Biochemical Analysis of the Lipoprotein Lipase Truncation Variant, LPLS447X, Reveals Increased Lipoprotein Uptake.

Lipoprotein lipase (LPL) is responsible for the hydrolysis of triglycerides from circulating lipoproteins. Whereas most identified mutations in the LPL gene are deleterious, one mutation, LPLS447X, causes a gain of function. This mutation truncates two amino acids from LPL's C-terminus. Carriers of LPLS447X have decreased VLDL levels and increased HDL levels, a cardioprotective phenotype. LPLS447X is used in Alipogene tiparvovec, the gene therapy product for individuals with familial LPL deficiency. It is unclear why LPLS447X results in a serum lipid profile more favorable than that of LPL. In vitro reports vary as to whether LPLS447X is more active than LPL. We report a comprehensive, biochemical comparison of purified LPLS447X and LPL dimers. We found no difference in specific activity on synthetic and natural substrates. We also did not observe a difference in the Ki for ANGPTL4 inhibition of LPLS447X relative to that of LPL. Finally, we analyzed LPL-mediated uptake of fluorescently labeled lipoprotein particles and found that LPLS447X enhanced lipoprotein uptake to a greater degree than LPL did. An LPL structural model suggests that the LPLS447X truncation exposes residues implicated in LPL binding to uptake receptors.

Structural and functional changes in high-density lipoprotein induced by chemical modification.

Reconstituted high-density lipoprotein (rHDL), a natural nanoparticle consisting of apolipoprotein A-I and phospholipids, was modified with a hydrophobic fluorescent dye before (pre-rHDL) and after (post-rHDL) reconstitution. Pre-rHDL particles had a similar size to unmodified rHDL, but post-rHDL particles were significantly larger and their avidity for a HDL receptor was 2.6 times of that shown by pre-rHDL.

Effect of relative weight group change on nuclear magnetic resonance spectroscopy derived lipoprotein particle size and concentrations among adolescents.

OBJECTIVE: To examine whether longitudinal changes in relative weight category (as indicated by change in body mass index [BMI] classification group) were associated with changes in nuclear magnetic resonance (NMR)-derived lipoprotein particles among US youth. STUDY DESIGN: Secondary analysis of data from a clustered randomized controlled trial. BMI and fasting blood samples were obtained from 2069 participants at the start of the 6th grade and end of the 8th grade. BMI was categorized as normal weight, overweight, or obese at both time points. Lipoprotein particle profiles were measured with NMR spectroscopy at both time points. Regression models were used to examine changes in relative weight group and change in lipoprotein variables. RESULTS: A total of 38% of participants changed relative weight category (BMI group) during the 2.5-year study period. Low-density lipoprotein (LDL) cholesterol and non-high-density lipoprotein (HDL) cholesterol decreased almost universally, but more with improved BMI category. There were adverse effects on LDL size and total LDL particles, HDL size, and cholesterol for participants who remained obese or whose relative weight group worsened. Changes in relative category had no impact on HDL particles. CONCLUSION: Improvement in relative weight group from 6th to 8th grade was associated with favorable changes in non-HDL cholesterol, very low-density lipoprotein size, LDL size, HDL size, and LDL particles but had no effect on HDL particles. Findings indicate that an improvement in relative weight group between 6th and 8th grade had an effect on NMR-derived particles sizes and concentrations among a large group of adolescents, which overrepresented low-income minorities.

Olive oil phenols modulate the triacylglycerol molecular species of human very low-density lipoprotein. A randomized, crossover, controlled trial.

Virgin olive oil phenolic compounds have been revealed to be potent antioxidants as part of the Mediterranean diet. To test the hypothesis that these phenolics can modulate the serum and very low-density lipoprotein (VLDL) triacylglycerol concentrations in humans, a double-blind, randomized, crossover trial was designed. Thirty-three participants received 25 mL/d of refined olive oil (devoid of phenolic content [PC]), common olive oil (PC = 370 mmol/kg), and virgin olive oil (PC = 825 mmol/kg) in a Latin square design. The 3 olive oils were administered over 3 periods of 3 weeks, each one preceded by 2-week washout periods. All analyses were carried out on an intention-to-treat basis. The interventions did not modify the concentrations of serum and low-density lipoprotein cholesterol and triacylglycerol; but they exerted changes in the cholesterol, triacylglycerol, and phospholipid content of VLDL. The virgin olive oil consumption led to increased oleic and palmitic acids, as well as decreased linoleic acid, in VLDL. The main outcome was the significant dose-dependent linear trend between the PC in the olive oils and the palmitic (16:0) and linoleic (18:2 n-6) acid and their corresponding triacylglycerol molecular species in VLDL.

NMR-determined lipoprotein subclass profile is associated with dietary composition and body size.

BACKGROUND AND AIMS: Dyslipidemia is influenced by diet and body habitus. The Nuclear Magnetic Resonance spectroscopy lipoprotein subclass profile (NMR-LSP) is associated with diabetes and its vascular complications; and an NMR-LSP featuring large VLDL particles and small LDL and HDL particles is linked with cardiovascular disease (CVD). Thus interventions which favourably modify NMR-LSP may reduce risk for diabetes, its complications and CVD. The study aim was to investigate the associations between NMR-LSP, dietary composition and body size measures using data from the Melbourne Collaborative Cohort Study (MCCS). METHODS AND RESULTS: NMR-LSP was assessed in 313 men and 403 women (median age 54 years) randomly selected from a community-based cohort study. Diet was assessed using a specifically developed food frequency questionnaire (FFQ), and body size was assessed by body mass index (BMI) or waist:hips ratio (WHR). To simplify the 15 NMR-LSP variables, factor analysis was used to derive a single factor. Multivariate linear regression with this factor score as the dependent variable demonstrated that in men, total PUFA and n-6 dietary fat intake and BMI were associated with a more atherogenic NMR-LSP pattern; while in women dietary glycemic index and WHR demonstrated positive associations, and n-3 fat intake an inverse association. CONCLUSIONS: We developed a single factor score to summarize the NMR-LSP that has the benefit of combining all aspects of the NMR-LSP and accounting for correlations between them. We have shown correlations between the NMR-LSP and body size and dietary composition.

Human luteinized granulosa cells secrete apoB100-containing lipoproteins.

Thus far, liver, intestine, heart, and placenta have been shown to secrete apolipoprotein (apo)B-containing lipoproteins. In the present study, we first investigated lipoproteins in human follicular fluid (FF), surrounding developing oocytes within the ovary, as well as in corresponding plasma samples (n = 12). HDL cholesterol within FF correlated well with plasma HDL cholesterol (r = 0.80, P < 0.01), whereas VLDL cholesterol did not, indicating that VLDL in FF might originate directly from the granulosa cells producing FF. Primary human granulosa cells expressed apoB, microsomal triglyceride transfer protein, and apoE, but not the apoB-editing enzyme apobec-1. Using (3)H-leucine, we show that granulosa cells secrete apoB100-containing lipoproteins and that secretion can be stimulated by adding oleate to the medium (+83%). With electron microscopy, apoB-containing lipoproteins within the secretory pathway of human granulosa cells were directly visualized. Finally, we found a positive relationship between apoB levels in FF and improved fertility parameters in a population of 27 women undergoing in vitro fertilization. This study demonstrates that human granulosa cells assemble and secrete apoB100-containing lipoproteins, thereby identifying a novel cell type equipped with these properties. These results might have important implications for female infertility phenotypes as well as for the development of drugs targeting the VLDL production pathway.

Optimization of N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide as a derivatization agent for determining isotopic enrichment of glycerol in very-low density lipoproteins.

Stable isotope kinetic studies play an important role in the study of very-low density lipoprotein (VLDL) metabolism, including basic and clinical research. Today, [1,1,2,3,3-(2)H(5)]glycerol is the most cost-effective alternative to measure glycerol and triglyceride kinetics. Recycling of glycerol from glycolysis and gluconeogenesis may lead to incompletely labelled tracer molecules. Many existing methods for the measurement of glycerol isotopic enrichment involve the production of glycerol derivatives that result in fragmentation of the glycerol molecule after ionization. It would be favourable to measure the intact tracer molecule since incompletely labelled tracer molecules may be measured as fully labelled. The number of methods available to measure the intact tracer in biological samples is limited. The aim of this project was to develop a gas chromatography/mass spectrometry (GC/MS) method for glycerol enrichment that measures the intact glycerol backbone and is suitable for electron ionization (EI), which is widely available. A previously published method for N-methyl-N-[tert-butyldimethylsilyl]trifluoroacetamide (MTBSTFA) derivatization was significantly improved; we produced a stable derivative and increased recovery 27-fold in standards. We used the optimized MTBSTFA method in VLDL-triglyceride and found that further modification was required to take matrix effects into account. We now have a robust method to measure glycerol isotopic enrichment by GC/EI-MS that can be used to rule out the known problem of tracer recycling in studies of VLDL kinetics.

NMR analysis of lipoprotein particle size does not increase sensitivity to the effect of soy protein on CVD risk when compared with the traditional lipid profile.

The traditional lipid profile compared with nuclear magnetic resonance (NMR) may underestimate the risk for cardiovascular disease and may explain some of the discrepancies in results between studies analyzing the salubrious effects of soy. Our purpose was to compare the traditional lipid profile with NMR quantification of the number of lipoprotein particles, subclasses, and diameters or sizes in 30 sedentary males, between 18 and 30 years of age, consuming 1 of the following 3 supplements daily for 28 days: milk protein (Milk), isoflavone-poor soy protein (Soy-), or isoflavone-rich soy protein (Soy+). The study used a double-blind, parallel-arm design with random assignment to 1 of the 3 protein supplement groups. Fasting EDTA blood samples were collected at baseline and after 28 days of supplementation and analyzed for the number and size of very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particles, respectively. Fasting serum samples were analyzed for concentrations of total cholesterol (TC), LDL cholesterol (LDL-C), total HDL cholesterol (HDL-C), HDL(2)-C, HDL(3)-C, triglycerides (TGs), free fatty acids (FFAs), and glucose. Fasting heparin blood samples were collected at baseline and after supplementation and analyzed for apolipoproteins A-I, A-II, B, C-II, C-III, and E, as well as hepatic and lipoprotein lipase concentrations. HDL3-C increased by 47.2% after Soy+ supplementation and hepatic lipase decreased 19.2% after Soy- supplementation (p < 0.05). HDL-C and apolipoproteins A-I and A-II were found to increase in all 3 groups (p < 0.05). Results support that NMR analysis of lipoprotein particle number and size are not more sensitive to the effect of soy protein on CVD risk compared with the traditional lipid profile. Furthermore, the lack of isoflavones in soy protein seems to have a deleterious effect on hepatic lipase.

Mild elevations of urine albumin excretion are associated with atherogenic lipoprotein abnormalities in the Multi-Ethnic Study of Atherosclerosis (MESA).

OBJECTIVE: Elevated urine albumin excretion is an established risk factor for cardiovascular disease. Increased cardiovascular risk may be partly mediated by abnormalities in lipoprotein metabolism. We examined cross-sectional associations of urine albumin-creatinine ratio (ACR) with standard lipid measurements and with lipoprotein particle concentrations measured by nuclear magnetic resonance (NMR) in the Multi-Ethnic Study of Atherosclerosis. METHODS AND RESULTS: Among 5633 participants who were not using lipid-lowering medications, greater ACR was associated with greater triglyceride concentration and lesser high density lipoprotein cholesterol concentration (women only), but not with low density lipoprotein (LDL) cholesterol calculated using conventional methods. In contrast, unadjusted mean small LDL particle concentrations measured by NMR were 770, 827 and 935 nmol/L for women (p<0.001) and 996, 1030 and 1040 nmol/L for men (p=0.037) among participants with normal, high normal and elevated ACR. Adjusting for age, race/ethnicity, diabetes, impaired fasting glucose, hypertension, smoking, medications, body mass index and serum creatinine, each two-fold greater ACR was associated with an increase in small LDL particle concentration of 27 nmol/L for women (p<0.001) and 14 nmol/L for men (p=0.008). Greater ACR was also associated with greater intermediate density lipoprotein particle concentration and smaller mean LDL particle size. CONCLUSIONS: Mild elevations of urine ACR are associated with atherogenic lipoprotein abnormalities that are not directly observed with a standard lipid panel.

Factors influencing the formation of small dense low-density lipoprotein particles in dependence on the presence of the metabolic syndrome and on the degree of glucose intolerance.

BACKGROUND: Small dense low-density lipoprotein (LDL) particles are known to be especially atherogenic. Several mechanisms are involved in this atherogenicity. AIMS: We wanted to look for the presence of small dense LDL particles depending on gender, metabolic syndrome (MS) and different degrees of glucose intolerance. Moreover, we looked for anthropometric factors and factors of lipid and carbohydrate metabolism that are associated with changes in the LDL size. RESULTS: We studied 752 persons (330 males, 422 females; age 40 +/- 17 years). LDL particle size was estimated with polyacrylamide gel electrophoresis. Males had smaller LDL particles than females. Probands with the MS had smaller LDL particles than those without this syndrome. With rising plasma triglyceride (TG) levels more small dense LDL particles were seen. The highest proportion of these small dense LDL particles was observed in the subgroup of type 2 diabetic patients. In the whole material, the mean LDL diameter was correlated negatively with plasma TG and very low-density lipoprotein components (TG, cholesterol and proteins) and positively with high-density lipoprotein cholesterol. In a linear stepwise regression analysis different significant factors influencing the LDL size were found in the whole population, in normoglycaemic probands, in persons with impaired glucose tolerance, in type 2 diabetic patients and in type 2 diabetic patients injecting insulin. CONCLUSIONS: Our data point to different mechanisms of the formation of small dense LDL particles in dependence on the degree of glucose intolerance. Moreover, the target values for plasma TG should be set lower.

Enzymatic O-methylation of flavanols changes lag time, propagation rate, and total oxidation during in vitro model triacylglycerol-rich lipoprotein oxidation.

3'-O-Methyl derivatives of flavan-3-ols, (+)-catechin (C), (-)-epicatechin (EC), and (-)-catechin gallate (CG) were prepared enzymatically. Hexanal (EC and CG family, 5 mmol/L) and conjugated diene (C and EC family, 0.25-10 mmol/L) formation following CuSO4-mediated triacylglycerol-rich lipoprotein oxidation was measured. All EC and CG compounds significantly reduced hexanal formation (p < 0.02). O-Methylation improved the ability of CG (more polar) while reducing the ability of EC (less polar) to limit hexanal formation. 3'-O-methyl EC was 18% (p < 0.001) and 4'-O-methyl 65% (p < 0.001) less able than EC to suppress hexanal formation. At >1 micromol/L all EC and C compounds significantly increased lag time. Parent compounds were more effective (> 4-fold increase) than metabolites (1.5-fold increase). Parent compounds did not influence propagation rate (DeltaOD/min). At >1 mmol/L O-methylated EC and C reduced propagation by 20-40% (p < 0.01). Notably, at 0.25 mmol/L O-methylated EC and C increased propagation rates 22% (p < 0.01) despite prolonging lag time.

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 hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis in rat: relation with changes in the VLDL composition.

AIMS: To study if the course of cerulein-induced pancreatitis in rats changes in a state of triglyceride-rich lipoprotein metabolism alteration. METHODS: Two groups of rats received control diet during a 90-day period (A) and sucrose-rich diet to induce endogenous hypertriglyceridemia (B). Subgroups A2 and B2 received i.p. 45 microg cerulein/kg body weight (to induce acute pancreatitis). Histological examination of pancreas tissue, serum pancreatic lipase, lipoprotein profile and VLDL chemical composition were assessed. Then, pancreatic lipase hydrolytic activity on VLDL-triglycerides was evaluated in vitro. RESULTS: Cellular vacuolization was observed in all of the cerulein-injected rats, but only in subgroup B2 fat necrosis was present. Serum triglycerides were higher in subgroup B1 than in subgroup A1 (mean +/- SEM, mg/dl 123,77 +/- 25.7 vs. 65.8 +/- 7, p < 0.01). Triglycerides from rats fed with sucrose-rich diet, decreased after cerulein-induced pancreatitis (80.38 +/- 11.3 vs. 123,77 +/- 25.7, p < 0.02). Moreover, the endogenous hypertriglyceridemic rats showed an increment of VLDL triglyceride content, which decreased when rats were injected with cerulein. A negative correlation was found between VLDL-triglyceride content and serum pancreatic lipase activity (r = 0.58, p < 0.02). The in vitro assay showed a decrease in VLDL-triglyceride content post incubation with pancreatic lipase enriched serum (mean +/- SD: 59.2 +/- 27.7%, p < 0.01). CONCLUSIONS: The endogenous hypertriglyceridemia intensifies the course of cerulein-induced pancreatitis and it could be related to the decrease in VLDL-triglycerides as a consequence of pancreatic lipase hydrolytic activity.

Apolipoprotein A-V deficiency results in marked hypertriglyceridemia attributable to decreased lipolysis of triglyceride-rich lipoproteins and removal of their remnants.

OBJECTIVE: ApoAV, a newly discovered apoprotein, affects plasma triglyceride level. To determine how this occurs, we studied triglyceride-rich lipoprotein (TRL) metabolism in mice deficient in apoAV. METHODS AND RESULTS: No significant difference in triglyceride production rate was found between apoa5(-/-) mice and controls. The presence or absence of apoAV affected TRL catabolism. After the injection of 14C-palmitate and 3H-cholesterol labeled chylomicrons and (125)I-labeled chylomicron remnants, the disappearance of 14C, 3H, and (125)I was significantly slower in apoa5(-/-) mice relative to controls. This was because of diminished lipolysis of TRL and the reduced rate of uptake of their remnants in apoa5(-/-) mice. Observed elevated cholesterol level was caused by increased high-density lipoprotein (HDL) cholesterol in apoa5(-/-) mice. VLDL from apoa5(-/-) mice were poor substrate for lipoprotein lipase, and did not bind to the low-density lipoprotein (LDL) receptor as well as normal very-low-density lipoprotein (VLDL). LDL receptor levels were slightly elevated in apoa5(-/-) mice consistent with lower remnant uptake rates. These alterations may be the result of the lower apoE-to-apoC ratio found in VLDL isolated from apoa5(-/-) mice. CONCLUSIONS: These results support the hypothesis that the absence of apoAV slows lipolysis of TRL and the removal of their remnants by regulating their apoproteins content after secretion.

Competitive displacement of clozapine from plasma proteins in normolipidemic and hyperlipidemic plasma samples: clinical implications.

OBJECTIVE: This study assesses whether competitive displacement of clozapine by warfarin affects clozapine's overall plasma distribution. METHODS: Warfarin sodium was preincubated in normolipidemic and hyperlipidemic plasma samples in varying concentrations. Following the preincubation with warfarin, [3H]clozapine mixed with unlabeled clozapine was added to the plasma samples. The plasma was separated into its lipoprotein and lipoprotein-deficient fractions by density gradient ultracentrifugation, and clozapine distribution was determined. RESULTS: When normolipidemic plasma was preincubated with various concentrations of warfarin, no significant redistribution of clozapine was noted among the various plasma lipoprotein fractions. However, in the case of the hyperlipidemic plasma, preincubating with warfarin did result in a significant redistribution of clozapine from the lipoprotein-deficient fraction to the very-low-density and low-density fractions of lipoproteins. Based on pharmacokinetic principles, the steady-state unbound concentration of clozapine in normolipidemic and hyperlipidemic plasma is not expected to change. CONCLUSION: Although no change in the steady-state unbound (active) concentration of clozapine would predict no change in clinical status, it is possible that this may only apply to the individuals with a normal lipid profile. We believe clozapine's association with lipoproteins (particularly triglycerides) may actually increase clozapine's effectiveness.

Diurnal and acute patterns of postprandial apolipoprotein B-48 in VLDL, IDL, and LDL from normolipidemic humans.

Smaller size particles of intestinal origin may be more relevant to atherosclerosis than larger particles because of easier transendothelial transport. We examined the diurnal pattern in response to low-fat high-carbohydrate meals and the effect of a single high-fat milkshake on apoB-48 in very light Sf degrees >400, light Sf degrees 60-400, and dense Sf degrees 20-60 VLDL, IDL, and LDL in 24 subjects (12 in each study). After the high-fat meal, apoB-48 concentrations peaked at 3 h for very light VLDL, 4 h for light VLDL, and 5 h for dense VLDL and IDL, suggesting a delipidation cascade. The diurnal pattern was characterized by a sharp increase in light and dense VLDL apoB-48 after breakfast, reaching the highest concentration 4-8h after breakfast. Less response was observed after lunch, dinner or snack, despite 75% of energy supplied by these meals. The proportion of apoB-48 in LDL in the fasting state ranged between 48 and 54%. LDL apoB-48 concentration decreased by 35% after the high-fat meal, and slowly recovered to near fasting concentrations after 10-12 h. In contrast, in the diurnal study, LDL apoB-48 concentration steady increased peaking 13 h after breakfast, followed by a sharp decrease. In sum, most intestinally synthesized apoB lipoproteins circulate in LDL, and are likely formed in the late postprandial period from larger VLDL-size particles. Although small apoB-48 lipoproteins may be atherogenic remnant particles, their very low concentration may diminish their clinical significance.

Sphingomyelinase induces aggregation and fusion of small very low-density lipoprotein and intermediate-density lipoprotein particles and increases their retention to human arterial proteoglycans.

OBJECTIVE: Infiltration of low-density lipoprotein (LDL) into subendothelial space is an early step in atherosclerosis. In addition to LDL particles, small very low-density lipoprotein (sVLDL) and intermediate-density lipoprotein (IDL) particles are also able to enter the arterial intima and be retained within the subendothelial extracellular matrix. Here we compared how proteolysis with alpha-chymotrypsin and phospholipid hydrolysis with phospholipase A2 or sphingomyelinase (SMase) of sVLDL, IDL, and LDL particles can influence their aggregation, fusion, and binding to human arterial proteoglycans in vitro. METHODS AND RESULTS: In each of the 3 lipoprotein classes, the particles became only slightly aggregated with alpha-chymotrypsin or phospholipase A2. However, the particles strongly aggregated when treated with SMase. The aggregated/fused particles were found to bind to proteoglycans in proteoglycan affinity chromatography more tightly than the native-sized counterparts. In addition, in a microtiter well assay, the binding of SMase-treated lipoproteins was enhanced: the amounts of proteoglycan-bound SMase-treated LDL, IDL, and sVLDL were 4-, 5-, and 20-fold higher, respectively, than the amounts of proteoglycan-bound native lipoproteins. CONCLUSIONS: These results imply a specific role for SMase as an sVLDL- and IDL-modifying enzyme and also suggest a novel mechanism of lipid accumulation in atherogenesis, namely enhanced retention of atherogenic triglyceride-rich lipoprotein particles in intimal areas expressing extracellular SMase activity.

Nephrotic livers secrete normal VLDL that acquire structural and functional defects following interaction with HDL.

BACKGROUND: Binding of very low-density lipoprotein (VLDL) isolated from serum of nephrotic rats VLDL to endothelial cells is defective. This defect is conferred on normal VLDL by prior incubation with high-density lipoprotein (HDL) from nephrotic, but not control rats. It is not known whether the defect is present in nascent VLDL (nVLDL) or is acquired after secretion. We test the hypothesis that VLDL is normal at the time of secretion from the liver and the defect in binding to endothelium is conferred following secretion through interaction with HDL. METHODS: nVLDL was synthesized by and collected from isolated perfused livers from either control or nephrotic rats. nVLDL was labeled with 3H-oleate to measure binding and 35S methionine to evaluate apolipoprotein exchange and composition. To test whether HDL conferred a binding defect, nVLDL was incubated with HDL obtained either from control or nephrotic rats prior to measurement of binding. To distinguish the effects of proteinuria from reduced albumin concentration we additionally incubated nVLDL with HDL obtained from rats with hereditary analbuminemia. Both HDL and VLDL were reisolated by centrifugation prior to subsequent binding and lipolysis determination. Exchange of 35S-labeled apolipoprotein E (apoE) among the subsequent VLDL and HDL fractions was determined. To determine the effect of HDL on lipolysis, HDL-treated VLDL was exposed to lipoprotein lipase-coated 96-well plates and 3H-oleate release measured. To establish whether differences in apoE content could explain the differences in binding and lipolysis, apoE was restored to nephrotic VLDL and lipolysis and binding were subsequently measured. RESULTS: Binding of nephrotic nVLDL was greater than control nVLDL (0.58 +/- 0.13 vs. 0.75 +/- 0.07 ng protein bound/mg cell protein) (P= 0.04, N= 6). Lipolysis was similarly elevated (0.091 +/- 0.010 vs 0.064 +/- 0.002 nmol NEFA released/well/hour) (P < 0.05). Prior incubation with nephrotic HDL reduced binding of nVLDL obtained from either nephrotic or control livers (P= 0.02, N= 6). Treatment with nephrotic (vs. control) HDL reduced both binding (control nVLDL + control HDL, 0.64 +/- 0.02; control + nephrotic, 0.43 +/- 0.06; nephrotic + control, 0.69 +/- 0.05; and nephrotic + nephrotic, 0.62 +/- 0.04 mg VLDL protein/mg cell protein) and lipolysis (control nVLDL + control HDL, 0.053 +/- 0.004; control + nephrotic, 0.038 +/- 0.004; nephrotic + control, 0.069 +/- 0.004; and nephrotic + nephrotic, 0.062 +/- 0.004 nmol NEFA/well/hour) (P < 0.05 vs. nVLDL + control HDL) of nVLDL from either source. The apoE content of nVLDL coincubated with control HDL or analbuminemic HDL was increased compared nVLDL incubated with either no HDL or nephrotic HDL (P < 0.05). Similarly, the apoE/apoA-I ratio was reduced in HDL from nephrotic rats but not in HDL from controls (P < 0.05). Reintroduction of apoE to nephrotic VLDL resulted in increased binding. CONCLUSION: Unlike circulating VLDL, binding of nVLDL from isolated livers from nephrotic rats to endothelial cells is greater and its lipolysis is increased compared to control nVLDL. Decreased binding and lipolysis is conferred following incubation with HDL isolated from control, but not nephrotic rats and binding can be restored by reintroduction of apoE. Thus both defects are conferred on VLDL by exposure to HDL obtained from nephrotic animals, possibly a consequence of a failure of nephrotic HDL to enrich VLDL with apoE during clearance.

The influence of dyslipidemia on the plasma protein and lipoprotein distribution of haloperidol.

The objective of this study is to determine the effect of dyslipidemia on the plasma protein and lipoprotein distribution of haloperidol. In doing so, total plasma cholesterol and triglyceride levels were adjusted by adding differing volumes of concentrated low-density lipoproteins and very-low-density lipoproteins to normolipidemic plasma. Following the incubation of [3H]-haloperidol plus cold haloperidol (18 microg/ml) the plasma was separated into its lipoprotein and lipoprotein deficient fractions by density gradient ultra centrifugation and [3H]-haloperidol distribution was determined. Our results revealed a significant positive correlation between total cholesterol concentration and haloperidol recovery in the low-density lipoprotein fraction (r=0.975, df=13, P<0.005). A positive correlation was also observed between total plasma triglyceride concentration and haloperidol recovery from both the very low-density lipoprotein (r=0.965, df=16, P<0.002) and low-density lipoprotein fractions (r=0.935, df=16, P<0.005). This redistribution was the result of haloperidol leaving the lipoprotein-deficient fraction were it is primarily bound to albumin and alpha(1)-acid glycoprotein. This redistribution of haloperidol may influence its clinical efficacy.