Analysis of eicosanoid synthesis by isotope-labelled arachidonic acid

An ex vivo examination of the eicosanoid synthesis of platelets was carried out in Medium 199, which consists of mineral salts, nucleotides, amino acids, vitamins, carbohydrates and Ca²⁺ in the same quantity as the extracellular concentration but without fibrinogen or other plasma proteins. Rat platelets (2.5×10⁸ platelets/mL in each sample) from the four diabetic treatment groups were pre-incubated at 37°C for 3 min, while the rat abdominal aortic rings (15 mg wet mass/mL) were pre-incubated for 10 min in Medium 199 (Merck). The enzyme reaction examined started after pipetting the tracer substrate 1-¹⁴C-arachidonic acid (3.7 kBq, 0.172 nM in each sample; American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) into the incubation mixture. The enzyme reaction was stopped by bringing the pH to 3 with formic acid after 13 minutes of incubation in the case of the platelet samples and after 30 minutes of incubation in the case of the aortic samples. All of the samples were extracted with ethyl acetate, and the organic phases were evaporated. The residues were reconstituted in ethyl acetate and quantitatively applied to silica gel G thin-layer plates (Kieselgel G 60/DC-Fertigplatten, Merck, Art. 5721). Although the eluent front is straight and the wind effect is insignificant when using positive-pressure thin-layer chromatography, the order of loading the samples onto the thin-layer plate was changed for each new series of tests to prevent these technical errors. The plates were developed to a distance of 16 cm in the organic phase of ethyl acetate:acetic acid:2,2,4-trimethylpentane:water (110:20:30:100) using overpressure thin-layer chromatography (Chrompress 25, Labor MIM, Hungary) [31–34].

A semi-quantitative analysis of labelled AA metabolites was performed with a BIOSCAN AR-2000 Imaging Scanner (Eckert & Ziegler Radiopharma, Berlin, Germany) using Win-Scan 2D Imaging Software. The radiolabelled products of AA were identified with unlabelled authentic standards [29]. Assuming that the exogenously administered labelled AA as a tracer is converted in the same way as the endogenous source, our method allows us to measure the relative quantity of various prostanoids [19, 31].