Studies on the Metabolic Error in Refsum's Disease Daniel Steinberg, … , Oddvar Stokke, Sigvald Refsum J Clin Invest. 1967;46(3):313-322. https://doi.org/10.1172/JCI105533. Research Article 14 Studies utilizing mevalonic acid-2- C and D2O as precursors failed to provide evidence for an appreciable rate of endogenous biosynthesis of phytanic acid in a patient with Refsum's disease. Orally administered tracer doses of phytol-U-14C were well absorbed both by seven normal control subjects (61 to 94%) and by two patients with Refsum's disease (74 and 80%). 14 The fraction of the absorbed dose converted to CO2 in 12 hours was 3.5 and 5.8% in Refsum's disease patients and averaged 20.9% in seven control subjects. Labeled phytanic acid was demonstrated in the plasma of both control subjects and patients given phytol-U1-4C, establishing phytol in the diet as a potential precursor of phytanic acid. This labeled phytanic acid had disappeared almost completely from the plasma of the seven control subjects by 24 to 48 hours, whereas it persisted at high concentrations in the plasma of the two patients for many days. We conclude that the phytanic acid accumulating in Refsum's disease is primarily of exogenous origin and that patients with Refsum's disease have a relative block in the degradation of phytanic acid and possibly other similar branched-chain compounds. This may relate to a deficiency in mechanisms for release of phytanic acid from stored ester forms or, more probably, to reactions essential to oxidative degradation of the carbon skeleton. Find the latest version: https://jci.me/105533/pdf Journal of Clinical Investigation Vol. 46, No. 3, 1967 Studies on the Metabolic Error in Refsum's Disease * DANIEL STEINBERG,t CHARLES E. MIZE, JOEL AVIGAN, HENRY M. FALES, LORENTZ ELDJARN, KENNETH TRY, ODDVAR STOKKE, AND SIGVALD REFSUM (From the Laboratory of Metabolism, National Heart Institute, Bethesda, Md., and the Institute of Clinical Biochemistry and Department of Neurology, University of Oslo Rikshospitalet, Oslo, Norway) Summary. Studies utilizing mevalonic acid-2-14C and D20 as precursors failed to provide evidence for an appreciable rate of endogenous biosynthesis of phytanic acid in a patient with Refsum's disease. Orally administered tracer doses of phytol-U-14C were well absorbed both by seven normal control subjects (61 to 94%) and by two patients with Refsum's disease (74 and 80%). The fraction of the absorbed dose converted to 14COQ in 12 hours was 3.5 and 5.8% in Refsum's disease patients and averaged 20.9% in seven control subjects. Labeled phytanic acid was demonstrated in the plasma of both control subjects and patients given phytol-U-_4C, establishing phytol in the diet as a potential precursor of phytanic acid. This labeled phytanic acid had disap- peared almost completely from the plasma of the seven control subjects by 24 to 48 hours, whereas it persisted at high concentrations in the plasma of the two patients for many days. We conclude that the phytanic acid accumulating in Refsum's disease is primarily of exogenous origin and that patients with Refsum's disease have a relative block in the degradation of phytanic acid and possibly other similar branched-chain compounds. This may relate to a deficiency in mechanisms for release of phytanic acid from stored ester forms or, more probably, to reac- tions essential to oxidative degradation of the carbon skeleton. Introduction In 1963 Klenk and Kahlke (5) found that the blood and tissues of patients with Refsum's dis- Heredopathia atactica polyneuritiformis was first ease contained high concentrations of an unusual reported as a clinical entity by Refsum in 1945 (1). The branched-chain fatty acid-phytanic acid (3,7,11, prominent features of the syndrome include 15-tetramethylhexadecanoic acid). In the liver of atypical retinitis pigmentosa, hypertrophic periph- the first patient studied chemically post-mortem, eral neuropathy, cerebellar ataxia, nerve deafness, phytanic and acid accounted for over 50% of total elevated cerebrospinal fluid protein concentra- fatty acids; the relative concentration of phytanic tion with albuminocytologic dissociation (2). Al- acid in kidney lipids was almost as great (5). though only some 40 cases have been reported, it Very similar results in tissues of another patient seems fairly certain that the disease is genetic in have been reported by Hansen (6). Phytanic origin and transmitted in an autosomal recessive acid in the plasma can account for anywhere from manner (2-4). 3 to 25%o of total fatty acids (7-9). The present studies were undertaken to explore the origins of * Submitted for publication August 1, 1966; accepted November 3, 1966. the phytanic acid and the nature of the metabolic t Address requests for reprints to Dr. Daniel Stein- error leading to its accumulation. berg, Laboratory of Metabolism, National Heart Insti- The polyisoprenoid structure of phytanic acid tute, Bethesda, Md. 20014. suggested that it might be biosynthesized by way 313 314 STEINBERG AND CO-WORKERS of mevalonic acid in analogy with the biosynthesis Acetyl CoA _,Mevolonic Acid _I.lsopentenyl.PP of the branched-chain skeleton of farnesyl pyro- (CH3 C = CH -CH2 -PP ) Fornesyl- PP I phosphate (10) (Figure 1). The formation of c)P ((CH33'-CC1=CH -CH2 -)-PP geranylgeranyl pyrophosphate by addition of a fourth isopentenyl pyrophosphate unit to farnesyl / sopentenyl -PP Squalene Geronylgeranyl - PP pyrophosphate is a normal step in plants and in CH3, ( CCH-C3-1PC=CH-CH2 pp yeast on the pathway to carotene formation (11). CH 4 .1 Reduction of Nandi and Porter have shown that a pig liver double bonds, Lano terol loss of - PP, preparation can catalyze this reaction (12). As ond oxidation of olcohol to shown in Figure 1, phytanic acid could be formed ocid elimina- from geranylgeranyl pyrophosphate (after Phytonic Acid tion of the pyrophosphate group) by reduction of Cholesterol its four double bonds and oxidation of the alcohol CH3Nc~~~c~IcH(3-., ccH CH3, CH3 CH - CH2 - CH2(CH2 - CH - CH2 - CH -CH2 - CH - CH2 - COOH function to a carboxylic acid. Although there is CH3 2 no evidence that geranylgeranyl pyrophosphate is FIG. 1. SCHEMATIC REPRESENTATION OF THE BIOSYN- formed in significant amounts by mammals under THETIC ROUTE TO CHOLESTEROL FROM ACETYL-CoA, AND OF ordinary circumstances, it seemed possible that a A POSSIBLE PATHWAY TO PHYTANIC ACID VIA GERANYL- genetic error could evoke or accentuate such a GERANYL PYROPHOSPHATE. pathway, e.g., by inhibiting the rate of condensa- tion of farnesyl pyrophosphate with itself to form Table I. The patients were under observation at Riks- squalene. If there were a metabolic block in the hospitalet in Oslo; seven normal volunteers were stud- breakdown of phytanic acid, then even a limited ied at the Clinical Center in Bethesda. At the time of the phytol-U-"C and D20 1 studies, the rate of synthesis could lead to accumulation of the patients were on a vegetable-free, low fat diet; on the day product. One purpose of the present studies was preceding the study and for 7 days thereafter, the normal to test for biosynthesis. subjects were given a diet lacking green vegetables and Animal studies established that phytol, ubiqui- dairy products but containing adequate amounts of vita- mins and iron. The radioactive preparations were ad- tously present in the diet as a component of the ministered after a 12-hour overnight fast, food being chlorophyll molecule, can readily be converted to withheld for an additional 3 hours after administration. phytanic acid (13-16). Phytanic acid itself is Phytol-U-"C was given orally, dissolved in 10 ml of corn also present in the diet as a minor component in oil or olive oil. The preparation of uniformly labeled butterfat (17) and ruminant body fats (18, 19). phytol-lC has been described elsewhere (13, 16) ; the samples used were 93 to 98% pure by radioassay. Meva- Normal human and animal sera contain phytanic lonate-2-"C (98.5% pure by radioassay) was obtained as acid but only at very low concentrations (20, 21). the dibenzylethylenediamine salt,2 converted to the potas- However, when either phytol or phytanic acid is sium salt, and given intravenously. fed to animals in sufficiently large dosage, phytanic At specified times, respiratory gases were collected acid accumulates in blood and tissues, and the over accurately measured 2-minute intervals into evacu- ated Douglas bags with subjects in a resting state. The concentrations can reach levels comparable to those total content of each Douglas bag was passed through observed in patients with Refsum's disease (13- 5 N NaOH for absorption of C02. The gas train in- 15, 22). If there were a metabolic block in the cluded a Ba(OH)2 trap to detect any loss of C02 in the degradation of phytanic acid, even a low intake of initial absorption; any BaCO3 formed was also analyzed these potential precursors could cause accumula- for radioactivity. The solution containing Na2"CO3 was counted directly on anthracene crystals (26) or was tion in affected patients. A second major purpose treated with saturated barium hydroxide or barium chlo- of the present studies was to explore the metabo- ride to precipitate Ba"'CO3 quantitatively. The barium lism of phytol and phytanic acid in patients with carbonate was either finely ground and suspended in 5% Refsum's disease. Results of these studies have Cab-O-Sil 3 for radioassay (27) or was treated with been in preliminary form (23-25). sulfuric acid with quantitative collection of the released reported "CO2 into phenethylamine 4 scintillation medium (28). Methods 1 Deuterium oxide was a generous gift of Norsk Hy- The case histories of our two patients with Refsum's dro-Electrisk Kvaelstofaktieselskab, Oslo, Norway.