Cholesteryl ester transfer protein inhibitory oxoacetamido-benzamide derivatives: Glide docking, pharmacophore mapping, and synthesis

Abstract Dyslipidemia is an abnormal lipid profile associated with many common diseases, including coronary heart disease and atherosclerosis. Cholesteryl ester transfer protein (CETP) is a hydrophobic plasma glycoprotein that is responsible for the transfer of cholesteryl ester from high-density lipoprotein athero-protective particles to pro-atherogenic very low-density lipoprotein and low-density lipoprotein particles. The requirement for new CETP inhibitors, which block this process has driven our current work. Here, the synthesis as well as the ligand-based and structure-based design of seven oxoacetamido-benzamides 9a-g with CETP inhibitory activity is described. An in vitro study demonstrated that most of these compounds have appreciable CETP inhibitory activity. Compound 9g showed the highest inhibitory activity against CETP with an IC50 of 0.96 µM. Glide docking data for compounds 9a-g and torcetrapib provide evidence that they are accommodated in the CETP active site where hydrophobic interactions drive ligand/CETP complex formation. Furthermore, compounds 9a-g match the features of known CETP active inhibitors, providing a rationale for their high docking scores against the CETP binding domain. Therefore, these oxoacetamido-benzamides show potential for use as novel CETP inhibitors

HDL Particle Subspecies and Their Association With Incident Type 2 Diabetes: The PREVEND Study.

CONTEXT: High-density lipoproteins (HDL) may be protective against type 2 diabetes (T2D) development, but HDL particles vary in size and function, which could lead to differential associations with incident T2D. A newly developed nuclear magnetic resonance (NMR)-derived algorithm provides concentrations for 7 HDL subspecies. OBJECTIVE: We aimed to investigate the association of HDL particle subspecies with incident T2D in the general population. METHODS: Among 4828 subjects of the Prevention of Renal and Vascular End-Stage Disease (PREVEND) study without T2D at baseline, HDL subspecies with increasing size from H1P to H7P were measured by NMR (LP4 algorithm of the Vantera NMR platform). RESULTS: A total of 265 individuals developed T2D (median follow-up of 7.3 years). In Cox regression models, HDL size and H4P (hazard ratio [HR] per 1 SD increase 0.83 [95% CI, 0.69-0.99] and 0.85 [95% CI, 0.75-0.95], respectively) were inversely associated with incident T2D, after adjustment for relevant covariates. In contrast, levels of H2P were positively associated with incident T2D (HR 1.15 [95% CI, 1.01-1.32]). In secondary analyses, associations with large HDL particles and H6P were modified by body mass index (BMI) in such a way that they were particularly associated with a lower risk of incident T2D, in subjects with BMI < 30 kg/m2. CONCLUSION: Greater HDL size and lower levels of H4P were associated with a lower risk, whereas higher levels of H2P were associated with a higher risk of developing T2D. In addition, large HDL particles and H6P were inversely associated with T2D in nonobese subjects.

High-Density Lipoproteins as Homeostatic Nanoparticles of Blood Plasma.

It is well known that blood lipoproteins (LPs) are multimolecular complexes of lipids and proteins that play a crucial role in lipid transport. High-density lipoproteins (HDL) are a class of blood plasma LPs that mediate reverse cholesterol transport (RCT)-cholesterol transport from the peripheral tissues to the liver. Due to this ability to promote cholesterol uptake from cell membranes, HDL possess antiatherogenic properties. This function was first observed at the end of the 1970s to the beginning of the 1980s, resulting in high interest in this class of LPs. It was shown that HDL are the prevalent class of LPs in several types of living organisms (from fishes to monkeys) with high resistance to atherosclerosis and cardiovascular disorders. Lately, understanding of the mechanisms of the antiatherogenic properties of HDL has significantly expanded. Besides the contribution to RCT, HDL have been shown to modulate inflammatory processes, blood clotting, and vasomotor responses. These particles also possess antioxidant properties and contribute to immune reactions and intercellular signaling. Herein, we review data on the structure and mechanisms of the pleiotropic biological functions of HDL from the point of view of their evolutionary role and complex dynamic nature.

ApoE and apoC-III-defined HDL subtypes: a descriptive study of their lecithin cholesterol acyl transferase and cholesteryl ester transfer protein content and activity.

BACKGROUND: The functionality of high-density lipoproteins (HDL) is a better cardiovascular risk predictor than HDL concentrations. One of the key elements of HDL functionality is its apolipoprotein composition. Lecithin-cholesterol acyl transferase (LCAT) and cholesterol-ester transfer protein (CETP) are enzymes involved in HDL-mediated reverse cholesterol transport. This study assessed the concentration and activity of LCAT and CETP in HDL subspecies defined by their content of apolipoproteins E (apoE) and C-III (apoC-III) in humans. METHODS: Eighteen adults (ten women and eight men, mean age 55.6, BMI 26.9 Kg/m2, HbA1c 5.4%) were studied. HDL from each participant were isolated and divided into four subspecies containing respectively: No apoE and no apoC-III (E-C-), apoE but not apoC-III (E + C-), apoC-III but no apoE (E-C+) and both apoE and apoC-III (E + C+). The concentration and enzymatic activity of LCAT and CETP were measured within each HDL subspecies using immunoenzymatic and fluorometric methods. Additionally, the size distribution of HDL in each apolipoprotein-defined fraction was determined using non-denaturing electrophoresis and anti-apoA-I western blotting. RESULTS: HDL without apoE or apoC-III was the predominant HDL subtype. The size distribution of HDL was very similar in all the four apolipoprotein-defined subtypes. LCAT was most abundant in E-C- HDL (3.58 mg/mL, 59.6% of plasma LCAT mass), while HDL with apoE or apoC-III had much less LCAT (19.8, 12.2 and 8.37% of plasma LCAT respectively for E + C-, E-C+ and E + C+). LCAT mass was lower in E + C- HDL relative to E-C- HDL, but LCAT activity was similar in both fractions, signaling a greater activity-to-mass ratio associated with the presence of apoE. Both CETP mass and CETP activity showed only slight variations across HDL subspecies. There was an inverse correlation between plasma LCAT activity and concentrations of both E-C+ pre-beta HDL (r = - 0.55, P = 0.017) and E-C- alpha 1 HDL (r = - 0.49, P = 0.041). Conversely, there was a direct correlation between plasma CETP activity and concentrations of E-C+ alpha 1 HDL (r = 0.52, P = 0.025). CONCLUSIONS: The presence of apoE in small HDL is correlated with increased LCAT activity and esterification of plasma cholesterol. These results favor an interpretation that LCAT and apoE interact to enhance anti-atherogenic pathways of HDL.

Dihydro-sphingosine 1-phosphate interacts with carrier proteins in a manner distinct from that of sphingosine 1-phosphate.

Dihydro-sphingosine 1-phosphate (DH-S1P) is an analog of sphingosine 1-phosphate (S1P), which is a potent lysophospholipid mediator. DH-S1P has been proposed to exert physiological properties similar to S1P. Although S1P is known to be carried on HDL via apolipoprotein M (apoM), the association between DH-S1P and HDL/apoM has not been fully elucidated. Therefore, in the present study, we aimed to elucidate this association and to compare it with that of S1P and HDL/apoM. First, we investigated the distributions of S1P and DH-S1P among lipoproteins and lipoprotein-depleted fractions in human serum and plasma samples and observed that both S1P and DH-S1P were detected on HDL; furthermore, elevated amounts of DH-S1P in serum samples were distributed to the lipoprotein-depleted fraction to a greater degree than to the HDL fraction. Concordantly, a preference for HDL over albumin was only observed for S1P, and not for DH-S1P, when the molecules were secreted from platelets. Regarding the association with HDL, although both S1P and DH-S1P prefer to bind to HDL, HDL preferentially accepts S1P over DH-S1P. For the association with apoM, S1P was not detected on HDL obtained from apoM knockout mice, while DH-S1P was detected. Moreover, apoM retarded the degradation of S1P, but not of DH-S1P. These results suggest that S1P binds to HDL via apoM, while DH-S1P binds to HDL in a non-specific manner. Thus, DH-S1P is not a mere analog of S1P and might possess unique clinical significance.

Activity of paraoxonase 1 (PON1) on HDL2 and HDL3 subclasses in renal disease.

INTRODUCTION: Cardiovascular complications, as the main cause of mortality in renal patients, are followed with altered lipoproteins composition. Considering that paraoxonase-1 (PON1) is an anti-oxidative enzyme located mainly on HDL particles, the current study has aim to investigate whether failure of kidney function leads to changes in the distribution of PON1 activity between different HDL subclasses. MATERIALS AND METHODS: In 77 renal patients (21 chronic kidney disease (CKD) and 56 end stage renal disease (ESRD) patients on dialysis) and 20 healthy subjects PON1 activity on HDL2 and HDL3 subclasses was determined by zymogram method that combines gradient gel electrophoresis separation of HDL subclasses and measurement of PON1 activity in the same gel. RESULTS: Serum paraoxonase (p<0.01) and arylesterase activity (p<0.001) of PON1 as well as its concentration (p<0.01) were significantly lower in CKD and ESRD patients compared to controls. Relative proportion of HDL3 subclasses was higher in ESRD patients than in healthy participants, while HDL2 subclasses was significantly decreased in CKD (p<0.05) and ESRD (p<0.001) patients, as compared to controls. Furthermore, control subjects had higher PON1 activity on HDL2 (CKD and ESRD patients p<0.001) and HDL3 (CKD p<0.05; ESRD patients p<0.001) subclasses in comparison with the both patients groups. Also, significant negative correlation was found between paraoxonase activity of PON1 in serum and creatinine concentration (ρ=-0.373, p<0.01). CONCLUSIONS: This study showed that altered HDL subclasses distribution, changed PON1 activities on different HDL subclasses as well as diminished anti-oxidative protection could be important factors in atherosclerosis development in CKD and ESRD patients.

Association of High-Density Lipoprotein Subclasses with Carotid Intima-Media Thickness: Shimane CoHRE Study.

AIMS: Recent studies suggested that subclasses of high-density lipoprotein (HDL) may be a better biomarker to predict the risk of atherosclerotic disorders. We aimed to examine the association of HDL2- and HDL3-cholesterol (HDL2-C and HDL3-C) with carotid intima-media thickness (IMT) using a new method to quantify the HDL-C subclasses. METHODS: Participants were 657 Japanese subjects (434 women) who received a health examination (mean age: 73 years). Serum samples were analyzed by the homogenous assay for HDL-C and HDL3-C. HDL2-C was calculated indirectly by subtracting HDL3-C from HDL-C. HDL3-C measured by this assay was well correlated with that measured by ultracentrifugation (r=0.898, p＜0.001). The maximum IMT (max-IMT) and plaque score (PS) were evaluated by ultrasonography following the standard protocol. RESULTS: HDL3-C was associated with age both in men (r=－0.322, p＜0.0001) and women (r=－0.315, p＜0.0001). In a simple regression analysis, max-IMT showed an inverse association with HDL3-C, whereas no significant association was observed with HDL2-C. A multiple linear regression analysis indicated, however, that the association between HDL3-C and max-IMT was not significant in both aged and younger populations when age was included in the analysis. Further, not only HDL2-C but also HDL3-C was not a significant predictor of 'atherosclerotic arteries' defined as the max-IMT ≥1.5 mm. Similar results were observed in the analysis on PS. CONCLUSIONS: Neither HDL3-C nor HDL2-C was significantly associated with carotid atherosclerosis in the Japanese population in this study.

Relapsing-remitting multiple sclerosis patients display an altered lipoprotein profile with dysfunctional HDL.

Lipoproteins modulate innate and adaptive immune responses. In the chronic inflammatory disease multiple sclerosis (MS), reports on lipoprotein level alterations are inconsistent and it is unclear whether lipoprotein function is affected. Using nuclear magnetic resonance (NMR) spectroscopy, we analysed the lipoprotein profile of relapsing-remitting (RR) MS patients, progressive MS patients and healthy controls (HC). We observed smaller LDL in RRMS patients compared to healthy controls and to progressive MS patients. Furthermore, low-BMI (BMI ≤ 23 kg/m2) RRMS patients show increased levels of small HDL (sHDL), accompanied by larger, triglyceride (TG)-rich VLDL, and a higher lipoprotein insulin resistance (LP-IR) index. These alterations coincide with a reduced serum capacity to accept cholesterol via ATP-binding cassette (ABC) transporter G1, an impaired ability of HDL3 to suppress inflammatory activity of human monocytes, and modifications of HDL3's main protein component ApoA-I. In summary, lipoprotein levels and function are altered in RRMS patients, especially in low-BMI patients, which may contribute to disease progression in these patients.

Optical Properties of Europium Tetracycline Complexes in the Presence of High-Density Lipoproteins (HDL) Subfractions.

Standard lipoprotein measurements of triglycerides, total cholesterol, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) fail to identify many lipoprotein abnormalities that contribute to cardiovascular heart diseases (CHD). Studies suggested that the presence of CHD is more strongly associated with the HDL subspecies than with total HDL cholesterol levels. The HDL particles can be collected in at least three subfractions, the HDL2b, HDL2a, and HDL3. More specifically, atherosclerosis is associated with low levels of HDL2. In this work, the optical spectroscopic properties of europium tetracycline (EuTc) complex in the presence of different HDL subspecies was studied. The results show that the europium spectroscopic properties in the EuTc complex are influenced by sizes and concentrations of subclasses. Eu3+ emission intensity and lifetime can discriminate the subfractions HDL3 and HDL2b.

Effects of the cholesteryl ester transfer protein inhibitor, TA-8995, on cholesterol efflux capacity and high-density lipoprotein particle subclasses.

BACKGROUND: TA-8995 is a potent inhibitor of cholesteryl ester transfer protein (CETP) with beneficial effects on lipids and lipoproteins. The effect of TA-8995 on cholesterol efflux capacity (CEC), a measure of high-density lipoprotein (HDL) function, and HDL subparticle distribution is largely unknown. OBJECTIVE: To assess the effect of the CETP inhibitor TA-8995 on ABCA1- and non-ABCA1-driven CEC and on HDL particle distribution. METHODS: Total, non-ABCA1-, and ABCA1-specific CEC from J774 cells and HDL subclass distribution assessed by two-dimensional gel electrophoresis were measured at baseline and after 12-week treatment in 187 mild-dyslipidemic patients randomized to placebo, 1 mg, 5 mg, 10 mg TA-8995, or 10 mg TA-8995 combined with 10 mg rosuvastatin (NCT01970215). RESULTS: Compared with placebo, total, non-ABCA1-, and ABCA1-specific CEC were increased dose dependently by up to 38%, 72%, and 28%, respectively, in patients randomized to 10 mg of TA-8995. PreBeta-1 HDL, the primary acceptor for ABCA1-driven cholesterol efflux, was increased by 36%. This increase in preBeta-1 HDL correlated significantly with the total and the ABCA1-driven CEC increase, whereas the high-density lipoprotein cholesterol (HDL-C) increase did not. CONCLUSION: TA-8995 dose dependently increased not only total and non-ABCA1-specific CEC but also ABCA1-specific CEC and preBeta-1 HDL particle levels. These findings suggest that TA-8995 not only increases HDL-C levels but also promotes functional properties of HDL particles. This CETP inhibitor-driven preBeta-1 HDL increase is an important predictor of both ABCA1 and total CEC increase, independent of HDL-C increase. Whether these changes in HDL particle composition and functionality have a beneficial effect on cardiovascular outcome requires formal testing in a cardiovascular outcome trial.

High-density lipoprotein subclass measurements improve mortality risk prediction, discrimination and reclassification in a cardiac catheterization cohort.

BACKGROUND AND AIMS: Recent failures of HDL cholesterol (HDL-C)-raising therapies to prevent cardiovascular disease (CVD) events have tempered the interest in the role of HDL-C in clinical risk assessment. Emerging data suggest that the atheroprotective properties of HDL depend on specific HDL particle characteristics not reflected by HDL-C. The purpose of this study was to determine the association of HDL particle concentration (HDL-P) and HDL subclasses with mortality in a high-risk cardiovascular population and to examine the clinical utility of these parameters in mortality risk discrimination and reclassification models. METHODS: Using nuclear magnetic resonance spectroscopy, we measured HDL-P and HDL subclasses in 3972 individuals enrolled in the CATHGEN coronary catheterization biorepository; tested for association with all-cause mortality in robust clinical models; and examined the utility of HDL subclasses in incremental mortality risk discrimination and reclassification. RESULTS: Over an average follow-up of eight years, 29.6% of the individuals died. In a multivariable model adjusted for ten CVD risk factors, HDL-P [HR, 0.71 (0.67-0.76), p = 1.3e-24] had a stronger inverse association with mortality than did HDL-C [HR 0.93 (0.87-0.99), p = 0.02]. Larger HDL size conferred greater risk and the sum of medium- and small-size HDL particles (MS-HDL-P) conferred less risk. Furthermore, the strong inverse relation of HDL-P levels with mortality was accounted for entirely by MS-HDL-P; HDL-C was not associated with mortality after adjustment for MS-HDL-P. Addition of MS-HDL-P to the GRACE Risk Score significantly improved risk discrimination and risk reclassification. CONCLUSION: HDL-P and smaller HDL subclasses were independent markers of residual mortality risk and incremental to HDL-C in a high-risk CVD population. These measures should be considered in risk stratification and future development of HDL-targeted therapies in high-risk populations.

High-density lipoprotein synthesis and metabolism (Review).

High density lipoproteins (HDL) are heterogeneous particles regarding their size and composition. They have vital functions in reverse cholesterol transport (RCT). RCT occurs when lipid-free apolipoprotein AI recruits cholesterol and phospholipid to form nascent HDL particles. Adenosine triphosphate­binding cassette transporters and scavenger receptor class B type I were found to be associated with the synthesis of HDL. Experimental studies have identified several potential anti­atherogenic effects of HDL, including promotion of macrophage cholesterol outflow as well as anti­inflammatory and anti­thrombotic effects. HDL can also transport microRNAs. This review mainly summarizes the present knowledge of HDL synthesis and metabolism.

Effects of established hypolipidemic drugs on HDL concentration, subclass distribution, and function.

The knowledge of an inverse relationship between plasma high-density lipoprotein cholesterol (HDL-C) concentrations and rates of cardiovascular disease has led to the concept that increasing plasma HDL-C levels would be protective against cardiovascular events. Therapeutic interventions presently available to correct the plasma lipid profile have not been designed to specifically act on HDL, but have modest to moderate effects on plasma HDL-C concentrations. Statins, the first-line lipid-lowering drug therapy in primary and secondary cardiovascular prevention, have quite modest effects on plasma HDL-C concentrations (2-10%). Fibrates, primarily used to reduce plasma triglyceride levels, also moderately increase HDL-C levels (5-15%). Niacin is the most potent available drug in increasing HDL-C levels (up to 30%), but its use is limited by side effects, especially flushing.The present chapter reviews the effects of established hypolipidemic drugs (statins, fibrates, and niacin) on plasma HDL-C levels and HDL subclass distribution, and on HDL functions, including cholesterol efflux capacity, endothelial protection, and antioxidant properties.

Structure of HDL: particle subclasses and molecular components.

A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.

HDL biogenesis, remodeling, and catabolism.

In this chapter, we review how HDL is generated, remodeled, and catabolized in plasma. We describe key features of the proteins that participate in these processes, emphasizing how mutations in apolipoprotein A-I (apoA-I) and the other proteins affect HDL metabolism. The biogenesis of HDL initially requires functional interaction of apoA-I with the ATP-binding cassette transporter A1 (ABCA1) and subsequently interactions of the lipidated apoA-I forms with lecithin/cholesterol acyltransferase (LCAT). Mutations in these proteins either prevent or impair the formation and possibly the functionality of HDL. Remodeling and catabolism of HDL is the result of interactions of HDL with cell receptors and other membrane and plasma proteins including hepatic lipase (HL), endothelial lipase (EL), phospholipid transfer protein (PLTP), cholesteryl ester transfer protein (CETP), apolipoprotein M (apoM), scavenger receptor class B type I (SR-BI), ATP-binding cassette transporter G1 (ABCG1), the F1 subunit of ATPase (Ecto F1-ATPase), and the cubulin/megalin receptor. Similarly to apoA-I, apolipoprotein E and apolipoprotein A-IV were shown to form discrete HDL particles containing these apolipoproteins which may have important but still unexplored functions. Furthermore, several plasma proteins were found associated with HDL and may modulate its biological functions. The effect of these proteins on the functionality of HDL is the topic of ongoing research.

Plasma lipidomics discloses metabolic syndrome with a specific HDL phenotype.

Lipidomics reveals a remarkable diversity of lipids in human plasma. In this study, we have performed an in-depth lipidomic analysis of human plasma from healthy individuals and subjects with metabolic syndrome (MetS) in order to determine the lipidomic profile that allows prognosis of a pathological subpopulation with altered high-density lipoprotein (HDL) metabolism. The MetS population was categorized as having pathological or nonpathological HDL. Anthropometric parameters, cardiovascular risk markers, and lipoprotein subclasses of HDL and low-density lipoproteins were also evaluated. Lipidomic analysis revealed 357 differential molecules that were clustered (k means) in the two groups. The molecules identified in the whole lipidome showed that MetS subjects presented lower levels of glycerolipids and higher levels of glycerophospholipids with respect to control subjects. In contrast, when only statistically differential lipids were taken into account, differences were found between the two groups in almost cases. Furthermore, levels of saturated fatty acids were higher in patients with pathological HDL levels than in controls, whereas levels of unsaturated fatty acids were lower. These results highlight the potential of lipidomics as a clinical tool for risk assessment and monitoring of disease.

Hepatic ACAT2 knock down increases ABCA1 and modifies HDL metabolism in mice.

OBJECTIVES: ACAT2 is the exclusive cholesterol-esterifying enzyme in hepatocytes and enterocytes. Hepatic ABCA1 transfers unesterified cholesterol (UC) to apoAI, thus generating HDL. By changing the hepatic UC pool available for ABCA1, ACAT2 may affect HDL metabolism. The aim of this study was to reveal whether hepatic ACAT2 influences HDL metabolism. DESIGN: WT and LXRα/ß double knockout (DOKO) mice were fed a western-type diet for 8 weeks. Animals were i.p. injected with an antisense oligonucleotide targeted to hepatic ACAT2 (ASO6), or with an ASO control. Injections started 4 weeks after, or concomitantly with, the beginning of the diet. RESULTS: ASO6 reduced liver cholesteryl esters, while not inducing UC accumulation. ASO6 increased hepatic ABCA1 protein independently of the diet conditions. ASO6 affected HDL lipids (increased UC) only in DOKO, while it increased apoE-containing HDL in both genotypes. In WT mice ASO6 led to the appearance of large HDL enriched in apoAI and apoE. CONCLUSIONS: The use of ASO6 revealed a new pathway by which the liver may contribute to HDL metabolism in mice. ACAT2 seems to be a hepatic player affecting the cholesterol fluxes fated to VLDL or to HDL, the latter via up-regulation of ABCA1.

A systematic approach to obtain validated partial least square models for predicting lipoprotein subclasses from serum NMR spectra.

A systematic approach is described for building validated PLS models that predict cholesterol and triglyceride concentrations in lipoprotein subclasses in fasting serum from a normolipidemic, healthy population. The PLS models were built on diffusion-edited (1)H NMR spectra and calibrated on HPLC-derived lipoprotein subclasses. The PLS models were validated using an independent test set. In addition to total VLDL, LDL, and HDL lipoproteins, statistically significant PLS models were obtained for 13 subclasses, including 5 VLDLs (particle size 64-31.3 nm), 4 LDLs (particle size 28.6-20.7 nm) and 4 HDLs (particle size 13.5-9.8 nm). The best models were obtained for triglycerides in VLDL (0.82 < Q(2) <0.92) and HDL (0.69 < Q(2) <0.79) subclasses and for cholesterol in HDL subclasses (0.68 < Q(2) <0.96). Larger variations in the model performance were observed for triglycerides in LDL subclasses and cholesterol in VLDL and LDL subclasses. The potential of the NMR-PLS model was assessed by comparing the LPD of 52 subjects before and after a 4-week treatment with dietary supplements that were hypothesized to change blood lipids. The supplements induced significant (p < 0.001) changes on multiple subclasses, all of which clearly exceeded the prediction errors.

Development of a homogeneous assay for measurement of high-density lipoprotein-subclass cholesterol.

BACKGROUND: Several studies have suggested that measurement of high-density lipoprotein (HDL) 2 and HDL3 subfractions might be more useful for evaluating coronary risk than total HDL-cholesterol (C). However, methods of measuring HDL2 and HDL3 are quite laborious for general clinical use. Development of a quick and easy method of measuring HDL subfractions has been long-awaited. METHODS: Triglyceride (TG) rich lipoproteins (TRLs), low-density lipoprotein (LDL), HDL2, and HDL3 were used for screening of surfactants and enzymes to react selectively with HDL3-C and to decompose other lipoproteins. RESULTS: In order to develop HDL3-C homogeneous assay, polyoxyethylene styrenated phenyl ether derivative, for which the hydrophilic lipophilic balance (HLB) value is 13.6, was adopted as the most effective and specific surfactant for selection of HDL3 from HDL. Sphingomyelinase (SMase) reacted with TRLs and LDL preferentially, and decomposed them. HDL2-C was estimated by subtracting measured HDL3-C from total HDL-C, directly measured by homogeneous method. The homogeneous assay exhibited excellent correlations with the results of HDL3-C and HDL2-C measured by standard ultracentrifugation (R(2)=0.848 and 0.982, respectively). CONCLUSIONS: We established a rapid and effective, fully-automated assay for the measurement of HDL3-C. Furthermore, the subtraction of HDL3-C from total HDL-C allows concurrent determination of HDL2-C.

Distribution of low-density lipoprotein and high-density lipoprotein subclasses in patients with sarcoidosis.

CONTEXT: Systemic inflammatory diseases are associated with proatherogenic lipoprotein profile, but there is a lack of information regarding overall distributions of lipoprotein subclasses in sarcoidosis. OBJECTIVE: To investigate whether patients with sarcoidosis have altered distributions of plasma low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles. DESIGN: Seventy-seven patients with biopsy-proven sarcoidosis (29 with acute and 48 with chronic sarcoidosis) treated with corticosteroids and 77 age- and sex-matched controls were included in the study. Low-density lipoprotein and HDL subclasses were determined by gradient gel electrophoresis, while inflammatory markers and lipid parameters were measured by standard laboratory methods. RESULTS: Compared to controls, patients had fewer LDL I subclasses (P < .001), but more LDL II and III (P < .001) subclasses. This pattern was evident in both acute and chronic disease groups. Patients also had smaller HDL size (P < .001) and higher proportions of HDL 2a (P = .006) and 3a particles (P = .004). Patients with chronic sarcoidosis had smaller LDL size than those with acute disease (P = .02) and higher proportions of HDL 3a subclasses (P = .04) than controls. In acute sarcoidosis, relative proportions of LDL and HDL particles were associated with levels of inflammatory markers, whereas in chronic disease an association with concentrations of serum lipid parameters was found. CONCLUSIONS: The obtained results demonstrate adverse lipoprotein subfraction profile in sarcoidosis with sustained alterations during disease course. Evaluation of LDL and HDL particles may be helpful in identifying patients with higher cardiovascular risk, at least for prolonged corticosteroid therapy due to chronic disease course.