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Free access

Angela E Taylor, Brian Keevil, and Ilpo T Huhtaniemi

Summary

The recent onslaught of mass spectrometry (MS) to measurements of steroid hormones, including demands that they should be the only acceptable method, has confused clinicians and scientists who have relied for more than 40 years on a variety of immunoassay (IA) methods in steroid hormone measurements. There is little doubt that MS methods with their superior specificity will be the future method of choice in many clinical and research applications of steroid hormone measurement. However, the majority of steroid measurements are currently, and will continue to be, carried out using various types of IAs for several reasons, including their technical ease, cost and availability of commercial reagents. Speedy replacement of all IAs with MS is an unrealistic and unnecessary goal, because the availability of MS measurements is limited by cost, need of expensive equipment, technical demands and lack of commercial applications. Furthermore, IAs have multiple well-known advantages that vindicate their continuing use. The purpose of this article is to elucidate the advantages and limitations of the MS and IA techniques from two angles, i.e. promotion of MS and defence of IA. The purpose of the text is to give the reader an unbiased view about the current state and future trends of steroid analysis and to help him/her choose the correct assay method to serve his/her diagnostic and research needs.

Open access

Irina Bancos, Jon Hazeldine, Vasileios Chortis, Peter Hampson, Angela E Taylor, Janet M Lord, and Wiebke Arlt

Open access

Irina Bancos, Jon Hazeldine, Vasileios Chortis, Peter Hampson, Angela E Taylor, Janet M Lord, and Wiebke Arlt

Objective

Mortality in patients with primary adrenal insufficiency (PAI) is significantly increased, with respiratory infections as a major cause of death. Moreover, patients with PAI report an increased rate of non-fatal infections. Neutrophils and natural killer (NK) cells are innate immune cells that provide frontline protection against invading pathogens. Thus, we compared the function and phenotype of NK cells and neutrophils isolated from PAI patients and healthy controls to ascertain whether altered innate immune responses could be a contributory factor for the increased susceptibility of PAI patients to infection.

Design and methods

We undertook a cross-sectional study of 42 patients with PAI due to autoimmune adrenalitis (n = 37) or bilateral adrenalectomy (n = 5) and 58 sex- and age-matched controls. A comprehensive screen of innate immune function, consisting of measurements of neutrophil phagocytosis, reactive oxygen species production, NK cell cytotoxicity (NKCC) and NK cell surface receptor expression, was performed on all subjects.

Results

Neutrophil function did not differ between PAI and controls. However, NKCC was significantly reduced in PAI (12.0 ± 1.5% vs 21.1 ± 2.6%, P < 0.0001). Phenotypically, the percentage of NK cells expressing the activating receptors NKG2D and NKp46 was significantly lower in PAI, as was the surface density of NKG2D (all P < 0.0001). Intracellular granzyme B expression was significantly increased in NK cells from PAI patients (P < 0.01).

Conclusions

Adrenal insufficiency is associated with significantly decreased NKCC, thereby potentially compromising early recognition and elimination of virally infected cells. This potential impairment in anti-viral immune defense may contribute to the increased rate of respiratory infections and ultimately mortality in PAI.

Open access

Lina Schiffer, Alicia Bossey, Punith Kempegowda, Angela E Taylor, Ildem Akerman, Dagmar Scheel-Toellner, Karl-Heinz Storbeck, and Wiebke Arlt

Objective: Androgens are important modulators of immune cell function. The local generation of active androgens from circulating precursors is an important mediator of androgen action in peripheral target cells or tissues. We aimed to characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs).

Methods: PBMCs were isolated from healthy male donors and incubated ex vivo with precursors and active androgens of the classic and 11-oxygenated androgen pathways. Steroids were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid-metabolizing enzymes was assessed by quantitative PCR.

Results: PBMCs generated 8-fold higher amounts of the active 11-oxygenated androgen 11-ketotestosterone than the classic androgen testosterone from their respective precursors. We identified the enzyme AKR1C3 as the major reductive 17β-hydroxysteroid dehydrogenase in PBMCs responsible for both conversions and found that within the PBMC compartment natural killer cells are the major site of AKRC13 expression and activity. Steroid 5α-reductase type 1 catalyzed the 5α-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood increased serum 11-ketotestosterone concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection.

Conclusions: 11-oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11-ketotestosterone the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the results of serum 11-ketotestosterone measurements, if samples are not separated in a timely fashion.

Open access

Silvia Parajes, Angel OK Chan, W M But, Ian T Rose, Angela E Taylor, Vivek Dhir, Wiebke Arlt, and Nils Krone

Context

Cytochrome P450 side-chain cleavage enzyme (CYP11A1) catalyses the first and rate-limiting step of steroidogenesis, the conversion of cholesterol to pregnenolone. CYP11A1 deficiency is commonly associated with adrenal insufficiency, and in 46,XY individuals, with variable degrees of disorder of sex development (DSD).

Patient and methods

The patient was born with hyperpigmentation, micropenis, penoscrotal hypospadias, and mild cryptorchidism. Biochemical and hormonal findings were normal except for low testosterone and low-borderline cortisol. However, no short synacthen test was undertaken. Development was unremarkable apart from an episode labeled as sepsis with documented hyperkalemia and elevated C-reactive protein at age 15 days. Diagnosis of 46,XY DSD was made at age 2.5 months. Progression of hyperpigmentation prompted further investigations and the diagnosis of adrenal insufficiency was established at 2 years with raised ACTH, normal renin activity, and failure of cortisol to respond to short synacthen test. Genetic analyses were performed. The novel CYP11A1 mutations were characterized in vitro and in silico.

Results

The patient was compound heterozygous for two novel CYP11A1 mutations, p.R360W and p.R405X. p.R360W retained 30–40% of wild-type activity. In silico analyses confirmed these findings and indicated that p.R405X is severe.

Conclusions

This study demonstrates the pathogenicity of two novel CYP11A1 mutations found in a patient with delayed diagnosis of CYP11A1 deficiency. Patients with partial deficiencies of steroidogenic enzymes are at risk to be misdiagnosed if adrenal function is not assessed. The adrenocortical function should be routinely assessed in all patients with DSD including severe hypospadias of unknown origin to prevent life-threatening adrenal crises.