Brian Keevil

Age-associated changes in hypothalamic-pituitary-testicular function in middle-aged and older men are modified by weight change and lifestyle factors: longitudinal results from the European Male Ageing Study.

OBJECTIVE Health and lifestyle factors are associated with variations in serum testosterone levels in ageing men. However, it remains unclear how age-related changes in testosterone may be attenuated by lifestyle modifications. The objective was to investigate the longitudinal relationships between changes in health and lifestyle factors with changes in hormones of the reproductive endocrine axis in ageing men. DESIGN A longitudinal survey of 2736 community-dwelling men aged 40-79 years at baseline recruited from eight centres across Europe. Follow-up assessment occurred mean (±S.D.) 4.4±0.3 years later. RESULTS Paired testosterone results were available for 2395 men. Mean (±S.D.) annualised hormone changes were as follows: testosterone -0.1±0.95  nmol/l; free testosterone (FT) -3.83±16.8  pmol/l; sex hormone-binding globulin (SHBG) 0.56±2.5  nmol/l and LH 0.08±0.57  U/l. Weight loss was associated with a proportional increase, and weight gain a proportional decrease, in testosterone and SHBG. FT showed a curvilinear relationship to weight change; only those who gained or lost ≥15% of weight showed a significant change (in the same direction as testosterone). Smoking cessation was associated with a greater decline in testosterone than being a non-smoker, which was unrelated to weight change. Changes in number of comorbid conditions or physical activity were not associated with significant alterations in hypothalamic-pituitary-testicular (HPT) axis function. CONCLUSIONS Body weight and lifestyle factors influence HPT axis function in ageing. Weight loss was associated with a rise, and weight gain a fall, in testosterone, FT and SHBG. Weight management appears to be important in maintaining circulating testosterone in ageing men, and obesity-associated changes in HPT axis hormones are reversible following weight reduction.

Adverse Reactions to Voriconazole

Voriconazole is a new antifungal agent effective in the treatment of invasive aspergillosis. Interpatient variation in plasma concentrations is considerable--more than 100-fold. We describe 3 patients with diverse manifestations of toxicity (e.g., hallucinations, hypoglycemia, electrolyte disturbance, and pneumonitis) possibly attributable to high voriconazole concentrations. Measurement of plasma concentrations could be helpful in optimizing voriconazole dosages.

State-of-the-Art of Serum Testosterone Measurement by Isotope Dilution–Liquid Chromatography– Tandem Mass Spectrometry

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.

Salivary Cortisone Is a Potential Biomarker for Serum Free Cortisol

CONTEXT Salivary cortisol measurement is used as a practical surrogate for serum free cortisol. However, parotid tissue harbors 11β-hydroxysteroid dehydrogenase (11β-HSD2) activity converting cortisol to cortisone. OBJECTIVE This study was designed to assess the impact of parotid 11β-HSD2 activity on the measurement of salivary cortisol. PATIENTS, DESIGN, AND OUTCOME MEASURES: Study participants with changes in circulating corticosteroid-binding globulin (CBG) (±oral contraceptive, functionally CBG null) and controls were studied during adrenal stimulation by ACTH and postoral and iv hydrocortisone administration. Simultaneous serum and saliva samples were collected for the measurement of total serum cortisol (SerF) by immunoassay, and unbound cortisol and cortisone in serum (FreeF and FreeE) and saliva (SalF and SalE) by liquid chromatography-tandem mass spectrometry. RESULTS ACTH stimulation increased SerF, FreeF, SalF, SalE, but not FreeE in all individuals. SerF significantly decreased after stopping oral contraceptive administration, but FreeF, SalF and SalE remained unchanged. In the hydrocortisone administration study, individual FreeF and SalE curves were nearly identical and SalE closely reflected FreeF in all participants, irrespective of CBG changes. The highest correlation in all (n = 537) matched serum-saliva samples was between SalE and FreeF (r = 0.95, P < 0.0001), and there was no evidence of 11β-HSD2 saturation. CONCLUSION Salivary cortisol is a useful surrogate for circulating free cortisol, but its concentration is determined both by serum free cortisol and parotid metabolism to cortisone. We have shown that salivary cortisone closely reflects free serum cortisol after adrenal stimulation and hydrocortisone administration and is unaffected by CBG changes. Salivary cortisone has potential as a useful surrogate for serum free cortisol in research and clinical assessment, and further research in states of chronic glucocorticoid excess is now needed.

Biological Variation of Glycated Hemoglobin: Implications for diabetes screening and monitoring

OBJECTIVE To assess the inherent potential of glycated hemoglobin as a screening test for type 2 diabetes by determining the biological variation in nondiabetic subjects. RESEARCH DESIGN AND METHODS HbA1c values were measured by high-performance liquid chromatography (HPLC) in 12 nondiabetic subjects (7 men and 5 women; median age, 40 years [range, 21–55 years]) on 10 fortnightly occasions. The nondiabetic index of individuality (IOI) for HbA1c (i.e., the square root of the ratio of intra- to interindividual variance) was determined. Any test with an IOI of 1.4 has the most potential in disease screening, while one of 0.6 will be of little value. RESULTS The analytical variance contributed to 9% of the total test variance, intraindividual variance, 6%; and interindividual variance, 85%. The IOI was, therefore, only 0.27. Thus, nondiabetic HbA1c values vary markedly between subjects, while values in the same individual change little with time. As such, to lie outside the assay reference range, the HbA1c values of some nondiabetic subjects must exceed 12 SD from their usual mean value, while in others a change of only 2 SD would be sufficient. CONCLUSIONS This fundamental characteristic of HbA1c means that even if analytical methods improve, glycated hemoglobin measurements will always be of limited value when screening for type 2 diabetes. If similar interindividual differences also exist in diabetic subjects, then patients with the same glycemic control may vary by at least 1–2%, which has implications in setting glycated hemoglobin targets.

Age-Specific Reference Ranges for Serum Testosterone and Androstenedione Concentrations in Women Measured by Liquid Chromatography-Tandem Mass Spectrometry

OBJECTIVE RIA-based sex hormone measurements offer only limited precision and specificity in the low concentration range of women. Therefore, we aimed to establish age-specific reference ranges for serum sex hormone concentrations in women using mass spectrometry and quantile regression. METHODS AND RESULTS Data from 985 women aged 20-80 yr, recruited for the prospective Study of Health in Pomerania, were included in the analyses. Quantile regressions models were performed to calculate the age-specific 2.5th and 97.5th percentiles for sex hormone concentrations in women. Serum total testosterone (TT) and androstenedione (AD) concentrations were measured by liquid chromatography-tandem mass spectrometry. Measured concentrations of SHBG and TT were used to calculate free testosterone (free T). TT, AD, and free T concentrations showed a distinct age-related decline across 10-yr age groups (one way ANOVA P < 0.001). Sex hormone reference ranges for TT, AD, and free T were determined across each single year of age and for 10-yr age groups. Reference ranges over the whole age range of 20-80 yr were 0.35-1.97 nmol/liter for TT, 0.89-4.56 nmol/liter for AD, and 0.0025-0.0253 nmol/liter for free T. Separate reference ranges were provided for pre- and postmenopausal women as well as after inclusion of women using oral contraceptives or hormone therapy (n = 1357). CONCLUSION This is the first study to establish age-specific reference ranges for liquid chromatography-tandem mass spectrometry-measured TT and AD and calculated free T concentrations based on quantile regression analyses, accurately accounting for the observed low concentration range and the strong age dependency of these sex hormones in women.

Simultaneous measurement of cortisol and cortisone in human saliva using liquid chromatography-tandem mass spectrometry: Application in basal and stimulated conditions

Immunoassays used for the measurement of salivary cortisol are limited by variable interference from cortisone. Salivary cortisone is a consequence of the salivary glands expressing 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) which converts cortisol to cortisone. We report a combined salivary cortisol and cortisone (SalF and SalE respectively) liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to address the cortisone cross-reactivity in cortisol immunoassays and as a tool to study 11beta-HSD2 activity. The method was linear up to 400 nmol/L for SalF and 200 nmol/L for SalE and the lower limits of quantitation were 0.39 nmol/L (SalF) and 0.78 nmol/L (SalE). No evidence of ion suppression was found and precision, accuracy and recovery were within internationally accepted limits. No interference was identified from 13 structurally related steroids. SalF, SalE and SalF/SalE were significantly greater in the morning than at bed-time and following stimulation of the adrenal glands. As serum cortisol increased, an exponential rise was observed in SalF and a linear increase in SalE which reached a plateau at higher SalF concentrations. We have developed a novel, robust LC-MS/MS assay for the combined measurement of SalF and SalE. Our results confirm the 11beta-HSD2 activity of the salivary glands resulting in high SalE concentrations and the enzyme saturation at high substrate concentrations. This method can be used as a simple, non-invasive and highly specific tool to assess the value of salivary cortisol as a surrogate for free serum cortisol and as a potential novel way to assess 11beta-HSD2 activity.

Beta-blockers are associated with lower C-reactive protein concentrations in patients with coronary artery disease

PURPOSE C-reactive protein is an important risk factor for coronary artery disease, and plasma concentrations are lowered by treatment with pravastatin and aspirin. We examined whether other cardiovascular drugs that are used in the treatment of ischemic heart disease affect C-reactive protein concentrations. SUBJECTS AND METHODS Plasma C-reactive protein concentration was measured by high sensitivity immunonephelometric assay in 333 consecutive patients with stable angina and confirmed coronary artery disease who underwent diagnostic angiography. RESULTS Patients prescribed beta-blockers had significantly lower mean C-reactive protein concentrations than did patients in whom these were not prescribed (by 1.2 mg/L, or 40% difference in geometric mean concentration; P <0.001). This association remained significant (P = 0.03) after excluding patients with contraindications to the use of beta-blockers, and adjusting for the probability of beta-blocker therapy (propensity score) and other clinical predictors of C-reactive protein concentration, including body mass index, high-density lipoprotein cholesterol level, family history of coronary artery disease, and angiographic severity. No differences among types or dosages of beta-blockers were evident. CONCLUSION Beta-blockers may affect C-reactive protein concentrations. Randomized studies are required to confirm these findings.

The analysis of dried blood spot samples using liquid chromatography tandem mass spectrometry

Assays using dried blood samples have been in use for a number of years particularly for the diagnosis of inborn errors of metabolism. This sampling technique has been greatly helped by the sensitivity and specificity offered by liquid chromatography tandem mass spectrometry because of the limited amount of sample available for analysis. There are advantages for patients to take samples at home but these must be weighed against potential problems with the sampling technique. Some assays appear to work very well whilst others suffer from poor recovery because of adsorption of the analyte onto the filter paper. Some newer strategies including impregnation of the filter paper and filter filtration will be discussed. Calibration of assays and the problems with matrix matching of standards are also important issues that need to be addressed before an assay can be used routinely.

Paradoxical Changes in Cystatin C and Serum Creatinine in Patients with Hypo- and Hyperthyroidism

In the ongoing search for improved serum markers of impaired renal function (1), the low-molecular-weight protein cystatin C has been advocated as a promising and probably superior alternative to creatinine when used to assess glomerular filtration rate (GFR) (2)(3). Cystatin C possesses most of the properties of an ideal GFR test in that it is produced by all nucleated cells at an apparently constant rate, is freely filtered at the glomerulus, and is then fully destroyed in the proximal renal tubule (4). Its rate of production is not influenced by inflammation or malignancy and, unlike creatinine, is unaffected by the muscle mass, sex, or age of a patient (5). Indeed, studies to date would seem to confirm the potential of the marker in many clinical situations in which an accurate estimate of GFR is required in both adults (5)(6)(7) and children (8)(9). Before cystatin C measurement became widespread, another low-molecular-weight protein, β2-microglobulin, was promoted as a marker of GFR for similar reasons (10)(11)(12), but in contrast to cystatin C, its usefulness was subsequently found to be limited by the increased serum values found in inflammatory and neoplastic conditions (13). Thyroid disease …