Application of liquid chromatography-mass spectrometry(n) analyses to the characterization of novel glyburide metabolites formed in vitro.

The application of bench-top ion-trap atmospheric pressure ionization mass spectrometry in the characterization of in vitro metabolites of glyburide is discussed. The metabolites formed in vitro by rat, dog, monkey and human liver microsomes were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (MS)n experiments. The utility of data dependent MS1-MS2-MS3 analyses, where the mass spectrometer makes "real-time" decisions about the experiment to be performed, are described using the characterization of two novel metabolites of glyburide as an example. The metabolite profiles from each species were similar. Six cyclohexyl hydroxylation products were detected, as well as two novel monooxygenation products formed via hydroxylation of the ethyl chain at the benzylic position, and alpha to the amide nitrogen. The ion-trap with electrospray ionization proved to be a sensitive and reliable HPLC detection system that provided important chemical structure information.

Lauric acid as a model substrate for the simultaneous determination of cytochrome P450 2E1 and 4A in hepatic microsomes.

In vitro techniques have been utilized to investigate the microsomal enzymes involved in the metabolism of lauric acid and to establish conditions in which it can be used as a model substrate for both cytochrome P450 4A and cytochrome P450 2E1 in human liver microsomes. Studies of enzyme kinetics of lauric acid omega-hydroxylation in human liver microsomes indicated the involvement of more than one enzyme in this pathway, a relatively low Km enzyme with a Km of 22 microM +/- 12 (n = 8) and a high Km enzyme with a Km an order of magnitude higher (550 microM +/- 310, n = 7). The apparent Vmax for this component correlated with the rate of cyclosporin metabolism and was highly sensitive to ketoconazole inhibition. These results indicated that this enzyme was a member of the 3A subfamily. The activity associated with the low Km enzyme (P450 4A) did not correlate with P450 1A2, 2A6, 2C9/8, 2C19, 2D6, 2E1, or 3A activities in a bank of human liver microsomes and was not appreciably inhibited by ketoconazole, furafylline, quinidine, sulfaphenazole, or diethyldithiocarbamate (DDC). Lauric acid omega-1 hydroxylation demonstrated simple Michaelis-Menten kinetics in each of the human liver microsomal samples examined, with a Km of 130 microM +/- 42 (n = 8). This activity was highly correlated with chlorzoxazone 6-hydroxylation in human liver microsomes (r = 0.98, n = 14, p < 0.001) and was inhibited by both DDC and chlorzoxazone. Additionally, rats treated with the P450 2E1 inducer isoniazid demonstrated a 3-fold increase in lauric acid omega-1 hydroxylation relative to the control group. Thus, the lauric acid hydroxylation assay, at a substrate concentration of 20 microM, appears to be an effective and specific P450 model substrate capable of determining simultaneously P450 4A and P450 2E1 related activities in hepatic microsomal samples.

Temporal relationship of changes in hepatobiliary function and morphology in rats following alpha-naphthylisothiocyanate (ANIT) administration.

These studies were designed to evaluate ANIT-induced changes in both hepatobiliary function and morphology during the onset, progression, and recovery of ANIT-induced cholestasis. A single oral dose of 150 mg/kg of ANIT or vehicle was administered by gavage to male Sprague-Dawley rats and hepatobiliary structure and function were evaluated 16, 24, 48, 72, and 168 hr later. Increased hepatocellular tight junction permeability, increased serum bile acids, and decreased bile acid excretion were observed 16 hr after ANIT administration. At 24 hr, bile flow was decreased in ANIT-treated rats, an effect accompanied by increased tight junction permeability, decreased bile acid excretion, and decreased erythritol clearance (estimate of canalicular flow). In addition, scattered small loci of hepatocellular necrosis accompanied by an inflammatory cell response were observed in ANIT-treated rats at this time, with no microscopic evidence of bile duct obstruction (BDO). These data suggest that the onset of ANIT-induced cholestasis was associated with hepatocanalicular changes and not BDO. In contrast, at 48 and 72 hr after ANIT treatment, cholestasis was more profound and was accompanied by mild hepatocellular necrosis and widespread BDO. Hepatocyte tight junction permeability in ANIT-treated rats was not different from controls at 72 hr. These data suggest that the pathogenesis of ANIT-induced cholestasis is biphasic; the onset of cholestasis appears to be associated with changes in hepatocanalicular function and increased tight junction permeability whereas the later and more profound phase of cholestasis appears to be related to a combination of BDO and hepatocellular dysfunction. The time course of biochemical and morphologic changes following ANIT treatment further suggests that the pathophysiologic changes during the onset or initiation phase of cholestasis differ from those involved in the later and more profound phase of ANIT-induced cholestasis.

Characterization of the renal effects and renal elimination of sulotroban in the dog.

The renal effects and renal handling of the nonprostanoid thromboxane receptor antagonist, sulotroban (4-[2-(phenylsulfonylamino)ethyl]phenoxyacetic acid), were characterized in dogs. Sulotroban was infused i.v. at 0.06, 0.2, 0.6 and 1.0 mg kg-1 min-1 (plus prime) for 180 min. Arterial blood pressure was reduced significantly during infusion of the 1.0 mg kg-1 min-1 dosage only. Diuresis, characterized by increases in both fractional and absolute urinary excretion of sodium, potassium, chloride and calcium, and decreases in urine osmolality occurred at each of the sulotroban dosages tested. The renal clearance of sulotroban exceeded the glomerular filtration rate, suggesting renal secretion of sulotroban. The transport maximum for sulotroban secretion was approximately 160 micrograms kg-1 min-1. Renal cortical slices from naive dogs accumulated [14C]sulotroban against a concentration gradient. Sulotroban accumulation was blocked by metabolic inhibitors (dinitrophenol and sodium azide) and inhibitors of organic anion transport (probenecid and p-aminohippurate), but not by inhibitors of organic cation transport (cyanine and tetraethylammonium), suggesting that tubular secretion of sulotroban is mediated by an organic anion transport system. It was concluded that: 1) decreases in blood pressure occurred only after high dosages and were associated with high plasma sulotroban concentrations; 2) diuresis occurred at all dosages and may represent a separate pharmacological action unrelated to thromboxane receptor antagonism; and 3) renal excretion of sulotroban in the dog occurs by both filtration and tubular secretion with secretion occurring via an organic acid transporter.

Hepatic and renal conjugation (Phase II) enzyme activities in young adult, middle-aged, and senescent male Sprague-Dawley rats.

Acetaminophen (APAP)-induced nephrotoxicity is age dependent in male Sprague-Dawley rats: nephrotoxicity occurs at lower dosages of APAP in 12- to 14-month olds compared with 2- to 3-month olds. The mechanisms responsible for enhanced nephrotoxicity in 12-month-old Sprague-Dawley rats are not entirely clear, but may be related to age-dependent differences in APAP metabolism in liver and/or kidney. Major pathways of hepatic APAP metabolism include sulfation and glucuronidation; glutathione conjugation represents a pathway for detoxification of reactive oxidative APAP metabolites. The present studies were designed to quantify in vitro activity of three Phase II enzyme activities: glutathione S-transferase using 1-chloro-2,4-dinitrobenzene as substrate, UDP-glucuronyl transferase using APAP as substrate, and sulfotransferase using APAP as substrate, in subcellular fractions of liver and kidney of 3-, 12-, 18-, and 30-month-old naive male Sprague-Dawley rats. In liver, glutathione S-transferase, UDP glucuronyl transferase, and sulfotransferase activities were not significantly different in rats from 3 through 30 months of age. Renal UDP glucuronyl transferase and sulfotransferase activities were similar in rats from 3 through 30 months of age. In contrast, renal glutathione S-transferase activity was characterized by a lower Km in 12- and 30-month olds when compared with 3-month olds. These data suggest that the reduced total systemic clearance of APAP in 12-month-old male Sprague-Dawley rats previously observed cannot be attributed to age-dependent differences in hepatic APAP metabolism. In addition, it is unlikely that differences in renal APAP metabolism contribute to age-dependent APAP nephrotoxicity.

Evaluation of the fit and strength of an all-ceramic fixed partial denture.

A laboratory investigation of the fit and strength of three-unit all-ceramic FPDs was performed with a metal-ceramic FPD control. The conclusions were: 1. No difference was found between the marginal seal of the metal-ceramic and all-ceramic FPDs. 2. The all-ceramic FPDs had a more uniform cement space and their occlusal seat was significantly (99.9%) better than the metal-ceramic restorations. 3. The metal-ceramic FPDs were significantly stronger than the all-ceramic FPDs. 4. Load calculations suggest that short-span all-ceramic FPDs may be strong enough to resist normal masticatory forces for selected patients. However, generalized usage will probably result in an unacceptable failure rate.

Shrink-free ceramic.

An all-ceramic crown employing the Cerestore system utilizes a unique shrink-free alumina ceramic as its substrate. This core renders the restoration exceptional fit and strength. A ceramic layering technique employed with the aluminous porcelain veneers offers the capability of constructing a full crown restoration incorporating the optical properties present in a natural tooth.

The effect of various finish line preparations on the marginal seal and occlusal seat of full crown preparations.

The influences of the marginal design of a full crown on the occlusal seat and marginal seal of a cemented full crown restoration was examined. Under the conditions of the study, the featheredge and parallel bevel preparations demonstrated the best marginal seal, followed in order by the full shoulder, 45-degree shoulder, and finally the 90-degree shoulders with 30-degree and 45-degree bevels. With regard to seating of the restoration, the 90-degree full shoulder demonstrated the best seat, followed in order by the 45-degree shoulder, 90-degree shoulder with 45-degree bevel, featheredge, 90-degree shoulder with 30-degree bevel, chamfer with parallel bevel, and finally 90-degree shoulder with parallel bevel.

A histologic and electronic evaluation of electrosurgical currents: nonfiltered full-wave modulated vs. filtered current.

A controlled system for examining the tissue alteration and healing of electrosurgically produced wounds was established. Standardization of width, depth, stroke, amperage, average voltage, and wave form was achieved. A histologic comparison of tissue alteration and healing produced by a conventional blade and two electrosurgical currents was carried out. The need for standarization of instrumentation and clinical procedures is discussed.