Increased basal oxidation of peroxiredoxin 2 and limited peroxiredoxin recycling in glucose-6-phosphate dehydrogenase-deficient erythrocytes from newborn infants.

Cheah FC1, Peskin AV2, Wong FL1, Ithnin A3, Othman A4, Winterbourn CC5.
  • 1Department of Paediatrics and.
  • 2Centre for Free Radical Research, Department of Pathology, and.
  • 3Department of Pathology, Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia;
  • 4Department of Pathology, Universiti Sains Islam Malaysia, Kuala Lumpur, Malaysia.
  • 5Centre for Free Radical Research, Department of Pathology, and Gravida National Centre for Growth and Development, University of Otago Christchurch, Christchurch, New Zealand; and



Erythrocytes require glucose-6-phosphate dehydrogenase (G6PD) to generate NADPH and protect themselves against hemolytic anemia induced by oxidative stress. Peroxiredoxin 2 (Prx2) is a major antioxidant enzyme that requires NADPH to recycle its oxidized (disulfide-bonded) form. Our aims were to determine whether Prx2 is more highly oxidized in G6PD-deficient erythrocytes and whether these cells are able to recycle oxidized Prx2 after oxidant challenge. Blood was obtained from 61 Malaysian neonates with G6PD deficiency (average 33% normal activity) and 86 controls. Prx2 redox state was analyzed by Western blotting under nonreducing conditions. Prx2 in freshly isolated blood was predominantly reduced in both groups, but the median level of oxidation was significantly higher (8 vs 3%) and the range greater for the G6PD-deficient population. When treated with reagent H2O2, the G6PD-deficient erythrocytes were severely compromised in their ability to recycle oxidized Prx2, with only 27 or 4% reduction after 1 h treatment with 0.1 or 1 mM H2O2 respectively, compared with >97% reduction in control erythrocytes. The accumulation of oxidized Prx2 in oxidatively stressed erythrocytes with common G6PD variants suggests that impaired antioxidant activity of Prx2 could contribute to the hemolysis and other complications associated with the condition.

KEYWORDS: antioxidant defense; hemolytic anemia; hydrogen peroxide

PMID: 24636884



G6PD deficiency is the commonest human disease caused by an enzyme defect because of a  genetic mutation that is carried by an estimated 4 million people worldwide. It is a Mendelian inherited X-linked recessive disease affecting predominantly males but random X-inactivation (lyonization) could also result in disease in females. In its classical form, G6PD deficiency manifests as favism with hemolysis causing severe anemia and jaundice when affected individuals are exposed to oxidizing agents present in fava beans, certain sulfur-containing medications and antimalarial drugs. G6PD deficiency is also recognized  as a major cause of severe jaundice in newborn infants that could lead to kernicterus (bilirubin-induced brain damage), especially affecting populations in Asia, the Mediterranean and Africa.  Ironically, this enzyme deficiency presumably protects against severe forms of malaria which may be through the process of destruction of infected red blood cells during hemolysis and sequestration in the spleen that perhaps diminishes the parasitic load. A rather simplistic understanding of the hemolysis occurring in this enzyme deficiency primarily attributes it to the limited ability of glutathione (GSH) to handle excess reactive oxygen species in red blood cell (RBC), causing oxidation of sulfhydryl groups of hemoglobin (Hb), precipitation of denatured Hb and damage to RBC membranes resulting in lysis. However, emerging evidence shows a more complex interactive biochemical pathway that could be involved in this process. While NADPH is required for the recycling of GSH after its oxidation by metabolites such as hydrogen peroxide (H2O2), mice with targeted disruption of the gene encoding glutathione peroxidase (GPx) has little effect on the oxidation of hemoglobin (Hb) in murine RBC challenged with peroxide. This implies that different pathways requiring GSH, such as the thioredoxin (Trx) reaction, might also be involved (Figure 1).

Recently, peroxiredoxin 2 (Prx2), the third most abundant RBC protein, has been shown to be a major scavenger of oxidants e.g. hydrogen peroxide (H2O2). The RBC, being the major “oxygen shuttle” in the human body is constantly having to limit oxygen-mediated radicals and metabolic by-products as its function. Prx2 becomes oxidized in RBCs exposed to very low concentrations of H2O2, even though these cells contain active catalase and GPx. Prx2 also scavenges endogenous peroxide generated through Hb autoxidation. Prx2 is implicated as a chaperone to protect Hb denaturation because Prx2-knockout mice became anemic and their RBCs contained higher amounts of Hb oxidation products than wild type mice. Some of these hematological attributes appear as well in G6PD deficiency, and therefore Prx2 may contribute to the phenotype exhibited by G6PD deficient patients when exposed to oxidants.

We studied how newborn infants transitioning from a relatively hypoxic state in utero (adapting with predominantly fetal hemoglobin) to a more oxygenated extrauterine environment, dealt with “physiologic” exposure to oxidative stress postnatally. We asked the question if G6PD deficient infants may actually show a difference in the basal Prx2 function shortly after birth compared to normal infants.  To test this hypothesis, we compared RBCs obtained from newborn infants in the first week of life with and without G6PD deficiency to determine whether Prx2 is more highly oxidized in this basal condition. Furthermore, we studied whether Prx2 responds differently in deficient and normal infants when exposed to oxidizing agents.  For this, we compared G6PD-deficient RBCs with normal cells in their ability to regenerate reduced Prx2 after in vitro exposure to H2O2. To explore the role of Prx2 in the development of jaundice in G6PD deficient infants, we screened for any discerning relationship between the extent of Prx2 oxidation and elevated bilirubin in the blood of deficient versus normal infants.

Our results showed that basal oxidation of Prx2 is significantly greater (about 3-fold) in G6PD deficient infants. The median percentage of oxidized Prx2 in the G6PD-deficient RBCs was 8% compared to 3%  in cells with normal G6PD activity. This difference was not accounted for by whether the mode of delivery was vaginal or Cesarean section. There was also no difference by gender, gestation or birth weight. The extent of Prx2 oxidation for all infants was negatively correlated with G6PD enzyme activity, which indicates that the lower the enzyme level, the more oxidized was the Prx2. When challenged with H2O2 in vitro for an hour, normal RBCs had almost complete recycling (97-99%) of the oxidized Prx2 into its basal reduced form but G6PD-deficient RBCs showed markedly impaired recovery, which was more extensive with greater oxidant challenge (H2O2 of 0.1 mM, 29% vs. H2O2 of 1 mM, 4%). We could not detect any significant relationship between Prx2 oxidation, bilirubin levels and severity of jaundice in G6PD-deficient or normal infants, but this could have been confounded by our clinical policy of aggressive and early therapeutic intervention with phototherapy to limit any rise in bilirubin.

In conclusion, the results of our study have added to the fundamental understanding of the balance in basal and regeneration of Prx2 in RBCs with limited G6PD enzyme activity. While Prx2 is advancing as a marker of oxidative stress in the RBC, fine-tuning its balance in oxidation-reduction of this abundant antioxidant protein may have potential for adjunct therapy that modulates RBC health in diseases related to hemolysis, membrane fragility or  ineffective erythropoeisis.

This figure illustrates the interactive processes between oxidants and antioxidants with glucose-6-phosphate dehydrogenase (G6PD) in the red blood cell (RBC). Superoxide dismutase (SOD) catalyzes oxygen-derived radical such as superoxide to form H2O2. The peroxide is removed by catalase, glutathione peroxidase (GPx) and peroxiredoxin 2 (Prx2). At the center shows the oxidation of Prx2 by H2O2. Prx2 exists as a homodimer. Oxidation occurs at a highly reactive active-site Cys residue, which condenses with the resolving Cys on the adjacent chain to form a disulfide-linked dimer. This is recycled by thioredoxin (Trx) through thioredoxin reductase (TrxR), with electrons coming from NADPH. NADPH is supplied by G6PD and the pentose phosphate pathway. Recycling of glutathione (GSH) from oxidized glutahione disulfide (GSSG) by glutathione reductase also requires NADPH.