Phillip J. Monaghan

Comparison of serum cortisol measurement by immunoassay and liquid chromatography-tandem mass spectrometry in patients receiving the 11β-hydroxylase inhibitor metyrapone

Background The accurate measurement of cortisol by immunoassay is compromised by the potential for cross-reactivity of reagent antibodies with structurally related steroids present in serum. This susceptibility is potentiated when normal steroid metabolism is altered pharmaceutically by antisteroidogenic drugs utilized in the management of Cushings syndrome to moderate cortisol production. The clinical implications of falsely elevated cortisol results include over-treatment and unrecognized hypoadrenalism. To investigate the effect of the 11β-hydroxylase inhibitor metyrapone on serum cortisol assay, a comparison of measurement by immunoassay versus liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted. Methods Cortisol was measured in serum from three patient groups: (1) patients receiving metyrapone (n = 112 samples from 10 patients); (2) control group of patients diagnosed with Cushings syndrome currently receiving no treatment (n = 31); and (3) control group of patients with no adrenal pathology and not receiving medication known to interfere in cortisol immunoassay (n = 67). Results Bland–Altman plots showed agreement between methods for the control group (bias = 1.1% [−4.3 nmol/L]) and Cushings control group (bias = 1.3% [−3.7 nmol/L]). This was in stark contrast to the metyrapone therapy group (bias = 23% [59 nmol/L]). The difference between LC-MS/MS versus immunoassay in the metyrapone therapy group positively correlated with metyrapone dose and serum 11-deoxycortisol concentration (Pearsons correlation coefficient r = 0.47, 95% CI 0.32–0.61; P < 0.0001). Conclusions These data show that liability of immunoassay measurement of serum cortisol to interference in patients receiving metyrapone may lead to erroneous clinical decisions concerning dose titration.

Case for the Wider Adoption of Mass Spectrometry-Based Adrenal Steroid Testing, and Beyond

Department of Clinical Biochemistry (B.G.K.), University Hospital of South Manchester, Manchester M23 9LT, United Kingdom; Medical School (P.M.S.), University of Leeds, Leeds LS2 9NL, United Kingdom; The Christie Pathology Partnership (P.J.M.), Manchester, M20 4BX, UK; Department of Endocrinology (P.J.T.), The Christie NHS Foundation Trust, Manchester M20 4BX, United Kingdom; and Centre for Endocrinology & Diabetes (P.J.T.), Institute of Human Development, University of Manchester, Manchester Academic Health Science Centre, United Kingdom

Secondary kinetic isotope effects as probes of environmentally-coupled enzymatic hydrogen tunneling reactions.

The secondary kinetic isotope effect for hydride transfer from NADPH to dihydrofolate catalyzed by dihydrofolate reductase (see traces) is neither temperature dependent nor exalted. In environmentally coupled models of H-tunneling, the secondary isotope effects do not report on promoting motions, but reflect the active site geometry attained immediately prior to H transfer (i.e. the ‘tunnelling ready configuration′).

The use of mass spectrometry to improve the diagnosis and the management of the HPA axis

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is becoming a standard analytical tool in the clinical laboratory for the measurement of small molecules, including steroid hormones. Endocrinologists are coming to acknowledge the superior quality of measurement that is achievable by LC-MS/MS through the enhanced analytical specificity and high sensitivity that this technique offers over conventional immunoassay (IA) methodologies. Additionally, LC-MS/MS overcomes many of the problems encountered in immunoassays, such as anti-reagent antibody interferences and cross-reactivity with structurally related compounds. The potential benefits of applying LC-MS/MS for the assessment of the hypothalamic-pituitary-adrenal (HPA) axis are beginning to be realised. This review critically evaluates recent developments in the application of LC-MS/MS for measurement of glucocorticoids and mineralocorticoids towards the diagnosis and management of HPA axis disorders and aims to address the current unmet need in this expanding field of endocrinology for which future studies into the potential applications of LC-MS/MS should be directed.

Serum Cortisol: An Up-To-Date Assessment of Routine Assay Performance

BACKGROUND Accurate serum cortisol quantification is required for the correct diagnosis and management of adrenal pathologies. Presently, most laboratories use immunoassay to measure serum cortisol with proficiency schemes demonstrating a wide dispersion of results. Here, we investigate the effects of sex, matrix, and antibody specificity on serum cortisol quantification in 6 routine assays. METHODS Surplus serum was obtained before disposal and the following cohorts were created: males, nonpregnant females, pregnant patients, and patients prescribed either metyrapone or prednisolone. Samples were anonymized and distributed to collaborating laboratories for cortisol analysis by 6 routine assays. Cortisol was also measured in all samples using an LC-MS/MS candidate reference measurement procedure (cRMP); cortisol-binding globulin (CBG) was measured in the nonpregnant and pregnant female cohorts. RESULTS Considerable inter- and intraassay variation was observed across the male and nonpregnant female cohorts relative to the cRMP. Four immunoassays underrecovered cortisol in the pregnancy cohort, and CBG was found to be significantly higher in this cohort than in the nonpregnant females. In the metyrapone and prednisolone cohorts, all immunoassays overestimated cortisol. The first generation Roche E170 and Siemens Centaur XP were particularly prone to overestimation. In all cohorts the routine LC-MS/MS assay aligned extremely well with the cRMP. CONCLUSIONS Despite the clinical importance of serum cortisol, the performance of routine immunoassays remains highly variable. Accurate quantification is compromised by both matrix effects and antibody specificity. Underpinning this study with a cRMP has highlighted the deficiencies in standardization across routine cortisol immunoassays.

Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase⁄oxidase

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines l‐proline, l‐pipecolic acid and sarcosine, with optimal catalytic activity towards l‐proline. The holoenzyme contains one FAD, FMN and ATP per αβ complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with l‐proline, for which a bell‐shaped dependence (pKa values 7.0 ± 0.2 and 7.6 ± 0.2) for kcat/Km as a function of pH was observed. The pH dependence of kcat is sigmoidal, described by a single macroscopic pKa of 7.7 ± 0.1, tentatively attributed to ionization of l‐proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion‐type mechanism. The midpoint potential of enzyme‐bound FAD has a linear temperature dependence (− 3.1 ± 0.05 mV·C°−1), and extrapolation to physiologic growth temperature for P. furiosus (100 °C) yields a value of − 407 ± 5 mV for the two‐electron reduction of enzyme‐bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic l‐proline dehydrogenase. Implications for its mechanism of action are discussed.

Serum and plasma 5-hydroxyindoleacetic acid as an alternative to 24-h urine 5-hydroxyindoleacetic acid measurement

Background Neuroendocrine tumours are slow growing tumours known to secrete a variety of vasoactive peptides which give rise to symptoms of the carcinoid syndrome. The diagnosis and monitoring of patients with neuroendocrine tumours is undertaken in many centres using 24 h urinary measurement of 5-hydroxyindoleacetic acid. However, 5-hydroxyindoleacetic acid can also be quantified in plasma and serum. Methods We measured 5-hydroxyindoleacetic acid concentration in 134 paired EDTA plasma and urine samples from 108 patients with known neuroendocrine tumours and 26 healthy volunteers. We also compared 5-hydroxyindoleacetic acid concentrations in paired serum and plasma samples (n = 63), then analysed paired urine and serum samples (n = 97). Furthermore, we examined the impact of renal impairment on serum 5-hydroxyindoleacetic acid by analysing 5-hydroxyindoleacetic acid in patients without neuroendocrine tumours in different stages of chronic kidney disease, as indicated by the estimated glomerular filtration rate. Results Plasma and urine 5-hydroxyindoleacetic acid had very similar diagnostic sensitivities and specificities, with areas under the curve on ROC analysis of 0.917 and 0.920, respectively. Serum and plasma 5-hydroxyindoleacetic acid values showed good correlation but serum results demonstrated a positive bias, indicating the necessity for different serum and plasma reference intervals. There was an inverse correlation between estimated glomerular filtration rate and serum 5-hydroxyindoleacetic acid concentration, with 5-hydroxyindoleacetic acid increasing once the estimated glomerular filtration rate falls below 60 mL/min/1.73 m2. Conclusion The measurement of both serum and plasma 5-hydroxyindoleacetic acid can be used for the diagnosis and monitoring of patients with neuroendocrine tumours. Provided renal function is taken into consideration, either of these tests should be incorporated into standard practice as an alternative assay to urinary 5-hydroxyindoleacetic acid.

Mass spectrometry for the endocrine clinic – much to digest†

The application of mass spectrometry (MS) is a growing presence in the endocrine laboratory; an elegant analytical technique that discriminates and quantifies analytes by mass:charge (m:z) ratio, akin to a ‘molecular sieve’. In contrast, the basic principle of immunoassay (IA) is founded on the molecular interaction between reagent antibody and antigen (analyte of interest). Driven largely by the limitations of conventional IA methodology, including the ever-present risk of interference from antireagent antibodies and endogenous autoantibodies, susceptibility to cross-reactivity with structurally related compounds (e.g. prednisolone cross-reactivity in cortisol assay), and the highdose hook effect; MS is now the method of choice for the routine measurement of steroid hormones. The capacity of MS to accurately quantify these small molecules with exquisite specificity and enhanced sensitivity has contributed to improved patient care. Indeed, the measurement of steroid hormones using MS is on the verge of supplanting conventional IA; however, there is a current lack of awareness that MS has a wider role in the application of peptide and protein quantification, as demonstrated in this issue of Clinical Endocrinology by Kay et al. Recent applications of MS for the endocrine clinic have included the measurement of serum cortisol for patients receiving metyrapone therapy for the medical management of Cushing’s syndrome. Metyrapone increases circulating 11-deoxycortisol, which cross-reacts in cortisol IA evoking positive interference, thus, a more accurate analysis using MS mitigates the potential for erroneous clinical decisions concerning dose titration and obviates the risk of unrecognized hypoadrenalism in the face of spuriously raised cortisol results by IA. In addition, the improved analytical sensitivity of MS has permitted the accurate and simultaneous low level quantification of cortisol and cortisone in saliva, affording endocrinologists a surrogate measurement of circulating bioavailable cortisol. The parotid glands harbour 11b-hydroxysteroid dehydrogenase type II activity, which converts cortisol to cortisone and results in greater levels of cortisone in comparison to cortisol in saliva. Due to the close structural homology between these two compounds, IA-based techniques for salivary cortisol measurement are prone to crossreactivity with the more abundant cortisone; implications for the accurate measurement of salivary cortisol by IA are therefore clear. Saliva steroid measurement also has immense utility in circumstances where the concentration of cortisol binding globulin (CBG) is perturbed, as is the case during pregnancy and in patients receiving the oral contraceptive pill or the adrenolytic agent mitotane, which has an oestrogenic influence on CBG resulting in spurious elevation of total serum cortisol measurement. Another example of a high sensitivity MS application has recently been reported for the quantification of oestrone and oestradiol in serum that exploits a negative electrospray ionization mode with ammonium fluoride aqueous phase to achieve high analytical sensitivity; potential clinical applications, including low oestrogen measurement in postmenopausal women, premenopausal amenorrheic women, prepubertal children and in patients receiving aromatase inhibitor therapy. Furthermore, MS has permitted the accurate determination of salivary testosterone at picomolar levels that could have utility as a surrogate marker of bioavailable (biologically active) testosterone for the assessment of androgen status in men. This MS method showed improved specificity over radioimmunoassay (RIA), which is liable to cross-reactivity from related steroids. The problem of bias in IA and the subsequent necessity to adopt method-specific reference ranges has been highlighted by the Endocrine Society in its guidelines on the diagnosis of Cushing’s syndrome. The source of bias in IA is likely multifactorial, including the use of different proprietary antibodies and antibody specificity, strategies employed for elimination of binding protein problems and assay calibration. An increasing awareness of the need for accurate, reliable, and comparable assays for testosterone measurement has been highlighted by an Endocrine Society Position Statement and recent Consensus Statement. Five testosterone assays are currently certified by the Centers for Disease Control and Prevention (CDC) that satisfy bias criteria, of which four are MS methods. The continuing transition from conventional IA methodology to rigorously validated and highly specific MS methods for testosterone measurement is of immense benefit for the investigation of conditions, including polycystic ovarian syndrome and male hypogonadism where the *Please see related paper on pages 424–430 of this issue.

Spuriously raised serum creatinine associated with an excipient present in an intravenous dexamethasone formulation

Although the active pharmaceutical ingredient remains constant, the excipients used will vary according to the manufacturer. This case report is of spuriously raised serum creatinine due to an excipient in one particular intravenous dexamethasone formulation. A patient had three serum creatinine measurements of 102, 369 and 91 μmol/L over a four-hour period. The second result was believed to be spurious and appropriate investigations were instigated. The patient had received dexamethasone intravenously between the first and second blood samples. This was administered as a bolus via a cannula in the dorsum of the hand, and the blood sample was taken by venepuncture of the antecubital fossa of the same arm approximately five minutes later. The dexamethasone used (Hospira UK Ltd) contained creatinine at a concentration of 70,720 μmol/L, with a total of 170 μmol of creatinine given to the patient. Assuming a volume of distribution of 40 L in a 70-kg man, an increase in serum creatinine of 4–5 μmol/L would be expected once equilibrated. It is thought that the serum creatinine result observed was a consequence of the creatinine excipient in the dexamethasone not having completely distributed throughout the body and still being at relatively high concentrations within the limb into which it had been administered. Intravenous dexamethasone can lead to spurious creatinine results, not due to analytical interference but rather the analytically correct measurement of creatinine added as an excipient. This case clearly demonstrates the impact preanalytical factors can have on the accuracy of results.

Proopiomelanocortin interference in the measurement of adrenocorticotrophic hormone: a United Kingdom National External Quality Assessment Service study.

It is recognized that measurement of ACTH‐precursor peptides including proopiomelanocortin (POMC) has clinical utility in identifying the aetiology of Cushings syndrome. Recent data have also demonstrated cross‐reactivity of POMC in ACTH immunoassays used in clinical laboratories. The aim of this study was to assess the cross‐reactivity of POMC in the main commercial immunoassays for ACTH and to survey the awareness of laboratory professionals to this potential interference.