Role of vitamin E supplementation in improving blood hemoglobin levels in apparently healthy mildly anemic Pakistani adults and the probable mechanism of this action of vitamin E

Prevalence of mild anemia is high in apparently healthy populations in the developing countries of South Asia. A few human studies have shown that use of antioxidant vitamins such as vitamin E could correct anemia. However, the exact molecular mechanism(s) of this correction is still unclear. One of the possible mechanisms of action of vitamin E in enhancing blood hemoglobin levels in humans could be through inhibition of apoptosis of erythroid stem cells. A few studies have shown that the inhibition of proapoptotic proteins by various intra- and extracellular factors may cause increased survival of human erythroid progenitor cells (EPCs). Some of the animal studies have also suggested the possible role of vitamin E in the prevention and/ or correction of defective erythropoiesis. Moreover, it has also been reported that vitamin E decreases the experimentally-induced apoptosis in bone marrow hematopoietic stem cells in animals. The objectives of this study were to find out the effect of vitamin E supplementation on blood hemoglobin levels in apparently healthy but mildly anemic Pakistani adults and to investigate whether any positive effect of vitamin E on hemoglobin levels in apparently healthy humans could be due to inhibition of apoptosis of EPCs. Methods: To study the effect of vitamin E on hemoglobin levels, a single blinded and placebo-controlled trial was carried out on 124 apparently healthy mildly anemic adult human subjects recruited from the General Practioners\u27 Clinics and also included personnel from the Aga Khan University, Karachi. All the subjects were recruited with informed consent. The study subjects were randomly assigned to the Intervention group (n=82) and the Control group (n=42). In the Intervention group, each subject was given vitamin E (400 mg) every day for three consecutive months, while Control group subjects received a placebo. Eighty six subjects completed the trial. Fasting venous blood was collected at baseline and after three months of supplementation. Blood hemoglobin levels and. serum/plasma concentrations of vitamin E, erythropoietin, total antioxidant status (TAS), vitamin B12, folate, ferritin, serum Transferrin Receptor (sTfR), glucose, creatinine and lipid profile were determined, compared between Intervention and Control groups and analyzed using repeated measures ANOVA and multiple linear regression. To find out the probable mechanism of action of vitamin E on blood hemoglobin levels, CD34+-derived EPCs were isolated from human peripheral blood mononuclear cells (PBMNCs) of apparently healthy Pakistani adult volunteers by density gradient centrifugation followed by magnetic activated cell sorting (MACS) using CD34+ selection kit. Purity of the isolated EPCs was assessed through immune fluorescence microscopy using Fluorescein-5-isothiocyanate (FITC)-conjugated monoclonal antibodies against various EPCs surface antigens. The purity of CD34+-derived EPCs was found to be 95-98%. CD34+-derived EPCs were then cultured for 7-14 days in the recommended medium supplemented with erythroid expansion supplement. To study the effect of vitamin E on apoptosis of erythroid stem cells, EPCs were treated in vitro with various concentrations of Tumor necrosis factor (TNF)-a to induce apoptosis. The concentration of TNF-a inducing maximum apoptosis was then selected to study any protective effect of vitamin E. EPCs were then incubated at various concentrations of vitamin E (zero, 10, 50 and 100 µg/ml) or erythropoietin (zero, 10, 50 and 100 IU/ml) followed by addition of 100 ng/ml concentration of TNF-a which produced the maximum apoptosis under the experimental conditions. The percentage of apoptosis of the treated EPCs was measured through flow cytometry by using annexin V and propidium iodide (PI) staining. Two-way ANOVA with replacement was used to find out the mean difference between the effects of increasing concentrations of drug (vitamin E or erythropoietin) treatment and the interaction of these drugs on TNF-a-induced apoptosis of EPCs. Results: In the study population, there was a significant increase in post-supplemental concentrations of both vitamin E and hemoglobin (p value= 0.045 and p value= 0.049, respectively) when compared with their baseline concentrations. However, when the mean post-supplemental levels of both vitamin E and hemoglobin levels were compared, significant increase in vitamin E (p value= 0.01) and hemoglobin (p value \u3c0.001) levels were observed in the intervention group (vitamin E supplemented) as compared to the control (placebo) group. The adjusted regression coefficients 03) and standard error [SEW)] of the significant determinants of post-supplemental hemoglobin levels were serum concentration of vitamin E (0.983 [0.095]), baseline hemoglobin levels (0.768[0.077]), sTfR(-0.06[0.02]) and gender (male or female, -0.656[0.224]). Regarding the second objective of the study pertaining to the possible underlying mechanism of the action of vitamin E on apoptosis of CD34±-derived EPCs, the mean percentages of early and late apoptotic CD34+-derived EPCs after treatment with TNF-a (100 ng/ml) alone were 58.7± 4.87% and 8.9± 3.43%, respectively. Vitamin E and erythropoietin in highest used concentrations (100 µg/m1 and 100 IU/ml, respectively) decreased the percent cell early apoptosis of treated EPCs to 25.2± 4.70% and 5.3± 1.81%, respectively. However, there was a significant difference in the mean percentage of TNF-a-induced apoptotic CD34+-derived EPCs in early and late apoptosis by vitamin E treatment (p value= 0.008) which was higher in early as compared to late apoptosis. There was a significant difference in the mean percentage of TNF-a-induced apoptotic CD34+-derived EPCs by vitamin E treatment (p value \u3c 0.001) when analyzed for a statistical interactive effect on early and late apoptotic phases and vitamin E concentration. Conclusion: The study showed a positive role of vitamin E supplementation in improving blood hemoglobin levels in apparently healthy mildly anemic Pakistani adults. The study also showed that one of the possible mechanisms of action of vitamin E appears to be through inhibition of apoptosis of the CD34±-derived EPCs