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Mol Genet Metab. 2011 Nov;104(3):241-8. doi: 10.1016/j.ymgme.2011.05.012. Epub 2011 May 19.

Mechanisms regulating superoxide generation in experimental models of phenylketonuria: an essential role of NADPH oxidase.

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Department of Pediatric Endocrinology and Genetical Metabolism, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Institute for Pediatric Research, 1665 Kongjiang Road, Shanghai 200092, China.


This study was designed to investigate whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a superoxide-producing enzyme, could be involved in phenylketonuria (PKU)-associated oxidative stress. A Pah(enu2)-BTBR PKU mouse model, and an in vitro cell culture model of PKU mimicking high phenylalanine insults in PKU, were employed for this study. The concentration of phenylalanine in mouse cerebral cortex was determined by liquid chromatography-tandem mass spectrometry. Superoxide production was displayed with dihydroethidium staining. NADPH oxidase expression level was measured by real-time RT-PCR, Western blotting and immunofluorescence. NADPH oxidase activity was measured by the colorimetric method. The phenylalanine concentrations in cerebral cortices of PKU mice were significantly higher than those in wild-type control mice. Similar results concerning superoxide production and NADPH oxidase protein expression and activity, were also found in this brain region. In addition, it was found that cerebral cortical neurons subjected to an in vitro high phenylalanine insult, displayed increased superoxide production accompanied by increases of NADPH oxidase protein expression and activity. Pretreatment with the inhibitor of this oxidase (diphenylene iodonium or apocynin) prevented this superoxide-increasing effect. Collectively, these findings provide evidence that NADPH oxidase might be a key enzyme involved in enhanced superoxide production in PKU and suggest that it may be a potential therapeutic target in neuroprotective strategies against phenylalanine-evoked oxidative brain injury in PKU.

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