Testosterone (T), alone or in combination with progestin, provides a promising approach to hormon... more Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR ؊/؊ ) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR ؊/؊ mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.-Oduwole, O. O., Vydra, N., Wood, N. E. M., Samanta, L., Owen, L., Keevil, B., Donaldson, M., Naresh, K., Huhtaniemi, I. T. Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception. FASEB J. 28, 000 -000 (2014). www.fasebj.org
Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Altho... more Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ioniza...
Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical... more Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography--tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 x 3.0 mm; 3- microm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 microL. We use...
BACKGROUND: The plasma renin activity (PRA) assay measures the ability of renin to generate angio... more BACKGROUND: The plasma renin activity (PRA) assay measures the ability of renin to generate angiotensin I (AngI) from angiotensinogen. It is used to monitor mineralocorticoid therapy and to screen hypertensive individuals for primary aldosteronism (PA). METHODS: Samples were incubated in the presence of protease inhibitors for 6.5 and 24 h. The reaction was stopped by the addition of 2% ammonium hydroxide. AngI was then quantified by liquid chromatography tandem mass spectrometry using online solid-phase extraction (XLC-MS/MS). RESULTS: This method requires a sample volume of 50 μL and has an inter-assay precision <14% across the working range. A 6.5-h incubation gave a lower limit of quantification (LLOQ) of 0.3 nmol/L/h and this can be reduced to 0.08 nmol/L/h using a 24-h incubation. Comparison to a radioimmunoassay revealed excellent correlation (r(2) = 0.98), but a 37% negative bias. We also found that renin is stable in whole blood for up to 24 h at room temperature. In con...
Background: Carcinoid heart disease (CHD) is an important complication of metastatic neuroendocri... more Background: Carcinoid heart disease (CHD) is an important complication of metastatic neuroendocrine disease, requiring regular monitoring to enable intervention prior to right heart failure. We aimed to identify the most appropriate echocardiographic scoring systems for the quantitative assessment of CHD. Methods: In this prospective study conducted between April and October 2012 in two European Neuroendocrine Tumor Society (ENETS) Centres of Excellence, patients with neuroendocrine tumours with liver metastases and/or carcinoid syndrome underwent transthoracic echocardiography and blood sampling for serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and plasma 5-hydroxyindoleacetic acid (5-HIAA). Each patient was assessed according to six echocardiographic scoring systems. The individual scoring systems' feasibility, observer variability, sensitivity, specificity and correlation with the concentration biomarkers were determined. Results: 100 patients were included; 21% ...
Despite observational evidence from epidemiological and clinical studies associating sex hormones... more Despite observational evidence from epidemiological and clinical studies associating sex hormones with various cardiometabolic risk factors or diseases, pathophysiological explanations are sparse to date. To reveal putative functional insights, we analyzed associations between sex hormone levels and whole blood gene expression profiles. We used data of 991 individuals from the population-based Study of Health in Pomerania (SHIP-TREND) with whole blood gene expression levels determined by array-based transcriptional profiling and serum concentrations of total testosterone (TT), sex hormone-binding globulin (SHBG), free testosterone (free T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (AD), estradiol (E2), and estrone (E1) measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay. Associations between sex hormone concentrations and gene expression profiles were analyzed using sex-specific regression models adjusted for age, body mass index, and technical covariables. In men, positive correlations were detected between AD and DDIT4 mRNA levels, as well as between SHBG and the mRNA levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B. No additional significant associations were observed. Besides the associations between AD and DDIT4 expression and SHBG and the transcript levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B, the present study did not indicate any association between sex hormone concentrations and whole blood gene expression profiles in men and women from the general population.
The Journal of Clinical Endocrinology & Metabolism, 2015
Secondary hypogonadism is common in ageing men; its natural history and predisposing factors are ... more Secondary hypogonadism is common in ageing men; its natural history and predisposing factors are unclear. 1) To identify factors which predispose eugonadal men (T ≥10.5nmol/L) to develop biochemical secondary hypogonadism (T&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10.5nmol/L, LH≤9.4U/L) and secondary hypogonadal men to recover to eugonadism. 2) To characterize clinical features associated with these transitions. Prospective observational general population cohort survey. Clinical research centres. 3369 community-dwelling men aged 40-79 yr in eight European centres. Observational follow-up of 4.3 years. Subjects were categorised according to change/no change in biochemical gonadal status during follow-up into persistent eugonadal (n=1909), incident secondary hypogonadal (n=140), persistent secondary hypogonadal (n=123) and recovered from secondary hypogonadism to eugonadism (n=96). Baseline predictors and changes in clinical features associated with incident secondary hypogonadism and recovery from secondary hypogonadism were analysed by regression models. The incidence of secondary hypogonadism was 155.9/10,000/year, while 42.9% of men with secondary hypogonadism recovered to eugonadism. Incident secondary hypogonadism was predicted by obesity [BMI≥30kg/m(2): odds ratio (OR)=2.86 (95% confidence interval 1.67;4.90); p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001], weight gain [OR=1.79 (1.15;2.80);p=0.011] and increased waist circumference [OR=1.73 (1.07;2.81); p=0.026 and 2.64 (1.66;4.21);p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001, for waist circumference 94-102 and ≥102 cm, respectively]. Incident secondary hypogonadal men experienced new/worsening sexual symptoms [low libido, erectile dysfunction and infrequent spontaneous erections]. Recovery from secondary hypogonadism was predicted by non-obesity [OR=2.28 (1.21;4.31); p=0.011], weight loss [OR=2.24 (1.04;4.85); p=0.042], normal waist circumference [OR=1.93 (1.01;3.70); p=0.048], younger age [&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;60yr OR=2.32 (1.12;4.82); p=0.024] and higher education [OR=2.11 (1.05;4.26); p=0.037], but symptoms did not show significant concurrent improvement. Obesity-related metabolic and lifestyle factors predispose older men to the development of secondary hypogonadism, which is frequently reversible with weight loss.
Aims. ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enh... more Aims. ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enhanced production of growth factors leading to increased arterial medial layer thickness, which is a characteristic of transplant arteriosclerosis. ACE inhibition is known to be of benefit to patients with cardiovascular risk factors. We aimed to determine the effect of ACE inhibitor therapy on ACE enzymatic activity and serum ANGII levels following cardiac transplantation. Methods. A total of 43 serum samples from eight transplant recipients were used for analysis. Samples were taken monthly from the date of transplant for the initial 6 months. ANGII was measured using sandwich ELISA. ACE enzymatic activity was measured using spectrophotometric kinetic analysis.
A simple and rapid liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the ana... more A simple and rapid liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the analysis of voriconazole has been developed. For comparison, serum voriconazole was measured using HPLC and bioassay. For the HPLC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of serum to 40 microL of 0.1 M zinc sulfate solution. Proteins were precipitated by adding 100 microL acetonitrile containing ketoconazole as internal standard. After vigorous mixing and centrifugation, 3 microL of the supernatant was injected into the HPLC-MS/MS system. An HPLC system was used to elute a C18 cartridge (2 mm x 4 mm) at 0.6 mL/min with a step gradient of 50% to 100% methanol containing 2 mM ammonium acetate and 0.1% (vol/vol) formic acid. The column was maintained at 55 degrees C, and the retention times were voriconazole 1.50 minutes and ketoconazole 1.47 minutes. Cycle time was 3 minutes, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: voriconazole m/z 350.0 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 224.1 and ketoconazole m/z 531.1 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 489.1. Within- and between-batch CVs were &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 5% and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 8%, respectively, over the range 0.38 to 15.3 mg/L. The lower limit of quantification was 0.1 mg/L. Regression analysis showed HPLC-MS/MS = 1.06 +/- 0.02 (HPLC-UV) - 0.07 +/- 0.1, R2 = 0.95, n = 99.
A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simul... more A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of cyclosporin A (CsA) and creatinine using capillary blood has been developed. Venous and capillary blood samples were taken predose and at C2 from 65 heart and lung transplant recipients (65 x 4 samples). For comparisons, serum creatinine and blood CsA concentrations were measured by the Jaffe and EMIT methods, respectively, using an Olympus AU600 analyzer. For the LC-MS/MS assay, samples were prepared in a 96 x 700-microL well block by adding 10 microL of blood (or serum) to 40 microL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 microL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 microL of the supernatant was injected into the LC-MS/MS system. A Waters 2795 high-performance liquid chromatography (HPLC) system was used to elute a C18 cartridge (3 mm x 4 mm) at 0.6 mL/min with a step gradient of 50-100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. The column was maintained at 55 degrees C, and the retention times were creatinine, 0.4 minutes; ascomycin, 0.98 minutes; and CsA, 1.2 minutes. Cycle time was 2.5 minutes, injection to injection. The analytes were monitored using a Quattro microtandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: creatinine, m/z 114&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;44; d3-creatinine (IS), m/z 117&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;47; ascomycin (IS), m/z 809&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;756; and CsA, m/z 1,220&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;1,203. Assay characteristics were CsA intraassay CV, 3.6-3.0% (33-1,500 microg/L); CsA interassay CV, 6.7-2.5% (10-5,000 microg/L); LC-MS/MS capillary [CsA] = 0.99 x LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 x EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 micromol/L); interassay CV, 5.7-3.3% (80-590 micromol/L); LC-MS/MS capillary [creatinine] = 0.99 Jaffe plasma [creatinine] -42.6, R = 0.87. Total time for the preparation and analysis of 30 samples was approximately 2 hours. This assay will provide a flexible, robust, and cost-effective solution for the monitoring of CsA and creatinine in transplant recipients with potential applications in pediatric medicine and pharmacokinetic studies, in which frequent sampling is required.
Testosterone (T), alone or in combination with progestin, provides a promising approach to hormon... more Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR ؊/؊ ) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR ؊/؊ mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.-Oduwole, O. O., Vydra, N., Wood, N. E. M., Samanta, L., Owen, L., Keevil, B., Donaldson, M., Naresh, K., Huhtaniemi, I. T. Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception. FASEB J. 28, 000 -000 (2014). www.fasebj.org
Scandinavian Journal of Clinical & Laboratory Investigation, 2014
An interlaboratory comparison study for melatonin, cortisol and testosterone in saliva in which f... more An interlaboratory comparison study for melatonin, cortisol and testosterone in saliva in which five laboratories participated is reported in this study. Each laboratory blindly measured eight samples prepared from natural saliva spiked with melatonin, cortisol and testosterone in the range 0-579 pmol/L for melatonin, 0-90 nmol/L for cortisol, and 0-622 pmol/L for testosterone. The recovery of spiked material for melatonin ranged from 91-110%, from 83-100% for cortisol and from 80-94% for testosterone. The content of natural hormone in saliva was estimated to be between 0.278 and 6.90 pmol/L for melatonin, 0.56 and 6.72 nmol/L for cortisol and 11.9 and 73.8 pmol/L for testosterone. This indicates a large interlaboratory variation. The present study emphasizes the importance of external quality control for the analysis of melatonin, cortisol and testosterone in saliva.
Background. Cardiovascular mortality in end-stage renal failure patients is high and early risk s... more Background. Cardiovascular mortality in end-stage renal failure patients is high and early risk stratification in these patients may aid clinical management improving outcomes. Cardiac troponin T (cTnT) is a component of the cardiac myocyte which is released into the circulation following myocardial necrosis. It has been shown to be of prognostic significance in patients with unstable angina. The role of cTnT in patients with renal disease remains unclear. The aim of this investigation, therefore, was to assess the prognostic significance of cTnT in chronic renal impairment patients, pre-dialysis. Methods. Ninety-six patients with chronic renal impairment were followed prospectively after cTnT determination by a quantitative laboratory method. The clinical outcomes after 2 years were determined. The measured cTnT values were correlated with biochemical parameters and clinical end-points. Results. A cut-off of 0.1 ng/ml was used in assessing the prognostic significance of cTnT. Twenty-five patients had a cTnT >0.1 ng/ml, whilst 71 had a cTnT 0.1 ng/ml. Twenty-one patients died during the follow-up period. Eleven of these had elevated cTnT at entry into the study. Death rate in the patients with cTnT >0.1 ng/ml was 42% compared with 14% in those with levels below the cut-off. Thirty-three patients died or had a vascular event. The rate of death or a vascular event in the elevated group was 64% compared with 24% in those with levels below the cut-off. At the end of the study, 23 patients were treated by continuous ambulatory peritoneal dialysis, 29 by haemodialysis, 22 had functioning renal transplants and one patient was not on renal replacement therapy. Factors that were found to significantly affect cTnT were diabetes, age and urea. cTnT was found to be a significant predictor of survival in these patients. Patients with high cTnT values were more likely to end up on haemodialysis. No relation of renal function to cTnT level was found. Conclusions. These results show that in patients with renal impairment, the measurement of cTnT prior to commencing renal replacement is a significant independent predictor of survival. cTnT did show potential as a prognostic test to stratify patients with a high cardiovascular risk and may enable intensive risk factor modification in this patient group. This may be of further use in selection of patients' suitability for renal transplantation.
The Journal of Heart and Lung Transplantation, 2008
Mannose-binding lectin (MBL) deficiency (or the common opsonic defect) has been reported as a ris... more Mannose-binding lectin (MBL) deficiency (or the common opsonic defect) has been reported as a risk factor for several immunologically controlled conditions, including infection and graft rejection. However, the effects of MBL deficiency on acute rejection after heart transplantation are unknown. Ninety heart transplant recipients were included in this study. Plasma MBL quantification was performed using a commercially available enzyme-linked immunoassay (ELISA). Acute rejection was diagnosed via endomyocardial biopsy and histologic assessment. MBL concentration was controlled for demographics, immunosuppression and anti-viral therapy. Individuals with dysfunctional MBL had significantly fewer rejection episodes (6.3 Ϯ 3.8%) than those with functional MBL (12.9 Ϯ 11.6%) (p ϭ 0.016). We found no significant difference between MBL concentrations among those with (1,232 Ϯ 58 ng/ml) and those without (1,216 Ϯ 161 ng/ml) GCAD (p ϭ 0.841). There was also no significant difference between the incidence of GCAD in those with "normal" concentrations (p ϭ 0.782). Heart transplant recipients with MBL deficiency had fewer acute graft rejection episodes compared to patients with functional MBL. This suggests the lectin pathway of complement activation is an important process in graft rejection. Furthermore, functional MBL may indicate a greater likelihood of rejection.
The Journal of Clinical Endocrinology & Metabolism, 2010
Context: Salivary cortisol measurement is used as a practical surrogate for serum free cortisol. ... more Context: Salivary cortisol measurement is used as a practical surrogate for serum free cortisol. However, parotid tissue harbors 11-hydroxysteroid dehydrogenase (11-HSD2) activity converting cortisol to cortisone.
In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyin... more In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 micorL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters Quattro Premier XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;145.6 and 193.9&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97-113%) and the method showed good linearity to 10,000 nmol/L (r(2)=0.999). Within-batch and between-batch imprecision was &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10% and bias &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC-MS/MS and HPLC showed good agreement between the two methods but this LC-MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.
Immunoassays used for the measurement of salivary cortisol are limited by variable interference f... more 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.
Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory i... more Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis(®) HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;189.1 for aldosterone and 366.0&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10%. Comparison of the UPLC-MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC-MS/MS=0.789(RIA)-41.7, linear regression r(2)=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.
Testosterone (T), alone or in combination with progestin, provides a promising approach to hormon... more Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR ؊/؊ ) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR ؊/؊ mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.-Oduwole, O. O., Vydra, N., Wood, N. E. M., Samanta, L., Owen, L., Keevil, B., Donaldson, M., Naresh, K., Huhtaniemi, I. T. Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception. FASEB J. 28, 000 -000 (2014). www.fasebj.org
Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Altho... more Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ioniza...
Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical... more Cyclosporin A (CsA) is commonly measured by immunoassay techniques that have a limited analytical range. The consequence of this is that low CsA concentrations that may be clinically significant are difficult to measure and that high concentrations require sample dilution, which introduces error and increases cost. More specific assays, such as HPLC, do not have the required turnaround times for busy transplant clinics. CsA was measured in whole blood from 180 cardiac and lung transplant recipients by a liquid chromatography--tandem mass spectrometry (MS) assay, and the results were compared with the Dade Behring Emit assay. Proteins were precipitated with acetonitrile containing ascomycin as internal standard. We used isocratic elution on a Supelco CN column (33 x 3.0 mm; 3- microm bead size) with a mobile phase of 65% aqueous acetonitrile containing ammonium acetate (2 mmol/L) and formic acid (1 g/L), at a flow rate of 0.5 mL/min, with a sample injection volume of 6 microL. We use...
BACKGROUND: The plasma renin activity (PRA) assay measures the ability of renin to generate angio... more BACKGROUND: The plasma renin activity (PRA) assay measures the ability of renin to generate angiotensin I (AngI) from angiotensinogen. It is used to monitor mineralocorticoid therapy and to screen hypertensive individuals for primary aldosteronism (PA). METHODS: Samples were incubated in the presence of protease inhibitors for 6.5 and 24 h. The reaction was stopped by the addition of 2% ammonium hydroxide. AngI was then quantified by liquid chromatography tandem mass spectrometry using online solid-phase extraction (XLC-MS/MS). RESULTS: This method requires a sample volume of 50 μL and has an inter-assay precision <14% across the working range. A 6.5-h incubation gave a lower limit of quantification (LLOQ) of 0.3 nmol/L/h and this can be reduced to 0.08 nmol/L/h using a 24-h incubation. Comparison to a radioimmunoassay revealed excellent correlation (r(2) = 0.98), but a 37% negative bias. We also found that renin is stable in whole blood for up to 24 h at room temperature. In con...
Background: Carcinoid heart disease (CHD) is an important complication of metastatic neuroendocri... more Background: Carcinoid heart disease (CHD) is an important complication of metastatic neuroendocrine disease, requiring regular monitoring to enable intervention prior to right heart failure. We aimed to identify the most appropriate echocardiographic scoring systems for the quantitative assessment of CHD. Methods: In this prospective study conducted between April and October 2012 in two European Neuroendocrine Tumor Society (ENETS) Centres of Excellence, patients with neuroendocrine tumours with liver metastases and/or carcinoid syndrome underwent transthoracic echocardiography and blood sampling for serum N-terminal pro-brain natriuretic peptide (NT-proBNP) and plasma 5-hydroxyindoleacetic acid (5-HIAA). Each patient was assessed according to six echocardiographic scoring systems. The individual scoring systems' feasibility, observer variability, sensitivity, specificity and correlation with the concentration biomarkers were determined. Results: 100 patients were included; 21% ...
Despite observational evidence from epidemiological and clinical studies associating sex hormones... more Despite observational evidence from epidemiological and clinical studies associating sex hormones with various cardiometabolic risk factors or diseases, pathophysiological explanations are sparse to date. To reveal putative functional insights, we analyzed associations between sex hormone levels and whole blood gene expression profiles. We used data of 991 individuals from the population-based Study of Health in Pomerania (SHIP-TREND) with whole blood gene expression levels determined by array-based transcriptional profiling and serum concentrations of total testosterone (TT), sex hormone-binding globulin (SHBG), free testosterone (free T), dehydroepiandrosterone sulfate (DHEAS), androstenedione (AD), estradiol (E2), and estrone (E1) measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay. Associations between sex hormone concentrations and gene expression profiles were analyzed using sex-specific regression models adjusted for age, body mass index, and technical covariables. In men, positive correlations were detected between AD and DDIT4 mRNA levels, as well as between SHBG and the mRNA levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B. No additional significant associations were observed. Besides the associations between AD and DDIT4 expression and SHBG and the transcript levels of RPIA, RIOK3, GYPB, BPGM, and RAB2B, the present study did not indicate any association between sex hormone concentrations and whole blood gene expression profiles in men and women from the general population.
The Journal of Clinical Endocrinology & Metabolism, 2015
Secondary hypogonadism is common in ageing men; its natural history and predisposing factors are ... more Secondary hypogonadism is common in ageing men; its natural history and predisposing factors are unclear. 1) To identify factors which predispose eugonadal men (T ≥10.5nmol/L) to develop biochemical secondary hypogonadism (T&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10.5nmol/L, LH≤9.4U/L) and secondary hypogonadal men to recover to eugonadism. 2) To characterize clinical features associated with these transitions. Prospective observational general population cohort survey. Clinical research centres. 3369 community-dwelling men aged 40-79 yr in eight European centres. Observational follow-up of 4.3 years. Subjects were categorised according to change/no change in biochemical gonadal status during follow-up into persistent eugonadal (n=1909), incident secondary hypogonadal (n=140), persistent secondary hypogonadal (n=123) and recovered from secondary hypogonadism to eugonadism (n=96). Baseline predictors and changes in clinical features associated with incident secondary hypogonadism and recovery from secondary hypogonadism were analysed by regression models. The incidence of secondary hypogonadism was 155.9/10,000/year, while 42.9% of men with secondary hypogonadism recovered to eugonadism. Incident secondary hypogonadism was predicted by obesity [BMI≥30kg/m(2): odds ratio (OR)=2.86 (95% confidence interval 1.67;4.90); p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001], weight gain [OR=1.79 (1.15;2.80);p=0.011] and increased waist circumference [OR=1.73 (1.07;2.81); p=0.026 and 2.64 (1.66;4.21);p&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001, for waist circumference 94-102 and ≥102 cm, respectively]. Incident secondary hypogonadal men experienced new/worsening sexual symptoms [low libido, erectile dysfunction and infrequent spontaneous erections]. Recovery from secondary hypogonadism was predicted by non-obesity [OR=2.28 (1.21;4.31); p=0.011], weight loss [OR=2.24 (1.04;4.85); p=0.042], normal waist circumference [OR=1.93 (1.01;3.70); p=0.048], younger age [&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;60yr OR=2.32 (1.12;4.82); p=0.024] and higher education [OR=2.11 (1.05;4.26); p=0.037], but symptoms did not show significant concurrent improvement. Obesity-related metabolic and lifestyle factors predispose older men to the development of secondary hypogonadism, which is frequently reversible with weight loss.
Aims. ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enh... more Aims. ANGII mediates vascular neointimal formation through smooth muscle cell stimulation and enhanced production of growth factors leading to increased arterial medial layer thickness, which is a characteristic of transplant arteriosclerosis. ACE inhibition is known to be of benefit to patients with cardiovascular risk factors. We aimed to determine the effect of ACE inhibitor therapy on ACE enzymatic activity and serum ANGII levels following cardiac transplantation. Methods. A total of 43 serum samples from eight transplant recipients were used for analysis. Samples were taken monthly from the date of transplant for the initial 6 months. ANGII was measured using sandwich ELISA. ACE enzymatic activity was measured using spectrophotometric kinetic analysis.
A simple and rapid liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the ana... more A simple and rapid liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the analysis of voriconazole has been developed. For comparison, serum voriconazole was measured using HPLC and bioassay. For the HPLC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of serum to 40 microL of 0.1 M zinc sulfate solution. Proteins were precipitated by adding 100 microL acetonitrile containing ketoconazole as internal standard. After vigorous mixing and centrifugation, 3 microL of the supernatant was injected into the HPLC-MS/MS system. An HPLC system was used to elute a C18 cartridge (2 mm x 4 mm) at 0.6 mL/min with a step gradient of 50% to 100% methanol containing 2 mM ammonium acetate and 0.1% (vol/vol) formic acid. The column was maintained at 55 degrees C, and the retention times were voriconazole 1.50 minutes and ketoconazole 1.47 minutes. Cycle time was 3 minutes, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: voriconazole m/z 350.0 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 224.1 and ketoconazole m/z 531.1 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 489.1. Within- and between-batch CVs were &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 5% and &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 8%, respectively, over the range 0.38 to 15.3 mg/L. The lower limit of quantification was 0.1 mg/L. Regression analysis showed HPLC-MS/MS = 1.06 +/- 0.02 (HPLC-UV) - 0.07 +/- 0.1, R2 = 0.95, n = 99.
A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simul... more A simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of cyclosporin A (CsA) and creatinine using capillary blood has been developed. Venous and capillary blood samples were taken predose and at C2 from 65 heart and lung transplant recipients (65 x 4 samples). For comparisons, serum creatinine and blood CsA concentrations were measured by the Jaffe and EMIT methods, respectively, using an Olympus AU600 analyzer. For the LC-MS/MS assay, samples were prepared in a 96 x 700-microL well block by adding 10 microL of blood (or serum) to 40 microL of 0.1 mol/L zinc sulphate solution containing deuterated creatinine internal standard. Proteins were precipitated by adding 100 microL acetonitrile containing ascomycin internal standard. After vigorous mixing and centrifugation, 5 microL of the supernatant was injected into the LC-MS/MS system. A Waters 2795 high-performance liquid chromatography (HPLC) system was used to elute a C18 cartridge (3 mm x 4 mm) at 0.6 mL/min with a step gradient of 50-100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. The column was maintained at 55 degrees C, and the retention times were creatinine, 0.4 minutes; ascomycin, 0.98 minutes; and CsA, 1.2 minutes. Cycle time was 2.5 minutes, injection to injection. The analytes were monitored using a Quattro microtandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions: creatinine, m/z 114&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;44; d3-creatinine (IS), m/z 117&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;47; ascomycin (IS), m/z 809&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;756; and CsA, m/z 1,220&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;1,203. Assay characteristics were CsA intraassay CV, 3.6-3.0% (33-1,500 microg/L); CsA interassay CV, 6.7-2.5% (10-5,000 microg/L); LC-MS/MS capillary [CsA] = 0.99 x LC-MS/MS venous [CsA] - 4.2, R = 0.98; and LC-MS/MS venous [CsA] = 0.93 x EMIT venous [CsA] + 2.9, R = 0.98. Creatinine intraassay CV, 6.6-2.5% (20-720 micromol/L); interassay CV, 5.7-3.3% (80-590 micromol/L); LC-MS/MS capillary [creatinine] = 0.99 Jaffe plasma [creatinine] -42.6, R = 0.87. Total time for the preparation and analysis of 30 samples was approximately 2 hours. This assay will provide a flexible, robust, and cost-effective solution for the monitoring of CsA and creatinine in transplant recipients with potential applications in pediatric medicine and pharmacokinetic studies, in which frequent sampling is required.
Testosterone (T), alone or in combination with progestin, provides a promising approach to hormon... more Testosterone (T), alone or in combination with progestin, provides a promising approach to hormonal male contraception. Its principle relies on enhanced negative feedback of exogenous T to suppress gonadotropins, thereby blocking the testicular T production needed for spermatogenesis, while simultaneously maintaining the extragonadal androgen actions, such as potency and libido, to avoid hypogonadism. A serious drawback of the treatment is that a significant proportion of men do not reach azoospermia or severe oligozoospermia, commensurate with contraceptive efficacy. We tested here, using hypogonadal luteinizing hormone/choriongonadotropin receptor (LHCGR) knockout (LHR ؊/؊ ) mice, the basic principle of the T-based male contraceptive method, that a specific T dose could maintain extragonadal androgen actions without simultaneously activating spermatogenesis. LHR ؊/؊ mice were treated with increasing T doses, and the responses of their spermatogenesis and extragonadal androgen actions (including gonadotropin suppression and sexual behavior) were assessed. Conspicuously, all dose responses to T were practically superimposable, and no dose of T could be defined that would maintain sexual function and suppress gonadotropins without simultaneously activating spermatogenesis. This finding, never addressed in clinical contraceptive trials, is not unexpected in light of the same androgen receptor mediating androgen actions in all organs. When extrapolated to humans, our findings may jeopardize the current approach to hormonal male contraception and call for more effective means of inhibiting intratesticular T production or action, to achieve consistent spermatogenic suppression.-Oduwole, O. O., Vydra, N., Wood, N. E. M., Samanta, L., Owen, L., Keevil, B., Donaldson, M., Naresh, K., Huhtaniemi, I. T. Overlapping dose responses of spermatogenic and extragonadal testosterone actions jeopardize the principle of hormonal male contraception. FASEB J. 28, 000 -000 (2014). www.fasebj.org
Scandinavian Journal of Clinical & Laboratory Investigation, 2014
An interlaboratory comparison study for melatonin, cortisol and testosterone in saliva in which f... more An interlaboratory comparison study for melatonin, cortisol and testosterone in saliva in which five laboratories participated is reported in this study. Each laboratory blindly measured eight samples prepared from natural saliva spiked with melatonin, cortisol and testosterone in the range 0-579 pmol/L for melatonin, 0-90 nmol/L for cortisol, and 0-622 pmol/L for testosterone. The recovery of spiked material for melatonin ranged from 91-110%, from 83-100% for cortisol and from 80-94% for testosterone. The content of natural hormone in saliva was estimated to be between 0.278 and 6.90 pmol/L for melatonin, 0.56 and 6.72 nmol/L for cortisol and 11.9 and 73.8 pmol/L for testosterone. This indicates a large interlaboratory variation. The present study emphasizes the importance of external quality control for the analysis of melatonin, cortisol and testosterone in saliva.
Background. Cardiovascular mortality in end-stage renal failure patients is high and early risk s... more Background. Cardiovascular mortality in end-stage renal failure patients is high and early risk stratification in these patients may aid clinical management improving outcomes. Cardiac troponin T (cTnT) is a component of the cardiac myocyte which is released into the circulation following myocardial necrosis. It has been shown to be of prognostic significance in patients with unstable angina. The role of cTnT in patients with renal disease remains unclear. The aim of this investigation, therefore, was to assess the prognostic significance of cTnT in chronic renal impairment patients, pre-dialysis. Methods. Ninety-six patients with chronic renal impairment were followed prospectively after cTnT determination by a quantitative laboratory method. The clinical outcomes after 2 years were determined. The measured cTnT values were correlated with biochemical parameters and clinical end-points. Results. A cut-off of 0.1 ng/ml was used in assessing the prognostic significance of cTnT. Twenty-five patients had a cTnT >0.1 ng/ml, whilst 71 had a cTnT 0.1 ng/ml. Twenty-one patients died during the follow-up period. Eleven of these had elevated cTnT at entry into the study. Death rate in the patients with cTnT >0.1 ng/ml was 42% compared with 14% in those with levels below the cut-off. Thirty-three patients died or had a vascular event. The rate of death or a vascular event in the elevated group was 64% compared with 24% in those with levels below the cut-off. At the end of the study, 23 patients were treated by continuous ambulatory peritoneal dialysis, 29 by haemodialysis, 22 had functioning renal transplants and one patient was not on renal replacement therapy. Factors that were found to significantly affect cTnT were diabetes, age and urea. cTnT was found to be a significant predictor of survival in these patients. Patients with high cTnT values were more likely to end up on haemodialysis. No relation of renal function to cTnT level was found. Conclusions. These results show that in patients with renal impairment, the measurement of cTnT prior to commencing renal replacement is a significant independent predictor of survival. cTnT did show potential as a prognostic test to stratify patients with a high cardiovascular risk and may enable intensive risk factor modification in this patient group. This may be of further use in selection of patients' suitability for renal transplantation.
The Journal of Heart and Lung Transplantation, 2008
Mannose-binding lectin (MBL) deficiency (or the common opsonic defect) has been reported as a ris... more Mannose-binding lectin (MBL) deficiency (or the common opsonic defect) has been reported as a risk factor for several immunologically controlled conditions, including infection and graft rejection. However, the effects of MBL deficiency on acute rejection after heart transplantation are unknown. Ninety heart transplant recipients were included in this study. Plasma MBL quantification was performed using a commercially available enzyme-linked immunoassay (ELISA). Acute rejection was diagnosed via endomyocardial biopsy and histologic assessment. MBL concentration was controlled for demographics, immunosuppression and anti-viral therapy. Individuals with dysfunctional MBL had significantly fewer rejection episodes (6.3 Ϯ 3.8%) than those with functional MBL (12.9 Ϯ 11.6%) (p ϭ 0.016). We found no significant difference between MBL concentrations among those with (1,232 Ϯ 58 ng/ml) and those without (1,216 Ϯ 161 ng/ml) GCAD (p ϭ 0.841). There was also no significant difference between the incidence of GCAD in those with "normal" concentrations (p ϭ 0.782). Heart transplant recipients with MBL deficiency had fewer acute graft rejection episodes compared to patients with functional MBL. This suggests the lectin pathway of complement activation is an important process in graft rejection. Furthermore, functional MBL may indicate a greater likelihood of rejection.
The Journal of Clinical Endocrinology & Metabolism, 2010
Context: Salivary cortisol measurement is used as a practical surrogate for serum free cortisol. ... more Context: Salivary cortisol measurement is used as a practical surrogate for serum free cortisol. However, parotid tissue harbors 11-hydroxysteroid dehydrogenase (11-HSD2) activity converting cortisol to cortisone.
In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyin... more In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 micorL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters Quattro Premier XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/z 191.9&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;145.6 and 193.9&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97-113%) and the method showed good linearity to 10,000 nmol/L (r(2)=0.999). Within-batch and between-batch imprecision was &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10% and bias &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC-MS/MS and HPLC showed good agreement between the two methods but this LC-MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.
Immunoassays used for the measurement of salivary cortisol are limited by variable interference f... more 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.
Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory i... more Aldosterone is a mineralocorticoid steroid hormone whose measurement in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Traditionally measurement of aldosterone has been performed by radioimmunoassay, however these assays lack specificity as they are prone to interference from structurally related steroid hormones. Herein, we have developed a novel, sensitive and specific method utilising solid phase extraction and quantitation of aldosterone from human plasma by UPLC-MS/MS. Standards, quality controls and samples (250μL) were extracted using Oasis(®) HLB 96-well plates. Extract (30μL) was injected onto a Krudcatcher UPLC In-Line Filter, 0.5μm guard column, coupled to a Kinetex PFP, 100mm×2.1mm, 2.6μm column with methanolic mobile phase gradient elution. Eluant was connected to a Waters(®) Xevo TQS tandem mass spectrometer operating in electrospray negative mode. We detected multiple reaction monitoring (MRM) transitions of m/z 359.0&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;189.1 for aldosterone and 366.0&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;194.1 for d7-aldosterone respectively, which co-eluted at 2.65min. Ion suppression was negligible. Mean recovery was 89.6%, limit of detection and lower limit of quantitation were 26pmol/L and 30pmol/L respectively. The assay was linear up to 3200pmol/L (r(2)=0.9999). Mean intra- and inter-assay imprecision and bias were all &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;10%. Comparison of the UPLC-MS/MS method with an immunoassay in routine clinical use in the UK yielded the equation UPLC-MS/MS=0.789(RIA)-41.7, linear regression r(2)=0.88, n=54. We have developed a sensitive and specific method for the extraction and measurement of aldosterone from human plasma. The method features a simple 96-well plate solid phase extraction procedure, highly selective column chemistry and short chromatographic run times.
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