Effect of beta-carotene on ultraweak chemiluminescence of UVB-irradiated squalene.

Abstract

An emission spectral analysis was carried out on chemiluminescence emitted from UVB-irradiated Squalene, The main emission species produced by the transition of ('dg) ('dg) and ('dg) ('gi to (3g_) (3g_) were found by spectroscopic analysis of the chemiluminescence. When betacarotene was added to the irradiated squalene, its spectral pattern changed drastically and many peaks disappeared. ---squalene; chemiluminescence; beta-carotene The chemiluminescence (CL) analyzer has been developed originally for estimation of the value of autoxidized oil. Miyazawa and Kaneda (1980), who applied this technique to living tissues, successfully showed that the increase of CL in intensity was closely related to the peroxidation process of the tissue. We measured the intensity of CL in rat skin with UV-irradiation and it was concluded that the weak CL would be attributable to singlet oxygen (Torinuki and Miura 1981). The light-emitting species of UVB-irradiated squalene, lipid derived from sebum, were analyzed spectroscopically and the effect of betacarotene on CL was also examined.First of all, qualitative analysis of squalene (Eastman Kodak Co., USA) was carried out on a thin layer chromatography plate. Two drops of squalene were resolved with a small amount of n-hexane, and an aliquot of the squalene solution was spotted on a Merk silica gel 60 coated plate (gel layer 0.25 mm thick). The plate was developed in a tank containing 100 ml of n-hexane, and only one spot (Rf value 0.64) was detected on the plate with I2. After the spot portion was scraped and added to 30 times volume of methanol, the absorption spectrum of squalene extracted in methanol solution was determined with a Hitachi Model 124 spectrophotometer. Absorption peak was observed only at 215 nm.Emission spectral analysis of CL of UVB-irradiated squalene was performed as previously described (Nakano et al. 1975). Twenty millilitres of squalene in Petri dish was irradiated with a sunlamp tube (Toshiba FL-205E, peaking at 310 nm) at a distance of 4 cm with stirring at room temperature. Irradiation times was 168 hr and the energy output of this instrument was 1.0 mW/sq cm, with a target distance of 4 cm. After irradiation, the light emission of the sample was recorded with a CL detector (Research Institute of Electrical Communication, Tohoku University). The result is shown in Fig. 1 (solid curve). The location of the emission peak at 478 nm (B), 520 nm (C), 588 nm (D) and 650 nm (E) are assigned to ('dg) (l~g+)-+(3~g-) (3Egi transition of 02 pairs (0, 0), ('dg) (idgY (3Eg-) (3~g-) transition of 02 pairs (2, 0), ('dg) (idg)-*(3>g-) (3~g-) transition of 02