Adult Refsum Disease: Case Series of Reducing Circulating Phytanic Acid Levels With Dietary Interventions

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.

Phytanic acid (PA) accumulates in patients with adult Refsum disease (ARD) and with peroxisomal disorders. In three related patients with ARD, PA levels were moderately increased in plasma, whereas phytanic oxidation was severely deficient in the fibroblasts. Two of these patients had a significant increase of pipecolic acid in plasma, a finding not reported in ARD, and a fourth related patient, a brother, died at age 17 from a progressive neurologic disorder with unusual clinical and neuropathologic (a spongy degeneration of the white matter) abnormalities for ARD. The first step of L-pipecolic acid degradation occurs in peroxisome. In these patients, the accumulation of PA could have resulted from an impaired capacity to degrade pristanic acid rather than PA. The activity of pristanic oxidase, measured in fibroblasts, was normal, as were two other peroxisomal enzymes, lignoceric acid oxidase and dihydroxyacetone phosphate transferase. Since both mitochondria and peroxisomes are involved in PA alpha-oxidation, we propose that these four related patients presented various phenotypical variants of a novel peroxisomal disease with impairment of PA and pipecolic acid oxidation.

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.

In humans the oxidation of phytanic acid is a peroxisomal function. To understand the possible mechanisms for the pathognomic accumulation of phytanic acid in plasma and body fluids of Refsum disease (RD) and rhizomelic chondrodysplasia punctata (RCDP), we investigated activities of various steps (activation, transport, and oxidation) in the metabolism of phytanic acid in peroxisomes isolated from cultured skin fibroblasts from control, RD, and RCDP subjects. Activation of phytanic acid was normal in peroxisomes from both RD and RCDP. Transport of phytanic acid or phytanoyl-CoA in the absence or presence of fatty acid activating cofactors (ATP, MgCl2, and CoASH) into peroxisomes isolated from RD and RCDP skin fibroblasts was also similar to that of peroxisomes from control fibroblasts. Defective oxidation of [(2,3)-3H]- or [1-14C]phytanic acid, or [1-14C]phytanoyl-CoA (substrate for the first step of alpha-oxidation) but normal oxidation of [1-14C] alpha-hydroxyphytanic acid (substrate for the second step of the alpha-oxidation pathway) in peroxisomes from RD clearly demonstrates that excessive accumulation of phytanic acid in plasma and body fluids of RD is due to the deficiency of phytanic acid alpha-hydroxylase in peroxisomes. However, in RCDP peroxisomes, in addition to deficient oxidation of [1-14C]phytanic acid or phytanoyl-CoA or [(2,3)-3H]phytanic acid, the oxidation of [1-14C] alpha-hydroxyphytanic acid was also deficient, indicating that in RCDP the activities both of alpha-hydroxylation of phytanic acid and decarboxylation of alpha-hydroxyphytanic acid are deficient. These observations indicate that peroxisomal membrane functions (phytanic acid activation and transport) in phytanic acid metabolism are normal in both RD and RCDP. The defect in RD is in the alpha-hydroxylation of phytanic acid; whereas in RCDP both alpha-hydroxylation of phytanic acid as well as decarboxylation of alpha-hydroxyphytanic acid are deficient.

Clinical and biochemical heterogeneity in conditions with phytanic acid accumulation

Refsum disease is a rare, inherited neurodegenerative disorder characterized by accumulation of the dietary branched-chain fatty acid phytanic acid in plasma and tissues caused by a defect in the alphaoxidation pathway. The accumulation of phytanic acid is believed to be the main pathophysiological cause of the disease. However, the exact mechanism(s) by which phytanic acid exerts its toxicity have not been resolved. In this study, the effect of phytanic acid on mitochondrial respiration was investigated. The results show that in digitonin-permeabilized fibroblasts, phytanic acid decreases ATP synthesis, whereas substrate oxidation per se is not affected. Importantly, studies in intact fibroblasts revealed that phytanic acid decreases both the mitochondrial membrane potential and NAD(P)H autofluorescence. Taken together, the results described here show that unesterified phytanic acid exerts its toxic effect mainly through its protonophoric action, at least in human skin fibroblasts.

Mutations in the Gene Encoding Peroxisomal Sterol Carrier Protein X (SCPx) Cause Leukencephalopathy with Dystonia and Motor Neuropathy

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

Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca 2+ signaling through activation of free fatty acid receptor GPR40

Refsum's disease (hereditary motor and sensory neuropathy type IV) is a rare autosomal recessive condition first characterized by Sigvald Refsum in 1945. He initially chose the name heredoataxia hemeralopica polyneuritiformis,1 subsequently amending this to heredopathia atactica polyneuritiformis.2 Thankfully, the eponymous version now predominates in the literature, and these earlier terms have been largely abandoned. Refsum's disease is caused by defective alpha oxidation of phytanic acid (3,7,11,15 tetramethylhexadecanoic acid), a branched‐chain fatty acid present in a wide range of foodstuffs including dairy products, some meats and fish.3 The defective enzyme is phytanoyl‐coenzyme A hydroxylase, which normally catalyses the second step in the breakdown of phytanic to pristanic acid using the CoA derivative as a substrate (the first step in alpha oxidation is the conversion of phytanic acid to phytanoyl CoA by Phytanoyl CoA ligase).4 This results in accumulation of phytanic acid, with elevated levels in blood and other tissues including fat and neurons (Figure 1). Phytanic acid can also be catabolized from the non‐carboxyl end by omega oxidation, but the capacity of this pathway is severely limited to ∼10 mg of phytanic acid per day.5 The average diet contains ∼50 mg/day, and this factor, in combination with limited excretory mechanisms (via the kidneys and skin), leads to phytanic acid accumulation. The mechanism of phytanic acid toxicity is unclear, but it may be incorporated into tissue lipids and result in impaired myelin function. An alternative hypothesis is that excess levels affect the metabolism of fat‐soluble vitamins. Levy has suggested that high phytanic acid levels interfere with vitamin A esterification in the retinal pigment epithelium leading to the production of a toxic substance and progressive visual failure.6 The physiological role of phytanic acid is unknown. Figure 1. The biochemistry of Refsum's disease. The onset of symptoms is typically in late childhood or …

Adult Refsum disease is an inherited disorder in which phytanic acid accumulates in tissues and serum. Two hypotheses have been proposed to explain the pathogenesis of this condition. The molecular distortion hypothesis suggests that phytanic acid may alter membrane composition and structure, thereby affecting membrane function(s). The anti-metabolite hypothesis suggests that an accumulation of phytanic acid in membranes may interfere with vitamin E function. These two hypotheses were investigated by studying the effects of modulating phytanic acid and alpha-tocopherol concentrations on the fatty acid composition and certain physical parameters of cultured retinal cells. Results showed that (a) the phospholipid fraction of retinal cells readily incorporated phytanic acid, (b) the incorporation of phytanic acid increased membrane fluidity, (c) there was no competition for uptake between phytanic acid and alpha-tocopherol, and (d) the incorporation of phytanic acid did not increase the susceptibility of membranes to lipid peroxidation in vitro. These results obtained with cultured retinal cells suggest that the molecular distortion hypothesis, but not the anti-metabolite hypothesis, could explain the pathogenesis of adult Refsum disease. In vitro tissue culture models can, however, only approximate to the much more complex situation that occurs in vivo.

Peroxisomes play an important role in the degradation of, among others, very long chain fatty acids (VLCFA), phytanic and pristanic acids, and pipecolic acid, and in the formation of primary bile acids, cholic acid and chenodeoxycholic acid, by {j-oxidation of diand trihydroxycholestanoic acids (DHCA and THCA). Recently, two cases have been described of di/trihydroxycholestanoic acidaemia due to a presumed THCA-CoA oxidase deficiency.L? In the first patient! next to accumulation of DHCA and THCA in plasma, an elevated plasma phytanic acid level was observed as the only additional biochemical abnormality. Since phytanic acid a-oxidation, measured in cultured fibroblasts as the production of 14COZ from [1_l4C] phytanic acid, was normal, accumulation of phytanic acid was explained by an inhibitory effect of the accumulating bile acid intermediates on phytanic acid a-oxidation. In the second patientaccumulation of DHCA and THCA in plasma was associated with undetectable THCACoA oxidase activity in post mortem liver, although the significance of this finding may be questioned since activities of palmitoyl-CoA oxidase and catalase were also found to be decreased, probably due to the long delay between the moment of death and the time of tissue sampling. Although both patients were assumed to suffer from a common enzyme defect, their clinical presentation was different. Since accumulation of phytanic acid (which was observed only in patient 1) may not only result from a primary defect in phytanic acid a-oxidation, but may also be secondary to a defective pristanic acid {j-oxidation,3

Refsum's disease is a rare, autosomal recessive neurometabolic disease, characterised biochemically by accumulation of phytanic acid in blood and tissues.1 This is due to deficiency of the peroxisomal enzyme phytanoyl-CoA-hydroxylase...

The saturated C20 isoprenoid phytanic acid is physiologically derived from phytol released in the degradation of chlorophyll. The presence of a C-3 methyl group in this substrate blocks normal beta-oxidation, so phytanic acid degradation primarily occurs by initial peroxisomal alpha-oxidation to shift the register of the methyl group. However, individuals with Refsum's disease are genetically deficient in the required phytanoyl-CoA alpha-hydroxylase and suffer from neurological pathologies caused by the accumulation of phytanic acid. Recent work has shown that phytanic acid can also be catabolized by a pathway initiated by omega-hydroxylation of the hydrocarbon chain, followed by oxidation of the alcohol to the acid and conventional beta-oxidation. However, the enzymes responsible for the omega-hydroxylation of phytanic acid have not been identified. In this study, we have determined the activities of all of the rat and human CYP4A enzymes and two of the rat CYP4F enzymes, with respect to the omega-hydroxylation of phytanic acid. Furthermore, we have shown that the ability to omega-hydroxylate phytanic acid is elevated in microsomes from rats pretreated with clofibrate. The results support a possible role for CYP4 enzyme elevation in the elimination of phytanic acid in Refsum's disease patients.

Patients with generalized peroxisomal disorders, rhizomelic chondrodysplasia punctata, and Refsum disease are all unable to alpha-oxidize 3,7,11,15-tetramethylhexadecanoic (phytanic) acid. The exact cause of the oxidation defect in these patients is not well characterized, in part because there is only limited knowledge of the biochemical pathway. In 1969, the alpha-oxidation of phytanic acid was reported [Tsai, S.-C., Avigan, J. & Steinberg, D. (1969) Studies on the alpha-oxidation of phytanic acid by rat liver mitochondria, J. Biol. Chem. 244, 2682-2692] to involve the formation of an alpha-hydroxyphytanic acid intermediate prior to removal of the alpha carbon. Subsequently, most researchers have had difficulty detecting this intermediate. In the present study, cofactors known to form hydroxy intermediates by both monooxygenase and dioxygenase reaction mechanisms were incubated with purified rat liver peroxisomes and either [2,3-3H]phytanic acid or [1-14C]phytanic acid. Reaction products were separated by reverse-phase HPLC. A single reaction product, identified as alpha-hydroxyphytanoyl-CoA rather than the free fatty acid, was detected when 2-oxoglutarate/Fe+2/ascorbate, cofactors associated with a dioxygenase reaction mechanism, were present. Concomitant with alpha-hydroxyphytanoyl-CoA production, there was an increased accumulation of formate and CO2. This increase in alpha-oxidation products is evidence that alpha-hydroxyphytanoyl-CoA is a true pathway intermediate and that the entire pathway functions in peroxisomes. In contrast, alpha-hydroxyphytanoyl-CoA was not formed in any quantity in mitochondria. These studies suggest that the alpha-hydroxylation step of phytanic acid oxidation, which has been shown to be defective in Refsum disease, is located in peroxisomes.