Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh > t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE (E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.