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Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma
Arne Gessner1, Maren Mieth2, Daniel Auge2
1Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054, Erlangen, Germany. arne.gessner@fau.de.
Insights
A new method simultaneously quantifies ten biomarkers for cardiovascular disease (CVD) and chronic kidney disease (CKD) using minimal plasma. This validated technique enables large-scale studies and establishes reference values for Nɛ-acetyllysine (Nɛ-AcLys).
Area of Science:
- Biochemistry
- Analytical Chemistry
- Clinical Diagnostics
Background:
- Cardiovascular disease (CVD) and chronic kidney disease (CKD) represent significant global health challenges.
- Accurate identification and validation of risk markers are crucial for epidemiological studies of CVD and CKD.
- Existing analytical methods often require adaptation or development for comprehensive biomarker analysis.
Purpose of the Study:
- To describe and validate an analytical procedure for the simultaneous quantification of ten endogenous biomarkers relevant to CVD and CKD.
- To assess the method's scalability for application in large-scale epidemiological cohorts.
- To establish reference values for Nɛ-acetyllysine (Nɛ-AcLys) in a population of healthy volunteers.
Main Methods:
- Development of a streamlined sample preparation protocol requiring only 20 µL of human plasma.
- Utilization of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for simultaneous detection.
- Rigorous method validation according to established guidelines, assessing accuracy, precision, linearity, selectivity, and quantification limits.
Main Results:
- The developed LC-MS/MS method was successfully validated, meeting all required performance criteria.
- The method demonstrated scalability, successfully analyzing plasma samples from 391 healthy volunteers.
- Reference values for Nɛ-acetyllysine (Nɛ-AcLys) were established, alongside values for creatinine, β-aminoisobutyric acid (β-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine.
Conclusions:
- The described analytical method is suitable for the simultaneous quantification of ten key CVD and CKD biomarkers in large cohorts.
- The study validates Nɛ-acetyllysine (Nɛ-AcLys) as a quantifiable biomarker in large-scale studies.
- This approach facilitates improved risk assessment and understanding of CVD and CKD pathogenesis.
Abstract:
Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20 µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, β-aminoisobutyric acid (β-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.
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