Plasma 6ß-hydroxycortisol to cortisol ratio as a less invasive cytochrome P450 3A phenotyping method.

AIM: A less invasive evaluation method of cytochrome P450 3A (CYP3A) activity provides an important tool for personalized medicine. We aimed to clarify the usefulness of the plasma 6ß-hydroxycortisol to cortisol concentration (6ß-OHF/F) ratio as a minimally invasive CYP3A phenotyping method. METHODS: Plasma 6ß-OHF and cortisol concentrations were measured via liquid chromatography/tandem mass spectrometry. The plasma 6ß-OHF/F ratio was compared with 6ß-hydroxylation clearance of endogenous cortisol (CLm(6ß); which we previously developed as an index of CYP3A activity) before, during and after oral contraceptive administration in 3 healthy women. The plasma 6ß-OHF/F ratio was observed during oral clarithromycin administration. The plasma 6ß-OHF/F ratio was also measured in 39 healthy participants. RESULTS: The plasma 6ß-OHF/F ratio in 3 healthy women on Day 21 of starting oral contraceptive administration decreased by 39, 49 and 61% compared with Day 0. These values were similar to CLm(6ß) values (43, 54 and 59%, respectively). Plasma 6ß-OHF/F ratio and CLm(6ß) exhibited a good correlation (r = .9053). The 6ß-OHF/F ratio decreased from 0.00921 to 0.00577 only 3 h following clarithromycin administration. The plasma 6ß-OHF/F ratio ranged 0.00565-0.01556 in 39 healthy participants. CONCLUSION: Based on its close relationship with CLm(6ß) and its decrease upon inhibition by clarithromycin, the plasma 6ß-OHF/F ratio serves as an index of CYP3A activity. Using this minimally invasive index, we can identify patients with extremely low CYP3A activity before treatment initiation and optimize the initial drug dose, thereby mitigating the risk of severe adverse reactions.

Quantification of fluticasone propionate in human plasma by LC-MS/MS and its application in the pharmacokinetic study of nasal spray at clinical doses.

We developed a method for quantifying fluticasone propionate (FP) using general-purpose liquid chromatography-tandem mass spectrometry equipment to measure the plasma concentration of FP for the pharmacokinetic study of FP following the administration of a prescribed nasal spray dose (100 µg). Using ammonium acetate (0.01 M)-formic acid (pH 2.9; 499:1, v/v) and methanol as the mobile phase, 3 pg/mL of FP was quantified. The relative error and standard deviation of the lower limit of quantification were <3.1%. The intra- and interday assay reproducibility was <3.5%. After 15 min of administering 200 µg FP nasal spray as the first dose, the FP concentration detected in the plasma of the two participants was 3.99 and 3.69 pg/mL. Subsequent doses of 100 µg FP were administered twice daily. The area under the plasma concentration-time curve values after 8-10 days of repeated administration of 100 µg of FP were approximately 1.6-fold higher than those achieved following a single administration of 200 µg of FP, which confirmed drug accumulation. The bioavailability of nasal FP was estimated to be 2% and 4%. This knowledge might help in reducing anxiety among patients who avoid using FP nasal spray, fearing its adverse effects.

Calculation of Human Melatonin Partial Metabolic Clearance in Healthy Adult Volunteers for Investigation of a Novel in Vivo CYP1A2 Phenotyping Method: A Pilot Study.

The method of administering caffeine as a probe to evaluate the phenotypic activity of the CYP1A2, has not yet been applied clinically. In contrast, if endogenous melatonin (MEL) metabolism can be used to assess CYP1A2 activity, it could be a simple method that does not require substance administration. The study aim was to calculate the MEL partial metabolic clearance (CLm(MEL)) from plasma MEL and its urinary metabolites and to test the potential of this approach as a novel CYP1A2 phenotyping method. Nine subjects were included in the study; 3 had 6 blood and 4 urine samples collected between 10:00 and 18:00 (collectively, the intraday sample). Nine subjects had 3 blood samples and 2-h urine samples collected between 10:00 and 12:00 once a week for 3 weeks (interday sample). The CLm(MEL) was calculated from the plasma area under the curve (AUC) of MEL (AUCMEL) and urinary MEL metabolites excretion (X6MEL). Among the intraday samples, the AUCMEL ranged from 6.45-13.17 pmol·h/L and X6MEL ranged from 0.204-0.899 nmol/2 h, showing a decrease in concentration over time. In contrast, the CLm(MEL) ranged from 30.52-69.57 L/h (within-individual percent relative standard deviation: 9.2-20.1%), showing no time-dependent variation. Large interindividual variability was observed in AUCMEL and X6MEL in the interday sample, but CLm(MEL) showed small interindividual variabilities. The CLm(MEL) was 1.8-fold higher for smokers than for nonsmokers. The results obtained in this study may be valuable in future studies of evaluating novel CYP1A2 phenotyping method.

Development and Validation of an LC-MS/MS-Based Method for Quantifying Urinary Endogenous 6-Hydroxymelatonin.

Evaluation of endogenous melatonin (MEL) secretion using its urinary metabolites is useful for the treatment of circadian rhythm sleep disorders. The primary melatonin metabolites excreted in the urine are 6-hydroxymelatonin (6-O-MEL) sulfate (S-O-MEL) and 6-O-MEL glucuronate, which result from sequential MEL metabolism by phases I and II drug metabolizing enzymes. To determine the accurate MEL secretion level, these urinary metabolites should be enzymatically deconjugated and converted into MEL. Furthermore, the use of LC-tandem mass spectrometry (LC-MS/MS) is preferable for the precision of this determination. Therefore, as part of our ongoing efforts to ultimately determine the level of MEL secretion, we herein aimed to develop an LC-MS/MS-based quantification method for 6-O-MEL and optimize deconjugation conditions. We determined the LC-MS/MS conditions of 6-O-MEL measurement and optimized the conditions of enzymatic reactions. The most efficient S-O-MEL deconjugation (102.1%) was achieved with Roche Glucuronidase/Arylsulfatase (from Helix pomatia) at 37 °C, pH-4.0 reaction buffer, and 60 min of reaction time. For human urine samples, the minimum amount of the enzyme required was 5944 units. Under these conditions, the accuracy and precision values of the 6-O-MEL determination (relative errors and standard deviation) were -3.60--0.47% and <6.80%, respectively. Finally, we analyzed the total amount of MEL metabolites excreted in 24-h urine samples; it was 6.70-11.28 µg in three subjects, which is comparable with the values reported till date. Thus, we have established a new method of measuring the total 6-O-MEL in human urine samples using an LC-MS/MS coupled with the prerequisite deconjugation reaction.

Dried blood spots analysis of 6ß-hydroxycortisol and cortisol using liquid chromatography/tandem mass spectrometry for calculating 6ß-hydroxycortisol to cortisol ratio.

Dried blood spot (DBS) sampling is a minimally invasive method used to collect blood samples of any population for personalized medicine. We aimed to develop a sensitive and reliable analytical method for measuring 6ß-hydroxycortisol (6ß-OHF) and cortisol concentrations in DBS by liquid chromatography/tandem mass spectrometry so as to utilize DBS as a less invasive blood sampling method for calculating the ratio of 6ß-OHF/cortisol. The lower limits of quantification obtained using four DBS were 1.08 pg/50 µl for 6ß-OHF and 1.01 pg/50 µl for cortisol. The 6ß-OHF and cortisol in DBS were stable for 28 days at room temperature. The intraday and interday accuracy and precision of the method was <12%. Additionally, the 6ß-OHF and cortisol in DBS were measured before, during, and after 3 days of clarithromycin administration to two of the subjects. Then, their concentration was compared in the plasma and whole blood collected simultaneously. The concentrations of 6ß-OHF and cortisol in four DBS ranged from 0.007 to 0.079 ng/50 µl and from 1.15 to 6.66 ng/50 µl, respectively. The 6ß-OHF/cortisol ratio in DBS decreased by approximately 50% on administering clarithromycin compared with that before the administration of clarithromycin. The 6ß-OHF/cortisol ratio in DBS also showed a strong correlation with that in whole blood (r = 0.9694) and plasma (r = 0.9383). This method provides high accuracy and precision for measuring 6ß-OHF and cortisol in DBS. It also allows the use of DBS instead of plasma for calculating the 6ß-OHF/cortisol ratio. The 6ß-OHF/cortisol ratio could be an index of CYP3A activity in clinical setting.

Effect of UGT1A1, CYP3A and CES Activities on the Pharmacokinetics of Irinotecan and its Metabolites in Patients with UGT1A1 Gene Polymorphisms.

BACKGROUND AND OBJECTIVES: Irinotecan (CPT-11) is metabolized to an active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterase (CES). SN-38 is then converted to the inactive metabolite SN-38 glucuronide (SN-38G) by glucuronosyltransferase 1A1 (UGT1A1). Genetic polymorphisms in UGT1A1 have been associated with altered SN-38 pharmacokinetics, which increase the risk of toxicity in patients. CPT-11 is also converted to 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC) by cytochrome P450 3A (CYP3A), and this route also affects the plasma concentration of SN-38. We evaluated the activities of UGT1A1, CYP3A, and CES and the factors affecting the pharmacokinetics of plasma SN-38 in patients with UGT1A1 gene polymorphisms. METHODS: Three male patients aged 56, 65, and 49 years were recruited for the analysis. All patients had pancreatic cancer, received FOLFIRINOX, and had UGT1A1*6/*6 (patients 1 and 3) or *6/*28 (patient 2) genetic polymorphisms. The rate constants for evaluating the enzyme activity were determined from the measured plasma concentration of CPT-11 and its metabolites using a two-compartment model by WinNonlin. RESULTS: The area under the plasma concentration-time curve (AUC) of SN-38 was patient 1 > patient 2 > patient 3. The rate constants obtained from the model analysis indicated the respective enzyme activities of UGT1A1 (k57), CYP3A (k13 + k19), and CES (k15). The order of values for UGT1A1 activity was patient 2 > patient 3 > patient 1. Since UGT1A1 activity was low in patient 1 with a high AUC of SN-38, it can be said that the increase in plasma concentration was due to a decrease in UGT1A1 activity. Conversely, the order of values for CYP3A and CES activities was patient 3 > patient 1 > patient 2 and patient 2 > patient 1 > patient 3, respectively. Patient 3 had the lowest AUC of SN-38, caused by a lower level of CES activity and increased CYP3A activity. CONCLUSION: In this study, we indicated that the plasma AUC of SN-38 and AUC ratio of SN-38G/SN-38 may depend on changes in the activities of CYP3A, CES, and UGT1A1. Using pharmacokinetic analysis, it is possible to directly evaluate enzyme activity and consider what kind of enzyme variation causes the increase in the AUC of SN-38.

Midazolam Intoxication in a Premature Neonate.

PURPOSE: We report the case of a male neonate with a respiratory disorder who developed adverse cardiorespiratory symptoms after the continuous infusion of midazolam. METHODS: To clarify the cause of cardiogenic shock, we performed whole exome sequencing and screened relative single-nucleotide variants of 2 cytochrome P450 (CYP) isoforms, CYP3A4 and CYP3A5, which play a dominant role in the metabolic elimination of midazolam. We measured endogenous cortisol 6ß-hydroxylation clearance to phenotypically assess CYP3A activity. FINDINGS: The CYP3A activity level in the patient was significantly lower than the mean CYP3A activity level in healthy adults. Three intronic mutations in the CYP3A4 and CYP3A5 isoforms were detected in the patient. IMPLICATIONS: Our findings suggest that the midazolam concentration in plasma was achieved at above the steady-state concentration during continuous infusion used to sedate neonates receiving mechanical ventilatory support. Evaluation of the drug-metabolizing ability based on CYP3A might be useful if adverse electrophysiologic variables or the induction of tachycardia occur because of delayed elimination.

Identification of equol-7-glucuronide-4'-sulfate, monoglucuronides and monosulfates in human plasma of 2 equol producers after administration of kinako by LC-ESI-MS.

Equol is a product formed during the biotransformation of the naturally occurring isoflavone daidzein by intestinal bacteria. The role of equol in the prevention of several hormone-dependent diseases such as prostate cancer and osteoporosis as well as vasomotor symptoms has been extensively investigated. Equol primarily occurs in the form of major metabolites such as glucuronides and sulfates, while intact equol has been detected at only ca. 1% in human plasma. However, to date, conjugated metabolites have been evaluated by measuring the free equol obtained after selective enzymatic hydrolysis. Thus, the precise types of conjugates circulating in vivo and the position(s) of the conjugation sites on the equol skeleton have yet to be clarified. Our study describes the identification of polar equol metabolites in the plasma of 2 equol-producers obtained at 8 hours after consuming 50 g of kinako (approximately 37 mg of daidzein). The structural identification of these conjugated metabolites in plasma was performed by comparison to the LC-ESI-MS n and 1H-NMR spectral data of the corresponding chemically synthesized compounds. The results of the LC-ESI-MS/MS analysis indicated that the main conjugated metabolite in plasma was (S)-equol-7-glucuronide-4'-sulfate along with lower amounts of 7- and 4'-monoglucuronides as well as 7- and 4'-monosulfates.

Sensitive and simultaneous quantitation of 6ß-hydroxycortisol and cortisol in human plasma by LC-MS/MS coupled with stable isotope dilution method.

CYP3A phenotyping provides a means for personalized drug therapy. We focused our attention on the plasma 6ß-hydroxycortisol (6ß-OHF) to cortisol ratio as an index for CYP3A phenotyping. In the present study, we developed a sensitive and reliable method for the simultaneous determination of 6ß-OHF and cortisol in human plasma using high-performance liquid chromatography/tandem mass spectrometry together with picolinylester derivatization or nonderivatization methods and 6ß-[9,11,12,12-2 H4 ]hydroxycortisol and [1,2,4,19-13 C4 ]cortisol as internal standards for in vivo CYP3A phenotyping in humans. The lower limits of quantification were 38.513 pg/mL for 6ß-OHF and 38.100 pg/mL for cortisol. The relative error and relative standard deviation of the lower limits of quantification were <5% for both methods. The intra-day and inter-day assay reproducibilities of the determined 6ß-OHF and cortisol concentrations were consistent with the actual amounts added as relative errors and relative standard deviations for both methods, which were <5.4% and <3.9%, respectively. Both methods were applied for the quantification of plasma 6ß-OHF and cortisol concentrations in healthy subjects taking oral contraceptives. The absolute concentrations and time course of 6ß-OHF and cortisol were found to be consistent when measured using the 2 methods. The ratio as an index for in vivo CYP3A activity decreased after 21 days of taking oral contraceptives for both methods. To the best of our knowledge, this is the first study reporting the detailed investigation of accuracy and precision in the simultaneous measurement of 6ß-OHF and cortisol in human plasma using liquid chromatography/tandem mass spectrometry coupled with stable isotope dilution method, which can be applied to CYP3A phenotyping.

Use of endogenous cortisol 6ß-hydroxylation clearance for phenotyping in vivo CYP3A activity in women after sequential administration of an oral contraceptive (OC) containing ethinylestradiol and levonorgestrel as weak CYP3A inhibitors.

The present study was undertaken to evaluate the time courses of in vivo cytochrome P450 3A (CYP3A) inhibition in four healthy women after sequential administration of an oral contraceptive (OC) containing ethinylestradiol and levonorgestrel, using 6ß-hydroxylation clearance of endogenous cortisol (CLm(6ß)) as a new index for CYP3A phenotyping. The 6ß-hydroxylation clearance (CLm(6ß)) was followed every 2h from 9:00 or 11:00 to 17:00 on days 0 (baseline), 1, 2, 21, and 28 during a single menstrual cycle. The serum concentrations of endogenous estradiol and progesterone were also measured. The time course data of CLm(6ß) clearly demonstrated 43-64% inhibition of CYP3A activity in women taking a low daily dose of the OC for 21days. The average CLm(6ß) levels that were suppressed by the OC in four women were extremely low (0.60-1.23mL/min) compared with the normal CLm(6ß) range (1.5-3.5mL/min) that was obtained from 49 healthy subjects in our previous study. The in vivo inhibitory potencies (43-64%) obtained in this study were stronger than expected from reported in vitro studies (∼20%). Furthermore, it would take at least seven days to return to the baseline activity of CYP3A after discontinuation of the OC. The results presented here should provide important information on the inhibitory effect of OC on the CYP3A activities in women, which are involved in the metabolism of a number of drugs with a narrow therapeutic range.

Intraindividual and interindividual variabilities in endogenous cortisol 6ß-hydroxylation clearance as an index for in vivo CYP3A phenotyping in humans.

This study was designed to evaluate the in vivo activity of cytochrome P450 3A (CYP3A) in 49 healthy Japanese subjects aged 22-58 years using endogenous cortisol 6ß-hydroxylation clearance [CLm(6ß)], a novel biomarker for CYP3A phenotyping. CLm(6ß) in the 49 healthy subjects was 2.40 ± 0.79 ml/min with an approximately 4-fold interindividual variability of CYP3A activity. The mean clearance in the 24 women was 2.50 ± 0.89 ml/min; the value in the women was higher than in the 25 men (2.30 ± 0.69 ml/min) by approximately 9%. We also measured the change of CLm(6ß) in 14 healthy subjects in the morning at 10:00-12:00 every 2 or 3 days over a period of 36-53 days and observed a 1.5-fold to 3.4-fold day-to-day intraindividual variability in the CYP3A activity. The mean value for CLm(6ß) in each subject for 36-53 days was 2.54 ± 0.76 ml/min (n = 14). We also evaluated the CLm(6ß) every 2 hours from 8:00-20:00 in 26 healthy subjects. The within-day intraindividual clearance variability was 1.1-fold to 2.5-fold (2.45 ± 0.91 ml/min, n = 26). No characteristic diurnal rhythms were observed in the in vivo activity of CYP3A.

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11ß-hydroxysteroid dehydrogenase 2 activity.

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11ß-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11ß-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11ß-HSD2 activity. The findings indicate that 11ß-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11ß-HSD2.

Separation and quantitative determination of 6α-hydroxycortisol and 6ß-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection.

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6ß-hydroxycortisol (6ß-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6ß-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C(18) plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and (B) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH/acetonitrile (2:3, v/v). 6α-OHF and 6ß-OHF were well separated on an XTerra MS C(18) 5 µm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6ß-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6ß-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6ß-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6ß-OHF determined were in good agreement with the actual amounts added, the relative errors being -5.37% and -3.73% (gradient 2) and -5.69% and -3.96% (gradient 3) for both 6α-OHF and 6ß-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6ß-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6ß-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6ß-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.

Methane dehydroaromatization over Mo-modified H-MFI for gas to liquid catalysts.

For direct gas to liquid (GTL), a novel process producing energy sources for methane dehydroaromatization is needed. Supporting MoO3 on H-MFI zeolite shows the high catalytic capacity and a selective activity for dehydroaromatization of methane to benzene at 973 K in a fixed bed reactor. On the other hand, deactivation by coke on the active sites in all the catalysts is formed during the reaction. H2 co-feed suppressed the deactivation, which is probably due to the decrease in coking amount. Mo K-edge X-ray absorption fine structure (XAFS) results showed the formation of dispersed Mo2C species with low crystallinity after dehydroaromatization. Mo L(III)-edge XANES (X-ray absorption near-edge structure) indicated the formation of active Mo species including Mo2C and Mo-oxycarbide (MoOxCy), where the redox state should be independent in the absence/presence of H2. It is concluded that Mo-oxycarbide species act as highly active species, and their stability affected the durable activity in the presence of H2.

Simultaneous determination of androstenedione, 11beta-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry.

This study describes a GC-MS method for the simultaneous determination of androstenedione (AD), 11beta-hydroxyandrostenedione (11beta-OHAD), and testosterone (TS) in human plasma. [19,19,19-(2)H(3)]Androstenedione (AD-(2)H(3)), 11beta-hydroxy-[1,2,4,19-(13)C(4)]androstenedione (11beta-OHAD-(13)C(4)), and [1,16,16,17-(2)H(4)]testosterone (TS-(2)H(4)) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC-MS analysis of the three steroids. The detection limit of the present GC-MS-SIM method was found to be 1 pg per injection for AD (S/N ratio=4.5), 5 pg for 11beta-OHAD (S/N ratio=5.0), and 1 pg for TS (S/N ratio=4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL (r=0.9998) for AD, from 0.56 to 3.19 ng/mL (r=0.9996) for 11beta-OHAD, and from 2.05 to 10.3 ng/mL (r=0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were -3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11beta-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11beta-OHAD and the interconversion of AD and TS in humans.

Simultaneous determination of prednisolone, prednisone, cortisol, and cortisone in plasma by GC-MS: estimating unbound prednisolone concentration in patients with nephrotic syndrome during oral prednisolone therapy.

Individual variability of the pharmacokinetics of prednisolone based on the unbound concentration in plasma is of significant clinical consideration. The unbound concentrations of prednisolone were measured in 10 patients with nephrotic syndrome, two patients with systemic lupus erythematosus, and one patient with dermatomyositis by examining protein bindings of prednisolone on one or more occasions during prednisolone treatment. In this study, plasma concentrations of prednisolone, prednisone, cortisol, and cortisone were simultaneously analyzed by GC-MS by using stable isotope-labeled internal standards. Equilibrium dialysis was employed to accurately estimate the unbound fractions of prednisolone in plasma. The unbound fraction of prednisolone changed depending on plasma total prednisolone concentration and plasma albumin concentration. The unbound fraction of prednisolone (Y) is calculated: Y=(-0.0101x' + 0.0736) x + 10.23, where x' is the plasma albumin concentration and x is the total prednisolone concentration. The estimated concentrations of unbound prednisolone by using the above equation were in good agreement with the measured concentrations of unbound prednisolone. Since the protein binding of prednisolone did not change in the presence of prednisone (114.0 ng/ml), it appeared that prednisone produced from the therapeutic dose of prednisolone did not affect the unbound fraction of prednisolone.

Validation of the plasma half-life of 11alpha-deuterium cortisol as a sensitive index for the analysis of human 11beta-HSD2 activity in vivo.

This study is concerned with validating the measurement of the plasma half-life of 11alpha-(2)H cortisol in an attempt to accurately assess the in vivo activity of 11beta-HSD2 in man. Oral administration of 5mg of cortisol-(13)C(4),(2)H(1) to a human subject after repeated ingestions of 130mg/day of glycyrrhetinic acid for 5 days resulted in a decrease in the rate constant of the cortisol-(13)C(4),(2)H(1) to cortisone-(13)C(4) conversion, a direct index reflecting 11beta-HSD2 activity. The reduced 11beta-HSD2 activity led to an increase in the elimination half-life of cortisol-(13)C(4),(2)H(1), indicating that the loss of 11alpha-(2)H is a sensitive in vivo means of assessing 11beta-HSD2 activity. A simultaneous oral administration of 3mg each of [1,2,4,19-(13)C(4),11alpha-(2)H]cortisol (cortisol-(13)C(4),(2)H(1)) and 11alpha-(2)H cortisol to another human subject confirmed the bioequivalency of the two labeled cortisols. The information obtained from the kinetic analysis of the 11beta-HSD2-catalyzed conversion of cortisol-(13)C(4),(2)H(1) to cortisone-(13)C(4) indicated that the elimination half-life of 11alpha-(2)H cortisol was a sensitive index of renal 11beta-HSD2 activity. The use of 11alpha-(2)H cortisol as a tracer appears to offer a significant advance in evaluating human 11beta-HSD2 activity in vivo.

Use of 11alpha-deuterium labeled cortisol as a tracer for assessing reduced 11beta-HSD2 activity in vivo following glycyrrhetinic acid ingestion in a human subject.

This study describes an oral administration of 5 mg of [1,2,4,19-13C4,11alpha-2H]cortisol (cortisol-13C4,2H1) to a human subject performed on two separate occasions, one with cortisol-13C4,2H1 alone and the other with cortisol-13C4,2H1 plus 130 mg per day of glycyrrhetinic acid for 6 days. The stable isotope methodology employed allowed for the evaluation of the individual in vivo activities of the two isozymes of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD1 and 11beta-HSD2, and to demonstrate the sensitivity of changes in cortisol elimination half-life for detecting inhibition of 11beta-HSD2 activity induced with glycyrrhetinic acid. The kinetic analysis associated with the loss of 11alpha-2H during the conversion of cortisol-13C4,2H1 to cortisone-13C4 by 11beta-HSD2 clearly indicated reduced 11beta-HSD2 activity with glycyrrhetinic acid ingestion, as observed by an increase in the elimination half-life of cortisol-13C4,2H1. The elimination half-life of cortisol-13C4,2H1 provided sensitive in vivo measures of 11beta-HSD2 activity and was more sensitive for detecting changes in renal 11beta-HSD2 activity than the measurement of the urinary ratio of free cortisol and free cortisone (UFF/UFE). The 2H-labeling in the 11alpha-position of cortisol served as an appropriate tracer for assessing the reduced 11beta-HSD2 activity in vivo induced by glycyrrhetinic acid.

Simultaneous determination of 6beta-hydroxycortisol and cortisol in human urine by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity determined by the cortisol 6beta-hydroxylation clearance.

This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH with acetonitrile (4:6, v/v). 6beta-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6beta-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6beta-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6beta-OHF and cortisol. The method was compared with the GC/MS method by measuring 6beta-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard.

Evidence for the validity of cortisol 6 beta-hydroxylation clearance as a new index for in vivo cytochrome P450 3A phenotyping in humans.

This study uses stable isotope methodology to evaluate the validity of 6beta-hydroxylation clearance of endogenous cortisol as a new index for in vivo CYP3A phenotyping in humans. Important factors contradictory to the use of a conventional index of urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) to evaluate in vivo CYP3A activity are also discussed. Stable isotopically labeled cortisol (3-5 mg) was orally administered to three healthy adult subjects to accurately determine the fractional metabolic clearance specific for the 6beta-hydroxylation of cortisol. Plasma concentrations of labeled cortisol and urinary excreted amounts of labeled cortisol and 6beta-OHF were analyzed by gas chromatography-mass spectrometry simultaneously with their endogenous counterparts. There was a good correlation between endogenous and exogenous 6beta-hydroxylation clearances in the three subjects tested (r = 0.7733, 0.9112, and 0.9534 for 2-, 4-, and 6- to 8-h urine collection periods, respectively). This strongly suggests that the endogenous 6beta-hydroxylation clearance can be used as an appropriate index for phenotyping the in vivo CYP3A activity. Furthermore, observed intra- (2.1- to 4.6-fold) and interindividual variabilities (ca. 5-fold) in the labeled cortisol renal clearance suggest that the urinary ratio 6beta-OHF/F, a function of 6beta-hydroxylation clearance and renal clearance of cortisol, does not always reflect the in vivo CYP3A activity. When a macrolide antibiotic, clarithromycin, was administered to a healthy volunteer in a dose of 200 mg every 12 h for 6 days, the inhibitory effects of clarithromycin on the in vivo CYP3A activity were clearly seen by the 6beta-hydroxylation clearance of endogenous cortisol but not by the urinary ratio 6beta-OHF/F.