顺铂对HeLa细胞6-磷酸葡萄糖脱氢酶表达和活性的影响

目的：探讨化疗药物顺铂在体外对HeLa细胞6-磷酸葡萄糖脱氢酶(G6PD)mRNA表达和活性的影响。 方法:5 mg/L顺铂作用于HeLa细胞一段时间后，MTT法检测细胞的生长抑制情况。Beutler改良法测定胞内还原型谷胱甘肽的含量，速率法测定G6PD活性，采用实时荧光定量RT-PCR观察G6PD mRNA的表达水平。结果:顺铂在体外能明显抑制HeLa细胞的生长。顺铂作用4 h和24 h后，G6PD mRNA的表达水平增强，且G6PD活性高于未加药对照组（P＜0.05）。4 h实验组GSH含量低于对照组。结论:顺铂能诱导HeLa细胞G6PD mRNA表达增加，G6PD活性增强。     

云南省两种G6PD基因突变的检测

红细胞葡萄糖 - 6 -磷酸脱氢酶 (glucose- 6 - phosphatedehydrogenase,G6 PD) ,是磷酸戊糖旁路中产生还原型辅酶 (reduced nicotinamide adenine dinucleotide phosphate,NADPH)的关键酶。G6 PD的缺陷可导致蚕豆病、药物性溶血、新生儿高胆红素血症乃至核黄疸、感染性溶血及非球形细胞溶血性贫血等 ,G6 PD缺乏症是一种常见的人类遗传性酶缺陷疾病。G6 PD基因突变是导致 G6 PD缺乏症的主要分子基础 ,目前已发现 DNA突变类型 78种 ,其中在中国人中发现了 13种[1 ] 。云南是 G6 PD缺乏症高发区 ,部分少数民族地区该病发生率达16 .…     

人类葡萄糖-6-磷酸脱氢酶的分子生物学研究进展

葡萄糖-6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PD)缺乏症作为一种全球范围内最常见的酶缺乏症之一,受到研究者们的广泛关注.G6PD催化磷酸戊糖途径的第一步,由此酶催化生成的NADPH+H+对于对抗氧化性损伤是极其重要的.本文将从G6PD的结构与功能,SNP的研究与单体型的建立,抗疟疾选择优势与新的G6PD基因突变检测方法这几方面的研究进展综述如下.     

海南汉族、黎族人葡萄糖-6-磷酸脱氢酶缺乏症的基因突变型分析及一种新的G6PD基因突变型的鉴定

目的 阐明海南汉族、黎族人群中葡萄糖－6－磷酸脱氢酶缺乏症的分子基础。方法 用聚合酶链反应、限制性内切酶消化筛查了1388G→A、1360C→T、1024C→T、517T→C、493A→G、487G→A、392G→T和95A→G突变。用单链构象多态性分析筛查其它突变;用核苷酸顺序分析鉴定具有SSCP异常区带样品的突变;。结果 在59例汉族G6PD缺乏症患者中,发现1388G→A14例（23．7％）、871G→A3例（5．1％）、835A→T1例（1．7％）517T→C1例（1．7％）、392G→T3例（5．1％）95A→G4例（6．8％）;在32例黎族G6PD缺乏症患者中,发现1388G→A6例（18．8％）、871G→A3例（9．4％）和95A→G2例（6．3％）;在1例汉族患者中发现了一种新的G6PD基因突变－835A→G突变,此突变导致第279位的苏氨酸被丙氨酸取代,将此突变型命名为G6PD－海口,其酶活性约是正常的10％,此835A→T突变的活性低,后者的酶活性约是正常的40％。分析人G6PD的三维结构模型表明,第279位苏氨酸残基的羟基是维持G6PD亚基相互作用的基团。结论 海南汉族、黎族人群中具有共同的常见G6PD基因突变型;与中国其它地区人群的G6PD基因突变谱比较,结果表明某些G6PD基因突变广泛分布于中国南方不同地区人群中;G6PD第279位苏氨酸残基的可能是维持G6PD亚基相互作用及酶活性的必需基团。     

盐酸小檗碱对G6PD酶缺陷患者红细胞的影响

目的本文研究盐酸小檗碱是否会诱发红细胞G6PD酶活性缺陷患者发生溶血,为G6PD酶缺陷患者使用黄连的安全性提供参考。方法采用G6PD酶缺陷患者静脉全血,以生理盐水为空白对照,观察不同浓度盐酸小檗碱（Ber）对红细胞形态、红细胞计数（RBC）、血红蛋白（HGB）、红细胞G6PD酶活性及血浆游离血红蛋白（F-HB）的影响。结果血中Ber浓度为0.1mg/L和0.5mg/L时,红细胞形态、RBC、HGB及G6PD酶活性均无明显改变（P〉0.05）;Ber药物浓度为1mg/L时,红细胞形态有轻度改变,RBC、HGB下降（P〈0.05）,G6PD酶活性无明显变化（P〉0.05）;Ber浓度为5mg/L时,红细胞形态明显改变,RBC、HGB显著下降（P〈0.01）,红细胞G6PD酶活性显著增高（P〈0.01）;各浓度Ber溶液均可使F-HB显著下降（P〈0.01）。结论①按正常剂量使用黄连对G6PD酶缺乏者可能是安全的,大剂量Ber能使G6PD酶缺陷患者红细胞遭到损害,提示避免超剂量使用黄连。②Ber能使血浆游离血红蛋白测定值降低,可能的原因有待进一步研究分析。     

脐带血葡萄糖6磷酸脱氢酶定量测定的应用研究

为了探讨G6PD缺乏与新生儿发生高间接胆红素血症（HIB）的相关性，该文对611例足月产新生儿脐带血做G6PD定量测定后进行跟踪检验，结果：G6PD缺乏的52例新生儿中，发生HIB17例（32．6％）；在G6PD正常的559例新生儿中发生HIE40例（7．1％）两组病例经统计学分析，新生儿HIB的发病率存在极显著的差异（U＝637、P＜0．01）。对52例G6PD缺乏的患儿统计分析发现；23例G6PD明显缺乏（G6PD＜7．0U/g Hb之间）的新生儿发生HIB的只有3例（10．3％）；两组病例的新生儿HIB发病率有极显著差异（U＝3．89，P＜0．01）。上述检验数据分析表明：G6PD缺乏是新生儿HIB的主要病因之一，并且G6PD测定值越低，新生儿发生HIB的可能性越大。     

应用G6PD／6PGD比值法检测云南勐腊县6-磷酸脱氢酶缺乏症的基因频率

采用G6PD／6PGD法对云南勐腊县123例随机采集的男性血液标末进行了红细胞6-磷酸脱氢酶缺陷的基因频率检测。先用四氮唑蓝纸片法筛选疑诊病例，对筛选出的病例进一步应用四氮唑蓝定量测定法测定G6PD／6PGD比值。结果在检测的123人中，G6PD缺乏者20人，即在此人群中G6PD缺乏的基因频率为0．1626，仍属国内G6PD缺陷高发地区，但显著低于过去报道的0.669。以前国内多数实验室均采用高铁血红蛋白还原试验来进行筛选．由于只能间接测定酶的活性，很易造成假阳性，致报告发病率偏高。本研究使用的四氮唑蓝法特异性较高，结果较可靠。     

红细胞葡糖6-磷酸脱氢酶缺乏症检测的G6PD/6PGD比值法

红细胞萄糖6-磷酸脱氢酶（G6PD）缺乏症的检测,目前国内外已建立和推荐了十余种方法。这些方法大致可分为定性和定量两类。前者适用于群体筛查;后者适用于临床确诊、变异型鉴定及群体筛查出可疑者的确认。由于G6PD杂合子的表现度范围很广,可以表现为G6PD活性接近正常或正常,故检测特别困难。除组织化学法外,其他方法都不敏感。而各组织化学方法都比较繁琐,特别是在群体中进行杂合子筛查时不适用。然而对于预防G6PD缺乏所致新生儿黄疸来说,从孕妇中早期查出杂合子是预防的关键之一。为此,我们建立了一种G6PD/6PGD比值法,配合高铁血红蛋白还原试验（MHb法）,进行群体筛查取得了较好效果,提高了杂合子检出率。现将此法介绍如下。     

中药复方对X线照射小鼠T淋巴细胞亚群和抗氧化能力的作用

目的：通过观察X线照射后小鼠T淋巴细胞亚群和抗氧化酶的变化，探讨辐射小鼠抗氧化酶和T淋巴细胞亚群的相互关系及中药养阴抗毒散的放射防护作用机理。方法：采用间接免疫荧光法和流式细胞仪测定小鼠外周血T淋巴细胞亚群，采用化学比色法测定总抗氧化能力（T－aoc)、过氧化氢酶（Cat)、超氧化物歧化酶（SOD）及丙二醛（MDA）。结果：和正常对照组比较，两个照射组CD4、CD8亚群的比例无显著下降（P＜0．01），照射对照组CD4／CD8小于正常对照组（P＜0．01）；照射中药组CD4亚群比例及CD4／CD8均高于照射对照组（P＜0．01），CD8亚群比例两者无显著差异。与正常对照组比较，照射对照组的抗氧化能力或酶类均显著降低（P＜0．01，P＜0．05），而丙二醛显著升高（P＜0．01）；照射中药组仅有过氧化氢酶下降（P＜0．01），丙二醛也显著升高（P＜0．01）；照射中药组与照射对照组比较，过氧化氢酶升高（P＜0．01），而丙二醛降低（P＜0．01）。结论：养阴抗毒散对X线照射小鼠T淋巴细胞亚群的减少有一定改善，它可能通过提高小鼠抗氧化能力达到此作用。     

siRNA沉默G6PD对人结肠癌细胞HIF-1α表达的影响

目的:研究siRNA干扰葡萄糖-6-磷酸脱氢酶(G6PD)基因表达的LoVo细胞在低氧环境中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平的变化及其对低氧诱导因子1α(HIF-1α)表达的影响。方法:构建靶向G6PD基因的siRNA干扰质粒载体,并转染人结肠癌细胞株(LoVo),用限制性内切酶法结合DNA测序鉴定特异重组子,RT-PCR检测G6PD mRNA表达,结合G6PD活性测定判断其干扰效率;以未转染LoVo细胞为对照,进一步观察G6PD沉默的瘤细胞在低氧环境下NADPH水平的变化,RT-PCR检测HIF-1αmRNA表达水平,West-ern blotting检测HIF-1α蛋白水平。结果:成功构建靶向G6PD的siRNA重组质粒载体并稳定转染LoVo细胞。与未转染组细胞相比,RNA干扰G6PD使mRNA表达下降43%,酶活性下降63.5%,细胞内NADPH水平显著下降(41%vs 100%,P<0.05),但HIF-1αmRNA表达水平无明显变化,而HIF-1α蛋白显著降低。结论:低氧环境下,G6PD基因可能通过下调NADPH水平从而降低HIF-1α的稳定性,导致其水平下降,进而影响肿瘤细胞的低氧...     

Pro-oxidative effects of tea and polyphenols, epigallocatechin-3-gallate and epigallocatechin, on G6PD-deficient erythrocytes in vitro

Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are vulnerable to chemical-induced hemolysis if exposed to oxidative agents. Recent studies reported that green tea and its constituents might act as pro-oxidants. Our objective was to investigate effects of tea and its polyphenolic components on the oxidative status of human G6PD-deficient erythrocytes. Erythrocytes of G6PD-deficient (n = 8) and normal (n = 8) subjects were incubated with water extracts of 3 types of tea samples (black tea, green tea and decaffeinated green tea extract) and 6 polyphenols. The resulting levels of reduced glutathione (GSH) and glutathione disulphide (GSSG), methemoglobin and plasma hemoglobin were quantified by HPLC and biochemical assays. The tea extracts significantly reduced GSH and increased GSSG levels in G6PD-deficient erythrocytes in a dose-dependent manner (0.5-10 mg/ml), but not in normal erythrocytes. Similar dose-dependent responses to (-)-epigallocatechin (EGC) and (-)-epigallocatechin-3-gallate (EGCG), but not to the other polyphenols, were observed. In G6PD-deficient cells, GSH was reduced by 43.3% (EGC at 0.05 mg/ml) and 33.3% (EGCG at 0.5 mg/ml), compared with pre-challenged levels. The concentration of methemoglobin was increased significantly when challenged with tea extracts, and EGC. Plasma hemoglobin levels were higher in G6PD-deficient samples after exposure to tea extracts, EGCG, EGC and gallic acid, compared with those in normal blood. Tea extracts and polyphenols significantly altered the oxidative status of G6PD-deficient erythrocytes in vitro as demonstrated by the decrease of GSH, and increased GSSG, methemoglobin and plasma hemoglobin. Our data caution against the excessive consumption of concentrated tea polyphenolic products by G6PD-deficient subjects.     

Glutathione metabolism in red cell aging

Normal human red cells were centrifugally separated according to age by discontinuous density gradient of Percoll. Reduced glutathione (GSH), GSH stability and glucose-6-phosphate dehydrogenase (G6PD) activity in fractionated red cells decreased with age, while oxidized glutathione (GSSG) and methemoglobin (MetHb) increased with age.     

Modulatory mechanisms of chemical carcinogenesis: the role of the NADPH pool in the benzo(a)pyrene activation

Human lymphocytes and human skin fibroblasts isolated in vitro from subjects carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit an 86-87% decrease of this enzymatic activity. This is coupled with 51% and 61% decreases of the NADPH/NADP+ ratio in the G6PD-deficient human lymphocytes (HL) and human skin fibroblasts (HSF), respectively. There also occurs a 63-67% decrease of the hexose monophosphate shunt (HMS) in the deficient cells. Incubation with 0.1 mM methylene blue stimulates the HMS of normal HL 15-fold and that of deficient lymphocytes only 2.4-fold. These figures are, respectively, 7 and 2.2 in the case of HSF. This behavior of G6PD-deficient HL and HSF is coupled with an increase of the resistance to the cell death induced by benzo(a)pyrene (BP). This effect is mimicked by the incubation of normal HSF with dehydroepiandrosterone (DEA) which strongly inhibits G6PD. In contrast, no differences between normal and deficient HSF occur as a result of the effect of methylnitrosourea (MNU), a carcinogen that does not need metabolic activation. The NADPH-cytochrome c (P450) reductase of G6PD-deficient HL and HSF homogenates becomes lower than that of controls when endogenous G6PD and exogenous glucose 6-phosphate (G6P) and NADP+ are used as a hydrogen donor system in place of NADPH. Normal and G6PD-deficient HL, having comparable BP-hydroxylating activities, in the presence of exogenous G6P, NADP+, and G6PD, were studied to determine the effect of the absence of exogenous G6PD in the reaction system.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetyl cysteine, L-cysteine, and beta-mercaptoethanol augment selenium-glutathione peroxidase activity in glucose-6-phosphate dehydrogenase-deficient human erythrocytes

Abstract.   In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and β-mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se- GSH-Px activity can facilitate the detoxification of reactive oxygen species.     

Control of glucose-6-phosphate dehydrogenase deficiency on the formation of mutagenic and carcinogenic metabolites derived from benzo(a)pyrene

It has been observed that human lymphocytes (HL) and fibroblasts, isolated in vitro from donors carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD), show a great decrease in this enzymatic activity, the hexose monophosphate shunt, and the NADPH/NADP+ ratio. This effect is associated with a decreased sensitivity of G6PD-deficient cells to the benzo(a)pyrene (BaP) cytotoxic effect and to a decreased in vitro transformation of BaP-treated fibroblasts. Further, benzo(a)anthracene (BaA)-induced BaP hydroxylase activity is lower in G6PD-deficient cells, when measured in the presence of endogenous NADPH. It has been hypothesized that the NADPH level could be rate-limiting for the NADPH-dependent steps of BaP metabolic activation. To test this hypothesis, the formation of BaP metabolites was studied in normal and G6PD-deficient HL incubated with the carcinogen. HPLC profiles of organic-soluble metabolites revealed that both types of HL produced all the following known BaP metabolites: 9,10-, 4,5- and 7,8-dihydrodiols, quinones, 9- and 3-hydroxy and two peaks of more polar metabolites. There was a great decrease of the various metabolites in the deficient HL. A decrease of total water-soluble BaP metabolites also occurred. HL formed mutagenic metabolites for S. typhimurium TA100 (his-) when incubated with the rat liver S9 fraction. When intact HL substituted S9 fraction, a significant reversed mutation occurred only with normal HL. This could indicate that the NADPH pool is inadequate in G6PD-deficient HL for active BaP metabolism. Accordingly, deficient HL formed lower amounts of BaP:DNA adducts than control during incubation with BaP.     

N-Acetyl cysteine, L-cysteine, and ?-mercaptoethanol augment seleniumglutathione peroxidase activity in glucose-6-phosphate dehydrogenasedeficient human erythrocytes

Abstract. In glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes, failure to maintain normal levels of reduced glutathione (GSH) due to decreased NADPH regeneration in the hexose monophosphate pathway results in acute hemolytic anemia following exposure to oxidative insults, such as ingestion of Vicia fava beans or use of certain drugs. GSH is a source of protection against oxidative attack, used by the selenium-dependent glutathione peroxidase (Se-GSH-Px)/reductase (GR) system to detoxify hydrogen peroxide and organic peroxides, provided that sufficient GSH is made available. In this study, Se-GSH-Px activity was analyzed in G6PD-deficient patients in the presence of reducing agents such as N-Acetyl cysteine, L-cysteine, and -mercaptoethanol. Se-GSH-Px activity was decreased in G6PD-deficient red blood cells (RBCs). N-Acetyl cysteine, L-cysteine, and -mercaptoethanol increased Se-GSH-Px activity in G6PD-deficient human erythrocytes, indicating that other reducing agents can be utilized to complement Se-GSH-Px activity in G6PD deficiency. Based on the increased susceptibility of G6PD-deficient patients to oxidative stress, the reported increase in Se- GSH-Px activity can facilitate the detoxification of reactive oxygen species.     

Elevated activity of the oxidative and non-oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver

(Pre)neoplastic lesions in livers of rats induced by diethylnitrosamine are characterized by elevated activity of the first irreversible enzyme of the oxidative branch of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PD), for production of NADPH. In the present study, the activity of G6PD, and the other NADPH-producing enzymes, phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (ICD) and malate dehydrogenase (MD) was investigated in (pre)neoplastic lesions by metabolic mapping. Transketolase (TKT), the reversible rate-limiting enzyme of the non-oxidative branch of the PPP, mainly responsible for ribose production, was studied as well. Activity of G6PD in (pre)neoplastic lesions was highest, whereas activity of PGD and ICD was only 10% and of MD 5% of G6PD activity, respectively. Glucose-6-phosphate dehydrogenase activity in (pre)neoplastic lesions was increased 25 times compared with extralesional parenchyma, which was also the highest activity increase of the four NADPH-producing dehydrogenases. Transketolase activity was 0.1% of G6PD activity in lesions and was increased 2.5-fold as compared with normal parenchyma. Transketolase activity was localized by electron microscopy exclusively at membranes of granular endoplasmic reticulum in rat hepatoma cells where G6PD activity is localized as well. It is concluded that NADPH in (pre)neoplastic lesions is mainly produced by G6PD, whereas elevated TKT activity in (pre)neoplastic lesions is responsible for ribose formation with concomitant energy supply by glycolysis. The similar localization of G6PD and TKT activity suggests the channelling of substrates at this site to optimize the efficiency of NADPH and ribose synthesis.     

Mechanism of action of divicine in a cell-free system and in glucose-6-phosphate dehydrogenase-deficient red cells

Favism is an acute hemolysis occurring in glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) individuals after intake of fava beans. Divicine (D), 2,6-diamino-4,5-dihydroxypyrimidine, is present in high amounts in the beans, and is suspected to play a role in hemolysis. Its mechanism of action was studied in a cell-free system and in G6PD (Mediterranean variant)-deficient red cells (RBC). Upon hydrolysis of the inactive beta-glucoside vicine, reduced divicine is formed. Oxygen acts as a one- or two-electron acceptor; superoxide anion and hydrogen peroxide are formed, respectively, together with the semiquinoid free-radical form of D. This free radical gives an electron spin resonance (ESR) signal, which is similar to that of the alloxan free radical. Added reduced glutathione (GSH) is rapidly oxidized with a stoichiometry of one to one, and the ESR signal is abolished. Additional GSH is oxidized by hydrogen peroxide and by a slow redox cycle which continuously regenerates oxidized D. The fast-direct and the slow-indirect oxidation result in nonstoichiometric oxidation of GSH. D added to G6PD-deficient RBC rapidly oxidizes GSH with an end point kinetics and a stoichiometry of one to one. Hydrogen peroxide and superoxide anion are scavenged in the RBC and no redox cycling is taking place. No GSH is regenerated even after long incubation periods. After the primary event, i.e., oxidation of GSH and--SH groups, a number of metabolic, rheologic, and membrane modifications, together with increased erythrophagocytosis take place in G6PD-deficient, D-treated RBC only.(ABSTRACT TRUNCATED AT 250 WORDS)     

G6PD缺乏不同检测方法的临床应用

目的对G6PD缺乏的不同检测方法进行比较和评价。方法以高铁法、杜传书教授提出的NBT定量比值法、WHO提出的NADPH定量比值法,分别对1236例来我院婚检和产检人群进行G6PD检测。结果以NADPH定量比值法为标准,1236例受检者中共检出G6PD缺乏100例,总检出率8.1%,其中男性6.8%,女性8.6%。NBT定量比值法和高铁法总检出率分别为6.1%、11.5%,两种方法对男性G6PD缺乏能全部检出。但对G6PD缺乏女性NBT定量比值法检出率较低（5.9%）,漏诊率32%。高铁法检出率较高（12%）,但假阳性率3.8%。结论高铁法适合大批量标本筛查,NBT定量比值法适合检测G6PD正常及严重缺乏标本。在全自动生化分析仪上使用NADPH定量比值法测定G6PD,与传统方法相比简便、快捷、用量微,优于NBT定量比值法。可以提高女性G6PD缺乏的检出率。