Red Blood Cell Enzymes and Membrane Disorders Laboratory, Eijkman Institute of Molecular Biology, Jakarta, 10430, Indonesia.
Master Programme in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia.
Malar J. 2020 Jun 17;19(1):208. doi: 10.1186/s12936-020-03272-y.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder in the world. Its main function is to generate NADPH that is required for anti-oxidative pathway in the cells especially in red blood cells (RBC). G6PD deficiency is X-linked and thus subject to random X-chromosome inactivation in women giving them mosaic expression of G6PD activities in their individual cells. This phenomenon makes it difficult for diagnosis with the currently available G6PD qualitative diagnostic tests. With the rolling out of newly marketed anti-malarial drug tafenoquine, which has a long half-life, screening for G6PD deficiency becomes a necessity where those with < 70% G6PD activity cannot receive this drug. Thus, evidence for a quantitative cut-off for G6PD activity is needed to ensure safe drug administration.
RBC models were developed to analyse the effect of oxidant on RBC oxidative markers namely total glutathione (GSH)and malondialdehyde (MDA). G6PD activity was measured using quantitative assay from Trinity Biotech and was correlated with cytofluorometric assay. RBC from two G6PD heterozygous women with different G6PD activities were also analysed for comparison.
There was a negative correlation between G6PD activity and CuCl concentration and a strong association between G6PD activities and proportion of G6PD normal RBC in CuCl-treated models and in ex vivo RBC. However, in terms of oxidative stress markers analyses, unlike the hypothesis where the lower G6PD activity, the higher MDA and the lower GSH level, the CuCl RBC model showed that in low G6PD activities (10-30%) cells, the MDA level is lower compared to the rest of the models (p < 0.05). The ex vivo models however were in line with the hypothesis, although the result was not significant (p = 0.5). There was a significant difference between RBC with < 60% and those with > 80% G6PD activities in CuCl RBC model, but not in ex vivo RBC (p = 0.5). Genotyping heterozygous subjects showed G6PDViangchan variant with 2.97 U/gHb (33% activity) and 6.58 U/gHb (74% activity).
The GSH analysis has pointed to the 60% G6PD activity cut-off and this data is supportive of the old World Health Organization threshold for intermediate upper limit of 60% G6PD activity. However, there are significant limitations in using MDA assay with CuCl RBC model because the RBC was already stressed due to the copper treatment and thus present a different result when compared to the ex vivo model.
葡萄糖-6-磷酸脱氢酶（G6PD）缺乏症是世界上最常见的酶缺陷症。它的主要功能是生成 NADPH，这是细胞中抗氧化途径所必需的，尤其是在红细胞（RBC）中。G6PD 缺乏症是 X 连锁的，因此女性的 X 染色体随机失活，导致她们个体细胞中 G6PD 活性呈嵌合表达。这种现象使得目前可用的 G6PD 定性诊断测试难以进行诊断。随着新型抗疟药物tafenoquine 的推出，其半衰期较长，因此需要对 G6PD 缺乏症进行筛查，那些 G6PD 活性<70%的患者不能使用这种药物。因此，需要有定量的 G6PD 活性截止值来确保安全用药。
建立 RBC 模型来分析氧化剂对 RBC 氧化标志物的影响，即总谷胱甘肽（GSH）和丙二醛（MDA）。使用 Trinity Biotech 的定量测定法测定 G6PD 活性，并与细胞荧光测定法相关联。还分析了来自两个 G6PD 杂合子女性的 RBC，她们的 G6PD 活性不同，作为比较。
G6PD 活性与 CuCl 浓度呈负相关，与 CuCl 处理模型和体外 RBC 中 G6PD 正常 RBC 的比例有很强的相关性。然而，在氧化应激标志物分析方面，与假设中 G6PD 活性越低，MDA 越高，GSH 水平越低的情况相反，CuCl RBC 模型显示在低 G6PD 活性（10-30%）的细胞中，MDA 水平低于其他模型（p<0.05）。然而，体外模型符合假设，尽管结果没有统计学意义（p=0.5）。在 CuCl RBC 模型中，G6PD 活性<60%的 RBC 与 G6PD 活性>80%的 RBC 之间存在显著差异，但在体外 RBC 中没有差异（p=0.5）。对杂合子受试者的基因分型显示 G6PDViangchan 变体的活性为 2.97 U/gHb（33%）和 6.58 U/gHb（74%）。
GSH 分析指出了 60%的 G6PD 活性截止值，这一数据支持世界卫生组织（WHO）中间上限为 60%G6PD 活性的旧阈值。然而，使用 CuCl RBC 模型进行 MDA 分析存在显著的局限性，因为铜处理已经使 RBC 受到压力，因此与体外模型相比，结果会有所不同。
