scholarly journals Determining a Critical Threshold for G6PD Activity below which RBC Response to Oxidative Stress is Poor

2020 ◽  
Author(s):  
Maria Swastika ◽  
Alida R Harahap ◽  
Lydia V Panggalo ◽  
Sri Widia A Jusman ◽  
Ari W Satyagraha
Keyword(s):  
Oxidative Stress ◽  
G6pd Deficiency ◽  
Ex Vivo ◽  
Strong Association ◽  
G6pd Activity ◽  
Critical Threshold ◽  
Main Function ◽  
Ex Vivo Model ◽  
Comparison Results ◽  
Significant Difference

Abstract Background: 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 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. Methods: RBC models were developed to analyze 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 analyzed for comparison. Results: 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, our 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). Our ex vivo model 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 G6PD Viangchan variant with 2.97 U/gHb (33% activity) and 6.58 U/gHb (74% activity). Conclusions: The GSH analysis has pointed to the 60% G6PD activity cut-off and this data is supportive of the old WHO 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.

scholarly journals Determining a Critical Threshold for G6PD Activity below which RBC Response to Oxidative Stress is Poor

2020 ◽  
Author(s):  
Maria Swastika ◽  
Alida R Harahap ◽  
Lydia V Panggalo ◽  
Sri Widia A Jusman ◽  
Ari W Satyagraha
Keyword(s):  
Oxidative Stress ◽  
G6pd Deficiency ◽  
Ex Vivo ◽  
Strong Association ◽  
G6pd Activity ◽  
World Health ◽  
Critical Threshold ◽  
Main Function ◽  
Oxidative Markers ◽  
Significant Difference

Abstract Background 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.Methods 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 twoG6PD heterozygous women with different G6PD activities were also analysed for comparison.Results 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 2.97U/gHb (33% activity) and 6.58 U/gHb (74% activity). Conclusions 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.

scholarly journals Determining Quantitative Diagnostic Test Cut-off in G6PD Heterozygous Women for Tafenoquine Therapy

2019 ◽  
Author(s):  
Maria Swastika ◽  
Alida R Harahap ◽  
Lydia V Panggalo ◽  
Sri Widia A Jusman ◽  
Ari W Satyagraha
Keyword(s):  
G6pd Deficiency ◽  
Ex Vivo ◽  
Strong Association ◽  
G6pd Activity ◽  
Main Function ◽  
Ex Vivo Model ◽  
Stress Markers ◽  
Oxidative Markers ◽  
Significant Difference ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Background 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.Methods RBC models were developed to analyze 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 G6PD heterozygous women with different G6PD activities were also analyzed for comparison.Results 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, our 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). Our ex vivo model 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 G6PD Viangchan variant with 2.97 U/g Hb (33% activity) and 6.58 U/g Hb (74% activity).Conclusions The MDA and GSH analyses have pointed to the 60% G6PD activity cut-off. This provides an evidence of possible cut-off for tafenoquine administration in G6PD heterozygous women.

scholarly journals PO 8609 PREVALENCE AND RISK FACTORS FOR GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD) DEFICIENCY IN TWO P. VIVAX MALARIA-ENDEMIC AREAS IN SUDAN

2019 ◽  
Vol 4 (Suppl 3) ◽  
pp. A62.2-A62
Author(s):  
Muzamil Mahdi Abdel Hamid ◽  
Musab Albsheer ◽  
Mohamed Muneer ◽  
Lina Altinae ◽  
Andrew A Lover
Keyword(s):  
Risk Factors ◽  
G6pd Deficiency ◽  
Demographic Data ◽  
Univariate Analysis ◽  
G6pd Activity ◽  
Radical Cure ◽  
Major Health Problem ◽  
Significant Difference ◽  
Endemic Areas ◽  
Glucose 6 Phosphate Dehydrogenase

BackgroundPlasmodium vivax malaria is a major health problem in Sudan and the parasite has become widely distributed in the recent years. The WHO recommends the use of primaquine as radical cure for liver dormant stage, the hypnozoite. However, prior its use, a test for Glucose-6-phosphate Dehydrogenase (G6PD) should be performed. The objective of the current study was to determine prevalence and risk factors for G6PD deficiency in two P. vivax malaria-endemic areas in Sudan.MethodsA cross-sectional study recruiting 557 subjects from two malaria-endemic areas in Sudan was conducted. Demographic data and blood samples were collected. G6PD activity was measured by spectrometry using SPINREACT enzymatic-UV kit.ResultsThe measured G6PD activities for both sites ranged from 0.6 to 37.7 U/g Hb, with a median value of 12.8 U/g Hb. There was a significant difference in enzyme activity by study site (p<0.001), but not by sex (p=0.91). Overall, across the two study sites, 22 (3.9%) is G6PDd (<30%). Prevalence of G6PDd (<30%) in Khartoum is 1.8% (4/230) compared to 4.8% (16/327) in New Hafla. In univariate analysis predictors of G6PDd were study site (odds ratio of G6PD activity <3.8, Khartoum relative to New Halfa=0.22 (95% CI: 0.08 to 0.66), p=0.006), and recent antibiotic use (OR=2.45 (95% CI: 1.1 to 5.5), p=0.027). In multivariate analysis, the only factor that was significant was the individual’s weight in kilograms, with an OR of 0.97 (95% CI 0.95 to 0.99, p=0.014).ConclusionG6PD deficiency is less prevalent among Sudanese population and this indicates that the use of primaquine for radical cure of P. vivax malaria is safe.

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

2020 ◽  
Author(s):  
Vahid Pourshafiei ◽  
Vahide Jamshidi ◽  
Ameneh Khodarahmi ◽  
Mahmood Vakili
Keyword(s):  
Oxidative Stress ◽  
G6pd Deficiency ◽  
Paraoxonase 1 ◽  
Pon1 Activity ◽  
Stress Markers ◽  
Genotype Frequencies ◽  
Q192r Polymorphism ◽  
Significant Difference ◽  
And Control

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.

scholarly journals Determining a critical threshold for G6PD activity below which red blood cell response to oxidative stress is poor

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Maria Swastika ◽  
Alida R. Harahap ◽  
Lydia V. Panggalo ◽  
Sri Widia A. Jusman ◽  
Ari W. Satyagraha
Keyword(s):  
Oxidative Stress ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Cell Response ◽  
G6pd Activity ◽  
Critical Threshold

scholarly journals Oxidative stress‐induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas

2020 ◽  
Vol 48 (5) ◽  
pp. 666-681 ◽  
Author(s):  
Ana M. Mueller‐Buehl ◽  
Hannah Doepper ◽  
Sven Grauthoff ◽  
Tobias Kiebler ◽  
Laura Peters ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mild Hypothermia ◽  
Ex Vivo ◽  
Retinal Damage ◽  
Ex Vivo Model

scholarly journals Comparison of Extracapsular Stabilization Techniques Using an Ultrasonically Implanted Absorbable Bone Anchor (Weldix) after Cranial Cruciate Ligament Rupture in Cats—An In Vitro Study

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1695
Author(s):  
Lydia Koch ◽  
Barbara Bockstahler ◽  
Alexander Tichy ◽  
Christian Peham ◽  
Eva Schnabl-Feichter
Keyword(s):  
Internal Rotation ◽  
Ex Vivo ◽  
Cruciate Ligament ◽  
Joint Stability ◽  
Bone Anchor ◽  
Ex Vivo Model ◽  
Cranial Cruciate Ligament ◽  
Stifle Joint ◽  
Significant Difference

Background: This study evaluated joint stability after surgical repair of cranial cruciate ligament (CrCL)-deficient stifle joints in cats using a novel absorbable polylactide bone anchor in an ex vivo model. Methods: Thirty-six hindlimbs from cats with intact (Gi group) and transected CrCLs were treated with fabellotibial suture alone (GFW group), suture combined with an absorbable polylactide bone anchor (GWD group), or suture combined with a nonabsorbable bone anchor (GFT group), positioned in a limb press with predefined joint angles (stifle joint: 120 ± 5°; hock joint: 120 ± 5°) and loaded with 10%, 20%, and 30% of body mass (BM). Predefined points were measured on lateral radiographs and with a coordinate measurement machine. Distances on radiographs (mm) were measured and angles (°) were calculated to represent the craniocaudal movement and the internal rotation of the tibia. Results: There were no differences for craniocaudal movement between Gi and GFW or GFT, but for GWD regarding angle measurement at 30% BM. For internal rotation, there was no significant difference between Gi and GFW or GWD, but for GFT. Conclusion: The used absorbable polylactide bone-anchor was able to stabilize the stifle joint regarding internal rotation and craniocaudal movement as calculated from distance measurements.

scholarly journals Assessing acute and chronic Staphylococcus aureus growth and virulence in an ex vivo model of cystic fibrosis lung infection

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Branagh Crealock-Ashurst ◽  
Freya Harrison ◽  
Esther Sweeney
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Ex Vivo ◽  
Bacterial Load ◽  
Clinical Results ◽  
Lung Model ◽  
Clinical State ◽  
Ex Vivo Model ◽  
Significant Difference ◽  
Factor Assay

Staphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable. There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation. Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.

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