Assessment of Hepatic Cytochrome P450 3A Activity Using Metabolic Markers in Patients with Renal Impairment.

BACKGROUND: The renal function of individuals is one of the reasons for the variations in therapeutic response to various drugs. Patients with renal impairment are often exposed to drug toxicity, even with drugs that are usually eliminated by hepatic metabolism. Previous study has reported an increased plasma concentration of indoxyl sulfate and decreased plasma concentration of 4ß-hydroxy (OH)-cholesterol in stable kidney transplant recipients, implicating indoxyl sulfate as a cytochrome P450 (CYP) inhibiting factor. In this study, we aimed to evaluate the impact of renal impairment severity-dependent accumulation of indoxyl sulfate on hepatic CYP3A activity using metabolic markers. METHODS: Sixty-six subjects were enrolled in this study; based on estimated glomerular filtration rate (eGFR), they were classified as having mild, moderate, or severe renal impairment. The plasma concentration of indoxyl sulfate was quantified using liquid chromatography-mass spectrometry (LC-MS). Urinary and plasma markers (6ß-OH-cortisol/cortisol, 6ß-OH-cortisone/cortisone, 4ß-OH-cholesterol) for hepatic CYP3A activity were quantified using gas chromatography-mass spectrometry (GC-MS). The total plasma concentration of cholesterol was measured using the enzymatic colorimetric assay to calculate the 4ß-OH-cholesterol/cholesterol ratio. The correlation between variables was assessed using Pearson's correlation test. RESULTS: There was a significant negative correlation between MDRD eGFR and indoxyl sulfate levels. The levels of urinary 6ß-OH-cortisol/cortisol and 6ß-OH-cortisone/cortisone as well as plasma 4ß-OH-cholesterol and 4ß-OH-cholesterol/cholesterol were not correlated with MDRD eGFR and the plasma concentration of indoxyl sulfate. CONCLUSION: Hepatic CYP3A activity may not be affected by renal impairment-induced accumulation of plasma indoxyl sulfate.

HPLC-ESI-MS/MS assessment of the tetrahydro-metabolites of cortisol and cortisone in bovine urine: promising markers of dexamethasone and prednisolone treatment.

The effects of long-term administration of low doses of dexamethasone (DX) and prednisolone (PL) on the metabolism of endogenous corticosteroids were investigated in veal calves. In addition to cortisol (F) and cortisone (E), whose interconversion is regulated by 11ß-hydroxysteroid dehydrogenases (11ßHSDs), special attention was paid to tetrahydrocortisol (THF), allo-tetrahydrocortisol (aTHF), tetrahydrocortisone (THE) and allo-tetrahydrocortisone (aTHE), which are produced from F and E by catalytic activity of 5α and 5ß-reductases. A specifically developed HPLC-ESI-MS/MS method achieved the complete chromatographic separation of two pairs of diastereoisomers (THF/aTHF and THE/aTHE), which, with appropriate mass fragmentation patterns, provided an unambiguous conformation. The method was linear (r(2) > 0.9905; 0.5-25 ng ml(-1)), with LOQQ of 0.5 ng ml(-1). Recoveries were in range 75-114%, while matrix effects were minimal. The experimental study was carried out on three groups of male Friesian veal calves: group PL (n = 6, PL acetate 15 mg day(-1) p.o. for 31 days); group DX (n = 5, 5 mg of estradiol (E2) i.m., weekly, and 0.4 mg day(-1) of DX p.o. for 31 days) and a control group (n = 8). Urine was collected before, during (twice) and at the end of treatment. During PL administration, the tetrahydro-metabolite levels decreased gradually and remained low after the suspension of treatment. DX reduced urinary THF that persisted after the treatment, while THE levels decreased during the experiment, but rebounded substantially after the DX was withdrawn. Both DX and PL significantly interfered with the production of F and E, leading to their complete depletion. Taken together, the results demonstrate the influence of DX and PL administration on 11ßHSD activity and their impact on dysfunction of the 5-reductase pathway. In conclusion, profiling tetrahydro-metabolites of F and E might serve as an alternative, indirect but reliable, non-invasive procedure for assessing the impact of synthetic glucocorticosteroids administration.

Simultaneous measurements of cortisol and cortisone in urine and hair for the assessment of 11ß-hydroxysteroid dehydrogenase activity among methadone maintenance treatment patients with LC-ESI-MS/MS.

The activity of 11ß-hydroxysteroid dehydrogenases (11ß-HSD) is traditionally assessed using the ratio of cortisol to cortisone in urine or saliva. However, these biomarkers only reflect the local activity of 11ß-HSD, and are easily affected by circadian variation of cortisol secretion. The shortcomings might be overcome by hair analysis. The present study aimed to develop an enhanced assay for simultaneous measurements of cortisol and cortisone in both hair and urine samples. The samples were collected from 29 patients under methadone maintenance treatment. The cortisol and cortisone were extracted either by solid phase extraction from a 20-mg milled hair sample after a 14-h incubation in 1ml of methanol, or by twice liquid-liquid extraction from a 20-fold diluted urine sample. The analyses were performed using high performance liquid chromatography tandem mass spectrometry with electrospray ionization in negative mode. Limits of detection and quantification were 0.5 and 1.25pg/mg for hair steroids and 0.2 and 0.5ng/ml for urinary steroids, respectively. The recoveries were more than 97%. The intra- and inter-day coefficients of variation were less than 10%. The ratios of cortisol to cortisone in hair and urine were both less than one, but did not correlate with each other. A possible reason for the lack of correlation was that the ratios in hair and urine might mostly reflect the activity of 11ß-HSD type 2 in the eccrine sweat gland and in the kidney, respectively. Additionally, a significant correlation was observed between results obtained using external standard quantification and internal standard quantification.

Surface plasmon resonance immunosensor for cortisol and cortisone determination.

In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regeneration and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spectrometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are characterized by a detection limit less than 10 microg l(-1), sufficiently sensitive for both clinical and forensic use.

Recent advances in the assessment of the ratios of cortisol to cortisone and of some of their metabolites in urine by LC-MS-MS.

A previously reported method for the assessment of the ratio of tetrahydrocortisol (THF) + allo-tetrahydrocortisol (A-THF) to tetrahydrocortisone (THE) by HPLC-MS-MS has been significantly improved, in order to increase either ruggedness and reliability. That was achieved by the introduction of an on-line sample cleanup stage, which made use of a perfusion column as a solid phase microextraction (SPE) cartridge. The set of analytes was expanded, by introducing cortisol and cortisone, whose ratio supply additional diagnostic information. The response factors of both THF and A-THF has been checked, resulting almost identical, as well as the influence of the matrix on the calibration curves which, although different for water and urine, had similar effect on the ratios of interest. As a consequence, the calibration solutions can be prepared in pure water. The influence of several different storage procedures has also been tested, resulting in no substantial effect on the final result. Finally, the improved method has been used to run real samples from healthy volunteers, with satisfactory results.

Suppression of adrenal function by low-dose prednisone: assessment with 24-hour urinary steroid hormone profiles--a review of five cases.

The impact of the synthetic glucocorticoid prednisone on adrenal steroid hormone production was examined using 24-hour urinary steroid hormone profiling. Five women, who were chronically taking low-dose prednisone, were tested, and the relevant literature was reviewed. As expected, adrenal glucocorticoid production, measured by urinary terminal cortisol and cortisone metabolites, was markedly suppressed compared to reference range values (p=0.03). Urinary cortisol and cortisone, reflecting circulating glucocorticoids, were decreased to a lesser extent than their terminal metabolites. Urinary dehydroepiandrosterone (DHEA) excretion was dramatically suppressed (p=0.03), while the downstream androgen metabolites androsterone and etiocholanolone were suppressed to a lesser extent. Aldosterone and tetrahydrocorticosterone production demonstrated modest suppression after prednisone administration, but allo-tetrahydrocorticosterone, which is highly sensitive to adrenocorticotropic hormone (ACTH) secretion, was suppressed to a greater extent. Prednisone administration results in a decrease in ACTH secretion by the anterior pituitary, suppressing synthesis of glucocorticoids, DHEA, and DHEA metabolites. Decreased glucocorticoid synthesis is adaptive, because prednisone is active at the glucocorticoid receptor, but suppression of DHEA synthesis is not mitigated by prednisone. DHEA is an important sex hormone precursor, neurosteroid, and endocrine and immune modulator; therefore, DHEA depletion may have significant adverse consequences in terms of sex hormone production, bone health, endocrine and immune system function, and neuropsychiatric status. Studies of DHEA replacement in patients taking prednisone for lupus demonstrate amelioration of some of these adverse effects.

Urinary excretion of catecholamines, cortisol and their metabolites in Meishan and large white sows: validation as a non-invasive and integrative assessment of adrenocortical and sympathoadrenal axis activity.

Urinary free corticoids (cortisol and cortisone), catecholamines (norepinephrine or NE, epinephrine or E, dopamine or DA, and their O-methoxylated metabolites) as well as creatinine (Cr) were analysed in 42 spontaneously voided urine samples from Large White (LW, n = 20), Meishan (MS, n = 6), and LW x MS (F1, n = 16) lactating sows. The cortisol concentration in the urine of MS (28.1 pg/micrograms Cr) was five-fold greater than that of LW sows (6.2 pg/micrograms Cr, P < 10(-4)). F1 were intermediate (12.0 pg/micrograms Cr). Mean cortisone concentration was also larger in MS (13.5 pg/micrograms Cr) compared to LW (7.1 pg/micrograms Cr, P < 0.01). Although the differences were less pronounced, the concentrations of the catecholamines were also greater in MS than in LW sows (norepinephrine: 25.4 versus 5.9 pg/micrograms Cr, epinephrine: 8.7 versus 2.8 pg/micrograms Cr and dopamine: 59.2 versus 17.8 pg/micrograms Cr, P < 10(-4)). These results confirmed the hypercortisolism state of MS pigs previously shown by plasma cortisol assay and supported the hypothesis that the sympathetic nervous system is hyperactive in this breed. These urinary investigations may offer possible applications for the assessment of chronic stress.

Urinary free cortisone and the assessment of 11 beta-hydroxysteroid dehydrogenase activity in man.

OBJECTIVE: Two isoforms of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) catalyse the interconversion of cortisol to hormonally inactive cortisone; defects in the 11 beta-HSD2 isoform result in hypertension. The kidney, expressing high levels of 11 beta-HSD2, is the principal source of cortisone in man. We have validated the measurement of urinary free cortisone (UFE) excretion in normals and in patients with disorders of the pitultary-adrenal axis in an attempt to more accurately measure the activity of 11 beta-HSD2 in vivo. SUBJECTS: Forty-one normal adults, 12 normal children < 12 years of age, 15 patients with Cushing's syndrome, 12 with hypopitultarism on replacement hydrocortisone, 12 with the syndrome of apparent mineralocorticoid excess (AME) and 7 volunteers consuming liquorice. MEASUREMENTS: A complete 24-hour urine collection was analysed by gas chromatography/mass spectrometry for "A-ring' reduced cortisol and cortisone metabolites, i.e. tetrahydrocortisols (THF and allo-THF) and tetrahydrocortisone (THE). In addition, urinary free cortisol (UFF) and urinary free cortisone were quantified using deuterium-labelled internal standards. RESULTS: In normal adults and children, UFE excretion exceeded that of UFF (UFF 30.4 +/- 2.4 micrograms/24h (mean +/- SE), UFE 54.6 +/- 4.1 micrograms/24h, adults) (for conversion to nmol/24h multiply E by 2.78 and F by 2.76 respectively). Thus the normal UFF/UFE ratio was 0.54 +/- 0.05 in contrast to the (THF + allo-THF)/THE ratio of 1.21 +/- 0.06. UFE excretion was normal in hypopituitary patients on replacement hydrocortisone. Although UFE was elevated in all forms of Cushing's syndrome, the UFF/UFE ratio was grossly elevated in patients with the ectopic ACTH syndrome (14.0 +/- 6.7, n = 6). UFE was below the lower limit of the assay (< 1 microgram/24h) in most patients with the so-called type 1 variant of AME and significantly reduced in 4 patients described as having the type 2 variant of AME (10.5 +/- 3.5 micrograms/h, P < 0.05) and in 7 volunteers consuming liquorice (26.8 +/- 10.0 micrograms/24h, P < 0.01). In ectopic ACTH syndrome, AME, and liquorice ingestion the UFF/UFE ratio was more deranged than the (THF + allo-THF)/THE ratio. CONCLUSION: In normals the discrepant THF + allo-THF/ THE and UFF/UFE ratio suggests that much more of the UFE is derived from the kidney. Reduction in UFE excretion is seen following liquorice ingestion and in both variants of AME, though it is more profound in AME1. The high UFF/UFE ratio in the mineralocorticoid excess state seen in the ectopic ACTH syndrome is compatible with substrate-saturation of renal 11 beta-HSD2. The measurement of UFE and the UFF/UFE ratio is a significant advance in the analysis of human 11 beta-HSD activity in vivo; in particular, the UFF/UFE ratio appears to be a more sensitive index than the (THF + allo-THF)/THE ratio of renal 11 beta-HSD2 activity.