Oxidation of Pristanic Acid in Fibroblasts and Its Application to the Diagnosis of Peroxisomal ␤-Oxidation Defects Barbara C. Paton,* Peter C. Sharp,* Denis I. Crane,‡ and Alf Poulos* *Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006; and ‡Faculty of Science and Technology, Griffith University, Nathan, Queensland 4111, Australia Abstract phology of peroxisomes in their tissues is accompanied by a series of biochemical abnormalities. Subsequent complemen- Pristanic acid oxidation measurements proved a reliable tation analyses have indicated that the three clinical pheno- tool for assessing complementation in fused heterokaryons types can arise from defects in a single gene (1, 2). In addition, from patients with peroxisomal biogenesis defects. We, in spite of all of these patients sharing the same series of bio- therefore, used this method to determine the complementa- chemical abnormalities, it is now clear that the peroxisomal tion groups of patients with isolated defects in peroxisomal biogenesis disorders, as a group, can result from defects in any ␤-oxidation. The rate of oxidation of pristanic acid was re- of at least 10 genes (2, 3). Recent evidence suggests that three duced in affected cell lines from all of the families with in- of these complementation groups have defects in the import of herited defects in peroxisomal ␤-oxidation, thus excluding peroxisomal matrix proteins via both of the known targeting the possibility of a defective acyl CoA oxidase. Complemen- mechanisms (peroxisomal targeting signals [PTS]1 1 and 2), tation analyses indicated that all of the patients belonged to but that for one of the complementation groups the defect is the same complementation group, which corresponded to restricted to proteins targeted via PTS1 (4). Recently, the gene cell lines with bifunctional protein defects. Phytanic acid (PXR1) associated with the latter complementation group has oxidation was reduced in fibroblasts from some, but not all, been identified (5). Apart from the difference in uptake of of the patients. Plasma samples were still available from six PTS1 and PTS2 targeted proteins, the different complementa- of the patients. The ratio of pristanic acid to phytanic acid tion groups are currently biochemically indistinguishable. was elevated in all of these samples, as were the levels of sat- Since several biochemical and immunocytochemical markers urated very long chain fatty acids (VLCFA). However, the are affected in this group of disorders, a range of techniques levels of bile acid intermediates, polyenoic VLCFA, and has been used to assess peroxisomal integrity, and hence com- docosahexaenoic acid were abnormal in only some of the plementation, in fused heterokaryons. These include the activ- samples. Pristanic acid oxidation measurements were help- ity of dihydroxyacetonephosphate acyltransferase (DHAPAT) ful in a prenatal assessment for one of the families where and the percentage of catalase that is in the particulate fraction previous experience had shown that cellular VLCFA levels (6), the immunocytochemical localization of catalase (7, 8), the were not consistently elevated in affected individuals. (J. oxidation of phytanic acid (9) and very long chain fatty acids Clin. Invest. 1996. 97:681–688.) Key words: bile acids and (VLCFA, i.e., fatty acids with more than 22 carbons) (10), salts • fatty acids • phytanic acid • prenatal diagnosis • Zell- plasmalogen biosynthesis (1), and sensitivity to pyrene fatty weger syndrome acid–mediated ultraviolet damage (11). It is now clear that some patients with the clinical features Introduction of a peroxisomal biogenesis disorder actually have an isolated defect in peroxisomal ␤-oxidation. This can result from a de- Complementation analyses have proved particularly useful in fect in one of the enzyme proteins of the peroxisomal ␤-oxida- helping to delineate the underlying genetic defects in patients tion pathway, namely acyl CoA oxidase, the bifunctional pro- with abnormalities in peroxisomal biogenesis. Initially, pa- tein, with its enoyl-CoA hydratase and 3-hydroxyacyl-CoA tients with these disorders were classified, on the basis of their dehydrogenase activities, or peroxisomal 3-oxoacyl-coenzyme clinical features, as having the relatively severe disorder of A thiolase. Initially, the defective gene in such patients was Zellweger’s syndrome or the progressively milder syndromes identified on the basis of a lack of immunologically cross-react- of neonatal adrenoleukodystrophy and infantile Refsum’s dis- ing material to one of these proteins, namely thiolase (12), acyl ease. The reduction in the number and/or changes in the mor- CoA oxidase (13), and the bifunctional protein (14), in the pa- tient’s tissues. However, many patients with an isolated defect A preliminary report of a portion of this work was presented at the in the peroxisomal ␤-oxidation pathway still have normal lev- “Peroxisomes: Biology and Role in Toxicology and Disease” confer- els of cross-reacting material for the three proteins (15–19). ence in Aspen, CO on 28 June to 2 July 1995. Since there are technical difficulties in assaying the individual Address correspondence to: Barbara Paton, Department of peroxisomal ␤-oxidation enzymes in cultured fibroblasts, com- Chemical Pathology, Women’s and Children’s Hospital, 72 King Wil- liam Road, North Adelaide, South Australia 5006, Australia. Phone: 1. Abbreviations used in this paper: C22:0, docosanoic acid; C24:0, tetra- 61-8-204-6733; FAX: 61-8-204-7100. cosanoic acid; C26:0, hexacosanoic acid; C22:6, docosahexaenoic acid; Received for publication 20 July 1995 and accepted in revised form C28:5, octacosapentaenoic acid, C29-dicarboxylic acid, 3␣,7␣,12␣-tri- 1 November 1995. hydroxy-27-carboxymethyl-5␤-cholestan-26-oic acid; DHAP, dihy- droxyacetonephosphate; DHAPAT, dihydroxyacetonephosphate acyl- J. Clin. Invest. transferase; DHCA, dihydroxycoprostanic acid, 3␣,7␣-dihydroxy-5␤- © The American Society for Clinical Investigation, Inc. cholestan-26-oic acid; PTS, peroxisomal targetting signal; THCA, tri- 0021-9738/96/02/0681/08 $2.00 hydroxycoprostanic acid, 3␣,7␣,12␣-trihydroxy-5␤-cholestan-26-oic Volume 97, Number 3, February 1996, 681–688 acid; VLCFA, very long chain fatty acids. Pristanic Acid Oxidation in Peroxisomal Bifunctional Protein Deficiency 681 plementation assays are proving invaluable in pinpointing the Table I. Clinical Findings in Patients with Isolated Defects in defective enzyme in such patients. Initial studies (20, 21) used Peroxisomal ␤-Oxidation the ␤-oxidation of VLCFA in complementation analyses, while Suzuki et al. (22) also determined the resistance to ultra- Patients violet damage after treating with 1-pyrene dodecanoic acid FA RS TS AE WE JG CN JS IC and the normalization of peroxisomal morphology determined using catalase immunofluorescence in their complementation Clinical feature sibs sibs studies. Seizures ϩϩϩ ϩ ϩ ϩ ϩ ϩ ϩ An elevation in plasma pristanic acid (2,6,10,14-tetrameth- Dysmorphic features ϩϪ ϩ ϩ ϩ Ϫ ϩ ylpentadecanoic acid) was first observed in patients with de- Hypotonia ϩϩϩϩϩϩϩ fects in peroxisomal biogenesis (23). More recently increased Hepatomegaly ϩϩϩϩ pristanic acid levels have been reported for patients with iso- Development delay ϩϩϩ ϩϩ lated defects in peroxisomal ␤-oxidation due to defects in the Poor feeding ϩϩϩϩ bifunctional protein and/or peroxisomal thiolase (24) but not Retinal haemorrhages ϩϩ when the defect in peroxisomal ␤-oxidation has been at the Cataracts ϩ level of acyl CoA oxidase (25). The latter observation is con- Adrenal abnormalities ϩϩϩ ϩ sistent with the identification of two acyl CoA oxidases in hu- Neuronal migration defect ϩ man liver and kidney, one which oxidizes the CoA esters of Demyelination ϩ straight chain fatty acids and prostaglandins and the other Parents consanguineous ϩϩϩϩ ϩ which oxidizes the CoA esters of 2-methyl–branched fatty ac- Survival time in months 6 6 2 4 8 11 16 Ͼ 198 5 ids and also bile acid intermediates (26). Not surprisingly, Poll- The et al. (13) found no bile acid abnormalities in their pa- tients with acyl CoA oxidase deficiency, and detection of bile ϩ, indicates feature observed; Ϫ, indicates feature not present; blank, acid precursors in patients with isolated ␤-oxidation defects is indicates no information supplied; sibs, siblings. indicative of a defect at the level of the bifunctional protein or thiolase (14, 27). as having a bifunctional protein defect (Shimozawa, N., personal In this study on Australian patients with peroxisomal disor- communication). Another of our cell lines (No. 14) was assessed in Professor Hugo Moser’s laboratory at the Kennedy Krieger Institute, ders we have investigated the use of pristanic acid oxidation Baltimore, MD, and was also shown to have a bifunctional protein estimations in the diagnosis of these patients and the applica- defect (Moser, H., personal communication). tion of this assay for complementation analyses using cells The liver samples used for the immunoblotting experiment were from patients with peroxisomal biogenesis and ␤-oxidation de- collected post-mortem. Patients D1, D2, and D3 had clinical features fects. While cells from the latter patients all had reduced rates of neonatal adrenoleukodystrophy, infantile Refsum’s disease, and of pristanic acid oxidation, considerable heterogeneity in the Zellweger’s syndrome, respectively. The biochemical findings for other biochemical findings for this group of patients was these three