Abstract
To investigate the involvement of reactive oxygen species in extracorporeal photoimmunotherapy (photopheresis), we have introduced two highly sensitive and specific techniques for the detection and quantitative measurement of oxygenated nonenzymatically formed arachidonic acid isomers [mono-hydroxyeicosatetraenoic acids (HETEs) and F<sub>2</sub>-isoprostanes] by gas chromatography-mass spectrometry/negative ion chemical ionization (GC-MS/NICI) in plasma samples of patients suffering from cutaneous T-cell lymphoma and progressive systemic scleroderma II. The analysis of HETEs involved hydrogenation, solid phase extraction on a C<sub>18</sub> cartridge, formation of pentafluorobenzyl bromide and trimethylsilyl ether derivatives. In the case of F<sub>2</sub>-isoprostanes, the analytical procedure was similar to that of HETEs except that the hydrogenation step was omitted. In the plasma of healthy volunteers picomole amounts of 2-, 5-, 8–12-, 15-HETEs, 8-iso-PGF<sub>2α</sub> and 9α,11α-PGF<sub>2α</sub> were quantified by using 12-hydroxy-heptadecatrienoic acid and PGF<sub>2α</sub>-d<sub>4</sub> as internal standards of HETEs and isoprostanes, respectively. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed characteristic increases in both, HETE and isoprostane levels. The enhancement of indicators of lipid peroxidation is in correspondence with a moderate loss of α-tocopherol, the most important lipid-soluble antioxidant in human plasma. Thus, our data confirm the involvement of lipid peroxidation in extracorporeal photoimmunotherapy.
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