A sensitive gas chromatography-mass spectrometry assay reveals increased levels of monohydroxyeicosatetraenoic acid isomers in human plasma after extracorporeal photoimmunotherapy and under in vitro ultraviolet A exposure

Extracorporeal photoimmunotherapy (photopheresis) is a highly effective the rapy in the treatment of various disorders. Although extracorporeal photoim munotherapy has been successfully used for more than 10 y, its mechanism of action is still unclear. The formation of reactive oxygen species have bee n implicated in extracorporeal photoimmunotherapy, but malonyl dialdehyde a s a marker of systemic lipid peroxidation did not increase significantly du ring treatment. To investigate further the involvement of reactive oxygen s pecies in extracorporeal photoimmunotherapy, we have introduced a highly se nsitive negative ion gas chromatography-mass spectrometry based method for quantitating oxygenated arachidonic acid isomers (hydroxyeicosatetraenoic a cids) in plasma samples of patients treated with extracorporeal photoimmuno therapy. In the plasma of healthy volunteers pmole amounts of 2-, 3-, 5-, 8 -12-, and 15-hydroxyeicosatetraenoic acid were detected and we observed a d ose-dependent augmentation in these metabolites when the blood was irradiat ed with increasing doses of ultraviolet A in the presence of the photosensi tizer 8-methoxypsoralen, Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed a characteristi c increase in total hydroxyeicosatetraenoic acid levels, particularly of 5- hydroxyeicosatetraenoic acid which contributed 80% to the sum of all hydrox yeicosatetraenoic acid isomers, Chiral phase high-performance liquid chroma tography indicated almost equal amounts of 5S- and 5R-hydroxyeicosatetraeno ic acid suggesting that the majority of lipid peroxidation products are for med via nonenzymatic oxidation reactions.