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 therapy in the treatment of various disorders. Although extracorporeal photoimmunotherapy has been successfully used for more than 10 y, its mechanism of action is still unclear. The formation of reactive oxygen species have been implicated in extracorporeal photoimmunotherapy, but malonyl dialdehyde as a marker of systemic lipid peroxidation did not increase significantly during treatment. To investigate further the involvement of reactive oxygen species in extracorporeal photoimmunotherapy, we have introduced a highly sensitive negative ion gas chromatography-mass spectrometry based method for quantitating oxygenated arachidonic acid isomers (hydroxyeicosatetraenoic acids) in plasma samples of patients treated with extracorporeal photoimmunotherapy. In the plasma of healthy volunteers pmole amounts of 2-, 3-, 5-, 8-12-, and 15-hydroxyeicosatetraenoic acid were detected and we observed a dose-dependent augmentation in these metabolites when the blood was irradiated with increasing doses of ultraviolet A in the presence of the photosensitizer 8-methoxypsoralen. Analysis of plasma samples obtained from patients before and after extracorporeal photoimmunotherapy revealed a characteristic increase in total hydroxyeicosatetraenoic acid levels, particularly of 5-hydroxyeicosatetraenoic acid which contributed 80% to the sum of all hydroxyeicosatetraenoic acid isomers. Chiral phase high-performance liquid chromatography indicated almost equal amounts of 5S- and 5R-hydroxyeicosatetraenoic acid suggesting that the majority of lipid peroxidation products are formed via nonenzymatic oxidation reactions.

Evaluation of UVA-mediated oxidative damage to proteins and lipids in extracorporeal photoimmunotherapy.

The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.

[Clinical and genetic findings in a patient with fundus albipunctatus]. / Klinische und molekulargenetische Befunde bei einer Patientin mit Fundus albipunctatus.

METHODS: The 38-year-old index patient was examined by visual acuity testing, perimetry, dark adaptometry, funduscopy, electroretinogram (ERG), and multifocal ERG. She was screened for mutations in exons 2-5 and exon/intron boundaries of the 11- cis retinol dehydrogenase gene by direct sequencing. RESULTS: Visual acuity was 1.0, but perimetry revealed paracentral scotomas associated with reading problems. The optic discs were normal. After 45 min of darkness there was nearly no increase of light sensitivity. After 30 min of dark adaptation, the scotopic ERG showed reduced amplitudes, but after 60 min a nearly normal level was reached. The 30-Hz flicker response of the cone ERG showed borderline implicit times, but no reduction of amplitudes. However, multifocal ERG clearly disclosed a paracentral amplitude reduction as the reason for the visual field defects. The fundus was typical for fundus albipunctatus. The patient is a compound heterozygote carrying a Ile33Asn and a Arg157Trp mutation. CONCLUSIONS: The paracentral visual field defects were due to cone dysfunction. So far the patient exhibits no cone dystrophy.

Protection against UVA damage and effects on neutrophil-derived reactive oxygen species by beta-carotene.

OBJECTIVE: Phagocyte-derived reactive oxygen species (ROS) are involved in microbicidal activities as well as in tissue damage at sites of inflammation. Carotenoids play an important function in protecting cells from oxidant damage. We investigated the in vitro and in vivo effect of 13-cis and 9-cis-beta-carotene on human neutrophils. METHODS: Neutrophils from healthy donors in the presence of 0.25 mumol/L-1 mumol/l beta-carotene and from subjects under beta-carotene supplementation and UVA or UVA/B exposure were stimulated by opsonized zymosan and the generation of ROS was measured by electron spin resonance spectroscopy. RESULTS: Our in vitro results show different effects of the two isomers on stimulated neutrophils. 9-cis-beta-carotene did not produce any change, whereas 13-cis-beta-carotene significantly and concentration-dependent inhibited the ROS generation by stimulated neutrophils. Further, in a controlled study, we were able to demonstrate an in vivo protective effect of beta-carotene on neutrophils against UVA damage by beta-carotene supplemented subjects.

Effects of retinoids on the generation of neutrophil-derived reactive oxygen species studied by EPR spin trapping techniques.

OBJECTIVE: To study the potential efficacy of retinoids on granulocytes with regard to their role in inflammatory dermatoses. METHODS: We investigated the in vitro effect of acitretin and isotretinoin on the generation of reactive oxygen species (ROS) by stimulated human neutrophils using EPR spin trapping techniques. RESULTS: The effects of the two retinoids on ROS generation by neutrophils were different. Acitretin increased the generation of hydroxyl radicals, whereas isotretinoin showed an antioxidant activity against the superoxide anion. CONCLUSIONS: Our study demonstrates retinoid type-dependent effects on ROS production by stimulated neutrophils.

Investigation of annexin V binding to lymphocytes after extracorporeal photoimmunotherapy as an early marker of apoptosis.

BACKGROUND: Induction of programmed cell death is assumed to be a possible effect of extracorporeal photoimmunotherapy (ECPI). OBJECTIVE: In the present study lymphocytes of patients with cutaneous T cell lymphoma undergoing ECPI were investigated for early apoptotic events. METHODS: Annexin V, known for its selective affinity to phospholipids, was used to detect early phases of apoptosis. Simultaneous staining with propidium iodide binding to DNA allowed detection of late apoptotic/necrotic cells. RESULTS: At 1 h after ECPI, an increase in early apoptotic cells was found indicating a direct effect of ECPI. At 20 h after each ECPI session, a delayed increase in the number of apoptotic lymphocytes was observed in early apoptotic annexin-stained cells and in late apoptotic cells, whereas in nonirradiated cells no remarkable changes were found. Apoptosis was confirmed by altered light scattering properties and DNA fragmentation. CONCLUSION: The apoptotic cell death of reinfused lymphocytes is supposed to be a therapeutic effect of ECPI.