Subjects and Sample Preparations
The RBC studies were developed from two normal males having normal G6PD activities and normal hematological profiles and two G6PD heterozygous women having normal hematological profiles. Informed consent was obtained from all subjects prior to obtaining 8 mL of venous blood into ACD tubes from two normal males and 2 mL of venous blood into EDTA tubes from two G6PD heterozygous women. Complete blood count was performed for each subject immediately after blood collection using automated hematology analyzer Cell Dyne pocH-100i (Sysmec, USA) and the blood was aliquoted accordingly for CuCl treatment, H2O2 incubation, G6PD activity measurement, flow cytometer analyses, MDA and GSH tests and were stored at 40C until used. Ethical clearance for the study was approved by The Ethics Committee of The Faculty of Medicine, Universitas Indonesia (No. 640/UN2.F1/ETIK/2016).
Red Blood Cells G6PD Deficiency Models using CuCl
Both Cu+ and Cu2+ can inhibit G6PD activity in RBC [21,22]. Slight modification to the method previously described was made . Procedures were done as soon as blood was collected where 400 µL of whole blood in ACD was treated with 10 mM CuCl in water (Fluka, Germany) in various final concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1.0 mM, 1.5 mM and 2.0 mM CuCl to mimic 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 100% G6PD activities in heterozygous women respectively. All samples were incubated in a 370C water bath for 24 hours without shaking.
G6PD Quantitative Test
G6PD activity was measure for each sample after treatment with CuCl using quantitative test from Trinity Biotech (Trinity Biotech, Ireland) to determine the enzyme activity. The assay measured the rate of reduction of NADP+ to NADPH spectrophotometrically at 340 nm using ultraviolet spectrophotometer (Shimadzu UV-VIS 800, Japan). The rate of NADPH formation is proportional to G6PD activity. Hemoglobin was measured using HemoCue (Hemocue® Hb 301 System, Sweden). G6PD activity is calculated in relation to hemoglobin level in U/g Hb according to the manufacturer’s manual.
H2O2 Oxidative Stress Treatment
Prior to incubation with H2O2, the RBC previously treated with CuCl, as well as the RBC from heterozygous G6PD subjects and normal controls, were washed twice with phosphate buffer pH 8.0. Plasma and buffy coat were discarded during the washing steps. After the first wash, complete blood count was performed for all samples to calculate the amount of RBC in each sample. Final volume of 200 µL of RBC suspension from each sample (each sample contained 108 RBC/200 µL suspension) was incubated with H2O2 (30%, Merck, Germany) to final concentration of 300 mM at 370C for 2 hours, to induce oxidative stress in these cells. These would then be measured for glutathione and TBARS assays. Meanwhile, H2O2-untreated RBC from G6PD heterozygous subjects and G6PD normal controls were also incubated at 370C for 2 hours alongside treated RBC samples.
Malondialdehyde Measurement Assay
Lipid peroxidation is an indicator of cellular damage caused by oxidative stress and MDA is a good marker of such lipid peroxidation. To measure MDA, OxiSelect TBARS Assay (Cell Biolabs, USA, Cat.# STA-330) was used where thiobarbituric acid reactive substances (TBARS) is a rapid and direct quantitative measurement of MDA in biological samples. MDA forms a 1:2 adduct with TBA and can be measured colorimetrically. The procedure followed the manufacturer’s protocol where an additional step was added to get rid of the interfering hemoglobin and its derivatives by adding n-Butanol. All samples and MDA standards were transferred to cuvettes including a blank control and read at 532 nm absorbance. All samples and standards were done in duplicates. All MDA standards were plotted into a standard curve and MDA concentration for every sample was measured in pmol/µg total protein where the protein was measured according to Lowry protein quantitation method .
Reduced GSH, as the major anti-oxidant in cells, especially in RBC, is normally present around 90-95% of total glutathione in the cells. Intracellular level of GSH is used as an indicator of the overall redox state of the cell. In normal cells, increased level of GSH indicates that there is an oxidative pressure within the cell. The Glutathione Assay kit (Sigma-Aldrich, Germany, Cat.# CS0260) provides the means to measure level of total glutathione (GSSG and GSH) in a biological sample. The kit uses a kinetic assay where the catalytic amounts of GSH cause a continuous reduction of 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) to TNB and the generating GSSG is recycled by glutathione reductase and NADPH. The rate of reaction is proportional to the concentration of GSH up to 2 µM where the yellow product, TNB is measured spectrophotometrically at 405 nm and level of GSH is measured in nmol/mg of protein in the sample as stated in the manufacturer’s protocol with slight modification, wherein a 405 nm wavelength was used instead of 412 nm.
G6PD Cytofluorometric Assay
Prior to treatment with H2O2, percentage of G6PD deficient cells were analyzed cytochemically to see whether they coincided with G6PD activities. The assay involved nitrite which oxidized all oxyhemoglobin to methemoglobin. The methemoglobin was reduced back to oxyhemoglobin by glucose as substrate and nile blue as redox catalyst. This reaction depended on NADPH and G6PD normal RBC would have oxyhemoglobin converted faster than G6PD deficient RBC. The addition of cyanide reacted with methemoglobin and produced cyan-methemoglobin, while oxyhemoglobin remained inactive. Afterward, H2O2 introduction generated fluorescence only in oxyhemoglobin thus distinguishing G6PD normal from G6PD deficient RBC. Fifty µL of packed RBC was mixed with 90 µL of phosphate buffer pH 8.0 for model cells and only 10 µL of packed RBC and 90 µL of phosphate buffer pH 8.0 for G6PD heterozygous subjects prior to applying the protocol from Shah et al. where the assay assessed G6PD activity at the level of individual RBC . The samples were analyzed in BD Acuri C6+ cytometer (BD Biosciences, USA), using setting system 10,000 total events with FSC-H > 30,000 collected. Samples were excited using an Arion laser (488 nm) and fluorescence emission was measured in the FL1 channel (533 ± 30 nm).
DNA Extraction and Genotyping
DNA of G6PD heterozygous subjects were extracted using Wizard Genomic Purification DNA Kit (Promega, USA, Cat.# A1120) with a small modification, whereas DNA isolated from 300 µL of whole blood was diluted in 50 µL of DNA Rehydration Solution. DNA was PCR amplified and cut at various restriction enzyme sites to detect for various known G6PD variants that are common in Indonesia [25,26]. The fragments of PCR/RFLP product were then analyzed using agarose gel electrophoresis.
To measure the correlation between CuCl concentration and G6PD activities, we used Spearman test. Linear regression test was used to analyze the association between G6PD activities with proportion of normal RBC in RBC models. Wilcoxon test was used to test the significance between RBC models in MDA and G6PD activities, as well as MDA and GSH levels in heterozygous G6PD subjects. Meanwhile, Kruskal-Wallis test was performed to analyze the differences between GSH and G6PD activity in RBC models. The data analysis was completed using RStudio version R i386 3.3.1. (www.rstudio.com).