Very-long-chain polyunsaturated fatty acids accumulate in phosphatidylcholine of fibroblasts from patients with Zellweger syndrome and acyl-CoA oxidase1 deficiency

Highlights

• Ether phospholipids are markedly reduced in peroxisome-deficient cells.

• DHA-containing lipids are decreased in peroxisome- and AOx-deficient fibroblasts.

• VLC-PUFAs accumulate in cells from peroxisome- and AOx-deficient patients.

• VLC-PUFA is not accumulated in cells from a patient with X-ALD.

Abstract

Peroxisomes are subcellular organelles that function in multiple anabolic and catabolic processes, including β-oxidation of very-long-chain fatty acids (VLCFA) and biosynthesis of ether phospholipids. Peroxisomal disorders caused by defects in peroxisome biogenesis or peroxisomal β-oxidation manifest as severe neural disorders of the central nervous system. Abnormal peroxisomal metabolism is thought to be responsible for the clinical symptoms of these diseases, but their molecular pathogenesis remains to be elucidated. We performed lipidomic analysis to identify aberrant metabolites in fibroblasts from patients with Zellweger syndrome (ZS), acyl-CoA oxidase1 (AOx) deficiency, D-bifunctional protein (D-BP) and X-linked adrenoleukodystrophy (X-ALD), as well as in peroxisome-deficient Chinese hamster ovary cell mutants. In cells deficient in peroxisomal biogenesis, plasmenylethanolamine was remarkably reduced and phosphatidylethanolamine was increased. Marked accumulation of very-long-chain saturated fatty acid and monounsaturated fatty acids in phosphatidylcholine was observed in all mutant cells. Very-long-chain polyunsaturated fatty acid (VLC-PUFA) levels were significantly elevated, whilst phospholipids containing docosahexaenoic acid (DHA, C22:6n-3) were reduced in fibroblasts from patients with ZS, AOx deficiency, and D-BP deficiency, but not in fibroblasts from an X-ALD patient. Because patients with AOx deficiency suffer from more severe symptoms than those with X-ALD, accumulation of VLC-PUFA and/or reduction of DHA may be associated with the severity of peroxisomal diseases.

Introduction

Peroxisomes are subcellular organelles that participate in various metabolic processes, such as β-oxidation of very-long-chain fatty acids (VLCFA) and the biosynthesis of ether phospholipids and bile acids [1], [2]. The functional importance of peroxisome metabolism in humans is demonstrated by the symptoms of peroxisomal diseases, including peroxisome biogenesis disorders (PBDs) and single peroxisomal enzyme deficiencies [3], [4].

Generalized PBDs, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD), are classified into thirteen complementation groups (CGs) by cell-fusion assay using skin fibroblasts [3]. The primary cause of PBDs is impaired biogenesis of peroxisomes, and genetic complementation analysis using peroxisome-deficient Chinese hamster ovary (CHO) cell mutants led to the identification of PEX genes essential for peroxisome biogenesis [3], [5]. Patients with ZS, the most severe PBD, are characterized by seizures, facial dysmorphism, severe hypotonia, and brain dysfunction, and die before 1 year of age. Neural migration defects, dysmyelination, and neural heterotopia are observed in the central nervous system (CNS) of ZS patients [3], as are biochemical abnormalities, including VLCFA accumulation, marked depletion of ether phospholipids, and reduced levels of docosahexaenoic acid (DHA) [4]. Although deficiencies in peroxisomal metabolism are thought to be responsible for the pathology of ZS, the underlying pathogenic mechanism is still unclear.

Severe defects in the CNS are also observed in X-linked adrenoleukodystrophy (X-ALD) and peroxisomal β-oxidation-deficient diseases, including acyl-CoA oxidase1 (AOx) deficiency and D-bifunctional protein (D-BP) deficiency [6], [7]. These disorders are diagnosed by an elevated plasma VLCFA ratio, such as C26:0/C22:0 and C26:1/C22:0. Of these, X-ALD is the most common single peroxisome enzyme deficiency caused by mutations in the ATP-binding cassette transporter subfamily D member 1 (ABCD1), which is essential for the translocation for CoA-activated VLCFAs across the peroxisomal membrane [4], [6], [8]. AOx is the first step in and rate-limiting enzyme of peroxisomal fatty acid β-oxidation, while D-BP catalyzes in second and third steps [9]. The patients with AOx and D-BP deficiency exhibit more severe symptoms than patients with X-ALD [10], [11], [12], [13]. Thus, peroxisomal fatty acid β-oxidation is of particular importance in the proper development and maintenance of the CNS.

The relationship between the biochemical and pathological abnormalities observed in patients with ZS and those with β-oxidation deficiencies remains to be defined. Since the majority of peroxisomal processes play a role in lipid metabolism, it is conceivable that aberrant peroxisomal lipid metabolites affect cellular function. Therefore, the identification of these aberrant lipid metabolites is essential for elucidating the pathogenesis of PBDs and β-oxidation disorders. Although the biochemical phenotypes of AOx deficiency and X-ALD are similar, their pathological severities are significantly different, indicating that additional metabolic abnormalities in patients with AOx deficiency may cause the severe dysfunction in the CNS.

To date, the phospholipid compositions of the brain [14], [15], [16] and skin fibroblasts [14], [17], [18] from ZS patients and in peroxisome-deficient CHO mutant cell lines [19], [20], [21] have been reported. Recently, liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) was utilized to detect small amounts of lipid metabolites and discriminate the precise fatty acid composition of individual phospholipid classes. Thus, in the present study, we used LC-ESI-MS/MS to analyze the lipid composition of skin fibroblasts from patients with ZS and peroxisomal fatty acid β-oxidation deficiency diseases.