High-performance liquid chromatographic assay with electrochemical detection for azithromycin in serum and tissues.

High-performance liquid chromatographic methods using reversed-phase chromatography and electrochemical detection have been developed for the quantitation of azithromycin in serum and tissues of laboratory animals and humans. Serum sample preparation involved addition of internal standard, alkalinization, and solvent extraction. Tissue sample preparation involved Polytron homogenization in acetonitrile containing internal standard, evaporation of the supernatant, alkalinization of the residue, and solvent extraction. Serum samples were chromatographed on an alkylphenyl-bonded silica column eluted with pH 6.8-7.2 mobile phase with a dual-electrode coulometric detector operated in the oxidative screen mode. Serum and tissue samples were chromatographed on a gamma RP-1 alumina column with pH 11 mobile phase with a glassy carbon amperometric detector. Recovery of azithromycin was 87% from serum and 85% from tissues. Linear standard curves were prepared in serum over two concentration ranges (0.01-0.20 and 0.20-2.0 micrograms/ml) and in tissues over several concentration ranges (0.1-2, 1-10, 10-100, and 100-1000 micrograms/g). In serum and tissues, intra- and inter-assay precision ranged from 1 to 8% and 4 to 11%, respectively. The tissue assay has been applied to liver, kidney, lung, spleen, muscle, fat, brain, tonsil, lymph nodes, eye, prostate and other urological tissues, and gynecological tissues.

Interspecies correlation of the pharmacokinetics of erythromycin, oleandomycin, and tylosin.

Knowledge of the disposition of macrolides in a single animal species has been insufficient for the prediction of the pharmacokinetics of macrolides in humans. To better understand the species differences in the pharmacokinetics of macrolide antibiotics, the disposition of erythromycin, oleandomycin, and tylosin in several mammalian species was examined. Generally, the serum concentration versus time profiles of these drugs after intravenous administration were described by two-compartment kinetic models and were similar within each species. These drugs were rapidly cleared, resulting in terminal half-lives of less than 2 h. Comparison of their pharmacokinetics showed greater variation in antibiotic disposition among animal species than noted for the differences within a species. When the pharmacokinetic data was fitted to an allometric model, the logarithms of volume of distribution, clearance, and half-life were linearly related to the logarithms of body weight. From these relationships, the human pharmacokinetics of erythromycin and oleandomycin were extrapolated and found to approximate observed human pharmacokinetics.

The defects in all classes of aryl hydrocarbon hydroxylase-deficient mutant of mouse hepatoma line, Hepa-1, are restricted to activities catalyzed by cytochrome P-450.

The extent of phenotypic derangement was investigated in representative aryl hydroxylase-deficient mutants of Hepa-1. One mutant is dominant while the others are recessive and are mutated in 3 different genes. All the mutants lacked ethoxyresorufin-O-deethylase activity as well as aryl hydrocarbon hydroxylase. However all had normal activities of NADPH-cytochrome P-450 reductase, epoxide hydrolase and ornithine decarboxylase. They also retained 2 liver-specific functions possessed by the parental line, namely albumin secretion and transferrin secretion. The phenotypic alterations in the mutants were therefore restricted to the cytochrome P-450 activities measured.

Characterization of NADPH-cytochrome P-450 reductase in a mouse hepatoma cell line.

NADPH-cytochrome c reductase in Hepa-1 cells was induced 2-fold by phenobarbital, but was not induced by benz[a]anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The apparent Km of the enzyme for NADPH was 0.57 microM; the activity was inhibitable by NADP; and segregated primarily to the microsomal fraction. Cytoplasm of Hepa-1 cells bound antibody to rabbit cytochrome P-450 reductase. 3T3 cells, which possessed one sixth of the cytochrome c reductase activity of Hepa-1 cells, bound correspondingly less cytochrome P-450 reductase antibody. This supports the notion that cytochrome P-450 reductase was responsible for the cytochrome c reductase activity that was measured.

A long-lived human diploid fibroblast line for cellular aging studies: applications in cell hybridization.

We have isolated a diploid fibroblast culture from human fetal lung with an in vitro lifespan of about 100 population doublings. The culture grows very well at clonal densities and long-lived clones can be isolated for use in cellular aging studies. The longer in vitro lifespan of the culture has allowed us to isolate from it a clone, containing a dominant and recessive mutation, having significant remaining proliferative potential. The nature of the mutations will allow for hybrid selection, after fusion of the mutant clone with wild type human cells. The mass culture and clones derived from it provide a valuable resource for cell aging studies.

In vitro proliferation and lifespan of bovine aorta endothelial cells: effect of culture conditions and fibroblast growth factor.

The effect of culture conditions on calf dorsal aorta endothelial cells was studied. Population doubling time varied as a function of the cell seeding density, growth Medium, serum supplement, and concentration of fibroblast growth factor (FGF). The shortest population doubling time was found for cells (population doubling level 0--30) grown in Eagle's Minimal Essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml FGF. The stimulatory effect of FGF on bovine endothelial cell proliferation was dependent on cell inoculation density. FGF significantly increased cell division rate at cell inocula less than 1 x 10(4) cells/cm2 but not at higher densities. The population doubling time and cell size increased as the mass culture population doubling level increased. The replicative lifespans of bovine endothelial cells grown in medium supplemented with 20% FBS were 10--15% greater than parallel cultures supplemented with 10% FBS. Cultures grown in medium supplemented with 10% FBS and 50 ng/ml FGF showed a 50% increase in replicative lifespan compared to cultures grown in medium supplemented with 10% FBS alone. When FGF was used the increase in the number of doublings was a function of the length of time the cells were grown in the presence of FGF. This report extends comparable observations on the in vitro aging of human diploid fibroblasts to bovine endothelial cells.