1. Dietary Vitamin C may be less effective than supplements in reducing hemoglobin oxidation in humans even with 400 iu of vitamin E Leta Moser and Alfred B. Ordman* Biochemistry Program, Beloit College, Beloit, WI 53511 ABSTRACT Even when vitamin E is taken at high levels, we expected to measure a significant effect in oxidative damage as a function of vitamin C intake. A pilot study was conducted to determine a useful design for a larger study. In addition to determining useful dosage levels, it was used to select appropriate endpoints to measure antioxidant effectiveness in vivo. A clinical trial of 10 participants was conducted. Participants took 400mg of vitamin E while consuming vitamin C for 3 weeks. Vitamin C dosage was either 200mg daily in food, 500 mg twice a day as supplements, or 1,000 mg twice a day as supplements, the upper level determined by the Food and Nutrition Board. Blood samples were then drawn on consecutive days between 4-8 hrs after vitamin C consumption. Participants then avoided vitamins C and E for at least a week and again provided blood. Heinz bodies in RBCs result from oxidative damage to hemoglobin. They have been shown to be especially sensitive for monitoring vitamin C effectiveness in vivo. Heinz bodies were significantly fewer with 500 mg of vitamin C taken twice a day. The other two dosages were less effective, even in the presence of high intake of vitamin E. INTRODUCTION Purpose: This pilot study was conducted to determine a useful design for a larger study. In addition to determining useful dosage levels, it was used to select appropriate endpoints to measure antioxidant effectiveness in vivo. Even when vitamin E is taken at high levels, we expected to measure significant protection from oxidative damage as a function of vitamin C intake. Dosage levels/Original studies:Beyond Dr. Linus Pauling’s recommendation of up to 16 g of vitamin C per day, three “pharmacologic dosages” of vitamin C have been recognized. The first was 500 mg twice a day, the dosage at which vitamin C is continually excreted in the urine (1). The second was 200 mg in the diet, the dosage at which a certain type of white blood cells is saturated with vitamin C (2). The third is the latest Food and Nutrition Board identification of an Upper Limit of vitamin C consumption (3). This study compared the effect of those three dosages. For oral consumption of vitamin C, either 500 or 1,000 mg twice a day results in statistically identical, maximal plasma saturation. Heinz bodies: Heinz bodies in red blood cells (RBC) result from oxidative damage to hemoglobin. They have been shown to be especially sensitive for monitoring vitamin C effectiveness in vivo(4). METHODS Beloit College IRB approval was granted before experimentation. Ten participants from Beloit College volunteered for the study. The median group characteristics were 23yr for age, 70 in for height, and145 lb for weight. All were healthy, though one smoked approximately 5 filtered cigarettes per day. Vitamin regimens and timeline are shown in Figures 1 and 2 resp. Samples were analyzed for Heinz bodies. Stained plasma smears were labelled in code and counted by three people before data was unblinded for analysis. Data was analyzed using ANOVA in JMP. RESULTS Heinz bodies were significantly fewer with 500 mg of vitamin C taken twice a day. Figure 4 shows the number of Heinz bodies found in participants taking different levels of vitamin C. Except for the group labeled minimal, all were taking 400 iu of vitamin E daily, in addition to the vitamin C dosage shown. The highest level of Heinz bodies was in the group avoiding vitamins E and C. The group taking 1,000 mg of vitamin C twice a day had higher levels of Heinz bodies than those taking 500 mg twice a day. For data pooled from all blood samples taken, ANOVA analysis of diet vs. Heinz bodies observed showed that the 500 mg twice a day diet resulted in significantly fewer Heinz bodies than the low vitamin C diet (  >0.0020). In Figure 5, data is analyzed by controlling for individual differences. An individual first took 400 iu of vitamin E and the vitamin C dosage indicated daily and Heinz bodies were counted. Then the individual avoided vitamins E and C and blood was analyzed again. This way the change in Heinz bodies could be interpreted for each individual. High and low doses of vitamin C produced higher levels of Heinz bodies. This pilot study indicates 500mg twice a day produced fewer Heinz bodies. DISCUSSION The study design demonstrated critical points for an effective study of antioxidants: 1. Time between intake of vitamin C and sampling must be carefully controlled. 2. Water-soluble vitamin C and fat-soluble vitamin E may have synergistic or competitive effects on oxidative processes. 3. Selection of appropriate vitamin C dosage should be justified. 4. A variety of convenient endpoints (urinary excretion, white blood cell saturation, plasma concentration, TBARs, protein carbonyls, Heinz bodies, arterial compliance) may measure different effects. Results are consistent with the hypothesis that 500mg of vitamin C taken twice daily may be substantially more effective in reducing hemoglobin damage. Figure 4 demonstrates that there may be significant disadvantage in consumption of 200 or 1,000x2 mg per day. The pro-oxidative effect of the higher dosage is similar to what has been observed in dosage comparison studies of vitamin E (5-6). REFERENCES 1. King, G., Beins, M., Larkin, J., Summers, B., and Ordman, A.B., "Rate of Excretion of Vitamin C in Human Urine", AGE 17:87-92 (1994) 2. Levine, M., Conry-Cantilena, C., Wang, Y., Welch, RW, and Cantilena, LR,"Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance", PNAS USA 93: 3704-9 (1996) 3. Panel on Dietary Antioxidants and Related Compounds, Food and Nutrtion Board: Vitamin C. In "Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids." Washington DC: National Academy Press, pp 95-185, 2000 4. Johnston, C.S. and Cox, S.K., "Plasma-saturating intakes of vitamin C confer maximal antioxidant protection to plasma", JACN 20:623-27, (2001) 5. Brown, K.M., Morrice, P.C., and Duthie, G.G., "Erythrocyte vitamin E and plasma ascorbate concentrations in relation to erythrocyte peroxidation in smokers and nonsmokers: Dose response to vitamin E supplementation", Am. J. Clin. Nutr. 65: 496-502 (1997) 6. Meydani S. N., Meydani M., Blumberg J. B., Leka L.S., Siber G., Loszewski R., Thompson C., Pedrosa M. C., Diamond R. D., Stollar B. D., "Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial", JAMA277:1380-6 (1997) ACKNOWLEDGEMENTS The authors wish to thank the following: Patrice Leahy for research assistance, Nikki Reading for statistical analysis, Elif Alpoge for computer aid, Beloit College for financial assistance, Balz Frei, Carol Johnston, Keith Richards for technical advice/support, And the students of Beloit College for their compliance and blood Figure 3: Timeline A. B. C. D. E. --------|----------------------|---|----------|--|-------- days 5d 19d 2d 7d 1d A. Avoided vitamin C enriched foods and all supplements B. Randomly assigned to regimen 1, 2, or 3 for 3 weeks C. On day 19 and 21, between 4-8 hours after morning supplements, blood was drawn by finger prick. Dietary logs were maintained for the four days ending with the last blood sampling. Continuous reminders were sent by email and telephone. D. Vitamin C and E-rich foods and all supplements were avoided. E. Two blood samples were taken 24 hours apart. Heinz Bodies Per RBC