Dennis Orton (Presenter)
University of Calgary
Bio: I completed my undergraduate (Double Major in Biochemistry and Immunology) and graduate (PhD in Pathology) degrees at Dalhousie University in Nova Scotia, Canada. My PhD focused on biomarker discovery of a pediatric kidney disease using proteomics methodology by LC-MS/MS. Currently, I am completing my Fellowship in Clinical Biochemistry at the University of Calgary in Calgary, Alberta, Canada. My program is focused on developing my skills as a Clinical Biochemist while developing new methods for LC-MS/MS analysis of hormones and drugs.
Authorship: Orton, D.J. (1,2), Buse, J.M. (1), Sadrzadeh, S.M.H. (1,2), Chin, A.C. (1,2)
(1) Calgary Laboratory Services, Calgary, AB, Canada; (2) University of Calgary, Calgary, AB, Canada
The ratio of aldosterone to plasma renin activity (PRA) is used to help diagnose hyperaldosteronism (Conn's syndrome). This study presents a novel combined LC-MS/MS method for determination of the aldosterone/PRA ratio. PRA is measured following the formation of the product angiotensin I (angI) over time. Following solid phase extraction, aldosterone and angI were separated on a reversed phase column using both a methanol and pH gradient. The pH gradient is required to promote ionization of each analyte in positive (angI) and negative (aldosterone) modes. Combined analysis of aldosterone and PRA by LC-MS/MS provides an improved workflow in the clinical laboratory.
Introduction: A well-studied pathway in physiological regulation of blood pressure is the renin-angiotensin-aldosterone system (RAAS). Under normal conditions, the protease renin is produced by the kidney in response to reduced blood pressure, which cleaves circulating angiotensinogen to angiotensin I (angI). AngI is subsequently processed into angiotensin II (angII) by angiotensin-converting enzyme (ACE). AngII then acts to increase synthesis and release of aldosterone by the adrenal cortex, which contributes towards increasing blood pressure. This process is normally under tight physiological regulation, therefore measurement of disruptions of the ratio of aldosterone to renin in circulation can be used to help diagnose hyperaldosteronism (Conn’s syndrome), a disease characterized by inappropriately hypersecretion of aldosterone. Because renin is a protease enzyme, it may be measured by either total mass or activity. Plasma renin activity (PRA) generally yields higher diagnostic accuracy than renin mass and was historically measured using radioimmunoassay (RIA). Unfortunately, RIA is an undesirable method, as radioactive substances are problematic for clinical routine application (safety training, disposal of waste, etc…). The challenges with measuring PRA have caused many labs to switch to total renin mass by sandwich immunoassay for diagnosis, which can cause problems for physician interpretation of results. Aldosterone is also generally measured by a traditional immunoassay, and the results may be used to determine a ratio, indicating if there is a disruption of normal physiological regulation of the RAAS pathway. This study presents a novel method for quantification of the aldosterone/PRA ratio by liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a single run to streamline the diagnosis of hyperaldosteronism and re-implement PRA in the clinical lab.
Methods: AngI and aldosterone were quantified from EDTA plasma following angI generation, or from blank stripped serum spiked with angI and aldosterone as standards/calibrators. Isotopically labelled angI (AS-61182) from Anaspec, and d4-aldosterone (S11214) from Isosciences were used as internal standards and were added before the angI generation phase. PRA was quantified by inhibition of ACE in vitro to allow generation of angI, followed by measurement of the concentration of angI in ng/mL/min. Briefly, 400 µL of EDTA patient plasma was spiked with 40 µL of 50 mM AEBSF in 250 mM maleate buffer (pH 1.2) to inhibit endogenous ACE activity. Following one hour incubation at 37 °C, samples were subject to solid-phase extraction with Waters HLB extraction cartridges and dried completely under nitrogen. Chromatographic separation was accomplished on a Phenomenex EVO C18 column with an Agilent 1260 quaternary pump connected to an Agilent 6460 triple quadrupole mass spectrometer with an electrospray ionization source operating in positive and negative mode sequentially. The solvent gradient employed 0.05% formic acid (FA; pH 3), methanol, and 1.0 mM ammonium formate (pH 6.7) to allow efficient separation and ionization. AngI was eluted from the column at pH 3 at 20 % methanol with 80 % FA in the mobile phase and detected in positive mode. The mobile phase was then switched to 80 % 1.0 mM ammonium formate and equilibrated for 2 minutes, followed by a linear increase in methanol from 20 to 65 % over 2 minutes to elute and detect aldosterone in negative mode. AngI eluted at 2.1 minutes, and aldosterone at 5.7 minutes. Total run time was 9 minutes. Precursor >> fragment ions for aldosterone and angI were 359.2 >> 189.1 / 332.2 and 432.9 >> 647.4 / 394.6 respectively.
Results and Discussion: Analysis of both angI and aldosterone in a single run has not been described previously. This is likely due to the fundamental biochemical differences between these two types of molecules. Aldosterone is a steroid hormone synthesized from cholesterol, and extraction for LC-MS analysis normally employs organic solvents such as methyl tertbutylethyl ether. Peptides such as angI are not soluble in such extraction matrices, and are therefore more commonly extracted into solid phase media, eluted, dried, and reconstituted. This method employs a solid phase extraction using Waters HLB cartridges that capture both types of analytes and allow analysis of each in a single run. In addition to optimization of the extraction parameters, these analytes ionize at different pH when analyzed by LC-MS/MS. Aldosterone is normally monitored in negative mode and requires a higher pH for ionization, while angI is ionized at low pH. Here, modulation of the methanol/water solvent gradient allows effective separation and elution of aldosterone and angI while a pH switch provides a means for effective ionization of both compounds throughout the analysis. The chromatographic conditions were started at pH 3 in FA and increased to 6.7 by converting the aqueous phase to that containing ammonium formate. The analysis range for aldosterone gave quantifiable results similar to that of immunoassay (50 - 2000 pmol/L), although the detection limit is currently unstable in some patients. This is attributed to phospholipid contamination causing signal suppression of aldosterone. Work is currently underway to optimize the extraction protocol and limit this interference. For PRA, angI was readily quantifiable down to 0.3 ng/mL in patient samples following a one hour incubation in the presence of the ACE-inhibitor AEBSF.
Conclusions: By employing a quaternary pump and a three solvent system, we demonstrate a novel LC-MS/MS method for combined analysis of aldosterone and PRA, improving the workflow for reporting results from the clinical laboratory. Current methods are unable to analyze both aldosterone and angI in a single run due to differences in extraction protocols as well as ionization pH for each analyte, therefore optimization of both the extraction procedure and chromatographic conditions were required for method development.
References & Acknowledgements:
IP Royalty: no
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