Plasma F(2)-isoprostanes in healthy and diabetic subjects: Analysis by mass spectrometry.

摘要

Increased oxidative stress is considered as an important contributing factor in aging and in pathogenesis of human diseases. Lipid peroxidation is one of the markers for oxidative stress. Measurement of F 2-isoprostanes (F2-iPs) or one specific biologically active isomer, iPF2alpha-III, is increasingly being used as a novel marker of in vivo lipid peroxidation. In this thesis, the lipid peroxidation status in Chinese subjects, both healthy and with diabetes mellitus (DM) were studied.;Gas chromatography-negative ion chemical ionisation-mass spectrometry (GC-NICI-MS) methods for measuring iPF2alpha-III and the precursor arachidonic acid (AA) were established. To study total iPF2alpha-III in plasma and erythrocyte membrane samples, two purification procedures prior to GC-NICI-MS were developed. A novel one-step solid phase extraction method to extract all F2-iPs isomers improved efficiency and recovery. An adapted immunoaffinity extraction method for iPF2alpha-III improved specificity.;Relationships of circulating total iPF2alpha-III with cigarette smoking, sex status, and age were studied in 89 healthy subjects. Plasma total iPF2alpha-III was significantly higher in heavy smokers, with a significant correlation between the lipid peroxidation marker and daily cigarette consumption. There was no sex-related difference of plasma total iPF2alpha-III in 38 age-matched non-smoking subjects. However, plasma total iPF2alpha-III was significantly higher in the older subjects amongst 65 non-smokers, with a significant correlation with advancing age in those over 40 years old. Erythrocyte membrane iPF2alpha-III/AA ratio was found to be 3-fold higher and significantly correlated with that in the plasma. This might provide an improved tissue marker for oxidative stress in vivo.;There were 294 type 2 DM patients recruited from a DM specialist clinic for the study. Controlling the effects of age and medications, plasma total iPF2alpha-III concentrations in the patients were significantly higher than that in the healthy controls by 1.6-fold. In a subgroup of 29 patients who had not been on any DM-related medication, their plasma total iPF2alpha-III did not associate with glycaemic status, but strongly correlated to low-density lipoprotein (LDL) levels. Such a correlation was not found in a group of age-match healthy controls. DM patients on different medications were found to have reduced oxidative stress, especially those receiving anti-DM therapy. However, logistic regression analysis on risk factors and complications showed that increased lipid peroxidation was not an independent risk factor for DM complications.;It can be concluded that the analytical methods developed were important tools for the understanding of lipid peroxidation status in healthy subjects and patients with type 2 DM of Chinese origin.