Detection of unusual very-long-chain fatty acid and ether lipid derivatives in the fibroblasts and plasma of patients with peroxisomal diseases using liquid chromatography-mass spectrometry.

Metabolic changes occur in patients with peroxisomal diseases owing to impairments in the genes involved in peroxisome function. For diagnostic purposes, saturated very-long-chain fatty acids (VLCFAs) such as C24:0 and C26:0, phytanic acid, pristanic acid, and plasmalogens are often measured as metabolic hallmarks. As the direct pathology of peroxisomal disease is yet to be fully elucidated, we sought to explore the fatty acid species that accumulate in patients with peroxisomal diseases. We developed a method for detecting a range of fatty acids implicated in peroxisomal diseases such as Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD). To this end, we employed an ultra-performance liquid chromatography-mass spectrometry (LC-MS) coupled with negatively charged electrospray ionization. Fatty acids from patients and control subjects were extracted from total lipids by acid-hydrolysis and compared. In accordance with previous results, the amounts of VLCFAs, phytanic acid, and pristanic acid differed between the two groups. We identified extremely long and highly polyunsaturated VLCFAs (ultra-VLC-PUFAs) such as C44:12 in ZS samples. Moreover, three unknown molecules were prominent in control samples but scarcely detectable in ZS samples. LC-MS/MS analysis identified these as 1-alkyl-sn-glycerol 3-phosphates derived from ether lipids containing fatty alcohols such as C16:0, C18:0, or C18:1. Our method provides an approach to observing a wide range of lipid-derived fatty acids and related molecules in order to understand the metabolic changes involved in peroxisomal diseases. This technique can therefore be used in identifying metabolic markers and potential clinical targets for future treatment.

Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.

Peroxisomes catalyze a range of essential metabolic functions, mainly related to lipid metabolism. However, their roles in obesity have yet to be clarified. The aim of this study was to investigate the correlation between obesity and peroxisomal lipid metabolism, particularly very long-chain fatty acid (VLCFA) metabolism, gene expression of peroxisomal ß-oxidation enzymes, peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy (ABCD1) gene and its related gene, ABCD2, the elongation of the VLCFA (ELOVL) gene family and the transcriptional factors involved in the regulation of these genes, including peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein. These factors were analyzed in livers from mice fed a high-fat diet (HFD) or a regular diet (RD) for 20 weeks. Furthermore, the amounts of plasma saturated and unsaturated fatty acids, including VLCFAs, were measured. A HFD induced hepatic gene expression of not only hydroxysteroid 17-ß dehydrogenase 4 (HSD17b4) and sterol carrier protein 2 (SCP2) in peroxisomal ß-oxidation enzymes but also of ELOVL1, 2, 5 and 6, which are involved in the elongation of saturated and unsaturated VLCFAs. Furthermore, ABCD2 mRNA prominently increased in the HFD mice. The transcriptional regulator of these genes, PPARα, was also up-regulated in the HFD mice. VLCFA ratios including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0 are the most significant diagnostic markers of inherited peroxisomal diseases. These ratios were found to be low in the plasma of the HFD mice compared with the RD mice. The results suggest that HFD activates hepatic peroxisomal VLCFA metabolism, and may provide useful fundamental information to explain the role of peroxisomal function in obesity and lifestyle-related diseases.

X-linked adrenoleukodystrophy: diagnostic and follow-up system in Japan.

X-linked adrenoleukodystrophy (ALD) is an intractable neurodegenerative disease associated with the accumulation of very long-chain saturated fatty acids (VLCFA) in tissues and body fluids. We have established a Japanese referral center for the diagnosis of ALD, using VLCFA measurements and mutation analysis of the ABCD1 gene, and have identified 60 kinds of mutations in 69 Japanese ALD families, which included 38 missense mutations, 6 nonsense mutations, 8 frame-shift mutations, 3 amino acid deletions, 2 exon-skip mutations and 3 large deletions. A total of 24 kinds of mutations (40%) were identified only in Japanese patients by referring to the current worldwide ALD mutation database. There was no clear correlation between these mutations and phenotypes of 81 male patients in these 69 families. About 12% of the individuals with ALD had de novo mutations by mutation analysis in the male probands and their mothers, which should be helpful data for genetic counseling. The only effective therapy for the cerebral form of ALD should be hematopoietic stem cell transplantation at the early stages of the cerebral symptoms, therefore, we performed presymptomatic diagnosis of ALD by extended familial screening of the probands with careful genetic counseling, and established a long follow-up system for these patients to prevent the progression of brain involvement and to monitor the adrenocortical insufficiency. Further elucidation of pathology in ALD, especially concerning the mechanisms of the onset of brain involvement, is expected.