Combination of LC–MS- and GC–MS-based Metabolomics to Study the Effect of Ozonated Autohemotherapy on Human Blood

Ozonated autohemotherapy (O₃-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O₃-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O₃-AHT, LC–MS- and GC–MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC–MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC–MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC–QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood