Department of Pediatrics, Faculty of Medicine, Kuwait University, Kuwait.
Exp Biol Med (Maywood). 2013 Jul;238(7):779-86. doi: 10.1177/1535370213480692. Epub 2013 Jul 4.
Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.
半乳糖血症是一种常染色体隐性遗传疾病，具有广泛的临床异常。细胞氧化应激被认为是半乳糖血症的发病机制之一。在这项研究中，我们检查了外周血淋巴细胞（PBL）中 NADPH 氧化酶（NOX）的活性，NOX 是一种主要的超氧化物生成酶系统。从半乳糖血症患者和健康对照者中分离出 PBL 进行细胞培养研究和生化测定。在存在或不存在半乳糖或半乳糖-1-磷酸（Gal-1-P）的情况下培养 PBL，并在细胞匀浆中测量 NOX、过氧化氢酶、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GPx）的酶活性和/或基因表达。从半乳糖血症患者中分离出的 PBL 显示出明显降低的（P <0.01）过氧化氢酶和 GPx 活性；然而，SOD 活性保持不变。半乳糖血症患者血液淋巴细胞中的丙二醛（MDA）水平显著升高（P <0.01）。NOX 的酶活性在半乳糖血症患者中显著降低（P <0.001）；然而，Western blot 显示 NOX-1 蛋白没有明显改变。有趣的是，当在缺乏半乳糖的培养基中培养两周时，从半乳糖血症患者中分离出的淋巴细胞中的 NOX 活性显著增加但仍低于正常水平，表明半乳糖对半乳糖血症患者的 NOX 具有抑制作用。当在存在半乳糖或 Gal-1-P 的情况下培养两周时，从对照者中分离出的淋巴细胞的 NOX 活性显著降低（P <0.01）。这些结果表明，半乳糖诱导的细胞氧化应激不是由 NOX 介导的。然而，NOX 系统的损伤可能是半乳糖血症患者一些临床并发症的原因。
