Trisodium citrate → 12-Hydroxy-5,8,10 heptadecatrienoic acid

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Reaction Steps

Eight ml of blood was gathered from the abdominal aorta of a rat (JcI: Wistar, male, 12-15 weeks age) subjected to abdominal incision under anesthesia by the use of 3.2% sodium citrate (1 volume of sodium citrate solution to 9 volume of whole blood). The blood was centrifuged at 800 rpm for 10 minutes at room temperature to gather PRP (platelet rich plasma), and the residual blood was further centrifuged at 3000 rpm for 10 minutes to gather PPP (platelet poor plasma). The platelet number in PRP was measured and the PRP was adjusted by diluting with PPP so that the concentration of platelet was one million/μl. To 0.25 ml of the PRP, 125 μg of arachidonic acid and the test compound (the final concentration of 100 μM, 10 μM, 1 μM and 0.1 μM) were added, and the mixture was incubated at 37° C. for 15 minutes. After the reaction, 1.1 ml of ethanol was added, well shaken and centrifuged at 2000 rpm for 10 minutes to separate the supernatant. To 1 ml of the supernatant 1 ml of water was added and subjected to high performance liquid chromatography to determine HHT. The absorption at 240 nm due to HHT was measured by an ultraviolet absorption monitor. The inhibition rate of HHT formation calculated from the peak area is shown in Table 2.

Provenance & Evidence