Enzymatic assay of hydroperoxides in plasma

Peroxide-free arachidonic acid Haemin solution 

Platelet-poor plasma is prepared from venous blood by centrifugation at 1000 xg for 15 min, and an equal volume of ethanol is added. The mixture is left on ice for 10 min and then centrifuged at 6500 xg for 15 min. Cyclooxygenase activity is measured polarographically, using an oxygen electrode and an incubation buffer (total volume 3 ml) containing potassium phosphate (pH 7.2, 0.1 M), 100 pM arachidonic acid, 1 mM phenol and 2.5 mM sodium cyanide. The reaction is 

Measurements of the lag time are made in the absence of exogenous hydroperoxide in a cyanide-treated system (LagcN) a control system not including cyanide (LagcontroiX and a cyanide-inhibited system to which hydroperoxide had been added (Lag(CN+ROOH))- A fractional activation of the enzyme response to the added hydroperoxide is calculated as  

This allows normalization of responses so that results from different experiments can be compared. 

A standard curve of fractional activation against the concentration of pure 15-HPETE is also prepared, and the concentration in plasma calculated by reference to this calibration curve. 

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