Biomedical subjects

Thomas F Kalhorn

Publications and source records attributed to Thomas F Kalhorn.

12 recordsLinked to original sources

PubMed2006

Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography-mass spectrometry.

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is approximately 20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.

Chromatography, High Pressure Liquid↗

PubMed2005

Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1alpha,25-dihydroxyvitamin D(3): implications for drug-induced osteomalacia.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Base Sequence↗

PubMed2005

A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells.

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of approximately 50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.

Antineoplastic Agents↗

PubMed2005

Metabolism-based cyclophosphamide dosing for hematopoietic cell transplant.

When cyclophosphamide (120 mg/kg) is used for hematopoietic cell transplant, the increased area under the curve of carboxyethylphosphoramide mustard (AUC(CEPM)) is related to liver toxicity and death. We determined the feasibility of dose-adjusting cyclophosphamide to a preset metabolic endpoint (AUC(CEPM), 325 +/- 25 micromol/L.h). In 20 patients blood sampling was done over a 16-hour period after administration of 45 mg/kg cyclophosphamide; AUC(CEPM) from 0 to 16 hours was calculated by noncompartmental analysis. The expected AUC(CEPM) for 0 to 48 hours was estimated, and the second cyclophosphamide dose was determined. The mean second cyclophosphamide dose was 42 mg/kg, and the mean total cyclophosphamide dose was 86 mg/kg (range, 54-120 mg/kg). The mean AUC(CEPM) for the time from 0 to 48 hours was 296 micromol/L.h (95% confidence interval, 275-317 micromol/L.h). A retrospective analysis indicated that AUC(CEPM) could be more accurately predicted by use of a population pharmacokinetic model. We conclude that metabolism-based dosing of cyclophosphamide is feasible and that a lower cyclophosphamide dose does not affect engraftment.

Adolescent↗

PubMed2004

Frequency of soy food consumption and serum isoflavone concentrations among Chinese women in Shanghai.

OBJECTIVE: The food-frequency questionnaire (FFQ) can be an efficient tool to evaluate dietary intake in large, population-based studies, especially for specific foods. The objective of this study was to validate the assessment of soy and isoflavone (daidzein and genistein) intakes, measured by an FFQ, by comparing intakes with serum isoflavone concentrations. DESIGN AND SETTING: Soy and isoflavone intakes and serum isoflavone concentrations were determined as part of a case-control study of dietary factors and risks of benign breast disease and breast cancer. The FFQ, administered during an in-person interview, included six soy-specific line items. Blood was drawn within one week of FFQ completion. SUBJECTS: In total, 1823 women living in Shanghai, People's Republic of China. RESULTS: In this population, soybean milk, fresh bean curd and other bean foods were eaten once per week, and fermented bean curd, fried bean curd puff and soybeans were eaten less than once per week. A significant linear trend (P<0.01) in serum isoflavone concentrations across increasing categories of soy and isoflavone intakes was observed, indicating that soy and isoflavone intakes, measured by the FFQ, well distinguished serum isoflavone concentrations. Linear trends were also observed in both case and control groups in stratified analyses, suggesting little differential bias by case-control status. CONCLUSIONS: The results suggest that the FFQ provides a useful marker of soy food consumption and isoflavone exposure in this population.

Adult↗

PubMed2003

ABCC2-mediated biliary transport of 4-glutathionylcyclophosphamide and its contribution to elimination of 4-hydroxycyclophosphamide in rat.

Hematopoietic stem cell transplantation patients conditioned with cyclophosphamide (CY) and total body irradiation have substantially greater risk of nonrelapse mortality when plasma area under the concentration-time curve (AUC) of O-carboxyethylcyclophosphoramide mustard (CEPM) is high. The discovery was paradoxical because CEPM is a nontoxic elimination route of the protoxic CY metabolite hydroxycyclophosphamide (HCY). CY was administered to Wistar and TR- rats (a Wistar strain lacking functional ABCC2) at doses of 100 and 200 mg/kg CY, respectively. After either dose, Wistar rats excreted 4-glutathionylcyclophosphamide (GSCY) abundantly in bile; GSCY was absent from bile of TR- rats. Liver AUC(GSCY) was 2- to 2.5-fold greater in TR- than Wistar rats after 100 and 200 mg/kg CY, respectively. Liver AUC(HCY) was 24-46% greater in TR- rats than in Wistar rats after the respective CY doses. Plasma AUC(CEPM) of TR- rats was approximately twice that of Wistar rats after 100 mg/kg, but did not differ between the two strains after 200 mg/kg. Conversely, plasma AUC(HCY) was not different after 100 mg/kg CY, but was 40% greater in TR- rats after 200 mg/kg. The dose dependence of plasma AUC(CEPM) and AUC(HCY) was explained by the concentrations of HCY attained and the in vitro K(m) of aldehyde dehydrogenase and inhibition of aldehyde dehydrogense in TR- rats. We conclude that GSCY is a substrate of ABCC2, and plasma AUC(CEPM) functions as a reporter of liver exposure to HCY and toxins formed from it when HCY concentration is below the K(m) of aldehyde dehydrogenase and the activity is not compromised.

Animals↗

PubMed2003

Transiently altered acetaminophen metabolism after liver transplantation.

BACKGROUND AND OBJECTIVES: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in drug metabolism occur after organ transplantation. This study was designed to characterize acetaminophen disposition during the first 6 months after liver transplantation. METHODS: Thirteen liver transplant patients received an oral dose of acetaminophen (500 mg) on days 2, 10, 90, and 180 after transplantation. Serial blood samples were collected for 8 hours, and urine was collected for 24 hours. Liver biopsy specimens were obtained from the donor liver during transplantation (day 0) and on days 10, 90, and 180 after transplantation. RESULTS: There were significant time-dependent changes in acetaminophen metabolism after liver transplantation. When day 2 and day 10 were compared with day 180, the respective mean urinary recovery was 137% and 81% higher for thioether conjugates derived from NAPQI (P =.0002 and P =.01, respectively); 31% and 22% lower for acetaminophen sulfate (P =.0006 and P =.008, respectively); and 22% and 27% lower for acetaminophen glucuronide (P =.05 and P =.004, respectively). Metabolite formation clearances changed in concordance with the fractional urinary recovery. It was surprising that hepatic CYP2E1 content on day 10 after transplantation was only 20% higher, on average, than that found on day 180 (not significant). In contrast, hepatic CYP3A4 content was 984% higher, on average, when tissue from days 10 and 180 was compared after transplantation (P =.007). CONCLUSIONS: Increased recovery of acetaminophen thioether conjugates during the first 10 days after liver transplantation was a result of impaired glucuronidation and sulfation and enhanced NAPQI formation.

Acetaminophen↗

PubMed2003

Validation of a soy food-frequency questionnaire and evaluation of correlates of plasma isoflavone concentrations in postmenopausal women.

BACKGROUND: Soy foods may have various health benefits, but little is known about the patterns and correlates of soy consumption among postmenopausal women in the United States. OBJECTIVE: We assessed the reliability and validity of a soy food-frequency questionnaire (FFQ) and examined demographic, lifestyle, and dietary correlates of plasma isoflavone concentrations in postmenopausal women. DESIGN: In this cross-sectional study, soy isoflavone intake and plasma isoflavone concentration were analyzed in 96 postmenopausal women aged 50-79 y; the data were obtained at 2 visits that were 1 wk apart. Intake was determined with a 20-item soy FFQ and a comprehensive FFQ that included questions about tofu and soymilk. Fasting plasma daidzein and genistein concentrations were determined with liquid chromatography-mass spectrometry. RESULTS: Intraclass correlations between week 1 and week 2 values were >0.98 for both the soy and comprehensive FFQs. Median reported isoflavone intake was <2 mg/d. Pearson's product-moment correlation coefficients relating isoflavone intakes with plasma isoflavone concentrations ranged from 0.35 to 0.43. Plasma isoflavone concentrations were positively associated with age, fiber consumption, servings of fruit and vegetables, and dietary supplement use and were inversely associated with caffeine consumption; no associations with body mass index, education, dietary beliefs, activity level, alcohol intake, or fat intake were observed. CONCLUSIONS: Within a population with low soy consumption, the soy FFQ and comprehensive FFQ showed good reliability and moderate validity. Associations of plasma isoflavone concentrations with other dietary behaviors suggest that these compounds may serve as biomarkers of health behaviors in populations with low soy consumption.

Aged↗

PubMed2003

Effect of route of administration on the pharmacokinetic behavior of enantiomers of nefopam and desmethylnefopam.

Nefopam hydrochloride is a non-narcotic analgesic used parenterally and orally as a racemic mixture for the relief of postoperative pain. However, no information is presently available on the oral kinetics of (+) and (-) nefopam in humans. Also, nefopam is metabolized by N-demethylation but it is not known whether the desmethylnefopam enantiomers (DES1 and DES2) are present in plasma following intravenous (I.V.) or oral administration of parent drug. To address these issues, 24 healthy white male subjects received two treatments using a double-blind, placebo-controlled crossover design: oral administration of 20 mg nefopam hydrochloride solution or a placebo solution on a sugar cube, simultaneously with a continuous infusion of 20 mg nefopam hydrochloride or placebo infusion. A chiral assay using LC-MS was developed for the simultaneous determination of both enantiomers of the parent drug and its metabolite in plasma and urine. Following I.V. administration, the kinetics of (+) and (-) nefopam could be fitted to a bi-exponential equation but exhibited no stereoselectivity. Both enantiomers had large clearances (53.7 and 57.5 L/hr) and volumes of distribution (390 and 381 L) and half-lives around 5 hours. Following oral administration, (+) and (-) nefopam were rapidly absorbed with bioavailabilities of 44% and 42%, respectively, probably due to a first-pass effect. After I.V. administration, the enantiomers of desmethylnefopam exhibited lower concentrations and longer half-lives (20.0 h for DES1 and 25.3 h for DES2) relative to nefopam enantiomers. Following oral administration, desmethylnefopam enantiomers' plasma concentrations peaked earlier and higher than after I.V. administration (P < 0.05). Following I.V. and oral administration, desmethylnefopam enantiomers showed stereoselectivity in AUC and Cmax values. Urinary excretion of parent and metabolite enantiomers was less than 5% of dose. This study shows that desmethylnefopam enantiomers can contribute to the analgesic effect of racemic nefopam only when it is administered orally.

Administration, Oral↗

PubMed2002

Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation.

Liver toxicity caused by high-dose myeloablative therapy leads to significant morbidity after hematopoietic cell transplantation. We examined the hypothesis that liver toxicity after cyclophosphamide and total body irradiation is related to cyclophosphamide through its metabolism to toxins. Cyclophosphamide was infused at 60 mg/kg over 1 to 2 hours on each of 2 consecutive days, followed by total body irradiation. Plasma was analyzed for cyclophosphamide and its major metabolites. Liver toxicity was scored by the development of sinusoidal obstruction syndrome (veno-occlusive disease) and by total serum bilirubin levels. The hazards of liver toxicity, nonrelapse mortality, tumor relapse, and survival were calculated using regression analysis that included exposure to cyclophosphamide metabolites (as the area under the curve). Of 147 patients, 23 (16%) developed moderate or severe sinusoidal obstruction syndrome. The median peak serum bilirubin level through day 20 was 2.6 mg/dL (range, 0.5-41.1 mg/dL). Metabolism of cyclophosphamide was highly variable, particularly for the metabolite o-carboxyethyl-phosphoramide mustard, whose area under the curve varied 16-fold. Exposure to this metabolite was statistically significantly related to sinusoidal obstruction syndrome, bilirubin elevation, nonrelapse mortality, and survival, after adjusting for age and irradiation dose. Patients in the highest quartile of o-carboxyethyl-phosphoramide mustard exposure had a 5.9-fold higher risk for nonrelapse mortality than did patients in the lowest quartile. Engraftment and tumor relapse were not statistically significantly related to cyclophosphamide metabolite exposure. Increased exposure to toxic metabolites of cyclophosphamide leads to increased liver toxicity and nonrelapse mortality and lower overall survival after hematopoietic cell transplantation.

Adolescent↗

PubMed2002

Validation of a soy food frequency questionnaire with plasma concentrations of isoflavones in US adults.

OBJECTIVE: To validate assessment of soy intake using food frequency questionnaires (FFQs) compared with plasma isoflavone (genistein and daidzein) concentrations. DESIGN: Cross-sectional analysis of soy isoflavone intake and plasma analysis of isoflavones. SUBJECTS: 77 men and women, age range 20 to 40 years, recruited from the Seattle metropolitan area. MAIN OUTCOME MEASURES: Isoflavone intake was determined from responses to a 40-item soy FFQ and from tofu and soymilk intake assessed as part of a comprehensive FFQ used for the Women's Health Initiative (WHI FFQ). Isoflavone concentrations in fasting blood samples were determined by liquid chromatography-mass spectrometry. STATISTICAL ANALYSES: Correlation coefficients were calculated for: a) isoflavone intake assessed by the soy FFQ and the WHI FFQ, b) intake assessed by the soy FFQ and plasma isoflavone concentrations, and c) intake assessed by the WHI FFQ and plasma isoflavone concentrations. RESULTS: Isoflavone intake was highly correlated between the soy FFQ and the WHI FFQ (r = 0.84). Genistein and daidzein intakes determined by the soy FFQ were significantly correlated with plasma concentrations (r = 0.53 and 0.45, respectively). Isoflavone intake assessed from the WHI FFQ was also correlated with plasma concentration (r = 0.46 and 0.45). Soymilk and tofu were the two major contributors to isoflavone intake (38.6%). CONCLUSIONS: A soy-specific, 40-item FFQ assessed isoflavone intake with good validity. Isoflavone intake assessed by the WHI FFQ (tofu and soymilk) had lower correlations with plasma concentrations compared with the soy FFQ. Nonetheless, assessment of the two foods is a reasonably good marker for soy food consumption in this sample.

Adult↗

PubMed2002

Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver.

Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-S-transferase alpha released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by approximately 2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were approximately 50% more susceptible to NAPQI (25-165 micromol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH.

Acetaminophen↗