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

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

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

Aim Using a novel micronutrient supplement containing physiological levels of folate, B, C and E -vitamins, and micronutrientsTo determine if a 3 month placebo controlled intervention would effect levels of DNA damage

Data in our lab showed HD patients had significantly elevated levels of DNA damage compared to age and gender matched controls

Background

Study Design

Baseline Clinical History & Blood Sample

Treatment

n =18

Treatment

n =18

Thirty Nine Participants RecruitedThirty Nine Participants Recruited

3 month intervention 3 month intervention

n=16n=16

Placebo

n=19

Placebo

n=19

n = 15n = 15

Post Intervention Clinical History & Blood Sample

Randomised to treatment

Randomised to treatment

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Supplements were provided monthly in a bottle by the pharmacist Volunteers were withdrawn if less than 90% of the supplements were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

The Comet Assay

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).

Characteristic Placebo

(n=19)

Treatment

(n=18)

Age (years) 62.58 ± 10.95 64.89 ± 8.29

Male/Female

(n/n)

12/7 10/8

Diabetes (n (%)) 6 (31.6%) 7 (38.9%)

Dialysis Duration

(months)

27.00 ± 17.75 27.33 ± 38.09

BMI (kg/m2) 27.08 ± 6.43 26.29 ± 4.80

Table 1: Volunteer Baseline Characteristics

Values are presented as mean ± SD

Figure 2 Changes in DNA Damage in Treatment Group Post Intervention

% T

ail D

NA

*** *** ***

Values are mean ± SD *p>0.001

Table 2: DNA Damage in Placebo Group

% Tail DNA Alkaline FPG ENDO III

Baseline 19.42 ± 8.64 23.38 ± 9.28 23.33 ± 9.64

3 Months 33.81** ± 9.52 30.44* ± 9.77 35.26* ± 9.01

.

Values are mean ±SD **p>0.001;p>0.05 compared to baseline

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

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

Acknowledgements Supported by grant from WHCST Amgen / Irish Nephrological Society Research

Award

Thanks to the research group: Dr Peter Garrett Ms Twyla Moffitt

Supplement Prescription: Patent Protected Supplement Dose Vitamin C (ascorbate) 60mg Vitamin E (tocopherol) 10mg Vitamin K 65g Folic acid (mg) 800g B2 Riboflavin (mg) 1.6mg *B6 (mg) 10mg *B12 (g) 12 g B5 (Pantothenic acid, mg) 1mg B1 Thiamin (mg) 1mg Zinc 15mg Copper 1.5mg Selenium 75g