Self-administered vitamin C increased red blood cell survival in a case of class A G6PD deficiency Open Access

TO THE EDITOR:

Polymorphic X-chromosome–linked glucose-6-phosphate dehydrogenase (G6PD) deficiency variants are the most common inherited enzymatic disorders affecting some 500 million persons of Middle Eastern, East Asian, and African ancestry.^1,2 The recognition of G6PD deficiency was the result of investigations of the hemolytic effect of the antimalarial primaquine.³ 

G6PD is the first enzyme of the glucose pentose monophosphate shunt converting glucose-6-phosphate into 6-phosphogluconolactone and reducing nicotinamide adenine dinucleotide phosphate (NADP) to NADPH.⁴ NADPH is required in glutathione metabolism, a major antioxidant pathway neutralizing reactive oxygen species (ROS) to maintain redox balance. The glutathione antioxidant defense involves ∼99% of glutathione in reduced form, using NADPH for continuous reduction of oxidized glutathione by glutathione reductase. Although G6PD is expressed ubiquitously, it is especially important in red blood cells during hemoglobin release of oxygen to the tissues as some ROS are generated. In red blood cells, G6PD is the only enzyme reducing NADP to NADPH.^1,5 Cells deficient in G6PD are at a greater risk of hemolysis upon oxidative stress.

G6PD deficiency is organized into 4 classes: A, B, C, and U.⁶ The polymorphic class B G6PD deficiency variants are enriched in areas where malaria is endemic, with G6PD enzyme activity reduced to between 20% and 40%. Most people who are affected (predominantly males) do not experience hemolysis unless they are exposed to hemolysis-precipitating drugs or ingest fava beans. Class A G6PD deficiency is very rare and defined by chronic hemolytic anemia with enzyme activity reduced to <20%. Complete G6PD deficiency is not compatible with life.^1,5 The G6PD gene is found on the X chromosome, making males significantly more likely than heterozygous females to experience hemolysis.⁷ In females, the severity of symptoms is dependent on the proportion of red blood cells undergoing X-chromosome inactivation, generating mosaicism of the deficient and normal red blood cells. Quantitative analysis of the proportion of cells in adult heterozygous females expressing an active maternal or paternal X chromosome was unchanged in >2 years.⁸ Thus, females heterozygous for G6PD deficiency have enzyme activity ranging between normal to the reduced levels depending on the relative proportion of cells bearing the same G6PD variant.^1,5 

For individuals with G6PD deficiency, avoiding oxidating medications and fava beans is important to prevent hemolysis.^1,5,15,16 Vitamin C is listed among contraindicated medications in G6PD deficiency in various sources such as g6pd.org because of a case report of the effect of IV administration of very high doses of vitamin C exceeding 4 g.^16-18 The original case study for which this recommendation was made is based upon an individual with G6PD deficiency who received 75 g of vitamin C IV.¹⁸ Upon a later review of contraindicated factors for G6PD deficiency, a group of experts withdrew this recommendation and labeled the supplement as likely posing “low-to-no” risk.¹⁵ Although we hypothesize that vitamin C’s antioxidant properties could serve as a preventive treatment against chronic hemolytic anemia in class A G6PD deficiency, and possibly even ameliorate acute hemolysis in class B G6PD–deficient variants, no evidence for this hypothesis exists. Vitamin C could act as a free-radical scavenger, directly neutralizing ROS and/or acting indirectly via the oxidation of glutathione.¹⁹ 

A member of a previously described large multigeneration family, as of now composed of 42 affected males, with class A G6PD contacted us for his family care.¹⁴ He was concerned that 1 of his brothers has significant neurological impairment after suffering kernicterus as an infant, and 1 of his sister’s sons was recently hospitalized with aplastic crisis and hemoglobin of 6 g/dL, requiring red blood cell transfusions. He was familiar with current literature on vitamin C in G6PD deficiency as well as the fact that vitamin C is listed among contraindicated medications. However, he was also aware that vitamin C and other antioxidants may have potential activity to reduce hemolysis and that we previously described a patient with methemoglobinemia from CYB5R mutation who could not be treated with methylene blue because of concomitant G6PD deficiency (Betica/A⁻). That patient was successfully treated with vitamin C, which ameliorated his sporadic cyanosis and did not cause hemolysis.²⁰ He decided to take 500 mg of over-the-counter vitamin C and requested that our team measure his hemolysis. We decided to perform end tidal carbon monoxide (ETCO) corrected to ambient CO (ETCOc) measurements, routine nonquantitative laboratory measurement of hemolysis, and blood counts immediately before, 1 hour after, and 24 hours after he took this dietary supplement. He was counseled not to proceed and was advised that we were planning to do formal studies using pharmaceutically approved vitamin C preparations under requested formal institutional review board approval. However, he did not follow our advice and decided to proceed anyway.

Previously routine quantitation of red blood cell survival using radioisotope labels is no longer available in the United States. A nonradioactive method to detect hemolysis is to measure ETCOc. Although not widely used, we have chosen ETCOc because of its simplicity, rapidity of hemolysis assessment in real time, and noninvasive nature permitting its use in neonates and adults.^9-12 

In every individual, there is some amount of CO exhaled as a byproduct from heme catabolism by heme oxygenase.¹³ The baseline level of ETCOc for a person who is not experiencing hemolysis is ∼0.80 ppm.¹³ Individuals with class A G6PD deficiency experiencing chronic hemolytic anemia have a higher baseline level of ETCOc, and the severity of their hemolysis can be quantitated.¹⁴ We measured ETCOc using Sensalyze (CoSense) to measure the patient’s level of hemolysis immediately before he took 500 mg of the vitamin C supplement, 1 hour after, and 24 hours after.

The 44-year-old man’s baseline ETCOc was 5.2 ppm, 3.9 ppm 1 hour after taking the supplement, and 4.6 ppm 24 hours later. These ETCOc measurements are highly significant, as variance for CoSense measurements is ±0.2 ppm. His hemoglobin data are difficult to interpret because of wide variation; however, there was a suggestion that reticulocyte number and bilirubin slightly improved after vitamin C administration. We previously demonstrated that increased ROS resulting from excessive expansion of mitochondria in reticulocytes may contribute to hemolysis. In a phenomenon coined neocytolysis, mitochondria in reticulocytes are expanded and young red cells have a shorter half-life. In neocytolysis, mitochondrial ROS diffuse into plasma and interact with red blood cells with low antioxidant capacity, causing hemolysis that is attenuated by antioxidants.²¹ Thus, we further measured ROS and mitochondrial mass in his red blood cells and reticulocytes; however, these levels showed no significant differences after vitamin C administration, indicating that its benefit is from protection of reduced glutathione rather than mitochondrial ROS. This result is encouraging and supports the hypothesis of vitamin C as a preventive treatment against chronic hemolysis in class A G6PD deficiency or therapy for acute hemolysis in class B G6PD deficiency.

We have submitted an investigational new drug application to the US Food and Drug Administration to begin a phase 1 clinical trial and use pure vitamin C under controlled conditions to establish a safe and effective dose of vitamin C to increase red blood cell survival. If these planned studies proceed, we hope that vitamin C in yet-to-be-determined dose and frequency of administration becomes a useful inexpensive treatment for hemolytic anemia due to G6PD deficiency.

Contribution: C.V. wrote manuscript and performed research; J.S. performed laboratory research and edited the manuscript; A.S. edited the manuscript and performed vitamin C research; W.T.S. edited the manuscript, referred patient to clinic, and provided background clinical information; B.P. was the institutional review board liaison and coauthored the manuscript; R.D.C. edited the manuscript, provided the ETCOc instrument, and performed research; and J.T.P. edited the manuscript and provided medical expertise and oversight.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Josef T. Prchal, The University of Utah and Huntsman Cancer Institute, 2000 Circle of Hope Dr, HCI RN 4126, Salt Lake City, UT 84112; email: [email protected].