JCL Roundtable: Global Think Tank on Lipoprotein(a).

Lipoprotein(a) operates in causal pathways to promote atherosclerosis, arterial thrombosis, and aortic stenosis. It has been associated with rare cases of nonatherosclerotic arterial thrombotic stroke at any age. Inherited variation of lipoprotein(a) levels substantially increases cardiovascular risk in 20% of people worldwide. Recent progress in identifying the risk associated with lipoprotein(a) and in pursuing effective treatment has led to a recent Global Think Tank including representatives from the European Atherosclerosis Society, American Heart Association, Preventive Cardiovascular Nurses Association, National Lipid Association, and other groups. The need for standardized laboratory measurement in nanomoles per liter met with unanimous consensus. Atherosclerotic risk is linearly associated with plasma lipoprotein(a) levels, so that persons with the highest levels may have risk similar to other severe inherited lipoprotein disorders. Universal once-in-lifetime screening has been recommended by European and Canadian cardiovascular societies, but not by U.S. organizations. Current pharmacologic therapies are limited to 20-30% lowering of lipoprotein(a) levels, and no pharmacologic treatment for lowering lipoprotein(a) has yet been proven to reduce risk in a cardiovascular outcomes trial. Treatment for high-risk patients focuses on reducing low density lipoprotein cholesterol and other risk factors. New therapies targeting messenger RNA for apolipoprotein(a) can achieve 80-90% reduction of lipoprotein(a) levels. One such therapy using a liver-directed antisense oligonucleotide is currently being tested in a large cardiovascular outcomes trial. Increased recognition of lipoprotein(a)-associated risk and emergence of potentially effective therapy together lead to a mandate for a unified global effort on education, standardization, and clinical management.

Development of an LC-MS/MS Proposed Candidate Reference Method for the Standardization of Analytical Methods to Measure Lipoprotein(a).

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.

Analytical Performance Specifications for Lipoprotein(a), Apolipoprotein B-100, and Apolipoprotein A-I Using the Biological Variation Model in the EuBIVAS Population.

BACKGROUND: With increased interest in lipoprotein(a) (Lp[a]) concentration as a target for risk reduction and growing clinical evidence of its impact on cardiovascular disease (CVD) risk, rigorous analytical performance specifications (APS) and accuracy targets for Lp(a) are required. We investigated the biological variation (BV) of Lp(a), and 2 other major biomarkers of CVD, apolipoprotein A-I (apoA-I) and apolipoprotein B-100 (apoB), in the European Biological Variation Study population. METHOD: Serum samples were drawn from 91 healthy individuals for 10 consecutive weeks at 6 European laboratories and analyzed in duplicate on a Roche Cobas 8000 c702. Outlier, homogeneity, and trend analysis were performed, followed by CV-ANOVA to determine BV estimates and their 95% CIs. These estimates were used to calculate APS and reference change values. For Lp(a), BV estimates were determined on normalized concentration quintiles. RESULTS: Within-subject BV estimates were significantly different between sexes for Lp(a) and between women aged <50 and >50 years for apoA-I and apoB. Lp(a) APS was constant across concentration quintiles and, overall, lower than APS based on currently published data, whereas results were similar for apoA-I and apoB. CONCLUSION: Using a fully Biological Variation Data Critical Appraisal Checklist (BIVAC)-compliant protocol, our study data confirm BV estimates of Lp(a) listed in the European Federation of Clinical Chemistry and Laboratory Medicine database and reinforce concerns expressed in recent articles regarding the suitability of older APS recommendations for Lp(a) measurements. Given the heterogeneity of Lp(a), more BIVAC-compliant studies on large numbers of individuals of different ethnic groups would be desirable.

Lipoprotein(a) mass: a massively misunderstood metric.

The importance of lipoprotein (a)-Lp(a)-as a cardiovascular (CV) risk marker has been underscored by recent findings that CV risk is directly related to baseline Lp(a) levels, even in well-treated patients. Although there is currently little that can be done pharmacologically to lower Lp(a) levels, knowledge of its serum concentration is important in overall risk assessment. This review focuses on 1 aspect of Lp(a) that is rarely discussed directly: how to express its levels in serum. There is considerable confusion on this point, and a fuller understanding of what the concentration units mean will help improve study-to-study comparisons and thereby advance our understanding of the pathobiology of this lipoprotein particle. As discussed here, the term Lp(a) mass refers to the entire mass of the particle: lipids, proteins, and carbohydrates combined. At present, there are no commercially available assays that are completely insensitive to the variability in particle mass, which arises not only from differences in apo(a) isoform mass but also from variations in lipid mass. Because lipoprotein "particle number" (molar concentration) has been found to be superior to component-based metrics (ie, low-density lipoprotein particle vs cholesterol concentrations) for CV disease risk prediction, the development of a mass-insensitive Lp(a) assay should be a high priority.

Abnormalities of triglyceride rich lipoproteins (TRLs) in type 2 diabetes and insulin resistance

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Lipoproteínas de alta densidad y riesgo cardiovascular: revisión de las evidencias y de las dudas / High-density lipoproteins and cardiovascular risk: review of the evidence and doubts

Las lipoproteínas de alta densidad (HDL) transportan colesterol desde la periferia hasta el hígado. Los estudios transversales relacionando las concentraciones bajas de colesterol unido a las HDL (cHDL) con una mayor prevalencia de enfermedad coronaria (EC) datan de los años cincuenta del siglo pasado. Posteriores estudios poblacionales establecieron que el cHDL bajo es un predictor independiente de EC, y así se reconoce en las guías clínicas de prevención cardiovascular. Muchas publicaciones, pero no todas, han establecido una correlación inversa entre incidencia de ictus isquémicos, mortales o no. La proteína transferidora de ésteres de colesterol (CETP) intercambia cHDL por triglicéridos de lipoproteínas de muy baja densidad. Algunas familias con trastornos genéticos de CETP tienen cHDL elevados y menor incidencia de EC. Posteriores estudios observacionales, aunque no todos, han mostrado que sujetos con las anomalías funcionales de CETP tienen cHDL elevado y menor incidencia de EC. Eso ha despertado interés por la inhibición de CETP como intervención para reducir la enfermedad coronaria (AU)

High-density lipoproteins (HDL) transport cholesterol from the periphery to the liver. Cross-sectional studies relating low HDL-cholesterol (HDL-c) concentrations to a higher prevalence of cardiovascular disease (CVD) date back to the 1950s. Subsequent populationbased studies established that low HDL-c levels are an independent predictor of CVD, a finding that is recognized in clinical guidelines for cardiovascular prevention. Many publications, although not all, have established an inverse correlation between the incidence of ischemic stroke, whether fatal or non-fatal, and HDL-c. Cholesteryl ester transfer protein (CETP) facilitates the exchange of triglyceride (for cholesteryl ester) from very low density lipoprotein (VLDL) particles to HDL particles. Some families with genetic CETP alterations have high HDL-c concentrations and a lower incidence of CVD. Some observational studies, but not all, have shown that persons with functional CETP anomalies have high HDL-c levels and a lower incidence of CVD. This observation has prompted interest in CETP inhibition as an intervention to reduce coronary heart disease (AU)

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.

Successful utilization of lyophilized lipoprotein(a) as a biological reagent.

Lipoprotein(a) [Lp(a)] represents a class of lipoprotein particles having as a protein moiety apoB-100 linked by a single disulfide bond to apolipoprotein(a) [apo(a)], a multikringle structure with a high degree of homology with plasminogen. A recognized feature of Lp(a) is its instability on storage caused by attendant protein and lipid modifications that affect the structural, functional, and immunological properties of this lipoprotein. Here we present data showing that, under appropriate conditions of cryopreservation, Lp(a) retains the properties of the freshly isolated product, and we provide examples supporting the stability of this cryopreserved product as a primary standard in immunoassay settings and in cell culture systems.

Standardization of immunoassays for measurement of high-sensitivity C-reactive protein. Phase I: evaluation of secondary reference materials.

BACKGROUND: Inflammation contributes to the development and progression of atherosclerosis, and C-reactive protein (CRP) can be used as a marker to assess risk for cardiovascular diseases. As variability among existing high-sensitivity CRP (hsCRP) assays can lead to misclassification of patients and hamper implementation of population-based medical decision points, standardization of hsCRP assays is needed. METHODS: We evaluated five proposed secondary reference materials, including two diluted preparations of Certified Reference Material 470 (CRM470), two preparations of a serum-based material with recombinant CRP added, and one serum-based material with isolated CRP added. Twenty-one manufacturers participated in the comparison with 28 different assays. We examined imprecision, linearity, and parallelism with these materials and with fresh serum. RESULTS: All materials had similar imprecision; CVs for the undiluted materials were 2.1-3.7%. None of the materials was linear across all assays. Each had between one and three cases of nonlinearity, with one preparation of CRM470 having the fewest cases of nonlinearity. Although none of the materials was parallel across all assays, the differences in slope from fresh serum were similar across all assays. CONCLUSIONS: All materials performed similarly with regard to imprecision, linearity, and parallelism. As one preparation of CRM470 had slightly better characteristics than the other materials and because CRM470 had been certified previously as a reference material for the acute-phase reactant range, it will be used in the next phase to standardize hsCRP assays.

Use of a reference material proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to evaluate analytical methods for the determination of plasma lipoprotein(a).

BACKGROUND: As part of the NIH/National Heart, Lung and Blood Institute Contract for the Standardization of Lipoprotein(a) [Lp(a)] Measurements, a study was performed in collaboration with the IFCC Working Group for the Standardization of Lp(a) Assays. The aims of the study, performed with the participation of 16 manufacturers and 6 research laboratories, were to evaluate the IFCC proposed reference material (PRM) for its ability to transfer an accuracy-based value to the immunoassay calibrators and to assess concordance in results among different methods. METHODS: Two different purified Lp(a) preparations with protein mass concentrations determined by amino acid analysis were used to calibrate the reference method. A Lp(a) value of 107 nmol/L was assigned to PRM. After uniformity of calibration was demonstrated in the 22 evaluated systems, Lp(a) was measured on 30 fresh-frozen sera covering a wide range of Lp(a) values and apolipoprotein(a) [apo(a)] sizes. RESULTS: The among-laboratory CVs for these samples (6-31%) were, in general, higher than those obtained for PRM (2.8%) and the quality-control samples (14%, 12%, and 9%, respectively), reflecting the broad range of apo(a) sizes in the 30 samples and the sensitivity of most methods to apo(a) size heterogeneity. Thus, although all of the assays were uniformly calibrated through the use of PRM, no uniformity in results was achieved for the isoform-sensitive methods. CONCLUSIONS: Linear regression analyses indicated that to various degrees, apo(a) size heterogeneity affects the outcome of the immunochemical methods used to measure Lp(a). We have also shown that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease.

Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard.

LP[a] is one of the most atherogenic lipoproteins consisting of an LDL-like core particle and a covalently linked glycoprotein of variable size. Due to its structural features, its heterogeneity and instability, there are great difficulties in standardizing quantitative immunochemical Lp[a] assays. One particular problem is the preparation of a pure primary standard, which is sufficiently stable to be used for value assignment of secondary reference material. Here we describe a method to purify Lp[a] to virtual homogeneity. When mixed with glycerol at a ratio of 1:1, the preparation is stable in the deep frozen state for more than 12 months. This latter material gave dose;-response curves in several immunochemical assays that were parallel to fresh or frozen sera, freshly prepared Lp[a], and other proposed reference materials. After determination of the protein content by amino acid analysis, it was possible to assign concentrations in molar and mass units to these preparations considering the theoretical molecular weights of the particular apo[a] isoform. Thus we propose to use this procedure for preparation of a "gold standard" for Lp[a] assays.

International Federation of Clinical Chemistry standardization project for the measurement of lipoprotein(a). Phase I. Evaluation of the analytical performance of lipoprotein(a) assay systems and commercial calibrators.

A secondary reference material for lipoprotein(a) is required to standardize the measurement of lipoprotein(a) in clinical laboratories worldwide. Towards this aim, the International Federation of Clinical Chemistry Working Group for the Standardization of Lipoprotein(a) Assays has initiated a standardization project involving a total of 33 diagnostic company and clinical chemistry laboratories from 12 countries. In Phase 1, the analytical performance of 40 lipoprotein(a) assay systems was evaluated by testing sera and manufactured lipoprotein(a) calibrator materials for precision, linearity, and parallelism. Twenty test systems were nonoptimized according to the results for a pooled serum, which tested nonlinear in 16 systems and imprecise in 4. Acceptable analytical properties and harmonization of lipoprotein(a) values were shown by some commercial calibrators, suggesting their possible use as reference materials. This study highlights the problems that currently occur for lipoprotein(a) measurement in existing assay systems.

Standardization and clinical management of lipoprotein(a) measurements.

The present article proposes personal suggestions to improve determinations and clinical interpretation of results of lipoprotein(a) assays. Methods and procedures for sampling and quantification of the various isoforms of lipoprotein(a) in serum, plasma and urine are reviewed with the aim of improving the reliability and reproducibility of results and reinforcing the clinical utility of lipoprotein(a) measurements.

Standardization of Lp(a) measurements.

The standardization of immunoassays for Lp(a) is a major challenge to clinical chemists. In order to establish a reference material acknowledged by the International Federation of Clinical Chemistry, the Center of Disease Control and possibly the World Health Organization, a working group with participants from four continents was put together. With the aid of 34 companies, eight proposed reference materials have been tested in the last 3 years and two of them have been selected for value assignment. A reference method based on dissociation-enhanced lanthanide fluorescence immunoassays was therefore developed which gives linear and parallel response curves by assaying freshly prepared primary reference standards, fresh plasma or serum as well as lyophilized or frozen proposed reference material. For value assignment, four laboratories simultaneously prepare primary reference standards with known isoforms and molecular weights. By assaying the amino acid composition of these primary reference standards, the molar concentration which is the basis of value transfer to the lyophilized proposed reference material can be calculated. In a final step, harmonization of all commercially available Lp(a) kits is to be tested by assaying 50 Lp(a) samples with increasing Lp(a) concentrations and varying isoforms. We hope to be able in the near future to create a basis for comparable results in epidemiological studies in different laboratories and also to help to improve future long-term precision in clinical chemical laboratories.

Lipoprotein(a) in health and disease.

Lipoprotein(a) [Lp(a)] represents an LDL-like particle to which the Lp(a)-specific apolipoprotein(a) is linked via a disulfide bridge. It has gained considerable interest as a genetically determined risk factor for atherosclerotic vascular disease. Several studies have described a correlation between elevated Lp(a) plasma levels and coronary heart disease, stroke, and peripheral atherosclerosis. In healthy individuals, Lp(a) plasma concentrations are almost exclusively controlled by the apo(a) gene locus on chromosome 6q2.6-q2.7. More than 30 alleles at this highly polymorphic gene locus determine a size polymorphism of apo(a). There exists an inverse correlation between the size (molecular weight) of apo(a) isoforms and Lp(a) plasma concentrations. The standardization of Lp(a) quantification is still an unresolved task due to the large particle size of Lp(a), the presence of two different apoproteins [apoB and apo(a)], and the large size polymorphism of apo(a) and its homology with plasminogen. A working group sponsored by the IFCC is currently establishing a stable reference standard for Lp(a) as well as a reference method for quantitative analysis. Aside from genetic reasons, abnormal Lp(a) plasma concentrations are observed as secondary to various diseases. Lp(a) plasma levels are elevated over controls in patients with nephrotic syndrome and patients with end-stage renal disease. Following renal transplantation, Lp(a) concentrations decrease to values observed in controls matched for apo(a) type. Controversial data on Lp(a) in diabetes mellitus result mainly from insufficient sample sizes of numerous studies. Large studies and those including apo(a) phenotype analysis came to the conclusion that Lp(a) levels are not or only moderately elevated in insulin-dependent patients. In noninsulin-dependent diabetics, Lp(a) is not elevated. Conflicting data also exist from studies in patients with familial hypercholesterolemia. Several case-control studies reported elevated Lp(a) levels in those patients, suggesting a role of the LDL-receptor pathway for degradation of Lp(a). However, recent turnover studies rejected that concept. Moreover, family studies also revealed data arguing against an influence of the LDL receptor for Lp(a) concentrations. Several rare diseases or disorders, such as LCAT- and LPL-deficiency as well as liver diseases, are associated with low plasma levels or lack of Lp(a).

Significant reduction of the bias among commercial immunoassays for lipoprotein(a) after use of a uniform calibrator.

Despite the increasing interest in the measurements of lipoprotein(a) (Lp(a) in serum or plasma, at present there is no effective standardization for Lp(a) assays; the main problems to solve are represented either by the lack of a suitable primary standard or by the absence of a reliable and widely available reference method. A first step is hence the uniformity of calibration of different immunoassays. We calibrated three commercial immunoassays for Lp(a) (enzyme linked immunosorbent assay (ELISA), latex-enhanced immunonephelometric assay (LINA), and immunonephelometric assay (INA) with either proprietary standards or purified Lp(a) material obtained with a rapid and simple procedure. Final results of purified Lp(a) calibration were reported in terms of protein Lp(a) mass whereas we were able to quantify the exact protein concentration of our purified lipoprotein. The uniformity of the calibration of the different assays led to a significant improvement of regression slopes (from 1.88 to 0.90 ELISA vs. LINA, from 1.45 to 0.95 ELISA vs. INA and from 1.27 to 0.96 INA vs. LINA) and correlation coefficients (from 0.990 to 0.994 ELISA vs. LINA, from 0.987 to 0.990 ELISA vs. INA and from 0.985 to 0.987 INA vs. LINA). Furthermore, the significant differences among Lp(a) values obtained after calibration with proprietary standards were minimized, becoming non-significant in two out of three cases. In conclusion, we demonstrated that a better agreement of Lp(a) values obtained with different commercial assays could be simply reached by uniformity of calibration and by employing standards with values accurately measured.

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)

International Lp(a) standardization.

Two international surveys for Lp(a) measurements were organized from 1989 to 1991. The results of the first survey led to the conclusion that the lack of a common primary standard was the main cause of the large inter-laboratory variation observed. No major effects of techniques or antisera were observed. The same findings were confirmed during the second survey, which was extended to include more samples and a larger number of participants. During the second survey, no consistent effect due to freezing or lyophilization could be demonstrated, although there was a trend towards lower Lp(a) values in lyophilized samples. The inter- and intra-laboratory coefficients of variation did not vary significantly for the different Lp(a) phenotypes, and variability was comparable for lyophilized, liquid and frozen materials. Large intra-assay coefficients of variation were observed during both surveys. Results obtained in different laboratories using the same commercial reagents and standards also showed a large variation. These initial results demonstrate that the lack of a primary standard and poor assay precision are the main factors responsible for the high inter-laboratory variation observed during these surveys.