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Determining a Critical Threshold for G6PD Activity below which RBC Response to Oxidative Stress is Poor
Maria Swastika
Eijkman Institute for Molecular Biology
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8811-0498
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This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Malaria Journal.
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.
Keywords
G6PD deficiency, glutathione, heterozygous women, malondialdehyde, oxidative stress, diagnostic
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CURRENT STATUS: Published
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Posted 04 Jun, 2020
Published
On 17 Jun, 2020
No community comments so far
Editorial decision: Accept
On 28 May, 2020
Editor assigned
On 27 May, 2020
Submission checks complete
On 26 May, 2020
Editor invited
On 26 May, 2020
No comments provided
Editorial decision: Minor Revision
On 11 May, 2020
Review #1 received
Received 17 Mar, 2020
Reviewer #1 agreed
On 13 Mar, 2020
Reviewers invited
Invitations sent on 12 Mar, 2020
Editor assigned
On 25 Feb, 2020
Submission checks complete
On 24 Feb, 2020
Editor invited
On 24 Feb, 2020
No comments provided
Editorial decision: Major Revision
On 27 Jan, 2020
Review #1 received
Received 26 Jan, 2020
Review #2 received
Received 25 Jan, 2020
Review #3 received
Received 25 Jan, 2020
Reviewer #3 agreed
On 22 Jan, 2020
Reviewer #2 agreed
On 19 Jan, 2020
Reviewer #1 agreed
On 10 Jan, 2020
Reviewers invited
Invitations sent on 09 Jan, 2020
Editor assigned
On 22 Dec, 2019
Submission checks complete
On 21 Dec, 2019
Editor invited
On 21 Dec, 2019
First submitted
On 19 Dec, 2019
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This preprint is under consideration at Malaria Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Determining a Critical Threshold for G6PD Activity below which RBC Response to Oxidative Stress is Poor
Maria Swastika
Eijkman Institute for Molecular Biology
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8811-0498
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 3
Posted 04 Jun, 2020
Published
On 17 Jun, 2020
No community comments so far
Editorial decision: Accept
On 28 May, 2020
Editor assigned
On 27 May, 2020
Submission checks complete
On 26 May, 2020
Editor invited
On 26 May, 2020
No comments provided
Editorial decision: Minor Revision
On 11 May, 2020
Review #1 received
Received 17 Mar, 2020
Reviewer #1 agreed
On 13 Mar, 2020
Reviewers invited
Invitations sent on 12 Mar, 2020
Editor assigned
On 25 Feb, 2020
Submission checks complete
On 24 Feb, 2020
Editor invited
On 24 Feb, 2020
No comments provided
Editorial decision: Major Revision
On 27 Jan, 2020
Review #1 received
Received 26 Jan, 2020
Review #2 received
Received 25 Jan, 2020
Review #3 received
Received 25 Jan, 2020
Reviewer #3 agreed
On 22 Jan, 2020
Reviewer #2 agreed
On 19 Jan, 2020
Reviewer #1 agreed
On 10 Jan, 2020
Reviewers invited
Invitations sent on 09 Jan, 2020
Editor assigned
On 22 Dec, 2019
Submission checks complete
On 21 Dec, 2019
Editor invited
On 21 Dec, 2019
First submitted
On 19 Dec, 2019
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Malaria Journal.
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.
Figure 1
Figure 2
Figure 3
Figure 4