Anwendung der Elektronenspinresonanzspektrometrie und der Spin-Trapping-Methode für die Untersuchung der Wirkung von Retinoiden und Carotinoiden auf die Bildung von Sauerstoffradikalen in stimulierten neutrophilen Granulozyten
Reactive oxygen species (ROS) released during phagocytosis were measured in human white blood cells (neutrophilic granulocytes) using electron spin resonance spectroscopy (EPR) and the spin trapping techniques.
ROS in biologic systems are highly toxic and transformed to less reactive products very fast by cell protection mechanisms. Therefore they have a very short half-life that makes their detection and measurement difficult. In the spin trapping techniques, spin trap molecules are used to react with the ROS and form intermediate products that are chemically more stable and can be detected by EPR. Spin trap molecules are diamagnetic substances that cause absorption lines in the EPR spectrum only after reaction with ROS. The shape of the absorption spectrum depends on the kind of the spin trap molecules and on the properties of the bound radical. Especially useful for the identification of the spectrum is the splitting of the spectral lines that is caused by the hyperfine interaction.
The circulating granulocytes react to chemotactic stimuli from local infections etc. by infiltrating the tissue and fight infection agents by the bactericide effects of the phagocytosis, which are essentially based on the production of ROS.
Systemic retinoids are widely used in the treatment of severe types of psoriasis, of acne, and dermatoses with inborn errors of keratinization. Their application is mainly based on their regulating effect on the basal and differentiated keratinocytes. They lead to the diminution of the inflammation and the inhibition of the migration of neutrophilic granulocytes. Carotenoids are used in the prevention of the skin from UV induced inflammation and aging and from cancer.
We investigated the in vitro and in vivo effects of retinoids and carotenoids on the generation of ROS by stimulated neutrophils using EPR spin trapping techniques. In addition, in a cellular free xanthine/xanthine oxidase system we looked for direct interactions between the retinoids or carotenoids and the generated radicals. The effects of retinoids were investigated in patients with Psoriasis vulgaris under an acitretin/PUVA combined therapy, carotenoids were measured in the scope of a placebo controlled double blind study under sun light simulating UV radiation.
An isomer specific behaviour was found in both the retinoids and the carotenoids. A clear antioxidant activity in the xanthine/xanthine oxidase system was measured for 13-cis and 9-cis retinoic acid, acitretin and all-trans-ß-carotene. In the cellular system some retinoids and carotenoids (all-trans-, 9-cis retinoic acid, acitretin, 13-cis-acitretin, all-trans-ß-carotene) showed a stimulating effect on the ROS release, especially on low concentrations. Others (13-cis retinoic acid, 13-cis- and 9-cis-ß-carotene) caused a concentration dependent inhibition of the ROS production. In the scope of the acitretin/PUVA combined therapy no clear inhibition of the ROS production could be found in vitro and in vivo. During the application of carotenoids for UV damage protection a protective effect on the cellular defence mechanisms was found in vivo. After the radiation the ROS production returned to normal values, while in the placebo group it remained increased.