Oxidative DNA Damage is Reduced by a Daily Micronutrient Supplement in HD Patients

1. Oxidative DNA Damage is Reduced by a Daily Micronutrient Supplement in HD Patients Dr Mary Hannon-Fletcher 2nd International Vitamin Conference 23rd May 2012 Copenhagen

2. Background • End stage renal disease is associated with an increase in oxidative stress, cardiovascular disease and cancer • Haemodialysis induces repetitive bouts of oxidative stress • Resulting in increased levels of reactive oxygen species • Leading to increased levels of oxidative DNA damage and thus genomic instability • May impact on the elevated levels of cancer reported in HD patients

3. Diet in HD Patients • Malnutrition is prevalent in 40-50% • Very restricted diets resulting in regulation of certain nutrients such as: • sodium, potassium, phosphate, protein & fluids • Reduction or exclusion of certain foods increases the risk of inadequate intakes • Under-nutrition exacerbates oxidative stress • Together with the increased losses of essential minerals and water-soluble vitamins via HD • Many studies have reported HD patients deficient in several important vitamins

4. Background • Data in our lab showed HD patients had significantly elevated levels of DNA damage compared to age and gender matched controls Aim • Using a novel micronutrient supplement containing physiological levels of folate, B, C and E -vitamins, and micronutrients • To determine if a 3 month placebo controlled intervention would effect levels of DNA damage

5. Study Design Thirty Nine Participants Recruited Baseline Clinical History & Blood Sample Treatment n =18 Placebo n=19 Randomised to treatment 3 month intervention n=16 n = 15 Post Intervention Clinical History & Blood Sample

6. Participants and Methods • Ethical permission was obtained from ORECNI and Governance was obtained from the WHSCT • DNA Damage was measured using the Comet assay (% tail DNA) • Oxidative specific DNA damage measured using bacterial enzymes: • Endonuclease III (Endo III) • Formamidepyrimidine DNA glycosilase (FPG) • Supplements were provided monthly in a bottle by the pharmacist • Volunteers were withdrawn if less than 90% of the supplements were taken

7. The Comet Assay

8. Assessment of DNA Damage The Comet assay is a Single Cell Microelectrophoretic method for the quantitative measurement of DNA damage and alkali labile sites. Damaged cells have the appearance of a comet with a tail owing to extension of the DNA towards the anode (Mc Kelvey Martin et al., Mut Res 288 (1993) 47-63).

9. Table 1: Volunteer Baseline Characteristics Values are presented as mean ± SD

10. Figure 2 Changes in DNA Damage in Treatment Group Post Intervention % Tail DNA *** *** *** Values are mean ± SD *p>0.001

11. Table 2: DNA Damage in Placebo Group . Values are mean ±SD **p>0.001;p>0.05 compared to baseline

12. Summary • A significant reduction in Alkaline, EndoIII and FPG DNA damage post intervention in the treatment group • Placebo DNA damage levels significantly increased from baseline

13. Conclusion • These data show for the first time that physiological levels of a novel supplement reduces levels of DNA damage • This novel treatment had a protective effect on the patient blood levels of DNA damage • In addition, the most significant reduction in DNA damage levels was observed in those patients with higher baseline levels • Additional research is urgently required

14. Acknowledgements • Supported by grant from WHCST • Amgen / Irish Nephrological Society Research Award • Thanks to the research group: • Dr Peter Garrett • Ms Twyla Moffitt

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