Neurological disorders and phytanic acid metabolism

Fifty patients with neurological symptoms and signs resembling those of Refsum's disease were analyzed for phytanic acid in serum. In addition, the phytanic acid oxidase capacity in skin fibroblasts was determined. The patients suffered from retinitis pigmentosa, cerebellar ataxia and/or chronic polyneuropathy of unknown cause. The serum level of phytanic acid was not increased, and the alpha-oxidation of (1-14C) phytanic acid was found to be normal in all patients. The present investigation lends no support to the theory that so-called atypical or mild cases of Refsum's disease exist. This disorder appears to be a disease entity with a specific genetic phenotype.     

Mechanism of toxicity of the branched-chain fatty acid phytanic acid, a marker of Refsum disease, in astrocytes involves mitochondrial impairment

Phytanic acid is a saturated branched-chain fatty acid, which is formed by bacterial degradation of chlorophyll in the intestinal tract of ruminants. The methyl group in beta-position prevents degradation of phytanic acid by the beta-oxidation pathway. Therefore, degradation of phytanic acid is initiated by alpha-oxidation in peroxisomes. The inherited peroxisomal disorder Refsum disease is characterised by accumulation of phytanic acid. Unusually high concentrations of phytanic acid can be found in the plasma of Refsum disease patients, who suffer from neurodegeneration and muscle dystrophy. Phytanic acid has been suggested to be causally involved in the clinical symptoms. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid in rat hippocampal astrocytes by monitoring the cytosolic Ca(2+) concentration, the mitochondrial membrane potential (Deltapsi(m)), the generation of reactive oxygen species as well as the cellular ATP level. In response to phytanic acid (100 microM) cytosolic Ca(2+) was quickly increased. The phytanic acid-evoked Ca(2+) response was transient and involved activation of intracellular Ca(2+) stores. In isolated rat brain mitochondria, phytanic acid dissipated Deltapsi(m) in a reversible and dose-dependent manner. Moreover, phytanic acid released cytochrome c from mitochondria. Depending on the mitochondrial activity state, phytanic acid either stimulated or inhibited the electron flux within the respiratory chain. In addition, phytanic acid induced substantial generation of reactive oxygen species in isolated mitochondria as well as in intact cells. Phytanic acid caused cell death of astrocytes within a few hours of exposure. In conclusion, we suggest that phytanic acid initiates astrocyte cell death by activating the mitochondrial route of apoptosis.     

Peroxisomal functions in classical Refsum's disease: comparison with the infantile form of Refsum's disease

The infantile and classical forms of Refsum's disease are generally considered to belong to the newly recognized group of peroxisomal disorders. In this study we carried out a detailed investigation into different peroxisomal functions in classical Refsum's disease by analyses of plasma (very long chain fatty acids, di- and trihydroxycoprostanoic acid and pipecolic acid) and cultured skin fibroblasts from the patients (de novo plasmalogen biosynthesis, very long chain fatty acid oxidation and amount of particle-bound catalase). The results obtained indicate that, except for a deficient phytanic acid oxidation, peroxisomal functions were found to be normal in classical Refsum's disease in contrast with the findings in infantile Refsum's disease, in which there is a general impairment of peroxisomal functions. Based on these results it is concluded that peroxisomal biogenesis is normal in classical (but not in infantile) Refsum's disease and that the classical and infantile form of Refsum's disease hence represent distinct entities. Since available evidence suggests that phytanic acid is oxidized in mitochondria rather than in peroxisomes, at least in rat liver, it remains to be established whether classical Refsum's disease is a peroxisomal disorder or not.     

New approaches in peroxisomal disorders

The peroxisome is a subcellular organelle with important functions in plants and protozoa, which during the last decade has also been shown to have a role in mammalian lipid and amino acid metabolism. These functions include steps in the synthesis of ether lipids and bile acids and fatty acid beta-oxidation, particularly those of very long chain fatty acids. The proposition that the peroxisome carries out significant functions in man is highlighted by the fact that lack of this organelle is associated with severe abnormalities in many human organs. Human peroxisomal disorders are now grouped into three general categories. In the first group, peroxisomes are lacking or reduced in number. This group includes the Zellweger cerebro-hepato-renal syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease and hyperpipecolic acidemia. These patients lack the capacity to synthesize ether lipids and to oxidize very long chain fatty acids or phytanic acid, and they show abnormally high levels of pipecolic acid and bile acid intermediates. These patients rarely survive early childhood, have severe neurological deficits and multiple malformations. A second group includes the 'pseudo-Zellweger' syndrome and the rhizomelic form of chondrodysplasia punctata. Here the peroxisomal structure is intact, but there is deficient function of several peroxisomal enzymes. The third group includes X-linked adrenoleukodystrophy, acatalasemia and 'adult' Refsum's disease. The peroxisomal structure is intact, and the defect in each instance is thought to involve a mutation which affects a single peroxisomal enzyme. Peroxisomal disorders are of current interest because they occur more commonly than had been recognized and show phenotypic and genotypic heterogeneity. Their study provides the opportunity to learn more about the role of the peroxisome in normal brain function and development.     

Biochemical markers predicting survival in peroxisome biogenesis disorders

OBJECTIVE: To identify prognostic markers reflecting the extent of peroxisome dysfunction in primary skin fibroblasts from patients with peroxisome biogenesis disorders (PBD). BACKGROUND: PBD are a genetically heterogeneous group of disorders due to defects in at least 11 distinct genes. Zellweger syndrome is the prototype of this group of disorders, with neonatal adrenoleukodystrophy and infantile Refsum disease as milder variants. Common to these three disorders are liver disease, variable neurodevelopmental delay, retinopathy, and perceptive deafness. Because genotype-phenotype studies are complicated by the genetic heterogeneity among patients with PBD, the authors evaluated a series of biochemical markers as a measure of peroxisome dysfunction in skin fibroblasts. METHODS: Multiple peroxisomal functions including de novo plasmalogen synthesis, dihydroxyacetonephosphate acyltransferase (DHAPAT) activity, C26:0/C22:0 ratio, C26:0 and pristanic acid beta-oxidation, and phytanic acid alpha-oxidation were analyzed in fibroblasts from a series of patients with defined clinical phenotypes. RESULTS: A poor correlation with age at death was found for de novo plasmalogen synthesis, C26:0/C22:0 ratio, and phytanic acid alpha-oxidation. A fairly good correlation was found for pristanic acid beta-oxidation, but the best correlation was found for DHAPAT activity and C26:0 beta-oxidation. A mathematic combination of DHAPAT activity and C26:0 beta-oxidation showed an even better correlation. CONCLUSIONS: DHAPAT activity and C26:0 beta-oxidation are the best markers in predicting life expectancy of patients with PBD. Combination of both markers gives an even better prediction. These results contribute to the management of patients with PBD.     

Refsum's disease. Epidemiologic, clinical and biological correlation. 6 cases

Nine patients with symptoms and signs of Refsum's disease are reported. In 6 a systemic accumulation of phytanic acid was demonstrated, together with low phytanic acid oxidase activity in skin fibroblasts in 5 of them. In 3, no disorder of phytanic acid metabolism was demonstrated. In 3, the diagnosis was made during the pre-clinical period. The disease seems more frequent in Northern France, which agrees with the hypothesis of a genetic mutation which would have taken place in Scandinavia some centuries ago and was subsequently spread by the Vikings. The effects of a dietary treatment on serum phytanic acid levels and clinical disorders are reported. The general condition of the patients improved remarkably but only partially. The diet is unpalatable and in some patients the level of serum phytanic acid increased, due to the mobilization of body fat. Patients with very high levels of phytanic acid might be initially treated by plasmapheresis. For the same reason, the diet should supply enough calories to keep body weight unchanged, and body weight loss whatever its cause should be avoided.     

Neonatal adrenoleukodystrophy.

Nine cases of neonatal adrenoleukodystrophy are described. All patients had abnormal facial features, moderate to severe hypotonia, hepatomegaly, and retinitis pigmentosa. The clinical course was rapidly progressive in six cases and more protracted in three others. Biological signs of adrenal insufficiency were present in five cases. CT scan showed a demyelinating process in four patients. Trilamellar inclusions were found in the liver of four cases and dark and complex lipidic inclusions in three other cases. In the three necropsied patients there was severe alteration of the white matter involving particularly the cerebellum in two cases. Gyral and cytoarchitectonic disturbances were absent in all three cases. Increased plasma levels of very long chain fatty acids (8/8), phytanic acid (7/8) and bile fluid trihydroxycoprostanic acid (2/4) confirmed the deficiency of multiple peroxisomal enzymes. Clinical, histopathological and biochemical findings of these nine cases are compared to those reported in other neonatal adrenoleukodystrophy cases and to those of other neonatal peroxisomal disorders, that is cerebro-hepato-renal syndrome of Zellweger and infantile Refsum's disease.     

The Refsum disease marker phytanic acid, a branched chain fatty acid, affects Ca2+ homeostasis and mitochondria, and reduces cell viability in rat hippocampal astrocytes

The saturated branched chain fatty acid, phytanic acid, a degradation product of chlorophyll, accumulates in Refsum disease, an inherited peroxisomal disorder with neurological clinical features. To elucidate the pathogenic mechanism, we investigated the influence of phytanic acid on cellular physiology of rat hippocampal astrocytes. Phytanic acid (100 microM) induced an immediate transient increase in cytosolic Ca2+ concentration, followed by a plateau. The peak of this biphasic Ca2+ response was largely independent of extracellular Ca2+, indicating activation of cellular Ca2+ stores by phytanic acid. Phytanic acid depolarized mitochondria without causing in situ swelling of mitochondria. The slow decrease of mitochondrial potential is not consistent with fast and simultaneous opening of the mitochondrial permeability transition pore. However, phytanic acid induced substantial generation of reactive oxygen species. Phytanic acid caused astroglia cell death after a few hours of exposure. We suggest that the cytotoxic effect of phytanic acid seems to be due to a combined action on Ca2+ regulation, mitochondrial depolarization, and increased ROS generation in brain cells.     

Plasma and skin fibroblast C26 fatty acids in infantile Refsum's disease

In infantile and adult Refsum's disease, the activity of phytanic acid oxidase is low in skin fibroblasts, but plasma phytanic acid levels are high. Cultured skin fibroblasts and plasma from patients with the infantile, but not the adult, disorder show marked increases in the concentration of the long-chain fatty acid, hexacosanoic acid (C26), a feature once thought pathognomonic of adrenoleukodystrophy or Zellweger's syndrome.     

Gas chromatography/mass spectrometry analysis of very long chain fatty acids, docosahexaenoic acid, phytanic acid and plasmalogen for the screening of peroxisomal disorders

Very long chain fatty acids (VLCFAs) and docosahexaenoic acid (DHA), phytanic acid, and plasmalogens are usually measured individually. A novel method for the screening of peroxisomal disorders, using gas chromatography/mass spectrometry (GC/MS), was developed. Saturated and unsaturated fatty acids, including VLCFAs and DHA, phytanic acid, and plasmalogen were detected by a selected ion monitoring-electron impact method, using 100 microl of serum or plasma. Methyl-esterification and extraction could be done in one tube, and data were obtained within 4 h. All patients with Zellweger syndrome (ZS), X-linked adrenoleukodystrophy (ALD), isolated deficiency of peroxisomal beta-oxidation enzyme, and most ALD carriers showed increased VLCFA ratios, including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0. The ratio of DHA to palmitic acid (C16:0) and plasmalogen (measured as hexadecanal dimethyl acetal) to C16:0 in ZS patients was significantly lower than for the controls (P<0.001 for healthy high school students, P<0.05 for infants with other disorders). Plasmalogen was also decreased in patients with isolated deficiency of plasmalogen biosynthesis. Two of eight patients with ZS, two of four with RCDP, and all of three classical Refsum patients showed increased levels of phytanic acid. This method will simplify the screening for peroxisomal disorders.     

Long-term plasma exchange in a case of Refsum's disease

Summary Refsum's disease (Heredopathia atactica polyneuritiformis) is caused by accumulation of phytanic acid in all body tissues due to an inherited failure of alphaoxidation of branched chain fatty acids. Plasmapheresis has been reported to be beneficial by removal of phytanic acid from the blood. We describe a patient with Refsum's disease in whom long-term plasmapheresis did not improve clinical, biochemical or electrophysiological parameters.     

Refsum's disease may mimic familial Guillain Barre syndrome

Refsum's disease is a rare autosomal recessive disorder with clinical features including retinitis pigmentosa, anosmia, deafness, chronic sensory-motor neuropathy, ataxia and the accumulation of phytanic acid in blood plasma and body tissues. We report the occurrence of Refsum's disease in two sisters, both presenting with acute demyelinating polyneuropathy mimicking the familial Guillain Barre syndrome. Thus, when GBS is suspected, particularly in cases of familial recurrence as well as in atypical cases of acute polyneuropathy, the diagnosis of Refsum's disease should be considered, looking for other features of the disease and, if appropriate, testing plasma phytanic acid levels.     

Refsum's disease: evolution 35 years after diagnosis

Refsum's disease (Heredopathia atactica polyneuritiformis) is an autosomal recessive disease caused by a defective alpha oxidation of a C20 fatty acid: the phytanic acid. Deficiency of a peroxysomal enzyme called "Phytanoyl-Co-A alpha hydroxylase" leads to an accumulation of phytanic acid. The clinical picture include retinitis pigmentosa, peripheral neuropathy, ataxia and elevated cerebrospinal fluid protein concentration. Firstly described in 1946 by Sigvald Refsum, dietary treatment leads to an improvement of neurological symptoms but does not affect retinal changes. To our knowledge, there is no data in the literature on long term follow-up. A patient with Refsum's disease diagnosed in 1965 presented with facial paralysis. The phytanic acid concentration was low, CSF protein level was normal leading to diagnosis of Bell's palsy. This observation is of particular interest because after 35 years evolution of the disease, the only handicap was visual impairment, with no loss of muscle strength or sensory deficit.     

Heredopathia atactica polyneurotiformis: therapeutic and pathogenetic aspects.

None of the single manifestations is in itself specific or pathognomonic for this disease except for the disturbance of the metabolism of phytanic acid. Therefore, clinical syndromes that involve similar manifestations without accumulations of phytanic acid should not be included under the term heredopathia atactica polyneuritiformis, or Refsum's disease. Whatever the biochemical mechanism that links phytanic acid accumulation with the clinical manifestations of heredopathia atactica polyneuritiformis, it appears that the observations made on patients following successful dietary treatment leave little doubt that most if not all of the manifestations in this disease are caused in some way by the accumulation of phytanic acid.     

The significance of plasma phytanic acid levels in adults.

The presence of phytanic acid in tissues and plasma has been considered diagnostic of heredopathia atactica polyneuritiformis (Refsum's disease), but recently slightly raised plasma phytanic acid levels have been reported in other conditions. Forty two normal people were found to have a phytanic acid level of 0-33 mumol/l. Fourteen patients with heredopathia atactica polyneuritiformis had a plasma phytanic acid level before treatment of 992-6400 mumol/l. Five patients with retinitis pigmentosa but not heredopathia atactica polyneuritiformis had plasma levels of 38-192 mumol/l. It was concluded that some patients with retinitis pigmentosa without heredopathia atactica polyneuritiformis but a raised plasma phytanic acid may represent a group of patients with a disease or diseases as yet uncharacterised apart from the retinal condition.     

REFSUM'S SYNDROME WITH NORMAL PHYTATE METABOLISM

A case of "Refsum's syndrome" is described, in which phytanic acid was not detected in serum, nerve and muscle. The existence of cases with identical phenotypic manifestations and different underlying metabolic error is postulated.     

Smell testing: an additional tool for identification of adult Refsum's disease

BACKGROUND: Adult Refsum's disease (ARD) is characterised by the presence of retinitis pigmentosa, ataxia, deafness, sensory neuropathy, and bony changes. The diagnosis is confirmed by the presence of phytanic acidaemia. Although reduced smell function has been described in ARD, its value in the diagnosis of the condition has not been fully evaluated. OBJECTIVE: To investigate the prevalence and degree of olfactory dysfunction in patients with ARD. METHOD: The olfactory function of 16 patients with ARD was assessed using the quantitative University of Pennsylvania Smell Identification Test (UPSIT). RESULTS: All patients had complete anosmia or grossly impaired smell function with a mean UPSIT score of 14.7 (SD 4.7) (normal > 34) despite having been treated with an appropriate diet for a median of 15 years (range 1-25). CONCLUSIONS: Identification of ARD patients can be facilitated by using the UPSIT in combination with the presence of retinitis pigmentosa, even if they have no neurological or bony features. Phytanic acid screening should be performed in any patient manifesting these two signs.     

Infantile phytanic acid storage disease, a disorder of peroxisome biogenesis: a case report.

The infantile and classic forms of phytanic acid storage disease belong to the newly recognized group of peroxisomal disorders. In this paper we report the full clinical, morphological and biochemical results in a patient with infantile phytanic acid storage disease. The results indicate a generalized loss of peroxisomal functions due to a deficiency of peroxisomes as demonstrated in hepatocytes and cultured skin fibroblasts.     

Lipapheresis: an immunoglobulin-sparing treatment for Refsum's disease

Introduction – Toxic phytanic acid concentrations in patients with Refsum's disease can be reduced by plasma separation, performed either as plasmapheresis, or as cascade filtration. The latter procedure is as efficient and safe as plasmapheresis, and eliminates the need for albumin replacement. This study investigates the loss of immunoglobulins associated with the procedure. Material and methods — Immunoglobulin- and phytanic acid serum concentrations before and after cascade filtration (n=16) were measured in a patient with Refsum's disease and their removal determined. Filters with sieving coefficients for immunoglobulin G of 70% and 30% were compared with each other and with historical data on plasmapheresis. Results — While differences in immunoglobulin M loss are negligible, the loss of immunoglobulin G in cascade filtration is significantly less than that reported for plasmapheresis and depends upon the pore size of the employed filters. The loss is least with larger pore size, but this advantage becomes statistically insignificant if immunoglobulin G loss is related to the lesser decrease in phytanic acid concentration that was achieved simultaneously in this study. Conclusion — Unless transplantation of a-hydroxylase containing tissue can be established as treatment for Refsum's disease, cascade filtration appears to be the treatment of choice in order to avoid loss of albumin and to reduce the loss of immunoglobulin G.     

Clinical and biochemical follow up of Refsum's disease (author's transl)]

Within a family refered here, two sisters are presenting heredopathia atactica polyneuritiformis (Morbus Refsum) with the complete clinical, electromyographical and serological findings (increased phytanic acid level, accumulation of mono- and diphytanyl, triglycerides). In addition, some symptoms of Refsum's disease are clinically apparent in the eight year old daughter of one of the patients, but significant principal characteristics are absent (dissociation of the spinal fluid, retinopathia pigmentosa, increased phytanic acid level). The phytanic acid level in the serum of ten clinically normal blood relations (three of which are also obligatory heterozygotes) is normal. In both patients with manifest illness we found not only the usual spinal fluid changes with highly increased protein levels (gammaglobulin, IgA- and IgG-fraction) but also a remarkably large number of macrophages with vacuoles. Within almost two years of keeping to a strict diet with low phytanic acid and phytol content, both patients showed a distinct clinical improvement in parallel with a decrease of the phytanic acid level. Even a temporary increase of the phytanic acid level in one of the patients did not cause clinical relapse. Regarding the biochemical control of the course of the disease, the phytanic acid containing triglycerides proved to be a highly sensitive parameter.