765 (2001) 199–203 Journal of Chromatography B, www.elsevier.com / locate / chromb Technical note Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides * Junji Terao , Mariko Miyoshi, Sayuri Miyamoto Department of Nutrition, School of Medicine, The University of Tokushima, Kuramoto-Cho 3, Tokushima 770-8503 Japan Received 1 July 2001; received in revised form 7 September 2001; accepted 17 September 2001 Abstract A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phos- phatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.  2001 Elsevier Science B.V. All rights reserved. Keywords: Phospholipid hydroperoxides; Cholesteryl ester hydroperoxides 1. Introduction tive and reproducible. On the other hand, Taki et al. [8,9] have developed thin-layer chromatography Lipid peroxidation attracts much attention because (TLC) blotting as a simple isolation method for of its possible role in a variety of pathophysiological glycolipids from biological tissues. We therefore events. Lipid hydroperoxides are the primary prod- developed this TLC blotting technique for detecting ucts of lipid peroxidation and their detection has lipid hydroperoxides and succeeded in the specific been frequently used as an effective tool for moni- detection of lipid hydroperoxides on the blotting toring lipid peroxidation in biological systems in membranes. vivo and in vitro. Several high-performance liquid chromatography (HPLC) methods for the separation and quantification of lipid hydroperoxides have been 2. Experimental reported in the literature [1–5]. Akasaka et al. [6,7] determined lipid hydroperoxides using HPLC with 2.1. Materials fluorescence detection. However, the HPLC tech- nique is time-consuming and requires special instru- Egg yolk phosphatidylcholine (PC) and choles- mentation, although fluorescence detection is sensi- teryl linoleate were obtained from Sigma (St. Louis, MO, USA). Phosphatidylcholine hydroperoxides (PC-OOH) were prepared from egg yolk PC by *Corresponding author. Tel.: 181-88-633-7087; fax: 181-88- reaction with soybean lipoxygenase and were iso- 633-7089. E-mail address: terao@nutr.med.tokushima-u.ac.jp (J. Terao). lated using reversed-phase column chromatography 0378-4347 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0378-4347(01)00426-1