Hidekuni Takahagi

Drug monitoring by a fully automated high-performance liquid chromatographic technique, involving direct injection of plasma.

A procedure involving direct injection of whole plasma for analyses of drugs by an automated high-performance liquid chromatograph was developed. This system comprised two columns, two pumps, one detector, two programmable switching valves, an automatic sample injector with a cooling device for sample tubes and a microprocessor. Effluents from the first column, containing a drug of interest, were selectively introduced into the second column for further separation. The columns used were an aqueous gel chromatography column (column 1) and an ODS column (column 2). The solvent for column 1 must be weaker than that for column 2, so that the solutes from the former will be enriched at the top of the latter. The validity and applicability of this procedure for the study of drug metabolism were demonstrated with the antibiotic cefmetazole, the anticoagulant warfarin, the antitumour agent carboquone and the anaesthetic ketamine.

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.

Determination of platelet activating factor using immunoaffinity mini-columns. Stimulation of platelet activating factor production by thapsigargin and effects of dexamethasone.

Thapsigargin, a hexaoxygenated tetraacylated sesquiterpene lactone isolated from the roots of Thapsia garganica L., is one of non-TPA type tumor promoters that stimulate prostaglandin E2 production at very low concentrations and induce inflammation when applied topically. Effects of thapsigar-gin on platelet activating factor (PAF) production by rat peritoneal macrophages were examined. Levels of PAF in the conditioned medium and in the cells were determined by radioimmunoassay after extraction of PAF using immunoaffinity mini-columns. It was demonstrated that thapsigargin at concentrations of 3 to 100 ng/ml stimulated production of cell-associated PAF when measured 10 min after incubation. Levels of PAF in the conditioned medium were less than the detectable amount.The stimulative effect by thapsigargin reached a plateau at a concentration of 30 ng/ml. Time course experiments revealed that the levels of cell-associated PAF showed a peak 10 to 15 min after incubation with thapsigargin at a concentration of 30 ng/ml. The levels were then declined until 40 min after incubation. When macrophages were preincubated for 3 h with dexamethasone at concentra-tions of 0.01, 0.1 and l μg/ml, thapsigargin (30 ng/ ml) -stimulated PAF production in the cells at 10 min were decreased. However, the maximum inhibition was about 50% even at 1μg/ml of dexamethasone. These date suggest that by treatment with thapsigargin, PAF is partly produced through formation of lyso-PAF by activated phospholipase A2 since dexamethasone shows partial inhibition of thapsigargin-induced PAF production, and thap-sigargin-stimulated PAF production may partly account for proinflammatory activity of thapsigar-gin.