BackgroundPlasma renin levels were determined in the academia-driven, EU-funded “Labeling of Enalapril from Neonates up to Adolescents” (LENA) project to evaluate its role in pediatric heart failure. Quality-controlled bioanalysis is crucial to ensure reliable data generation. However, a comprehensive bioanalytical quality control (QC) concept to monitor the method performance within an academic environment was lacking.MethodsThus, a QC concept was designed encompassing regulatory guidance, international recommendations and current scientific discussions. The concept included (1) a system-suitability test, (2) verification of single bioanalytical runs by calibration curve performance and evaluation of QCs, (3) assessment of the inter-run accuracy according to Clinical Laboratory Standards Institute (CLSI) guideline, (4) monitoring of reproducibility by pediatric incurred samples, (5) blank-sample analysis and (6) participation in interlaboratory testing.ResultsThe concept was successfully applied to the academic project. About 11% of single runs were identified as invalid and triggered a re-analysis of unknown samples being included in those runs. The usefulness of the customized inter-run monitoring was demonstrated and proved the good accuracy from the first to the last run. All 147 reanalyzed incurred sample pairs complied with regulatory requirements.ConclusionsThe regulatory complied QC concept was customized for the demands of academia-driven pediatric trials and contributed to the reliable quantification of 965 pediatric renin samples.
As part of the extended renin-angiotensin-aldosterone system, active renin and its precursor prorenin have been an area of research interest for decades. Although several studies showed a correlation with disease, other studies found no significant association, e.g. attributed to limited sample size or pharmacological effects of antihypertensive drugs. Since the measurement of both proteins has typically been carried out in adult populations, the data in paediatrics is limited. This review aimed to collate the current data on plasma renin and prorenin levels in children and compare the levels of healthy vs. the diseased state. A literature search using Medline resulted in 213 publications of which 15 studies were classified as relevant. In the extant studies in the literature, an age-dependent decline of renin plasma concentration was observed in newborns compared to adolescents. For children with cardiovascular disease, five studies were identified that provide limited insight into the pathophysiological regulation of renin. In general, sample handling is still a crucial step, which might particularly affect measured active renin concentrations due to conformational changes of its precursor prorenin. A reliable assessment for prorenin levels in the maturating population is yet not possible due to the low number of available publications. Three different approaches to quantify prorenin were found and raise the question on the comparability of these methods. The review emphazised several weaknesses and highlights the need for an accurate procedure to determine levels of active renin as well as prorenin in its closed and open form.
Rationale Human prorenin, representing the precursor of mature renin, has been discussed as a potential biomarker, e.g. in diagnosing primary hyperaldosteronism or diabetes‐induced nephropathy. Currently, only immunoassays are available for prorenin quantification. As the similarity of prorenin to active renin impedes its accurate determination by immunoassay, mass spectrometry appears as an accurate alternative for differentiation of that protein. Methods Immunoaffinity purification plus a mixed‐solvent‐triggered digestion was combined with liquid chromatography/mass spectrometry (LC/MS) to enable a fast, sensitive, and less laboratory‐intensive approach to the quantification of prorenin. Statistical experimental planning, which is known as Design of Experiments (DOE), was used to identify the optimal conditions for the generation of the signature peptides within a manageable number of experiments. The efficiency of the mixed‐solvent‐triggered digestion by trypsin was investigated using four different organic solvents: acetonitrile, acetone, tetrahydrofuran and methanol. Results By utilizing a D‐optimal design, we found that the optimal mixed‐solvent type for the generation of both signature peptides was acetonitrile at a concentration of 84% and an incubation temperature of 16°C. Using the mixed‐solvent‐triggered digestion, the procedure time allowed a fast analysis of active renin and prorenin with a short digestion time of 98 min. This optimized mixed‐solvent‐triggered digestion procedure was applied to detect renin and prorenin successfully in human plasma by the newly developed hybrid approach. Conclusions The identification of unique surrogates for human prorenin enabled the mass spectrometric differentiation between the two similar proteins. The novel hybrid approach successfully proved its ability to purify, detect and distinguish between prorenin and active renin in human plasma.
BackgroundSince sample volume is limited in children, innovative bioanalytical methods and enrichment procedures are highly required. The analysis of endogenous substances by liquid chromatography coupled to mass spectrometry is a highly specific method because of its selectivity and accuracy. However reliable detection of endogenous substances can only be achieved by a hybrid assay approach combining immunocapture and mass spectrometry. Key element of the immunocapture procedure is the selection of the appropriate antibody for capturing the desired antigen. This study is meant to identify the most suitable antibody that facilitates the development of an hybrid assay approach concerning reliable detection of endogenous prorenin in paediatric samples.MethodsDynabeads magnetic beads were coupled to three different antibodies from three different vendors (GeneTex, Molecular Innovations, R&D systems). 500 µL human plasma which was spiked with 20 ng recombinant human prorenin (Cayman chemicals). The immunocapture step was followed by protease digestion and a custom-made µelution solid-phase extraction protocol. The digest was analyzed by Shimadzu Nexera LC-system coupled with Sciex TripleTOF 6600 mass spectrometer.ResultsThe analysis of the captured prorenin was performed by the surrogate peptide approach. In this case the surrogate peptide was identified as unique. The comparison of the three available antibodies showed that one antibody did not ensure reliable binding properties in human matrix. Among the two remaining antibodies only one showed sufficient binding capacities to be applied in small sample volumes commonly available in paediatric samples. Using this hybrid approach enabled the enrichment of the required volume by factor of 20.ConclusionThis study identified the most suitable antibody for the immunocapture procedure of the prorenin hybrid approach. This is now followed by further mass spectrometric method development and validation prior to its application in paediatric samples.Disclosure(s)Declaration of interest: none Ilja Burdman and Bjoern B. Burckhardt declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors
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