Abstract
The purpose of this study is to establish an assay method to screen for chemical compounds that stimulate peroxisomal fatty acid β-oxidation activity in X-linked adrenoleukodystropy (X-ALD) fibroblasts. In this investigation, we used 12-(1-pyrene)dodecanoic acid (pyrene-C12:0), a fluorescent fatty acid analog, as a substrate for fatty acid β-oxidation. When human skin fibroblasts were incubated with pyrene-C12:0, β-oxidation products such as pyrene-C10:0 and pyrene-C8:0 were generated time-dependently. These β-oxidation products were scarcely detected in the fibroblasts from patients with Zellweger syndrome, a peroxisomal biogenesis disorder. In contrast, in fibroblasts with mitochondrial carnitine-acylcarnitine translocase deficiency, the β-oxidation products were detected at a level similar to control fibroblasts. These results indicate that the β-oxidation of pyrene-C12:0 takes place in peroxisomes, but not mitochondria, so pyrene-C12:0 is useful for measuring peroxisomal fatty acid β-oxidation activity. In X-ALD fibroblasts, the β-oxidation activity for pyrene-C12:0 was approximately 40 % of control fibroblasts, which is consistent with previous results using [1-14C]lignoceric acid as the substrate. The present study provides a convenient procedure for screening chemical compounds that stimulate the peroxisomal fatty acid β-oxidation in X-ALD fibroblasts.
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Acknowledgements
This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (23590072, 26460063). Pacific Edit reviewed the manuscript prior to submission.
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Masashi Morita, Shun Matsumoto, Airi Okazaki, Kaito Tomita, Shiro Watanabe, Kosuke Kawaguchi, Daishiro Minato, Yuji Matsuya, Nobuyuki Shimozawa and Tsuneo Imanaka declare that they have no conflict of interest.
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All procedures followed were in accordance with the ethical standards of the responsible committee on human studies (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all patients for being included in the study.
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Communicated by: Ronald JA Wanders
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Supplementary Fig. 1
The structure of 12-(1-pyrene)dodecanoic acid (PPTX 66 kb)
Supplementary Fig. 2
Uptake of pyrene-C12:0 by various patient fibroblasts. Human skin fibroblasts (Cont, X-ALD, CADDS, CACT-D, and Zell) were incubated in serum-free medium containing pyrene-C12:0 (50 μM). The fluorescence of the cellular lipid extracts was quantified as described in the Materials and methods section. The data represents the mean of three samples (PPTX 62 kb)
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Morita, M., Matsumoto, S., Okazaki, A. et al. A novel method for determining peroxisomal fatty acid β-oxidation. J Inherit Metab Dis 39, 725–731 (2016). https://doi.org/10.1007/s10545-016-9952-y
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DOI: https://doi.org/10.1007/s10545-016-9952-y