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Background: Aldosterone and renin are pivotal hormones in the regulation of salt and water homeostasis and blood pressure. Measurement of renin and aldosterone in serum/plasma is essential for the investigation of primary hyperaldosteronism (PA) and monitoring of glucocorticoid replacement therapy., Methods: We report 2 LC-MS/MS methods developed to measure aldosterone and plasma renin activity (PRA). PRA was determined by endogenous enzymatic generation of angiotensin I using 150 µL of sample. Generated angiotensin I was purified by solid phase extraction prior to chromatographic separation and mass spectrometry. Aldosterone measurement required 300 μL of sample extracted with MTBE prior to LC-MS/MS analysis., Results: The PRA method was linear (1.2-193 nmol/L), sensitive (LLOQ = 1.2 nmol/L), precise (CV = 4.1%), and specific (no cross reactivity for a number of structurally similar steroids). Dilutional linearity and recovery (84%) were acceptable. Accuracy was confirmed by comparison against our current RIA method. The aldosterone method had equally acceptable performance characteristics. Reference ranges in 110 healthy normotensive subjects were: PRA 0.2-3.7 nmol/L/h and aldosterone 50-950 pmol/L. Consecutive patients (n = 62) with adrenal incidentalomas shown to have no functional adrenal disease; their post overnight 1 mg dexamethasone test values were: PRA 0.2-2.6 nmol/L/h and aldosterone 55-480 pmol/L. Serum aldosterone values after 2 liter saline suppression were-normal subjects (n = 17): 78-238 pmol/L and confirmed primary hyperaldosteronism (n = 25): 131-1080 pmol/L., Conclusions: We have developed robust assays for PRA and aldosterone with appropriate clinical evaluation. These assays are now in routine practice in the UK., (© Crown copyright 2020.)

Objective: To examine the relationship between anti-Müllerian hormone (AMH) and the severity of the phenotype of patients with polycystic ovary syndrome (PCOS) and whether AMH can act as a diagnostic marker for PCOS?, Design: A prospective diagnostic utility study of AMH as a marker of PCOS., Patients: A consecutive series of women presenting to a tertiary infertility clinic (n = 164) plus a second series of women prepared for assisted conception treatments (n = 89) recruited between June 2012 and May 2013., Measurements: Polycystic ovary syndrome was diagnosed using the Rotterdam criteria. AMH was measured using the Generation II assay (Beckman Coulter). The diagnostic utility of AMH was established using receiver operator characteristic (ROC) curves. Cut-off values for the individual features of PCOS are proposed., Results: There was a significant difference in serum AMH concentration in women with normal ovaries (13·2 pmol/l), polycystic ovary morphology (PCOM) alone (37·8 pmol/l) and PCOS (53·2 pmol/l). Follicle number, increasing cycle length and evidence of hyperandrogenism were all independently associated with serum AMH concentration (P < 0·01). AMH was significantly affected by the different phenotypic presentations of PCOS with those with all components (PCOM, HA and OA) having the highest mean value [72·7 pmol/l (P < 0·01)]., Conclusions: Serum AMH has the capacity to act as a diagnostic test for PCOS. Moreover, since its value rises with the more marked phenotypes, different cut-off values need to be used to differentiate those patients with polycystic ovarian morphology (PCOM), hyperandrogenism (HA) and oligoanovulation (OA)., (© 2016 John Wiley & Sons Ltd.)

Mass spectrometry methods have the potential to measure different hormones during the same analysis and have improved specificity and a wide analytical range compared with many immunoassay methods. Increasingly in clinical laboratories liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are replacing immunoassays for the routine measurement of testosterone, 17-hydroxyprogesterone, and other steroid hormones. Reference LC-MS/MS methods for steroid, thyroid, and peptide hormones are being used for assessment of the performance and calibration of commercial immunoassays. In this chapter, the general principles of tandem mass spectrometry and examples of hormone assays are described.

Unlabelled: Type 1 pseudohypoaldosteronism (PHA) is a rare heterogeneous group of disorders characterised by resistance to aldosterone action. There is resultant salt wasting in the neonatal period, with hyperkalaemia and metabolic acidosis. Only after results confirm isolated resistance to aldosterone can the diagnosis of type 1 PHA be confidently made. Type 1 PHA can be further classified into i) renal type 1 (autosomal dominant (AD)) and ii) multiple target organ defect/systemic type 1 (autosomal recessive (AR)). The aim of this case series was to characterise the mode of presentation, management and short-term clinical outcomes of patients with PHA type 1. Case notes of newly diagnosed infants presenting with PHA type 1 were reviewed over a 5-year time period. Seven patients were diagnosed with PHA type 1. Initial presentation ranged from 4 to 28 days of age. Six had weight loss as a presenting feature. All subjects had hyperkalaemia, hyponatraemia, with elevated renin and aldosterone levels. Five patients have renal PHA type 1 and two patients have systemic PHA type, of whom one has had genetic testing to confirm the AR gene mutation on the SCNN1A gene. Renal PHA type 1 responds well to salt supplementation, whereas management of patients with systemic PHA type 1 proves more difficult as they are likely to get frequent episodes of electrolyte imbalance requiring urgent correction., Learning Points: Patients with type 1 PHA are likely to present in the neonatal period with hyponatraemia, hyperkalaemia and metabolic acidosis and can be diagnosed by the significantly elevated plasma renin activity and aldosterone levels.The differential diagnosis of type 1 PHA includes adrenal disorders such as adrenal hypoplasia and congenital adrenal hyperplasia; thus, adrenal function including cortisol levels, 17-hydroxyprogesterone and a urinary steroid profile are required. Secondary (transient) causes of PHA may be due to urinary tract infections or renal anomalies; thus, urine culture and renal ultrasound scan are required respectively.A differentiation between renal and systemic PHA type 1 may be made based on sodium requirements, ease of management of electrolyte imbalance, sweat test results and genetic testing.Management of renal PHA type 1 is with sodium supplementation, and requirements often decrease with age.Systemic PHA type 1 requires aggressive and intensive fluid and electrolyte management. Securing an enteral feeding route and i.v. access are essential to facilitate ongoing therapy.In this area of the UK, the incidence of AD PHA and AR PHA was calculated to be 1:66 000 and 1:166 000 respectively.

A number of androgens are measured for clinical purposes. Most laboratories will run a testosterone assay but the requirement for other androgens may be too small for a laboratory to set up their own assay. In these cases samples would be sent to a specialized laboratory. In the routine laboratory testosterone is analyzed on automated systems but these lack the sensitivity to accurately measure the hormone in children and normal women. Many laboratories analyzed such samples by radioimmunoassay, but tandem mass spectrometry is now replacing these assays. This chapter provides simple methods for measuring the androgens in serum and saliva by radioimmunoassay and tandem mass spectrometry.

Background: There is growing epidemiological evidence linking serum 25 hydroxy-vitamin D (25(OH)D) concentrations to outcome in cardiovascular and other diseases. We have studied patients with acute myocardial infarction (AMI) to determine if they exhibit an acute phase reaction affecting 25(OH)D., Methods: Patients (n=32) with first AMI who had been treated with primary percutaneous coronary intervention within 12 h of symptom onset had venous blood samples taken two days, one week, one month and three months after presentation. Samples were analysed for troponin I, C-reactive protein (CRP) and 25(OH)D., Results: All patients had significant rises in troponin confirming the myocardial damage and CRP, both of which resolved by 28 days. In contrast, 25(OH)D remained unchanged throughout the 90-day observation period with a median concentration of 46 nmol/L., Conclusion: Serum 25(OH)D does not change after AMI and is likely to be a reliable marker of vitamin D status in patients with cardiovascular disease.

Objective: Hyperandrogenism is one of the diagnostic criteria for the polycystic ovary syndrome (PCOS) despite no agreed definition of hyperandrogenism. In part, this is due to the quality of testosterone immunoassays. We have developed liquid chromatography-tandem mass spectrometry methods for analysing testosterone and androstenedione (Ad) to study their reference ranges and diagnostic utility in PCOS., Design, Setting and Subjects: A consecutive series of 122 women attending a reproductive medicine clinic., Methods: Blood samples were taken during the early follicular phase for measurement of LH, FSH, oestradiol, Ad, testosterone and sex hormone-binding globulin (SHBG). Retrospective case note analysis was used to determine the clinical features and ultrasound findings., Results: The incidence of PCOS was 13.9%. The reference interval for testosterone was <1.8 nmol/l and for Ad was 1.4-7.4 nmol/l. There were significant differences in total testosterone (P=0.001), Ad (P<0.05) and free androgen index (FAI; P<0.0001) between the women with and without PCOS. Diagnostic performance using receiver operator characteristic plots showed area under the curve (AUC) for FAI 0.81, testosterone 0.75 and Ad 0.66. The AUC for the LH:FSH ratio was 0.72., Conclusions: Our analysis of a consecutive series of women attending a reproductive clinic has provided an appropriate series on which to construct reference ranges for key androgens in women. Secondly, it has allowed us to conclude that early follicular serum testosterone measured using tandem mass spectrometry, FAI and the LH:FSH ratio are valuable laboratory tests in the diagnosis of PCOS.

Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine., Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs., Results: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%., Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

Background: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory., Methods: We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h., Results: Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak., Conclusions: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.

Results of the direct leucocyte migration inhibition (LMI) test using gluten fraction III as antigen were unaffected by incorporation of puromycin into the culture medium at concentrations shown to prevent lymphokine mediated inhibition. Results of the LMI test performed with purified polymorphs were similar to and correlated with results of the standard LMI test using mixed leucocytes in both coeliacs and controls. The addition of purified T lymphocytes did not increase migration inhibition. Normal leucocytes incubated with serum from coeliac patients and washed showed marked migration inhibition when incubated with gluten fraction III. This sensitisation of normal leucocytes was prevented by preincubation with aggregated human IgG. These results suggest that leucocyte migration inhibition by gluten in coeliac disease is not due to lymphokine production by sensitised lymphocytes but is caused by cytophilic antibody.

The test for pancreatic exocrine function using N-benzoyl-L-tyrosyl-p-aminobenzoic acid (BTP test) does not require duodenal intubation, but misleadingly abnormal results often occur in patients with liver or bowel disease because the p-aminobenzoic acid (PABA) released by chymotrypsin hydrolysis of the peptide either is not conjugated or is malabsorbed. This study evaluated a modified BTP test, using a tracer dose of 14C-PABA to eliminate misleading results, to assess exocrine function from a single six-hour collection of urine. The test clearly distinguished all patients with pancreatic steatorrhoea from normal subjects and identified patients with less severe pancreatitis as often as did the Lundh test. Furthermore, in patients with bowel or liver disease the misleadingly abnormal results of the unmodified BTP test were eliminated by the modified test in all but one case. These findings suggest that the modified BTP test provides a practical alternative to conventional tests of pancreatic function that entail duodenal intubation.

Zinc status and the effect of zinc supplementation were assessed in groups of patients with non-Hodgkin's lymphoma and Hodgkin's disease; patients were either untreated or in remission. In the patients in remission, plasma zinc was normal; and whereas 30% of untreated patients had low plasma zinc, the group as a whole did not differ from normal. For mononuclear cell zinc, the range of values in the disease group was far wider than in controls, but there was no significant difference between the means of the groups. Granulocyte zinc was significantly lower in both the groups of patients in remission from non-Hodgkin's lymphoma and Hodgkin's disease compared with the control group. Significant increases were found in the plasma copper, ceruloplasmin, and the copper-to-zinc ratio in several of the patient groups. Plasma zinc increased by 23% with zinc supplementation (50 mg elemental Zn/day), but there was no effect on mononuclear cell or granulocyte zinc. Apart from granulocyte zinc, there is little evidence of zinc deficiency in non-Hodgkin's lymphoma or Hodgkin's disease. However, the presence of depleted granulocyte zinc levels could modify the immune function of this cell population.

Plasma, mononuclear cell and granulocyte zinc were assessed in a group of 15 institutionalised elderly female patients, and the effect of different levels of zinc supplements (50, 100, 150 mg elemental zinc/day) on these parameters was measured. Plasma but not cellular zinc was lower in the elderly compared with a young control group, but a diminished serum albumin may explain the plasma results. Plasma zinc increased with supplementation, but only when the daily intake was 100 or 150 mg/day; the greatest response was with the higher dose. Mononuclear cell zinc did not respond to zinc supplements. There was a decrease in granulocyte zinc at all levels of zinc supplements, the greatest decrease being found with the highest dose (150 mg/day). The decrease in granulocyte zinc at high intakes may have implications in phagocytosis and chemotaxis.

Serum calcium, magnesium, iron, copper and zinc were measured in 117 patients with cystic fibrosis. Apart from serum iron levels, all the other essential element levels were well maintained and there was no evidence of a need for supplementation. Serum iron was frequently low and the need for iron supplements must be considered after further investigation. The subjects studied covered a wide age range, had widely varying fat malabsorption and clinical grading, and also varying degrees of pulmonary involvement. We found no evidence that any of these parameters directly influenced essential element status as assessed by serum levels.