Development of a system adapted for the diagnosis and evaluation of peroxisomal disorders by measuring bile acid intermediates.

OBJECTIVE: Bile acid intermediates, 3α,7α,12α-trihydroxycholestanoic acid (THCA) and 3α,7α-dihydroxycholestanoic acid (DHCA), are metabolized in peroxisomes. Some peroxisomal disorders (PDs), such as Zellweger spectrum disorder (ZSD), show an accumulation of bile acid intermediates. In particular, ABCD3 deficiency and acyl-CoA-oxidase 2 deficiency are characterized by these metabolite abnormalities. In patients with ZSD, levels of bile acid intermediates can be lowered by a primary bile acid supplementation treatment; therefore, measuring their levels could help evaluate treatment effectiveness. Here, we established a method for the quantitative determination of bile acid intermediates (THCA/DHCA) for differentiating PDs and assessing bile acid treatment. METHODS: Serum samples, obtained from patients with several forms of ZSD as well as peroxisomal ß-oxidation enzyme deficiencies, were deproteinized and analyzed using liquid chromatography-mass spectrometry. RESULTS: Levels of the bile acid intermediates increased significantly in patients with Zellweger syndrome (ZS) and slightly in patients with neonatal adrenoleukodystrophy and infantile Refsum disease (IRD), reflecting the severity of these diseases. One patient with ZS treated with primary bile acids for 6 months showed slightly decreased serum DHCA levels but significantly increased serum THCA levels. One patient with IRD who underwent living-donor liver transplantation showed a rapid decrease in serum THCA and DHCA levels, which remained undetected for 6 years. In all controls, THCA and DHCA levels were below the detection limit. CONCLUSION: The analytical method developed in this study is useful for diagnosing various PD and validating bile acid treatment. Additionally, it can help predict the prognosis of patients with PD and support treatment strategies.

Hypomorphic mutation of PEX3 with peroxisomal mosaicism reveals the oscillating nature of peroxisome biogenesis coupled with differential metabolic activities.

Impaired peroxisome assembly caused by mutations in PEX genes results in a human congenital metabolic disease called Zellweger spectrum disorder (ZSD), which impacts the development and physiological function of multiple organs. In this study, we revealed a long-standing problem of heterogeneous peroxisome distribution among cell population, so called "peroxisomal mosaicism", which appears in patients with mild form of ZSD. We mutated PEX3 gene in HEK293 cells and obtained a mutant clone with peroxisomal mosaicism. We found that peroxisomal mosaicism can be reproducibly arise from a single cell, even if the cell has many or no peroxisomes. Using time-lapse imaging and a long-term culture experiment, we revealed that peroxisome biogenesis oscillates over a span of days; this was also confirmed in the patient's fibroblasts. During the oscillation, the metabolic activity of peroxisomes was maintained in the cells with many peroxisomes while depleted in the cells without peroxisomes. Our results indicate that ZSD patients with peroxisomal mosaicism have a cell population whose number and metabolic activities of peroxisomes can be recovered. This finding opens the way to develop novel treatment strategy for ZSD patients with peroxisomal mosaicism, who currently have very limited treatment options.

Zebrafish model of human Zellweger syndrome reveals organ-specific accumulation of distinct fatty acid species and widespread gene expression changes.

In Zellweger syndrome (ZS), lack of peroxisome function causes physiological and developmental abnormalities in many organs such as the brain, liver, muscles, and kidneys, but little is known about the exact pathogenic mechanism. By disrupting the zebrafish pex2 gene, we established a disease model for ZS and found that it exhibits pathological features and metabolic changes similar to those observed in human patients. By comprehensive analysis of the fatty acid profile, we found organ-specific accumulation and reduction of distinct fatty acid species, such as an accumulation of ultra-very-long-chain polyunsaturated fatty acids (ultra-VLC-PUFAs) in the brains of pex2 mutant fish. Transcriptome analysis using microarray also revealed mutant-specific gene expression changes that might lead to the symptoms, including reduction of crystallin, troponin, parvalbumin, and fatty acid metabolic genes. Our data indicated that the loss of peroxisomes results in widespread metabolic and gene expression changes beyond the causative peroxisomal function. These results suggest the genetic and metabolic basis of the pathology of this devastating human disease.

Positional determination of the carbon-carbon double bonds in unsaturated fatty acids mediated by solvent plasmatization using LC-MS.

Fatty acids (FAs) are the central components of life: they constitute biological membranes in the form of lipid, act as signaling molecules, and are used as energy sources. FAs are classified according to their chain lengths and the number and position of carbon-carbon double bond, and their physiological character is largely defined by these structural properties. Determination of the precise structural properties is crucial for characterizing FAs, but pinpointing the exact position of carbon-carbon double bond in FA molecules is challenging. Herein, a new analytical method is reported for determining the double bond position of mono- and poly-unsaturated FAs using liquid chromatography-mass spectrometry (LC-MS) coupled with solvent plasmatization. With the aid of plasma on ESI capillary, epoxidation or peroxidation of carbon-carbon double bond in FAs is facilitated. Subsequently, molecular fragmentation occurs at or beside the epoxidized or peroxidized double bond via collision-induced dissociation (CID), and the position of the double bond is elucidated. In this method, FAs are separated by LC, modified by plasma, fragmented via CID, and detected using a time-of-flight mass spectrometer in a seamless manner such that the FA composition in a mixture can be determined. Our method enables thorough characterization of FA species by distinguishing multiple isomers, and therefore can uncover the true diversity of FAs for their application in food, health, and medical sciences.

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.