Direct LDL-C Research Articles

Small Dense LDL Cholesterol and Coronary Heart Disease: Results from the Framingham Offspring Study

We sought to establish reference values for a new direct assay for small dense LDL cholesterol (sdLDL-C) and to measure sdLDL-C concentrations in patients with established coronary heart disease (CHD) vs controls. Direct LDL-C and sdLDL-C were measured in samples from 3188 male and female participants of the Framingham Offspring Study, including 173 men and 74 women with CHD. Postmenopausal status and male sex were associated with higher sdLDL-C concentrations (P < 0.0001). Cholesterol-lowering medication use was more frequent (P < 0.0001) in CHD patients than in controls (46.8% vs 11.4% in men; 35.1% vs 8.8% in women). In men, mean LDL-C was lower in CHD than in controls (3.22 vs 3.51 mmol/L, P < 0.0001), whereas mean sdLDL-C concentrations were similar (0.83 vs 0.84 mmol/L, P = 0.609). In women, mean LDL-C was similar in CHD and controls (3.53 vs 3.46 mmol/L, P = 0.543), but mean sdLDL-C was higher (0.83 vs 0.68 mmol/L, P = 0.0015). The mean percentage of LDL-C as sdLDL-C was higher in both men and women with CHD than controls (P < 0.01). Increased LDL-C and sdLDL-C were found in 10.4% and 22.0% of men and in 24.3% and 27.8% of women with CHD, respectively. Despite 4-fold greater cholesterol-lowering therapy use, CHD patients had mean LDL-C concentrations above the LDL-C goal of <2.6 mmol/L (<100 mg/dL). Although women with CHD had higher sdLDL-C concentrations than controls, this difference was not seen in men. These findings may explain some of the high residual risk of future CHD events in CHD patients.

Direct assessment of plasma low density lipoprotein and high density lipoprotein cholesterol levels and coronary heart disease: Results from the Framingham Offspring Study

Background We evaluated direct low density lipoprotein (LDL) cholesterol (C) and high density lipoprotein (HDL) cholesterol (C) versus standard methods using fasting plasma samples from participants in cycle 6 of the Framingham Offspring Study. Methods Direct LDL-C and HDL-C measurements were performed on fasting plasma from male (1335 controls, 173 CHD cases) and female (1606 controls, 74 cases) participants, and compared with LDL-C, as calculated with the Friedewald formula, and HDL-C, as measured after dextran-Mg 2+ precipitation. Results Values for direct LDL-C and HDL-C correlated well with standard methods (both about r 2 = 0.94, p < 0.001) with similar absolute values. Biases of >10% were present for 7.7% of samples for LDL-C, while for HDL-C this value was 8.5%. Despite higher use of cholesterol-lowering medication in CHD cases, calculated or direct LDL-C values were still well above recommended values [<2.6 mmol/L (100 mg/dL)] in CHD cases, especially in females. Conclusions Direct assays for both LDL-C and HDL-C provide an acceptable guide for lipid treatment. In Framingham Offspring Study participants most CHD cases had LDL-C levels above the recommended target.

Abstract 6214: Comparison of Adiponectin Levels in Healthy Americans and Japanese: Results from Framingham and Fukuoka

Background and Objectives: Adiponectin is an adipocytokine with insulin-sensitizing and anti-atherosclerotic effects. Serum adiponectin levels have been reported to be negatively correlated with body weight and waist circumference (WC). Low plasma adiponectin levels are known to predispose to the development of insulin resistance and type 2 diabetes mellitus. Adiponectin levels may vary according to ethnic group. The aim of this study was to investigate potential differences in plasma adiponectin levels between Japanese and Americans. Methods: Subjects consisted of 2,060 healthy American male and female participants in exam 6 of the Framingham Offspring Study (mean age 57.0 ± 9.0 years, 57.4 ± 10.5 years, respectively), and 1131 healthy Japanese male and female participants in the Fukuoka Study (mean age 56.3 ± 5.5 years, 55.6 ± 5.4 years, respectively). Frozen plasma samples (at −80 degree C) from both studies were shipped to Tufts University, Boston. Plasma concentrations of direct LDL-C, fasting plasma glucose and adiponectin were measured using the same assay kits on the same machine (Hitachi 911) by the same staff. Anthropometric measurements including weight, height, body mass index (BMI) and WC, were also used in the analysis. Results: Plasma adiponectin levels were significantly and inversely correlated with BMI, WC, glucose, insulin, triglycerides, and systolic and diastolic blood pressure. Moreover adiponectin levels were significantly and positively correlated with HDL-C in both populations. Mean BMI and WC of Japanese men and women were significantly lower (23.8 kg/m 2 and 22.1 kg/m 2 , 86.0 cm and 78.7 cm, respectively) than in US men and women (28.2 kg/m 2 and 27.0 kg/m 2 , 100.4 cm and 92.8 cm, respectively) (all p&lt;0.05). However, mean adiponectin levels (± SD) in Japanese men and women were significantly lower (9.3 ± 4.5 micro g/mL and 14.7 ± 7.3 micro g/mL, respectively) than in US men and women (10.3 ± 5.2 micro g/mL and 16.3 ± 7.7 micro g/mL, respectively)(p&lt;0.05). Conclusion: Our data indicate a substantially lower prevalence of adiposity, but not higher adiponectin levels, in a healthy Japanese population as compared to an American population. The data indicate that population specific norms may have to be used for different ethnic groups.

Multicentric evaluation of the homogeneous LDL-cholesterol Plus assay: Comparison with beta-quantification and Friedewald formula

Objectives To evaluate the analytical and clinical performance of a new version of the LDL-C Plus assay and compare it with the beta-quantification (BQ) method in a multicenter study. Design and methods Direct LDL-C was measured in 169 fresh pooled samples and in 830 clinical samples with known LDL-C by BQ. The reactivity of lipoproteins and the effect of hemoglobin, bilirubin and chylomicrons (CM) were studied. Results Direct LDL-C total imprecision was < 2.2%; inaccuracy < ± 2.5% (unaffected by triglycerides up to 9.5 mmol/L); and total error 9.8%. Direct assay reacted with 95%, 50% and 25% of the cholesterol in the LDL, intermediate (IDL) and VLDL fractions, respectively. A significant association was observed with BQ. Icteric samples showed a negative bias and the effect of CM was variable. A positive bias was observed when VLDL-cholesterol/triglyceride ratio was > 0.57. Conclusions LDL-C Plus assay represents a valid alternative to BQ for clinical laboratories.

A simple and precise method for measuring HDL-cholesterol subfractions by a single precipitation followed by homogenous HDL-cholesterol assay

HDL consists of two major subfractions, HDL2 and HDL3. This paper describes a simple method for assaying HDL subspecies by combining a single precipitation with a direct high density lipoprotein-cholesterol (HDL-C) assay. A precipitation reagent (0.06 ml) containing 1,071 U/ml heparin, 500 mmol/l MnCl2) and 12 mg/ml dextran sulfate was added to a serum (0.3 ml). The sample was incubated and centrifuged at 10,000 rpm for 10 min. HDL3-C was measured by a homogenous HDL-C assay in the supernatant, and HDL2-C was estimated by subtracting the HDL3-C from the direct HDL-C. The HDL3-C and HDL2-C values determined by the precipitation method were identical to those determined by ultracentrifugation, and there were excellent correlations between the methods in the measurements of HDL3-C and HDL2-C (r = 0.933 and 0.978, respectively; n = 102). The two methods also proved to be highly correlated in the measurement of apolipoprotein A-I and A-II in HDL subfractions. The HDL-C subfractions determined by ultracentrifugation were more closely associated with the homogenous HDL-C assay than with the total cholesterol assay, especially in the hypertriglyceridemic samples. Our method is far simpler and more precise than the classical dual precipitation method for HDL-C subfractions, and it can be easily performed in a routine chemical laboratory.

Abstract 193: Lapaquistat Acetate, A Novel Squalene Synthase Inhibitor, Co-administered with Atorvastatin Reduces Plasma Lipids and C-Reactive Protein Levels in Subjects with Primary Hypercholesterolemia

Background/Objective: Lapaquistat acetate (LAPA), a novel lipid-lowering agent, inhibits squalene synthase, an enzyme distal to HMG-CoA reductase in the cholesterol biosynthetic pathway. We evaluated the effects of LAPA on LDL-C, other plasma lipid variables, and high-sensitivity C-reactive protein (hs-CRP) levels when co-administered with a stable dose of the HMG-CoA reductase inhibitor, atorvastatin (ATV), in subjects with primary hypercholesterolemia already on ATV therapy. Methods: This multicenter, randomized, double-blind, placebo-controlled, Phase III trial was conducted at 79 centers in the US. After a 4-wk dietary run-in and ATV (10, 20, or 40 mg/d) stabilization, 448 subjects (LDL-C ≥100 mg/dL, TG ≤400 mg/dL) were randomized to 1 of 3 treatments: LAPA 50 mg, 100 mg, or placebo co-administered with ongoing ATV dosed once daily for 24 wks. The primary endpoint was percent (%) change from baseline in direct LDL-C at 24 wks; secondary endpoints included % changes in calculated LDL-C, total cholesterol (TC), non-HDL-C, Apo B, TG, HDL-C, VLDL-C, Apo A1, and hs-CRP levels. Safety was monitored using clinical and laboratory examinations. Results: Baseline clinical and lipid characteristics were comparable among the 3 treatment groups. Compared with placebo, LAPA treatment resulted in significant reductions (see Table ) in LDL-C, other lipid variables, and hs-CRP. The enhanced LDL-C-lowering effect was independent of ATV dose. LAPA co-administration was well tolerated. Overall, adverse event (AE) incidences were similar in the 3 treatment groups. Serious AEs in 12 subjects (5 in placebo, 3 in 50 mg LAPA, and 4 in 100 mg LAPA) were reported. Conclusion: Lapaquistat acetate, a novel squalene synthase inhibitor, was well tolerated and effective in reducing LDL-C, hs-CRP, and in improving the atherogenic lipid profile when co-administered with atorvastatin. This combination may prove to be a useful therapeutic option for patients with hypercholesterolemia. Least Squares Mean % Change from Baseline (±SE)

Abstract 682: Lapaqistat Acetate Monotherapy: Effects of a Novel Squalene Synthase Inhibitor on LDL-C Levels and Other Lipid Parameters in Patients with Primary Hypercholesterolemia

Background: Lapaquistat acetate (LAPA) inhibits squalene synthase, an important enzyme in the cholesterol biosynthetic pathway. Early studies showed that LAPA reduced LDL-C and improved lipid profiles in animals and humans. Methods: This multicenter, phase III, randomized, double-blind, parallel-group, placebo-controlled study assessed LAPA monotherapy in nondiabetic patients ≥ 18 yrs old with primary hypercholesterolemia (mean LDL-C &gt; 130 mg/dL and &lt; 220 mg/dL; mean triglyceride [TG] ≤400 mg/dL). Treatment-naïve patients or patients discontinuing previous lipid-lowering therapy underwent a 6-wk dietary stabilization period, then were randomized to 100 mg LAPA (n = 241) or Placebo (n = 120) once daily for 12 wks. The primary endpoint was percent (%) change from baseline compared with Placebo in direct LDL-C at Wk 12 or last on-treatment value. Secondary endpoints included: % change in calculated LDL-C, non-HDL-C, total cholesterol (TC), Apo B, HDL-C, VLDL-C, Apo A1, TG, and hs-CRP. Safety was monitored using clinical and laboratory examinations. Results: LAPA significantly reduced LDL-C levels (see Table ) within 2– 4 wks of treatment initiation that were maintained throughout the study. LDL-C remained relatively unchanged in the Placebo group. Compared with Placebo, LAPA lowered LDL-C levels 20.09% by end of study (p &lt; 0.001). Non-HDL-C, TC, Apo B, VLDL-C, Apo A1, TG, and hs-CRP were also significantly reduced vs Placebo. Both the overall adverse event (AE) profile and the rate of AEs considered potentially related to study drug (~24%), were similar between treatment groups. Two serious AEs occurred in each group; the 2 SAEs in lapaquistat-treated patients were considered not related to study drug. Conclusion: LAPA (100 mg/day) monotherapy provided significant improvements in LDL-C, non-HDL-C, TC, Apo B, VLDL-C, TG, and hs-CRP levels, was generally safe and well tolerated, and potentially represents a novel treatment for hypercholesterolemic patients.

Comparison of Direct and Indirect Measurement of LDL-C in HIV-Infected Individuals: ACTG 5087

Background: Hypertriglyceridemia is common in HIV-infected individuals on antiretroviral therapy. Triglyceride (TG) levels >400 mg/dL interfere with the accurate determination of low-density lipoproteins (LDL-C) by the Friedewald equation, making it difficult to assess coronary heart disease risk. Objective: The objective of this study is to compare the agreement of the direct LDL-C assay and the Friedewald equation with a reference ultracentrifugation method in the estimation of LDL-C concentrations. Method: Samples from ACTG 5087 were assayed by ultracentrifugation and a direct enzymatic assay and calculated using the Friedewald equation. Results: In subjects with TG <400 mg/dL (n = 271), 90% of the direct LDL-C values and Friedewald calculations were within 30 mg/dL and 32 mg/dL of the ultracentrifugation values, respectively. With TG ⩾400 mg/dL (n = 186), 90% of the direct assay and Friedewald observations were within 68 mg/dL and 120 mg/dL of the ultracentrifugation results, respectively. Only 27% of the LDL-C values were within 15 mg/dL of the ultracentrifugation LDL-C results for direct assay and 16.3% for the Friedewald equation. Conclusion: The direct LDL-C assay and the calculated LDL-C values did not display adequate agreement with the reference ultracentrifugation method. In subjects with TG >400 mg/dL, the direct assay overestimates the actual LDL-C whereas the Friedewald calculation underestimates the actual LDL. Clinical usage of these methods may lead to misclassification of the severity of dyslipidemia, resulting in improper management.

The effect of VLDL particles on the accuracy of a direct LDL-cholesterol method in type 2 diabetic patients

Objective To assess the accuracy of the direct method LDL-c Plus, in type 2 diabetic patients. Methods LDL-c Plus was measured in 64 consecutive samples of type 2 diabetic patients and compared with betaquantification (BQ), Friedewald’s and an alternative formula. LDL-c Plus was also measured in the VLDL (d<1.006 Kg/L) fraction of these samples and in total serum and the VLDL fraction of a phenotype III patient, before and after diluting it with saline or VLDL from normolipidemic subjects. Results LDL-c Plus showed a significant, constant bias (-8.5 ± 5.6%) against BQ which correlated with VLDL-cholesterol/total triglyceride ratio (r = 0.760, p < 0.0005); bias decreased to zero when the ratio increased. In the VLDL fraction of the diabetic patients and the phenotype III patient LDL-c Plus measured 20.7 ± 11.6% and 56.2% of the cholesterol, respectively. Dilution with saline did not alter the latter percentage, whereas dilution with normolipidemic VLDL reduced it showing that LDL-c Plus recognized cholesterol-enriched particles in the d<1.006 Kg/L. Friedewald’s formula also showed a significant, constant bias (-3.1 ± 6.4%) against BQ, whereas the alternative formula did not (0.5 ± 6.1%). Both calculations classified patients better than LDL-c Plus did at NCEP cut-off points. Conclusions In type 2 diabetic patients, LDL-c Plus underestimates LDL-c but measures cholesterol associated to IDL particles in the d<1.006 Kg/L fraction. Although LDLc-Plus might be a better cardiovascular risk estimator when well standardized, at the moment, it does not seem to be superior to calculations.

Evaluation of a Homogeneous Direct LDL-Cholesterol Assay in Diabetic Patients: Effect of Glycemic Control

Increased serum LDL-cholesterol (LDL-C) is associated with increased risk for ischemic heart disease, and lowering of LDL-C has been shown to decrease mortality in patients with known coronary heart disease (1)(2)(3). Persons with diabetes have a greatly increased risk for atherosclerosis and its complications. Many of these patients have increased plasma cholesterol (including LDL-C) and triglycerides (TGs). Additionally, glycation and oxidation of circulating LDL particles may further increase the risk for atherosclerotic disease in patients with diabetes (4)(5). Currently used methods for LDL-C include calculations [based on total cholesterol (TC), HDL-cholesterol (HDL-C), and TG concentrations], ultracentrifugation, and most recently, direct LDL-C assays. Calculation methods, such at the Friedewald formula (6), are well known to have a prominent negative bias in patients with TG concentrations >4.5 mmol/L (400 mg/dL) (6)(7). In addition, because TGs are measured, fasting blood samples are preferred. This can present a problem for certain patient populations, including many people with diabetes. Ultracentrifugation methods are time-consuming and expensive and generally are performed only in reference laboratories. Direct LDL-C assays have been developed recently and have been shown to provide accurate and precise measurements of LDL-C (8)(9)(10)(11)(12). They overcome the TG and fasting limitations of calculation methods, are readily adapted to clinical laboratories, are less expensive than ultracentrifugation methods, and provide greatly improved turnaround time. Direct LDL-C assays make use of either an immunoseparation step or specific detergents to separate LDL particles from other lipoproteins, followed by measurement of cholesterol by conventional enzymatic reactions. The N-geneousTM LDL-C assay is a solute-based homogeneous assay that agrees well with ultracentrifugation (11). The purpose of the present study was to evaluate the effect of glycemic control (as gauged by hemoglobin A1c) on the …

Reflex testing I: Algorithm for lipid and lipoprotein measurement in coronary heart disease risk assessment

We reviewed the current literature in order to construct a reflex testing algorithm that maximizes clinical utility and cost-effectiveness of lipid and lipoprotein testing. The algorithm was based on the 2nd Report of the National Cholesterol Education Program Adult Treatment Panel guidelines for use of total cholesterol (TC), triglycerides (TG), HDL-C, and LDL-C, and published reports describing the clinical use of apolipoprotein B and lipoprotein (a). The success of this algorithm was tested in a low-risk general and a high-risk hyperlipidemic patient population. Lipid data and non-lipid risk factors were obtained from a national database and from patients seen at two lipid clinics. A total of 16 968 individuals from the National Health and Nutrition Examination Survey III database comprised the low-risk group, and 239 patients examined in the Hartford Hospital and Washington University Lipid Clinics comprised the high-risk group. We found a solid scientific base to support the NCEP guidelines and reasonable support for limited testing of apoB and Lp(a). According to the algorithm, the direct LDL-C assay was deemed unnecessary in 98% and 91% of low- and high-risk subjects, respectively, if one assumes that the Friedewald equation is adequate with TG≤4.00 g/l. With a more conservative cutoff of TG≤2.50 g/l, the algorithm canceled 92% and 81% of direct LDL tests, respectively. The algorithm also limited TG to 20 and 64%, apoB to 6 and 20%, and Lp(a) to 15 and 56%, of low- and high-risk groups, respectively. Use of a comprehensive, reflex algorithm for coronary heart disease risk assessment will substantially reduce the utilization of laboratory services without diminishing the clinical value of these tests. The algorithm will prevent the overuse of certain expensive tests (direct LDL) while promoting the limited use of underutilized tests [apoB and Lp(a)].

Analytical and clinical performance of a homogeneous enzymatic LDL-cholesterol assay compared with the ultracentrifugation-dextran sulfate-Mg2+ method

LDL-cholesterol (LDL-C) concentration is currently determined in most clinical laboratories by the Friedewald calculation. This approach has several limitations and may not meet the current total error requirement in LDL-C measurement of < or = 12%. We evaluated the analytical and clinical performance of the direct N-geneous LDL-C assay (Equal Diagnostics). The N-geneous method correlated highly with the modified beta-quantification assay (r = 0.95; y = 0.91x + 70.6 mg/L; n = 199), showed no significant effect of increased triglyceride or other common interferants, and performed adequately in serum samples from nonfasting individuals. This assay demonstrated a mean total error of 6.75% over a wide range of LDL-C concentrations. In addition, at the medical decision cutoff points, this LDL-C assay showed positive predictive values of 78-95% and negative predictive values of 84-99%. We conclude that the N-geneous LDL-C meets the currently established analytical performance goals and appears to have a role in the diagnosis and management of hypercholesterolemic patients.