Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: effect of a high-dose statin on VLDL receptor expression

BackgroundComparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated.MethodsLDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit β-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined.ResultsBoth LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, β-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-β-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and β-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than β-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration.ConclusionsRLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.     

Linkage between cholesterol 7alpha-hydroxylase and high plasma low-density lipoprotein cholesterol concentrations.

Interindividual differences in plasma low-density lipoprotein cholesterol (LDL-C) levels reflect both environmental variation and genetic polymorphism, but the specific genes involved and their relative contributions to the variance in LDL-C are not known. In this study we investigated the relationship between plasma LDL-C concentrations and three genes with pivotal roles in LDL metabolism: the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and cholesterol 7alpha-hydroxylase (CYP7). Analysis of 150 nuclear families indicated statistically significant linkage between plasma LDL-C concentrations and CYP7, but not LDLR or APOB. Further sibling pair analyses using individuals with high plasma LDL-C concentrations as probands indicated that the CYP7 locus was linked to high plasma LDL-C, but not to low plasma LDL-C concentrations. This finding was replicated in an independent sample. DNA sequencing revealed two linked polymorphisms in the 5' flanking region of CYP7. The allele defined by these polymorphisms was associated with increased plasma LDL-C concentrations, both in sibling pairs and in unrelated individuals. Taken together, these findings indicate that polymorphism in CYP7 contributes to heritable variation in plasma LDL-C concentrations. Common polymorphisms in LDLR and APOB account for little of the heritable variation in plasma LDL-C concentrations in the general population.     

K562和HL-60细胞低密度脂蛋白胆固醇受体及3-羟基-3-甲基戊二酸单酰辅酶A还原酶活性变化的意义

目的:寻找白血病细胞和正常细胞之间生物特性的差异,进而利用这种差异进行靶向治疗,在最大限度地杀伤白血病细胞的同时保护正常细胞是近年来白血病药物治疗一直在探索的领域.测定白血病细胞K562、HL-60细胞低密度脂蛋白胆固醇受体和3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMG-CoA)活性,并与正常细胞系HK-2比较,分析其活性变化,并寻找抑制K562、HL-60细胞增殖的有效方法.方法:实验于2006-07/2007-05在青岛大学医学院附属医院中心实验室及血液免疫研究室完成.实验过程中应用的K562(慢粒急变的的白血病)、HL-60(原髓细胞白血病)细胞株为中国医学科学院血液病研究所惠赠.参照孟凡青方法并加以改进,采用低浓度阿托伐他汀抑制细胞内源性胆固醇合成途径,提供外源性低密度脂蛋白胆固醇供细胞生长,应用MTT比色法检测细胞分裂生长抑制率,进而判断低密度脂蛋白受体活性.应用紫外分光光度法测定细胞3-羟基-3-甲基戊二酸单酰辅酶A还原酶活性;采用MTT法观察HMG-CoA抑制剂阿托伐他汀对 K562、HL-60和HK-2细胞增殖作用的影响:将对数期生长的细胞按每孔100 μL接种于96孔培养板中,阿托伐他汀用RPMI1640分别配成10,20,40,80,160 μmol/L加入培养体系,每孔100 μL,以不加药物而加含10%胎牛血清的RPMI1640 100 μL及上述细胞100 μL的培养体系为对照.结果:与正常细胞HK-2比较,白血病细胞K562、HL-60低密度脂蛋白胆固醇受体活性增高、HMG-CoA 还原酶活性增高 (P < 0.05);阿托伐他汀以时间和剂量依赖性的方式抑制K562、HL-60细胞增殖,即随着阿托伐他汀处理浓度的加大、作用时间的延长,细胞存活率逐渐下降.阿托伐他对HK-2细胞增殖抑制作用与时间及剂量无关.结论:白血病细胞系K562、HL-60细胞低密度脂蛋白胆固醇受体和HMG-CoA 还原酶活性较正常细胞HK-2增高;阿托伐他汀具有抑制K562、HL-60细胞增殖的作用.     

High-density lipoprotein cholesterol, hepatic lipase and lipoprotein lipase activities in thyroid dysfunction--effects of treatment

We have investigated the effects of hyper- and hypothyroidism (clinical and subclinical) on lipid metabolism, with special emphasis on serum high-density lipoprotein cholesterol, post-heparin plasma hepatic lipase and lipoprotein lipase activities. In 16 patients with hyperthyroidism, increased post-heparin plasma hepatic lipase activity, decreased serum total cholesterol and serum high-density lipoprotein cholesterol were found while lipoprotein lipase activity and serum triglyceride were normal. In six patients with overt hypothyroidism serum total cholesterol and triglyceride were increased, post-heparin plasma hepatic lipase and lipoprotein lipase were decreased while serum high-density lipoprotein cholesterol was normal. In six patients with subclinical hypothyroidism, serum total cholesterol was increased, serum high-density lipoprotein cholesterol was decreased, while serum triglyceride, post-heparin plasma hepatic lipase and lipoprotein lipase were normal. When the three groups of patients became euthyroid, serum total cholesterol, serum triglyceride, post-heparin plasma hepatic lipase, lipoprotein lipase, and serum high-density lipoprotein cholesterol reverted to normal except for serum high-density lipoprotein cholesterol in the hyperthyroid group which showed no significant change with treatment. A positive correlation was found between serum T3 and post-heparin plasma hepatic lipase while negative correlations were found between serum total cholesterol and serum T3, post-heparin plasma hepatic lipase and serum total cholesterol, lipoprotein lipase and serum triglyceride respectively. Thus in these patients with thyroid dysfunction, significant reversible alterations in serum total cholesterol, triglyceride and high-density lipoprotein cholesterol were found and could be correlated with the observed changes in the activities of hepatic lipase and lipoprotein lipase.     

Serum LDL cholesterol,the LDL/HDL cholesterol ratio and liver microsomal enzyme induction evaluated by antipyrine kinetics

The association of serum LDL and HDL cholesterol with hepatic microsomal enzyme induction, assessed by plasma antipyrine kinetics was investigated in 30 epileptics. Patients on enzyme-inducing anticonvulsants had reduced LDL/HDL cholesterol ratios and elevated HDL cholesterol concentrations and HDL/total cholesterol ratios, indicating a cholesterol transfer from LDL to HDL. Strong hepatic microsomal enzyme induction was associated with reduced LDL cholesterol. The LDL/HDL cholesterol ratio was negatively proportional and the HDL/total cholesterol ratio positively proportional to the antipyrine clearance rate. Epileptics, particularly those with a high antipyrine clearance, had a cholesterol distribution pattern characteristic of a low probability of developing coronary atherosclerosis. The results support the view that hepatic microsomal enzyme induction favourably alters the cholesterol distribution in the body.     

Serum LDL cholesterol, the LDL/HDL cholesterol ratio and liver microsomal enzyme induction evaluated by antipyrine kinetics

The association of serum LDL and HDL cholesterol with hepatic microsomal enzyme induction, assessed by plasma antipyrine kinetics was investigated in 30 epileptics. Patients on enzyme-inducing anticonvulsants had reduced LDL/HDL cholesterol ratios and elevated HDL cholesterol concentrations and HDL/total cholesterol ratios, indicating a cholesterol transfer from LDL to HDL. Strong hepatic microsomal enzyme induction was associated with reduced LDL cholesterol. The LDL/HDL cholesterol ratio was negatively proportional and the HDL/total cholesterol ratio positively proportional to the antipyrine clearance rate. Epileptics, particularly those with a high antipyrine clearance, had a cholesterol distribution pattern characteristic of a low probability of developing coronary atherosclerosis. The results support the view that hepatic microsomal enzyme induction favourably alters the cholesterol distribution in the body.     

Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations

We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.     

Different VLDL apo B, and HDL apo AI and apo AII metabolism in two heterozygous carriers of unrelated mutations in the lipoprotein lipase gene

BACKGROUND: Lipoprotein lipase (LPL) deficiency has been suggested as a cause of low HDL-cholesterol (HDL-C) plasma levels, by a mechanism that involves an enhanced catabolism of HDL apolipoprotein (apo) AI. To verify the role of 2 different LPL gene mutations on HDL metabolism, we studied the in vivo turnover of the apo AI and apo AII in heterozygous carriers of LPL deficiency. METHODS: Apo AI and AII kinetics were studied by a 10-h primed constant infusion of 5,5,5-2H3-leucine approach in 2 carriers, 1 man (patient 1) and 1 woman (patient 2), and 5 control subjects. The rates of HDL apolipoproteins production (PR) and catabolism (FCR) were estimated using a one-compartment model-based analysis. RESULTS: Both carriers had low HDL-C plasma levels and only patient 1 was hypertriglyceridemic. VLDL apo B was 4-times slower in patient 1 as compared to patient 2. The FCRs of apo AI in both carriers was within the range of the controls (0.200, 0.221 and 0.211+/-0.051 day(-1), respectively). Apo AII FCR in patient 1 was about 20% lower than the mean of the control group whereas being normal in patient 2. Apo AI PR in patient 1 (9.20 mg kg(-1) day(-1)) was below the lowest value in controls (range, 10.52-13.24 mg kg(-1) day(-1)) whereas in patient 2 it was normal. Apo AII PR in both patients was similar to controls. CONCLUSION: The heterozygous carriers of 2 different mutations in the LPL gene had different VLDL apo B FCR, and from normal to slightly low HDL apolipoprotein FCR and PR. These results disagree with the putative enhanced apo AI FCR in LPL deficient patients and suggest the need to reconsider the effects of LPL activity on HDL metabolism.     

Coenzyme Q10, alpha-tocopherol and free cholesterol in HDL and LDL fractions.

Twenty-three randomly selected plasma samples from apparently healthy, middle aged men were analysed for coenzyme Q10 (CoQ10), alpha-tocopherol (AT) and free cholesterol (FC) in: 1) whole plasma, 2) the HDL lipoprotein fraction after LDL precipitation (VLDL + LDL). CoQ10, AT and FC in plasma averaged 0.69 +/- .11, 6.74 +/- 1.78 micrograms x ml-1 and 0.59 +/- .11 mg x ml-1 and in HDL 0.17, 3.24 micrograms x ml-1 and 0.17 mg x ml-1 or 29, 48 and 29% of plasma values. Amounts of CoQ10 and AT were correlated to that of FC in all pools. The amount of HDL-CoQ10 but not of HDL-AT fell, with the HDL-FC expressed as the fraction of plasma FC. In all pools, N-AT versus AT initially increased and then levelled off, indicating saturation like conditions in contrast to CoQ10. Thus, CoQ10 and AT are differently allocated in HDL and LDL. This might have a bearing both on the suggested lipoprotein protection against peroxidation by these two antioxidants, but also on the distribution and allocation in different organs of CoQ10 and AT by HDL and LDL transportation.     

Reagent-free, simultaneous determination of serum cholesterol in HDL and LDL by infrared spectroscopy

BACKGROUND: The purpose of this study was to assess the feasibility of infrared (IR) spectroscopy for the simultaneous quantification of serum LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) concentrations. METHODS: Serum samples (n = 90) were obtained. Duplicate aliquots (5 microL) of the serum specimens were dried onto IR-transparent barium fluoride substrates, and transmission IR spectra were measured for the dry films. In parallel, the HDL-C and LDL-C concentrations were determined separately for each specimen by standard methods (the Friedewald formula for LDL-C and an automated homogeneous HDL-C assay). The proposed IR method was then developed with a partial least-squares (PLS) regression analysis to quantitatively correlate IR spectral features with the clinical analytical results for 60 randomly chosen specimens. The resulting quantification methods were then validated with the remaining 30 specimens. The PLS model for LDL-C used two spectral ranges (1700-1800 and 2800-3000 cm(-1)) and eight PLS factors, whereas the PLS model for HDL-C used three spectral ranges (800-1500, 1700-1800, and 2800-3500 cm(-1)) with six factors. RESULTS: For the 60 specimens used to train the IR-based method, the SE between IR-predicted values and the clinical laboratory assays was 0.22 mmol/L for LDL-C and 0.15 mmol/L for HDL-C (r = 0.98 for LDL-C; r = 0.91 for HDL-C). The corresponding SEs for the test spectra were 0.34 mmol/L (r = 0.96) and 0.26 mmol/L (r = 0.82) for LDL-C and HDL-C, respectively. The precision for the IR-based assays was estimated by the SD of duplicate measurements to be 0.11 mmol/L (LDL-C) and 0.09 mmol/L (HDL-C). CONCLUSIONS: IR spectroscopy has the potential to become the clinical method of choice for quick and simultaneous determinations of LDL-C and HDL-C.     

Serum total, HDL, LDL cholesterol, and triglyceride levels in patients with rheumatoid arthritis

Patients with rheumatoid arthritis are at risk of increased prevalence of coronary heart disease. In general, the plasma level of high density lipoprotein cholesterol (HDL) correlates with the risk of incidence of ischemic heart disease. The levels of total, HDL, low density (LDL) cholesterol, and triglycerides were measured in sera of patients with rheumatoid arthritis and in healthy controls. In patients with rheumatoid arthritis (26 men and 103 women), the serum total and LDL cholesterol were higher, whereas the HDL cholesterol and triglycerides were lower (p less than 0.001) compared to the values observed in controls (625 men and 749 women). Similar patterns were seen when results of age and sex matched controls were compared to the results of patients suffering from rheumatoid arthritis. The lipid parameters of patients with rheumatoid arthritis were not different when the patients were treated with steroidal or nonsteroidal anti-inflammatory drugs.     

Multiple abnormalities in the transfer of phospholipids from VLDL and LDL to HDL in non-insulin-dependent diabetes

Transfers or exchanges of cholesterol esters and triglycerides between lipoproteins are mediated by a specialized protein referred to as cholesteryl ester transfer protein (CETP), whereas those of phospholipids (PLs) are facilitated by both CETP and a specific phospholipid transfer protein (PLTP). In the present study, the authors compared phospholipid transfer (PLT) in normal subjects and in patients with non-insulin-dependent diabetes (NIDD), which is associated with an increased risk of atherosclerosis. PLT was measured in different recombination experiments using an isotopic assay in which the transfer of labelled PLs from very low-density lipoprotein (VLDLs) and low-density lipoproteins (LDLs) to high-density lipoproteins (HDLs) was determined. This allowed discrimination between the roles of VLDLs+LDLs, HDLs, and plasma PLT activity (PLTA). VLDL+LDL-dependent PLT, HDL-dependent PLT and PLTA were decreased in NIDD. VLDL+LDL-dependent PLT was found to be negatively correlated with the PL/apolipoprotein B ratio, whereas HDL-dependent PLT was positively correlated with the HDL₂/HDL₃ and PL/apolipoprotein A-I ratios and negatively correlated with the flow activation energy at the HDL surface. The HDL₂/HDL₃ ratio was positively correlated with PLTA but not with CETP, which confirms previous reports suggesting that PLTP might act as an HDL conversion factor. These data show that several abnormalities in PLT occur in NIDD and raise the question as to whether a lowered PLT might be a new characteristic of dis factors associated with an increased risk of atherosclerosis.     

The lipoprotein lipase HindIII polymorphism: association with total cholesterol and LDL-cholesterol, but not with HDL and triglycerides in 342 females.

BACKGROUND: Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of core triglycerides in chylomicrons and VLDL. METHODS: We investigated the association between the HindIII polymorphism of the LPL gene and fasting glucose, lipid, and lipoprotein concentrations in 683 Caucasians. We first stabilized the study subjects, using an 8-day diet and exercise intervention program before obtaining blood samples. The use of this standardization period reduced the variance of all glucose and lipid concentrations. RESULTS: In our study, the HindIII allele frequencies for females and males were 0.29 and 0.34 for H- and 0.71 and 0.66 for H+, respectively. We found in females, but not in males, a significant association between the HindIII genotype and total cholesterol (P = 0.007) and LDL-cholesterol (P = 0.018), with females homozygous for the rare H- allele having the lowest, heterozygotes (H-/+) having intermediate, and women homozygous for the common H+ allele having the highest of each of these lipid traits. With regard to triglycerides, HDL-cholesterol, and glucose, no significant effect of the HindIII genotype was noted in either gender. CONCLUSIONS: These results suggest that in a gender-specific manner, the rare LPL HindIII H- allele has a cholesterol-lowering and, therefore, potentially cardioprotective effect compared with the common H+ allele.     

Effects of dextran sulfate on stabilization of milk lipoprotein lipase and VLDL triglyceride hydrolysis in vitro

The effects of dextran sulfate (DS), which has various molecular numbers, on hydrolysis of very low density lipoprotein triglyceride (VLDL-TG) by bovine milk lipoprotein lipase (LPL) and the stability of LPL were studied. VLDL-TG hydrolysis was increased by the addition of DS; DS caused linear increase in the Vmax for VLDL-TG with increase in its sulfate content, but did not change the Km value for VLDL-TG. DS also stabilized LPL, but this effect was not dependent on its sulfate content. These results suggest that the mechanism of action of DS in LPL stabilization may be different from that in enhancement of VLDL hydrolysis.     

Determination of LDL cholesterol and LDL apolipoprotein B following precipitation of VLDL in blood serum with phosphotungstic acid/MgCl2

A method is described for the selective precipitation of VLDL in blood serum using phosphotungstic acid/MgCl2. The method allows for the calculation of LDL apolipoprotein B as well as for the calculation of LDL cholesterol (following the additional determination of HDL cholesterol). Dependent on the triglyceride and the cholesterol content of the serum, three different procedures were developed using phosphotungstic acid and MgCl2 in different concentrations in the precipitation assay. Within the tested range of 3-10 mmol/l total cholesterol and 1-4 mmol/l triglyceride in blood serum the VLDL were nearly completely precipitated with negligible coprecipitation of LDL and HDL, but 40-50% coprecipitation of Lp(a). Regression analysis of the cholesterol values obtained by precipitation with phosphotungstic acid/MgCl2 (= serum cholesterol - LDL cholesterol), and the cholesterol values obtained by ultracentrifugation (d greater than 1.006 kg/l) revealed a good measure of agreement (r = 0.97, y = 0.93 X + 0.35, n = 76). An equally good measure of agreement was found for the corresponding apolipoprotein B values (r = 0.96, y = 1.03 X - 0.2, n = 61). In the determination of LDL cholesterol a variation coefficient of 4.3% (n = 20) was found in relation to the precision in the series, and a variation coefficient of 4.8% (n = 25) in relation to day to day precision.