Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counter-receptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10–2000 and 50–2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.

A high-performance liquid chromatographic (HPLC) assay was developed for the determination of the ratios of the (S)-(+) and (R)-(-) enantiomers of the anti-inflammatory drug carprofen in blood, urine, and feces. The procedure relies on: (a) extraction and purification of carprofen from biological fluids, (b) reaction of carprofen with (S)-(-)-α-methylbenzylamine to form the two diastereomeric (S)-(-)-α-methylbenzylamides via the 1, 1′-carbonyldiimidazole intermediate, (c) purification of the reaction mixture by extraction of the diastereomeric derivatives into hexane at pH 11, and (d) analysis of the diastereomeric derivatives by HPLC with UV detection. The (S)-(+):(R)-(-) ratios in the blood of three subjects receiving single 100-mg oral doses of carprofen were greater than unity up to 16 hr after dosing. The mean ± SD of the ratios in the early blood samples (0.5, 1, and 2 hr) was 1.21 ± 0.09, while the mean of the ratios in the later blood samples (4, 6, 8, 12, and 16 hr) was slightly higher (1.48 ± 0.17). The blood level fall-off curves for the (S)-(+) and (R)-(-) enantiomers were similar in each of the three subjects for the 4-16-hr period. The carprofen enantiomers were excreted stereoselectively by humans. An excess of the (S)-(+) enantiomer relative to the (R)-(-) enantiomer was excreted in the urine as the ester glucuronide, while unchanged (R)-(-) enantiomer predominated in the feces. The total urinary plus fecal excretion of the enantiomers (0-96 hr) revealed only a slight excess of the (S)-(+) enantiomer over the (R)-(-) enantiomer, which amounted to 2.1-4.9% of the dose. Since the amount of carprofen (free and glucuronide) excreted in 96 hr by the three subjects only accounted for 62-72% of the dose, no definitive statement could be made relative to the possible inversion of the carprofen chiral center.