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BackgroundEarly diagnosis of type 2 diabetes mellitus (T2DM) is still difficult. Screening of plasma biomarkers has great significance of optimizing diagnosis and predicting the complications of T2DM.MethodsWe used a special diet, Purina #5008, to induce diabetes in Zucker leptin receptor gene-deficient rats (fa/fa) to establish Zucker diabetic fatty (ZDF) rats, simulating the early stage of T2DM. The differentially expressed proteins (DEP) and lipids (DEL), as potential biomarkers, were screened to compare the plasma expression levels in ZDF rats and their basic diet-fed wild-type controls (fa/+) by Tandem Mass Tags (TMT) and liquid chromatography-tandem mass spectrometry.ResultsThese two groups had different plasma proteins and lipids profiles consisting of 84 DEPs and, 179 DELs identified in the positive ion mode and 178 DELs in the negative ion mode, respectively. Enrichment analysis of these different indicators showed that oxidative stress, insulin resistance and metabolic disorders of glycan and lipid played an important role in generating the difference. Some markers can be used as candidate biomarkers in prediction and treatments of T2DM, such as ceruloplasmin, apolipoprotein C-I, apolipoprotein C-II and apolipoprotein C-IV.ConclusionThese plasma differences help to optimize the diagnosis and predict the complications of T2DM, although this remains to be verified in the crowd. Trace elements related-metalloproteins, such as ceruloplasmin, and lipid metabolism and transport-related apolipoprotein C are expected to be candidate biomarkers of T2DM and should be given more attention.

High-density lipoprotein (HDL) subpopulations functional assessment is more relevant for HDL anti-atherogenic activity than cholesterol level. The aim of the study was to assess the impact of HDL-2 and HDL-3 on lipoprotein lipase (LPL)-mediated very-low-density lipoprotein (VLDL) catabolism related to hypertriglyceridemia development. VLDL and HDLs were isolated from serum by ultracentrifugation. VLDL was incubated with LPL in the absence and presence of total HDL or HDL subpopulations. Next, VLDL remnants were separated, and their composition and electrophoretic mobility was assessed. Both HDL subpopulations increased the efficiency of triglyceride lipolysis and apolipoprotein CII and CIII removal from VLDL up to ~90%. HDL-3 exerted significantly greater impact than HDL-2 on apolipoprotein E (43% vs. 18%, p < 0.001), free cholesterol (26% vs. 18%, p < 0.05) and phospholipids (53% vs. 43%, p < 0.05) removal from VLDL and VLDL remnant electrophoretic mobility (0.18 vs. 0.20, p < 0.01). A greater release of these components was also observed in the presence of total HDL with a low HDL-2/HDL-3 cholesterol ratio. Both HDL subpopulations affect VLDL composition during lipolysis, but HDL-3 exhibited a greater effect on this process. Altered composition of HDL related to significant changes in the distribution between HDL-2 and HDL-3 can influence the VLDL remnant features, affecting atherosclerosis progression.

Apolipoprotein C2 (ApoC2) is an important member of the apolipoprotein C family and functions as a major activator of lipoprotein lipase (LPL). In cardiovascular and cerebrovascular systems, the lipolytic activity of the LPL-ApoC2 complex is critical for the metabolism of triglyceride-rich lipoproteins and contributes to the pathogenesis of ischemic stroke (IS). However, the regulation of ApoC2 in IS development remains unclear. In this study, we first explored potential ApoC2-targeting microRNAs (miRNAs) by bioinformatics tool and compared the miRNA expression profiles in the blood cells of 25 IS patients and 25 control subjects by miRNA microarray. miR-1275 was predicted to bind with the 3' untranslated region of ApoC2, and a significant reduction of blood miR-1275 levels was observed in IS patients. Dual-luciferase reporter assay and quantitative RT-PCR confirmed the regulation of ApoC2 by miR-1275 in THP-1 derived macrophages. miR-1275 also inhibited cellular uptake of ox-LDL and suppressed formation of macrophage foam cell. Furthermore, the whole blood miR-1275 levels were validated in 279 IS patients and 279 control subjects by TaqMan assay. miR-1275 levels were significantly lower in IS cases and logistic regression analysis showed that miR-1275 level was negatively associated with the occurrence of IS (adjusted OR, 0.76; 95% CI, 0.69-0.85; p 0.001). Addition of miR-1275 to traditional risk factors showed an additive prediction value for IS. Our study shows that blood miR-1275 levels were negatively associated with the occurrence of IS, and miR-1275 might exert an athero-protective role against the development of IS by targeting ApoC2 and blocking the formation of macrophage foam cells.

High-density lipoprotein (HDL) proteomic study has identified substantial changes associated with various disease states. In the current study, the HDL proteomes in patients with cerebral lacunar infarction (LACI) and control subjects were investigated. A total of 12 LACI patients without evident large vessel occlusions and 12 controls were enrolled in the study. The HDL fraction from each sample was isolated from the plasma by ultracentrifugation. The protemics of the HDL were investigated using nano liquid chromatography coupled to tandem mass spectrometry. There were 55 proteins identified as differentially expressed in the LACI and control groups. Among the 55 proteins, 33 were upregulated and 22 were downregulated in the patients with LACI. The identified proteins were associated with numerous molecular functions, including lipid and cholesterol transport, lipid metabolism, inflammatory response, the complement and coagulation pathway, metal ion metabolism, hemostasis and endopeptidase inhibitory activity. Serum amyloid A, apolipoprotein C (apoC-III) and apolipoprotein A-II (apoA-II) were selected to confirm the proteomics results via western blotting. HDL from the LACI patients exhibited an impaired ability to inhibit the binding of THP-1 cells to endothelial cells compared with the controls (P

Patients with chronic kidney disease (CKD) have a considerably higher risk of death due to cardiovascular causes. Using an iTRAQ MS/MS approach, we investigated the alterations in plasma protein accumulation in patients with CKD and classical cardiovascular disease (CVD) without CKD. The proteomic analysis led to the identification of 130 differentially expressed proteins among CVD and CKD patients and healthy volunteers. Bioinformatics analysis revealed that 29 differentially expressed proteins were involved in lipid metabolism and atherosclerosis, 20 of which were apolipoproteins and constituents of high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Although dyslipidemia is common in CKD patients, we found that significant changes in apolipoproteins were not strictly associated with changes in plasma lipid levels. A lack of correlation between apoB and LDL concentration and an inverse relationship of some proteins with the HDL level were revealed. An increased level of apolipoprotein AIV, adiponectin, or apolipoprotein C, despite their anti-atherogenic properties, was not associated with a decrease in cardiovascular event risk in CKD patients. The presence of the distinctive pattern of apolipoproteins demonstrated in this study may suggest that lipid abnormalities in CKD are characterized by more qualitative abnormalities and may be related to HDL function rather than HDL deficiency.

Objective Circulating hepatitis C virus (HCV) virions are associated with triglyceride-rich lipoproteins, including very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL), designated as lipo-viro-particles (LVPs). Previous studies showed that lipoprotein lipase (LPL), a key enzyme for hydrolysing the triglyceride in VLDL to finally become LDL, may suppress HCV infection. This investigation considers the regulation of LPL by lipoproteins and LVPs, and their roles in the LPL-mediated anti-HCV function. Design The lipoproteins were fractionated from normolipidemic blood samples using iodixanol gradients. Subsequent immunoglobulin-affinity purification from the canonical VLDL and LDL yielded the corresponding VLDL-LVP and LDL-LVP. Apolipoprotein (apo) Cs, LPL activity and HCV infection were quantified. Results A higher triglyceride/cholesterol ratio of LDL was found more in HCV-infected donors than in healthy volunteers, and the triglyceride/cholesterol ratio of LDL-LVP was much increased, suggesting that the LPL hydrolysis of triglyceride may be impaired. VLDL, VLDL-LVP, LDL-LVP, but not LDL, suppressed LPL lipolytic activity, which was restored by antibodies that recognised apoC-III/-IV and correlated with the steadily abundant apoC-III/-IV quantities in those particles. In a cell-based system, treatment with VLDL and LVPs reversed the LPL-mediated inhibition of HCV infection in apoC-III/-IV-dependent manners. A multivariate logistic regression revealed that plasma HCV viral loads correlated negatively with LPL lipolytic activity, but positively with the apoC-III content of VLDL. Additionally, apoC-III in VLDL was associated with a higher proportion of HCV-RNA than was IgG. Conclusion This study reveals that LPL is an anti-HCV factor, and that apoC-III in VLDL and LVPs reduces the LPL-mediated inhibition of HCV infection.

Objective— Aberrations in apolipoprotein (apo) metabolism and increased systemic inflammation associate with the metabolic syndrome (MetS) and may contribute to its atherogenicity. We examined whether the association between carotid atherosclerosis and MetS in a population of young adults is mediated by apoB and apoA-I and/or by inflammatory markers C-reactive protein and type II secretory phospholipase A2. Methods and Results— We used cross-sectional and 6-year prospective data from the cardiovascular risk in young Finns study. In young adults (aged 24 to 39 years), apoB, C-reactive protein, and type II secretory phospholipase A2 enzyme activity were significantly higher and apoA-I lower in subjects with MetS (N=325) than in subjects without MetS (N=1858). In prospective analysis (N=1587), both MetS and high apoB predicted ( P 90th percentile and/or plaque. The association between MetS and incident high carotid intima-media thickness was attenuated by ≈40% after adjustment with apoB. Adjustments with apoA-I, C-reactive protein, or type II secretory phospholipase A2 did not diminish the association. Conclusion— High levels of apoB, C-reactive protein, and type II secretory phospholipase A2 and low levels of apoA-I associate with MetS in young adults. The atherogenicity of MetS in this population assessed by incident high carotid intima-media thickness appears to be substantially mediated by elevated apoB but not inflammatory markers.

Background— Here, we aim to identify defects of apolipoprotein (apo) B lipoprotein metabolism that characterize hypertriglyceridemia, focusing on apoC-III and apoE. Methods and Results— We studied the transport of plasma apoB within 21 distinct subfractions as separated by anti–apoC-III and anti–apoE immunoaffinity chromatography and ultracentrifugation in 9 patients with moderate hypertriglyceridemia and 12 normotriglyceridemic control subjects. Hypertriglyceridemia was characterized by a 3-fold higher liver secretion of very low-density lipoprotein (VLDL) that had apoC-III but not apoE and a 50% lower secretion of VLDL with both apoC-III and apoE (both P P Conclusions— These results support a central role for apoC-III in metabolic defects leading to hypertriglyceridemia. Triglyceride-rich lipoprotein metabolism shifts from an apoE-dominated system in normotriglyceridemic participants characterized by rapid clearance from circulation of VLDL to an apoC-III–dominated system in hypertriglyceridemic patients characterized by reduced clearance of triglyceride-rich lipoproteins and the formation of the dense LDL phenotype.

Objective— We aimed to clarify the influence of apolipoprotein C-III (apoCIII) on human apolipoprotein B metabolism. Methods and Results— We studied the kinetics of 4 very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) types containing: (1) otherApos−CIII−: none of apoCIII, apoAII, apoCI, apoCII, or apoE; (2) otherApos+CIII−: no apoCIII but at least one of the others; (3) otherApos−CIII+: apoCIII, but not any others; and (4) otherApos+CIII+: apoCIII and at least one other. VLDL and IDL otherApos−CIII+ and otherApos−CIII− had similar rates of lipolytic conversion to smaller particles. However, light LDL otherApos−CIII+ compared with otherApos−CIII− had much faster conversion to dense LDL as did light LDL otherApos+CIII+ compared with otherApos+CIII−. VLDL and IDL otherApos−CIII+ had minimal direct removal from circulation, whereas VLDL and IDL otherApos+CIII−, rich in apoE, showed fast clearance. Lipoproteins in fraction otherApos+CIII+ also rich in apoE had very low clearance. Conclusions— The results suggest that apoCIII strongly inhibits hepatic uptake of VLDL and IDL overriding the opposite influence of apoE when both are present. The presence of apoCIII on dense VLDL is not associated with slow conversion to IDL, a lipoprotein lipase-dependent process; but when on light LDL, apoCIII is associated with enhanced conversion to dense LDL, a process involving hepatic lipase.

High-density lipoprotein (HDL) subclasses have different antiatherogenic potentials and functional properties. This work presents our findings and discussions on their metabolic implications on apolipoprotein (apo) C-III together with other apolipoprotein levels and HDL subclass distribution profile. Apolipoprotein A-I contents of plasma HDL subclasses were quantitated by 2-dimensional gel electrophoresis coupled with immunodetection in 511 subjects. Concentrations of triglycerides and of apo B-100, C-II, and C-III were higher, whereas those of HDL cholesterol were lower, for subjects in the highest tertile of apo C-III levels group, which presented a typical hypertriglyceridemic lipid profile. Subjects in the middle and highest tertile of apo C-III levels groups had increased prebeta(1)-HDL, HDL(3c), HDL(3b) (only in the highest tertile of apo C-III group), and HDL(3a), but decreased HDL(2a) and HDL(2b) contents compared with subjects in the lowest tertile of apo C-III levels group. With the elevation of apo C-III together with apo C-II levels, contents of small-sized prebeta(1)-HDL increased successively and significantly; but those of large-sized HDL(2b) reduced successively and significantly. With a rise in apo C-III and apo A-I levels, those of prebeta(1)-HDL increased significantly. Moreover, subjects with high apo A-I levels showed a substantial increase in HDL(2b); on the contrary, HDL(2b) declined progressively and obviously for subjects in the low apo A-I levels with the elevation of apo C-III levels. Correlation analysis illustrated that apo C-III levels were positively associated with prebeta(1)-HDL, prebeta(2)-HDL, and HDL(3a). The particle size of HDL shifted toward smaller sizes with the increase of plasma apo C-III levels, and the shift was more remarkable when the elevation of apo C-III and apo C-II was simultaneous; and besides, higher apo A-I concentrations could modify the effect of apo C-III on HDL subclass distribution profile. Large-sized HDL(2b) particles decreased greatly for hypertriglyceridemic subjects who were characterized by elevated apo C-III and C-II accompanied with significantly lower apo A-I, which, in turn, blocked the maturation of HDL.

A patient with multiple myeloma presented with an accumulation of chylomicron-like particles. This rare finding resembled that of the type V hyperlipoproteinaemia phenotype. The lipid and lipoprotein concentration and composition were compared with values obtained from other patients with multiple myeloma, patients with the type V hyperlipoproteinaemia phenotype (accumulation of chylomicrons and very low density lipoproteins), and normal subjects. An immunoglobulin-lipid complex was demonstrated in our patient. This complex was found not to be associtated with the chylomicrons and was detected only in the lipoprotein-dificient plasma. Lipid and lipoprotein concentration and composition differed from the other groups. Very low density lipoprotein concentration was reduced, and there was thus a marked difference from the type V phenotype. The chylomicrons derived from this patient were also richer in apolipoprotein C compared to chylomicrons derived from the patients with type V hypolipoproteinaemia. It appears that the abnormal composition of the triglyceride-rich lipoproteins observed in this patient renders her refractory to the normal pathways of metabolism.

The metabolism of the apolipoproteins of very low density lipoprotein (VLDL) was studied in three subjects with primary type V hyperlipoproteinaemia (HLP) after injection of autologous 125I-labelled VLDL of Sf 100–400. The fractional disappearance rate of total radioactivity was stable during the two-week period of study. Transfer of apolipoprotein C (apo-C) from VLDL to high density lipoprotein (HDL) occurred both in vivo and in vitro upon mixing the labelled VLDL preparation with serum and significant amounts of 125I-labelled apoli-poprotein B (apo-B) and apo-C were recovered in the chylomicron fraction (Sf >400) in both situations. The time-courses of the disappearance of specific activity from VLDL (Sf 20–400) and its appearance in low density lipoprotein (LDL) are consistent with apo-B in VLDL (VLDL-B) being the precursor of that in LDL. In comparison to studies in which the whole VLDL class (Sf 20–400) was labelled, the rate of synthesis of VLDL-B was slightly decreased and the fractional rates of catabolism (FCRs) of apo-B in VLDL and in the whole spectrum of VLDL+chylomicrons were markedly depressed. In turn, the rate of formation of LDL was low, and its FCR was probably slightly decreased. The findings are consistent with the belief that in these three subjects the type V HLP is to a great extent due to impaired rates of formation and breakdown of apo-B in VLDL. They do not exclude the possibility that the disorder is also caused by an increased rate of formation of chylomicrons.

Background: Wet-wrap treatment (WWT) with diluted topical steroids is widely used in atopic dermatitis (AD). Mice with transgenic overexpression of human apolipoprotein C1 (APOC1) in the liver and the skin are not only characterized by hyperlipidaemia and raised IgE levels, but also by pruritic dermatitis and a disturbed skin barrier function, providing a novel in vivo mouse model for AD. Objectives: We investigated an adapted WWT method in the AD model in APOC1 mice in order to establish its efficacy. Methods: The effect of topical 0.1% and 0.03% tacrolimus ointment, tacrolimus base ointment, different dilutions of 0.05% fluticasone propionate (FP) cream and emollient on the development of dermatitis in APOC1 mice was investigated. WWT was performed with 0.03% tacrolimus ointment or 0.017% FP cream. Results: AD in APOC1 mice responded to topical treatment with tacrolimus or FP. In contrast to tacrolimus treatment, FP treatment was associated with loss of body weight. WWT reinforced several therapeutic aspects, notably improvements in transepidermal water loss and in epidermal thickness. WWT using tacrolimus 0.03% ointment was more effective than WWT using FP 0.017% cream. Conclusions: AD in APOC1 mice responds to treatment with (diluted) tacrolimus or FP; treatment with FP cream, but not tacrolimus ointment, was associated with weight loss. In this study, the adapted WWT using tacrolimus or FP in mice had a limited improving effect as compared with open application of tacrolimus or FP. © 2008 The Authors.

Portomicron remnant and very low density lipoprotein (VLDL) remnant in chickens were prepared by the method using ultracentrifugation (d

Objectives— The metabolic syndrome (MetS) is characterized by insulin resistance and dyslipidemia that may accelerate atherosclerosis. Disturbed apolipoprotein (apo) C-III metabolism may account for dyslipidemia in these subjects. Atorvastatin and fenofibrate decrease plasma apoC-III, but the underlying mechanisms are not fully understood. Methods and Results— The effects of atorvastatin (40 mg/d) and fenofibrate (200 mg/d) on the kinetics of very-low density lipoprotein (VLDL)-apoC-III were investigated in a crossover trial of 11 MetS men. VLDL-apoC-III kinetics were studied, after intravenous d 3 -leucine administration using gas chromatography-mass spectrometry and compartmental modeling. Compared with placebo, both atorvastatin and fenofibrate significantly decreased ( P P Conclusions— Both atorvastatin and fenofibrate have dual regulatory effects on VLDL-apoC-III kinetics in MetS; reduced production and increased fractional catabolism of VLDL-apoC-III may explain the triglyceride-lowering effect of these agents.

OBJECTIVE AND DESIGN The present study aims at evaluating the influence of genetic polymorphisms on antiretroviral therapy (ART)-associated lipodystrophy. We included in the study 255 ICoNA. patients and we assessed the distribution of Fas -670 AG polymorphism, ApoC3 -455 CT and -482 CT polymorphisms, C161T silent substitution in the PPAR gamma gene, the Adrenergic beta3 Receptor (ARbeta3) codon 64 TC variant, and two polymorphisms in the Adrenergic beta2 Receptor (ARbeta2) codon 16 AG and codon 27 CG. Crude rates of lipoatrophy and fat accumulation and adjusted relative rates were calculated using Poisson regression. RESULTS In a multivariate model after adjusting for gender, HIV exposure, age, current viral load, hepatitis C virus (HCV) serology, nucleoside reverse-transcriptase inhibitor (NRTI) pair/'third drugs' currently used, months of pre-highly active antiretroviral therapy (HAART) exposures to NRTI, the following genotypes resulted protective against lipoatrophy: ApoC3 -455 CC genotype [adjusted relative risks (ARR) 0.2, 95% confidence interval (CI) 0.046-0.91 vs CT/TT, P = 0.037], ARbeta3 codon 64 TT genotype (ARR 0.39, 95% CI 0.14-1.06 vs TC/CC, P = 0.066), and Fas -670 GG genotype (ARR 0.51, 95% CI 0.26-1.01 vs AG/AA, P = 0.053). With regard to fat accumulation, in the multivariate model, the ARbeta2 codon 27 CC genotype resulted protective (ARR 0.21, 95% CI 0.08-0.51 vs CG/GG, P = 0.0006), whereas the ARbeta2 codon 16 AA genotype resulted associated with higher risk (ARR 3.72, 95% CI 1.58-8.76 vs AG/GG, P = 0.0026). CONCLUSION Our study suggests that genetic polymorphisms of genes involved in apoptosis and adipocyte metabolism are significantly related to ART associated lipodystrophy. Particularly, we evidenced a role for ApoC3 -455 in lipoatrophy and for the two variants of ARbeta2 in fat accumulation.

Objective— Insulin resistant states are associated with increased fatty acid flux to liver and intestine, which stimulates the production of triglyceride-rich lipoproteins (TRL). ApoC-III production and plasma and TRL concentrations are increased in insulin resistance and may contribute to the hypertriglyceridemia of these conditions. The mechanism underlying that increase is not known, but because apoC-III and VLDL production are closely linked we hypothesized that FFAs may stimulate TRL apoC-III production. Methods and Results— We used Intralipid/heparin (IH) to raise plasma FFA in 12 healthy men in the fed state, and stable isotopes to examine apoC-III metabolism. TRL apoC-III concentration was significantly higher in the IH study, and this increase was associated with higher production (PR) and fractional catabolic rate (FCR). The increase in production was greater than in FCR (90% versus 30%, respectively), accounting for the elevated concentration. Glycerol infusion had no effect on apoC-III concentration, PR, or FCR compared to saline, indicating that the effect was not attributable to glycerol released from intralipid. Conclusion— These findings confirm that TRL apoC-III production is stimulated by an acute elevation of plasma FFAs, suggesting a novel regulatory pathway that may play a role in the overproduction of TRL apoC-III in insulin resistant states.

The rare apolipoprotein C-II (apoC-II) mutation, apoC-IILys19-->Thr, also known as apoC-II-v, has been found previously in association with hyperlipoproteinemia. From a lipid clinic screening we identified three unrelated individuals who had the apoC-IILys19-->Thr mutation. Among eight family members of one proband, we have found another four who were affected. None of the individuals in this kindred is dyslipidemic and there is no difference in lipid levels between affected and unaffected family members. Therefore, we conclude that the presence of this apolipoprotein variant by itself has no effect on lipoprotein levels. In addition, the apolipoprotein E (apoE) isoform, apoE4 does not have a synergistic effect on lipoprotein levels in this kindred, in contrast to observations on the interaction of apoE4 with another apoC-II mutant (apoC-IIToronto). The single nucleotide substitution-that causes the apoC-IILys19-->Thr variant introduces a previously unrecognized restriction site (for Mae III), that provides for easy screening.

We have used a common TaqI restriction fragment length polymorphism (RFLP) near the human apolipoprotein C-II (apoC-II) gene to study linkage with apolipoprotein E (apoE). The inheritance of the apoC-II RFLP was followed in seven families that were segregating for apoE protein variants. No recombinants were observed in 20 informative meioses, giving an overall lod score of > 4.0 at recombination fraction 0. We have also observed apparent linkage disequilibrium between apoE and the apoC-II RFLP. Taken together these results demonstrate that these two apolipoprotein genes are closely linked and confirm that the gene for apoC-II is on human chromosome 19.

The high-density lipoprotein (HDL) constituent apolipoprotein CI (apoCI) protects mice against mortality in bacterial sepsis. We assessed whether high plasma apoCI levels protect against mortality from infection in humans. We determined plasma levels of apoCI, lipids, and C-reactive protein in 85-year-old participants of the prospective population-based Leiden 85-Plus Study (n = 561). Participants were followed for specific causes of death. High apoCI levels were associated with 40% reduced risk of mortality from infection (hazard ratio [HR], 0.60; 95% confidence interval [CI], 0.42-0.86; p = .005) for every increase of 1 standard deviation in apoCI level. A similar association was observed for high HDL cholesterol (HR, 0.65; 95% CI, 0.46-0.94; p = .022), but not for LDL cholesterol, triglycerides, and C-reactive protein levels. The association of apoCI was independent of HDL cholesterol, as multivariate analysis did not alter the association for apoCI (HR, 0.63; 95% CI, 0.44-0.90; p = .013), whereas for HDL cholesterol significance was lost. We conclude that high apoCI levels are associated with reduced mortality from infection, in line with experimental evidence in rodents. Copyright 2008 by The Gerontological Society of America.

Objective Apolipoprotein E (ApoE) is a lipid transport protein with expanded functions in cellular responses to tissue injury, immune regulation and cell growth. ApoE directs vascular changes that contribute to arterial protection as evidenced by the fact that isoforms of ApoE and ApoE deficiency correlate closely with accelerated plaque growth. The N-terminus of the ApoE protein has well-characterized functions, displaying lipid-binding and anti-atherogenic activity, whereas the function of the C-terminus is only partially defined. We have assessed the effects of a 14 amino acid C-terminal ApoE peptide, termed Ep1.B (239–252), on intimal neoplasia in animal models. This peptide is a fragment of a naturally processed peptide (236–252) of murine ApoE. Methods and results Ep1.B injection reduced neointimal hyperplasia after vascular surgery in rats and mice. When given during early plaque progression in ApoE-deficient mice, Ep1.B injections also prevented plaque growth. Treatment with Ep1.B did not, however, reduce established plaque growth in older mice. Peptides with alanine substitution of amino acid 249, Ep1.N, and with complete sequence reversal, Ep1.R, did not consistently inhibit plaque growth. Conclusion A naturally processed C-terminal ApoE peptide, Ep1.B, has anti-atherogenic activity indicating a role for this naturally metabolized peptide in vascular wound healing and lipid homeostasis.

Major advances in cancer control depend upon early detection, early diagnosis and efficacious treatment modalities. Current existing markers of pancreatic ductal adenocarcinoma, generally incurable by available treatment modalities, are inadequate for early diagnosis or for distinguishing between pancreatic cancer and chronic pancreatitis. We have used a proteomic approach to identify proteins that are differentially expressed in sera from pancreatic cancer patients, as compared to control. Normal, chronic pancreatitis and pancreatic cancer serum samples were depleted of high molecular weight proteins by acetonitrile precipitation. Each sample was separated by chromatofocusing, and then further resolved by reversed-phase (RP)-HPLC. Effluent from the RP-HPLC column was split into two streams with one directly interfaced to an electrospray time-of-flight (ESI-TOF) mass spectrometer for molecular weight (MW) determination of the intact proteins. The remainder went through a UV detector with the corresponding peaks collected with a fraction collector, subsequently used for MS/MS analysis. The ion intensities of proteins with the same MW obtained from ESI-TOF-MS analysis were compared, with the differentially expressed proteins determined. An 8915 Da protein was found to be up-regulated while a 9422 Da protein was down-regulated in the pancreatic cancer sera. Both proteins were identified by MS and MS/MS as proapolipoprotein C-II and apolipoprotein C-III 1 , respectively. The MS/MS data of proapolipoprotein C-II was searched using “semi-trypsin” as the search enzyme, thus confirming that the protein at 8915 Da was proapolipoprotein C-II. In order to confirm the identity of the protein at 9422 Da, we initially identified a protein of 8765 Da with a similar mass spectral pattern. Based on MS and MS/MS, its intact molecular weight and “semi-trypsin” database search, the protein at 8765 Da was identified as apolipoprotein C-III 0 . The MS and MS/MS data of the proteins at 8765 Da and 9422 Da were similar, thus confirming the protein at 9422 Da as being apolipoprotein C-III 1 . The detection of differentially expressed proapolipoprotein C-II and apolipoprotein C-III 1 in the sera of pancreatic cancer patients may have utility for detection of this deadly malignancy.

Objective Both mildly modified LDL subfraction that carries a more-negative electric charge and remnant-like particles (RLP) are closely related to triglyceride (TG) levels. We examined the relation between the RLP-cholesterol (C) level and charge-based apolipoprotein (apo) B-containing lipoprotein subfractions as determined by capillary isotachophoresis (cITP) in patients with hypercholesterolemia. Methods and results cITP apo B lipoprotein subfractions were identified by analyzing plasma depleted of the related lipoproteins. While fast-migrating triglyceride-rich lipoprotein (fTRL) subfraction contained both chylomicrons and VLDL fraction, slow TRL (sTRL) only contained VLDL. cITP fLDL also contained VLDL fraction, i.e., β-VLDL. Levels of cITP fTRL and sTRL were significantly correlated with serum levels of TG, RLP-C, apo C-II, and C-III. Levels of cITP sTRL were also correlated with apo E. Levels of cITP fLDL were positively correlated with not only LDL-C levels but also levels of TG, RLP-C, apo C-II, C-III, and E. Conclusion cITP fast LDL correlated with RLP-C levels and modified the relation between RLP-C and TG levels.

Interleukin (IL)-6 is a circulatory, pleiotropic cytokine with multiple roles in the immune system. Both IL-6 and the IL6 -174G>C promoter polymorphism have been linked to various diseases associated with inflammation. However, the mechanism by which the polymorphism influences disease risk is unclear. We postulated that serum proteome analysis of individuals with different IL6 -174G>C genotypes would provide insight on genotype-phenotype associations of this polymorphism and its role in disease susceptibility. Serum from a random sample of control participants in an ongoing population-based case-control study of non-Hodgkin lymphoma was pooled by IL6 genotype and used to screen for the optimal SELDI-TOF MS arrays for analysis. We report differences in serum protein expression of individuals with specific genotypes based on pooled and individual sample analysis. In particular, we report an association of the -174C allele with increased apolipoprotein C-I (ApoC-I). Additionally, we corroborate previous findings of an association of the -174C allele with lower autoantibodies to heat shock protein 60 and confirm the absence of any association between the IL6 -174G>C genotype and serum IL-6 levels. This study illustrates that proteome analysis can enhance our understanding of genotype-phenotype relationships. Additional studies are needed to clarify the interaction between the IL6 -174G>C polymorphism and ApoC-I.

Introdução: O diabetes mellitus tipo 2 (DM2) é a doença metabólica mais comum. Objetivo: Pesquisar apresença do polimorfismo SstI da apolipoproteína C-III e verificar se existe associação com o perfil lipídicoem uma amostra de diabéticos do Sul do Brasil. Materiais e Métodos: 65 pacientes com DM2 foramsubmetidos à avaliação antropométrica e análise do perfil lipídico e do polimorfismo SstI da APO C-IIIutilizando o método de reação em cadeia da polimerase seguida de digestão enzimática (PCR-RFLP).Foram classificados como alelo G os fragmentos que apresentaram tamanho de 269 e 159 pares de bases(pb) e como alelo C os fragmentos que apresentaram tamanho de 428 pb. Utilizou-se o teste t de Studentpara comparar as médias do perfil lipídico entre homens e mulheres, e a análise do polimorfismo foi feitapor meio do teste qui-quadrado (?2) e do teste exato de Fischer. Resultados: A média de idade foi 64,67± 8,74 anos e o IMC foi 31,09 ± 4,86 kg/m2. 55% dos indivíduos não utilizaram medicação antilipêmica.Os valores do colesterol total, triglicerídeos, HDL e LDL foram normais. Com relação ao polimorfismo,80% apresentou genótipo normal (CC), 3,1% genótipo alterado (GG) e 16,9% genótipo alterado (CG).Não foi observada associação significativa entre o genótipo CC e os valores de triglicerídeos menores que200 mg/dL. Conclusão: Na população avaliada a maioria dos indivíduos apresentou genótipo normalpara o polimorfismo SstI sem haver associação com os níveis de triglicerídeos. Introduction: Type 2 diabetes mellitus (DM2) is the most common metabolic disease. Objective: Toinvestigate the presence of the SstI polymorphism of the C-III apolipoprotein and verify the associationwith the lipid profile in a diabetic sample from Southern Brazil. Materials and methods: Sixty-fivepatients with DM2 were subjected to anthropometric measurements, lipid profile and polymorphismanalyses. The SstI polymorphism of APO C-III was made using the polymerase chain reaction methodfollowed by enzymatic digestion (PCR-RFLP). Fragments that showed a size of 269 base pairs (bp) and159 bp were classified as G allele, and fragments that showed a size of 428 bp were classified as C allele.We used the Student t test to compare the means of the lipid profile between men and women while thepolymorphism analysis was performed using the chi-square test (?2) and the Fisher exact test Results:The mean age was 64.67±8.74 and BMI was 1.09±4.86 kg/m2. Of the 65 individuals, 55% did not useantilipemic medication. The rate of total cholesterol, triglycerides, HDL and LDL levels were withinnormal limits. Regarding polymorphism, 80% of the subjects had normal genotype (CC), 3.1% changedgenotype (GG), and 16.9% altered genotype (CG). There was no significant association between CCgenotype and triglyceride levels below 200 mg/dL. Conclusion: In the evaluated population, the majorityof the individuals presented a normal genotype to the SstI polymorphism with no association with thetriglycerides rate.

Lipoprotein lipase (LPL)-mediated triacylglycerol (TAG) hydrolysis in very low density lipoprotein (VLDL) is accompanied by the release of surface material containing phospholipids (PL), free cholesterol (FC) and apolipoproteins, E (apoE) and Cs (apoCII, apoCIII). The released molecules are accepted by high density lipoprotein (HDL), and new HDL-sized apoE-containing particles are also generated. A decrease in the number of HDL particles or abnormalities in their structure is associated with unfavourable changes in the features of VLDL remnants. Phosphatidylcholine liposomes (PC-L) can also act as acceptors of surface material components released from lipoproteins. Thus, the aim of this study was to assess the impact of liposomes on compositional changes of VLDL during its LPL-mediated lipolysis. VLDL isolated from human sera was incubated with LPL (LPL:VLDLTAG; 24 μg/ml:90 mg/dl) and/or PC-L (VLDLPL:PC-LPL; 1:30 weight ratio). After incubation (2h, 37 °C) VLDL was separated from other reaction products, and VLDL lipid and apolipoprotein content were analysed. Newly generated HDL-sized apoE-containing lipoproteins were separated by two-dimensional non-denaturing gradient gel electrophoresis (2D-PAGGE). The reaction of VLDL with PC-L in the presence or absence of LPL significantly affected the VLDL composition. The ratio of core (TAG+cholesteryl ester) to surface (PL+FC) lipids in VLDL decreased 1.8-fold with PC-L, 1.2-fold with LPL and 3-fold with PC-L+LPL. The reaction with PC-L and PC-L+LPL caused a 3.7-fold and 3.2-fold decrease of apoCs/apoE average weight ratio, respectively. Compositional changes in VLDL under the influence of PC-L were accompanied by an increase in the efficiency of VLDL lipolysis and the generation of apoE-containing HDL-sized particles, heterogeneous in size (from ∼ 9 to ∼ 18.8 nm) and mobility (γ and preβ). We conclude that PL-rich particles, similarly to HDL, promote the release of surface material components from VLDL during LPL-mediated lipolysis and positively influence VLDL features which can facilitate VLDL metabolism. Such PC-L activity may impact on its antiatherogenic properties.

Serum triglyceride levels (TG) are important independent risk factors for coronary heart disease. The apolipoproteins C-III (apoCIII) and A-V (apoAV) are central to normal TG metabolism and the complete sequence analysis of these genes was carried out in severe cases (TG9 mmol/l) and controls (TG2 mmol/l). A total of 53 SNPs were identified in these genes with 17 being novel to this study. Further analysis defined four APOC3 SNPs and three APOA5 SNPs showing strong association with TG levels. Analysis of the two major SNPs from APOA5 [c.56CG, c.-3AG] and from APOC3 [c.102CT, c.340CG] using THESIAS has identified two major haplotypes relative to the most common CACC haplotype showing very strong association with hypertriglyceridaemia, CGTG and GATC (odds ratio 7.45 and 5.26). Logistic regression analysis of these four SNPs revealed that, carriage of the APOA5 c.56 G allele (odd ratios 4.49) and the APOA5 c.-3 G allele (odds ratio 3.23) were strong independent predictors of hypertriglyceridaemia (P0.001), whereas in contrast, carriage of the APOC3 c102 T allele (odds ratio 1.35) and the APOC3 c.340 G allele (odds ratio 1.37), did not show any significant effects that were independent of APOA5.

Carefully controlled ZipTip extraction of diluted human plasma or serum was combined with MALDI-TOF-MS to produce highly reproducible protein profiles. Components detected included apolipoproteins CI, CII and CIII as well as transthyretin and several isoforms of each protein that are created by glycosylation or other modification and by proteolytic processing. Profiles of healthy individuals all contained the same 15 components. Others were found in plasma from individuals with disease. Profiles were analyzed by peak ratios within the same spectrum. Reproducibility for multiple assays was generally 4 to 10%. Within the healthy population, a given peak ratio occurred with a range of about fourfold. However, peak ratios of multiple samples from the same individual showed a much lower range, typically +/-10%. In fact, each individual displayed a personal protein profile that changed very little over time. Because of the stability of protein profiles over time within individuals, these results suggest further studies may discover that certain profile characteristics or changes in an individual's profile may be a sign of current or future disease, even when the altered profile remains within the range for healthy individuals.

Background: Apolipoprotein C-III (apo C-III) is a marker of cardiovascular disease risk associated with triglyceride (TG)-rich lipoproteins. The T−455C polymorphism in the insulin-responsive element of the APOC3 gene influences TG and apo C-III concentrations. Long-chain n-3 polyunsaturated fatty acids (PUFAs) contained in fish have well-known apo C-III-lowering properties.Methods: We investigated the possibility of an interactive effect between the APOC3 gene variant and erythrocyte n-3 PUFAs, suitable markers of dietary intake of fatty acids, on apo C-III concentrations in a population of 848 heart disease patients who had coronary angiography.Results: In the population as a whole, apo C-III concentrations were significantly inversely correlated with total erythrocyte PUFAs, but the correlation was not significant when only −455CC homozygous individuals were taken into account. In the total population and in subgroups with the −455TT and −455CT genotypes, the relative proportions of individuals presenting with increased apo C-III (i.e., above the 75th percentile value calculated on the entire population after exclusion of individuals taking lipids-lowering medications) decreased progressively as the n-3 PUFA and docosahexaenoic acid concentrations increased. The opposite situation was observed in the homozygous −455CC subgroup, in whom increasing erythrocyte n-3 PUFA and docosahexaenoic acid concentrations were associated with higher proportions of individuals with high apo C-III. A formal interactive effect between genotype and n-3 PUFAs was confirmed even after adjustment for possible confounding variables [age, sex, body mass index, smoking, coronary artery disease (CAD)/CAD-free status, or use of lipid-lowering medications] by logistic models.Conclusion: Patients homozygous for the −455C APOC3 variant are poorly responsive to the apo C-III-lowering effects of n-3 PUFAs.

Extracellular sphingosine 1-phosphate (S1P) has been shown to contribute to the action of high density lipoprotein (HDL) on endothelial and smooth muscle cells. We examined the relationship of lipoprotein-associated S1P concentrations with cholesterol (C) and apolipoprotein (apo) contents of lipoprotein and lipoprotein subfractions characterized by capillary isotachophoresis (cITP).Blood samples were drawn from 16 volunteers. S1P concentrations were quantified by bioassay based on the ability of S1P to stimulate its receptor. cITP was performed using plasma that had been prestained with NBD-ceramide.In plasma, S1P was concentrated in HDL and associated with LDL at a much lower concentration. HDL-S1P was the major determinant of the plasma S1P concentration. HDL-S1P was strongly and positively (p0.001) correlated with serum levels of HDL-C (r=0.82), apo A-I (r=0.91) and apo A-II (r=0.92). HDL-S1P was strongly and positively (p0.01) correlated with the apo A-I- and apo A-I/apo A-II-containing cITP HDL subfractions [fast HDL-C (r=0.66) and intermediate HDL-C (r=0.80)], but was not significantly correlated with apo E-containing slow HDL, suggesting that S1P is associated with both apo A-I HDL and apo A-I/A-II HDL. LDL-S1P was positively correlated (p0.01) with levels of LDL-C (r=0.65) and apo B (r=0.85).Lipoprotein-associated S1P was related to the lipoprotein composition of cholesterol and apolipoproteins, suggesting that extracellular S1P may play different roles depending on the particles with which it is associated.

Objectives— Apolipoprotein C-I (apoC-I) influences lipoprotein metabolism, but little is known about its cellular effects in aortic smooth muscle cells (ASMC). Methods and Results— In cultured human ASMC, apoC-I and immunoaffinity purified apoC-I–enriched high-density lipoproteins (HDL) markedly induced apoptosis (5- to 25-fold), compared with control cells, apoC-I–poor HDL, and apolipoprotein C-III (apoC-III) as determined by 4′, 6-diamidino-2-phenylindole dihydrochloride staining and DNA ladder assay. Preincubation of cells with GW4869, an inhibitor of neutral sphingomyelinase (N-SMase), blocked apoC-I–induced apoptosis, an effect that was bypassed by C-2 ceramide. The activity of N-SMase was increased 2- to 3-fold in ASMC by apoC-I, apoC-I–enriched HDL, and tumor necrosis factor α (TNF-α) (positive control) after 10 minutes and then decreased over 60 minutes, which is a kinetic pattern not seen with controls, apoC-III, and apoC-I–poor HDL. ApoC-I and apoC-I–enriched HDL stimulated the generation of ceramide, the release of cytochrome c from mitochondria, and activation of caspase-3 greater than that found in controls, apoC-III, and apoC-I–poor HDL. GW4869 inhibited apoC-I–induced production of ceramide and cytochrome c release. Conclusions— ApoC-I and apoC-I–enriched HDL activate the N-SMase-ceramide signaling pathway, leading to apoptosis in human ASMC, which is an effect that may promote plaque rupture in vivo.

By immunohistochemistry, we investigated pepsinogen C (pepC) and apolipoprotein D (apoD) expressions in 23 basal cell (BCC) and 25 squamous cell carcinomas (SCC) of the eyelids and analyzed the possible relationship to clinicopathological parameters. A total of 3 (13%) BCC and 7 (28%) SCC stained positively for pepC, whereas 11 (47.8%) BCC and 6 (24%) SCC stained positively for apoD. No significant correlation was found between pepC and apoD expressions and patients or tumor characteristics (p > 0.05). pepC and apoD, two androgen-inducible proteins, may be expressed by BCC and SCC of the eyelids. Further studies will be necessary to determine their clinical significance.

Probucol, an antioxidative and hypolipidemic agent, has been postulated to increase reverse cholesterol transport by enhancing cholesteryl ester transfer protein (CETP) activity. However, its clinical implication in CETP deficient patients has not been fully defined. To characterize the effects of probucol in the absence of CETP, we evaluated the changes in lipid profile, lipid peroxidation, and paraoxonase 1 (PON1) activity in two complete CETP deficient patients, caused by treatment with probucol. When the patients were not receiving probucol, low-density lipoprotein (LDL) particles were smaller and high-density lipoprotein (HDL) particles were larger in these patients than in controls. Treatment with probucol (500 mg/day) resulted in the decrease in the levels of HDL-C and apolipoprotein (apo) A-I up to 22%. The size of HDL particles became smaller. LDL cholesterol concentration did not change in one patient, while it decreased by 47% in the other. PON1 activity/HDL-C, which was about 40% lower in the patients before treatment than in controls with the matching PON1 genotype, increased by 30% during the treatment. Lag time for LDL and HDL in both cases became prolonged more than 1.8 times after administration of probucol. This study demonstrated for the first time that probucol reduces HDL-C even in humans with complete CETP deficiency. Probucol treatment in these patients was also associated with protection of lipoproteins against oxidative stress, suggesting a clinical benefit of this drug even in such a state.

It has been reported that human plasma sphingomyelin (SM) levels are positively and independently related to coronary artery disease. The lipoprotein surface is mainly formed by phosphatidylcholine (PC) and SM together with cholesterol and apolipoproteins. However, the influence of SM on the cell uptake of triglyceride-rich lipoproteins and remnants is poorly understood. To clarify the role of SM in lipoprotein uptake, we prepared lipid emulsions containing triolein, PC and SM as model particles of lipoproteins. Apolipoprotein E (ApoE) binding studies revealed that incorporation of SM into the emulsion surface reduced the binding capacity of apoE without changing the affinity. Surface SM reduced apoE-mediated uptake of emulsions by HepG2 cells because of the decreased amount of binding apoE. Apolipoproteins C-II and C-III inhibited the apoE-mediated uptake of SM containing emulsions more effectively. The stimulatory effect of lipoprotein lipase (LPL) on emulsion uptake was decreased by replacing surface PC with SM. These results suggest that SM-induced changes in the binding properties of apolipoproteins and LPL correlate with decreased hepatic uptake of lipid particles.

Apolipoprotein LPA, APOE, APOC1, and APOC2 genotype frequencies have been determined for the first time in a North African population. A sample of 140 Berber individuals from the Moroccan Moyen Atlas region has been analyzed. Allelic and haplotypic data have been used to compare our sample with other world populations and the results clearly differentiate Berbers from Europeans and Sub-Saharans, suggesting several distinctive features of Moroccan Berbers as the extreme high values of LPA PNR*11 pentanucleotide allele (10.5%) and the relatively high and low values of APOE*E4 (15.7%) and *E2 (4.5%) in comparison to other Mediterraneans. Another remarkable result is the frequency distribution of the two APOC2 alleles (70% vs 30%) in comparison with the European pattern (50% of each allele). The high values of APOE*E4 and LPA PNR*7 together with the intermediate linkage disequilibrium values between APOE and APOC1 alleles in comparison with Europeans and Africans suggest a certain degree of Sub-Saharan influence in the current Moroccan population.

The frequency distributions of apolipoprotein C alleles in native Asian sheep (Bengal, Kagi, Lampuchhre, Vietnamese, Myanmar, Baruwal, Bhyanglung, Khalkhas, and Kuwait) were determined using a one-dimensional polyacrylamide gel isoelectric-focusing immunoblotting technique. Among these Asian sheep, the genetic locus consisted mainly of two common APOC*1 and APOC*3 alleles. The Baruwal sheep most frequently possessed APOC*1 (0.9881), showing significant differences from all other Asian sheep in allele frequency (0.7195–0.9032, P < 0.02). Compared with European sheep tested previously, these Asian sheep had significantly higher frequencies of APOC*1 (0.7828 and 0.7127 averages, P < 0.0005) and APOC*3 (0.2162 and 0.11 averages, P < 0.0001). In particular, APOC*2 was detected at an extremely low frequency (0.0025) in the Khalkhas sheep only, and was uncommon in all Asian sheep (0.001 average), in contrast with the 0.1773 average of European sheep (P < 0.00001). This finding suggests that APOC*2 is exceedingly rare in Asian sheep. With the exception of triglycerides and VLDL cholesterol in the Vietnamese sheep, no phenotypic effects on plasma lipid and lipoprotein levels in the Asian sheep tested were seen.

The genetic polymorphism of apolipoprotein C in normal plasma of four European sheep breeds (Suffolk, Corriedale, Cheviot, and Finn) was first detected using one-dimensional polyacrylamide gel isoelectric focusing (pH 2.5–5.0) followed by immunoblotting with antihuman apolipoprotein CII antibody. Six phenotypes (1-1, 2-1, 2-2, 3-1, 3-2, and 3-3) were identified in the 4.3–4.8 pH range, consisting of the combination of three isoform groups. On the basis of family and population data, these phenotypes were controlled autosomally by three codominant alleles, designated APOC*1, APOC*2, and APOC*3, the first being the most common allele. The frequency distributions of these alleles were similar between the Suffolk and Corriedale sheep, and between the Cheviot and Finn sheep. The former breeds had a significantly lower APOC*2 frequency than the latter breeds (P< 0.001). The mean plasma total-, HDL- and LDL-cholesterol levels of type 3-1 animals were significantly higher compared to type 1-1 animals in the Suffolk sheep (P≦ 0.04). However, these differences were not seen in the Corriedale sheep

Patients with nephrotic-range proteinuria have apolipoprotein C and E deficient VLDL1.BackgroundImpaired very low-density lipoprotein (VLDL) clearance contributes to dyslipidemia in nephrotic-range proteinuria. VLDL can be subdivided into large light VLDL1 (Sf 60 to 400) and smaller, denser VLDL2 (Sf 20 to 60). In nephrotic-range proteinuria, the clearance of VLDL1 is delayed. VLDL1 lipolysis is influenced by apolipoprotein CII (apoCII) and apoCIII, whereas apoE regulates receptor-mediated clearance.MethodsTo ascertain whether impaired VLDL1 clearance was related to a deficiency in apolipoproteins on VLDL1, we measured VLDL subfraction concentrations and VLDL1 apolipoprotein and lipid compositions in 27 patients with glomerular disease and urinary albumin> 2 g/24 h along with 27 age- and sex-matched controls.ResultsProteinuric patients had increased plasma VLDL1, VLDL2, apoCII, apoCIII (all P < 0.001), and apoE concentration (P < 0.002). Patients appeared to have smaller VLDL1 particles, as assessed by triglyceride per particle (median + interquartile range, moles per VLDL1 particle): patients, 4.9 (3.0 to 7.9) ×103; controls, 7.0 (4.6 to 15.7) ×103, P < 0.05, with reduced apoCII, 4.2 (3.1 to 8.2) versus 9.9 (7.4 to 23.2), P < 0.0004; apoCIII, 16.6 (9.1 to 27.2) versus 29.3 (18.5 to 69.4), P < 0.02; and apoE content, 0.17 (0.08 to 0.44) versus 0.48 (0.31 to 1.31), P < 0.006. The VLDL1 surface free cholesterol to phospholipid results were increased in proteinuric patients (0.55 ± 0.17 vs. 0.40 ± 0.18, P < 0.002, all mean ± SD). For all patients, VLDL1 apoCII, apoCIII, and apoE contents per particle were related to particle size (apoCII, r2 = 61.5%, P < 0.001; apoCIII, r2 = 75.8%, P < 0.001; apoE, r2 = 58.2%, P < 0.001) and inversely to the free cholesterol to phospholipid ratio (apoCII, r2 = 41.6%, P < 0.001; apoCIII, r2 = 38.8%, P < 0.001; apoE, r2 = 11.7%, P < 0.05). Multivariate analysis suggested that the relative lack of apoCII and apoCIII on patients VLDL1 was related to smaller particle size and increased free cholesterol:phospholipid (FC:PL) ratio. Particle size but not free cholesterol determined the apoE content of VLDL1.ConclusionsWe postulate that impaired VLDL1 clearance in nephrotic-range proteinuria results from the appearance of particles deficient in apoCII, apoCIII, and apoE. VLDL1 apoC deficiency is associated with the formation of smaller particles with a high FC:PL ratio, and is likely to cause inefficient lipolysis. VLDL1 apoE deficiency is associated with smaller VLDL1 particles but not altered VLDL1 surface lipid content, and may reduce receptor-mediated clearance of this lipoprotein.

Background —Exaggerated postprandial triglyceridemia is common in normolipidemic patients with coronary artery disease (CAD). Alterations in the composition of triglyceride-rich lipoproteins (TRLs) are likely to underlie this metabolic disturbance. However, the composition of very-low-density lipoproteins (VLDLs), which are the most abundant postprandial TRLs, has never been defined in CAD patients. Methods and Results —We examined postprandial changes in the number and composition of VLDLs in middle-aged, normolipidemic CAD patients and control subjects. TRLs from 14 patients and 14 control subjects aged 45 to 55 years were subfractionated by density gradient ultracentrifugation into Svedberg flotation rate (Sf) fractions >400, 60 to 400, and 20 to 60. The VLDLs were separated from chylomicron remnants by immunoaffinity chromatography. In CAD patients, the postprandial concentrations of triglycerides and large (Sf 60 to 400) VLDL particles were elevated. In addition, their postprandial large VLDLs were enriched in apolipoprotein (apo) C-I and their postprandial small (Sf 20 to 60) VLDL remnants were enriched with apo C-I and cholesterol. Conclusions —Perturbed handling of postprandial triglycerides in normolipidemic CAD patients involves the accumulation of apo C-I–rich large VLDL particles and the generation of small, apo C-I– and cholesterol-rich VLDL remnants.

A 52-year-old Japanese woman was evaluated for severe hypertriglyceridemia and recurrent acute pancreatitis. This hypertriglyceridemia was found to be due to the absence of serum apolipoprotein C-II (apo C-II) which was identified by Western blotting using polyclonal anti-apo C-II antiserum. DNA sequence analysis of the apo C-II gene from the patient revealed a homozygous nucleotide change: a thymidine (T) to cytosine (C) substitution in codon 26 (TGG->CGG) at the third exon of the apo C-II gene, that resulted in a Trp 26 to Arg substitution. The mutation was also confirmed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzyme Hpa II. The same mutation has been found in a case previously reported in Japan, and was named apo C-II Wakayama. However, the case in Wakayama prefecture showed two concomitant point mutations at the 5'-flanking region upstream from the first exon, which were not identified in our case by RFLP analysis with the restriction enzyme BstXI. Considering that the prefectures of these two cases, Nara and Wakayama, are next to each other, the mutation in our case may be a genetic forebear of apo C-II Wakayama. However, no familial relationship between the two cases has been documented.

Background Hypertriglyceridaemia is a common metabolic disorder frequently found in patients with coronary heart disease. Numerous studies have revealed an association between the SstI polymorphism in the APOC3 gene and increased plasma apoC3 and triglyceride levels. In addition, two different variants within the promoter region have been recently suggested to be the mutations of the APOC3 gene leading to hypertriglyceridaemia. Methods In the present study, we have applied haplotype analysis to investigate whether these promoter polymorphisms are involved in the lipid disorders of patients with distinct types of hypertriglyceridaemia: combined hyperlipidaemia (CHL), familial dysbetalipoproteinaemia (FD) and endogenous hypertriglyceridaemia (HTG). Results The −482 and −455 polymorphisms were significantly more frequent in FD patients (P = 0.017) and endogenous HTG patients (P

Hypertriglyceridemia is a common metabolic disorder with a major inherited component. In some individuals the condition is suspected to occur as a result of overproduction of apolipoprotein (apo)CIII, a major constituent of triglyceride-rich lipoproteins. Population studies have established an association with the apoCIII gene but the identify of the causal mutation remains unknown. In the present study we have examined a series of six 5' polymorphic nucleotides (G-935 to A, C-641 to A, G-630 to A, deletion of T-625, C-482 to T, and T-455 to C) that lie within the promoter region of the apoCIII gene for evidence of possible involvement in disease susceptibility. The polymorphic nucleotides at positions -455 and -482 reside within a negative insulin-response element. We show, in a community-based sample of 503 school children, that a DNA polymorphism (S2 allele) within the 3'-noncoding region of the apoCIII gene was associated with elevated apoCIII and triglyceride levels, but that the polymorphic nucleotides of the promoter were not. In addition, no obvious effect of any extended apoCIII promoter haplotype on plasma apoCIII or triglyceride levels, over and above that conferred by the presence of the S2 polymorphic nucleotide, was detected. These results demonstrate that none of the 5' apoCIII polymorphisms can account for the association of the apoCIII gene locus with hypertriglyceridemia and, moreover, owing to linkage disequilibrium, raise the possibility that the region conferring susceptibility maps downstream, rather than upstream, of the apoCIII gene promoter sequences.

Transient increases in triglycerides and cholesterol were found in rat liver immediately after birth. Plasma VLDL and HDL increased after birth and reached a plateau after one week of life. The content of cholesterol ester was low at birth in all lipoproteins and increased in LDL and HDL during the first week of life. After birth, VLDL became enriched in apolipoproteins C and E, whereas HDL was enriched in apolipoprotein C and depressed in apolipoprotein E. The developmental changes in plasma lipoprotein levels and compositions in rats during the first week of life are comparable to those described in humans.

The objective of this study was to investigate whether compositional variation in apolipoprotein (apo) content of triglyceride-rich lipoproteins (TRLP) modulates binding of heparan sulfate proteoglycans (HSPG). Human TRLP was enriched with apoE and apoCs and the ability to bind biotin-conjugated heparan sulfate (b-HS) was studied in the presence or absence of heat-inactivated lipoprotein lipase (LPL). TRLP, associated with LPL, showed an increased capacity to bind b-HS compared with TRLP alone. Low density lipoproteins (LDL) bound both b-HS and LPL with a higher affinity than TRLP. ApoE enrichment of TRLP resulted in an enhanced binding of b-HS. Increased binding of b-HS to TRLP by the combination of apoE enrichment and LPL addition was found to be complementary, not affecting their individual binding capacity. TRLP enrichment with apoC led to the formation of an apoC-rich, apoE-poor particle; this alteration by itself did not change the ability to bind b-HS. ApoC enrichment of TRLP resulted in a reduced capacity to bind LPL and therefore a subsequently reduced capacity to bind b-HS, compared with control TRLP associated with LPL. Competition studies revealed that b-HS binding to TRLP was fully displaceable by lactoferrin but barely by heparan sulfate, dermatan sulfate, or chondroitin-4-sulfate. Using TRLP coated to microtiter wells and associated with LPL, the b-HS displacement patterns were comparable to those obtained with coated LDL in the presence or absence of LPL. The cell-free system that was used enabled us to identify the functions of apoC and apoE in the binding of TRLP to LPL and HSPG. Both LPL and apoE increased the ability of TRLP to bind HSPG. The apoC content of TRLP regulated the docking of TRLP to LPL. ApoC enrichment reduced the affinity or capacity of TRLP to LPL binding, and this has relevance for the lipolytic cascade.

The authors evaluated serum retinol, retinol-binding protein (RBP), and beta-carotene levels to elucidate the retinoid metabolism in non-insulin-dependent diabetes mellitus (NIDDM). The mean retinol levels by gender (1.83 mumol/L for females and 2.24 mumol/L for males) in diabetics were higher than those (1.31 mumol/L for females and 1.82 mumol/L for males) in control subjects (P0.0001, P0.01, respectively). The mean retinol/RBP ratios (0.95 for females and 0.97 for males) of diabetics were higher than those of the control subjects (0.60 for females and 0.64 for males) and of male patients having impaired glucose tolerance (0.55) (P0.0001). Lipid-lowering medication significantly decreased retinol, with decreasing apolipoprotein C-II but without a commensurate decrease in RBP. The retinol levels had a positive correlation with apolipoprotein C-II in all or normolipidemic patients with diabetes and control subjects. The high retinol/RBP ratio implies that an excessive or free retinol possibly exists in NIDDM. An alternative metabolism of retinol is inferred to underlie NIDDM without direct influences of cholesterol or triglyceride themselves.

Short term treatment of rats with beta,beta′-tetramethylhexadecanedioic acid (MEDICA 16) results in a pronounced decrease in plasma very-low-density-lipoprotein (VLDL) cholesterol and VLDL triacylglycerol, previously ascribed to a decrease in liver VLDL production [Bar-Tana, Rose-Kahn, Frenkel, Shafer and Fainaru (1988) J. Lipid Res. 29, 431-441]. The hypolipidaemic effect of MEDICA 16 was further analysed here by monitoring plasma VLDL clearance and its hepatic uptake. VLDL triacylglycerol and VLDL apolipoprotein (apo) B fractional clearance rates were increased 7-8-fold in MEDICA 16-treated rats. The increase in the fractional clearance rate of plasma VLDL was essentially eliminated by functional hepatectomy. It was accounted for by activation of plasma VLDL uptake by the liver being completed during the first 4 min after the injection of the VLDL label and before commencement of uptake in non-treated animals. The hypolipidaemic effect of MEDICA 16 was accompanied by a 3.5-fold decrease in plasma apoC-III, but plasma apoC-III clearance remained unaffected by MEDICA 16. MEDICA 16-induced premature hepatic uptake of plasma VLDL due to suppression of apoC-III production may thus account for enhancement of plasma VLDL clearance in treated animals.

A 56-year-old male with apolipoprotein C-II deficiency experienced a myocardial infarction without pancreatitis. A coronary angiogram showed complete occlusions of both the right and circumflex coronary arteries. His serum lipid levels were as follows: fasting total cholesterol 3.15 mmol/l; postprandial total cholesterol 3.62 mmol/l; fasting triglycerides 1.46 mmol/A; postprandial triglycerides 6.14 mmol/l; fasting high-density lipoprotein-cholesterol 0.47 mmol/l; and postprandial high-density lipoprotein cholesterol 0.36 mmol/l. His fasting level of plasma apolipoprotein C-II was 0.005 g/l, but his plasma levels of other apolipoproteins were within normal ranges. A DNA sequence analysis of the apolipoprotein C-II gene showed no mutations in exon 1, 2, 3, or 4, where most gene mutations related to apolipoprotein C-II deficiency occur. We report this patient's very rare heterozygous apolipoprotein C-II deficiency with coronary artery disease. Although this patient had some risk factors for coronary artery disease, coronary atherosclerosis in this patient might have occurred as a result of lipoprotein abnormalities caused by at least one mutation in the apolipoprotein C-II gene.

Preeclampsia is a hypertensive disorder unique to pregnancy. Although the pathogenesis of the disease begins with aberrant spiral artery invasion in the first trimester, clinical symptoms usually do not present until late in pregnancy. Apolipoprotein CII (ApoCII) and its negative regulator, apolipoprotein CIII (ApoCIII), have recently been described as atherogenesis biomarkers in models of cardiovascular disease. Given the similarities in pathology, etiology, and clinical presentation between cardiovascular disease and preeclampsia, we hypothesized that the ratio of ApoCIII to ApoCII in maternal first trimester plasma would predict preeclampsia later in pregnancy. To test this hypothesis, plasma was prospectively collected from 311 nulliparas at 8 to 12 weeks gestation. After delivery, patients were divided into cohorts based on preeclampsia diagnosis. Conditioning monocytes with preeclamptic plasma potentiated monocyte adhesion to endothelial cells in an in vitro model. The ratio of ApoCIII to ApoCII was significantly elevated in patients with severe preeclampsia relative to normotensive and gestational hypertensive individuals (P < .05) as determined by mass spectrometry and competitive enzyme-linked immunosorbent assay (ELISA) assays. These results support a predictive change in the ratio of ApoCIII to ApoCII in pregnancies complicated by severe preeclampsia.

Using artificial triglyceride emulsions, we have demonstrated the presence of non-equilibrating pools of apolipoproteins C-II and C-III in human plasma lipoproteins. As the concentrations of acceptor triglycerides were increased, a greater fraction of both apoC-II and apoC-III shifted away from the native plasma lipoproteins to the artificial lipid emulsions. All of the apoC-II and apoC-III in very low density and high density lipoproteins (VLDL and HDL), however, could not be removed from native plasma lipoproteins. The percent of total plasma apoC-II and apoC-III that could be recovered in the VLDL and HDL density fractions varied when plasma from different individuals was used. When plasma samples from normotriglyceridemic subjects were used, HDL was the primary donor of apoCs. The percent of total plasma apoCs associated with HDL decreased from 60% to 25% for apoC-II and from 65% to 15% for apoC-III. When plasma samples from hypertriglyceridemic subjects were incubated with artificial lipid emulsions, VLDL was the primary donor of apoCs. HDL from hypertriglyceridemic subjects only accounted for 5-10% of total fasting plasma apoCs and did not contribute significantly to the final apoC contents of the artificial triglyceride emulsions. To evaluate the significance of the depletion of exchangeable apoCs from plasma HDL, we also examined the ability of control and apoC-depleted HDL to serve as activator for bovine milk lipoprotein lipase (LPL) in vitro. When HDL depleted of exchangeable apoCs were used as the source of plasma apolipoproteins for the activation of LPL in vitro, only 5-10% of the maximal activity obtained with native HDL was demonstrated. In fact, in the presence of comparable concentrations of HDL apoC-II, activation of LPL was the least with HDL which lacked exchangeable apoCs. Our data thus indicated that the presence of exchangeable apoC-II on HDL is necessary for the activation of LPL in vitro. This finding is consistent with our data that suggest that HDL from hypertriglyceridemic subjects do not stimulate LPL as well as HDL from normolipidemic subjects.

A cloned rat apolipoprotein (apo) C-III cDNA was used as a hybridization probe to measure apo C-III mRNA levels in rats after induction of inflammation. Hepatic apo C-III mRNA levels decrease to a minimum value of 46% of normal 72 h after the induction of inflammation. The changes observed are very similar to the changes in mRNA levels of rat apo A-IV, while the hepatic mRNA levels for apo A-I and apo C-I during inflammation remain relatively constant. Alterations in apo C-III gene expression during inflammation were associated with changes in lipoprotein particle size and composition. A decrease of approximately twofold in the amount of apo C-III-containing HDL particles was found in rats 24 h after the induction of inflammation. The average size of apo C-III-containing HDL particles in rats during inflammation is significantly larger than that of the control group. A decrease in the concentration of apo A-I-containing HDL particles was also observed in these rats. The results indicate altered apolipoprotein gene expression associated with alterations in the size and composition of HDL particles.