Is AAGP™'s Bioactivity pH Dependent: Protective Effects of AAGP™ on Hydrogen Peroxide and Low temperature (15°C) Induced Cell Death in Human Adult Fibroblast at pHs of 5.3, 7.3 and 10.5

Hussein and Retamal, December 2005

Introduction

It is becoming increasingly apparent that AAGP™ has strong cell preservation qualities. With this in mind a series of experiments mimicking cell stress conditions (pH, oxidative stress, low temperatures and time) were conducted to challenge both human adult and neonatal fibroblasts exposed to AAGP™ in comparison to human adult and neonatal fibroblasts not exposed to AAGP™. This was done with the use of AAGP™ as a anti- aging cosmetic, protecting skin from the harsh environment, in mind. The particular experimental protocol aimed to elucidate the protective effects of AAGP™ against both temperature and oxidative stress induced cell death at different pHs.

Method

Hydrogen Peroxide - Adult fibroblasts were plated at a concentration of 1x105 cells/ml and grown on non-adherent plates in serum-free media at 37°C in the presence of 1mM hydrogen peroxide and a concentration of either 0 or 15mg/ml AAGP™. To test the affects of AAGP™ at different pHs this was repeated at a pH of 5.3 (the normal pH of AAGP™) and 7.3. Cell counts were taken at various time points using Trypan blue and two counts were performed per sample to calculate the percentage viability (from 0 hours) of the cell sample.

Temperature - Adult fibroblasts were plated at a concentration of 2x105 cells/ml and grown on non-adherent plates in low- serum media at 15°C in the presence of a concentration of either 0 or 15mg/ml AAGP™. To test the affects of AAGP™ at different pHs this was repeated at a pH of 5.3 (the normal pH of AAGP™), 7.3 and 10.5. Cell counts were taken at various time points using Trypan blue and two counts were performed per sample to calculate the percentage viability (from 0 hours) of the cell sample.

Results

Hydrogen peroxide - 61 hours post 1mM hydrogen peroxide exposure all control cells were dead. However, AAGP™ exposed cells still remained at 60% viability in both 5.3 and 7.3 pH conditions. Thus AAGP™ is affective at both acidic and basic pHs.

Temperature - 7 days post low temperature exposure the control cells kept at a pH of 5.3 were still 60% viable and the control cells kept at 7.3 were 24% viable. This suggests a lower pH is optimal for adult human fibroblasts. However, in terms of the functionality of AAGP™ at varying pH cells were 100% viable at both pH levels when exposed to AAGP™. Thus AAGP™ is able to function at both 5.3 and 7.3 pH. At a pH as high as 10.5 the AAGP™ was less effective. However, at day 2 20% of the AAGP™ exposed cells were still viable compared to 0% of the control cells showing it still has a protective affect at a pH as high as 10.5.


Figure 1: Effects of 15mg/ml AAGP™ on the survival of hydrogen peroxide treated Human Adult Fibroblasts at different pHs.


Figure 2: Effects of 15mg/ml AAGP™ on the survival of low temperature (15°C) treated Human Adult Fibroblasts at different pHs.


Figure 3: Effect of 15mg/ml AAGP™ on the survival of low temperature (15°C) treated Human Adult Fibroblasts at a pH of 10.5.

Conclusion

The protective affects of AAGP™ against temperature and oxidative stress induced cell death are apparent at varying pHs (5.3 -7.3) and still continue up to pH 10.5. Thus, it can be said that the protective effects of AGP are not pH dependant within the optimal pH range of 5.3-7.3.