Meeusen, Jeffrey W.

Abstract Plasma concentrations of low-density-lipoprotein cholesterol (LDL-C) are directly associated with risk for coronary artery disease (CAD). Multisociety guidelines define LDL-C>160mg/dL as a risk factor for CAD and LDL-C>190mg/dL as an indication for lipid lowering medication, regardless of other clinical factors. Subfractionation of LDL according to size (LDL-s) enables differentiation between two LDL phenotypes: large-buoyant LDL and small-dense LDL. The small-dense LDL phenotype reportedly conveys increased risk for CAD. Major societies do not recommend LDL subfractions be used for clinical decision making and most payers do not cover LDL subfraction testing. Despite these restrictions, LDL subfraction is routinely requested by clinicians. Nuclear magnetic resonance (NMR) spectroscopy measures LDL-C and LDL-s. Following inquiries regarding interpretation of conflicting LDL-C and LDL-s results, we investigated associations between LDL-C and LDL-s measured by NMR in order to determine how often they provide contradicting or additive information. Verification of NMR LDL-C accuracy was confirmed by ß-quantification in a subset of patient samples (n=250). The average bias was -4.5mg/dL and the correlation coefficient was 0.92. High-risk was defined as LDL-C>160mg/dL or LDL-s<20.5 nm (small-dense LDL); and low-risk was defined as LDL-C<70mg/dL or LDL-s>20.5nm (large-buoyant LDL). In 26,710 clinical NMR analyses, the median LDL-C was 94.0mg/dL (range:5-436mg/dL) with median LDL-s of 20.8 nm (range:19.4–23.0nm). LDL-s moderately correlated with LDL-C (Ï#129;=0.51;p<0.01). Small-dense-LDL was identified in only 18% (407/2,191) of patients with elevated LDL-C (>160mg/dL) and was more common (73.2% of 6,093) in patients with low LDL-C (<70mg/dL;p<0.001). Associations with CAD were investigated among patients without cholesterol-lowering medication treatment referred for angiography (n=356). CAD (defined as stenosis >50% in one or more coronary artery) was diagnosed in 14% (1/7) of subjects with low LDL-C (<70mg/dL) compared to 59% (47/80) of subjects with elevated LDL-C (p=0.01). When stratifying by LDL-s, CAD was diagnosed in 50% (57/115) of subjects with small-dense LDL compared to 43% (104/241) of subjects with large-buoyant LDL (p=0.2). Small-dense LDL was identified in only 33% (26/80) of cases with elevated LDL-C. Limiting to subjects with elevated LDL-C, CAD was diagnosed in 50% (13/26) of subjects with concordant (high-risk) small-dense LDL compared to 61% (33/54) of subjects with discordant (low-risk) large-buoyant LDL (LDL-s>20.5nm) (p=0.3). Our data confirm that LDL-s subfraction measured by NMR is reported discordantly in most cases when LDL-C is unequivocally high or low. Furthermore, CAD diagnosis was significantly associated with LDL-C, but not with LDL-s. Our data also show that in discrepant samples, elevated LDL-C correlates better with disease state compared to LDL-s. Therefore, LDL-s should not be used to justify treatment decisions in patients with elevated LDL-C. Laboratories should consider carefully whether or not to report LDL-s when it is known that misleading and discordant values will be reported in a majority of cases.

Abstract Lipoprotein(a) [Lp(a)] is a pro-atherogenic and pro-thrombotic LDL-like particle recognized as an independent risk factor for cardiovascular disease (CVD) that is resistant to typical lipid-lowering treatments. The cholesterol within Lp(a) (Lp(a)-C) contributes to the reported LDL-cholesterol (LDL-C) concentration by nearly all available methods including beta-quantification, direct homogenous assays, and all estimating equations. Accurate LDL-C measurements are critical for identification of genetic hyperlipidemia conditions such as familial hypercholesterolemia (FH). FH risk estimators such as the Dutch Lipid Clinic Network (DLCN) criteria utilize LDL-C concentration cut-offs and other clinical inputs to assess the likelihood of FH. Therefore, failure to adjust for Lp(a)-C can impact accurate FH classification, appropriate follow-up testing and treatments, and interpretation of cholesterol-lowering treatment efficacy. Lp(a)-C can be estimated from Lp(a) mass as measured by immunoassay using an average cholesterol content per particle. However, Lp(a)-C size and composition varies significantly within individuals resulting in inaccurate Lp(a)-C estimates. In this study, we use direct Lp(a)-C measurements to assess the potential misclassification of FH risk due to the contribution of Lp(a)-C to LDL-C in patient samples submitted for advanced lipoprotein profiling. A total of 28,200 samples submitted for lipoprotein profiling were included. The profiling included lipid testing in a CDC-certified laboratory on Roche cobas 501 (cholesterol and triglycerides by enzymatic method, high-density lipoprotein cholesterol by MgCl2/dextran sulfate precipitation). LDL-C was measured by beta-quantification, and Lp(a)-C by quantitative lipoprotein electrophoresis (SPIFE Vis Cholesterol, Helena Laboratories). The DLCN LDL-C cut-offs (155, 190, 250, and 330mg/dL) were applied to LDL-C results before and after accounting for Lp(a)-C contribution. Lp(a)-C was detected in 3,728 (13.2%) samples. The median (range) concentrations of Lp(a)-C and LDL-C were 11mg/dL (5-108mg/dL) and 121mg/dL (27-678mg/dL), respectively. Overall, subtracting Lp(a)-C would reclassify 6.5% of all samples into a lower LDL-C category within the DLCN algorithm. Within the LDL-C scoring categories, 7.0% (n=222) of subjects with LDL-C 155-189mg/dL, 5.6% (n=66) of subjects with LDL-C 190-249mg/dL, 5.2% (n=10) of subjects with LDL-C 250-329mg/dL, and 3.4% (n=4) of subjects with LDL-C >330mg/dL would be down-classified after adjusting for Lp(a)-C. Limiting to subjects with measurable Lp(a)-C, reclassification to a lower diagnostic threshold occurred in 47.4% of subjects with LDL-C 155-189mg/dL, 37.5% with LDL-C 190-249mg/dL, 41.6% with LDL-C 250-329mg/dL, and 33.3% with LDL-C >330mg/dL after adjustment. Current guidelines recommend screening for elevated Lp(a) in patients with family history of CVD. Our data show that a high percentage of samples evaluated for advanced lipid testing contain measurable Lp(a)-C that could cause mis-classification in FH prediction algorithms. If labeled high probability of FH, these mis-classifications could trigger inappropriate work-up for suspected FH. As clinical follow-up and therapeutic strategies differ between FH and elevated Lp(a), proper distinction between LDL-C and Lp(a)-C is needed to guide appropriate patient management.

Objective— Ceramides are sphingolipids involved with cellular signaling. Synthesis of ceramides occurs in all tissues. Ceramides accumulate within tissues and the blood plasma during metabolic dysfunction, dyslipidemia, and inflammation. Elevations of ceramides are predictive of cardiovascular mortality. We sought to verify the utility of plasma concentrations of 4 ceramides: N-palmitoyl-sphingosine [Cer(16:0)], N-stearoyl-sphingosine [Cer(18:0)], N-nervonoyl-sphingosine [Cer(24:1)], and N-lignoceroyl-sphingosine [Cer(24:0)] in predicting major adverse cardiovascular events in a diverse patient population referred for coronary angiography. Approach and Results— Plasma ceramides were measured in 495 participants before nonurgent coronary angiography. Coronary artery disease, defined as >50% stenosis in ≥1 coronary artery, was identified 265 (54%) cases. Ceramides were not significantly associated with coronary artery disease. Patients were followed for a combined primary end point of myocardial infarction, percutaneous intervention, coronary artery bypass, stroke, or death within 4 years. Ceramides were significantly predictive of outcomes after adjusting for age, sex, body mass index, hypertension, smoking, LDL (low-density lipoprotein) cholesterol, HDL (high-density lipoprotein) cholesterol, triglycerides, serum glucose, and family history of coronary artery disease. The fully adjusted per SD hazard ratios (95% confidence interval) were 1.50 (1.16–1.93) for Cer(16:0), 1.42 (1.11–1.83) for Cer(18:0), 1.43 (1.08–1.89) for Cer(24:1), and 1.58 (1.22–2.04) for the ceramide risk score. Conclusions— Elevated plasma concentrations of ceramides are independently associated with major adverse cardiovascular events in patients with and without coronary artery disease.