Santica M Marcovina1, Noémie Clouet-Foraison1,2, Marlys L Koschinsky3, Mark S Lowenthal4, Allen Orquillas5, Michael B Boffa6, Andrew N Hoofnagle2,5, Tomáš Vaisar2. 1. Division of Metabolism, Endocrinology, and Nutrition, Northwest Lipid Metabolism and Diabetes Research Laboratories, University of Washington, Seattle, WA, USA. 2. Division of Metabolism, Endocrinology, and Nutrition, University of Washington, Seattle, WA, USA. 3. Department of Physiology & Pharmacology, Robarts Research Institute, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, Canada. 4. National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA. 5. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA. 6. Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, Canada.
Abstract
BACKGROUND: Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. METHOD: A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. RESULTS: A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). CONCLUSIONS: Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays. Published by Oxford University Press on behalf of the American Association for Clinical Chemistry 2021. This work is written by US Government employees and is in the public domain in the US.
BACKGROUND: Use of lipoprotein(a) concentrations for identification of individuals at high risk of cardiovascular diseases is hampered by the size polymorphism of apolipoprotein(a), which strongly impacts immunochemical methods, resulting in discordant values. The availability of a reference method with accurate values expressed in SI units is essential for implementing a strategy for assay standardization. METHOD: A targeted LC-MS/MS method for the quantification of apolipoprotein(a) was developed based on selected proteotypic peptides quantified by isotope dilution. To achieve accurate measurements, a reference material constituted of a human recombinant apolipoprotein(a) was used for calibration. Its concentration was assigned using an amino acid analysis reference method directly traceable to SI units through an unbroken traceability chain. Digestion time-course, repeatability, intermediate precision, parallelism, and comparability to the designated gold standard method for lipoprotein(a) quantification, a monoclonal antibody-based ELISA, were assessed. RESULTS: A digestion protocol providing comparable kinetics of digestion was established, robust quantification peptides were selected, and their stability was ascertained. Method intermediate imprecision was below 10% and linearity was validated in the 20-400 nmol/L range. Parallelism of responses and equivalency between the recombinant and endogenous apo(a) were established. Deming regression analysis comparing the results obtained by the LC-MS/MS method and those obtained by the gold standard ELISA yielded y = 0.98*ELISA +3.18 (n = 64). CONCLUSIONS: Our method for the absolute quantification of lipoprotein(a) in plasma has the required attributes to be proposed as a candidate reference method with the potential to be used for the standardization of lipoprotein(a) assays. Published by Oxford University Press on behalf of the American Association for Clinical Chemistry 2021. This work is written by US Government employees and is in the public domain in the US.
Authors: Michael France; Alan Rees; Dev Datta; Gilbert Thompson; Nigel Capps; Gordon Ferns; Uma Ramaswami; Mary Seed; Dermot Neely; Robert Cramb; Carol Shoulders; Mahmoud Barbir; Alison Pottle; Ruth Eatough; Steven Martin; Graham Bayly; Bill Simpson; Julian Halcox; Ray Edwards; Linda Main; Jules Payne; Handrean Soran Journal: Atherosclerosis Date: 2016-11-05 Impact factor: 5.162