An improved capillary gasoline chromatography technique is described for the assay of urinary phenol for the evaluation of benzene publicity. Enzymatic hydrolysis with an extract of Helix pomatia is used to liberate phenol from its glucuronide and sulphate conjugates in urine and has the benefit in that full hydrolysis happens and clear chromatograms are produced.

Moreover, an inner customary (2,4-xylenol) has been integrated within the technique enabling fast and single step extraction of phenol. The microassay can be utilized to measure phenol concentrations as little as 1 mg/l in urine thus making it an appropriate technique for monitoring benzene publicity.

A microdetermination of propranolol enantiomers and of their glucuronide and sulphate conjugates in human plasma and urine by reversed-phase HPLC after chiral derivatization is described. After extraction from 100 microliters of plasma or urine with racemic 4-methylpropranolol as inner customary (I.S.), the enantiomers are derivatized with R(+)-phenylethylisocyanate as chiral derivatization reagent. Chromatography is carried out on Novapak C18 column with fluorescence detection. Glucuronide and sulphate conjugates are cleaved previous to extraction by incubating, respectively, the samples with glucuronidase-arylsulphatase and saccharic acid 1-Four lactone as particular glucuronidase inhibitor.

The retention instances of propranolol and I.S. enantiomer derivatives are brief (tR = 5.5-6.2 min and eight.8-10.1 min, respectively). The diastereomeric derivatives are very secure and present good peak symmetry and resolutions (RS = 2 and a pair of.2). The usage of 4-methylpropranolol as I.S. improves considerably relative customary deviations (RSD: 1.7-5.1). Sensitivity is about 1 ng ml-1 per enantiomer. The tactic is utilized to pharmacokinetic research of racemic propranolol in human plasma and urine. S-propranolol and its conjugates present larger concentrations than R-propranolol and its conjugates in plasma and urine.

The avidity of leukocyte integrin CR3 (Mac-1, CD11b/CD18, alpha m beta 2) on neutrophils (PMN) could also be quickly modulated by a number of agonists. We describe a technique for figuring out the avidity of those receptors by measuring the adhesion of PMN to fibrinogen-coated surfaces. Cells are loaded with a succinimidyl ester of carboxyfluorescein diacetate, which is deacetylated by intracellular esterases yielding a extremely fluorescent carboxyfluorescein that continues to be trapped inside the PMN.

The variety of cells adhering to fibrinogen-coated wells of Terasaki microplates is quantitated with a fluorescence plate reader. Stimulation of PMN with quite a few agonists, together with PMA, fNLLP, Ca-ionophore A23187, interleukin-8, tumor necrosis issue and lipopolysaccharide strongly elevated adhesion to fibrinogen, which was CD11b/CD18 dependent. The extent of cell adhesion trusted stimulus focus and incubation time. This assay requires little time, makes use of small numbers of cells and doesn’t require hazardous reagents.

This examine offers with the event of a delicate and easy microassay for measuring cytochrome P450IA1 and P450IIB1 actions in intact human and rat hepatocytes. The tactic relies on the metabolism by cells cultured in microwells of acceptable substrates at noncytotoxic concentrations (Eight microM 7-ethoxyresorufin and 15 microM 7-pentoxyresorufin). After incubation of the probes with the cells, the dealkylated resorufin shaped and launched into tradition medium was quantified.

To make sure the hydrolysis of the resorufin conjugates ultimately shaped, tradition supernatants had been incubated within the microwells with beta-glucuronidase and arylsulfatase. The fluorescence was then learn utilizing a microplate fluorescence reader. A excessive correlation between the monooxygenase exercise measured by this process and that measured by typical procedures within the microsomal fraction of the identical cells was discovered.

The main benefits of this technique are: (1) the small variety of cells required; (2) a drastic discount in assay time; (3) that the assay is carried out in intact cells; and (4) the potential of performing repeated assays with the identical cell monolayer over a interval of a number of days since no damage to cells is detectable in the course of the assay. This technique proved to be very handy for learning cytochrome P450 induction by xenobiotics in main cultures of human hepatocytes.