003 1-399819213205-0566$03.00/0 PEDIATRIC RESEARCH Val. 32, No. 5, 1992 Copyright 0 1992 International Pediatric Research Foundation, Inc Printed in (I.S. A. In Vivo Study of Phytanic Acid &-Oxidationin Classic Refsum's Disease and Chondrodysplasia Punctata HERMAN J. TEN BRINK, DANIELLE S. M. SCHOR, ROBERT M. KOK, FRANS STELLAARD, JOHANNES KNEER, BWEE TIEN POLL-THE, JEAN-MARIE SAUDUBRAY, AND CORNELIS JAKOBS Department of Pediatrics, Free University Hospital, Amsterclam, The Netherlands [H.J.t.B., D.S.M.S., R.M.K., C.J.J;Kinderklinik, Freie Universitat Berlin, Berlin, Germany[J.K.]; University Children's Hospital, Het Wilhe1)nina Kin~lmieltenhuis,Utrechf, The Netherlands /B.T.P.-T.]; and Clinique Medicale Infantile, H6pital des Enfants Malades, Paris, France [J-M.S.J ABSTRACT. A series of in vivo experiments is described in which [l-'3C]phytanic acid was given as an oral substrate to a healthy subject and two patients showing an impair- ment in phytanic acid degradation, one with Refsum's Phytanic acid (3,7,11,15-tetramethylhexadecanoicacid) is a disease and one with chondrodysplasia punctata. After 20-carbon branched chain fatty acid found only in trace amounts intake of the substrate by the control in a dose of 20 mg/ in healthy human beings. Endogenous de novo biosynthesis of kg body weight, the production of I3CO2 was measured in phytanic acid has not been demonstrated, and its origin appears exhaled breath air and the concomitant formation of la- to be exclusively exogenous. Dietary intake of the compound beled 2-hydroxyphytanic acid and of pristanic acid was itself is the major source of phytanic acid in man, and minor demonstrated by plasma analysis. After application of a additional amounts may result from dietary phytol. The presence substrate dose of 1 mg/kg body weight to the control, no of a 3-methyl group in the phytanic acid molecular structure substantial amounts of 13COz were measured, whereas prohibits its breakdown by P-oxidation, the usual degradation time-dependent analysis of labeled 2-hydroxyphytanic acid route for straight-chain fatty acids. Instead, phytanic acid metab- in plasma yielded a concentration curve superimposed upon olism is believed to involve an initial a-hydroxylation and de- the baseline value (0.2 pmollL) of the unlabeled substance. carboxylation, leading to pristanic acid (2,6,10,14-tetramethyl- Phytanic acid accumulated in plasma from the Refsum's pentadecanoic acid), which can be degraded through successive disease patient 1649 pmol/L, controls > 1 y (n = 100): c P-oxidation steps (see Ref. 1 for a review). Phytanic acid was 10 pmol/L], whereas the pristanic acid concentration was long thought to accumulate only in cases of classic RD. Later, within the control range 11.4 pmol/L, controls > 1 y (n = accunlulation of phytanic acid was demonstrated to occur in 100): c 3 pmol/L]. Low amounts of 2-hydroxyphytanic patients suffering from peroxisomal disorders characterized by a acid were found normally present 10.04 pmol/L, controls > general loss of peroxisomal functions, i.e., Zellweger syndrome, 1 y (n = 11): < 0.2 pmol/L], and formation of labeled 2- infantile RD, neonatal adrenoleukodystrophy, and hyperpipe- hydroxyphytanic acid could not be demonstrated after colic acidemia, and in the rhizomelic form of CDP (see Ref. 2 ingestion of [1-13C]phytanicacid in a dose of 1 mg/kg body for a review). In all these disorders, a concomitant impairment weight. In addition to phytanic acid accumulation (232 of phytanic acid a-oxidation was found, usually determined by pmol/L), the chondrodysplasia punctata patient showed an measurement of I4CO2 release after incubation of tissue prepa- elevated 2-hydroxyphytanic acid plasma concentration (0.4 rations with [l-'4C]phytanic acid. pmol/L), whereas the plasma pristanic acid level was in Studies on rat liver homogenates incubated with radioactively the control range (0.7 pmol/L). Oral administration of [l- labeled phytanic acid have shown the presence of labeled 2- I3C]phytanic acid (1 mg/kg body weight) to this patient hydroxyphytanic acid, indicating that in mammals a-hydroxyl- resulted in a plasma concentration/time profile of labeled ation is the first step in the phytanic acid a-oxidation (3-6). This 2-hydroxyphytanic acid similar to that of the control. leads to the proposed pathway for degradation of phytanic acid These findings demonstrate that a-hydroxylation of phy- shown in Figure 1. The occurrence of this pathway in man has tanic acid is a physiologic process and indicate that Ref- been confirmed in tissue cultures of skin fibroblasts (3, 7). The sum's disease and chondrodysplasia punctata patients have finding that i.v. injected [U-'4C]2-hydroxyphytanicacid was a different ability to convert phytanic acid into 2-hydroxy- metabolized rapidly in healthy humans and also in RD patients phytanic acid. (Pediatu Res 32: 566-570, 1992) provided evidence that the biochemical defect in RD is a deficient a-hydroxylation of phytanic acid (8). However, in vivo studies in Abbreviations man confirming the direct formation of 2-hydroxyphytanic acid from phytanic acid have not been reported, and recently the CDP, chondrodysplasia punctata formation of 2-hydroxyphytanic acid as an intermediate in the PFB, pentafluorobenzyl phytanic acid metabolism was questioned (9). RCDP, rhizomelic chondrodysplasia punctata In our investigation of phytanic acid a-oxidation in man, we RD, Refsum's disease developed an alternative in vivo approach, consisting of oral TMS, trimethylsilyl administration of [l-13C]phytanicacid to a healthy control and to selected patients with a deficiency in phytanic acid a-oxidation Received November 29, 199 1; accepted June 1, 1992. Correspondence and reprint requests: Dr. C. Jakobs, Dept. of Pediatrics, Free and measurement of accordingly labeled metabolites. To assess University Hospital, De Boelelaan 11 17, 1081 HV Amsterdam, The Netherlands. experimental conditions for approximation of a physiologic sit- 56 PHYTANIC ACID a-OXIDATION IN RD AND CDP 567 was applied (13). The product was purified by column chroma- COSCoA tography over silica gel, using a gradient of hexane and ethyl phytanic acid acetate that had been freshly distilled. Unlabeled 2-hydroxyphytanic acid was synthesized from phy- tanic acid. Bromine was introduced into the a-position by Hell- Volhard-Zelinsky reaction (17), and ethanolysis of the reaction COSCoA mixture yielded 2-bromophytanic acid ethyl ester. Bromide was substituted by acetate using sodium acetate in DMSO, and the OH 2-hydroxyphytanic acid resulting diester was saponified with KOH in ethanol, giving 2- hydroxyphytanic acid after acidification. In an analogous manner, [3-methyl-2H3]2-hydroxyphytanic acid (99 atom % 'H3) was synthesized from [3-methyl-'H3] ~cosco* phytanic acid. The synthesis of this latter compound, together pristanic acid with that of [2-methyl-2H3]pristanicacid, has been published before (1 8). Administration of [l-'3C]phytanic acid. In two separate exper- iments, [l-13C]phytanicacid was given to the healthy volunteer. Fig. I. Metabolism of phytanic acid. I, a-hydroxylation; 2, decarbox- After a fasting period of 12 h, the free acid, soaked in a piece of ylation; and 3, P-oxidation. bread, was administered orally in doses of 20 and 1 mg/kg body weight, respectively. Subsequently, the RD patient and the CDP uation, the substrate was first given in two different dosages to a patient (also after 12 h fasting) received the dose of 1 mg/kg healthy volunteer. Thereupon an amount of the substrate con- body weight, which was appropriate to allow the detection of sidered sufficient to detect labeled degradation products in reliably measurable amounts of parent compound and metabo- plasma was given to a classic RD patient and a CDP patient. lites in plasma. Administration of the substrate to the RD patient The finding of severe phytanic acid accumulation combined with was as to the control. For the CDP patient, the substrate was normal pristanic acid levels in plasma from such patients indi- embedded in a small amount of fruit yogurt, and complete intake cates that in these disorders the metabolic defect in the degra- was carefully controlled. For the control person, exhaled breath dation pathway of phytanic acid is situated within its a-oxidation air was collected every 15 min during the first 4 h, every 30 min (10). In RD, a deficient a-hydroxylation of phytanic acid is during the next 4 h, and subsequently every hour during 4 h. supposed to be the only biochemical abnormality (7, 8). In the Two additional breath samples were taken after 24 and 26 h. rhizomelic form of CDP, an impaired plasmalogen biosynthesis After initial blood sampling 1 h after administration, blood and the occurrence of an unprocessed peroxisomal 3-oxoacyl- samples were taken every 2 h during the first 13 h of the CoA thiolase are found next to an impaired phytanic acid a- experimental period. oxidation (1 1). Studies on phytanic acid a-oxidation by comple- Measurement of exhaled C02. Exhaled COz in breath air mentation analysis of RD and peroxisomal disorders revealed samples was purified by following a standard kryogenic proce- that RD and RCDP represent different complementation groups, dure (19). The isotopic enrichment of the purified COz (6A indicating that the enzyme defect in RCDP involves a different values) was determined by isotope ratio mass spectrometric step in the phytanic acid a-oxidation from that of the defect in measurement of m/z 44 and 45. Cumulative output of I3CO2(76 RD (12). of dose) was calculated and corrected for normal endogenous The present study was undertaken to investigate whether dif- C02production, which was determined independently. ferences in phytanic acid degradation deficiency would be dis- Plasma analysis. Analyses of pristanic acid and unlabeled and cernible by detection of specific metabolites.