Abstract
In this work we studied the inhibitory properties of derivatives of 1,4-dihydropyridine (DHP) in the oxidation of arachidonic acid to aliphatic hydroperoxides, catalyzed by soybean lipoxygenase. The test system used represented lipoxygenase enclosed in inverted micelles of the sodium salt of the di(2-ethyl)hexyl ester of sulfosuccinic acid (aerosol OT, AOT) in octane. The following DHP were tested: a coronary vasodilator, antagonist of Ca 2+ ions, foridon (riodipine) - 2,6-dimethyl-3,5-dimethoxycarbonyl-4-(o-difluoro methoxy)phenyl-l,4dihydropyridine (I) [i0] as a representative of 4-substituted 1,4-dihydropyridines, as well as the antioxidant diludin (diethone, ethidine) -- 2,6-dimethyl-3,5-diethoxycarbonyl-l,4dihydropyridine (II) [2, 3] and its oxidized form -- 3,5-diethoxycarbonyl-2,6-dimethylpyridine (III), which have no substituents in the 4-position. The DHP were synthesized earlier according to the methods described in [2, 3, ii], at the Institute of Organic Synthesis of the Academy of Sciences of the Latvian SSR, and were kindly provided for the investigations by Candidate of Chemical Sciences V. V. Kastron (foridon) and Candidate of Chemical Sciences Ya. R. Uldrikos (diludin and its oxidized form). EXPERIMENTAL (BIOLOGICAL) In the work we used soybean lipoxygenase (EC 1.13.11o12) and arachidonic acid from Serva (Federal Republic of Germany) and AOT from Fluka (Switzerland). The water content in the AOT preparations, determined by the Fischer method, was 0.7 mole of water per mole of AOT. Octane was treated with sulfuric acid, with aluminum oxide, dried over phosphorus pentoxide, and redistilled over metallic sodium. The activity of lipoxygenase in the oxidation of arachidonic acid was determined at 25~ by a spectrophotometric method, recording the increase in optical density (at the wavelength 250 nm) corresponding to the accumulation of the reaction product -- the conjugated diene of arachidonic acid~ The differential coefficient of extinction (i.eo, the difference of the coefficients of extinctioh:of the product and substrate) decreases by a factor of 1o9 in the transition from the wavelength 234 nm (e=34 = 2.5"i04 M-1"cm -I, absorption maximum of the product) to the wavelength 250 nm [12]. A solution of lipoxygenase (1.5 mg/ml) was prepared in 0.05 M Tris-HCl buffer, pH 8.5. To prepare the reaction mixture, 20 ~i of chloroform, containing various amounts of DHP, and 50 ~i of the lipoxygenase solution were added to 2.7 ml of 0.05 M AOT in octane, and the mixture was shaken for 30 sec until a transparent solution was obtained. The reaction was begun by introducing 50 ~i of a 3.2.10 -2 M solution of arachidonic acid into 0.05 M AOT in octane (0.6 m}{ in the reaction). Chloroform in the amounts used had no effect on the rate of the enzymatic reaction~ The change in the optical density was recorded in 2-millimeter cuvettes on a Cary-219 spectrophotometer from Varian (Switzerland), equipped with a thermostatically controlled cuvette compartment. All-Union Vitamin Scientific Research Institute. Vitaminy Scientific-Indus
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