Urinary 6β-hydroxycortisol Research Articles

Canine urinary lactate and cortisol metabolites in hypercortisolism, nonadrenal disease, congestive heart failure, and health.

Spontaneous hypercortisolism (HC) is a common endocrine disease of senior dogs, often overlapping in selected clinical signs and hematologic and blood biochemical abnormalities with nonadrenal diseases (NADs). HC and NAD could differentially affect cortisol metabolism, which is a complex 10-enzymatic pathway process. HC might also affect blood and urine lactate levels through its effects on mitochondrial function. We aimed to differentiate between HC and NAD via a urinary cortisol metabolites and lactate panel. We prospectively recruited 7 healthy dogs and 18 dogs with HC, 15 with congestive heart failure (CHF), and 9 with NAD. We analyzed urine by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. We normalized urinary lactate and cortisol metabolites to urine creatinine concentration, and then compared groups using a linear-mixed model and principal component (PC) analysis. A machine-learning classification algorithm generated a decision tree (DT) model for predicting HC. The least-squares means of normalized urinary 6β-hydroxycortisol and PC1 of the HC and CHF groups were higher than those of the healthy and NAD groups (p = 0.05). Creatinine-normalized urinary 6β-hydroxycortisol had better sensitivity (Se, 0.78; 95% CI: 0.55-0.91), specificity (Sp, 0.89; 95% CI: 0.57-0.99), and a likelihood ratio (LR; 7), than the Se (0.72; 95% CI: 0.49-0.88), Sp (0.89; 95% CI: 0.57-0.99), and LR (6.5) of PC1 for distinguishing HC from NAD. Lactate and dihydrocortisone had the highest decreasing node-weighted impurity value and were considered the most important features in the DT model; dihydrocortisol had no role in determining whether a dog had HC.

A background subtraction approach for determination of endogenous cortisol and 6β-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women

Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6β-hydroxycortisol/cortisol ratio (6β-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6β-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6β-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquid-liquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC®BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1–1.000 ng/mL and 2–1.000 ng/mL for C and 6β-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6β-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6β-OHF:C ranged from 2.57 to 51.69, with a mean ± standard deviation (SD) of 15.12 ± 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6β-OHF:C ranging from 3.48 to 44.54 with a mean ± SD of 14.37 ± 5.73 (n = 7). Even though the 6β-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability.

Cortisol and 11 beta-hydroxysteroid dehydrogenase type 2 as potential determinants of renal citrate excretion in healthy children.

In patients with Cushing disease, renal citrate excretion is reduced. A low urinary citrate concentration is a risk factor for nephrolithiasis. Since higher acid loading is one major determinant of reduced citrate excretion, we aimed to examine whether glucocorticoids still within the physiological range may already impact on urinary citrate excretion independently of acid-base status. Overall, 132 healthy prepubertal participants of the DOrtmund Nutritional and Anthropometric Longitudinally Designed (DONALD) Study who had collected two successive 24-h urine samples (at 1 and 2 years) before the start of their pubertal growth spurt were included in the study. Net acid excretion capacity (NAEC), urinary potential renal acid load (PRAL), creatinine, calcium, and various cortisol metabolites were measured in all samples. Glucocorticoid quantification was done by GC-MS and radioimmunoassay. In regression models multivariable-adjusted for 24-h urinary PRAL, NAEC, creatinine and calcium, urinary free cortisol (UFF), 6β-hydroxycortisol, and 20α-dihydrocortisol showed significant inverse relationships (P ≤ 0.02) with 24-h renal citrate output. By contrast, the estimate of renal 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), i.e., the ratio of urinary free cortisone/UFF, associated positively with urinary citrate (P = 0.04). In line with studies in hypercortisolic state, even moderately high cortisol levels in healthy children, still within the physiological range, may negatively impact on the kidney's citrate excretion. Besides, a higher 11β-HSD2 activity, favoring cortisol inactivation, is paralleled by an increased citrate excretion.

The cytochrome P450 isoenzyme and some new opportunities for the prediction of negative drug interaction in vivo.

Cytochrome (CYP) 450 isoenzymes are the basic enzymes involved in Phase I biotransformation. The most important role in biotransformation belongs to CYP3A4, CYP2D6, CYP2C9, CYP2C19 and CYP1A2. Inhibition and induction of CYP isoenzymes caused by drugs are important and clinically relevant pharmacokinetic mechanisms of drug interaction. Investigation of the activity of CYP isoenzymes by using phenotyping methods (such as the determination of the concentration of specific substrates and metabolites in biological fluids) during drug administration provides the prediction of negative side effects caused by drug interaction. In clinical practice, the process of phenotyping of CYP isoenzymes and some endogenous substrates in the ratio of cortisol to 6β-hydroxycortisol in urine for the evaluation of CYP3A4 activity has been deemed to be a quite promising, safe and minimally invasive method for patients nowadays.

Co-Administration of the CYP3A4 Inhibitor Diltiazem Counteracts Mitotane-Induced Clearance of Glucocorticoids and Antihypertensives in a Patient with Adrenocortical Carcinoma

ABSTRACT Objective: We report a case of adrenal insufficiency and resistant hypertension in a patient with adrenocortical cell carcinoma (ACC) under mitotane treatment, in which a cytochrome P450 (CYP)3A4 inhibitor, diltiazem, successfully reduced mitotane-induced clearance of glucocorticoids and antihypertensives. Methods: The patient underwent clinical, biochemical, and radiologic assessment. The urinary 6β-hydroxycortisol (6β-OHF) to cortisol (F) ratio, an index of CYP3A4 activity, was evaluated before and after administration of diltiazem. Results: A 63-year-old woman diagnosed with ACC and Cushing syndrome was treated with mitotane. After several months, she presented signs of adrenal insufficiency, despite adequate glucocorticoid replacement. She developed grade 3 hypertension, although treatment with 5 antihypertensives (amlodipine, eplerenone, olmesartan, carvedilol, and doxazosin) was continued. Her plasma mitotane concentrations were above the therapeutic range, suggesting mitotane-induced, CYP3...

Simultaneous Determination of Cortisol, Cortisone, 6 -Hydroxycortisol and 6 -Hydroxycortisone by HPLC

A specific and sensitive method based on high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) was developed for the simultaneous determination of urinary cortisol (F), cortisone (E), 6β-hydroxycortisol (6β-OHF) and 6β-hydroxycortisone (6β-OHE) using dexamethasone as the internal standard. The method involved solid-phase extraction of the five compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate-diethyl ether (5 mL; 4:1, v/v), followed by 1 mol/L of NaOH (1 mL) and 1.0% acetic acid (1 mL). Separation of the five analytes was achieved within 31 min by using a reversed-phase C18 analytical column (200 × 4.6 mm, 5 µm, Agilent). A UV detector operated at 245 nm was used. According to the method validation, inter-run and intra-run precision was below 9.45% and accuracy ranged from 98.16 to 115.50%. The lower limits of quantitation were 5 ng/mL for four analytes. This is the first HPLC method that can simultaneously determine F, E, 6β-OHF and 6β-OHE in human urine. The assay was applied to research the ratio of (6β-OHF + 6β-OHE)/(F + E) as a non-invasive biomarker for the metabolism of tacrolimus.

Comparison of two endogenous biomarkers of CYP3A4 activity in a drug-drug interaction study between midostaurin and rifampicin.

PurposeMidostaurin, a multitargeted tyrosine kinase inhibitor, is primarily metabolized by CYP3A4. This midostaurin drug–drug interaction study assessed the dynamic response and clinical usefulness of urinary 6β-hydroxycortisol to cortisol ratio (6βCR) and plasma 4β-hydroxycholesterol (4βHC) for monitoring CYP3A4 activity in the presence or absence of rifampicin, a strong CYP3A4 inducer.MethodsForty healthy adults were randomized into groups for either placebo or treatment with rifampicin 600 mg QD for 14 days. All participants received midostaurin 50 mg on day 9. Midostaurin plasma pharmacokinetic parameters were assessed. Urinary 6βCR and plasma 4βHC levels were measured on days 1, 9, 11, and 15.ResultsBoth markers remained stable over time in the control group and increased significantly in the rifampicin group. In the rifampicin group, the median increases (vs day 1) on days 9, 11, and 15 were 4.1-, 5.2-, and 4.7-fold, respectively, for 6βCR and 3.4-, 4.1-, and 4.7-fold, respectively, for 4βHC. Inter- and intrasubject variabilities in the control group were 45.6 % and 30.5 %, respectively, for 6βCR, and 33.8 % and 7.5 %, respectively, for 4βHC. Baseline midostaurin area under the concentration–time curve (AUC) correlated with 4βHC levels (ρ = −0.72; P = .003), but not with 6βCR (ρ = 0.0925; P = .6981).ConclusionsBoth 6βCR and 4βHC levels showed a good dynamic response range upon strong CYP3A4 induction with rifampicin. Because of lower inter- and intrasubject variability, 4βHC appeared more reliable and better predictive of CYP3A4 activity compared with 6βCR. The data from our study further support the clinical utility of these biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00228-014-1675-0) contains supplementary material, which is available to authorized users.

A comparison of 4β‐hydroxycholesterol : cholesterol and 6β‐hydroxycortisol : cortisol as markers of CYP3A4 induction

To compare plasma 4β-hydroxycholesterol : cholesterol with urinary 6β-hydroxycortisol : cortisol as markers of cytochrome P4503A4 activity before and after treatment with rifampicin for 2 weeks. 6β-hydroxycortisol and cortisol were determined by liquid chromatography tandem mass spectrometry and 4β-hydroxycholesterol was determined by gas chromatography-mass spectrometry in three groups of healthy volunteers. Induction ratios for 6β-hydroxycortisol : cortisol were 1.8, 3.9 and 4.5 for 20 mg day(-1) , 100 mg day(-1) or 500 mg day(-1) of rifampicin, respectively. The corresponding ratios for 4β-hydroxycholesterol : cholesterol were 1.5, 2.4 and 3.8. Plasma 4β-hydroxycholesterol : cholesterol gave similar induction ratios to urinary 6β-hydroxycortisol : cortisol.

Quantitative analysis of cortisol and 6β‐hydroxycortisol in urine by fully automated SPE and ultra‐performance LC coupled with electrospray and atmospheric pressure chemical ionization (ESCi)‐TOF‐MS

An ultra-performance LC TOF MS method for quantitative analysis of cortisol and 6β-hydroxycortisol in urine was developed. The method was used for determination of the ratio between 6β-hydroxycortisol and cortisol in urine received from autopsy cases and living persons as a measure of cytochrome P450 3A enzyme activity. Urine samples (0.25 mL) were extracted with an in-house developed fully automated 96-well SPE system. The compounds were quantified using a Waters ACQUITY UPLC system coupled to a Waters SYNAPT G2. The MS sensitivity was optimized by using negative ionization in sensitivity mode (resolution >10 000 full-width at half-maximum), and further optimized by using the enhanced duty cycle around the 410 m/z. ESCi (simultaneous electrospray and atmospheric pressure chemical ionization) mode was used to compensate for the matrix effects of postmortem urine. Finally, the SYNAPT G2 was tested as a quantitative instrument. The developed method has a measurement range from 2.5-300 ng/mL for cortisol to 10-1200 ng/mL for 6β-hydroxycortisol. Mean overall process efficiencies were 29.4 and 23.0% for cortisol and 6β-hydroxycortisol, respectively. In 20 forensic reference cases, the range of the 6β-hydroxycortisol/cortisol ratio was 0.29-14.2 with a median of 3.04.

Effects of mitotane treatment on human steroid metabolism: implications for patient management.

Mitotane (o,p'-DDD), an oral adrenolytic agent for treatment of advanced adrenocortical carcinoma (ACC), is reported to inhibit cortisol biosynthesis in vitro and enhance production from exogenous cortisol of urinary 6β-hydroxycortisol and unidentified polar unconjugated metabolites. We examined urinary steroid profiles by gas chromatography–mass spectrometry of patients with histologically confirmed ACC following surgery, receiving a) hydrocortisone alone (three males and three females) and b) mitotane and hydrocortisone (six males and 11 females). Samples were collected after plasma mitotane had reached the therapeutic range of 14–20 mg/l. Increased excretion of polar unconjugated steroids during mitotane treatment was confirmed, with 6β-hydroxycortisol and 6β-hydroxy-20-dihydrocortisols predominating. The proportion of additionally hydroxylated metabolites was <2% in untreated controls and 52, 35–52% (mean, range) in the mitotane plus hydrocortisone group. Ratios of 5α-/5β- and 20β-/20α-metabolites of administered cortisol were decreased 50-, 15-fold, and 14-, 8-fold respectively (males, females – mean values) but with no change in metabolite ratios that reflect oxidoreduction at C11 or C20. Patterns of decrease in 5α- relative to 5β-reduced metabolites were similar to those of patients with 5α-reductase 2 deficiency or on treatment with the 5α-reductase 2 inhibitor finasteride but different from those of patients on dutasteride, indicating specific inhibition of 5α-reductase 2. We conclude that mitotane causes consistent changes in cortisol catabolism, most of which have not been previously recognised. These need not interfere with early detection of ACC recurrence. Induction of 6β-hydroxylation offers an explanation for a reported decrease in cortisol bioavailability. Mitotane also has potential as a unique steroid metabolic probe for 20β-reduction.

Quantitation of Urinary 6β-Hydroxycortisol and Free Cortisol by Ultra- Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry

A new UPLC-MS method that combined 0.01% formic acid/isopropanol as the mobile phase and an ESI-QTOF instrument has been developed for reliable quantitation of urinary 6β-hydroxycortisol (6βHC) and free cortisol (FC). The use of 0.01% formic acid/isopropanol for binary gradient elution on a reversed-phase C18 column resulted in 3.1 and 5.0 times the peak areas for 6βHC and FC, respectively, as compared to 0.1% formic acid/acetonitrile. Liquid-liquid extraction with ethyl acetate at pH 5 was superior to solid-phase extraction for sample preparation. A mass window of 50 ppm was used for quantitative monitoring of 6βHC (m/z 379.212) and FC (m/z 363.217) using full-mass detection, with the on-column limits of detection as 4.0 and 1.4 fmol, and the lower limits of quantitation as 13.6 and 6.9 fmol for 6βHC and FC. The accuracy of quantitation ranged from 93.3% to 102.3% recoveries at three spiking levels, with the maximum intraday and interday %CV being 3.7 and 5.3 for both analytes. This LC-MS method was then applied to the quantitation of 6βHC and FC in the urine pairs collected during the follicular and premenstrual phases of menstrual cycle from each of sixty-one premenopausal women — forty-one with ovulatory cycles and twenty with subclinical anovulatory cycles. Paired two-tailed T-tests showed no significant difference (p>0.05) between the metabolic ratios of urinary 6βHC/FC during the follicular and premenstrual phases in both ovulatory and anovulatory subjects, indicating no correlation of the urinary 6βHC/FC ratios between the two physiological phases of menstrual cycle in these subjects.

Effects of Rifampin and Ketoconazole on the Pharmacokinetics of Nilotinib in Healthy Participants

Nilotinib (Tasigna), an orally bioavailable second-generation BCR-ABL tyrosine kinase inhibitor, is approved for use in patients with chronic myeloid leukemia in chronic phase and accelerated phase who are resistant or intolerant to prior therapy, including imatinib. Previous in vitro studies indicated that nilotinib metabolism is primarily mediated by CYP3A4. To investigate the effect of CYP3A4 induction and inhibition on nilotinib pharmacokinetics, 2 studies were conducted in healthy volunteers prior to and following treatment with a strong inducer (rifampin) or inhibitor (ketoconazole). In the induction study, administration of rifampin 600 mg once daily for 8 days significantly increased urinary 6β-hydroxycortisol/ cortisol ratio, from a preinduction baseline of 5.8 ± 2.7 to 18.0 ± 10.2 after 8 days of rifampin treatment, confirming an inductive effect on CYP3A4. Nilotinib oral clearance was increased by 4.8-fold, and the maximum serum concentration (C(max)) and area under the serum concentration-time curve (AUC) were decreased by 64% and 80%, respectively, in the induced state compared with baseline. In the inhibition study, ketoconazole 400 mg once daily for 6 days increased the C(max) and AUC of nilotinib by 1.8- and 3-fold, respectively, compared with nilotinib alone. These results indicate that concurrent use of strong CYP3A4 inducers or inhibitors may necessitate dosage adjustments of nilotinib and should be avoided when possible.

Effects of dexamethasone, administered for growth promoting purposes, upon the hepatic cytochrome P450 3A expression in the veal calf

Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (−20.64% and −16.07% with Q RT-PCR; −30.55% and −34.31% with Northern blotting); at the post-translational level, decreased TAT activity (−19.84% and 44.34%), CYP3A apoprotein (−27.65% and −42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, −78.89% and −23.87%; ethylmorphine N-demethylase, −44.26% and −28.37%; testosterone 6β-hydroxylase, −44.60% and −18.07%; testosterone 2β-hydroxylase, −43.95% and −11.69%); by contrast, an increase (about 2-fold) of the urinary 6β-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR–NRs–CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6β-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.

Authors' response: Poor correlation between 6β‐hydroxycortisol:cortisol molar ratios and midazolam clearance as measure of hepatic CYP3A activity: a comment

We appreciate the comments of Dr Fenske [1] in the above letter. However, we disagree with his conclusions regarding the utility of the cortisol ratio once time of day and fluid intake are controlled. First, it is incorrect to assume that a population has similar corticosteroid production patterns. To this end, morning spot urine collections have been utilized for the cortisol ratio with little success. For example, Tran et al. [2] demonstrated a high intra-individual variability (>50%) in the cortisol ratio obtained from a morning spot urine. Sleep–wake cycles affect the timing of peak cortisol concentrations [3] and episodic cortisol secretion occurs from adrenal glands in response to such varied factors as stress, nutrition and environment [4, 5]. Therefore, we disagree with Dr Fenske and believe that our collection of a 24-h urine sample for the cortisol ratio in fact assisted in normalizing for variable peak cortisol concentration times. Although Dr Fenske is correct in stating that the variability of urinary 6β-hydroxycortisol:cortisol ratios can be influenced by fluid intake [6], urine production and elimation can also be influenced by such factors as insensible losses and fluid shifts from positioning [7]. To this end, it would be unlikely that a group of individuals given the same amount of fluid over 24 h would have the same urine flow and volume. Although Furuta et al. [8] have stated that the urinary ratio of 6β-hydroxycortisol:cortisol was valid when the renal clearance of cortisol was constant, even within the same subject there were 2.1–4.6-fold variations in the renal clearance of cortisol. It has also been demonstrated that higher fluid intake (5 l) increases the secretion of free cortisol disproportional to metabolites (17-hydroxycorticosteroids), whereas normal fluid intake does not [6]. In our study, the average urine volume was 530 ml and ranged from 375 ml to 915 ml. This is within the range of normal fluid intake and urine production. Median (range) intra-individual variability (as measured by the coefficient of variation) in urine volume was 25.5% (12.2–26.3%). Therefore, the high inter- or intra-individual variability of the urinary 6β-hydroxycortisol:cortisol ratio found in our study was probably a result of other factors than just fluid intake. In summary, Dr Fenske has highlighted the impracticality of controlling all factors that can possibly contribute to high inter- or intra-individual variability of the urinary 6β-hydroxycortisol:cortisol ratio. Therefore, we believe Dr Fenske's comments further endorse our conclusion that the urinary 6β-hydroxycortisol:cortisol ratio is a suboptimal phenotyping tool.

Exaggerated adrenarche and altered cortisol metabolism in Type 1 diabetic children

Reported literature data strongly suggest that steroid metabolism is dysregulated in Type 1 diabetes mellitus. The aim of this study was to non-invasively examine the cortisol metabolism in children with Type 1 diabetes mellitus (T1DM) in detail and to test the hypothesis that adrenarche is affected under conventional intensive insulin therapy. In 24-h urine samples of 109 patients aged 4–18 years with T1DM of more than 1 year, steroids were profiled using gas chromatography–mass spectrometry. Additionally, urinary free cortisol (UFF) and cortisone (UFE) were quantified by RIA after extraction and chromatographic purification. Data on urinary steroids from 400 healthy controls served as reference values. Enzyme activities were assessed by established steroid metabolite ratios, e.g. 5α-reductase and 11beta-hydroxysteroid dehydrogenase Type 2 (11beta-HSD2) by 5α-tetrahydrocortisol/tetrahydrocortisol and UFE/UFF, respectively. Urinary markers of adrenarche, especially dehydroepiandrosterone and its direct metabolites were elevated in patients, as were urinary 6β-hydroxycortisol, UFE, and 11beta-HSD2 activity. However, overall cortisol secretion, as reflected by the sum of major urinary cortisol metabolites, was mostly normal and activity of 5α-reductase clearly reduced. Our study provides evidence for an exaggerated adrenarche in T1DM children, which may help to understand reported sequelae in female patients like hyperandrogenic symptoms. The findings also suggest a reduced cortisol inactivation via 5α-reductase that is not compensated by a fall in cortisol secretion. Whether the elevated urinary 6β-hydroxycortisol and cortisone excretion, observed in the patients, are also present in other forms of hypercortisolism and may thus serve as non-invasive clinical stress markers deserves further study.

Within-person variability of urinary 6β-hydroxycortisol to urinaryl ratios in Caucasian women

Cortisol is metabolized to 6β-hydroxycortisol by human cytochrome P450-3A4 (CYP3A4), an important enzyme involved in the metabolism of a variety of exogenous and endogenous compounds. Both cortisol and 6β-hydroxycortisol are excreted in urine, and the ratio of these steroids has been proposed as an indicator of CYP3A4 activity. We evaluated within-person variability of this biomarker in 10 healthy Caucasian women, aged 23–58 years. Each study participant was asked to provide a fasting morning urine sample once a week consecutively for 8 weeks. Urinary cortisol and 6β-hydroxycortisol were determined by immunoassay kits purchased from the DiaSorin (Stillwater, MN) and the Stabiligen (Nancy, France), respectively. The coefficients of variation (CV) of urinary 6β-hydroxycortisol to cortisol ratios from study participants ranged from 16.7 to 51.4% (mean, 31.1%) over the study period. The level of the ratio measured in any single urine sample was correlated reasonably well with the average of the ratios over the 8-week study period from the same woman, with the mean correlation coefficient of 0.79. These results indicated that urinary 6β-hydroxycortisol to cortisol ratios measured in a spot urine sample may reflect the level of this biomarker over a relatively longer time period in Caucasian women, and thus, it can be used in epidemiologic studies as a biomarker to evaluate the association between CYP3A4 activity and disease risk.

Simple and sensitive high-performance liquid chromatography method for simultaneous determination of urinary free cortisol and 6β-hydroxycortisol in routine practice: For CYP 3A4 activity evaluation in basal conditions and after grapefruit juice intake

Cytochrome P450 3A4 activity displays a wide variability. The urinary 6β-hydroxycortisol to cortisol ratio, as a non-invasive assay, can be useful for its pretherapeutic characterization. We developed an HPLC–UV method preceded by liquid–liquid extraction for assessment of this ratio in clinical practice. Urine was collected on second void morning-spot sample. Percentage recoveries were high and reproducible. The 6β-hydroxycortisol to cortisol ratio ranged from 1.6 to 9.9 in 12 Caucasian healthy volunteers. It was reduced by 30 to 70% after ingestion of white grapefruit juice, a CYP3A4 inhibitor. Our method, simple, sensitive and accurate, could be helpful for determination of CYP 3A4 activity before oral chemotherapy, or for the monitoring of the use of grapefruit juice as a pharmacological modulator.

Reduced urinary 6β-hydroxycortisol to cortisol ratios in patients with liver cirrhosis

While the urinary concentration ratio of 6β-hydroxycortisol (6β-HC) to cortisol (C) in 24-h urine samples is an established parameter for the induction of hepatic cytochrome P450 (CYP) 3A activity, it remains unclear whether it would be reduced in association with chronic liver damages. To clarify this issue we measured this parameter in 12 patients with chronic hepatitis, 15 patients with liver cirrhosis and 12 healthy subjects. Urinary 6β-HC and C were assayed with high-performance liquid chromatography with ultraviolet absorption. Multiple regression analysis was performed to search for which biochemical or demographic parameter(s) would be associated with the overall variability of urinary 6β-HC/C ratios. Results showed that the mean (±S.D.) 6β-HC/C ratio obtained from the patients with liver cirrhosis (4.3±2.6), but not that from patients with chronic hepatitis (7.4±7.2), was significantly ( P<0.05) lower than that obtained from the healthy controls (11.8±8.3). It was also revealed that among the clinical parameters examined (e.g. serum transaminases concentrations) only serum albumin concentrations were correlated significantly ( R=0.61, P<0.05) with the urinary 6β-HC/C ratio. In conclusion, the present study implies that that the reduced urinary 6β-HC/C ratios in patients with liver cirrhosis may be associated with attenuated hepatic CYP3A activity in these patients.

URINARY 6β-HYDROXYCORTISOL/CORTISOL RATIO DOES NOT REFLECT &lt;I&gt;IN VIVO&lt;/I&gt; CYP3A ACTIVITY IN HUMANS

A rationale is described to explain that the urinary 6β-hydroxycortisol/cortisol (6β-OHF/F) ratio does not appropriately reflect in vivo CYP3A activity (phenotype). To evaluate CYP3A-mediated 6β-hydroxylation of cortisol in vivo, “fractional metabolic clearance” specific for the 6β-hydroxylation was calculated by measuring both urinary 6β-hydroxycortisol and plasma cortisol after administering stable isotopically labelled cortisol to humans. It is shown that the urinary 6β-OHF/F ratio is a function of metabolic clearance and renal clearance of cortisol. The observed intra- and interindividual variabilities of the renal clearance indicate that the urinary ratio is not appropriate for the CYP3A phenotyping. We propose a safe and reliable CYP3A-phenotyping.

Specific determination of urinary 6β-hydroxycortisol and cortisol by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

The urinary 6β-hydroxycortisol to cortisol ratio is believed to be a noninvasive index of cytochrome P450 3A activity. For precise assessment of the ratio in human urine, we have developed a reversed-phase high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry method. The selective method was accurate and reproducible with intra- and inter-day precision of variation coefficients of less than 8%. The 6β-hydroxycortisol to cortisol ratio ranged from 3.0 to 12.4 in healthy Japanese 24-h urine. With the recent popularization of LC–MS, our LC–MS method will be advantageous to detect human in vivo CYP3A activity for clinical investigation and routine measurement in various laboratories.