Quantification of lipoprotein lipase in mouse plasma with a sandwich enzyme-linked immunosorbent assay.

To support in vivo and in vitro studies of intravascular triglyceride metabolism in mice, we created rat monoclonal antibodies (mAbs) against mouse LPL. Two mAbs, mAbs 23A1 and 31A5, were used to develop a sandwich ELISA for mouse LPL. The detection of mouse LPL by the ELISA was linear in concentrations ranging from 0.31 ng/ml to 20 ng/ml. The sensitivity of the ELISA made it possible to quantify LPL in serum and in both pre-heparin and post-heparin plasma samples (including in grossly lipemic samples). LPL mass and activity levels in the post-heparin plasma were lower in Gpihbp1-/- mice than in wild-type mice. In both groups of mice, LPL mass and activity levels were positively correlated. Our mAb-based sandwich ELISA for mouse LPL will be useful for any investigator who uses mouse models to study LPL-mediated intravascular lipolysis.

Quantification of soluble very low-density lipoprotein receptor in human serum using a sandwich enzyme-linked immunosorbent assay.

To investigate the regulation of soluble very low-density lipoprotein receptor (sVLDL-R), which is cleaved mostly from the extracellular domain of VLDL-R II, we generated two rat monoclonal antibodies (mAbs) against human sVLDL-R, and used them to develop a sandwich enzyme-linked immunosorbent assay (ELISA) to measure sVLDL-R levels in human serum or plasma. The ELISA had a linear range from 0.20 ng/mL to 13.02 ng/mL and allowed for the quantification of sVLDL-R in serum and culture cell medium. The coefficient of variation (CV) was less than 10% for both the intra- and inter-assays. The bililubin F, and C, triglyceride (TG), and hemoglobin levels did not affect assay precision. The sVLDL-R concentration was negatively associated with body fat percentage, TG, and HbA1c, suggesting the possibility of obesity and diabetes in middle-aged Japanese women.

The lipoprotein lipase that is shuttled into capillaries by GPIHBP1 enters the glycocalyx where it mediates lipoprotein processing.

Lipoprotein lipase (LPL), the enzyme that carries out the lipolytic processing of triglyceride-rich lipoproteins (TRLs), is synthesized by adipocytes and myocytes and secreted into the interstitial spaces. The LPL is then bound by GPIHBP1, a GPI-anchored protein of endothelial cells (ECs), and transported across ECs to the capillary lumen. The assumption has been that the LPL that is moved into capillaries remains attached to GPIHBP1 and that GPIHBP1 serves as a platform for TRL processing. In the current studies, we examined the validity of that assumption. We found that an LPL-specific monoclonal antibody (mAb), 88B8, which lacks the ability to detect GPIHBP1-bound LPL, binds avidly to LPL within capillaries. We further demonstrated, by confocal microscopy, immunogold electron microscopy, and nanoscale secondary ion mass spectrometry analyses, that the LPL detected by mAb 88B8 is located within the EC glycocalyx, distant from the GPIHBP1 on the EC plasma membrane. The LPL within the glycocalyx mediates the margination of TRLs along capillaries and is active in TRL processing, resulting in the delivery of lipoprotein-derived lipids to immediately adjacent parenchymal cells. Thus, the LPL that GPIHBP1 transports into capillaries can detach and move into the EC glycocalyx, where it functions in the intravascular processing of TRLs.

Hypertriglyceridemia in Apoa5-/- mice results from reduced amounts of lipoprotein lipase in the capillary lumen.

Why apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia has remained unclear, but we have suspected that the underlying cause is reduced amounts of lipoprotein lipase (LPL) in capillaries. By routine immunohistochemistry, we observed reduced LPL staining of heart and brown adipose tissue (BAT) capillaries in Apoa5-/- mice. Also, after an intravenous injection of LPL-, CD31-, and GPIHBP1-specific mAbs, the binding of LPL Abs to heart and BAT capillaries (relative to CD31 or GPIHBP1 Abs) was reduced in Apoa5-/- mice. LPL levels in the postheparin plasma were also lower in Apoa5-/- mice. We suspected that a recent biochemical observation - that APOA5 binds to the ANGPTL3/8 complex and suppresses its capacity to inhibit LPL catalytic activity - could be related to the low intracapillary LPL levels in Apoa5-/- mice. We showed that an ANGPTL3/8-specific mAb (IBA490) and APOA5 normalized plasma triglyceride (TG) levels and intracapillary LPL levels in Apoa5-/- mice. We also showed that ANGPTL3/8 detached LPL from heparan sulfate proteoglycans and GPIHBP1 on the surface of cells and that the LPL detachment was blocked by IBA490 and APOA5. Our studies explain the hypertriglyceridemia in Apoa5-/- mice and further illuminate the molecular mechanisms that regulate plasma TG metabolism.

GPIHBP1 autoantibody is an independent risk factor for the recurrence of hypertriglyceridemia-induced acute pancreatitis.

BACKGROUND: GPIHBP1, a glycolipid-anchored protein of capillary endothelial cells, is a crucial partner for lipoprotein lipase (LPL) in plasma triglyceride metabolism. GPIHBP1 autoantibodies block LPL binding to GPIHBP1 and lead to severe hypertriglyceridemia (HTG) and HTG-induced acute pancreatitis (HTG-AP). We sought to define the incidence of GPIHBP1 autoantibodies in patients with HTG-AP. OBJECTIVE: We determined the incidence of GPIHBP1 autoantibody in HTG-AP patients, and compared the clinical features and long-term outcomes between GPIHBP1 autoantibody-positive and negative groups. METHODS: An enzyme-linked immunosorbent assay was used to screen for GPIHBP1 autoantibody in 116 HTG-AP patients hospitalized from Jan 1, 2015 to Aug 31, 2019. All patients were followed up for 24 months. The primary outcome was the recurrence rate of HTG-AP during the two-year follow-up period. The incidence of recurrent episodes was analyzed by the Kaplan-Meier method and multivariable Cox regression was used to identify risk factors. RESULTS: GPIHBP1 autoantibodies were present in 17 of 116 study patients (14.66%). The 2-year recurrence rate of HTG-AP was much higher in the GPIHBP1 autoantibody-positive group (35%, 6 in 17) than in the negative group (4%, 4 in 99). The multivariable Cox regression analysis showed that GPIHBP1 autoantibody was an independent risk factor for HTG-AP recurrence in two years. CONCLUSIONS: The presence of GPIHBP1 autoantibody is common in patients with HTG-AP, and is an independent risk factor for two-year recurrence of HTG-AP.

Electrostatic sheathing of lipoprotein lipase is essential for its movement across capillary endothelial cells.

GPIHBP1, an endothelial cell (EC) protein, captures lipoprotein lipase (LPL) within the interstitial spaces (where it is secreted by myocytes and adipocytes) and transports it across ECs to its site of action in the capillary lumen. GPIHBP1's 3-fingered LU domain is required for LPL binding, but the function of its acidic domain (AD) has remained unclear. We created mutant mice lacking the AD and found severe hypertriglyceridemia. As expected, the mutant GPIHBP1 retained the capacity to bind LPL. Unexpectedly, however, most of the GPIHBP1 and LPL in the mutant mice was located on the abluminal surface of ECs (explaining the hypertriglyceridemia). The GPIHBP1-bound LPL was trapped on the abluminal surface of ECs by electrostatic interactions between the large basic patch on the surface of LPL and negatively charged heparan sulfate proteoglycans (HSPGs) on the surface of ECs. GPIHBP1 trafficking across ECs in the mutant mice was normalized by disrupting LPL-HSPG electrostatic interactions with either heparin or an AD peptide. Thus, GPIHBP1's AD plays a crucial function in plasma triglyceride metabolism; it sheathes LPL's basic patch on the abluminal surface of ECs, thereby preventing LPL-HSPG interactions and freeing GPIHBP1-LPL complexes to move across ECs to the capillary lumen.

Lipoprotein Profiles before Heparin Administration in Patients with or without Coronary Thrombosis Following Atherosclerosis.

Objective: The correlation between lipoproteins and arterial thrombosis is not fully elucidated, and no data exist in terms of lipoprotein profiles before heparin administration in patients with coronary arterial thrombosis (CAT). This cross-sectional study aimed to evaluate the lipoprotein profile before heparin administration in 63 ST-segment elevation myocardial infarction (STEMI) patients with CAT. Methods: The lipoprotein profile was measured via polyacrylamide gel electrophoresis prior to heparin administration for primary percutaneous coronary intervention for STEMI. Age- and sex-matched subjects with <25% stenosis in stable coronary artery disease were enrolled as controls. Results: In the pre-heparin serum, the fraction of very-low-density lipoprotein (P=0.75) in STEMI patients was not different from that in controls, and the fraction of intermediate-density lipoprotein (P<0.01) in STEMI patients was significantly lower than that in controls. Although the fraction of small dense low-density lipoprotein (s-LDL) in STEMI patients was significantly higher than that in controls (P<0.01), 44% (28/63) of STEMI patients were negative for s-LDL. Conclusion: Although lipoproteins are a risk factor for atherosclerosis, lipoprotein profiles with CAT following atherosclerosis in STEMI are different from those profiles without CAT in stable coronary artery disease.

Endothelial Lipase Exerts its Anti-Atherogenic Effect through Increased Catabolism of ß-VLDLs.

AIM: Endothelial lipase (EL) plays an important role in lipoprotein metabolism. Our recent study showed that increased hepatic expression of EL attenuates diet-induced hypercholesterolemia, thus subsequently reducing atherosclerosis in transgenic (Tg) rabbits. However, it is yet to be determined whether increased EL activity itself per se is anti-atherogenic or whether the anti-atherogenic effect of EL is exclusively dependent on its lipid-lowering effect. METHODS: To determine the mechanisms underlying EL-mediated anti-atherogenic effect, we fed Tg and non-Tg rabbits diets containing different amounts of cholesterol to make their plasma cholesterol levels similarly high. Sixteen weeks later, we examined their lipoprotein profiles and compared their susceptibility to atherosclerosis. RESULTS: With Tg and non-Tg rabbits having hypercholesterolemia, the plasma lipids and lipoprotein profiles were observed to be similar, while pathological examinations revealed that lesion areas of both aortic and coronary atherosclerosis of Tg rabbits were not significantly different from non-Tg rabbits. Moreover, Tg rabbits exhibited faster clearance of DiI-labeled ß-VLDLs than non-Tg rabbits. CONCLUSION: The results of our study suggest that the enhancement of ß-VLDL catabolism is the major mechanism for atheroprotective effects of EL in Tg rabbits.

Association between high normal-range thyrotropin concentration and carotid intima-media thickness in euthyroid premenopausal, perimenopausal and postmenopausal women.

OBJECTIVE: There are currently no established cutoff levels for thyrotropin (TSH) within the reference intervals associated with carotid atherosclerosis to prevent the onset of cardiovascular diseases. The present study aimed to determine the TSH cutoff level associated with carotid maximum intima-media thickness (max IMT) in euthyroid premenopausal, perimenopausal and postmenopausal women. STUDY DESIGN: We conducted a cross-sectional study of 468 euthyroid women who had not been treated for or diagnosed with cardiovascular diseases and/or metabolic disorders among 1221 Japanese women who participated in a comprehensive medical examination at the Hidaka Hospital, Japan. Participants' weight, blood pressure, plasma glucose, serum lipoprotein, free thyroxine and TSH were measured and an interview about menstruation was conducted. Carotid ultrasonography was performed to determine max IMT. RESULTS: Max IMT significantly increased stepwise as menopausal status progressed (p < 0.001). Serum TSH levels were significantly higher in participants with carotid plaques, defined as max IMT ≥1.1 mm (p = 0.038), and were independently associated with the presence of carotid plaque using multivariate logistic regression analysis (ß =1.218, p = 0.036). In postmenopausal women, significantly higher carotid max IMT values were observed in women with serum TSH ≥2.5 µIU/mL compared with women with concentrations <2.5 µIU/mL (p = 0.018) without elevated total cholesterol and low-density lipoprotein cholesterol concentrations. These differences were not observed in premenopausal women. CONCLUSIONS: Laboratory finding of serum TSH concentration ≥2.5 µIU/mL may be useful to assess risk of atherosclerosis, especially in postmenopausal women.

Chylomicronemia from GPIHBP1 autoantibodies.

Some cases of chylomicronemia are caused by autoantibodies against glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1), an endothelial cell protein that shuttles LPL to the capillary lumen. GPIHBP1 autoantibodies prevent binding and transport of LPL by GPIHBP1, thereby disrupting the lipolytic processing of triglyceride-rich lipoproteins. Here, we review the "GPIHBP1 autoantibody syndrome" and summarize clinical and laboratory findings in 22 patients. All patients had GPIHBP1 autoantibodies and chylomicronemia, but we did not find a correlation between triglyceride levels and autoantibody levels. Many of the patients had a history of pancreatitis, and most had clinical and/or serological evidence of autoimmune disease. IgA autoantibodies were present in all patients, and IgG4 autoantibodies were present in 19 of 22 patients. Patients with GPIHBP1 autoantibodies had low plasma LPL levels, consistent with impaired delivery of LPL into capillaries. Plasma levels of GPIHBP1, measured with a monoclonal antibody-based ELISA, were very low in 17 patients, reflecting the inability of the ELISA to detect GPIHBP1 in the presence of autoantibodies (immunoassay interference). However, GPIHBP1 levels were very high in five patients, indicating little capacity of their autoantibodies to interfere with the ELISA. Recently, several GPIHBP1 autoantibody syndrome patients were treated successfully with rituximab, resulting in the disappearance of GPIHBP1 autoantibodies and normalization of both plasma triglyceride and LPL levels. The GPIHBP1 autoantibody syndrome should be considered in any patient with newly acquired and unexplained chylomicronemia.

The antagonic behavior of GPIHBP1 between EAT and circulation does not reflect lipolytic enzymes levels in the tissue and serum from coronary patients.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of morbidity and mortality worldwide. Recently, triglyceride rich lipoproteins are proposed to contribute to CAD risk; its concentrations would be partly determined by lipoprotein lipase (LPL) and endothelial lipase (EL). Epicardial adipose tissue (EAT), a visceral AT surrounding myocardium and coronary arteries, emerged as an important actor in CAD; the increase in its volume could be a consequence of LPL and EL. Circulating enzymes levels would be conditioned by local tissue factors. Our aim was to evaluate LPL, EL and their regulators levels in serum and EAT from CAD patients, searching for possible parallelisms and their role in the lipoprotein profile. METHODS: In serum, EAT and subcutaneous AT (SAT) from patients undergoing coronary artery bypass graft (CABG, n = 25) or valve replacement (No CABG, n = 25), LPL, EL and glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein-1 (GPIHBP1) expression were evaluated. Besides, Apoprotein (Apo)CII, CIII and AV were determined in serum, along with lipoprotein profile. RESULTS: Insulin-resistance markers were higher in CABG (p < 0.05). Serum LPL levels were decreased (p = 0.045), while EL levels increased (p < 0.001) in CABG, without differences in EAT or SAT. Circulating GPIHBP1 concentrations were decreased in CABG (p = 0.047), while EAT GPIHBP1 expression was increased (p < 0.001). ApoCII and ApoAV concentrations were higher in CABG (p = 0.016 and p = 0.047, respectively), without differences in ApoCIII concentrations between groups. CONCLUSIONS: In EAT, LPL and EL protein levels were not changed in CAD, although GPIHBP1 protein levels were higher. EAT would be a minor contributor to the circulating levels of the enzymes.

Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Intermittent chylomicronemia caused by intermittent GPIHBP1 autoantibodies.

Chylomicronemia caused by a deficiency in lipoprotein lipase (LPL) or GPIHBP1 (the endothelial cell protein that transports LPL to the capillary lumen) is typically diagnosed during childhood and represents a serious, lifelong medical problem. Affected patients have high plasma triglyceride levels (>1500 mg/dL) and a high risk of acute pancreatitis. However, chylomicronemia frequently presents later in life in the absence of an obvious monogenic cause. In these cases, the etiology for the chylomicronemia is presumed to be "multifactorial" (involving diabetes, drugs, alcohol, or polygenic factors), but on a practical level, the underlying cause generally remains a mystery. Here, we describe a 15-year-old female with chylomicronemia caused by GPIHBP1 autoantibodies (which abolish LPL transport to the capillary lumen). Remarkably, chylomicronemia in this patient was intermittent, interspersed between periods when the plasma triglyceride levels were normal. GPIHBP1 autoantibodies were easily detectable during episodes of chylomicronemia but were undetectable during periods of normotriglyceridemia. During the episodes of chylomicronemia (when GPIHBP1 autoantibodies were present), plasma LPL levels were low, consistent with impaired LPL transport into capillaries. During periods of normotriglyceridemia, when GPIHBP1 autoantibodies were absent, plasma LPL levels normalized. Because the chylomicronemia in this patient was accompanied by debilitating episodes of acute pancreatitis, the patient was ultimately treated with immunosuppressive drugs, which resulted in disappearance of GPIHBP1 autoantibodies and normalization of plasma triglyceride levels. GPIHBP1 autoantibodies need to be considered in patients who present with unexplained acquired cases of chylomicronemia.

Association of cholesterol uptake capacity, a novel indicator for HDL functionality, and coronary plaque properties: An optical coherence tomography-based observational study.

BACKGROUND: Cholesterol efflux from atherosclerotic lesion is a key function of high-density lipoprotein (HDL). Recently, we established a simple, high-throughput, cell-free assay to evaluate the capacity of HDL to accept additional cholesterol, which is herein referred to as "cholesterol uptake capacity (CUC)". OBJECTIVE: To clarify the cross-sectional relationship between CUC and coronary plaque properties. METHODS: We enrolled 135 patients to measure CUC and assess the morphological features of angiographic stenosis by optical coherence tomography (OCT). We estimated the extent of the lipid-rich plaque by multiplying the mean lipid arc by lipid length (lipid index). The extent of the OCT-detected macrophage accumulation in the target plaque was semi-quantitatively estimated using a grading system. RESULTS: Lipid-rich plaque lesions were identified in 125 patients (92.6%). CUC was inversely associated with the lipid index (R = -0.348, P < 0.0001). In addition, CUC was also inversely associated with macrophage score (R = -0.327, P < 0.0001). Conversely, neither circulating levels of HDL cholesterol nor apoA1 showed a similar relationship. CONCLUSIONS: We demonstrated that CUC was inversely related to lipid-rich plaque burden and the extent of macrophage accumulation, suggesting that CUC could be useful for cardiovascular risk stratification.

Activation of NK cells in male cancer survivors by fucoidan extracted from Cladosiphon okamuranus.

Cancer survivors are highly motivated to seek information about the use of dietary supplements and complementary nutritional therapies to improve their quality of life. Fucoidan, a sulfated polysaccharide extracted from brown marine alga, exhibits a wide range of bioactivities, including anticancer activity. As natural killer (NK) cells serve an important role in defenses against tumor cells, the present study examined the effects of fucoidan extracted from Cladosiphon Okamuranus on NK cell activity in cancer survivors. A prospective, open-label clinical study was conducted on cancer survivors treated with fucoidan via oral administration; 11 cancer survivors with a performance status of 0 or 1 participated and consumed 3 g of fucoidan for 6 months. No significant changes were observed in the mean activities of NK cells in total subjects following the ingestion of fucoidan. An analysis of each sex revealed that NK cell activity was significantly increased by the ingestion of fucoidan in male, yet not female subjects. Serum fucoidan levels were markedly increased following the ingestion of fucoidan and the peak levels ranged between 30 and 198 ng/ml. Tumor markers remained within the reference range during the trial period in subjects, in whom primary tumors were eradicated by treatment. The basal values of tumor markers were elevated in three cases; tumor markers were increased in two cases and decreased in one by the ingestion of fucoidan. These findings suggest that fucoidan enhances the activation of NK cells in male cancer survivors.

Serum concentration of full-length- and carboxy-terminal fragments of endothelial lipase predicts future cardiovascular risks in patients with coronary artery disease.

BACKGROUND: Endothelial lipase (EL), a regulator of plasma high-density lipoprotein cholesterol (HDL-C), is secreted as a 68-kDa mature glycoprotein, and then cleaved by proprotein convertases. However, the clinical significance of the circulating EL fragments remains unclear. OBJECTIVE: The objective of this study was to analyze the impact of serum EL fragments on HDL-C levels and major adverse cardiovascular events (MACE). METHODS: Using novel monoclonal antibodies (RC3A6) against carboxy-terminal EL protein, we have established a new enzyme-linked immunosorbent assay (ELISA) system, which can detect both full-length EL protein (full EL) and carboxy-terminal truncated fragments (total EL) in serum. The previous sandwich ELISA detected only full EL. The full and total EL mass were measured in 556 patients with coronary artery disease. Among them, 272 patients who underwent coronary intervention were monitored for 2 years for MACE. RESULTS: There was a significant correlation between serum full and total EL mass (R = 0.45, P < .0001). However, the total EL mass showed a stronger inverse correlation with serum HDL-cholesterol concentration than the full EL mass (R = -0.17 vs -0.02). Kaplan-Meier analysis documented an association of serum total EL mass and MACE (log-rank P = .037). When an optimal cutoff value was set at 96.23 ng/mL, total EL mass was an independent prognostic factor for MACE in the Cox proportional hazard model (HR; 1.75, 95% CI; 1.10-2.79, P = .018). CONCLUSION: Serum total EL mass could be a predictor for MACE in patients with coronary artery disease. This novel ELISA will be useful for further clarifying the impact of EL on HDL metabolism and atherosclerosis.

GPIHBP1 and Lipoprotein Lipase, Partners in Plasma Triglyceride Metabolism.

Lipoprotein lipase (LPL), identified in the 1950s, has been studied intensively by biochemists, physiologists, and clinical investigators. These efforts uncovered a central role for LPL in plasma triglyceride metabolism and identified LPL mutations as a cause of hypertriglyceridemia. By the 1990s, with an outline for plasma triglyceride metabolism established, interest in triglyceride metabolism waned. In recent years, however, interest in plasma triglyceride metabolism has awakened, in part because of the discovery of new molecules governing triglyceride metabolism. One such protein-and the focus of this review-is GPIHBP1, a protein of capillary endothelial cells. GPIHBP1 is LPL's essential partner: it binds LPL and transports it to the capillary lumen; it is essential for lipoprotein margination along capillaries, allowing lipolysis to proceed; and it preserves LPL's structure and activity. Recently, GPIHBP1 was the key to solving the structure of LPL. These developments have transformed the models for intravascular triglyceride metabolism.

Direct Versus Calculated LDL Cholesterol and C-Reactive Protein in Cardiovascular Disease Risk Assessment in the Framingham Offspring Study.

BACKGROUND: Increases in circulating LDL cholesterol (LDL-C) and high-sensitivity C-reactive protein (hsCRP) concentrations are significant risk factors for cardiovascular disease (CVD). We assessed direct LDL-C and hsCRP concentrations compared to standard risk factors in the Framingham Offspring Study. METHODS: We used stored frozen plasma samples (-80 °C) obtained after an overnight fast from 3147 male and female participants (mean age, 58 years) free of CVD at cycle 6 of the Framingham Offspring Study. Overall, 677 participants (21.5%) had a CVD end point over a median of 16.0 years of follow-up. Total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDL-C), direct LDL-C (Denka Seiken and Kyowa Medex methods), and hsCRP (Dade Behring method) concentrations were measured by automated analysis. LDL-C was also calculated by both the Friedewald and Martin methods. RESULTS: Considering all CVD outcomes on univariate analysis, significant factors included standard risk factors (age, hypertension, HDL-C, hypertension treatment, sex, diabetes, smoking, and TC concentration) and nonstandard risk factors (non-HDL-C, direct LDL-C and calculated LDL-C, TG, and hsCRP concentrations). On multivariate analysis, only the Denka Seiken direct LDL-C and the Dade Behring hsCRP were still significant on Cox regression analysis and improved the net risk reclassification index, but with modest effects. Discordance analysis confirmed the benefit of the Denka Seiken direct LDL-C method for prospective hard CVD endpoints (new-onset myocardial infarction, stroke, and/or CVD death). CONCLUSIONS: Our data indicate that the Denka Seiken direct LDL-C and Dade Behring hsCRP measurements add significant, but modest, information about CVD risk, compared to standard risk factors and/or calculated LDL-C.