dramatic Ca2+ deregulation, in situ mitochondrial depolarization and cell death. Interestingly, pristanic acid strongly induced generation of reactive oxygen species (ROS), whereas phytanic acid exerts weaker effects on ROS production. In conclusion, pristanic acid as well as phytanic acid induced a complex array of toxic activities with mitochondrial dysfunction and Ca2+ deregulation.