Daniel M. Hoefner

Apolipoprotein B and Cardiovascular Disease Risk: Position Statement from the AACC Lipoproteins and Vascular Diseases Division Working Group on Best Practices

BACKGROUND Low-density lipoprotein cholesterol (LDL-C) has been the cornerstone measurement for assessing cardiovascular risk for nearly 20 years. CONTENT Recent data demonstrate that apolipoprotein B (apo B) is a better measure of circulating LDL particle number (LDL-P) concentration and is a more reliable indicator of risk than LDL-C, and there is growing support for the idea that addition of apo B measurement to the routine lipid panel for assessing and monitoring patients at risk for cardiovascular disease (CVD) would enhance patient management. In this report, we review the studies of apo B and LDL-P reported to date, discuss potential advantages of their measurement over that of LDL-C, and present information related to standardization. CONCLUSIONS In line with recently adopted Canadian guidelines, the addition of apo B represents a logical next step to National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII) and other guidelines in the US. Considering that it has taken years to educate physicians and patients regarding the use of LDL-C, changing perceptions and practices will not be easy. Thus, it appears prudent to consider using apo B along with LDL-C to assess LDL-related risk for an interim period until the superiority of apo B is generally recognized.

Association of Apolipoprotein B and Nuclear Magnetic Resonance Spectroscopy–Derived LDL Particle Number with Outcomes in 25 Clinical Studies: Assessment by the AACC Lipoprotein and Vascular Diseases Division Working Group on Best Practices

BACKGROUND The number of circulating LDL particles is a strong indicator of future cardiovascular disease (CVD) events, even superior to the concentration of LDL cholesterol. Atherogenic (primarily LDL) particle number is typically determined either directly by the serum concentration of apolipoprotein B (apo B) or indirectly by nuclear magnetic resonance (NMR) spectroscopy of serum to obtain NMR-derived LDL particle number (LDL-P). CONTENT To assess the comparability of apo B and LDL-P, we reviewed 25 clinical studies containing 85 outcomes for which both biomarkers were determined. In 21 of 25 (84.0%) studies, both apo B and LDL-P were significant for at least 1 outcome. Neither was significant for any outcome in only 1 study (4.0%). In 50 of 85 comparisons (58.8%), both apo B and LDL-P had statistically significant associations with the clinical outcome, whereas in 17 comparisons (20.0%) neither was significantly associated with the outcome. In 18 comparisons (21.1%) there was discordance between apo B and LDL-P. CONCLUSIONS In most studies, both apo B and LDL-P were comparable in association with clinical outcomes. The biomarkers were nearly equivalent in their ability to assess risk for CVD and both have consistently been shown to be stronger risk factors than LDL-C. We support the adoption of apo B and/or LDL-P as indicators of atherogenic particle numbers into CVD risk screening and treatment guidelines. Currently, in the opinion of this Working Group on Best Practices, apo B appears to be the preferable biomarker for guideline adoption because of its availability, scalability, standardization, and relatively low cost.

New Automated Assay of Small Dense Low-Density Lipoprotein Cholesterol Identifies Risk of Coronary Heart Disease The Multi-Ethnic Study of Atherosclerosis

Objective— Coronary heart disease (CHD) is the leading cause of death in the United States, yet assessing risk of its development remains challenging. The present study evaluates a new automated assay of small dense low-density lipoprotein cholesterol content (sdLDL-C) and whether sdLDL-C is a risk factor for CHD compared with LDL-C or small LDL particle concentrations derived from nuclear magnetic resonance spectroscopy. Approach and Results— sdLDL-C was measured using a new automated enzymatic method, and small LDL concentrations were obtained by nuclear magnetic resonance in 4387 Multi-Ethnic Study of Atherosclerosis participants. Cox regression analysis estimated hazard ratios for developing CHD for 8.5 years after adjustments for age, race, sex, systolic blood pressure, hypertension medication use, high-density lipoprotein cholesterol, and triglycerides. Elevated sdLDL-C was a risk factor for CHD in normoglycemic individuals. Those in the top sdLDL-C quartile showed higher risk of incident CHD (hazard ratio, 2.41; P=0.0037) compared with those in the bottom quartile and indicated greater CHD risk than the corresponding quartile of LDL-C (hazard ratio, 1.75; P=0.019). The association of sdLDL-C with CHD risk remained significant when LDL-C (<2.57 mmol/L) was included in a multivariate model (hazard ratio, 2.37; P=0.012). Nuclear magnetic resonance–derived small LDL concentrations did not convey a significant risk of CHD. Those with impaired fasting glucose or diabetes mellitus showed higher sdLDL-C and small LDL concentrations but neither was associated with higher CHD risk in these individuals. Conclusions— This new automated method for sdLDL-C identifies risk for CHD that would remain undetected using standard lipid measures, but only in normoglycemic, nondiabetic individuals.

Myocardial Tissue Remodeling in Adolescent Obesity

Background Childhood obesity is a significant risk factor for cardiovascular disease in adulthood. Although ventricular remodeling has been reported in obese youth, early tissue‐level markers within the myocardium that precede organ‐level alterations have not been described. Methods and Results We studied 21 obese adolescents (mean age, 17.7±2.6 years; mean body mass index [BMI], 41.9±9.5 kg/m2, including 11 patients with type 2 diabetes [T2D]) and 12 healthy volunteers (age, 15.1±4.5 years; BMI, 20.1±3.5 kg/m2) using biomarkers of cardiometabolic risk and cardiac magnetic resonance imaging (CMR) to phenotype cardiac structure, function, and interstitial matrix remodeling by standard techniques. Although left ventricular ejection fraction and left atrial volumes were similar in healthy volunteers and obese patients (and within normal body size‐adjusted limits), interstitial matrix expansion by CMR extracellular volume fraction (ECV) was significantly different between healthy volunteers (median, 0.264; interquartile range [IQR], 0.253 to 0.271), obese adolescents without T2D (median, 0.328; IQR, 0.278 to 0.345), and obese adolescents with T2D (median, 0.376; IQR, 0.336 to 0.407; P=0.0001). ECV was associated with BMI for the entire population (r=0.58, P<0.001) and with high‐sensitivity C‐reactive protein (r=0.47, P<0.05), serum triglycerides (r=0.51, P<0.05), and hemoglobin A1c (r=0.76, P<0.0001) in the obese stratum. Conclusions Obese adolescents (particularly those with T2D) have subclinical alterations in myocardial tissue architecture associated with inflammation and insulin resistance. These alterations precede significant left ventricular hypertrophy or decreased cardiac function.

Race Is a Key Variable in Assigning Lipoprotein(a) Cutoff Values for Coronary Heart Disease Risk Assessment The Multi-Ethnic Study of Atherosclerosis

Objective— We aimed to examine associations of lipoprotein(a) (Lp(a)) concentrations with coronary heart disease (CHD) and determine whether current Lp(a) clinical laboratory cut points identify risk of disease incidence in 4 races/ethnicities of the Multi-Ethnic Study of Atherosclerosis (MESA). Approach and Results— A subcohort of 1323 black, 1677 white, 548 Chinese American, and 1044 Hispanic MESA participants were followed up during a mean 8.5-year period in which 235 incident CHD events were recorded. Lp(a) mass concentrations were measured using a turbidimetric immunoassay. Cox regression analysis determined associations of Lp(a) with CHD risk with adjustments for lipid and nonlipid variables. Lp(a) concentrations were continuously associated with risk of CHD incidence in black (hazard ratio [HR], 1.49; 95% confidence interval [CI], 1.09–2.04] and white participants (HR, 1.22; 95% CI, 1.02–1.45). Examining Lp(a) risk by the 50 mg/dL cut point revealed higher risks of incident CHD in all races except Chinese Americans: blacks (HR, 1.69; 95% CI, 1.03–2.76), whites (HR, 1.82; 95% CI, 1.15–2.88); Hispanics (HR, 2.37; 95% CI, 1.17–4.78). The lower Lp(a) cut point of 30 mg/dL identified higher risk of CHD in black participants alone (HR, 1.87; 95% CI, 1.08–3.21). Conclusions— Our findings suggest that the 30 mg/dL cutoff for Lp(a) is not appropriate in white and Hispanic individuals, and the higher 50 mg/dL cutoff should be considered. In contrast, the 30 mg/dL cutoff remains suitable in black individuals. Further research is necessary to develop the most clinically useful Lp(a) cutoff values in individual races/ethnicities.

Falsely Decreased Human Chorionic Gonadotropin (hCG) Results Due to Increased Concentrations of the Free β Subunit and the β Core Fragment in Quantitative hCG Assays

BACKGROUND Earlier studies have shown that increased concentrations of certain human chorionic gonadotropin (hCG) variants can cause false-negative results in some qualitative hCG devices. The objective of this study was to determine if increased concentrations of hCGβ and hCGβ core fragment (hCGβcf) cause falsely decreased results on 9 commercially available quantitative hCG assays. METHODS Several concentrations of purified hCGβ and hCGβcf were added to 2 sets of 6 serum samples with and without a fixed concentration of intact hCG. We examined 9 widely used immunoassays to measure immunoreactive hCG. Falsely decreased results were defined as those in which the measured hCG concentration was ≤50% of expected. RESULTS High concentrations of hCGβ (≥240 000 pmol/L) produced falsely decreased hCG measurements in 2 assays known to detect this variant. Similarly, high concentrations of hCGβcf (≥63 000 pmol/L) produced falsely decreased hCG measurements in 3 assays that do not detect purified hCGβcf. Two assays were identified that detected both hCGβ and hCGβcf, and neither produced falsely decreased results in the presence of high concentrations of these variants. CONCLUSIONS Extremely high concentrations of hCG variants can cause falsely decreased results in certain quantitative hCG assays. Of the 9 assays examined, none exhibited falsely decreased results in the presence of hCGβ concentrations typically associated with hCGβ-producing malignancies. Two assays exhibited decreased (>50%) hCG results in the presence of hCGβcf concentrations found during normal pregnancy.

Validation of a lipoprotein(a) particle concentration assay by quantitative lipoprotein immunofixation electrophoresis.

BACKGROUND Low-density lipoprotein (LDL) particle (P, or molar) concentration has been shown to be a more sensitive marker of cardiovascular disease (CVD) risk than LDL cholesterol. Although elevated circulating lipoprotein(a) [Lp(a)] cholesterol and mass have been associated with CV risk, no practicable method exists to measure Lp(a)-P. We have developed a method of determining Lp(a)-P suitable for routine clinical use. METHODS Lipoprotein immunofixation electrophoresis (Lipo-IFE) involves rigidly controlled electrophoretic separation of serum lipoproteins, probing with polyclonal apolipoprotein B antibodies, then visualization after staining with a nonspecific protein stain (Acid Violet). Lipo-IFE was compared to the Lp(a) mass assay for 1086 randomly selected patient samples, and for 254 samples stratified by apo(a) isoform size. RESULTS The Lipo-IFE method was shown to be precise (CV <10% above the 50 nmol/l limit of quantitation) and linear across a 16-fold range. Lipo-IFE compared well with the mass-based Lp(a) assay (r=0.95), but was not affected by variations in apo(a) isoform size. With a throughput of 100 samples in 90 min, the assay is suitable for use in the clinical laboratory. CONCLUSIONS The Lipo-IFE method will allow Lp(a)-P to be readily tested as a CVD risk factor in large-scale clinical trials.

Comparison of cardiometabolic risk biomarkers from a national clinical laboratory with the US adult population

BACKGROUND Clinical laboratory patient databases are an untapped source of valuable diagnostic and prognostic information. However, the lack of associated clinical and/or demographic information and questionable generalizability to nonpatient populations often limit utility of these data. OBJECTIVES This study compared levels of cardiometabolic biomarkers between a national clinical laboratory patient cohort (Health Diagnostic Laboratory [HD Lab]) and the US population as inferred from the National Health and Nutrition Examination Survey (NHANES, 2011-2012). METHODS Sample sizes for HD Lab ranged from 199,000 to 739,000 and for NHANES from 2200 to 5300. The latter were weighted to represent the adult US population (∼220 million). Descriptive statistics were compared for body mass index, 5 lipid biomarkers, and 3 glycemic biomarkers. RESULTS Using age- and sex-matched data, mean biomarker values (mg/dL unless noted) and percent differences (%) for HD Lab vs NHANES were body mass index (kg/m(2)), 29.1 vs 28.6 (1.7%); total cholesterol, 185 vs 193 (-4.1%); apolipoprotein B, 92 vs 90 (2.2%); low-density lipoprotein cholesterol, 107 vs 115 (-7%); high-density lipoprotein cholesterol, 53 vs 53 (0%); triglycerides, 128 vs 127 (0.8%); glucose, 99 vs 108 (-8.3%); insulin (uU/mL), 13.7 vs 13.4 (2.2%); and hemoglobin A1c (%), 5.6 vs 5.8 (-3.4%). Although all differences were statistically significant, only low-density lipoprotein cholesterol and glucose differed by more than 5%. These may reflect a greater use of medications among HD Lab patients and/or preanalytical factors. CONCLUSIONS Cardiometabolic risk markers from a national clinical laboratory were broadly similar to those of the US population; thus, with certain caveats, data from the former may be generalizable to the latter.

Hypertriglyceridaemia unresponsive to multiple treatments

A 52-year-old man with a longstanding history of hypertriglyceridaemia (approximately 7 mmol/L (600 mg/dL)), unresponsive to treatment, presented to a lipid-specialty clinic. Additional triglyceride-lowering therapies were added with no effect. It was then noted that despite the apparent hypertriglyceridaemia, his serum sample was clear. A ‘glycerol blank’ was then requested from an advanced lipid laboratory, which reported a triglyceride value of 0.7 mmol/L (62 mg/dL). These findings suggest isolated asymptomatic glycerol kinase deficiency (GKD) or ‘pseudohypertriglyceridaemia’. The falsely elevated triglyceride values in such individuals are a result of excess serum glycerol and clinical laboratories measuring glycerol to report triglyceride concentrations. After discontinuation or modification of the patients primary triglyceride-lowering agents, the lipid panels and triglyceride values remained comparable to previous readings. Recognition of asymptomatic GKD is important to prevent unnecessary treatment and overestimated cardiovascular risk.

Race Is a Key Variable in Assigning Lipoprotein(a) Cutoff Values for Coronary Heart Disease Risk Assessment

Objective— We aimed to examine associations of lipoprotein(a) (Lp(a)) concentrations with coronary heart disease (CHD) and determine whether current Lp(a) clinical laboratory cut points identify risk of disease incidence in 4 races/ethnicities of the Multi-Ethnic Study of Atherosclerosis (MESA). Approach and Results— A subcohort of 1323 black, 1677 white, 548 Chinese American, and 1044 Hispanic MESA participants were followed up during a mean 8.5-year period in which 235 incident CHD events were recorded. Lp(a) mass concentrations were measured using a turbidimetric immunoassay. Cox regression analysis determined associations of Lp(a) with CHD risk with adjustments for lipid and nonlipid variables. Lp(a) concentrations were continuously associated with risk of CHD incidence in black (hazard ratio [HR], 1.49; 95% confidence interval [CI], 1.09–2.04] and white participants (HR, 1.22; 95% CI, 1.02–1.45). Examining Lp(a) risk by the 50 mg/dL cut point revealed higher risks of incident CHD in all races except Chinese Americans: blacks (HR, 1.69; 95% CI, 1.03–2.76), whites (HR, 1.82; 95% CI, 1.15–2.88); Hispanics (HR, 2.37; 95% CI, 1.17–4.78). The lower Lp(a) cut point of 30 mg/dL identified higher risk of CHD in black participants alone (HR, 1.87; 95% CI, 1.08–3.21). Conclusions— Our findings suggest that the 30 mg/dL cutoff for Lp(a) is not appropriate in white and Hispanic individuals, and the higher 50 mg/dL cutoff should be considered. In contrast, the 30 mg/dL cutoff remains suitable in black individuals. Further research is necessary to develop the most clinically useful Lp(a) cutoff values in individual races/ethnicities.