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.

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.

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.

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.

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.

Type 1 aldosterone synthase deficiency presenting in a middle-aged man.

Aldosterone synthase deficiency due to mutations in the CYP11B2 gene usually presents in infancy with electrolyte abnormalities and failure to thrive, whereas affected adults are usually asymptomatic. We describe a patient who first came to medical attention in middle age when he developed hyperkalemia after preparation for a barium enema. Past medical history was notable for failure to thrive in infancy. He had elevated PRA with low serum and urinary levels of aldosterone and its metabolites and normal or slightly elevated levels of 18-hydroxycorticosterone. These findings suggested a diagnosis of type 1 aldosterone synthase deficiency. The patient had a homozygous duplication of six nucleotides at codon 143 in exon 3 of CYP11B2, leading to the insertion of two amino acid residues (Arg-Leu). When the corresponding mutant complementary DNA was expressed in cultured cells, the resulting enzyme was completely inactive, confirming the diagnosis. We conclude that aldosterone synthase deficiency represents an unusual cause of hyperreninemic hypoaldosteronism presenting in adult life, but it should be suspected if the past medical history is positive for failure to thrive in childhood or if the patient manifests no other recognized causes of hyperreninemic hypoaldosteronism.

Aldosterone synthase deficiency type I with no documented homozygous mutations in the CYP11B2 gene.

This case report concerns a girl born from non-consanguineous parents and hospitalized in another hospital at the age of 14 days because of a severe salt-losing syndrome (Na=125, K=8.6 mEq/l). In spite of normal genitalia, diagnosis of 21-hydroxylase deficiency was assessed on the basis of a slightly increased 17-OH-progesterone serum level (6.4 ng/ml). The onset of both hydrocortisone and 9alpha-fluorohydrocortisone therapy was followed by a resolution of the clinical picture. At the age of 60 days she was admitted to our clinic for a re-evaluation of the diagnosis. Steroid hormone serum levels were measured after withdrawal of therapy and diagnosis of corticosterone methyl oxidase (CMO) deficiency type I was definitely established in the light of the biochemical results: i.e. very low 18-hydroxycorticosterone (18-OH-B) serum levels (20 pg/ml), an abnormally high corticosterone/18-OH-B serum ratio (306.5) and an abnormally low 18-OH-B/aldosterone serum ratio (2.1). This autosomal recessively inherited disorder can be differentiated from CMO type II and other salt-wasting syndromes only on the basis of the serum steroid hormone pattern. After establishing the diagnosis of CMO I deficiency, hydrocortisone therapy was withdrawn whilst treatment with 9alpha-fluorohydrocortisone was begun again, with a satisfactory clinical and metabolic impact. Direct sequences of the patient's DNA were able to identify only a (heterozygous) amino acid substitution in exon 7 of that gene, which is known to have only a small effect on enzyme activity and cannot be the only cause of the patient's phenotype: valine-386-alanine (V386A) GTG-->GcG. No homozygous mutations in the CYP11B2 gene were observed. This is the first report of a patient with CMO type I who does not carry any homozygous mutation in the entire CYP11B2 alleles, whereas some cases with no mutations in this gene have already been reported in CMO II. The present study seems to be inconsistent with the previously reported correlation of the phenotype and genotype in CMO type I. A reasonable question that might be raised on the basis of our findings in this case report is whether other genes, apart from CYP11B2, are involved in the regulation of terminal aldosterone synthesis.

["High pouch output" syndrome. Role of mineralocorticoid diagnosis after restorative proctocolectomy]. / "High-Pouch-Output"-Syndrom. Stellenwert der Mineralocorticoiddiagnostik nach restaurativer Proktocolektomie.

UNLABELLED: The two-phase restorative proctocolectomy is the treatment of choice for surgical therapy of the familial adenomatous polyposis (FAP) and also for the ulcerative colitis (UC). Besides the well-known complications the entire removal of the colorectum leads to an impairment of fluid and electrolyte resorption. PATIENTS AND METHODS: Over a time period of two years we observed 320 proctocolectomized patients with ileal pouch-anal anastomosis (IPAA). All patients with high pouch output but without organic malfunction were identified. The organic reasons were excluded with the help of pouchoscopy, radiography or MR imaging. We evaluated routine parameters, the kidney function, the electrolyte changes, the acid-base balance and the urine pH, as well as the hormonal changes of the suprarenal glands. We identified seven patients with 'high pouch output' out of 320 patients observed. The control group consisted of 14 proctocolectomized patients without hints of complications in the endoscopic, radiographic and routine laboratory diagnostics. RESULTS: Neither group showed any significant differences in the analysis of the routine parameters. A significant drop of the urine sodium concentration of 40.5 +/- 18.7 mmol/l (control group 98 +/- 43.4 mmol/l) was observed in the group with 'high pouch output'. In this group the plasma aldosterone values were strongly increased with an average of 42.6 +/- 28.9 ng/dl (control group 13.2 +/- 6.8 ng/dl) as well as the plasma 18-hydroxycorticosterone with an average of 153.7 +/- 121.1 ng/dl (control group 153.7 +/- 121.1 ng/dl). Neither group of patients showed increased activity of free corticosterone and free cortisol. Only free 11-desoxycorticosterone was elevated in the group with 'high pouch output'. CONCLUSION: Our results prove that the mineralocorticoid adrenal activity plays a central role in order to preserve the volume and electrolyte homeostasis. The low frequency of 'high-pouch-output'-complications in realms of the restorative proctocolectomy proves the excellent compensation of the removal of the colon mucosa. Plasma aldosterone seems to be a diagnostic marker encapsulating the reabsorption problems of intestinal salt and volume losses after proctocolectomy.

Homozygous deletion of arginine-173 in the CYP11B2 gene in a girl with congenital hypoaldosteronism. Corticosterone methyloxidase deficiency type II.

The first child of consanguineous parents presented with failure to thrive and feeding problems at age 6 weeks. Important laboratory findings were low plasma sodium and elevated potassium and renin. Salt wasting was caused by an enzymatic defect in the terminal aldosterone biosynthesis. The biochemical diagnosis of corticosterone methyloxidase (CMO) deficiency type II was established on the basis of plasma multisteroid analysis, showing a pathologic increase of 18-OH-corticosterone/aldosterone ratio. Sequence analysis of the CYP11B2 gene which encodes aldosterone synthase (P450c11Aldo), the enzyme required for the terminal steps in aldosterone biosynthesis, revealed a hitherto undescribed homozygous deletion of codon 173. CYP11B2 is polymorphic at this position, encoding arginine or lysine. Both parents were heterozygous carriers of the mutation. Amino acid residue 173 in P450c11Aldo is positioned in alpha-helix D. We presume that the secondary structure of the enzyme is changed by the single amino acid deletion. This report describes a novel mutation in the CYP11B2 gene, the third known mutation associated with CMO deficiency type II.

Correlations between mood scores, LH, adrenocortical steroids, and urine volumes in a patient with a history of postpartum depression and monthly psychotic episodes.

A simple semantic differential test was administered twice daily and urinary hormones measured as described previously . The subjects included the patient and two controls, one of whom experienced premenstrual physical, but not mental, discomfort. The test scores of the two controls, administered for five weeks, did not differ significantly from one another but greatly differed from those of the patient. The highest scores in the patient (indicating improved mood) were obtained during the week containing the urinary LH peak, and daily ratings correlated significantly with LH values. Significant, positive, correlations were also found between mood and ratios for aldosterone/cortisol, 18-hydroxydeoxycorticosterone(18-OH-DOC)/cortisol, and 18-OH-DOC/18-hydroxycorticosterone. Urine volumes correlated positively with cortisol, negatively with 18-OH-DOC, and negatively with mood (P<0.01). Urinary hormone assays, affording the advantages of a non-invasive technique may thus reveal relationships of potential interest. Whether these are causal, rather than casual, remains to be assessed.

Hereditary defect in biosynthesis of aldosterone: aldosterone synthase deficiency 1964-1997.

We studied two of the three patients with a hereditary defect in the biosynthesis of aldosterone originally described by Visser and Cost in 1964. All three presented as newborns with salt-losing syndrome and failure to thrive. The original biochemical studies showed a defect in the 18-hydroxylation of corticosterone. According to the nomenclature proposed by Ulick, this defect would be termed corticosterone methyl oxidase deficiency type I. We measured plasma steroids in the untreated adult patients and performed molecular genetic studies. Aldosterone and 18-OH-corticosterone were decreased, whereas corticosterone and 11-deoxycorticosterone were elevated, thus confirming the diagnosis of corticosterone methyl oxidase deficiency type I. Cortisol and its precursors were in the normal range. Genetic defects in the gene CYP11B2 encoding aldosterone synthase (P450c11Aldo) have been described in a few cases. We identified a homozygous single base exchange (G to T) in codon 255 (GAG) causing a premature stop codon E255X (TAG). This mutation destroys a Aoc II restriction site. Digestion of a PCR fragment containing exon 4 of CYP11B2 (261 bp) with this restriction enzyme revealed in the two patients homozygous for the E255X mutation only a 261-bp fragment, whereas the heterozygous parents had three fragments (261 bp from the mutant allele and 194 and 67 bp from the wild-type allele). The mutant enzyme had lost the five terminal exons containing the heme binding site, and thus there was a loss of function enzyme. We conclude that the biochemical phenotype of these prismatic cases of congenital hypoaldosteronism can be explained by the patients genotype.

Adrenocortical adenoma producing 18-hydroxycorticosterone.

A 30-year-old man presented at our hospital with microscopic hematuria. Ultrasonography and computed tomography scanning revealed a right adrenal mass measuring 20 x 20 mm. The tumor was asymptomatic, but there was obvious accumulation on the right side when scintigraphy was performed with radioactive iodine (131I)-labeled adosterol. Endocrinology studies showed elevation of the plasma cortisol and renin concentrations, while the plasma aldosterone level was low. Right laparoscopic adrenalectomy was done on July 4, 1994. Histologic examination showed an adrenocortical adenoma. Serum levels of adrenocortical hormones were measured before and after surgery, and the tissue content for the same hormones was determined in the resected tumor. The hormonal studies showed that the tumor produced 18-hydroxycorticosterone.

[Adrenal adenoma or hyperplasia? Problems of differential diagnosis in an unusual case of primary hyperaldosteronism]. / Adenoma surrenalico o iperplasia? Problemi di diagnostica differenziale in un caso inusuale di iperaldosteronismo primitivo.

Although primary hyperaldosteronism is an uncommon cause of hypertension, it is the most common form of renin-independent hypermineralocorticoidism. The plasma aldosterone concentration and PRA with orthostatic test and saline infusion test are very useful aids to make a diagnosis. In this case the inconsistency between hormonal data and morphologic images (TC and NMR) led us to a dilemma: it was a question of adrenal adenoma or hyperplasia? Because it was impossible to dose the 18-OH-corticosterone, we had to perform a iodocholesterol scintigraphy NP 59. To distinguish an hyperplasia as cause of this kind of hyperaldosteronism made us able to define a therapeutic program useful to hypertension control.