Background Diabetes is a chronic disease associated with many problems and high costs. In recent decades, a lot of research has been carried out in order to improve methods of treatment of diabetic patients. One of the currently used complementary therapies for diabetes is ozone therapy or autohemotherapy. The beneficial effects of ozone has been proven in many diseases such as diabetes, but the critical issue is the determination of the safe and effective concentration of ozone reacting with blood and in particular hemoglobin. Methods A number of spectroscopic techniques including intrinsic fluorescence, circular dichroism and UV–VIS spectroscopies were used as well as SDS-PAGE, Native-PAGE and dynamic light scattering to analyze the effect of ozonation on hemoglobin of a non-diabetic individual and four diabetic patients in order to find the appropriate concentration(s) of ozone for personalized autohemotherapy. Results In this study, we determined the personalized concentration(s) for a safe and effective ozonation of a non-diabetic individual and four diabetic type II patients, based on blood hemoglobin analysis. Conclusions A number of techniques were used to determine the personalized ozone concentration(s) for a safe and effective autohemotherapy based on blood hemoglobin analysis. SDS-PAGE and dynamic light scattering were identified as the two main techniques needed for personalizing the ozone concentration(s) for each individual as otherwise hemoglobin in blood can oligomerise and cause serious damage if the inappropriate ozone concentration is used.

In autohemotherapy, it is important to find a way to lower the effects of oxidation, especially at high concentrations of ozone. One of the parameters, other than ozone concentration, which can have a significant effect on the stability and rate of decomposition of ozone at high concentrations, is the presence of ions in water. A number of spectroscopic techniques including intrinsic fluorescence, circular dichroism and UV-VIS were used as well as SDS-PAGE, Native-PAGE dynamic light scattering and water ion analysis, in order to investigate the effects of two relatively high concentrations of ozone on purified human hemoglobin (Hb) in phosphate buffer and diluted versions with deionized, double distilled and tap water in vitro. Purified human Hb and not whole blood human Hb was used in this study, since the addition of water to the whole blood would have caused the RBCs to lyse, affecting the purification of Hb for further analysis. Therefore, using purified Hb, it was possible to investigate the effects of dilution of 50 mM phosphate buffer to 10 mM phosphate buffer with different water types including non-ion containing deionized and double distilled water as well as ion-containing tap water, when ozonated at 55 and 80 µg/ml ozone. The fundamental changes in the secondary and tertiary structures of Hb were seen to be related to non-ozonated Hb samples diluted with deionized and double distilled waters, respectively. Generally, Hb oligomerization was more likely to occur at the higher concentration of ozone (80 µg/ml) and in samples where phosphate buffer was diluted with non-ion containing deionized and double distilled waters and not the ion-containing tap water. This could be explained by the presence of water alkalinity or bicarbonate ions in tap water, which can scavenge free radicals and reduce Hb oxidation/oligomerization. Therefore, it was concluded that