Donor glucose-6-phosphate dehydrogenase deficiency decreases blood quality for transfusion

Abstract

BACKGROUNDGlucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared with G6PD-normal RBCs.METHODSMale volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated 1 RBC unit. After 42 days of refrigerated storage, autologous 51-chromium 24-hour posttransfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed.RESULTSThe mean 24-hour PTR for G6PD-deficient subjects was 78.5% ± 8.4% (mean ± SD), which was significantly lower than that for G6PD-normal RBCs (85.3% ± 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and 3 G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways.CONCLUSIONBased on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs.TRIAL REGISTRATIONClinicalTrials.gov NCT04081272.FUNDINGThe Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation, and National Heart, Lung, and Blood Institute grants R01HL14644 and R01HL148151.

Keywords: Hematology; Monogenic diseases.

Conflict of interest statement

Conflict of interest: AD and TN are founders of Omix Technologies, Inc. and Altis Biosciences, LLC. SLS is a consultant for Tioma, Inc. AD and SLS are members of the Scientific Advisory Board of Hemanext, Inc.

Figures

Figure 1. CONSORT diagram.

Number of subjects who were screened or excluded, dropped out, or completed the study.

Figure 2. G6PD deficiency reduces 24-hour PTR.

(A) The 51-chromium PTRs for G6PD-normal (n = 27) and G6PD-deficient (n = 10) subjects are shown. The dotted gray line denotes the FDA criterion for acceptability (at outdate, on average >75% of transfused RBCs should still be circulating for 24 hours). (B) The 51-chromium PTRs for G6PD-normal (n = 6) and G6PD-deficient (n = 7) subjects of African origin and (C) non–African origin are shown (n = 21 and n = 3 for G6PD-normal and G6PD-deficient, respectively). Mean and SD are shown. Statistical significance calculated using an unpaired t test. *P < 0.05, ***P < 0.001. (D) Correlation between G6PD enzyme activity and PTR is shown. Results of linear regression for all subjects (N = 37) is shown with the 95% CI in dashed gray lines (R2 = 0.27; P = 0.001). Within-group results for G6PD-deficient (red) and G6PD-normal (blue) volunteers are depicted along with the results of the corresponding linear regression (red and blue lines, respectively), which are not statistically significant by Pearson correlation. The unfilled red circle represents the single subject with the Mediterranean variant.

Figure 3. Standard in vitro measures of RBC units throughout storage.

(A) Spontaneous hemolysis, (B) methemoglobin, (C) lactate, (D) pH, (E) glucose, and (F) sodium were measured in aliquots obtained from each unit at the designated storage time. G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) are represented. Mean and SD are shown. Statistical significance was calculated using 2-way repeated-measures ANOVA with Sidak’s multiple comparisons test; *P < 0.05. Thick dotted red line denotes the FDA criterion for the allowable spontaneous hemolysis rate during storage.

Figure 4. Differences in glycolysis between G6PD-deficient and G6PD-normal RBCs at baseline.

(A) Schematic of the glycolytic pathway. The levels of glycolytic intermediates were measured in RBCs collected directly from volunteers and measured before storage. (B) bisphosphoglycerate, (C) 2/3 phosphoglycerate, (D) phosphoenolpyruvate, (E) pyruvate, (F) lactate, and (G) ATP in G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) RBCs are shown. Median with interquartile range represented. Unfilled red circles represent the single subject with the Mediterranean variant. Statistical significance was assessed by the Mann Whitney U test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Defective activation of the PPP in response to methylene blue stimulation in G6PD-deficient RBCs.

(A) Fresh (unstored) or 6-week-stored RBC lysates were incubated with [1,2,3-13C3]-glucose and then treated with methylene blue to stimulate the oxidative phase of the PPP. The presence of 3-carbon moieties was measured and isotopologues compared. 13C2 3-carbon species indicate metabolism by the PPP and 13C3 species indicate metabolism by glycolysis. (B) Glycolytic pathway activity, as measured by [13C3]-glucose phosphate levels, (C) PPP pathway activity, as measured by [13C2]-lactate levels, (D) the ratio of [13C2]-lactate levels/[13C3]-glucose phosphate levels indicating the ratio of PPP/glycolysis, and (E) [13C3]-pentose phosphate demonstrating the pentose phosphate derived from glycolysis pathway activity in fresh and stored (as labeled) G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) RBCs, with either vehicle (circles) or methylene blue treatment (squares), were all determined. Medians with interquartile ranges are represented. Unfilled symbols represent the single subject with the Mediterranean variant. Statistical significance calculated using 2-way ANOVA with Sidak’s multiple comparisons test; *P < 0.05, ****P < 0.0001. (F) Schematic demonstrating that pentose phosphates are derived more from the contributions of glycolytic intermediates, rather than the oxidative phase of the PPP, in G6PD-deficient RBCs.

Figure 6. G6PD-deficient RBCs are characterized by altered glycolysis and PPP activity during storage.

(A) Schematic of glycolysis with branching to the PPP. Metabolite levels were measured in RBCs during weeks 1 to 6 of refrigerated storage. (B) PCA of the metabolites throughout storage. (C) Glucose, (D) glucose-6-phosphate, (E) 6-phosphogluconolactone, (F) fructose 1,6 bisphosphate, (G) glyceraldehyde-3-phosphate, (H) bisphosphoglycerate, (I) 2/3-phosphoglycerate, (J) pyruvate, (K) lactate, (L) pyruvate/lactate ratio, (M) ATP. Medians with interquartile ranges are shown. G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) are represented. The dotted red line with the unfilled circle represents the single subject with the Mediterranean variant. Statistical significance was calculated using 2-way repeated-measures ANOVA with Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significance for the effect of storage time and the interaction between G6PD status and time are as labeled, or not significant if not shown. Significant time points are shown and significant differences between G6PD-deficient and G6PD-normal subjects are denoted with a bracket to the right of the curves. When the interaction term was significant, the significance of the main effects was assessed in MetaboAnalyst.

Figure 7. NADPH-dependent/generating metabolic pathways affected by G6PD deficiency in stored RBCs.

Metabolite levels were measured in RBCs during weeks 1 to 6 of refrigerated storage. Schematic of the NADPH-dependent/generating pathway shown with graphs of relevant metabolites throughout storage to the right in each figure panel. (A) Reduction of oxidized glutathione (GSSG) to reduced glutathione (GSH) and glutathionylation of cysteine residues (S-glutathionyl-Cys), (B) conversion of biliverdin to bilirubin, (C) conversion of glucose to hexose sugar alcohol, (D) conversion of 4-hydroxynonenal (HNE) to dihydroxynonene (DHN), and (E) conversion of malate to pyruvate. Medians with interquartile ranges are shown. G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) are represented. The dotted red lines with the unfilled circles represent the single subject with the Mediterranean variant. Statistical significance was calculated using 2-way repeated-measures ANOVA with Sidak’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Significance for the effect of storage time and the interaction between G6PD status and time are as labeled, or not significant if not shown. Significant time points are shown and significant differences between G6PD-deficient and G6PD-normal subjects are denoted with a bracket to the right of the curves. When the interaction term was significant, the significance of the main effects was assessed in MetaboAnalyst.

Figure 8. The nonoxidative phase of the PPP is maintained in G6PD-deficient RBCs.

(A) Schematic of the nonoxidative phase of the PPP and its connection with glycolysis in gray. Metabolite levels were measured in RBCs before storage (fresh) and during weeks 1 to 6 of refrigerated storage. The metabolites (B) ribose phosphate, (C) sedoheptulose phosphate, and (D) erythrose 4-phosphate are shown. Medians with interquartile ranges are shown. G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) are represented. The dotted red lines with unfilled red circles represent the single subject with the Mediterranean variant. Statistical significance was calculated by Mann Whitney U test (fresh) or by 2-way repeated-measures ANOVA with Sidak’s multiple comparisons test (storage); *P < 0.05; **P < 0.01, ****P < 0.0001. Significance for the effect of time and the interaction between G6PD status and time are as labeled, or not significant if not shown. Significant differences between G6PD-deficient and G6PD-normal subjects are denoted with a bracket to the right of the curves.

Figure 9. Metabolic alterations at baseline and end of storage in G6PD-deficient RBCs correlate with G6PD activity and PTR.

Circos plots of metabolic correlates to PTR and G6PD activity in the total population of subjects at baseline (A) or after 6 weeks of storage (D), in only G6PD-deficient subjects at baseline (B) or week 6 (E), or only G6PD-normal subjects at baseline (C) or week 6 (F). Metabolites are represented as nodes and connected by an edge if their linear correlation (Spearman) is higher than 0.4. Highlighted nodes represent the most significant correlates (Spearman, FDR adjusted P values < 0.05) with PTR (black spheres) or G6PD activity (blue spheres). The correlation of baseline levels of octenoyl-carnitine (G), ribose phosphate (H), and epiandrosterone (I); and week 6 levels of α-ketoglutarate (J), hypoxanthine (K), and octenoyl-carnitine (L) in the total subject population, or limited to only G6PD-deficient or G6PD-normal subjects, respectively. *P < 0.05, **P < 0.01. G6PD-normal (blue; n = 27) and G6PD-deficient (red; n = 10) subjects are represented. The unfilled red circle represents the single subject with the Mediterranean variant.