Apolipoprotein CIII predicts cardiovascular events in patients with coronary artery disease: a prospective observational study.

BACKGROUND: Apolipoprotein CIII (apoCIII) is associated with triglyceride-rich lipoprotein metabolism and has emerged as independent marker for risk of cardiovascular disease. The objective was to test whether apoCIII is regulated postprandially and whether apoCIII concentrations in native and chylomicron-free serum predict future cardiovascular events in patients with stable coronary artery disease (CAD). METHODS: ApoCIII concentrations were measured in native and chylomicron-free serum in the fasting state and after a standardized oral fat load test in 195 patients with stable CAD. Clinical follow-up was 48 months. Chylomicron-free serum was prepared by ultracentrifugation (18,000 rpm, 3 h). The log-rank test and Cox regression analyses were used to investigate the association of apoCIII with recurrent cardiovascular events. RESULTS: Of the 195 patients included, 92 had a cardiovascular event, and 103 did not. 97% were treated with a statin. No significant changes in apoCIII concentration were observed after the oral fat load test. The apoCIII concentration was associated with event-free survival independent of conventional risk factors. This association reached statistical significance only for apoCIII concentration measured in chylomicron-free serum (hazard ratio [95% confidence interval] for apoCIII above the mean: postprandial: 1.67 (1.06-2.29), P = 0.028, fasting: 2.09 (1.32-3.32), P = 0.002), but not for apoCIII concentration measured in native serum (postprandial: 1.47 [0.89-2.43], P = 0.133, fasting: 1.56 [0.95-2.58], P = 0.081). The effects were independent of other risk factors. CONCLUSIONS: ApoCIII concentrations in chylomicron-free serum are independently associated with event-free survival in patients with CAD both in fasting and postprandial state. This findings support considering apoCIII for risk assessment and attempting to test the hypothesis that lowering apoCIII reduces residual cardiovascular risk. TAKE HOME MESSAGE: Apolipoprotein CIII concentration measured in chylomicron-free serum predicts recurrent cardiovascular events in patients with stable coronary artery disease. TRIAL REGISTRATION: The trial which included the participants of this study was registered at https://clinicaltrials.gov (NCT00628524) on March 5, 2008.

Impact of Novel Low-Density Lipoprotein-Cholesterol Assessment on the Utility of Secondary Non-High-Density Lipoprotein-C and Apolipoprotein B Targets in Selected Worldwide Dyslipidemia Guidelines.

BACKGROUND: Selected dyslipidemia guidelines recommend non-high-density lipoprotein-cholesterol (non-HDL-C) and apolipoprotein B (apoB) as secondary targets to the primary target of low-density lipoprotein-cholesterol (LDL-C). After considering 2 LDL-C estimates that differ in accuracy, we examined: (1) how frequently non-HDL-C guideline targets could change management; and (2) the utility of apoB targets after meeting LDL-C and non-HDL-C targets. METHODS: We analyzed 2518 adults representative of the US population from the 2011 to 2012 National Health and Nutrition Examination Survey and 126 092 patients from the Very Large Database of Lipids study with apoB. We identified all individuals as well as those with high-risk clinical features, including coronary artery disease, diabetes mellitus, and metabolic syndrome who met very high- and high-risk guideline targets of LDL-C <70 and <100 mg/dL using Friedewald estimation (LDL-CF) and a novel, more accurate method (LDL-CN). Next, we examined those not meeting non-HDL-C (<100, <130 mg/dL) and apoB (<80, <100 mg/dL) guideline targets. In those meeting dual LDL-C and non-HDL-C targets (<70 and <100 mg/dL, respectively, or <100 and <130 mg/dL, respectively), we determined the proportion of individuals who did not meet guideline apoB targets (<80 or <100 mg/dL). RESULTS: A total of 7% to 9% and 31% to 36% of individuals had LDL-C <70 and <100 mg/dL, respectively. Among those with LDL-CF<70 mg/dL, 14% to 15% had non-HDL-C ≥100 mg/dL, and 7% to 8% had apoB ≥80 mg/dL. Among those with LDL-CF<100 mg/dL, 8% to 10% had non-HDL-C ≥130 mg/dL and 2% to 3% had apoB ≥100 mg/dL. In comparison, among those with LDL-CN<70 or 100 mg/dL, only ≈2% and ≈1% of individuals, respectively, had non-HDL-C and apoB values above guideline targets. Similar trends were upheld among those with high-risk clinical features: ≈0% to 3% of individuals with LDL-CN<70 mg/dL had non-HDL-C ≥100 mg/dL or apoB ≥80 mg/dL compared with 13% to 38% and 9% to 25%, respectively, in those with LDL-CF<70 mg/dL. With LDL-CF or LDL-CN<70 mg/dL and non-HDL-C <100 mg/dL, 0% to 1% had apoB ≥80 mg/dL. Among all dual LDL-CF or LDL-CN<100 mg/dL and non-HDL-C <130 mg/dL individuals, 0% to 0.4% had apoB ≥100 mg/dL. These findings were robust to sex, fasting status, and lipid-lowering therapy status. CONCLUSIONS: After more accurately estimating LDL-C, guideline-suggested non-HDL-C targets could alter management in only a small fraction of individuals, including those with coronary artery disease and other high-risk clinical features. Furthermore, current guideline-suggested apoB targets provide modest utility after meeting cholesterol targets. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01698489.

The ACTN3 R577X polymorphism affects the lipid profile and the prognosis of nutritional intervention in HIV-positive patients.

The hypothesis of the present study is that the polymorphisms in the APOC3, CEPT, ACE, and ACTN3 genes can affect the outcome of nutritional intervention and the plasma lipid profile of HIV+ patients. To test the hypothesis, genetic material was collected from buccal cells, and serum was collected for biochemical analysis. Sixty-five patients were analyzed. The incorporation of protease inhibitor (PI) was more frequent in women (77% vs 33% in men). Nutritional intervention improved anthropometric parameters independent of the genotype. Patients with the RR genotype for the ACTN3 R577X polymorphism had lower glycemia (RR = 95.4 ± 6.5 mg/dL, RX = 102.6 ± 10.6 mg/dL, XX = 110.1 ± 16.3 mg/dL; P = .03) and a greater reduction in low-density lipoproteins (LDL) after intervention (LDL: RR = -23.7 ± 15.8 mg/dL, RX = 1.32 ± 5.13 mg/dL, XX = 30.21 ± 24.4 mg/dL; P = .01). Patients using PI had a negative response to dietary intervention regarding the levels of high-density lipoprotein (-2.4 ± 1.70 with PI, 2.56 ± 1.60 mg/dL without PI; P = .02), very low density lipoprotein (0.84 ± 2.73 with IP, -5.46 ± 3.37 mg/dL without PI; P = .03), and triglycerides (1.79 ± 13.22 with PI, -34.00 ± 17.67 mg/dL without PI; P = .052). This response was also independent of the genotype (P > 0.05) and suggested the need for oral lipid-lowering drugs in all HIV+ patients using PI. Our results indicate that the ACTN3 R577X polymorphism is a good predictor of both the lipid profile and the prognosis of nutritional intervention in reducing LDL in HIV+ patients.

Proteoform profiling of peripheral blood serum proteins from pregnant women provides a molecular IUGR signature.

Intrauterine growth restriction (IUGR) is an important cause of perinatal morbidity and mortality and contributes substantially to medically indicated preterm birth; preventing fetal death. Molecular profiling of the mothers' peripheral blood was desired to monitor the health conditions of the fetuses. To develop such a minimally invasive assay, we applied a protein affinity fractionation method to peripheral blood serum samples from pregnant women belonging to either the IUGR or to the control group. Proof-of-principle was shown by relative quantitation analysis of mixtures of intact proteoforms using MALDI-ToF mass spectrometry. The two best differentiating proteins and proteoforms, respectively, were apolipoprotein C-II and apolipoprotein C-III0. Together with three robustly expressed protein proteoforms proapolipoprotein C-II, apolipoprotein C-III1, and apolipoprotein C-III2, which served as landmarks for relative quantitation analysis, they constituted the maternal IUGR proteome signature. Separation confidence of our IUGR proteoform signature reached a sensitivity of 0.73 and a specificity of 0.87 with an area under curve of 0.86 in receiver operator characteristics. SIGNIFICANCE: Identification of IUGR newborns in the case room is required as children are severely diseased and need specialized care during infancy. Yet, at time of birth there is no readily applicable clinical test available. Hence, a molecular profiling assay is highly desired. It needs to be mentioned that current clinical definitions and recommendations for IUGR are unfortunately misleading and are not universally applicable. The most commonly adopted definition is an abdominal circumference (AC) or estimated fetal weight measurement <10th percentile. Although both, the American College of Obstetricians and Gynecologists (ACOG) and the Royal College of Obstetricians and Gynecologists (RCOG) agree that at this cut-off the risk of perinatal morbidity and mortality increases, this definition does not take into account the individualized growth potential of each fetus. In particular its sole use fails to identify larger fetuses that have not achieved their growth potential and may be at risk of adverse outcomes. Also, this definition, when solely applied, will result in the misdiagnosis of IUGR for some constitutionally small fetuses. It needs to be pointed out that the above mentioned criteria can only be determined during pregnancy in case mothers report from early on during pregnancy. We have developed a test that relies on mass spectrometric analysis of the mother's serum protein composition (IUGR signature) which can be determined just ahead of delivery and at date of delivery, respectively using a minimal invasive blood sampling approach. With this manuscript we describe the use of a mass spectrometric profiling method of 30 peripheral blood samples from pregnant women prior to giving birth of either unsuspicious newborns or IUGR-affected infants. We report for the first time that maternal blood sample analysis via affinity mass spectrometry differentiates IUGR infants from controls with high confidence.

Effects of the absence of apolipoprotein e on lipoproteins, neurocognitive function, and retinal function.

IMPORTANCE: The identification of a patient with a rare form of severe dysbetalipoproteinemia allowed the study of the consequences of total absence of apolipoprotein E (apoE). OBJECTIVES: To discover the molecular basis of this rare disorder and to determine the effects of complete absence of apoE on neurocognitive and visual function and on lipoprotein metabolism. DESIGN, SETTING, AND PARTICIPANTS: Whole-exome sequencing was performed on the patient's DNA. He underwent detailed neurological and visual function testing and lipoprotein analysis. Lipoprotein analysis was also performed in the Cardiovascular Research Institute, University of California, San Francisco, on blood samples from the proband's mother, wife, 2 daughters, and normolipidemic control participants. MAIN OUTCOME MEASURES: Whole-exome sequencing, lipoprotein analysis, and neurocognitive function. RESULTS: The patient was homozygous for an ablative APOE frameshift mutation (c.291del, p.E97fs). No other mutations likely to contribute to the phenotype were discovered, with the possible exception of two, in ABCC2 (p.I670T) and LIPC (p.G137R). Despite complete absence of apoE, he had normal vision, exhibited normal cognitive, neurological, and retinal function, had normal findings on brain magnetic resonance imaging, and had normal cerebrospinal fluid levels of ß-amyloid and tau proteins. He had no significant symptoms of cardiovascular disease except a suggestion of myocardial ischemia on treadmill testing and mild atherosclerosis noted on carotid ultrasonography. He had exceptionally high cholesterol content (760 mg/dL; to convert to millimoles per liter, multiply by 0.0259) and a high cholesterol to triglycerides ratio (1.52) in very low-density lipoproteins with elevated levels of small-diameter high-density lipoproteins, including high levels of prebeta-1 high-density lipoprotein. Intermediate-density lipoproteins, low-density lipoproteins, and very low-density lipoproteins contained elevated apoA-I and apoA-IV levels. The patient's apoC-III and apoC-IV levels were decreased in very low-density lipoproteins. Electron microscopy revealed large lamellar particles having electron-opaque cores attached to electron-lucent zones in intermediate-density and low-density lipoproteins. Low-density lipoprotein particle diameters were distributed bimodally. CONCLUSIONS AND RELEVANCE: Despite a profound effect on lipoprotein metabolism, detailed neurocognitive and retinal studies failed to demonstrate any defects. This suggests that functions of apoE in the brain and eye are not essential or that redundant mechanisms exist whereby its role can be fulfilled. Targeted knockdown of apoE in the central nervous system might be a therapeutic modality in neurodegenerative disorders.

Acute effects of interleukin-6 infusion on apo-B-containing lipoprotein subclasses in humans.

IL-6 is believed to mediate the elevation in plasma TG and VLDL lipids in patients with sepsis. Previous studies of lipoprotein density fractions do not reveal the extent to which cytokines change the immunochemically distinct TG-rich (LpB:C, LpB:C:E, LpAII:B:C:D:E) and cholesterol-rich (LpB, LpB:E) apoB-containing subclasses present in VLDL. Therefore, we have directly measured these subclasses following their isolation by sequential immunoprecipitation in seven healthy male subjects during a 3-h infusion with recombinant human (rh) IL-6. Though plasma TG and apoB-containing particle number were unchanged by IL-6, the distribution of TG-rich subclasses was significantly altered. Compared to baseline values, LpB:E + LpB:C:E increased significantly at 0.5 h (p < 0.02) and were higher than saline-infused controls at 0.5 and 1 h (p < 0.05). At 0.5 h LpAII:B:C:D:E reciprocally declined from baseline (p < 0.01). While the pattern of change for total apoB showed an overall decline (p < 0.05), these changes in LpB:E + LpB:C:E and LpAII:B:C:D:E in IL-6 subjects differed from controls (p < 0.05; p < 0.01, respectively). These findings indicate that physiologic concentrations of IL-6 rapidly and selectively regulate the transport of apoB particles that contain apoE. Since apoE has immunomodulatory and host defense functions, these changes may be a previously unrecognized early step in the innate immune response.

Mechanism of action of a peroxisome proliferator-activated receptor (PPAR)-delta agonist on lipoprotein metabolism in dyslipidemic subjects with central obesity.

CONTEXT: Dyslipidemia increases the risk of cardiovascular disease in obesity. Peroxisome proliferator-activated receptor (PPAR)-δ agonists decrease plasma triglycerides and increase high-density lipoprotein (HDL)-cholesterol in humans. OBJECTIVE: The aim of the study was to examine the effect of GW501516, a PPAR-δ agonist, on lipoprotein metabolism. Design, Setting, and Intervention: We conducted a randomized, double-blind, crossover trial of 6-wk intervention periods with placebo or GW501516 (2.5 mg/d), with 2-wk placebo washout between treatment periods. PARTICIPANTS: We recruited 13 dyslipidemic men with central obesity from the general community. MAIN OUTCOME MEASURES: We measured the kinetics of very low-density lipoprotein (VLDL)-, intermediate-density lipoprotein-, and low-density lipoprotein (LDL)-apolipoprotein (apo) B-100, plasma apoC-III, and high-density lipoprotein (HDL) particles (LpA-I and LpA-I:A-II). RESULTS: GW501516 decreased plasma triglycerides, fatty acid, apoB-100, and apoB-48 concentrations. GW501516 decreased the concentrations of VLDL-apoB by increasing its fractional catabolism and of apoC-III by decreasing its production rate (P < 0.05). GW501516 reduced VLDL-to-LDL conversion and LDL-apoB production. GW501516 increased HDL-cholesterol, apoA-II, and LpA-I:A-II concentrations by increasing apoA-II and LpA-I:A-II production (P < 0.05). GW501516 decreased cholesteryl ester transfer protein activity, and this was paralleled by falls in the triglyceride content of VLDL, LDL, and HDL and the cholesterol content of VLDL and LDL. CONCLUSIONS: GW501516 increased the hepatic removal of VLDL particles, which might have resulted from decreased apoC-III concentration. GW501516 increased apoA-II production, resulting in an increased concentration of LpA-I:A-II particles. This study elucidates the mechanism of action of this PPAR-δ agonist on lipoprotein metabolism and supports its potential use in treating dyslipidemia in obesity.

Dietary whey hydrolysate with exercise alters the plasma protein profile: a comprehensive protein analysis.

OBJECTIVE: It has been shown that dietary whey protein accelerates glucose uptake by altering glycoregulatory enzyme activity in skeletal muscle. In the present study, we investigated the effect of dietary whey protein on endurance and glycogen resynthesis and attempted to identify plasma proteins that reflected the physical condition by a comprehensive proteomics approach. METHODS: Male c57BL/6 mice were divided into four groups: sedentary, sedentary with whey protein hydrolysate, exercise, and exercise with whey protein hydrolysate. The mice in the exercise groups performed treadmill running exercise five times per week for 4 wk. Protein profiling of plasma sample obtained from individuals was performed, as were measurements of endurance performance and the glycogen content of gastrocnemius muscle. RESULTS: After the training period, the endurance of mice fed the whey diet was improved compared with that of mice fed the control diet. Muscle glycogen content was significantly increased after 4 wk of exercise, and intake of whey protein led to a further increase in glycogen. Apolipoproteins A-II and C-I and ß(2)-glycoprotein-1 were found to be altered by training combined with the intake of whey protein, without significant changes induced by exercise or whey protein alone. CONCLUSION: Results of the present study suggest that these three proteins may be potential biomarkers of improved endurance and glycogen resynthesis and part of the mechanism that mediates the benefits of whey protein.

APOE/C1/C4/C2 gene cluster genotypes, haplotypes and lipid levels in prospective coronary heart disease risk among UK healthy men.

The role of common APOE variants on plasma lipids, particularly low density lipoprotein (LDL) levels, and coronary heart disease (CHD) risk is well known; the influence of variation in the other nearby apolipoprotein genes APOC1, APOC4 and APOC2 is unclear. This study examines the association between APOE/C1/C4/C2 gene cluster variation using tagging SNPs and plasma lipid concentration along with risk of CHD in a prospective cohort. Genotypes for 11 common APOE/C1/C4/C2 SNPs were determined in 2,767 middle-aged (49 to 64 years) men from the Second Northwick Park Heart Study, with 275 CHD events over a 15-year follow-up period. Seven SNPs showed significant associations with one or more lipid trait in univariate analysis. Multivariate and haplotype analysis showed that the APOE genotypes are most strongly associated with effects on LDL-C and apoB concentration (explaining 3.4% of the LDL-C variance) while the other SNPs in this gene cluster explained an additional 1.2%. Haplotypes in APOC2 and APOC4 were associated with modest effects on HDL-C and apoAI (explaining respectively 1.4% and 1.2%). Carriers of the APOE ɛ2 SNP had a significantly lower risk of CHD hazard ratio (HR) of 0.63 (95% confidence interval [CI]: 0.42-0.95), as did carriers of the APOC2 SNP rs5127 (HR = 0.72, 95% CI: 0.56-0.93), while carriers of APOC1 SNP rs4803770 had higher risk of CHD (HR = 1.36, 95% CI: 1.04-1.78) compared with noncarriers. While the common APOE polymorphism explains the majority of the locus genetic determinants of plasma lipid levels, additional SNPs in the APOC1/C2 region may contribute to CHD risk, but these effects require confirmation.

Parallel increase of plasma apoproteins C-II and C-III in Type 2 diabetic patients.

AIMS: To determine plasma levels of apoprotein (apo) C-II and apoprotein C-III in Type 2 diabetic patients and to examine the clinical and biological factors that are associated with elevated apoC concentrations. METHODS: We measured apoC-II and apoC-III in total plasma and in non-high-density lipoprotein fractions by an immunoturbidimetric assay in 88 Caucasian Type 2 diabetic patients and in 138 healthy control subjects. RESULTS: Plasma levels of both apoC-II and apoC-III were increased in Type 2 diabetic patients. The clinical conditions associated with an increase of plasma apoC-II and apoC-III were abdominal obesity, body mass index, poor glycaemic control and lack of insulin treatment. However, when multivariate analysis was used, plasma apoCs levels correlated with triglyceride levels only. The apoC-III/apoC-II ratio was similar in the Type 2 diabetic and control subjects. CONCLUSIONS: Our study shows the parallel increase of apoC-II and C-III in Type 2 diabetic patients. This parallel increase is related to hypertriglyceridaemia only.

Antidiabetic and hypolipidemic effects of a novel dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, E3030, in db/db mice and beagle dogs.

We investigated the antidiabetic effects of E3030, which is a potent dual activator of peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma, in an animal model of diabetes, C57BL/KsJ-db/db mice (db/db mice), and the lipidemic effects of E3030 in beagle dogs, whose PPARalpha and PPARgamma transactivation responses to E3030 were similar to those of humans. E3030 activated human PPARalpha, mouse PPARalpha, dog PPARalpha, human PPARgamma, mouse PPARgamma, and dog PPARgamma with EC(50) values of 65, 920, 87, 34, 73, and 34 nM, respectively, in the chimeric GAL4-PPAR receptor transactivation reporter assay. In db/db mice orally administered E3030 decreased blood glucose, triglyceride (TG), non-esterified fatty acids (NEFA), and insulin levels and increased blood adiponectin levels during a 14-day experimental period. Significant effects on blood glucose and adiponectin levels were observed at a dose of 3 mg/kg or greater. Furthermore, significant effects on blood TG, NEFA, and insulin levels were observed at doses of 1 mg/kg or more. An oral glucose tolerance test (OGTT) performed on Day 15 showed that E3030 at 3 mg/kg improved glucose tolerance in this model. Fourteen days of oral treatment with E3030 at a dose of 0.03 mg/kg or greater showed remarkable TG- and non high-density lipoprotein (non-HDL) cholesterol-lowering effects in beagle dogs. These results were similar to those observed for the PPARalpha agonist fenofibrate. E3030 also reduced apo C-III levels on Days 7 and 14, and elevated lipoprotein lipase (LPL) levels on Day 15. These results indicate that the TG- and non-HDL cholesterol-lowering actions of E3030 involve combined effects on reduction of apo C-III and elevation of LPL, resulting in increased lipolysis. The experimental results in animals suggest that E3030 has potential for use in the treatment of various aspects of metabolic dysfunction in type 2 diabetes, including dyslipidemia, hyperglycemia, hyperinsulinemia, and impaired glucose disposal.

Plasma apolipoprotein CI protects against mortality from infection in old age.

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.

Association of apolipoproteins C3 and E with metabolic changes in HIV-infected adults treated with a protease-inhibitor-containing antiretroviral therapy.

OBJECTIVE: To examine the relationship between plasma levels of apolipoproteins C3 (APOC3) and E (APOE) and the presence of lipid and carbohydrate metabolism abnormalities or clinical signs of lipodystrophy in HIV-1-infected patients started with a protease-inhibitor-containing antiretroviral therapy. METHODS: The Aproco (Antiproteases Cohort) Study enrolled 1,181 HIV-1-infected adults in 47 French healthcare centres from May 1997 to June 1998. From December 1998 through July 1999, the APROCO-Metabolic Complications (APROCO-MC) cross-sectional study was performed at the month 20 visit for those patients enrolled in 1997 and at the month 12 visit for those enrolled in 1998. The current analysis presents results from a subset of patients who had undergone additional tests to measure APOC3 and APOE in order to study their relationship with metabolic syndrome (n=157) and abnormal results in an oral glucose tolerance test (n=135). RESULTS: Increases in triglycerides and non-high-density lipoprotein (HDL) cholesterol were associated with significantly higher levels of APOC3, in both Lp B (lipoproteins containing apolipoprotein B) and Lp non-B (lipoproteins free of apolipoprotein B), and a significant higher level of APOE Lp B. APOC3 and APOC3 Lp non-B were increased when glucose metabolism abnormalities were more severe. The presence of a metabolic syndrome was associated with increased plasma APOC3, APOC3 Lp B and APOC3 Lp non-B levels. In a multiple regression analysis, high levels of APOC3 in Lp B and APOC3 Lp non-B were associated with the presence of clinical signs of lipodystrophy, even after adjustment for triglycerides and HDL-cholesterol levels. CONCLUSIONS: Lipid and/or glucose metabolism abnormalities in treated HIV-1-infected patients are associated with increased levels of APOC3 and, to a lesser extent, APOE plasma concentrations. Increased values are also related to clinical signs of insulin resistance and lipodystrophy.

Abnormal glycosylation with hypersialylated O-glycans in patients with Sialuria.

Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder results in a loss of feedback control of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase by CMP-N-acetylneuraminic acid (CMP-NeuAc) resulting in a substantial overproduction of cytoplasmic free sialic acid. This study addresses fibroblast CMP-NeuAc levels and N- and O-glycan sialylation of serum proteins from Sialuria patients. CMP-NeuAc levels were measured with HPLC in fibroblasts. Isoelectric focusing (IEF) of serum transferrin and of apolipoprotein C-III (apoC-III) was performed on serum of three Sialuria patients. Isoforms of these proteins can be used as specific markers for the biosynthesis of N- and core 1 O-glycans. Furthermore, total N- and O-linked glycans from serum proteins were analyzed by HPLC. HPLC showed a clear overproduction of CMP-NeuAc in fibroblasts of a Sialuria patient. Minor changes were found for serum N-glycans and hypersialylation was found for core 1 O-glycans on serum apoC-III and on total serum O-glycans in Sialuria patients. HPLC showed an increased ratio of disialylated over monosialylated core 1 O-glycans. The hypersialylation of core 1 O-glycans is due to the increase of NeuAcalpha2,6-containing structures (mainly NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc). This may relate to KM differences between GalNAc-alpha2,6-sialyltransferase and alpha2,3-sialyltransferases. This is the first study demonstrating that the genetic defect in Sialuria results in a CMP-NeuAc overproduction. Subsequently, increased amounts of alpha2,6-linked NeuAc were found on serum core 1 O-glycans from Sialuria patients. N-glycosylation of serum proteins seems largely unaffected. Sialuria is the first metabolic disorder presenting with hypersialylated O-glycans.

In-vivo metabolism of VLDL-apolipoprotein-B, -CIII and -E in normolipidemic subjects.

BACKGROUND AND AIM: ApoE and apoC-III are important components of lipoprotein metabolism. While the function of both apoproteins is relatively well understood, little is known about the in vivo metabolism of these proteins, partly because of the lack of a standardized method to isolate these apoproteins in large sample numbers. METHODS AND RESULTS: We developed a new reverse phase HPLC method (acetonitril/phosphate gradient; Aquapore RP-300, 7 microm, 220 x 4.6 mm) to isolate a number of different apoproteins, including apoC-III and apoE from VLDL. This method was then used in a study which aimed at determining VLDL-apoE-3 and VLDL-apoC-III metabolism. In addition VLDL-apoB and LDL-apoB metabolism was determined. Endogenous labeling with d(3)-leucine, mass spectrometry and multicompartmental modeling was used in 6 normolipidemic healthy male subjects. Tracer/tracee ratios of free plasma leucine, VLDL-apoE, -apoC-III, -apoB, and LDL-apoB leucine were determined over 60 h following a bolus of d(3)-leucine (5 mg kg(-1)). In all subjects sufficient apoC-III could be isolated by reverse phase HPLC to derive metabolic parameters, while apoE metabolic parameters could only be determined if apoE plasma concentration was 0.75 mg dl(-1) or higher. Compared to VLDL-apoB (FCR 10.4 +/- 3.3 d(-1), production 17.8 +/- 4.5 mg kg(-1) d(-1)), VLDL-apoE-3 (FCR 1.03 +/- 0.11 d(-1), production 0.50 +/- 0.29 mg kg(-1) d(-1)) and VLDL-apoC-III (FCR 1.67 +/- 1.22 d(-1), production 0.44 +/- 0.24 mg kg(-1) d(-1)) parameters were much lower. This indicates that apoE-3 and apoC-III recirculate in plasma and that only a small fraction of apoE and apoC-III on VLDL is newly synthesized. CONCLUSIONS: We conclude that HPLC methodology can be used to isolate VLDL-apoC-III and apoE for metabolic studies and that the metabolic fate of apoC-III and apoE is different from that of apoB because both apoproteins recycle through the VLDL fraction.

Endogenous apoC-I increases hyperlipidemia in apoE-knockout mice by stimulating VLDL production and inhibiting LPL.

Previous studies have shown that overexpression of human apolipoprotein C-I (apoC-I) results in moderate hypercholesterolemia and severe hypertriglyceridemia in mice in the presence and absence of apoE. We assessed whether physiological endogenous apoC-I levels are sufficient to modulate plasma lipid levels independently of effects of apoE on lipid metabolism by comparing apolipoprotein E gene-deficient/apolipoprotein C-I gene-deficient (apoe-/-apoc1-/-), apoe-/-apoc1+/-, and apoe-/-apoc1+/+ mice. The presence of the apoC-I gene-dose-dependently increased plasma cholesterol (+45%; P < 0.001) and triglycerides (TGs) (+137%; P < 0.001), both specific for VLDL. Whereas apoC-I did not affect intestinal [3H]TG absorption, it increased the production rate of hepatic VLDL-TG (+35%; P < 0.05) and VLDL-[35S]apoB (+39%; P < 0.01). In addition, apoC-I increased the postprandial TG response to an intragastric olive oil load (+120%; P < 0.05) and decreased the uptake of [3H]TG-derived FFAs from intravenously administered VLDL-like emulsion particles by gonadal and perirenal white adipose tissue (WAT) (-34% and -25%, respectively; P < 0.05). As LPL is the main enzyme involved in the clearance of TG-derived FFAs by WAT, and total postheparin plasma LPL levels were unaffected, these data demonstrate that endogenous apoC-I suffices to attenuate the lipolytic activity of LPL. Thus, we conclude that endogenous plasma apoC-I increases VLDL-total cholesterol and VLDL-TG dose-dependently in apoe-/- mice, resulting from increased VLDL particle production and LPL inhibition.

Biomarker discovery in breast cancer serum using 2-D differential gel electrophoresis/ MALDI-TOF/TOF and data validation by routine clinical assays.

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.

Use of Intralipid for kinetic analysis of HDL apoC-III: evidence for a homogeneous kinetic pool of apoC-III in plasma.

Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used Intralipid(R) (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 +/- 0.7%) was not different from that of VLDL apoC-III (2.6 +/- 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 +/- 2.9%) (P < 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P < 0.001). In conclusion, this simple IL method can be used to isolate apoC-III from HDL with minimal interference from other HDL apoproteins, and it demonstrates that the kinetics of apoC-III in VLDL and HDL are similar, supporting the concept of a single kinetically homogeneous pool of apoC-III in plasma.

Proteomic analysis of rat plasma by SELDI-TOF-MS under the condition of prevention of progressive adriamycin nephropathy using oral adsorbent AST-120.

AIMS: To determine changes in relative peak intensities of mass-to-charge ratio (m/z) between 2,000 and 15,000, which are difficult to evaluate by 2-dimensional gel electrophoresis, SELDI-TOF-MS (surface-enhanced laser desorption/ionization time of flight-mass spectrometry) proteomic changes in rat models of adriamycin nephropathy with or without AST-120 were investigated. METHODS: A normal group (n = 5), an adriamycin nephropathy group (n = 9), and an adriamycin nephropathy + AST-120 group (4 g/head/day) (n = 9) were established in SD rats. Anion exchange chips, Q10, washed by 50 mM Tris-HCl pH 8 as a ProteinChip and sinapinic acid were used. The mass range between 2,000 and 15,000 Da was measured. Twenty to 34 weeks after adriamycin 3 mg/kg injection, the adriamycin nephropathy + AST-120 group (plasma creatinine value: 2.1 +/- 0.8 mg/dl) clearly demonstrated slight renal dysfunction compared with that in the adriamycin nephropathy group (5.4 +/- 2.0 mg/dl). RESULTS: The relative intensities in the adriamycin nephropathy group were significantly higher in 7 peaks (such as 8,640, and 8,822 Da) and lower in 8 peaks (such as 4,188, and 8,358 Da) than those in the normal group. The relationship between the relative intensity of peaks and the plasma creatinine value demonstrated a positive correlation in 11 peaks (such as 8,640, and 8,822 Da), and a negative correlation in 6 peaks (such as 4,188 and 8,358 Da). Although the relative intensities of peaks in the adriamycin nephropathy + AST-120 group were between that of the adriamycin nephropathy group and that of the normal group, the relative intensities of 4 peaks (such as 3,664 and 5,179 Da) in the adriamycin nephropathy + AST-120 group demonstrated higher values than in the two other groups. The m/z 3,664 peak was purified and identified as a C-terminal fragment of apolipoprotein C-III. CONCLUSION: Low-molecular proteins and peptides in plasma in this chronic renal failure model showed not only increases but also decreases in some peaks. The relative intensities in some peaks increased in the adriamycin nephropathy + AST-120 group more than in the two other groups. One of these peaks was identified as the apolipoprotein C-III fragment. The relationship between these changes and the prevention of progression of chronic renal failure by AST-120 remains to be established.

Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation.

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.