Glucose-6-phosphate Dehydrogenase Enzyme Activity Research Articles

The Reference Levels of Glucose-6-Phosphate Dehydrogenase Enzyme Activity for Infants in Taiwan

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzyme deficiency in Taiwan with incidence of 1.8-2.1%. Nationwide newborn screen has been implemented since 1987. To assess the lacking reference levels of G6PD enzyme activity between 7 to 90 days old infants, infants visited National Taiwan University Hospital Hsin-chu Branch from January 1, 2016 to June 30, 2017 were enrolled. G6PD mutation genotyping were carried out by using the Agena MassARRAY system (Agena, San Diego, CA), containing 21 mutations: c.95A>G, c.371A>G, c.392G>T, c.487G>A, c.493A>G, c.517T>C, c.519C>G, c.519C>T, c.592C>T, c.835A>T, c.835A>G, c.871G>A, c.1004C>A, c.1004C>T, c.1024C>T, c.1311C>T, c.1360C>T, c.1376G>T, c.1387C>T, c.1388G>A and c.1365-13T>C.Total 448 cases were studied. Assuming the newborn screen and gene mutation reports are compatible, 24 cases with mismatch reports were excluded. Finally, 424 cases were enrolled, 60.6% (n = 257) were males. The mean age was 37.9 ± 25.0 days old, and mean gestational age was 38 weeks and 6 days. Total 236 cases passed newborn screen and no gene mutation was found. Eighteen (20.5%) samples were collected while infants presented jaundice and the levels were significantly higher than those without jaundice (16.3 ± 4.9 vs. 13.6 ± 3.7 U/gHb, p= 0.014). Therefore, the cases with jaundice were excluded. The rest 218 cases were analyzed and the reference levels of G6PD enzyme activity were 13.6 ± 3.7 U/gHb (95% confidence interval 7.3-25.0 U/gHb), 12.2 ± 3.8 U/gHb (95% confidence interval 8.0-25.2 U/gHb) and 11.4 ± 3.0 U/gHb (95% confidence interval 7.5-19.3 U/gHb) for infants of age 7-30 days old (n = 70), 31-60 days old (n = 65), and 61-90 days old (n = 83), respectively (Table 1). There was no difference between males and females.Total 188 cases failed newborn screen and gene mutation was detected simultaneously. All cases harbored only 1 mutation. The 3 most common mutations, including c.1376G>T (p.R459L), c.1388G>A (p.R463H) and c.95A>G (p.H32R) represented 80.8% (n = 152) in our study. The G6PD level of deficiency cases was significantly lower than that of carrier (Table 2). In G6PD deficiency cases, the G6PD enzyme activity level of cases with c.1388G>A mutation presented the lowest level (p= 0.01). Otherwise, in G6PD carrier cases, the level of cases with c.95A>G mutation tended to present the lowest level (p= 0.056).In conclusion, the levels of G6PD enzyme activity were inversely corrected to age. From our results, we set up the reference levels of G6PD enzyme activity in infants of age between 7 to 90 days old, making diagnosis of G6PD deficiency more precisely in Taiwan. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Prevalence of Congenital Hypothyroidism and G6PD Deficiency in Newborns in a Tertiary Care Hospital of Central India

Objectives: The fundamental prerequisite of a screening program is the accessible epidemiological data regarding disease burden and awareness regarding the disorder. This is the foremost study in this state to estimate the burden of congenital hypothyroidism (CH) and glucose-6-phosphate dehydrogenase (G6PD) enzyme deficiency in infants born or attending a tertiary care hospital, pursuing skill development for the technical staffs, and creating awareness for parents. Study Design: A cross-sectional study for 21 months was conducted in the Department of Biochemistry in collaboration with the Department of Pediatrics and Department of Obstetrics and Gynecology. Methods: The screening was conducted on 1282 babies, 48 hours subsequent to birth and up to 8 weeks of age. The dried blood spot specimens collected were analyzed for thyroid stimulating hormone (TSH) level and G6PD enzyme activity by fluoroimmunoassay method. Prevalence was calculated by percentage. Results: The proficiency of the program for all live birth babies delivered in the institute was 94.6%. In consequence to improper sampling, 4.1% samples could not be analyzed. The screening tests reported elevated TSH levels in 1.6% of total population and G6PD enzyme deficiency in 2.6% of all babies enrolled. Confirmatory tests revealed that 4 (3.1/1000) babies were declared positive for CH and 8 (6.2/1000) were announced to be G6PD deficient. Conclusion: The new prevalence observed is one of the highest prevalence of CH published in India that urges mandatory and immediate need for development of newborn screening program in the state, and improvisation and upgradation of regional diagnostic center with facilities for primary- and secondary-level testing.

Management of glucose 6-phosphate dehydrogenase (G6PD) deficient patients undergoing open-heart surgery

Background: There are scarce studies on the management of glucose 6-phosphate dehydrogenase (G6PD) deficient patients during cardiac surgery. The purposes of this retrospective study were to present and evaluate our experience with G6PD deficient patients who underwent cardiac surgery with cardiopulmonary bypass (CPB).
 Methods: We included 20 patients with G6PD deficiency who had cardiac surgeries from 2015 to 2019. We used free radical scavenging strategy and careful perioperative management. The patients were compared to a control group of 20 patients with normal G6PD enzyme activity who underwent the same type of operations in the same period.
 Results: Males represented 80% of G6PD deficient patients. There were significant elevations in preoperative total bilirubin (1.03±0.33 vs. 0.57±0.11 mg/dl, p< 0.001) and reticulocytes (1.87±0.62 vs. 0.54±0.18%) in G6PD deficient patients. Valve surgery was done for 60% of G6PD deficient patients. There were no significant differences between both groups regarding the type of surgery, aortic cross-clamp, CPB, and total operative time. G6PD deficient patients had significantly lower postoperative hemoglobin levels (9.44±0.94 vs. 10.0±0.59 g/dl, p= 0.04) and significantly higher postoperative total bilirubin (1.51±0.51 vs. 0.98±0.45 mg/dl; p=0.002) and reticulocytes (1.85±0.51 vs. 0.57±0.13%; p< 0.001). There was no significant difference regarding postoperative urea and creatinine levels. Ventilation time (10.3±2.7 vs. 8.2±1.9 hours; p=0.01), ICU stay (3.1±0.87 vs. 2.3±0.71 days; p=0.004), and hospital stay (3.1±0.87 vs. 6.0±1.02 days; p<0.001) significantly increased in G6PD deficient patients. The mortality rate was 5% (one patient) in G6PD deficient patients.
 Conclusion: Despite the management strategy, G6PD deficient patients undergoing cardiac surgery are more liable to hemolysis and hypoxia with more need for blood transfusion and longer ventilation time, ICU, and hospital stays when compared to patients with normal G6PD enzyme activity. Further research to improve the outcomes in G6PD deficient patients is required.

The integrity and stability of specimens under different storage conditions for glucose-6-phosphate dehydrogenase deficiency screening using WST-8

Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37 °C, room temperature, 4 °C and -20 °C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20 °C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20 °C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine.

Prevalence and molecular characterization of glucose-6-phosphate dehydrogenase deficiency in the Lue ethnic group of northern Thailand

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzyme disorders. Prevalence and variant distribution of G6PD deficiency can vary in different regions and among differing ethnic groups. To reveal the G6PD frequency and molecular characterization among the Lue ethnic group of northern Thailand, blood samples of 296 unrelated individuals collecting from 6 Lue villages were analyzed. The observed G6PD enzyme activity ranged from 0.11 to 20.60 U/g Hb. Overall, 13.51% (40/296) of the individuals were identified as having G6PD deficiency status. The prevalence in males was 14.28% (20/140), while that of females was 12.82% (20/156). The most common G6PD variants in the Lue were the Kaiping 1388G > A (5.40%) and Canton 1376G > T (6.42%) types. Observed prevalence and variant types of the G6PD gene in the Lue population are similar to that of the Tai-Kadai speaking ethnic groups in southern China, which is consistent with their historically close line of ancestry. However, the founder effect that occurred during the Lue’s transboundary migration from China to Thailand showed its impact upon different patterns of G6PD distribution among each Lue village.

Integrative identification of the pathogenic role of a novel G6PD missense mutation c.697G>C.

BackgroundGlucose-6-phosphate dehydrogenase (G6PD) deficiency is a hereditary disease caused by pathogenic mutations of G6PD. While most of the pathogenic variants of G6PD have been annotated, hemolysis of unknown etiology but analogous to that in G6PD deficiency persists, implying the existence of undocumented pathogenic variants. In our previous study, we reported four novel G6PD variants in China, for which the pathogenicity remains to be verified.MethodsThe variants were verified by exogenous expression in HEK-293 cells, and their functions were predicted by PolyPhen-2 and SIFT. The CRISPR/Cas9 system was exploited to edit the G6PD c.697G>C variant in HEK-293 cells and K562 cells. The expression of G6PD was detected by quantitative PCR (qPCR) and western blotting. The cell growth capacity was detected by the CCK-8 assay and crystal violet staining. The G6PD enzyme activity was reflected by the G6P/6PG ratio test. The apoptosis of cells was detected by Annexin V-APC/7-AAD staining. The secondary and crystallographic structures were denoted according to the literature and PyMOL software. The G6PD protein was purified from lysis of transformed Escherichia coli (E. coli) cell with Ni-charged Resin Column. The enzymatic activity was detected at different temperatures.ResultsThe G6PD activity of exogenous G6PD c.697G>C in HEK-293 cells was significantly lower than that of wild type (WT) G6PD, a finding that was consistent with the observation in clinical samples. The functional predictions conducted by different algorithms indicated the damage role of the G6PD c.697G>C variant in its enzymatic activity. We recapitulated the G6PD c.697G>C variant both in HEK-293 cells and K562 cells by adapting the CRISPR/Cas9 strategy. Using distinct cell lines expressing the G6PD c.697G>C variant endogenously, we confirmed the deteriorative role of the G6PD c.697G>C variant in its enzymatic activity. Regarding the secondary and crystallographic structure, we found a mutated amino acid approaching the structural NADP+ binding site. Finally, we demonstrated the c.697G>C variant compromised the thermal stability of G6PD protein.ConclusionsOur data delineated the pathogenic role of G6PD c.697G>C variant for G6PD deficiency, implying the wide usage of CRISPR/Cas9 for genetic disease research.

Assessment of serum aflatoxin B1 levels in neonatal jaundice with glucose-6-phosphate dehydrogenase deficiency: a preliminary study.

Aflatoxin (AF) contamination of food products is still a major health issue globally. Prior studies suggest that exposure to AFs during pregnancy has harmful fetal outcomes. This preliminary study was designed to assess serum AFB1 levels in neonatal jaundice (NNJ) secondary to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Twenty-four full-term neonates with hemolytic jaundice secondary to G6PD deficiency were enrolled in the study. Erythrocyte G6PD status was assessed colorimetrically, and serum aflatoxin B1 (AFB1) concentrations were measured by high-performance liquid chromatography. The results revealed that AFB1 was detected in 58% (14/24) of the studied newborns while detected in 75% (18/24) of their mothers. AFB1 positive cases had a highly significantly lower birthweight and G6PD activity (P=0.001, each). Birthweight (r=-0.574, P=0.032) and G6PD activity (r=-0.585, P=0.028) negatively correlated with serum AFB1 levels while serum alanine aminotransferase activity positively correlated with serum AFB1 levels (r=0.536, P=0.048). Maternal AFB1 exposure is associated with adverse birth outcomes as verified by the low birthweight and the evident decline in the activity of G6PD enzyme with the resultant hemolytic NNJ.

Newborn Screening-A Bolstering Step towards Quality Health in Neonates

Introduction: Newborn Screening (NBS) is considered the need of the hour for quality health in neonates. It is also important to understand that the rising trend of prevalence of inherited metabolic disorders and the various maternal factors that might influence genetic changes in the foetus in- utero affecting the neonatal outcome. Aim: The primary objective of the study was to determine frequency of Congenital Hypothyroidism (CH) and Glucose- 6-Phosphate Dehydrogenase (G6PD) deficiency in a most approachable tertiary care hospital. The secondary objectives were to find out impact of maternal factors on the frequency of the disease and the impact of the disorder on the neonate’s health. Materials and Methods: The cross-sectional study was conducted on 1282 neonates of 48 hours upto eight weeks of age. The Dried Blood Spot (DBS) specimens collected were analysed for Thyroid Stimulating Hormone (TSH) level and G6PD enzyme activity. Details of neonatal characteristics and antenatal history were documented. Prevalence of CH and G6PD deficiency was calculated and maternal and neonatal variables were analysed for association using Statistical Package for the Social Sciences (SPSS) version 20. Results: The prevalence of CH and G6PD were respectively 3.3/1000 and 6.6/1000 making the overall prevalence of metabolic disorders as 9.8/1000. A 27.3% had Low Birth Weight (LBW) and 62% had low Ponderal Index (PI). The odds for raised TSH was 6.62 times in sick neonates. The probability for high TSH in LBW babies was more by 94% and in female neonates by 18%. The neonates with higher TSH values depicted significant association with maternal age (p=0.016), gestational age (p=0.019) and maternal anaemia (p<0.001). Babies born by caesarean section showed twice the chances for screening positive for TSH. Conclusion: The high prevalence estimated in this study and association with maternal factors urges new queries and recommends an obligatory need for NBS program in this region.

Prevalence and spectrum of mutations causing G6PD deficiency in Indian populations

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human erythroenzymopathy affecting around 10% of the world population. India is endemic for malaria and antimalarial drugs are known to induce haemolysis in G6PD deficient individuals. Here we report the prevalence as well as the molecular diversity of G6PD deficiency in geographical regions of India. A total of 20,896 individuals (11,838 males and 9058 females) were screened by DPIP dye decolorisation method followed by quantitation of G6PD enzyme activity on the suspected samples. Molecular analysis was undertaken in a total of 350 G6PD deficient individuals by PCR-RFLP and DNA sequencing. A structural characteristic of the novel variant was deduced by using DynaMut web-server. The prevalence rate of G6PD deficiency varied between 0.8 and 6.3% with an overall prevalence of 1.9%. A total of twelve mutations were identified. Of the total deleterious alleles detected G6PD Orissa (56.5%) was found to be the most predominant variant followed by G6PD Mediterranean (23.6%). G6PD Mediterranean, G6PD Kaiping and G6PD Mahidol were found to be severely deficient variant and 14.1% of them showed undetectable activity. A novel mutation c.544C➔G (R182G) in exon 6 was identified in one tribal male where substitution of arginine by glycine, likely causes the alteration in the alpha helix leading to disruption of secondary structure of the protein. There are large differences in the distribution of G6PD causal variants between Indian states, and this may have implications for the treatment in the malaria endemic areas.

Identification of the Cytosolic Glucose-6-Phosphate Dehydrogenase Gene from Strawberry Involved in Cold Stress Response.

Glucose-6-phosphate dehydrogenase (G6PDH) plays an important role in plant stress responses. Here, five FaG6PDH sequences were obtained in strawberry, designated as FaG6PDH-CY, FaG6PDH-P1, FaG6PDH-P1.1, FaG6PDH-P2 and FaG6PDH-P0, which were divided into cytosolic (CY) and plastidic (P) isoforms based on the bioinformatic analysis. The respective FaG6PDH genes had distinct expression patterns in all tissues and at different stages of fruit development. Notably, FaG6PDH-CY was the most highly expressed gene among five FaG6PDH members, indicating it encoded the major G6PDH isoform throughout the plant. FaG6PDH positively regulated cold tolerance in strawberry. Inhibition of its activity gave rise to greater cold-induced injury in plant. The FaG6PDH-CY transcript had a significant increase under cold stress, similar to the G6PDH enzyme activity, suggesting a principal participant in response to cold stress. Further study showed that the low-temperature responsiveness (LTR) element in FaG6PDH-CY promoter can promote the gene expression when plant encountered cold stimuli. Besides, FaG6PDH-CY was involved in regulating cold-induced activation of antioxidant enzyme genes (FaSOD, FaCAT, FaAPX and FaGR) and RBOH-dependent ROS generation. The elevated FaG6PDH-CY enhanced ROS-scavenging capability of antioxidant enzymes to suppress ROS excessive accumulation and relieved the oxidative damage, eventually improving the strawberry resistance to cold stress.

A pilot study of expanded newborn screening for 573 genes related to severe inherited disorders in China: results from 1,127 newborns.

BackgroundNewborn screening (NBS) in China is mainly aimed at detecting biochemical levels of metabolites in the blood, which may generate false-positive/negative results. Current biochemical NBS includes tandem mass spectrometry (MS/MS) screening for metabolites as well as phenylalanine (Phe), thyroid-stimulating hormone (TSH), 17-α-hydroxyprogesterone (17-OHP), and glucose-6-phosphate dehydrogenase (G6PD) test. This study intended to explore whether next-generation sequencing (NGS) for dried blood spots combining with biochemical screening could improve the current screening efficiency and to investigate the carrier frequencies of mutations in causative genes related to amino acid metabolism, organic acid metabolism, and fatty acid oxidation in this cohort.MethodsWe designed a panel of 573 genes related to severe inherited disorders and performed NGS in 1,127 individuals who had undergone biochemical NBS. The NGS screening results of neonates were used to compare with the biochemical results.ResultsNGS screening results revealed that all the four newborns with abnormal G6PD values carried hemizygous G6PD mutations, which were consistent with the decreased G6PD enzymatic activity. The NGS results revealed an individual with compound heterozygous mutations of SLC22A5, who was biochemically negative in 2016. The MS/MS screening results in 2019 showed free carnitine deficiency, which was consistent with the genetic findings. The top five genes with the highest carrier frequencies of mutations in these newborns were PAH (1:56, 1.79%), ETFDH (1:81, 1.23%), MMACHC (1:87, 1.15%), SLC25A13 (1:102, 0.98%), and GCDH (1:125, 0.80%).ConclusionsOur study highlighted that combining NGS screening with biochemical screening could improve the current NBS efficiency. This is the first study to investigate carrier frequencies of mutations in 77 genes causing inherited metabolic diseases (IMDs) in China.

Frequency of Human Paraoxonase-1 Q192R Polymorphism and Measurement of Oxidative Stress Parameters in Infants with G6PD Deficiency

Background and Aims: This study aimed to investigate the frequency of Q192R polymorphism and oxidative stress markers in infants with glucose-6phosphate dehydrogenase (G6PD) deficiency. Materials and Methods: This is a case-control study in which 60 male infants (2-4 months old) with G6PD deficiency along with 60 age- and sexmatched healthy neonates were included. The diagnosis of G6PD deficiency was made by Beutler test by which the G6PD enzyme activity is measured by the fluorescent spot test. The blood samples were taken from all infants, and the sera were isolated for the evaluation of Paraoxonase-1 (PON1) and malondialdehyde (MDA) using the spectrophotometric method. Restriction fragment length polymorphism was applied for determination of Q192R polymorphism (rs 662). Results: The frequencies of QQ, QR, and RR genotypes were 55%, 39%, and 6%, respectively in infants with G6PD deficiency while the above genotype frequencies were 45%, 49%, and 6%, respectively in healthy neonates. The frequency of R and T alleles failed to show any significant difference when G6PD deficient infants and healthy neonates were compared. The results indicated PON1 activity and MDA levels being significantly (p<0.05) higher in neonates with G6PD deficiency compared with their healthy counterparts. Conclusion: Contrary to previous studies, it was indicated that the presence of RQ and RR genotypes at Q192R position is associated with decreased activity of PON1 and increased oxidative stress. In this study, no significant differences were found in the genotype and allele frequency of PON1 Q192R polymorphism between the case and control groups. Also, this frequency was not consistent with the results obtained from oxidative stress conditions.

Genome-Wide Analysis of the Glucose-6-Phosphate Dehydrogenase Family in Soybean and Functional Identification of GmG6PDH2 Involvement in Salt Stress.

Glucose-6-phosphate dehydrogenase (G6PDH) is known as a critical enzyme responsible for nicotinamide adenine dinucleotide phosphate (NADPH) generation in the pentose phosphate pathway (PPP), and has an essential function in modulating redox homeostasis and stress responsiveness. In the present work, we characterized the nine members of the G6PDH gene family in soybean. Phylogenic analysis and transit peptide prediction showed that these soybean G6PDHs are divided into plastidic (P) and cytosolic (Cy) isoforms. The subcellular locations of five GmG6PDHs were further verified by confocal microscopy in Arabidopsis mesophyll protoplasts. The respective GmG6PDH genes had distinct expression patterns in various soybean tissues and at different times during seed development. Among them, the Cy-G6PDHs were strongly expressed in roots, developing seeds and nodules, while the transcripts of P-G6PDHs were mainly detected in green tissues. In addition, the activities and transcripts of GmG6PDHs were dramatically stimulated by different stress treatments, including salt, osmotic and alkali. Notably, the expression levels of a cytosolic isoform (GmG6PDH2) were extraordinarily high under salt stress and correlated well with the G6PDH enzyme activities, possibly implying a crucial factor for soybean responses to salinity. Enzymatic assay of recombinant GmG6PDH2 proteins expressed in Escherichia coli showed that the enzyme encoded by GmG6PDH2 had functional NADP+-dependent G6PDH activity. Further analysis indicated overexpression of GmG6PDH2 gene could significantly enhance the resistance of transgenic soybean to salt stress by coordinating with the redox states of ascorbic acid and glutathione pool to suppress reactive oxygen species generation. Together, these results indicate that GmG6PDH2 might be the major isoform for NADPH production in PPP, which is involved in the modulation of cellular AsA-GSH cycle to prevent the oxidative damage induced by high salinity.

A cross-sectional study on parental awareness for newborn screening and assessment of the burden of congenital hypothyroidism and glucose 6-phosphate dehydrogenase deficiency

Introduction: Newborn Screening (NBS) has been one of the most successful health programs and of most paramount importance worldwide but not so in India. Due to the lack of awareness and paucity of laboratory facilities, the disease burden in our population has not yet been established. Aim and Objective: The aim and objective of the study was to assess the burden of Congenital Hypothyroidism (CH) and Glucose 6-Phosphate Dehydrogenase (G6PD) deficiency in this area by NBS tests and to assess the impact of awareness sessions on the response rate of parents for NBS. Material and Methods: The screening was conducted in 474 babies of age 48 h up to 8 weeks. The dried blood spots collected were subjected to the following analytical protocol. Thyroid-stimulating hormone (TSH) level and G6PD enzyme activity were analyzed by immunofluorescence method-based neonatal kits. All babies with a positive screening test for G6PD deficiency and CH were asked for venous confirmatory testing after 7 days. Results: The efficiency of the program for all live birth babies delivered in the institute was 92% (n = 410/445). It was 82% in the first phase of the study period and 98.5% in the second phase. Repeated training of nursing professional reduced the sampling errors from 14.7% in the first phase to 6.1% in the second phase. A total of 11 samples reflected high TSH values, of which one baby confirmed for CH. Of the 24 babies who screened positive for G6PD, four were confirmed for the same. The prevalence for CH and G6PD deficiency was, respectively, 1 in 462 (2/1000) and 4 in 462 (8.7/1000). Conclusion: Development of expansion of NBS program in the state should be made mandatory for all newborns. The recommendations include awareness among parents, during antenatal and postnatal period and also to health professionals and provision for laboratory facilities for NBS testing at low cost.

Study of G6pd Deficit (Glucose-6-phosphate Deshydrogenase) in Blood Donors in Brazzaville

Glucose-6-Phosphate dehydrogenase (G6PD) is an enzyme present in the cytoplasm of all cells in the body and protects red blood cells from certain attacks. The anomaly is genetically inherited in a recessive manner, linked to the X chromosome. Currently, an estimated 420 million individuals lack G6PD worldwide and its incidence varies between 1-25% in some African populations. This was an analytical cross-sectional study with prospective data collection that took place from May to November 2018, i.e. six (6) months. Our study included 108 blood donors, 88 men and 20 women. The samples were stored at 2-8 ° C for 24 hours while awaiting handling. The determination of the enzymatic activity of G6PD was carried out by the spectrophotometric method using a commercially available kit. 02/108 subjects studied (ie a prevalence of 1.85%) were deficient in our study. These two deficit were mostly young and between 18-25 years old only male. They were originally from the plateau and observed in the blood transfusion station (PTS) of the Talangai referral hospital. The mean hemoglobin value in normal subjects was 12.53 g / dl in men and 11.3 g / dl in women. In contrast, the average hemoglobin value was 10.45 g / dl in the deficient patients. The mean value of G6PD enzyme activity in normal subjects was 11.7 IU / g hemoglobin in men, 11.54 IU / g hemoglobin in women and in deficient subjects was 2.83 IU / g hemoglobin. As the results of our study show, G6PD deficiency affects hemoglobin levels.

Evidence of positively selected G6PD A- allele reduces risk of Plasmodium falciparum infection in African population on Bioko Island.

BackgroundGlucose‐6‐phosphate dehydrogenase (G6PD) is an essential enzyme that protects red blood cells from oxidative damage. Although G6PD‐deficient alleles appear to confer a protective effect of malaria, the link with clinical protection against Plasmodium infection is conflicting.MethodsA case–control study was conducted on Bioko Island, Equatorial Guinea and further genotyping analysis used to detect natural selection of the G6PD A‐ allele.ResultsOur results showed G6PD A‐ allele could significantly reduce the risk of Plasmodium falciparum infection in male individuals (adjusted odds ratio [AOR], 0.43; 95% confidence interval [CI], 0.20–0.93; p < .05) and homozygous female individuals (AOR, 0.11; 95% CI, 0.01–0.84; p < .05). Additionally, the parasite densities were significantly different in the individuals with different G6PD A‐ alleles and individual levels of G6PD enzyme activity. The pattern of linkage disequilibrium and results of the long‐range haplotype test revealed a strong selective signature in the region encompassing the G6PD A‐ allele over the past 6,250 years. The network of inferred haplotypes suggested a single origin of the G6PD A‐ allele in Africans.ConclusionOur findings demonstrate that glucose‐6‐phosphate dehydrogenase (G6PD) A‐ allele could reduce the risk of P. falciparum infection in the African population and indicate that malaria has a recent positive selection on G6PD A‐ allele.

Effects of butylparaben on antioxidant enzyme activities and histopathological changes in rat tissues.

Butyl p-hydroxybenzoic acid, also known as butylparaben (BP), is one of the most common parabens absorbed by the skin and gastrointestinal tract and metabolised in the liver and kidney. Recent in vivo and in vitro studies have raised concern that BP causes reproductive, development, and teratogenic toxicity. However, BP-induced oxidative stress and its relation to tissue damage has not been widely investigated before. Therefore, we aimed to investigate the effects of butyl 4-hydroxybenzoate on enzyme activities related to the pentose phosphate pathway and on glutathione-dependent enzymes such as glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6-PGD), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in kidney, liver, brain, and testis tissues. Male rats were randomly divided into four groups to orally receive corn oil (control) or 200, 400, or 800 mg/kg/day of BP for 14 days. Then we measured G6PD, GR, GST, 6-PGD, and GPx enzyme activities in these tissues and studied histopathological changes. BP treatment caused imbalance in antioxidant enzyme activities and tissue damage in the liver, kidney, brain, and testis. These findings are the first to show the degenerative role of BP on the cellular level. The observed impairment of equivalent homeostasis and antioxidant defence points to oxidative stress as a mechanism behind tissue damage caused by BP.

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (&amp;gt; 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents &amp; Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents &amp; Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

STUDY OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE ENZYME AND ISOPROSTANE ON PREECLAMPSIA WITH NIFEDIPINE, METHYLDOPA, AND MAGNESIUM SULFATE THERAPY

Objective: The objective of this research is to measure erythrocyte glucose-6-phosphate dehydrogenase (G6PD) enzyme activity and isoprostane andto correlate enzyme activity of G6PD with proteinuria and isoprostane in pregnant with proteinuria after the administration of nifedipine, methyldopa,and magnesium sulfate.Methods: This cross-sectional study was held in Soewandi Hospital, Surabaya, East Java, Indonesia. This study used total sampling as much as 30pregnant women with proteinuria who got nifedipine, methyldopa, and magnesium sulfate administration, age ranged from 17 to 48 years during theirthird trimester (&gt;20 weeks). G6PD enzyme activity was measured from plasma by spectrophotometric method; plasma isoprostane was measuredby competitive-ELISA method; and proteinuria urine spot was analyzed by urine dipstick from standardized laboratory of the hospital. Statisticalanalysis used in this study was Spearman’s correlation coefficient.Results: In this research, the effect of proteinuria +1 (OR=0.056) is lower than proteinuria +3 level on the presence of high G6PD enzyme activity,and proteinuria +2 (OR=0.933) is lower than proteinuria +3 level on the presence of high G6PD enzyme activity in pregnant women with proteinuria.G6PD enzyme was positively correlated (p=0.08) with proteinuria, and the connection was statistically significant. There was no significant statisticcorrelation between G6PD enzyme activity and isoprostane concentration (p=0.797).Conclusion: This study found correlations between the enzyme activity of G6PD and proteinuria as the marker of renal damage in pre-eclampsia (PE)with the administration of nifedipine, methyldopa, and magnesium sulfate. However, it had no correlation with isoprostane as the marker of oxidativestress. This study suggests that there should be a concern about understanding the pathophysiology of proteinuria for possibility of drug target forindividuals with PE.

The Synthesis of Thiosemicarbazone-Based Aza-Ylides as Inhibitors of Rat Erythrocyte Glucose 6-Phosphate Dehydrogenase Enzyme

Glucose 6-phosphate dehydrogenase (G6PD) enzyme plays an important role in various biochemical processes such as synthesis of cholesterol, fatty acids, sphingosine, steroid hormones, NADPH, some amino acids and ribose 5-phosphate. In this study, thiosemicarbazone-based aza-ylide (TSCAs) derivatives 3a-3e, which form the main framework of many drugs such as thioacetazone, ambazone, and perchlozone, were synthesized with a green approach and in vitro inhibitor or activator effects on G6PD enzyme activity was investigated. As a result of studies, TSCAs derivatives 3a-3d inhibited the G6PD enzyme activity with IC50 in the range of 40.77 μM to 58.0 μM for G6PD.