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Abstract Introduction
Lipoprotein(a) (Lp(a)) is a lipoprotein particle that is causally related with atherosclerotic disease, myocardial infarction and aortic valve stenosis. The recent introduction of Lp(a) lowering medication has caused large interest in Lp(a) quantitation. The Lp(a) particle consists of an LDL particle to which an apolipoprotein (a) (apo(a)) protein is covalently bound via a disulfide bridge. Apo(a) contains a number of kringle IV repeats, a kringle V and a protease domain, and varies widely in size due to a size polymorphism in kringle IV-2. The Lp(a) particle holds a lipid core consisting of free cholesterol, phospholipids, cholesteryl esters and triglycerides, apoB and apo(a), and its concentration is traditionally expressed in mass units. Yet, apo(a) size heterogeneity and post-translational modifications such as N- and O-glycosylation of apo(a) affect the molecular mass of Lp(a) particles. We assessed the impact of defining the measurand at the molecular level using bottom-up proteomics, in relation to apo(a) size polymorphism and available PTMs.
Methods
The chemical composition of Lp(a) was assessed through literature study, and the masses of apoB and apo(a) were determined based on their amino acid sequence. A model for the molecular composition of Lp(a) was developed based on the assumption that the lipid composition of the particle is not affected by the apo(a) size polymorphism. The number of kringle IV-2 repeats reported in literature is 3-50, and chemical compositions were calculated for these values. Glycosylation patterns of both apoB and apo(a) were taken into account.
Results
The outcomes of the developed model correspond well with previously reported chemical compositions of Lp(a) [1,2]. An Lp(a) particle containing only three kringle IV-2 repeats per apo(a) is calculated to have a particle mass of 2,767 kDa with a lipid portion of 70% (w/w), apoB of 20% (w/w) and apo(a) of 10% (w/w). However, for a particle containing 50 kringles, the particle mass is 3,639 kDa with a lipid portion of 53% (w/w), apoB of 16% (w/w) and apo(a) of 31% (w/w). The huge mass variation of Lp(a) impedes Lp(a) standardization efforts, whereas molecular characterization of the apo(a) measurand and molar expression of apo(a) content may avert standardization problems.
Conclusions
The expression of Lp(a) particle concentrations in mass units is metrologically inappropriate and should be abandoned. Mass spectrometry using LC-MRM-MS allows molecular characterization of the apo(a) measurand and enables accurate quantitation in molar units, unaffected by the apo(a) size polymorphism and glycosylation. Future traceability of apo(a) to SI units can only be accomplished with an unequivocally molecularly defined protein measurand and the consistent use of molar units [3].
References
1. Kostner et al. J Lipid Res. 1999;40:2255-63.
2. Tsimikas et al. J Clin Lipidol. 2018;12:1313-23.
3. Cobbaert et al. Clin Chem Lab Med. 2018;56:1598-1602. |
= Discovery stage.
= Translation stage.
= Clinically available.
