Platelet-rich plasma was prepared by centrifugation of citrated blood at 200 g for 15 mins. To induce platelet aggregation, ADP was added at a concentration of 1.2×10-6 M. For the prostanoid assay, 10 mL of blood was placed in a tube containing 0.5 M EDTA 0.1 mL and ASA (0.4%) 0.1 mL, and centrifuged for 10 mins to obtain platelet-rich plasma and for 15 mins to obtain platelet-poor plasma. Plasma was stored at -70°C, and 6-keto-prostoglandin F1a and thromboxane B2 (the stable conversion products of prostoglandin I2 and thromboxane A2, respectively) levels in plasma were determined by radioimmunoassay, by means of commercial reagent kits from the Institute of Isotopes (Budapest, Hungary), according to the manufacturer’s instructions. 6-keto-prostaglandin F1a and thromboxane B2 levels were expressed in pg/mL of plasma.The level of the major metabolite of melatonin (6-sulphatoxymelatonin [aMT6s]) in urine was measured by enzymatic immunoassay by using a commercial reagent kits from DRG Instruments GmbH (Marburg, Germany).
The total excretion of nitrites and nitrates in urine was determined by means of a modified Griess method as follows: Total excretion of nitrites and nitrates = £(NO2″ + NO3″)
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