Daniel T. Holmes

Towards the development of an immuno MALDI (iMALDI) mass spectrometry assay for the diagnosis of hypertension

The renin-angiotensin-aldosterone system (RAAS) plays an essential role in the regulation of plasma volume and arterial blood pressure. One of the most common diseases of the RAAS is the autonomous production of aldosterone by the adrenal glands, caused by either bilateral adrenal hyperplasia or an aldosterone-producing adenoma. This condition, known as primary aldosteronism, is a treatable and often curable form of hypertension. The measurement of plasma renin activity (PRA), as determined by radioimmunoassay for angiotensin I is essential to the diagnosis of primary aldosteronism. However, accurate determination of PRA is often hampered by low plasma concentrations of angiotensin I. Here, we report the use of immuno-MALDI (iMALDI) as a highly sensitive and specific method for the absolute quantitation of angiotensin I in plasma. iMALDI permits concentration determination by affinity-capture of angiotensin I and a stable-isotopically labeled standard (SIS) peptide on immobilized anti-peptide antibodies. The affinity beads are placed on the MALDI target, permitting automated analysis of large numbers of patient samples. Pretreatment of the plasma is not required, and this method is suitable for the accurate determination of angiotensin I in whole plasma. The calibration curve generated using this method was linear over a 50-fold concentration range in plasma, with a correlation coefficient of 0.984. MS/MS sequence confirmation provides absolute specificity. The iMALDI angiotensin I assay, therefore, has the potential to be developed into a method for determining PRA that has advantages in time, in specificity, and in safety.

Plasma renin activity predicts cardiovascular mortality in the Heart Outcomes Prevention Evaluation (HOPE) study

AIMS Plasma renin activity (PRA) has been proposed as an independent predictor of cardiovascular (CV) risk, but there are limited data from large prospective studies, particularly in patients with stable vascular disease and/or diabetes, without heart failure. METHODS AND RESULTS We evaluated the predictive value of PRA as a marker of CV events and mortality in a large population of patients with stable chronic vascular disease and/or diabetes and one CV risk factor. Baseline PRA was measured in 2913 patients enrolled in the Heart Outcomes Prevention Evaluation (HOPE) study. Subjects were followed for a median of 4.5 years. Compared with the referent lowest fifth, subjects in the highest fifth of the PRA distribution had a hazard ratio (HR) of 1.38 (95% confidence interval, 1.03-1.86; P = 0.03) for the composite of major vascular events, with an HR of 1.89 for CV death. These associations remained statistically significant after full adjustment for clinical characteristics, background use of β-blockers, diuretics, allocation to ramipril, in addition to inflammatory biomarkers, high-sensitivity C-reactive protein, and N-terminal pro-brain natriuretic peptide. CONCLUSION High PRA is an independent predictor of major vascular events and mortality in a stable population of high-risk patients with atherosclerosis and/or diabetes. Although an increase in PRA could be a marker of more intense antihypertensive therapy, our results suggest that PRA may represent a risk marker and potential target for therapy in high-risk patients with atherosclerosis and/or diabetes.

Cohort profile: Canadian study of prediction of death, dialysis and interim cardiovascular events (CanPREDDICT)

BackgroundThe Canadian Study of Prediction of Death, Dialysis and Interim Cardiovascular Events (CanPREDDICT) is a large, prospective, pan-Canadian, cohort study designed to improve our understanding of determinants of renal and cardiovascular (CV) disease progression in patients with chronic kidney disease (CKD). The primary objective is to clarify the associations between traditional and newer biomarkers in the prediction of specific renal and CV events, and of death in patients with CKD managed by nephrologists. This information could then be used to better understand biological variation in outcomes, to develop clinical prediction models and to inform enrolment into interventional studies which may lead to novel treatments.Methods/DesignsCommenced in 2008, 2546 patients have been enrolled with eGFR between 15 and 45 ml/min 1.73m2 from a representative sample in 25 rural, urban, academic and non academic centres across Canada. Patients are to be followed for an initial 3 years at 6 monthly intervals, and subsequently annually. Traditional biomarkers include eGFR, urine albumin creatinine ratio (uACR), hemoglobin (Hgb), phosphate and albumin. Newer biomarkers of interest were selected on the basis of biological relevance to important processes, commercial availability and assay reproducibility. They include asymmetric dimethylarginine (ADMA), N-terminal pro-brain natriuretic peptide (NT-pro-BNP), troponin I, cystatin C, high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL6) and transforming growth factor beta 1 (TGFβ1). Blood and urine samples are collected at baseline, and every 6 monthly, and stored at −80°C. Outcomes of interest include renal replacement therapy, CV events and death, the latter two of which are adjudicated by an independent panel.DiscussionThe baseline distribution of newer biomarkers does not appear to track to markers of kidney function and therefore may offer some discriminatory value in predicting future outcomes. The granularity of the data presented at baseline may foster additional questions.The value of the cohort as a unique resource to understand outcomes of patients under the care of nephrologists in a single payer healthcare system cannot be overstated. Systematic collection of demographic, laboratory and event data should lead to new insights.The mean age of the cohort was 68 years, 90% were Caucasian, 62% were male, and 48% had diabetes. Forty percent of the cohort had eGFR between 30–45 mL/min/1.73m2, 22% had eGFR values below 20 mL/min/1.73m2; 61% had uACR < 30. Serum albumin, hemoglobin, calcium and 25-hydroxyvitamin D (25(OH)D) levels were progressively lower in the lower eGFR strata, while parathyroid hormone (PTH) levels increased. Cystatin C, ADMA, NT-proBNP, hsCRP, troponin I and IL-6 were significantly higher in the lower GFR strata, whereas 25(OH)D and TGFβ1 values were lower at lower GFR. These distributions of each of the newer biomarkers by eGFR and uACR categories were variable.

Clinical validation for the aldosterone-to-renin ratio and aldosterone suppression testing using simultaneous fully automated chemiluminescence immunoassays

Background: As larger numbers of hypertensive patients are screened for primary aldosteronism with the aldosterone-to-renin ratio (ARR), automated analyzers present a practical solution for many laboratories. We report the method-specific ARR cutoff determined with direct, automated chemiluminescence immunoassays allowing the simultaneous measurement of plasma aldosterone concentrations (PACs) and plasma renin concentrations (PRCs). Methods: Method comparisons to commonly employed assays and tandem mass spectrometry were undertaken. Patients were previously diagnosed based on the local ARR cutoff of 1.2 (ng/dl)/(&mgr;IU/ml) in samples collected in upright seated position. Lack of aldosterone suppression in response to salt load to less than 5 ng/dl confirmed primary aldosteronism. For the new assays, the optimal ARR cutoff was established in 152 patients with essential hypertension, 93 with primary aldosteronism and 147 normotensive patients. Aldosterone suppression was assessed in 73 essential hypertensive and 46 primary aldosteronism patients. Results: PAC and PRC were significantly correlated to values determined with currently available methods (P < 0.001). In patients with primary aldosteronism, patients with essential hypertension and controls, mean (95% confidence interval) PAC was 28.4 (25.4–31.8), 6.4 (5.9–6.9) and 6.2 (5.6–6.9) ng/dl, respectively. In the same groups, PRC was 6.6 (5.6–7.7), 12.9 (11.2–14.8) and 26.5 (22.2–31.5) &mgr;IU/ml. An ARR cutoff of 1.12 provided 98.9% sensitivity and 78.9% specificity. Employing the new assay aldosterone suppression confirmed the diagnosis of primary aldosteronism and essential hypertension using the cutoff of 5 ng/dl. Conclusion: Our data demonstrate that the new assays present a convenient alternative for the measurement of PAC and PRC on a single automated analyzer. Availability of these simultaneous assays should facilitate screening and diagnosis of primary aldosteronism.

Determination of serum aldosterone by liquid chromatography and tandem mass spectrometry: a liquid–liquid extraction method for the ABSCIEX API-5000 mass spectrometry system

Aims Accurate serum aldosterone determination is critical to the screening and diagnosis of primary aldosteronism, the localisation of aldosterone producing tumours, and the investigation of other disorders of the renin-angiotensin system. Mass spectrometry offers a means to overcome problems with method-dependent bias between competitive immunoassays for aldosterone. The authors have developed a simple, sensitive and precise liquid–liquid extraction aldosterone method for the ABSCIEX API-5000 liquid chromatography and tandem mass spectrometry (LC-MS/MS) system. Methods Using d7-aldosterone internal standard, 500 μl of sample is extracted with 2500 μl of methyl tertbutyl ether followed by dry-down, reconstitution and LC-MS/MS analysis in ESI negative mode. Method validation was undertaken using standard approaches and comparison made against a commercial radioimmunoassay. Accuracy was assessed using EQA material with assigned aldosterone concentrations. Results The assay was linear up to 3420 pmol/l (LOQ=50 pmol/l, LOD<22 pmol/l). Total CVs were ≤5% for concentrations ≥120 pmol/l and 10% at the LOQ. Mean accuracy was 98.5% against GCMS assigned material. Conclusion The authors present a precise, sensitive and simple aldosterone method suitable for routine clinical use that requires no solid phase extraction or specialised ion sources.

Laboratory challenges in primary aldosteronism screening and diagnosis.

BACKGROUND AND OBJECTIVE The laboratory has a critical role to play in the screening and diagnosis of primary aldosteronism. This review highlights some of the important analytical considerations and the new developments in the determination of aldosterone and renin. METHODS The review considered the published literature and clinical practice guidelines in the area of primary aldosteronism. RESULTS A brief introduction to primary aldosteronism is provided. A detailed description of the pre-analytical, analytical and post-analytical considerations for the laboratory determination of aldosterone, renin and the aldosterone to renin ratio follows. CONCLUSIONS The lack of internationally accepted standardized methodologies and standard reference material has impeded screening and diagnosis of primary aldosteronism. The development of more accurate and sensitive methods by LC-MS/MS has improved the reliability of aldosterone and renin testing and the availability of commercial chemiluminescent assays may improve the standardization of reporting. Laboratorians need to understand the strengths and weaknesses of their analytical approach and ensure that their interpretative reports are appropriate to their assays.

Diagnosis and management of testosterone deficiency syndrome in men: clinical practice guideline

See also page [1342][1] and [www.cmaj.ca/lookup/doi/10.1503/cmaj.151208][2] Testosterone deficiency syndrome, also known as late-onset hypogonadism, is a clinical and biochemical syndrome that can occur in men in association with advancing age. The condition is characterized by deficient testicular

Biomarkers of inflammation, fibrosis, cardiac stretch and injury predict death but not renal replacement therapy at 1 year in a Canadian chronic kidney disease cohort

BACKGROUND Newer biomarkers, reflective of biological processes, such as inflammation and fibrosis, cardiac stretch or damage and vascular health may be useful in understanding clinical events in chronic kidney disease (CKD). We assessed whether these newer biomarkers, alone or as a panel, improve risk prediction for renal replacement therapy or death, over and above conventional clinical, demographic and laboratory variables. METHODS We conducted a prospective observational Canadian cohort study in 2544 CKD patients with estimated glomerular filtration rate (eGFR) of 15-45 mL/min/1.73 m(2), under nephrology care, in urban and rural centers. We measured traditional clinical and laboratory risk factors, as well as newer biomarkers: cystatin C, high sensitivity c-reactive protein (hsCRP), interleukin 6 (IL6), transforming growth factor β1 (TGFβ1), fibroblast growth factor 23 (FGF23), N-terminal probrain natriuretic peptide (NT-proBNP), troponin I and asymmetric dimethylarginine (ADMA). Key outcomes were renal replacement therapy (RRT, dialysis or transplantation) and death, during the first year follow-up after enrollment: a time point important for clinical decision-making for patients and providers. RESULTS Newer biomarkers do not improve the prediction of RRT, when added to conventional risk factors such as eGFR, urine albumin to creatinine ratio, hemoglobin, phosphate and albumin. However, in predicting death within 1 year, cystatin C, NT-proBNP, hsCRP and FGF23 values significantly improved model discrimination and reclassification: c statistic increased by absolute 4.3% and Net Reclassification Improvement for categories of low, intermediate and high risk at 11.2%. CONCLUSIONS Our findings suggest that the addition of newer biomarkers may be useful in predicting death in patients with established CKD within a 1-year timeframe. This information may be useful in informing prognosis and redirect resources to serve patients at higher risk to improve outcomes and sustainability of the nephrology care system.

Steroid profiling reveals widespread local regulation of glucocorticoid levels during mouse development.

Glucocorticoids (GCs) are produced by the adrenal glands and circulate in the blood to coordinate organismal physiology. In addition, different tissues may independently regulate their local GC levels via local GC synthesis. Here, we find that in the mouse, endogenous GCs show tissue-specific developmental patterns, rather than mirroring GCs in the blood. Using solid-phase extraction, HPLC, and specific immunoassays, we quantified endogenous steroids and found that in tissues of female and male mice, (1) local GC levels can be much higher than systemic GC levels, (2) local GCs follow age-related patterns different from those of systemic GCs, and (3) local GCs have identities different from those of systemic GCs. For example, whereas corticosterone is the predominant circulating adrenal GC in mice, high concentrations of cortisol were measured in neonatal thymus, bone marrow, and heart. The presence of cortisol was confirmed with liquid chromatography-tandem mass spectrometry. In addition, gene expression of steroidogenic enzymes was detected across multiple tissues, consistent with local GC production. Our results demonstrate that local GCs can differ from GCs in circulating blood. This finding suggests that steroids are widely used as local (paracrine or autocrine) signals, in addition to their classic role as systemic (endocrine) signals. Local GC regulation may even be the norm, rather than the exception, especially during development.

Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension

Plasma renin activity (PRA) is an essential analytical tool for screening and diagnosis of secondary forms of hypertension. Typically, PRA is measured by competitive radioimmunoassay, but there are significant drawbacks to this technique including non-specificity, long analysis times, narrow calibration range, and the requirement for radionucleotides. In this paper, we report a method for plasma renin activity determination by immuno-MALDI mass spectrometry detection. This method overcomes the issues of non-specificity and long analytical times present with RIA, and does not require the use of radionucleotides. As an initial methodological evaluation, plasma renin activity results obtained by radioimmunoassay, LC/ESI-MS/MS, and immuno-MALDI on 64 samples from an outpatient primary aldosteronism screening program have been compared. A strong correlation was found between immuno-MALDI and radioimmunoassay (R2 = 0.9412, 62/64 within the 95% CI of the Bland-Altman plot), and iMALDI and LC/ESI-MS/MS (R2 = 0.9471, 62/64 within the 95% CI of the Bland-Altman plot). Technical replicates showed a 4.8% CV, while inter- and intra-day replicates showed CVs of 17.3% and 17.2% respectively. We have developed an assay capable of measuring PRA without the use of radionucleotides. This immuno-MALDI approach affords the specificity of MS while avoiding the long analytical run times and technical problems associated with HPLC. With the use of robotic sample preparation to optimize precision, this assay should be adaptable to clinical environments.