Towards an SI-Traceable Reference Measurement System for Seven Serum Apolipoproteins Using Bottom-Up Quantitative Proteomics: Conceptual Approach Enabled by Cross-Disciplinary/Cross-Sector Collaboration.

Current dyslipidemia management in patients with atherosclerotic cardiovascular disease (ASCVD) is based on traditional serum lipids. Yet, there is some indication from basic research that serum apolipoproteins A-I, (a), B, C-I, C-II, C-III, and E may give better pathophysiological insight into the root causes of dyslipidemia. To facilitate the future adoption of clinical serum apolipoprotein (apo) profiling for precision medicine, strategies for accurate testing should be developed in advance. Recent discoveries in basic science and translational medicine set the stage for the IFCC Working Group on Apolipoproteins by Mass Spectrometry. Main drivers were the convergence of unmet clinical needs in cardiovascular disease (CVD) patients with enabling technology and metrology. First, the residual cardiovascular risk after accounting for established risk factors demonstrates that the current lipid panel is too limited to capture the full complexity of lipid metabolism in patients. Second, there is a need for accurate test results in highly polymorphic and atherogenic apolipoproteins such as apo(a). Third, sufficient robustness of mass spectrometry technology allows reproducible protein quantification at the molecular level. Fourth, several calibration hierarchies in the revised ISO 17511:2020 guideline facilitate metrological traceability of test results, the highest achievable standard being traceability to SI. This article outlines the conceptual approach aimed at achieving a novel, multiplexed Reference Measurement System (RMS) for seven apolipoproteins based on isotope dilution mass spectrometry and peptide-based calibration. This RMS should enable standardization of existing and emerging apolipoprotein assays to SI, within allowable limits of measurement uncertainty, through a sustainable network of Reference Laboratories.

Pediatric reference intervals for lipids and apolipoproteins on the VITROS 5,1 FS Chemistry System.

OBJECTIVES: Lipid biomarkers are integral in the assessment of dyslipidemia and cardiovascular risk, conditions that have become increasingly prevalent in pediatric populations. A comprehensive set of pediatric reference intervals for traditional or recently established lipid analytes is not currently available. DESIGN AND METHODS: 525 outpatient samples from a pediatric population were categorized into five age groups ranging from 0 to 20 years of age. Groups were further partitioned by gender. Serum or plasma samples were analyzed on the VITROS 5,1 FS Chemistry System for cholesterol and triglycerides by dry-film methods, direct HDL-C and LDL-C by selective detergent elimination, and apolipoproteins AI and B by immunoturbidimetry. Reference intervals were established by non-parametric methods at the 2.5th and 97.5th percentiles. RESULTS: Lipid levels show age- and gender-related differences, particularly during the first year of life and in young adults following puberty. Concentrations of total cholesterol, LDL-C, and apo B were lowest in the 12 months after birth and remained relatively constant throughout childhood, but decreased for males in early adulthood. Triglyceride levels increased gradually throughout childhood and adolescence, and along with cholesterol, the upper limits of these intervals exceeded the recommended concentrations of lipid levels in children. For HDL-C and apo AI, no age- or sex-related differences were found until after puberty when values for males decreased slightly. CONCLUSIONS: Our current reference intervals in children and adolescents provide an important update for lipid markers and suggest earlier incidence of hypercholesterolemia when compared to previous ranges. Increased profiling of lipids is anticipated, and these will aid in the early assessment of cardiovascular risk in pediatric populations.

Lipid, lipoprotein, apolipoprotein and lipoprotein(a) levels: reference intervals in a healthy Indian population.

The role of lipids, lipoproteins and lipoprotein(a) [Lp(a)] in coronary artery disease (CAD) is known but the role of major apolipoproteins (apos) other than apo A-I and apo B remains unclear. In this study, using immunoturbidimetry we have estimated serum levels of total cholesterol, HDL-C, LDL-C, triglyceride, LDL-apoB and all major apos; A-I, A-II, B, C-II, C-III and E, in 751 healthy Indian subjects (470 men and 281 women, age 25-65 years), determined their percentiles, and established reference intervals. The effects of age, smoking and alcohol on all these analytes were also evaluated. This is the first study to provide reference intervals for all apos, in both sexes from a general population. The percentiles and the reference intervals have clinical relevance and will be useful in assessing the risk of CAD in patients with hyperlipidemia and other diseases.

Standardization of Lp(a) measurements.

Two direct binding ELISAs were developed in our laboratory for measuring Lp(a) in human plasma. The first ELISA was performed by using a monoclonal antibody to apo(a) bound to the solid phase and a second monoclonal antibody to apo(a) as detecting antibody. The second ELISA was performed by using the same monoclonal antibody bound to the solid phase and an anti-apo B polyclonal antibody as detecting antibody. The immunoreactivity of Lp(a) particles of different size, isolated from subjects with F, B or S2 isoform, were evaluated by the two ELISA methods. Superimposable standard curves were obtained by the three Lp(a) preparations when using the apo(a) detection system, but the Lp(a) containing the largest apo(a) isoform S2 had a significantly reduced slope by the apo B detection method. Lp(a) concentration was determined in plasma samples by the two ELISA methods. When Lp(a) with apo(a) isoform S2 was used to calibrate the assays, similar Lp(a) values were obtained by the two different detecting systems on samples from subjects with isoforms S2, S3 or S4, while values in the samples with B and S1 isoforms were significantly higher. When Lp(a) with isoform B was used as calibrator, comparable Lp(a) values were obtained by the two methods on samples with B isoform, while the values were lower in the samples with the higher molecular weight isoforms when measured by the apo B detection method. A pilot study was conducted to evaluate Lp(a) values obtained by different methods calibrated with a common fresh-frozen serum with a defined apo(a) isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-competitive enzyme-linked immunosorbent assay for human apolipoprotein SAA or S.

Purified antibodies against human apolipoprotein S were used to measure apo S levels in human plasma by a sandwich ELISA technique. The method developed was highly sensitive and specific. A reliable calibration of a plasma standard was obtained by reassociating the purified protein with plasma HDL. A significant correlation (r = 0.69) was found between plasma apo S and CRP values. This correlation coefficient reached 0.97 when the plasma samples were selected according to additional clinical and laboratory criteria.

Some considerations of methodology and standardization of apolipoprotein A-I immunoassays.

Apolipoprotein A-I (apo A-I) is the major protein of high-density lipoprotein. Compelling evidence suggests that measurement of concentrations of apo A-I in serum may be useful in predicting and assessing ischemic heart disease. The following review of methods of isolation, characterization, and assay of apo A-I has been developed to assess the possibility of standardizing apo A-I immunoassays. We consider the properties of apo A-I that have an effect on its quantification, such as self-association, polymorphism, and stability characteristics. We attempt to review critically the various methods presented, but more information about the physicochemical properties of the protein is required before definitive recommendations can be made.

Some considerations of methodology and standardization of apolipoprotein B immunoassays.

Apolipoprotein B, the major protein component of very-low-density (VLDL) and low-density (LDL) lipoproteins in serum, is being widely measured in serum, to help assess risk of cardiovascular disease. Standardization has proven difficult because of the physical and immunochemical heterogeneity of LDL and VLDL, the masking of antigenic determinants of LDL and VLDL, the insolubility of apolipoprotein B, the instability of lipoprotein reference materials, and the multiplicity of analytical methods. Here we have examined the literature and collected current information from lipoprotein scientists to determine the problems--and the ways investigators have attempted to solve them--in preparation, storage, and use of standards and anti-apolipoprotein B sera, analytical procedures and reagents, and the nature of apolipoprotein B in serum. Standardization appears possible with development of stable reference materials that are applicable to the various analytical methods, controlled immunochemical reaction conditions, and establishment of a selected interim reference method for use as a point of reference.