Effect of St. John's Wort extract on intestinal expression of cytochrome P4501A2: studies in LS180 cells.

BACKGROUND AND AIMS: St. John's Wort (SJW) is known to induce expression and activity of cytochrome P4503A4 (CYP3A4). However, its effects on other cytochrome P450 (CYP) are not well understood. Our objective was to characterise the effect of SJW on the expression of CYP1A2 in the LS180 intestinal cell model. STUDY DESIGN AND METHODS: LS180 cells were cultured in the presence and absence of SJW extract for 48 hours. CYP1A2 protein content was measured by Western blot analysis using monoclonal antibody. Time-dependent expression of CYP1A2 was assessed during exposure to SJW extract for 24 hours and following its removal for another 24 hours. RESULTS: SJW increased the expression of CYP1A2 in the LS180 cells in a concentration dependent manner. The induction was time-dependent, as enzyme levels returned to baseline within 4-8 hours after removal of SJW. CONCLUSIONS: SJW reversibly induces expression of CYP1A2 in LS180 cells. This induction may be responsible for reduced plasma theophylline concentrations upon co-administration of SJW, as reported earlier.

Characterization of tubular functional capacity in humans using para-aminohippurate and famotidine.

BACKGROUND: Renal drug excretion by glomerular filtration and active tubular secretion may be altered by factors such as acute and chronic renal disease, nephrotoxins, and drug interactions. Thus, accurate and reproducible methods for quantitation of glomerular filtration rate (GFR) and tubular functional capacity are critical. METHODS: We utilized a four-step sequential infusion method to characterize anionic [para-aminohippurate (PAH)] and cationic (famotidine) tubular functional capacity in healthy volunteers. Filtration and secretion rates were quantitated from renal clearance and iothalamate-derived GFR determinations. RESULTS: Concentration-dependent renal clearance of PAH was observed at plasma concentrations> 100 mg/L; renal clearances were 442 +/- 131 (mean +/- SD), 423 +/- 94, 233 +/- 45, and 152 +/- 18 mL/min/1.73 m2 at plasma concentrations of 18 +/- 2, 92 +/- 5, 291 +/- 47 and 789 +/- 28 mg/L, respectively. The apparent affinity (Km) and maximum secretory capacity (TmPAH) were 141 +/- 70 mg/L and 71 +/- 16 mg/min/1.73 m2, respectively. The unbound renal clearance and tubular secretory clearance of famotidine were 384 +/- 70 and 329 +/- 78 mL/min/1.73 m2, respectively, and were not significantly correlated with the unbound plasma concentrations, which ranged from 126 to 2659 ng/mL. The rate of tubular secretion was linear at unbound plasma concentrations up to 2659 ng/mL. CONCLUSIONS: These data indicate that a sequential infusion method using PAH may be used to characterize the anionic secretory component of proximal tubular function. The tubular clearance of famotidine may be a suitable index of the cationic secretory capacity of the proximal tubule in humans. Saturation of the cationic secretory pathway was not observed, and further investigation into parallel pathways of cationic secretion, such as p-glycoprotein, may be warranted.

Determination of famotidine in human plasma and urine by high-performance liquid chromatography.

An improved, rapid and specific high-performance liquid chromatographic assay was developed for the determination of famotidine in human plasma and urine. Plasma samples were alkalinized and the analyte and internal standard (cimetidine) extracted with water-saturated ethyl acetate. The extracts were reconstituted in mobile phase, and injected onto a C18 reversed-phase column; UV detection was set at 267 nm. Urine samples were diluted with nine volumes of a mobile phase-internal standard mixture prior to injection. The lower limits of quantification in plasma and urine were 75 ng/ml and 1.0 microg/ml, respectively; intra- and inter-day coefficients of variation were < or =10.5%. This method is currently being used to support renal function studies assessing the use of intravenously administered famotidine to characterize cationic tubular secretion in man.

Comparison of iothalamate clearance methods for measuring GFR.

STUDY OBJECTIVE: To evaluate the bias and precision of three methods of measuring glomerular filtration rate (GFR) relative to a standard method. DESIGN: Prospective, outpatient study. SETTING: University-affiliated general clinical research center. PATIENTS: Twenty-six patients with various degrees of renal function (GFR range 25-151 ml/min/1.73 m2). INTERVENTIONS: Each patient received iothalamate twice during the study visit, first as a bolus injection and then as a priming dose followed by a constant-rate infusion for 2.5 hours. MEASUREMENTS AND MAIN RESULTS: Plasma (ClpIVB) and renal clearances (ClrIVB) after bolus injection and plasma clearance during constant-rate infusion (ClpINF) were compared with standard renal clearance during constant-rate infusion (ClrINF). All three measures were highly correlated with ClrINF (r>0.90, p<0.001). The mean ClrIVB was not significantly different from ClrINF (106.3+/-30.4 vs 104.2+/-28.5 ml/min/1.73 m2) and provided a precise (8.8%, 95% CI 6.5-11.1%) and unbiased measure of GFR. Both ClpIVB and ClpINF were positively biased; values exceeded ClrINF by 11.8+/-11.1 (p=0.0001) and 10.5+/-12.5 ml/min/1.73 m2 (p=0.0003), respectively. Use of a nonrenal correction factor of 9.8 and 10.5 ml/min/1.73 m2 for infusion and bolus plasma clearance values, respectively, eliminated bias and improved the precision of these methods. CONCLUSIONS: Iothalamate renal clearance after bolus injection is a simple, accurate, and precise measurement of GFR and may be a useful alternative to the standard infusion method in clinical investigations. The corrected plasma clearance provides a simple index of GFR for clinical practice.

Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.

Enterococci resistant to multiple antimicrobial agents, including vancomycin. Establishment of endemicity in a university medical center.

OBJECTIVES: To determine the distribution of and risk factors for colonization and infection with vancomycin-resistant enterococci; to evaluate the molecular epidemiology of these strains; and to assess the effect of interventions, including 1) strict adherence to infection control procedures and 2) restricted use of vancomycin. DESIGN: Problem identification based on descriptive studies, point-prevalence surveys, and case-control studies and followed by specific interventions and evaluation of the response to these interventions. SETTING: University medical center. PARTICIPANTS: All patients hospitalized between May 1992 and June 1994 (59,196 admissions). MAIN RESULTS: 75 active infections attributed to vancomycin-resistant enterococci were identified. Thirty-one patients (41%) had bloodstream infections and 6 (8%) died. The incidence of active infection was highest in the organ transplantation unit (13.2 infections/1000 admissions). In the point-prevalence studies, vancomycin-resistant enterococci were isolated from 20% of a random sample of hospitalized patients in July, August, and September 1993 (adjusted prevalence, 16.9%). Case-control studies showed significant associations between colonization and infection and 1) receipt of antimicrobial agents, particularly vancomycin, and 2) severity of illness. Although several small case clusters had isolates with identical banding patterns on pulsed field gel electrophoresis, at least 45 different banding patterns were noted among medical center isolates. Interventions took place in November and December 1993. Vancomycin restriction policies resulted in a 59% decrease in intravenous vancomycin use and an 85% decrease in oral vancomycin use. Point-prevalence surveys done in April, May, and June 1994 showed a consistent 20% level of colonization with vancomycin-resistant enterococci strains (adjusted prevalence, 18.7%). No significant changes were seen in rates of vancomycin-resistant enterococci infection. CONCLUSIONS: Vancomycin-resistant enterococci are an important cause of illness and death in the study institution, particularly among organ transplant recipients and other seriously ill persons; they have also become a common intestinal colonizer among hospitalized patients. The diversity of isolates (based on molecular typing studies) suggests that resistant organisms have been introduced from multiple sources. Interventions that effectively lower the overall level of colonization with vancomycin-resistant enterococci must still be identified.