A LC-MS/MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine.

Aldosterone (ALD), its precursor 18-hydroxycorticosterone (18-OHB) and its metabolite tetrahydroaldosterone (TH-ALD) are important biomarkers for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in the detection of small molecules of hormones because it has advantages in terms of specificity and sensitivity. The objective of this study is to develop a new LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB, and TH-ALD in human urine and attempt to diagnose primary aldosteronism using different indicators. The urine samples were treated with a solid-phase extraction pretreatment technique and the three analytes were separated on a reversed-phase column and detected on a triple quadrupole mass spectrometer. The established method was validated according to CLSI C62-A standard guidelines. The calibration ranges from 25 pg/mL to 5000 pg/mL for aldosterone (free), 18-hydroxycorticosterone and tetrahydroaldosterone, and the lower limit of quantification for these three analytes was 25 pg/mL. The matrix effects and recoveries of these three analytes ranged from 85.1 % to 115 % and from 86.3 % to 114 %, respectively. The intra-day and inter-day precision ranged from 1.29 % to 6.78 % and from 1.77 % to 8.64 %, respectively. The performance of the method met the requirements of the guidelines. 40 clinical urine samples including 22 PA patients and 18 non-PA patients were detected, and the ROC curves of three diagnostic indicators were established. The area under the curve (AUC) of ALD (free) is the biggest, so ALD (free) was the best compound to be used as a diagnostic indicator in this study. When the cut-off point was taken as 141 ng/24-h, the sensitivity was 72.7 % and the specificity was 88.9 %. We developed and validated an LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB and TH-ALD in human urine. Our study provides a reference for the use of new biomarkers for the diagnosis of primary aldosteronism.

Serum steroid metabolome on the day of oocyte retrieval in women with polycystic ovarian syndrome and its association with pregnancy outcome of in vitro fertilization.

Steroid hormone level is a crucial factor affecting the outcomes of in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). The purpose of this study was to evaluate serum steroid metabolome on the day of oocyte retrieval in women with polycystic ovarian syndrome (PCOS) and explore whether specific steroids can be potential indicators to improve the prediction of pregnancy outcomes in PCOS patients undergoing IVF/ICSI. In this study, the serum levels of 21 steroids in 89 women with PCOS and 73 control women without PCOS on the day of oocyte retrieval of the first IVF/ICSI treatment cycle were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All patients subsequently received good-quality embryo transfer, and the correlation between their steroid profiles and pregnancy outcomes of the first embryo transfer (ET) was retrospectively analyzed. We found PCOS patients had aberrant levels of 11 out of 21 steroid hormones compared to control individuals, with androgen steroid hormones being considerably enhanced. Enzyme activity evaluation indicated that PCOS women might have abnormal activity of CYP17A1, CYP21A2, CYP11B2, CYP19A1, HSD3B, HSD11B, and HSD17B. Additionally, the level of 18-hydroxycorticosterone (p = 0.014), corticosterone (p = 0.035), and 17-hydroxypregnenolone (p = 0.005) were markedly higher in live birth group than in non- live birth group for PCOS women following frozen embryo transfer (FET). Multiple logistic regressions indicated that 18-hydrocorticosterone and 17-hydroxypregnenolone were independently associated with live birth outcomes of PCOS women following FET. Receiver operating characteristic (ROC) curve analysis revealed that 0.595 ng/mL for 18-hydrocorticosterone level (AUC: 0.6936, p = 0.014).and 2.829 ng/mL for 17-hydroxypregnenolone level (AUC: 0.7215, p = 0.005) were the best cutoff values to predict live birth outcomes of PCOS. In conclusion, the blood steroid metabolome was closely related to the IVF/ICSI outcomes of PCOS patients. 18-hydroxycorticosterone and 17-hydroxypregnenolone might be potential indicators to predict pregnancy outcomes of PCOS undergoing IVF/ICSI treatment.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay for the simultaneous quantification of 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol in human plasma.

18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF) and 18-oxocortisol (18-OXOF) are important biomarkers for the diagnosis of subtypes of primary aldosteronism. The detection of these three analytes by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is free from structurally similar compounds. The aim of this study was to develop and validate a new LC-MS/MS assay for the simultaneous quantification of 18-OHB, 18-OHF and 18-OXOF in plasma and to establish a reference intervals for apparently healthy population. Plasma samples were prepared by solid phase extraction and separated in an ultra-high performance reversed phase column. MS detection was achieved using a triple quadrupole mass spectrometer in both positive and negative ionization modes. The developed assay was then validated against standard guidelines. We collected 691 plasma samples from apparently healthy individuals (M:398, F:293) to establish the reference intervals. The analytes were separated and quantified within 5 min. The newly developed method demonstrated linearity for the detected steroid concentration in range of 5 to 3000 pg/ml for 18-OXOF (r2 = 0.999) and 20 to 3000 pg/ml for 18-OHB (r2 = 0.997) and 18-OHF (r2 = 0.997). The lower limit of quantification (LLOQ) was 2.5 pg/ml, 20 pg/ml and 20 pg/m for 18-OXOF, 18-OHB and 18-OHF respectively. Specificity, precision, accuracy and stability were tested, and met the requirements of the guidelines. 18-OHB was higher in females than in males, but 18-OHF were higher in males than females. The reference intervals of 18-OHB, 18-OHF and 18-OXOF for both genders together were 90.5-1040.6 pg/ml, 224.4-1685.2 pg/ml, 4.0-70.5 pg/ml, respectively. Age was also an important factor influencing the levels of these three hormones. We have developed a sensitive and reliable method for the simultaneous quantification of 18-OHB, 18-OHF, and 18-OXOF. Our work provides a reference interval for the clinical application of these three steroid hormones.

Human cytochrome P450 11B2 produces aldosterone by a processive mechanism due to the lactol form of the intermediate 18-hydroxycorticosterone.

Human cytochrome P450 (P450) 11B2 catalyzes the formation of aldosterone, the major endogenous human mineralocorticoid. Aldosterone is important for the regulation of electrolyte homeostasis. Mutations and overexpression of P450 11B2 (also known as aldosterone synthase) can lead to hypertension, congestive heart failure, and diabetic nephropathy. The enzyme is therefore a target for drug development to manage these various disorders. P450 11B2 catalyzes aldosterone formation from 11-deoxycorticosterone through three distinct oxidation steps. It is currently unknown to which degree these reactions happen in sequence without the intermediate products dissociating from the enzyme (i.e. processively) or whether these reactions happen solely distributively, in which the intermediate products must first dissociate and then rebind to the enzyme before subsequent oxidation. We present here a comprehensive investigation of processivity in P450 11B2-catalyzed reactions using steady-state, pre-steady-state, pulse-chase, equilibrium-binding titrations, and stopped-flow binding studies. We utilized the data obtained to develop a kinetic model for P450 11B2 and tested this model by enzyme kinetics simulations. We found that although aldosterone is produced processively, the enzyme preferentially utilizes a distributive mechanism that ends with the production of 18-OH corticosterone. This seemingly contradictory observation could be resolved by considering the ability of the intermediate product 18-OH corticosterone to exist as a lactol form, with the equilibrium favoring the ring-closed lactol configuration. In summary, our refined model for P450 11B2 catalysis indicates isomerization of the intermediate to a lactol can explain why P450 11B2 must produce aldosterone through a processive mechanism despite favoring a distributive mechanism.

Enabling insights into the maturation of the renin-angiotensin-aldosterone system in children-Development of a low-volume LC-MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite.

INTRODUCTION: As part of the renin-angiotensin-aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. METHODS: A HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight-mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. RESULTS: Using the Sciex TripleTOF 6600, a reliable determination in 50 µL serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between -1.21 and -6.99 % and 2.07 and -10.22 %, respectively, confirming compliance with international guidelines. CONCLUSION: A simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 µL serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.

Primary Neurolymphomatosis Presenting With Polyradiculoneuropathy Affecting One Lower Limb.

OBJECTIVES: We report a case of primary neurolymphomatosis (NL) with unusual presentation and excellent treatment response. METHODS: Chart review. RESULTS: A 64-year-old woman presented with 2 months of progressive pain, weakness, and numbness in her right leg. Nerve conduction study/electromyogram suggested a right lumbosacral radiculoplexus neuropathy with associated acute right peroneal neuropathy at the fibular head. L/S spine and right leg magnetic resonance imaging showed thickening and contrast enhancement of the right S1 nerve root and the right distal sciatic, tibial, and common peroneal nerves, as well as a lobular enhancing lesion of the right superficial peroneal nerve. Whole-body fludeoxyglucose-positron emission tomography scan showed no other lesions. A right superficial peroneal nerve lesion biopsy revealed infiltration of the nerve by diffuse large B-cell lymphoma. The lymphoma cells expressed BCL2 but not CD10, suggesting an origin in peripheral blood not lymph nodes. Despite the expression of BCL2, which is considered as a poor prognosis marker, our patient responded very well to the combined radiotherapy and chemotherapy with the R-MPV (rituximab, MTX, procarbazine, and vincristine) regimen. The patient showed marked clinical improvement and complete resolution of lymphoma lesions on the PET scan. CONCLUSIONS: Our case broadens the clinical spectrum and illustrates the importance of early diagnosis and aggressive treatment of primary NL.

Simultaneous determination of 18 tetrahydrocorticosteroid sulfates in human urine by liquid chromatography/electrospray ionization-tandem mass spectrometry.

A liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) method for the direct determination of eighteen tetrahydrocorticosteroid sulfates in human urine has been developed. The analytes were 3- and 21-monosulfates and 3,21-disulfates of tetrahydrocortisol (THF), tetrahydrocortisone (THE), tetrahydro-11-deoxycortisol (THS), and their corresponding 5α-H stereoisomers. The mass spectrometric behavior of these sulfates in negative-ion ESI-MS/MS revealed the production of intense structure specific product ions within the same group of sulfates and permitted distinction between regioisomeric sulfates by collision-induced fragmentation with the MS/MS technique using a linear ion-trap instrument. For the quantitative analysis, selected reaction monitoring analysis in the negative-ion detection mode using triple-stage quadrupole mass spectrometer was performed by monitoring transitions from [M-H](-) to the most abundant product ion of each tetrahydrocorticosteroid sulfate. After addition of 3- and 21-monosulfates of [2,2,3ß,4,4-d5]-THF, -THE, and -THS as internal standards, urine sample was applied to a solid phase extraction using a lipophilic-weak anion exchange cartridge column, and then analyzed by LC/ESI-MS/MS. The method had satisfactory performance in terms of intra- and inter-assay precision (less than 9.7% and 9.6%, respectively), and accuracy (91.2-108.2%). The limit of quantification was lower than 2.5 ng/mL for all sulfates examined. We applied this method to determine the concentration of eighteen tetrahydrocorticosteroid sulfates in the urine of healthy subjects. Thus, we have developed a sensitive, precise and accurate assay for urinary tetrahydrocorticosteroid sulfates that should be useful for clinical and biological studies.

18-hydroxycorticosterone, 18-hydroxycortisol, and 18-oxocortisol in the diagnosis of primary aldosteronism and its subtypes.

CONTEXT: Diagnosis of primary aldosteronism (PA) is made by screening, confirmation testing, and subtype diagnosis (computed tomography scan and adrenal vein sampling). However, some tests are costly and unavailable in most hospitals. OBJECTIVE: The aim of the study was to evaluate the role of serum 18-hydroxycorticosterone (s18OHB), urinary and serum 18-hydroxycortisol (u- and s18OHF), and urinary and serum 18-oxocortisol (u- and s18oxoF) in the diagnosis of PA and its subtypes, aldosterone-producing adenoma (APA) and bilateral adrenal hyperplasia (BAH). PATIENTS: The study included 62 patients with low-renin essential hypertension (EH), 81 patients with PA (20 APA, 61 BAH), 24 patients with glucocorticoid-remediable aldosteronism, 16 patients with adrenal incidentaloma, and 30 normotensives. INTERVENTION AND MAIN OUTCOME MEASURES: We measured s18OHB, s18OHF, and s18oxoF before and after saline load test (SLT) and 24-h u18OHF and u18oxoF. RESULTS: PA patients displayed significantly higher levels of s18OHB, u18OHF, and u18oxoF compared to EH and normal subjects; APA patients displayed s18OHB, u18OHF, and u18oxoF levels significantly higher than BAH patients. Similar results were obtained for s18OHF and s18oxoF. SLT significantly reduced s18OHB, s18OHF, and s18oxoF in all groups, but steroid reduction was much less for APA patients compared to BAH and EH. The s18OHB/aldosterone ratio after SLT more than doubled in EH but remained unchanged in APA patients. CONCLUSIONS: u18OHF, u18oxoF, and s18OHB measurements in patients with a positive aldosterone/plasma renin activity ratio correlate with confirmatory tests and adrenal vein sampling in PA patients. If verified, these steroid assays would refine the diagnostic workup for PA.

Synthesis, biological evaluation, and molecular modeling of 1-benzyl-1H-imidazoles as selective inhibitors of aldosterone synthase (CYP11B2).

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, this is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, we used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-yl)methyl)benzonitrile) displaying an IC(50) for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC(50) for CYP11B2 6.0 nM, selectivity 19.8). Molecular docking of the inhibitors in the models enabled us to generate posthoc hypotheses on their binding modes, providing a valuable basis for future studies and further design of CYP11B2 inhibitors.

Liver lesion detection and characterization in patients with colorectal cancer: a comparison of low radiation dose non-enhanced PET/CT, contrast-enhanced PET/CT, and liver MRI.

OBJECTIVES: To compare low-radiation dose non-enhanced fluorine 18 fluorodeoxyglucose (F-18 FDG) positron emission tomography (PET)/computed tomography (CT) (NE-PET/CT), contrast-enhanced fluorine 18 fluorodeoxyglucose PET/CT (CE-PET/CT), and gadolinium-enhanced liver magnetic resonance imaging (MRI) for the detection and characterization of liver lesions in patients with colorectal cancer (CRC). METHODS: In this retrospective review of imaging database of CRC patients with suspected liver metastases, 33 patients (22 men, 11 women; mean age, 63 years) evaluated with low-radiation dose NE-PET/CT, CE-PET/CT, and liver MRI were studied. The final diagnosis was established either by pathological examination or follow-up imaging over a period of at least 6 months for lesion stability or growth. The liver lesions were characterized on an ordinal scale of 0 to 6 (0 = absent, 1 = definitely benign, and 6 = definitely malignant). Receiver operating characteristic analysis was performed to compare performance of the 3 imaging methods. RESULTS: A total of 110 lesions were present on follow-up. The detection rate on low-radiation dose NE-PET/CT, CE-PET/CT, and MRI was 73.6%, 90.9%, and 95.4%, respectively. Magnetic resonance imaging (P < 0.001) and CE-PET/CT (P < 0.001) had a higher detection rate than low-radiation dose NE-PET/CT. There was no significant statistical difference in lesion detection between MRI and CE-PET/CT (P = 0.11). The sensitivity, specificity, and accuracy for characterization of detected liver lesions on low-radiation dose NE-PET/CT were 67%, 60%, and 66%, respectively; those on CE-PET/CT were 85%, 100%, and 86%, respectively; and those on MRI were 98%, 100%, and 98%, respectively. Comparative receiver operating characteristic analysis showed an area under curve of 0.74 for low-radiation dose NE-PET/CT, 0.86 for CE-PET/CT, and 0.97 for MRI. There were statistically significant differences in the accuracy of MRI, low-radiation dose NE-PET/CT, and CE-PET/CT for lesion characterization. CONCLUSIONS: When performing PET/CT, optimal detection and characterization of liver lesions require the use of a fused contrast-enhanced CT. Magnetic resonance imaging and CE-PET/CT have similar lesion detection rates. Magnetic resonance imaging is the best test for liver lesion characterization in patients with CRC.

Measurement of 18-hydroxycorticosterone during adrenal vein sampling for primary aldosteronism.

CONTEXT: In primary aldosteronism, elevated serum 18-hydroxycorticosterone (18OHB) suggests aldosterone-producing adenoma (APA) rather than bilateral, idiopathic hyperaldosteronism (IHA), but little is known about the relative production of 18OHB and aldosterone (A) in APAs compared with IHA. OBJECTIVES: We measured 18OHB, A, and cortisol (F) in blood from adrenal vein sampling (AVS) studies. We compared the discriminatory power of gradients in 18OHB/A and 18OHB/F ratios with A/F ratio gradients for distinguishing APA from IHA. DESIGN, SETTING, AND SUBJECTS: We measured 18OHB and A in excess serum from 23 AVS studies performed at our university hospitals. MAIN OUTCOME MEASURES: We calculated the ratios 18OHB/A, 18OHB/F, and A/F for all specimens, and determined the adrenal vein gradients for these ratios. RESULTS: The 18OHB/A ratios were much lower in blood draining APAs (2.17 +/- 0.62) than in blood draining the contralateral adrenals (12.96 +/- 12.76; P < 0.001) but similar to blood draining IHA adrenals (4.69 +/- 4.32; P = 0.02). In contrast, the 18OHB/F ratios were elevated in specimens from APAs (26.03 +/- 11.51) compared with IHA adrenals (9.22 +/- 5.18; P < 0.001) or the contralateral adrenals (6.23 +/- 2.97; P < 0.001). Using 18OHB/F gradient greater than two or 18OHB/A gradient less than 0.5 as criteria for lateralization, interpretations agreed with lateralizations based on A/F gradients in 21 of 23 cases. CONCLUSIONS: High serum 18OHB in APA reflects augmented production of both 18OHB and A, not disproportionate 18OHB secretion relative to A. The 18OHB/A and 18OHB/F gradients are useful adjuncts but not as reliable as A/F gradients for A lateralization during AVS.

Determination of corticosterone and 17-hydroxycorticosterone in plasma and urine samples by sweeping techniques using micellar electrokinetic chromatography.

The analysis of corticosterone in mouse blood serum (metabolic-stress experiment) and 17-hydroxycorticosterone in human urine (exercise-stress experiment) samples by means of capillary electrophoresis/UV absorbance in conjunction with online sample concentration techniques is described. The use of normal MEKC had an analyte detection limit of 7 microg/ml (S/N=3); whereas when online sample concentration methods, including sweeping-micellar electrokinetic chromatography (Sweeping-MEKC) and cation-selective exhaustive injection-sweep-micellar electrokinetic chromatography (CSEI-sweep-MEKC) were used, the detection limits could be improved to 3 and 5 ng/ml, respectively. In the analysis of actual samples from animal metabolic-stress experiments (39 mouse), chronically stressed animals showed a higher level (552+/-152 ng/ml) and acute stressed animals showed an intermediate level (375+/-105 ng/ml). In comparison, normal animals show a lower concentration level of corticosterone (153+/-109 ng/ml). In addition, based on a human exercise-stress experiment (seven volunteers), the acute stressed humans (after exercise, 800 m of running) show a higher concentration of 17-hydroxycorticosterone (113+/-55 ng/ml for males; 128+/-25 for females) and the non-stressed humans (before exercise) show a lower concentration (63+/-37 ng/ml for male; 60+/-20 for female), respectively.

Ghrelin, an endogenous ligand for the growth hormone-secretagogue receptor, is expressed in the human adrenal cortex.

Ghrelin is an endogenous ligand of the growth hormone secretagogue receptor (GHS-R), which was originally isolated from rat stomach. Ghrelin and GHS-R are also expressed in several peripheral tissues, including adrenal glands, and this prompted us to study ghrelin expression and ghrelin-binding site localization in the human adrenal cortex, and the possible effect of this peptide on corticosteroid-hormone secretion. Reverse transcription-polymerase chain reaction (RT-PCR) and radioimmune assay (RIA) showed sizeable expression of ghrelin mRNA and protein in six human adrenal cortexes. Autoradiography evidenced abundant [125I]ghrelin binding sites in the adrenal zona glomerulosa and outer zona fasciculata. However, ghrelin (10(-6) M) did not significantly affect either basal or agonist (ACTH and angiotensin-II)-stimulated early and late steps of steroid-hormone synthesis from adrenocortical slices (as measured by quantitative high pressure liquid chromatography). Since zona glomerulosa is the cambium layer involved in the growth maintenance of adrenal cortex, the present coupled RT-PCR, RIA and autoradiographic findings could suggest the involvement of ghrelin in the autocrine-paracrine regulation of human adrenal growth.

Norbormide enhances late steps of steroid-hormone synthesis in rat and mouse adrenal cortex.

Norbormide (N) is a vasoconstrictor agent, which acts selectively on the peripheral arteries of the rat, through the activation of the phospholipase C (PLC) cascade and the stimulation of Ca(2+) entrance in the vascular myocytes. Several endogenous vasoconstrictor agent (e.g. angiotensin-II (ANG-II) and endothelin-1 (ET-1)), that stimulate PLC pathway, are also able to enhance aldosterone secretion by the adrenal gland. Hence, we examined the effects of norbormide ((0.5, 1.0 or 5) x 10(-5)M) on corticosteroid-hormone secretion from adrenal slices of rats and mice. Quantitative HPLC assay showed that under basal conditions rat and mouse adrenal quarters secreted progesterone (PROG), 11-deoxycorticosterone (DOC), 18-hydroxy-DOC (18OH-DOC), corticosterone (CORT), 18-hydroxy-corticosterone (18OH-CORT) and aldosterone (ALDO), as well as large amounts of pregnenolone (PREG) when its metabolism was blocked by 10(-5)M cyanoketone. Norbormide concentration-dependently raised the secretion of all post-DOC steroids assayed, decreased progesterone and DOC production, and did not affect pregnenolone release. In conclusion, norbormide is able to enhance late steps of steroid synthesis, i.e. those leading to the transformation of DOC to corticosterone and aldosterone, without affecting early steps. This is an interesting finding because the other main endogenous adrenal secretagogues are known to stimulate both early and late steps of steroid synthesis. The mechanism underlying the selective activating action of norbormide on 11beta- and 18-hydroxylation remains to be investigated.

Chromatographic system for the simultaneous measurement of plasma 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone by radioimmunoassay: reference data for neonates and infants and its application in aldosterone-synthase deficiency.

A new chromatographic system for the steroid precursor separation and a sensitive radioimmunoassay system for the subsequent measurement of 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone has been developed. 18-Hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone were extracted with methylene chloride and separated from cross-reacting steroids by Sephadex LH-20 column chromatography. Anti-18-hydroxy-11-deoxycorticosterone and anti-18-hydroxycorticosterone antibodies raised in rabbits were used. The lower detection limit of the assay is 0.03 nmol/l and 0.128 nmol/l for 18-hydroxy-11-deoxycorticosterone and 18-hydroxycorticosterone, respectively. Normal values for this assay in 128 healthy neonates and infants aged 0-5 months were established as a basis for the early hormonal diagnosis of aldosterone synthase deficiency types I and II. Its application for the diagnosis of aldosterone synthase deficiency is demonstrated in two patients with homozygous mutation/deletion in the encoding CYP11B2 gene.

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography.

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml (S/N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.

Analysis of biological samples by gas chromatography-mass spectrometry without a reference standard: measurement of urinary 18-hydroxytetrahydro-11-dehydrocorticosterone excretion rate in human subjects.

Reference standards for some minor urinary steroid metabolites are sometimes unavailable. We describe a novel procedure to quantitate a urinary steroid metabolite of known structure and mass spectrum, using as a standard a compound which produces ions in common with it and has a similar retention time in gas chromatography-mass spectrometry. The steroid of interest was 18-hydroxy-11-dehydrotetrahydrocorticosterone (18-OH-THA), the major urinary metabolite of 18-hydroxycorticosterone (18-OH-B), a putative intermediate in the conversion of 11-deoxycorticosterone to aldosterone. The steroid used as an alternative to the authentic 18-OH-THA standard was beta-cortol which, like 18-OH-THA, produces a fragmentation ion at m/z 457. Allo-tetrahydrodeoxycorticosterone (5alpha-THDOC) was used as the internal standard. beta-Cortolone also has the fragmentation ion at m/z 449 (in common with beta-cortol) and an authentic standard is available commercially. To validate the procedure, we quantitated beta-cortolone urinary excretion rate against this alternative standard and also against authentic beta-cortolone standards. Both methods produced similar results (adjusted R(2): 0.998, P<0.001). The method was then used to measure urinary excretion of 18-OH-THA rate in healthy volunteers. The reference range obtained was 20-204 microgram/24 h (n=32). This is similar to the few results available by conventional assay. Method performance was also similar to other assays of urinary steroids. This procedure could be generally applicable for assays when authentic standards are not available but mass spectra are known or can be predicted.

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences.

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.