Simultaneous quantitation of urine aldosterone and tetrahydroaldosterone in healthy Chinese subjects using a validated LC-MS/MS method.

Aldosterone (ALD) is excreted in urine mainly as glucuronide conjugates of ALD and tetrahydroaldosterone. Measuring these urinary metabolites might be an alternative screening test to plasma ALD for primary aldosteronism. We report a validated LC-MS/MS method to measure both analytes simultaneously. Urine samples underwent enzymatic hydrolysis to release the analytes from their glucuronide conjugates followed by organic solvent extraction and LC-MS/MS. The analytical performance of this method was evaluated. The within-batch and between-batch coefficients of variation for urine ALD and urine THA were all ≤5.2 and ≤3.7%. The lower limit of quantification was 0.5 nmol/L, and the linearity was up to at least 2770 nmol/L for both analytes. No significant matrix interference and carryover were observed. Both analytes in urine were stable for at least 48 h at 10°C and at least 18 months at -80°C. Local reference intervals were established from 126 healthy normotensive Chinese subjects (53% women, age: 20-65 years). Reference intervals for urine ALD and tetrahydroaldosterone were 2-38 and 9-139 nmol/day, respectively. This validated method can be applied to screening and diagnosing primary aldosteronism.

Chinese normotensive and essential hypertensive reference intervals for plasma aldosterone and renin activity by liquid chromatography-tandem mass spectrometry.

OBJECTIVES: The renin-angiotensin-aldosterone system (RAAS) regulates blood pressure. Plasma renin activities (PRA) and plasma aldosterone concentrations (PAC) are biomarkers related to RAAS. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based measurements for PRA and PAC have become popular. Method-specific reference intervals (RIs) are required. METHODS: Routine PRA and PAC services in a Hong Kong teaching hospital were based on LC-MS/MS methods. PRA and PAC RIs were developed for normotensive subjects and essential hypertensive (EH) patients. Healthy volunteers were recruited to establish normotensive RIs. PRA and PAC results of hypertensive patients with urine aldosterone tests for primary aldosteronism (PA) screening were retrieved from the laboratory information system. Patients without PA were included. Patients with secondary hypertension and patients on medications affecting the RAAS were excluded. The central 95% RIs were established based on the recommendations of the Clinical and Laboratory Standards Institute guideline C28-A3. RESULTS: PRA and PAC of 170 normotensive volunteers and 362 EH patients were analysed. There was no sex-specific difference in PRA and PAC for normotensive and EH reference subjects. Differences for PRA and PAC were noted between normotensive subjects aged below 45 and their older counterparts. However, such a difference was only identified for PRA but not PAC in EH patients. Age-specific RIs were established accordingly. CONCLUSIONS: This study presented age-specific LC-MS/MS RIs of PRA and PAC for both normotensive and EH populations for local Chinese in Hong Kong.

Quantitation of plasma angiotensin II in healthy Chinese subjects by a validated liquid chromatography tandem mass spectrometry method.

Quantitation of plasma angiotensin (Ang) II, the active mediator of the renin-angiotensin system, is challenging owing to its low physiological concentration. We report a validated liquid chromatography-mass spectrometry (LCMS) method to overcome this challenge. Ang II was extracted from EDTA plasma by an offline solid-phase extraction procedure with a Waters MAX µElution plate. LCMS quantitation was performed on the Waters TQS system, monitoring the 3+ ions of the peptide. The analytical performance of the LCMS method was validated. The stability of Ang II was studied with or without the presence of a protease inhibitor. Local reference intervals were established from 143 healthy normotensive subjects (57% female, 21-60 years old). The Ang II LCMS method had a measurable range of 3.3-700 pmol/L. The between-batch precision coefficient of variation was <7% over Ang II concentrations of 8.6-110 pmol/L. No significant matrix interference and carryover were observed. There was no significant difference in Ang II concentration in EDTA blood and plasma for at least 2h and 1 h at room temperature, respectively. Ang II was stable for at least 1 year when stored at -80°C, with or without the protease inhibitor. Age-dependent Ang II reference intervals were established: 4.4-17.7 pmol/L (21-30 years) and 3.9-12.8 pmol/L (31-60 years). The present LCMS method is suitable for quantitation of plasma Ang II to study the renin-angiotensin system.

Aldosterone-producing Adenoma in Primary Aldosteronism: CT-guided Radiofrequency Ablation-Long-term Results and Recurrence Rate.

Purpose To evaluate the long-term biochemical, clinical, and recurrence outcomes of radiofrequency (RF) ablation in treating primary aldosteronism due to aldosterone-producing adenoma (APA). Materials and Methods Institutional review board approval and written informed consent were obtained. The use of computed tomographically (CT) guided percutaneous RF ablation was evaluated in 36 patients (19 men; mean age ± standard deviation, 52.1 years ± 10.4) with APA (17 right and 19 left side; mean size, 15.5 mm ± 5.0). Primary aldosteronism was confirmed by using the oral sodium-loading test. After RF ablation, CT images, aldosterone-to-renin ratio (ARR), serum potassium level, and blood pressure control were assessed at 3 months and at the latest follow-up examination. Long-term treatment success was defined as normalization of ARR at the latest assessment. Comparison of ARR, potassium, and blood pressure levels before and after RF ablation was performed by using the Wilcoxon signed-rank test. Results Primary technical success was achieved in 33 (92%) patients who underwent a single RF ablation session. Secondary technical success was achieved in three (8%) patients who required a second RF ablation. At 3-month follow-up, primary aldosteronism was resolved in 33 (92%) patients, with a starting median ARR of 8583 pmol/L per µg/(L · h) that normalized to 97 pmol/L per µg/(L · h) (P < .01). Mean serum potassium levels increased from 2.6 mmol/L ± 0.4 to 4.0 mmol/L ± 0.3 (P = .01). At long-term follow-up (mean, 6.2 years ± 2.5), treatment success was maintained in 33 patients (92%), all of whom had ARRs in the normal range (P < .01). The long-term recurrence rate was 0%. Hypokalemia was resolved in all patients (2.6 mmol/L ± 0.4 to 4.1 mmol/L ± 0.3, P = .01). Hypertension was resolved in 13 (36%) patients, and its control was improved in seven (19%) patients. One (3%) patient had major complications and six (17%) had minor complications. Conclusion CT-guided RF ablation is an effective treatment for APA, with high sustainable long-term treatment success. It may serve as a justifiable treatment alternative to surgery and medical therapy for APA. © RSNA, 2016.

Diagnosis of 5α-reductase 2 deficiency: is measurement of dihydrotestosterone essential?

BACKGROUND: 5α-Reductase 2 deficiency (5ARD) is a known cause of 46,XY disorders of sex development (DSD). Traditionally, the diagnosis relies on dihydrotestosterone (DHT) measurement, but the results are often equivocal, potentially leading to misdiagnosis. We reviewed alternative approaches for diagnosis of 5ARD. METHODS: We conducted a retrospective review of the results of urinary steroid profiling (USP) by GC-MS and mutational analysis of SRD5A2 [steroid-5-alpha-reductase, alpha polypeptide 2 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 2)] by PCR and direct DNA sequencing of all 46,XY DSD patients referred to our laboratory with biochemical and/or genetic findings compatible with 5ARD. We also performed a literature review on the laboratory findings of all 5ARD cases reported in the past 10 years. RESULTS: Of 16 patients diagnosed with 5ARD between January 2003 and July 2012, 15 underwent USP, and all showed characteristically low 5α- to 5ß-reduced steroid metabolite ratios. Four patients had DHT measured, but 2 did not reach the diagnostic cutoff. In all 12 patients who underwent genetic analysis, 2 mutations of the SRD5A2 gene were detected to confirm the diagnosis. Twenty-four publications involving 149 patients with 5ARD were published in the review period. Fewer than half of these patients had DHT tested. Nearly 95% of them had the diagnosis confirmed genetically. CONCLUSIONS: 5ARD can be confidently diagnosed by USP at 3 months postnatally and confirmed by mutational analysis of SRD5A2. Interpretation of DHT results may be problematic and is not essential in the diagnosis of 5ARD. We propose new diagnostic algorithms for 46,XY DSD.

A Hong Kong Chinese kindred with familial hypocalciuric hypercalcaemia caused by AP2S1 mutation.

Familial hypocalciuric hypercalcaemia (FHH) is a genetic disorder of altered calcium homeostasis. Mutations in the CASR, GNA11 and AP2S1 genes have been reported to cause FHH. We report a Hong Kong Chinese kindred with FHH type 3 (FHH3) caused by mutations in AP2S1. The proband, a 51-year-old woman with hypercalcaemia, was initially diagnosed to have primary hyperparathyroidism but repeated parathyroidectomy failed to normalize her plasma calcium concentrations. Later, FHH was suspected and yet no mutations were identified in the CASR gene which causes FHH type 1 (FHH1), the most common form of FHH. Genetic testing of AP2S1 revealed a heterozygous c.43C>T (p.Arg15Cys) mutation, confirming the diagnosis of FHH3. The elder brother and niece of the proband, who both have hypercalcaemia, were found to harbour the same mutation. To our knowledge, this is the first Chinese kindred of FHH3 reported in the English literature.