Evaluation of inhibitors of the arachidonic acid cascade with intact platelets using an on-line dilution and on-line solid phase extraction HPLC-MS method.

An automatic on-line dilution/on-line solid phase extraction (SPE) system has been developed for the detection of metabolites of the arachidonic acid cascade in platelets. The method allows the direct injection of larger quantities of centrifugates from cell suspensions previously treated with an equal volume of an acetonitrile/methanol mixture for protein precipitation. The method was used to study the effect of inhibitors of platelet arachidonic acid cascade enzymes (cytosolic phospholipase Aα, cyclooxygenase-1, thromboxane synthase, 12-lipoxygenase) and related targets (cyclooxygenase-2, microsomal prostaglandin E synthase-1, 5-lipoxygenase) in intact platelets after stimulation with calcium ionophore A23187.

HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase using crude bovine plasma.

Recently, we have described a method for evaluation of plasma amine oxidase (PAO) inhibitors, which monitors the formation of 6-(5-phenyl-2H-tetrazol-2-yl)hexanal from the corresponding amine substrate by HPLC with UV-detection using purified bovine PAO. We now investigated, whether crude bovine plasma can be used as enzyme source in this assay instead of the purified enzyme. With the aid of specific inhibitors, it was ensured that there was no detectable activity of other important amine oxidases in the plasma, namely monoamine oxidase (MAO) A and B and diamine oxidase (DAO).

HPLC-UV assays for evaluation of inhibitors of mono and diamine oxidases using novel phenyltetrazolylalkanamine substrates.

Recently, we have described an HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase (PAO) using 6-(5-phenyl-2H-tetrazol-2-yl)hexan-1-amine (4) as a new type of substrate. Now we studied, whether this compound or homologues of it can also function as substrate for related amine oxidases, namely diamine oxidase (DAO), monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B). Among these substances, 4 was converted by DAO with the highest rate.

HPLC-UV method for evaluation of inhibitors of plasma amine oxidase using derivatization of an aliphatic aldehyde product with TRIS.

Plasma amine oxidase (PAO), which is also designated as semicarbazide-sensitive amine oxidase (SSAO), copper-containing amine oxidase 3 (AOC3), or vascular adhesion protein-1 (VAP-1), catalyzes the oxidative deamination of primary amines to aldehydes using copper and a quinone as cofactors. Because it participates in the transmigration of inflammatory cells through the blood vessels into the tissue, PAO is attributed an important role in inflammatory diseases. Therefore, inhibitors of this enzyme could lead to new therapeutics for the treatment of inflammation-related conditions.