Closing the gaps in patient management of dyslipidemia: stepping into cardiovascular precision diagnostics with apolipoprotein profiling.

In persons with dyslipidemia, a high residual risk of cardiovascular disease remains despite lipid lowering therapy. Current cardiovascular risk prediction mainly focuses on low-density lipoprotein cholesterol (LDL-c) levels, neglecting other contributing risk factors. Moreover, the efficacy of LDL-c lowering by statins resulting in reduced cardiovascular risk is only partially effective. Secondly, from a metrological viewpoint LDL-c falls short as a reliable measurand. Both direct and calculated LDL-c tests produce inaccurate test results at the low end under aggressive lipid lowering therapy. As LDL-c tests underperform both clinically and metrologically, there is an urging need for molecularly defined biomarkers. Over the years, apolipoproteins have emerged as promising biomarkers in the context of cardiovascular disease as they are the functional workhorses in lipid metabolism. Among these, apolipoprotein B (ApoB), present on all atherogenic lipoprotein particles, has demonstrated to clinically outperform LDL-c. Other apolipoproteins, such as Apo(a) - the characteristic apolipoprotein of the emerging risk factor lipoprotein(a) -, and ApoC-III - an inhibitor of triglyceride-rich lipoprotein clearance -, have attracted attention as well. To support personalized medicine, we need to move to molecularly defined risk markers, like the apolipoproteins. Molecularly defined diagnosis and molecularly targeted therapy require molecularly measured biomarkers. This review provides a summary of the scientific validity and (patho)physiological role of nine serum apolipoproteins, Apo(a), ApoB, ApoC-I, ApoC-II, ApoC-III, ApoE and its phenotypes, ApoA-I, ApoA-II, and ApoA-IV, in lipid metabolism, their association with cardiovascular disease, and their potential as cardiovascular risk markers when measured in a multiplex apolipoprotein panel.

Biochemical risk factors of atherosclerotic cardiovascular disease: from a narrow and controversial approach to an integral approach and precision medicine.

INTRODUCTION: Guidelines of management of dyslipidemias and prevention of cardiovascular disease (CVD) are based on firm scientific evidence obtained by randomized controlled trials (RCTs). However, the role of elevated low-density lipoprotein-cholesterol (LDL-C)as a risk factor of CVD and therapies to lower LDL-C are frequently disputed by colleagues who disagree with the conclusions of the RCTs published. This review focuses on this dispute, and evaluates the current approach of management of dyslipidemias and CVD prevention to find modern alternatives for more precise diagnosis and therapy of dyslipidemic patients. AREAS COVERED: Recent interest in lipoprotein(a) (Lp(a)) and remnants lipoproteins and in therapies that do not influence LDL-C levels primarily, such as anti-inflammatory drugs and icosapent ethyl, has revitalized our concern to optimize the care for patients with increased CVD risk without focusing simply on reduction of LDL-C by therapy with statins, ezitemibe, and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors. EXPERT OPINION: The limited characterization of study populations by measurement of total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C) and triglycerides (TG) followed by measurement or calculation of LDL-C should be extended by a more integral approach in order to realize precision diagnostics and precision medicine, for the sake of personalized patient care.

Association of apolipoproteins C-I, C-II, C-III and E with coagulation markers and venous thromboembolism risk.

PURPOSE: Apolipoproteins C-I, C-II, C-III and E have been associated with risk of arterial thrombotic diseases. We investigated whether these apolipoproteins have prothrombotic properties and are associated with risk of venous thromboembolism (VTE). PATIENTS AND METHODS: A total of 127 VTE patients and 299 controls were randomly selected from the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis study (1999-2004), in the Netherlands. The apolipoproteins were quantified using mass spectrometry (LC/MS/MS), and their levels were analyzed as continuous variable (per SD increase). RESULTS: In controls, increases in levels of apolipoproteins were associated with increases in levels of vitamin K-dependent factors, factor XI, antithrombin and clot lysis time. Additionally, increasing apolipoproteins C-III and E levels were associated with higher factor VIII and von Willebrand factor levels. Levels of C-reactive protein were not associated with any apolipoprotein. The age- and sex-adjusted odds ratios of apolipoproteins E, C-III, CII and CI to the risk of venous thrombosis were 1.21 (95% CI, 0.98-1.49), 1.19 (95% CI, 0.99-1.44), 1.24 (95% CI, 0.95-1.61) and 1.06 (95% CI, 0.87-1.30) per SD increase, respectively. These odds ratios did not attenuate after adjustments for statin use, estrogen use, BMI, alcohol use, and self-reported diabetes. CONCLUSIONS: Levels of apolipoproteins C-I, C-II, C-III and E are associated with those of several coagulation factors. However, whether these apolipoproteins are also associated with an increased risk of VTE remains to be established.

Apolipoprotein profiling as a personalized approach to the diagnosis and treatment of dyslipidaemia.

An elevated low-density lipoprotein cholesterol concentration is a classical risk factor for cardiovascular disease. This has led to pharmacotherapy in patients with atherosclerotic heart disease or high heart disease risk with statins to reduce serum low-density lipoprotein cholesterol. Even in patients in whom the target levels of low-density lipoprotein cholesterol are reached, there remains a significant residual cardiovascular risk; this is due, in part, to a focus on low-density lipoprotein cholesterol alone and neglect of other important aspects of lipoprotein metabolism. A more refined lipoprotein analysis will provide additional information on the accumulation of very low-density lipoproteins, intermediate density lipoproteins, chylomicrons, chylomicron-remnants and Lp(a) concentrations. Instead of measuring the cholesterol and triglyceride content of the lipoproteins, measurement of their apolipoproteins (apos) is more informative. Apos are either specific for a particular lipoprotein or for a group of lipoproteins. In particular measurement of apos in atherogenic particles is more biologically meaningful than the measurement of the cholesterol concentration contained in these particles. Applying apo profiling will not only improve characterization of the lipoprotein abnormality, but will also improve definition of therapeutic targets. Apo profiling aligns with the concept of precision medicine by which an individual patient is not treated as 'average' patient by the average (dose of) therapy. This concept of precision medicine fits the unmet clinical need for stratified cardiovascular medicine. The requirements for clinical application of proteomics, including apo profiling, can now be met using robust mass spectrometry technology which offers desirable analytical performance and standardization.

Growth Differentiation Factor-15 Levels at Admission Provide Incremental Prognostic Information on All-Cause Long-term Mortality in ST-Segment Elevation Myocardial Infarction Patients Treated with Primary Percutaneous Coronary Intervention.

INTRODUCTION: To investigate the additive prognostic value of growth differentiation factor (GDF-15) levels in ST-segment elevation myocardial infarction (STEMI) patients treated with primary percutaneously coronary intervention (pPCI) with 10-year mortality on top of clinical characteristics and known cardiac biomarkers. METHODS: Baseline serum GDF-15 levels were measured in 290 STEMI patients treated with pPCI in the MISSION! intervention trial conducted from February 1, 2004 through October 31, 2006. The incremental prognostic value of GDF-15 and NTproBNP levels was evaluated on top of clinical characteristics using Cox proportional hazards analysis, Chi-square models and C-index. Outcome was 10-year all-cause mortality. RESULTS: Mean age was 59.0 ± 11.5 years and 65 (22.4) patients were female. A total of 37 patients died during a follow-up of 9.4 (IQR 8.8-10.0) years. Multivariable Cox regression revealed GDF-15 and NTproBNP levels above median to be independently associated with 10-year all-cause mortality [HR GDF-15, 2.453 (95% CI 1.064-5.658), P = 0.04; HR NTproBNP, 2.413 (95% CI 1.043-5.564), P = 0.04] after correction for other clinical variables. Stratified by median GDF-15 (37.78 pmol/L) and NTproBNP (11.74 pmol/L) levels, Kaplan-Meier curves showed significant better survival for patients with GDF-15 and NTproBNP levels below the median versus above the median. The likelihood ratio test showed a significant incremental value of GDF-15 (P = 0.03) as compared with a model with clinically important variables and NTproBNP. The C-statistics for this model improved from 0.82 to 0.84 when adding GDF-15. CONCLUSION: GDF-15 levels at admission in STEMI patients are independently associated with 10-year all-cause mortality rates and could improve risk stratification on top of clinical variables and other cardiac biomarkers.

Plasma LDL-Cholesterol Level at Admission is Independently Associated with Infarct Size in Patients with ST-Segment Elevation Myocardial Infarction Treated with Primary Percutaneous Coronary Intervention.

INTRODUCTION: Hypercholesterolemia is a well-known risk factor for developing atherosclerosis and subsequently for the risk of a myocardial infarction (MI). Moreover, it might also be related to the extent of damaged myocardium in the event of a MI. The aim of this study was to evaluate the association of baseline low density lipoprotein-cholesterol (LDL-c) level with infarct size in patients with ST-segment elevation myocardial infarction (STEMI) after primary percutaneously coronary intervention (pPCI). METHODS: Baseline blood samples were obtained from all patients admitted between 2004 and 2014 with STEMI who underwent pPCI. Patients were excluded in case of out of hospital cardiac arrest, treatment delay of at least 10 h or no complete reperfusion after pPCI in the culprit vessel. Peak creatine kinase (CK) level was used for infarct size estimation, defined as the maximal value during admission. RESULTS: A total of 2248 patients were included in this study (mean age 61.8 ± 12.2 years; 25.0% female). Mean LDL-c level was 3.6 ± 1.1 mmol/L and median peak CK level was 1275 U/L (IQR 564-2590 U/L). Baseline LDL-c level [ß = 0.041; (95% CI 0.019-0.062); p < 0.001] was independently associated with peak CK level. Furthermore, left anterior descending artery as culprit vessel, initial TIMI 0-1 flow in the culprit vessel, male gender, and treatment delay were also correlated with high peak CK level (p < 0.05). Prior aspirin therapy was associated with lower peak CK level [ß = - 0.073 (95% CI - 0.146 to 0.000), p = 0.050]. CONCLUSION: This study demonstrates that besides the more established predictors of infarct size, elevated LDL-c is associated with augmented infarct size in patients with STEMI treated with pPCI.

Apolipoproteins A1, B, and apoB/apoA1 ratio are associated with first ST-segment elevation myocardial infarction but not with recurrent events during long-term follow-up.

INTRODUCTION: The current way to assess the risk of cardiovascular disease (CVD) is to measure conventional lipid and lipoprotein cholesterol fractions. Despite the success of statin treatment, residual cardiovascular risk remains high. Therefore, the value of extensive serum apolipoprotein (apo) profiling to assess the risk of ST-segment elevation myocardial infarction (STEMI) and of major adverse cardiac events (MACE) in patients with STEMI was investigated in a case-control design. METHODS AND RESULTS: Serum apo levels were measured using liquid chromatography and mass spectrometry in 299 healthy individuals and 220 patients with STEMI. First, the association of apo profiles in baseline samples with risk of STEMI was examined, and second, the association of apo profiles at baseline with risk of recurrent MACE in patients with STEMI in a longitudinal study design was studied. High baseline (> 1.25 g/L) apoA1 levels were associated with a decreased risk of STEMI [odds ratio (OR) 0.17; 95% CI 0.11-0.26], whereas high apoB (> 1.00 g/L) levels (OR 2.17; 95% CI 1.40-3.36) and apoB/apoA1 ratio (OR per 1 SD (OR/SD): 2.16; 95% CI 1.76-2.65) were associated with an increased risk. Very-low-density-lipoprotein (VLDL)-associated apos gave conflicting results. Neither conventional lipid levels nor apo levels were associated with MACE in the STEMI group. CONCLUSION: In conclusion, apoA1, apoB, and apoB/apoA1 were strongly associated with risk of STEMI. No clear relation between VLDL-associated apos and the risk of STEMI was found. Neither baseline serum apos nor lipids predicted MACE in statin-treated patients during long-term follow-up after a first STEMI.

A Rapid (Differential) Effect of Rosuvastatin and Atorvastatin on High-Sensitivity Cardiac Troponin-I in Subjects With Stable Cardiovascular Disease.

Serum troponin within the normal range is an emerging predictor of cardiovascular mortality. We aimed to determine how rapidly high-sensitivity troponin-I (hs-cTnI) levels are lowered by statin therapy in patients with stable cardiovascular disease. In the RADAR substudy, patients were randomized to atorvastatin 20 mg/day (n = 39) or rosuvastatin 10 mg/day (n = 39) and up-titrated at 6-week intervals to 80 mg of atorvastatin or 40 mg of rosuvastatin. Hs-cTnI concentrations were measured at baseline and at 6 and 18 weeks of follow-up. Statin treatment resulted in a mean change of serum hs-cTnI of -8.2% (P = 0.010) after 6 weeks and -12.3% (P = 0.001) after 18 weeks. After 18 weeks, hs-cTnI levels were lowered by 21.8% with atorvastatin and by 4.1% with rosuvastatin (P = 0.001 and P = 0.133, respectively). During statin therapy, serum hs-cTnI levels decreased rapidly within weeks of treatment, suggesting an effect beyond long-term atherosclerosis regression. Mechanisms that mediate this effect require further study.

RGDfK-Peptide Modified Alginate Scaffold for Cell Transplantation and Cardiac Neovascularization.

Cell implantation for tissue repair is a promising new therapeutic strategy. Although direct injection of cells into tissue is appealing, cell viability and retention are not very good. Cell engraftment and survival following implantation are dependent on a sufficient supply of oxygen and nutrients through functional microcirculation as well as a suitable local microenvironment for implanted cells. In this study, we describe the development of a porous, biocompatible, three-dimensional (3D) alginate scaffold covalently modified with the synthetic cyclic RGDfK (Arg-Gly-Asp-D-Phe-Lys) peptide. Cyclic RGDfK peptide is protease resistant, highly stable in aqueous solutions, and has high affinity for cellular integrins. Cyclic RGDfK-modified alginate scaffolds were generated using a novel silicone sheet sandwich technique in combination with freeze-gelation, resulting in highly porous nonimmunogenic scaffolds that promoted both human and rodent cell survival in vitro, and neoangiogenesis in vivo. Two months following implantation in abdominal rectus muscles in rats, cyclic RGDfK-modified scaffolds were fully populated by host cells, especially microvasculature without an overt immune response or fibrosis, whereas unmodified control scaffolds did not show cell ingrowth. Importantly, modified scaffolds that were seeded with human mesenchymal precursor cells and were patched to the epicardial surface of infarcted myocardium induced myocardial neoangiogenesis and significantly improved cardiac function. In summary, purified cyclic RGDfK peptide-modified 3D alginate scaffolds are biocompatible and nonimmunogenic, enhance cell viability, promote angiogenesis, and may be used as a means to deliver cells to myocardial infarct areas to improve neovascularization and cardiac function.

Low levels of apolipoprotein-CII in normotriglyceridemic patients with very premature coronary artery disease: Observations from the MISSION! Intervention study.

BACKGROUND: While the overall acute myocardial infarction rates declined in women and men, premature acute myocardial infarction rates remained stable in men and increased in women. OBJECTIVE: The purpose of this study was to assess whether baseline apolipoprotein (apo) levels, clinical characteristics, and follow-up of patients with very premature coronary artery disease (CAD) could provide novel clues for the identification of high-risk individuals. METHODS: Apos were measured with a validated quantification liquid chromatography-mass spectrometry method in a well-defined cohort of 38 patients aged ≤45 years admitted with acute ST-segment elevation myocardial infarction. RESULTS: Mean age was 39.8 ± 4.6 years and 24% was female. Four of these patients (11%) had apoCII levels ≤5.0 mg/L. Compared with the very premature CAD group with apoCII > 5 mg/L, the patients with apoCII levels ≤5.0 mg/L were all females, tended to be younger (35.8 ± 8.4 years vs 40.3 ± 3.9 years, P = .063), had more often a family history of cardiovascular disease ≤65 years (P = .034) and a significantly lower Framingham risk score (P = .001). They presented with normal triglyceride levels, and had lower low-density lipoprotein cholesterol, apoB100, and apoE levels. Corrected for differences in risk profile, apoCII ≤ 5 mg/L was associated with increased risk of 10-years reinfarction or revascularization (hazard ratio 7.9 [95% confidence interval 1.5-41.6], P = .015). CONCLUSIONS: In 38 patients with very premature CAD, 11% were found to have low apoCII levels (≤5.0 mg/L) with normal triglyceride levels. Despite their low a priori risk for CAD, these patients presented with ST-segment elevation myocardial infarction and had a high relative risk of 10-year reinfarction or revascularization. This particular phenotype of relatively young female patients with CAD is not recognized earlier and deserves further study.

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds.

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 µg/g, and RNA by 61% down to less than 10 µg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria.

Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping.

BACKGROUND: Direct and calculated measures of lipoprotein fractions for cardiovascular risk assessment suffer from analytical inaccuracy in certain dyslipidemic and pathological states, most commonly hypertriglyceridemia. LC-MS/MS has proven suitable for multiplexed quantification and phenotyping of apolipoproteins. We developed and provisionally validated an automated assay for quantification of apolipoprotein (apo) A-I, B, C-I, C-II, C-III, and E and simultaneous qualitative assessment of apoE phenotypes. METHODS: We used 5 value-assigned human serum pools for external calibration. Serum proteins were denatured, reduced, and alkylated according to standard mass spectrometry-based proteomics procedures. After trypsin digestion, peptides were analyzed by LC-MS/MS. For each peptide, we measured 2 transitions. We compared LC-MS/MS results to those obtained by an immunoturbidimetric assay or ELISA. RESULTS: Intraassay CVs were 2.3%-5.5%, and total CVs were 2.5%-5.9%. The LC-MS/MS assay correlated (R = 0.975-0.995) with immunoturbidimetric assays with Conformité Européenne marking for apoA-I, apoB, apoC-II, apoC-III, and apoE in normotriglyceridemic (n = 54) and hypertriglyceridemic (n = 46) sera. Results were interchangeable for apoA-I ≤3.0 g/L (Deming slope 1.014) and for apoB-100 ≤1.8 g/L (Deming slope 1.016) and were traceable to higher-order standards. CONCLUSIONS: The multiplex format provides an opportunity for new diagnostic and pathophysiologic insights into types of dyslipidemia and allows a more personalized approach for diagnosis and treatment of lipid abnormalities.

Serum Cardiac Troponin-I is Superior to Troponin-T as a Marker for Left Ventricular Dysfunction in Clinically Stable Patients with End-Stage Renal Disease.

BACKGROUND: Serum troponin assays, widely used to detect acute cardiac ischemia, might be useful biomarkers to detect chronic cardiovascular disease (CVD). Cardiac-specific troponin-I (cTnI) and troponin-T (cTnT) generally detect myocardial necrosis equally well. In dialysis patients however, serum cTnT levels are often elevated, unlike cTnI levels. The present study aims to elucidate the associations of cTnI and cTnT with CVD in clinically stable dialysis patients. METHODS: Troponin levels were measured using 5th generation hs-cTnT assays (Roche) and STAT hs-cTnI assays (Abbott) in a cohort of dialysis patients. Serum troponin levels were divided into tertiles with the lowest tertile as a reference value. Serum troponins were associated with indicators of CVD such as left ventricular mass index (LVMI), left ventricular ejection fraction (LVEF) and the presence of coronary artery disease (CAD). Associations were explored using regression analysis. RESULTS: We included 154 consecutive patients, 68±7 years old, 77% male, 70% hemodialysis. Median serum cTnT was 51ng/L (exceeding the 99th percentile of the healthy population in 98%) and median serum cTnI was 13ng/L (elevated in 20%). A high cTnI (T3) was significantly associated with a higher LVMI (Beta 31.60; p=0.001) and LVEF (Beta -4.78; p=0.005) after adjusting for confounders whereas a high serum cTnT was not. CAD was significantly associated with a high cTnT (OR 4.70 p=0.02) but not with a high cTnI. Unlike cTnI, cTnT was associated with residual renal function (Beta:-0.09; p=0.006). CONCLUSION: In the present cohort, serum cTnI levels showed a stronger association with LVMI and LVEF than cTnT. However, cTnT was significantly associated with CAD and residual renal function, unlike cTnI. Therefore, cTnI seems to be superior to cTnT as a marker of left ventricular dysfunction in asymptomatic dialysis patients, while cTnT might be better suited to detect CAD in these patients.

Blood Leukocyte Count on Admission Predicts Cardiovascular Events in Patients with Acute Non-ST Elevation Myocardial Infarction.

We aim to test the hypothesis that blood leukocyte count adds prognostic information in patients with acute non-ST-elevation myocardial infarction (non-STEMI). A total of 585 patients with acute non-STEMI (thrombolysis in myocardial infarction risk score ≥ 3) were enrolled in this cohort retrospective study. Blood leukocyte count was measured immediately after admission in the emergency department. The composite of death, reinfarction, urgent revascularization, and stroke during hospitalization were defined as the primary end point of the study. The mean age of the patients was 61 ± 9.6 years and most of them were male (79%). Using multivariate Cox regression analysis involving seven variables (history of smoking, hypertension, heart rate > 100 beats/minute, serum creatinine level > 1.5 mg/dL, blood leukocyte count > 11,000/µL, use of ß-blocker, and use of angiotensin-converting enzyme inhibitor), leukocyte count > 11,000/µL demonstrated to be a strong predictor of the primary end point (hazard ratio = 3.028; 95% confidence interval = 1.69-5.40, p < 0.001). The high blood leukocyte count on admission is an independent predictor of cardiovascular events in patients with acute non-STEMI.

Stem and progenitor cell therapy for pulmonary arterial hypertension: effects on the right ventricle (2013 Grover Conference Series).

In experimental animals and in patients with pulmonary arterial hypertension (PAH), a wide spectrum of structural and functional conditions is known that may be responsible for the switch of a state of "compensated" right ventricular (RV) hypertrophy to a state of RV failure. In recent years, therapy with differentiated cells, endothelial progenitor cells, and mesenchymal stem cells has been shown to cause partial or complete reversal of pathological characteristics of PAH. The therapeutic effects of stem or progenitor cell therapy are considered to be (1) paracrine effects from stem or progenitor cells that had engrafted in the myocardium (or elsewhere), by compounds that have anti-inflammatory, antiapoptotic, and proangiogenic actions and (2) unloading effects on the right ventricle due to stem or progenitor cell-induced decrease in pulmonary vascular resistance and decrease in pulmonary artery pressure.

Quantifying protein measurands by peptide measurements: where do errors arise?

Clinically actionable quantification of protein biomarkers by mass spectrometry (MS) requires analytical performance in concordance with quality specifications for diagnostic tests. Laboratory-developed tests should, therefore, be validated in accordance with EN ISO 15189:2012 guidelines for medical laboratories to demonstrate competence and traceability along the entire workflow, including the selected standardization strategy and the phases before, during, and after proteolysis. In this study, bias and imprecision of a previously developed MS method for quantification of serum apolipoproteins A-I (Apo A-I) and B (Apo B) were thoroughly validated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP15-A2 and EP09-A3, using 100 patient sera and either stable-isotope labeled (SIL) peptides or SIL-Apo A-I as internal standard. The systematic overview of error components assigned sample preparation before the first 4 h of proteolysis as major source (∼85%) of within-sample imprecision without external calibration. No improvement in imprecision was observed with the use of SIL-Apo A-I instead of SIL-peptides. On the contrary, when the use of SIL-Apo A-I was combined with external calibration, imprecision improved significantly (from ∼9% to ∼6%) as a result of the normalization for matrix effects on linearity. A between-sample validation of bias in 100 patient sera further supported the presence of matrix effects on digestion completeness and additionally demonstrated specimen-specific biases associated with modified peptide sequences or alterations in protease activity. In conclusion, the presented overview of bias and imprecision components contributes to a better understanding of the sources of errors in MS-based protein quantification and provides valuable recommendations to assess and control analytical quality in concordance with the requirements for clinical use.

Impact of Timing of Eptifibatide Administration on Preprocedural Infarct-Related Artery Patency in Acute STEMI Patients Undergoing Primary PCI.

The appropriate timing of eptifibatide initiation for acute ST segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PCI) remains unclear. This study aimed to analyze the impact of timing of eptifibatide administration on infarct-related artery (IRA) patency in STEMI patients undergoing primary PCI. Acute STEMI patients who underwent primary PCI (n = 324) were enrolled in this retrospective study; 164 patients received eptifibatide bolus ≤ 30 minutes after emergency department (ED) admission (group A) and 160 patients received eptifibatide bolus > 30 minutes after ED admission (group B). The primary endpoint was preprocedural IRA patency. Most patients in group A (90%) and group B (89%) were late presenters (> 2 hours after symptom onset). The two groups had similar preprocedural thrombolysis in myocardial infarction 2 or 3 flow of the IRA (26 vs. 24%, p = not significant [NS]), similar creatine kinase-MB (CK-MB) levels at 8 hours after admission (339 vs. 281 U/L, p = NS), similar left ventricular ejection fraction (LVEF) (52 vs. 50%, p = NS), and similar 30-day mortality (2 vs. 7%, p = NS). Compared with group B, patients in group A had shorter door-to-device time (p < 0.001) and shorter procedural time (p = 0.004), without increased bleeding risk (13 vs. 18%, p = NS). Earlier intravenous administration of eptifibatide before primary PCI did not improve preprocedural IRA patency, CK-MB level at 8 hours after admission, LVEF and 30-day mortality compared with patients who received intravenous eptifibatide that was administered later.

Metrological traceability in mass spectrometry-based targeted protein quantitation: a proof-of-principle study for serum apolipoproteins A-I and B100.

In this study, we have followed up on previous liquid chromatography (LC) multiple reaction monitoring (MRM) mass spectrometry (MS) approaches for measurement of apolipoprotein (apo) A-I and apo B100 in serum aiming for implementation of a multiplexed assay in a clinical chemistry laboratory with full metrological traceability. Signature peptides were selected and detected by dynamic MRM, and stable isotope labeled (SIL)-peptides were used as internal standards. Five apo A-I and four apo B100 peptides were measured in serum digests with linearity (R(2)>0.992) in the physiologically relevant concentration ranges. Linearity with regard to protein concentration was ascertained at five concentration levels (R(2)>0.926 and R(2)>0.965, for the apo A-I and apo B100 peptides, respectively). Three native value-assigned sera were used as external calibrators for further method verification. Imprecision values on sample preparation and LC-MS/MS acquisition were below the established minimal specifications for apo A-I and apo B100 (5.0% and 5.3%, respectively). Correlation of LC-MS/MS results with immunoturbidimetric assay results, for normo- and hypertriglyceridemic samples, showed R(2)>0.944 for apo A-I and R(2)>0.964 for apo B100. This LC-MS/MS method has potential for clinical application in normo- and dyslipidemic patients. BIOLOGICAL SIGNIFICANCE: Measurement of apo A-I and apo B100 may offer an alternative to high and low density lipoprotein cholesterol (HDL-c and LDL-c) methods for cardiovascular disease risk assessment in dyslipidemic patients [1]. An LC-MS/MS method for apo A-I and apo B100 has the advantage of antibody independent and specific detection of protein signature peptides. The introduction of an LC-MS/MS method for apo A-I and apo B100 can serve as an example for many existing and newly developed (multiplex) biomarker methods in quantitative clinical chemistry proteomics (qCCP). Such LC-MS/MS methods should meet basic clinical chemistry principles with regard to test evaluation [2]. Criteria for imprecision should be pre-defined, e.g., based on biological variation. The use of commutable and traceable serum-based calibrators will improve inter-laboratory reproducibility of LC-MS/MS methods and may contribute to a more rapid transition of biomarker discovery to clinical utility with benefit for the patient treatment and improvement of general health care.