Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.

PURPOSE: In this study we examined the uptake of circulating lipoproteins into the retina, using a naturally fluorescent cholesterol analog for imaging and deuterated cholesterol for quantification by mass spectroscopy. The purpose of this study was to better understand cholesterol uptake, transport and homeostasis in the retina. METHODS: Human low density lipoprotein (LDL) and high density lipoprotein (HDL) were labeled with the fluorescent cholesterol analog cholesta-5,7,9(11)-trien-3beta-ol (CTL) and deuterated cholesterol (25,26,26,26,27,27,27-[2H]cholesterol, D7Ch). Rats were injected intravenously with CTL-LDL, CTL-HDL and D7Ch-LDL. Fluorescent confocal microscopy was used to image the uptake of CTL and mass spectroscopy was used to quantify D7Ch. Immunohistochemistry and fluorescent confocal microscopy were used to localize apoB (an LDL marker protein) and LDL receptor (LDLR) protein in rat and monkey retinas. RESULTS: CTL-specific fluorescence was imaged by confocal microscopy in the retinal pigment epithelium (RPE), choriocapillaris and parts of the neural retina within 2 h post-injection and was visualized in the photoreceptor outer segments by 4 h. Replacing LDL with HDL as the CTL carrier gave a less robust and more delayed labeling of retinal layers. Human apolipoprotein B (apoB) was also localized in the rat choriocapillaris and RPE by 4 h post-injection. Human apoB was detected by immunoblot analysis in the rat retina primarily as a about 70 kDa protein, suggesting proteolytic degradation. LDL-mediated uptake of cholesterol was quantified by mass spectroscopy using deuterated cholesterol in place of CTL. In addition, apoB and LDLR were localized in monkey retina by immunohistochemistry. CONCLUSIONS: The retina is capable of rapid uptake of circulating LDL via an LDLR-mediated process primarily occurring in the RPE and also possibly Müller cells. Despite the dominance of HDL over LDL in rat serum, LDL appears to be the preferred carrier for cholesterol transport to and uptake by the retina. The results also suggest that blood-borne LDL represents a significant contributor to the steady-state levels of cholesterol and possibly other lipids in the retina.

Effect of n-3 fatty acids on metabolism of apoB100-containing lipoprotein in type 2 diabetic subjects.

The effect of long-chain n-3 PUFA on the metabolism of apoB100-containing lipoprotein in diabetic subjects is not fully understood. The objective of the present study was to determine the effect of a daily intake of 1080 mg EPA and 720 mg DHA for diabetic subjects on the kinetics of apoB100-containing lipoprotein in the fasting state. A kinetic study was undertaken to determine the mechanisms involved in the effects of n-3 fatty acids in terms of a decrease in triacylglycerol level in type 2 diabetic patients. We have studied the effect of fish oils on the metabolism of apoB100 endogenously labelled by [5,5,5-2H3]-leucine in type 2 diabetic patients in the fasting state. The kinetic parameters of apoB100 in VLDL, intermediate-density lipoprotein and LDL were determined by compartmental modelling in five diabetic subjects before and 8 weeks after n-3 fatty acid treatment. Treatment did not change the plasma cholesterol level (0.801 (sd 0.120) v. 0.793 (sd 0.163) mmol/l) but lowered the plasma triacylglycerol level (1.776 (sd 0.280) v.1.356 (sd 0.595) mmol/l; P < 0.05). Treated patients showed a decrease in VLDL apoB100 concentration (0.366 (sd 0.030) v.0.174 (sd 0.036) g/l; P < 0.05) related to a decrease in VLDL 1 production (1.49 (sd 0.23) v.0.44 (sd 0.19) mg/kg per h; P < 0.05) and an increase in the VLDL conversion rate (0.031 (sd 0.024) v.0.052 (sd 0.040) per h; P < 0.05), with no change in fractional catabolic rates. Treatment led to a higher direct production of intermediate-density lipoprotein (0.02 (sd 0.01) v.0.24 (sd 0.12) mg/kg per h; P < 0.05). In conclusion, the present study, conducted in the fasting state, showed that supplementation with n-3 fatty acids in type 2 diabetic patients induced beneficial changes in the metabolism of apoB100-containing lipoprotein.

Cholesteryl esters of aggregated LDL are internalized by selective uptake in human vascular smooth muscle cells.

OBJECTIVE: Low-density lipoprotein (LDL) receptor-related protein (LRP1) mediates the internalization of aggregated LDL (agLDL)-LDL trapped in the arterial intima bound to proteoglycans-into human vascular smooth muscle cells (VSMC). LRP1-mediated agLDL uptake induces high-intracellular cholesteryl ester (CE) accumulation. The aim of this study was to characterize the mechanism of agLDL internalization in human VSMC. METHODS AND RESULTS: The lipidic component of LDL was labeled with [3H] and the apolipoprotein component with [(125)I]. We found that >90% of intracellular CE derived from agLDL uptake was not associated with apoB100 degradation but was selectively taken up from agLDL. The inhibition of LRP1 expression by small interfering RNA treatment led to a decrease of 80+/-0.05% in agLDL-CE selective uptake. AgLDL induced intracellular CE accumulation without a concomitant CE synthesis. Cytosolic and cytoskeletal proteins were not required for CE transport. Electron and confocal microscopy experiments indicate that CE derived from agLDL accumulated in adipophilin-stained lipid droplets that were not removable by high-density lipoprotein. CONCLUSIONS: Taken together, these results demonstrate that LRP1 mediates the selective uptake of CE from agLDL and that CE derived from agLDL is not intracellularly processed but stored in lipid droplets in human VSMC.

Effects of apolipoproteins on dalargin transport across the blood-brain barrier.

Antinociceptive activity of dalargin (7.5 mg/kg) adsorbed on poly(butyl)cyanoacrylate nanoparticles with different coating was studied on outbred albino mice by the tail-flick test. poly(butyl)cyanoacrylate nanoparticles without coating did not increase the antinociceptive activity of dalargin and hence, did not increase its transport across the blood-brain barrier. poly(butyl)cyanoacrylate nanoparticles coated with apolipoprotein B, apolipoprotein E, and polysorbate 80 increased the transport of dalargin across the blood-brain barrier. Delivery of dalargin to the brain was most effective in case of using poly(butyl)cyanoacrylate nanoparticles with polysorbate 80 coating and subsequent supercoating with apolipoprotein E.

Lipoprotein metabolism in subjects with hepatic lipase deficiency.

A heritable deficiency of hepatic lipase (HL) provides insights into the physiologic function of HL in vivo. The metabolism of apolipoprotein B (apoB)-100 in very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) and of apoA-I and apoA-II in high-density lipoprotein (HDL) particles lipoprotein (Lp)(AI) and Lp(AI:AII) was assessed in 2 heterozygous males for compound mutations L334F/T383M or L334F/R186H, with 18% and 22% of HL activity, respectively, compared with 6 control males. Subjects were provided with a standard Western diet for a minimum of 3 weeks. At the end of the diet period, apo kinetics was assessed using a primed-constant infusion of [5,5,5-(2)H(3)] leucine. Mean plasma triglyceride (TG) and HDL cholesterol levels were 55% and 12% higher and LDL cholesterol levels 19% lower in the HL patients than control subjects. A higher proportion of apoB-100 was in the VLDL than IDL and LDL fractions of HL patients than control subjects due to a lower VLDL apoB-100 fractional catabolic rate (FCR) (4.63 v 9.38 pools/d, respectively) and higher hepatic production rate (PR) (33.24 v 10.87 mg/kg/d). Delayed FCR of IDL (2.78 and 6.31 pools/d) and LDL (0.128 and 0.205 pools/d) and lower PR of IDL (3.67 and 6.68 mg/kd/d) and LDL 4.57 and 13.07 mg/kg/d) was observed in HL patients relative to control subjects, respectively. ApoA-I FCR (0.09 and 0.13 pools/d) and PR (4.01 and 6.50 mg/kg/d) were slower in Lp(AI:AII) particles of HL patients relative to control subjects, respectively, accounting for the somewhat higher HDL cholesterol levels. HL deficiency may result in a lipoprotein pattern associated with low heart disease risk.

Metabolic effects of Troglitazone in patients with diet-controlled type 2 diabetes.

BACKGROUND: In order to study the mechanisms of action of Troglitazone (TGZ) in vivo in Type 2 diabetes, its effects were studied on glucose metabolism, lipolysis and very low-density lipoprotein (VLDL) apolipoprotein B100 (apoB) kinetics. MATERIALS AND METHODS: A placebo-controlled, double-blind study was performed in 24 diet-treated patients randomized to receive TGZ 600 mg day(-1), TGZ 200 mg day(-1) or placebo for 8 weeks. Glucose and glycerol turnover were assessed after an overnight fast, and during sequential low-dose insulin infusions (0.01 U kg(-1) h(-1) followed by 0.015 U kg(-1) h(-1)) using 6,6-2H Glucose and 1,2,3-2H Glycerol. Very low-density lipoprotein apoB secretion was measured using l-13C-leucine, monitoring isotopic enrichment by gas chromatography-mass spectrometry. Treatment effects were analyzed by analysis of covariance, adjusting for baseline. RESULTS: Therapy resulted in a significant group differences in fasting plasma glucose adjusting for baseline (P=0.039). This was most evident at TGZ 600 mg daily [glucose decrease from (mean +/- SD) 9.2 +/- 2.7 to 6.6 +/- 0.9 mmol L(-1)]. HbA1c and insulin levels did not change significantly. Plasma nonesterified fatty acid (NEFA) levels decreased (P=0.045), most evidently at TGZ 200 mg daily, but glycerol was not significantly affected. Although no significant effects were observed on VLDL apoB or triglyceride concentrations, there were treatment differences in the absolute secretion rate of VLDL apoB of borderline (P=0.056) statistical significance, with a decrease observed at TGZ 600 mg daily [geometric mean, SD range, 0.94 (0.41-2.15) to 0.40 (0.14-1.13 mg kg(-1) h(-1))]. Very low-density lipoprotein apoB fractional secretion rate and pool size were unaffected. The VLDL triglyceride: apoB molar ratio differed between treatment groups (P=0.013), being higher in the TGZ 600 mg group [5714 (4128-7741) to 8092 (5669-11552)]. Neither glucose nor glycerol rates of appearance were significantly altered by TGZ and nor did TGZ affect their suppression by insulin. DISCUSSION: The PPARgamma agonist, troglitazone, decreases fasting glucose and NEFA levels in diet-treated Type 2 diabetes. It may also decrease VLDL particle secretion. These effects would be considered beneficial. The biological importance of the increase in VLDL-triglyceride enrichment warrants further study.

Human apolipoprotein (Apo) B-48 and ApoB-100 kinetics with stable isotopes.

The kinetics of apolipoprotein (apo) B-100 and apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within IDL and LDL were examined with a primed-constant infusion of (5,5,5-(2)H(3)) leucine in the fed state (hourly feeding) in 19 subjects after consumption of an average American diet (36% fat). Lipoproteins were isolated by ultracentrifugation and apolipoproteins by SDS gels, and isotope enrichment was assessed by gas chromatography/mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II. The pool sizes (PS) of TRL apoB-48, VLDL apoB-100, and LDL apoB-100 were 17+/-10, 273+/-167, and 3325+/-1146 mg, respectively. There was a trend toward a faster fractional catabolic rate (FCR) for VLDL apoB-100 than for TRL apoB-48 (6.73+/-3.48 versus 5.02+/-2.07 pools/d, respectively, P=0.06). The mean FCRs for IDL and LDL apoB-100 were 10.07+/-7.28 and 0.27+/-0.08 pools/d, respectively. The mean production rate (PR) of TRL apoB-48 was 6.5% of VLDL apoB-100 (1. 3+/-0.90 versus 20.06+/-6.53 mg. kg(-1). d(-1), P<0.0001). TRL apoB-48 PS was correlated with apoB-48 PR (r=0.780, P<0.0001) but not FCR (r=-0.1810, P=0.458). VLDL apoB-100 PS was correlated with both PR (r=0.713, P=0.0006) and FCR (r=-0.692, P=0.001) of VLDL apoB-100 and by apoB-48 PR (r=0.728, P=0.0004). LDL apoB-100 PS was correlated with FCR (r=-0.549, P=0.015). These data indicate that (1) the FCRs of TRL apoB-48 and VLDL apoB-100 are similar in the fed state, (2) TRL apoB-48 PS is correlated with TRL apoB-48 PR, (3) VLDL apoB-100 PS is correlated with both PR and FCR of VLDL apoB-100 and PR of TRL apoB-48, and (4) LDL apoB-100 PS is correlated with LDL FCR.

Effects of apolipoprotein B-100 on the metabolism of a lipid microemulsion model in rats.

In previous studies, it was shown that lipid microemulsions resembling LDL (LDE) but not containing protein, acquire apolipoprotein E when injected into the bloodstream and bind to LDL receptors (LDLR) using this protein as ligand. Aiming to evaluate the effects of apolipoprotein (apo) B-100 on the catabolism of these microemulsions, LDE with incorporated apo B-100 (LDE-apoB) and native LDL, all labeled with radioactive lipids were studied after intraarterial injection into Wistar rats. Plasma decay curves of the labels were determined in samples collected over 10 h and tissue uptake was assayed from organs excised from the animals sacrificed 24 h after injection. LDE-apo B had a fractional clearance rate (FCR) similar to native LDL (0.40 and 0.33, respectively) but both had FCR pronouncedly smaller than LDE (0.56, P<0.01). Liver was the main uptake site for LDE, LDE-apoB, and native LDL, but LDE-apoB and native LDL had lower hepatic uptake rates than LDE. Pre-treatment of the rats with 17alpha-ethinylestradiol, known to upregulate LDLR, accelerated the removal from plasma of both LDE and LDE-apoB, but the effect was greater upon LDE than LDE-apoB. These differences in metabolic behavior documented in vivo can be interpreted by the lower affinity of LDLR for apo B-100 than for apo E, demonstrated in in vitro studies. Therefore, our study shows in vivo that, in comparison with apo E, apo B is a less efficient ligand to remove lipid particles such as microemulsions or lipoproteins from the intravascular compartment.

High-density lipoprotein (HDL3)-associated alpha-tocopherol is taken up by HepG2 cells via the selective uptake pathway and resecreted with endogenously synthesized apo-lipoprotein B-rich lipoprotein particles.

alpha-Tocopherol (alphaTocH) is transported in association with lipoproteins in the aqueous milieu of the plasma. Although up to 50% of circulating alphaTocH is transported by high-density lipoproteins (HDLs), little is known about the mechanisms of uptake of HDL-associated alphaTocH. During the current study, human apolipoprotein (apo)E-free HDL subclass 3 (HDL3) labelled with [14C]alphaTocH was used to investigate uptake mechanisms of HDL3-associated alphaTocH by a permanent hepatoblastoma cell line (HepG2). HDL3-associated alphaTocH was taken up independently of HDL3 holoparticles in excess of apoA-I comparable with the non-endocytotic delivery of cholesteryl esters to cells termed the 'selective' cholesteryl ester uptake pathway. Experiments with unlabelled HDL3 demonstrated net mass transfer of alphaTocH to HepG2 cells. Time-dependent studies with [14C]alphaTocH-labelled HDL3 revealed tracer uptake in 80-fold excess of apoA-I and in 4-fold excess of cholesteryl linoleate. In addition to HLDs, low-density lipoprotein (LDL)-associated alphaTocH was also taken up in excess of holoparticles, although to a lesser extent. These findings were confirmed with unlabelled lipoprotein preparations, in which HDL3 displayed a 2- to 3-fold higher alphaTocH donor efficiency than LDLs (lipoproteins adjusted for equal amounts of alphaTocH). An important factor affecting particle-independent uptake of alphaTocH was the cellular cholesterol content (a 2-fold increase in cellular cholesterol levels resulted in a 2.3-fold decrease in uptake). Pulse-chase studies demonstrated that some of the HDL3-associated alphaTocH taken up independently of holoparticle uptake was resecreted along with a newly synthesized apoB-containing lipoprotein fraction.

SAAM II: Simulation, Analysis, and Modeling Software for tracer and pharmacokinetic studies.

Kinetic analysis and integrated systems modeling have contributed substantially to our understanding of the physiology and pathophysiology of metabolic systems and the distribution and clearance of drugs in humans and animals. In recent years, many researchers have become aware of the usefulness of these techniques in the experimental design. With this has come the recognition that the discipline of kinetic analysis requires its own expertise. The expertise can impact experimental design in many ways, from the collaborative and service activities in which individuals interact in formal ways to the development of software tools to aid in kinetic analysis. The purpose of this report is to describe one such software tool, Simulation, Analysis, and Modeling Software II (SAAM II). In the first part, we describe in general how the user can take advantage of the capabilities of the software system, and in the second part, we give three specific examples using multicompartmental models found in lipoprotein (apolipoprotein B [apoB] kinetics) and diabetes (glucose minimal model) research.

Three-fold effect of lovastatin treatment on low density lipoprotein metabolism in subjects with hyperlipidemia: increase in receptor activity, decrease in apoB production, and decrease in particle affinity for the receptor. Results from a novel triple-tracer approach.

To differentiate effects of lovastatin on low density lipoprotein (LDL) receptor activity from effects on LDL metabolic properties, LDL apolipoprotein B (apoB) turnover was studied in eight hyperlipidemic subjects during baseline and lovastatin treatment, in the latter case with LDL tracers isolated during both baseline (CLDL) and drug treatment (Rx-LDL) conditions. Lovastatin (40 mg/day) significantly lowered total plasma and LDL cholesterol levels (27% and 25%, respectively) as well as plasma triglyceride levels (30%). Using contemporaneous tracers (C-LDL before and Rx-LDL during treatment), lovastatin caused a modest increase in LDL fractional catabolic rate (FCR) (0.410+/-0.113 vs. 0.339+/-0.108 pools/day, P < 0.04 by paired t). The increase in LDL tracer FCR was higher when C-LDL tracer isolated during the untreated period was injected during lovastatin treatment (0.496+/-0.177 vs. 0.339+/-0.108 pools/day, P < 0.02). These in vivo studies in humans were confirmed by injecting LDL tracers from two patients into five guinea pigs. The C-LDL tracer was cleared consistently faster than the Rx-LDL tracer (0.082+/-0.018 vs. 0.057+/-0.015 pools/h, P< 0.001). The results demonstrate three important outcomes of lovastatin treatment in these subjects: LDL receptor activity increased by 49% (P < 0.02); LDL apoB production rate decreased by 17% (P < 0.03), and LDL particle in vivo affinity for the LDL receptor decreased by 15% (P < 0.01). The decrease in LDL particle affinity partially negated the expected effect of increased LDL receptors on LDL clearance. The present study provides an explanation for earlier observations by several investigators using contemporaneous tracers that treatment with HMG-CoA reductase inhibitors resulted in only modest increases in low density lipoprotein functional catabolic rate.

Gluconeogenesis measured with [U-13C]glucose and mass isotopomer analysis of apoB-100 amino acids in pigs.

Infant pigs (8.5 kg) were fasted for 16 h and infused for 6 h with [U-13C]glucose. The fractional abundances of all 13C mass isotopomers of plasma glucose, lactate, and pyruvate and of plasma, hepatic, and very low density lipoprotein apolipoprotein B-100 (apoB-100) alanine, glutamate, and aspartate were measured. The ratios of [13C3]aspartate. [13C3]glutamate, and [13C3]alanine in apoB-100 were used to estimate the positional equilibrium of [13C3]oxaloacetate, the fractional contribution of pyruvate carboxylase to the hepatic oxaloacetate flux, and the activity of hepatic pyruvate dehydrogenase. The values were compared with those based on glucose labeling and previously published equations. The two methods [Katz and Lee method (J. Katz, P.A. Wals., and W.-N. P. Lee. J. Biol. Chem. 264: 12994-13001, 1989) and apoB method] gave similar estimates of the positional equilibrium of [13C3]oxaloacetate (0.59, Katz and Lee method; 0.61, apoB method) but slightly different estimates of the contribution of pyruvate carboxylase to the oxaloacetate flux (0.36, Katz and Lee; 0.31 apoB). Gluconeogenesis apparently contributed between 71 (Katz and Lee method) and 80% (apoB method) of the glucose entry rate (25 mumol.kg-1.min-1), and pyruvate dehydrogenase contributed 20% of the hepatic acetyl-CoA. We conclude that the labeling of aspartate in apoB-100 provides a good estimate of the isotopomer distribution in hepatic oxaloacetate but may underestimate the absolute isotopic enrichment by 50%.

Lipoprotein kinetics in patients with analbuminemia. Evidence for the role of serum albumin in controlling lipoprotein metabolism.

In vitro data suggested that albumin is a key factor controlling apolipoprotein (apo) synthesis by hepatocytes. Studies in analbuminemic rats have shown an increase in secretion of apoB-containing lipoprotein from the liver. We studied the kinetic aspects of apoB- and apoAI-containing lipoprotein metabolism in two sisters with analbuminemia using a constant 14-hour infusion of leucine labeled with stable isotopes. Compared with control subjects, total cholesterol was higher in the two patients (432 and 461 versus 155 +/- 14 mg/dL), as was apoB (257 and 230 versus 72 +/- 7 mg/dL). Triglycerides were slightly increased (134 and 105 versus 89 +/- 9 mg/dL), whereas apoAI was lower (109 and 105 versus 124 +/- 6 mg/dL). VLDL-apoB production was higher, as was the production of IDL-apoB and LDL-apoB (32.8 and 36.0 versus 24.8 +/- 5.9, 32.1 and 27.2 versus 16.4 +/- 2.3, and 14.1 and 17.6 versus 10.3 +/- 1.2 mg.kg-1.d-1, respectively). The fractional catabolic rate of all the apoB-containing lipoproteins was decreased (0.23 and 0.37 versus 0.48 +/- 0.05, 0.27 and 0.28 versus 0.62 +/- 0.08, and 0.012 and 0.009 versus 0.022 +/- 0.002.h-1, respectively). A similar mechanism could explain the dyslipidemia observed in other conditions associated with low albumin levels, such as nephrotic syndrome.

Overexpression of human lecithin:cholesterol acyltransferase in cholesterol-fed rabbits: LDL metabolism and HDL metabolism are affected in a gene dose-dependent manner.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme well known for its involvement in the intravascular metabolism of high density lipoproteins; however, its role in the regulation of apolipoprotein (apo) B-containing lipoproteins remains elusive. The present study was designed to investigate the metabolic mechanisms responsible for the differential lipoprotein response observed between cholesterol-fed hLCAT transgenic and control rabbits. 131I-labeled HDL apoA-I and 125I-labeled LDL kinetics were assessed in age- and sex-matched groups of rabbits with high (HE), low (LE), or no hLCAT expression after 6 weeks on a 0.3% cholesterol diet. In HE, the mean total cholesterol concentration on this diet, mg/dl (230 +/- 50), was not significantly different from that of either LE (313 +/- 46) or controls (332 +/- 52) due to the elevated level of HDL-C observed in HE (127 +/- 19), as compared with both LE (100 +/- 33) and controls (31 +/- 4). In contrast, the mean nonHDL-C concentration for HE (103 +/- 33) was much lower than that for either LE (213 +/- 39) or controls (301 +/- 55). FPLC analysis of plasma confirmed that HDL was the predominant lipoprotein class in HE on the cholesterol diet, whereas cholesteryl ester-rich, apoB-containing lipoproteins characterized the plasma of LE and, most notably, of controls. In vivo kinetic experiments demonstrated that the differences in HDL levels noted between the three groups were attributable to distinctive rates of apoA-I catabolism, with the mean fractional catabolic rate (FCR, d-1) of apoA-I slowest in HE (0.282 +/- 0.03), followed by LE (0.340 +/- 0.01) and controls (0.496 +/- 0.04). A similar, but opposite, pattern was observed for nonHDL-C levels and LDL metabolism (h-1), such that HE had the lowest nonHDL-C levels with the fastest rate of clearance (0.131 +/- 0.027), followed by LE (0.057 +/- 0.009) and controls (0.031 +/- 0.001). Strong correlations were noted between LCAT activity and both apoA-I (r= -0.868, P < 0.01) and LDL (r = 0.670, P = 0.06) FCR, indicating that LCAT activity played a major role in the mediation of lipoprotein metabolism. In summary, these data are the first to show that LCAT overexpression can regulate both LDL and HDL metabolism in cholesterol-fed rabbits and provide a potential explanation for the prevention of diet-induced atherosclerosis observed in our previous study.

Dietary carbohydrate type and fat amount alter VLDL and LDL metabolism in guinea pigs.

The effects of low/high fat diets and simple/complex carbohydrate intake on specific aspects of plasma VLDL and LDL metabolism were evaluated. Guinea pigs were fed for 4 wk two different fat/carbohydrate concentrations: 2.5/58 (g/100 g) or 25/29 (g/100 g) with either sucrose or cornstarch as the sole carbohydrate source. Intake of high fat diets resulted in higher plasma cholesterol (P < 0.001), whereas sucrose intake resulted in higher plasma triacyglycerol (TAG) concentrations (P < 0.03). Intake of starch increased apolipoprotein (apo) B secretion rates (P < 0.001), and nascent VLDL were smaller and contained less TAG/apo B than particles from the sucrose-fed group (P < 0.01). Guinea pigs fed the starch diets had higher plasma VLDL apo B flux and faster VLDL apo B clearance than those fed sucrose diets (P < 0.01). In addition, more rapid VLDL removal from plasma in guinea pigs fed complex carbohydrate/high fat diets was associated with less conversion of VLDL to LDL and lower plasma cholesterol concentrations compared with the high fat/sucrose group (P < 0.01). Low fat compared with high fat intake resulted in 60% more rapid plasma LDL apo B fractional catabolic rates (FCR). The LDL apo B fractional catabolic rate of all dietary groups was inversely correlated with plasma cholesterol concentrations (r = -0.83, P < 0.001). These results demonstrate that in guinea pigs, low fat diets decrease plasma LDL cholesterol concentrations by increasing LDL turnover rates, and complex carbohydrates reduce plasma TAG by affecting the composition of nascent VLDL particles and by increasing VLDL apo B catabolism.

Sensitive methods to study human apolipoprotein B metabolism using stable isotope-labeled amino acids.

The objective of the study was to develop a sensitive method using stable isotope-labeled tracers that would permit the determination of apolipoprotein B (apoB) metabolism in very low-density lipoprotein subfractions (VLDL1, Sf 60-400; VLDL2, Sf 20-60), intermediate-density lipoprotein (IDL, Sf 12-20), and low-density lipoprotein (LDL, Sf 0-12). Six normolipidemic subjects were given trideuterated leucine, and its clearance from plasma and appearance in the four apoB-containing lipoprotein fractions were followed by use of a highly sensitive gas chromatography-mass spectrometry technique in which the m + 3-to-m + 2 ion ratio was selectively monitored. This analytic approach permitted the precise measurement of low enrichments in IDL and LDL and extension of the turnover out to 250-300 h. A compartmental model was developed to derive rate constants from the plasma and apoB enrichment curves. The model was uniquely identifiable once parameter dependencies had been introduced to reduce the number of unknowns. Values were obtained for apoB input into all lipoprotein density intervals, together with rates of interconversion and catabolism; these agreed well with results from radioiodinated tracer experiments. An alternative model structure was also explored in which input occurred only into VLDL1. Altering the protocol of tracer administration (bolus vs. primed constant infusion) and dose (over a 10-fold range) had no influence on the results obtained. The analytic and modeling approach described will permit stable isotopes to be used to elucidate key features of apoB metabolism in normal and pathological states.

[Influence of the primary membrane on lipid apheresis selectivity in cascade filtration]. / Influence de la première membrane sur la sélectivité de la lipaphérèse par filtration en cascade.

In apheresis, plasma separation from whole blood may be achieved by filtration. Some plasmapheresis membranes do not seem to be totally permeable to proteins: for instance, polyvinyl alcohol membrane presents apparent sieving coefficients significantly lower than 1, especially for apolipoproteins B (S' = 0.74 +/- 0.15). Moreover, severe membrane fouling is noticed during the treatment. The low permeability of the primary membrane for ApoB induces direct recirculation of this lipoprotein to the patient, without any chance of removal by the second membrane. In order to increase double filtration performance, it seems necessary to use plasmalfilters with a pore diameter of 0.5 microns. These filters are more permeable to plasma proteins and eliminate pathogenic protein better during the second stage of the process.

Apoliproteínas A-I y B-100 y enfermedad coronaria comprobada angiográficamente

Objetivo : establecer la correlación de las apolipoproteínas (APO) A-I y B-100 séricas , con la enferemdad coronaria (EC) y su severidad. Método: estudio abierto, prospectivo y análitico en 52 pacientes sometidos a angiocoronariografía de abril a septiembre de 1991, tomando previamente muestra de sangre para medición de colesterol (CT), triglicéridos (TG), HDL, APO-AI y APO B-100 y determinación de LDL, índice aterogénico (IA), LDL/HDL y relación APO A1/B-100. Sitio : Servicio universitario de referencia de pacientes de atención terciaria. Principales resulatdos : 65.4 por ciento de los pacientes fueron hombres y 34.6 por ciento, mujeres; 67.3 por ciento tenían EC y 32.7 por ciento nola tenían. Entre los grupos con y sin EC hubo diferencia significativa en todas las variables medidas, especialmente en el CT, cuya media era de 210+-51 mg por ciento en el grupo sin EC y de 255+-48 mg por ciento en el grupo con EC (p=0.0006), y en la relación APO-AI/B-100, que fue de 2.08+-0.49 para el grupo sin EC y de 1.18+-0.51 para elgrupo con EC (p=0001). Entre hombres y mujeres fueron significativamente diferentes las la APO-AI (p=0.004) y la relación APO-AI/B-100 (p=0.003). Ninguna variable diferenció el grupo con EC leve del grupo sin EC, pero sí diferenciaron la ausencia de EC de lapresencia de EC moderada y severa el CT, APO-AI, APO-AI/B-100, IA y LDL/HDL. En la regresión lineal el grado EC pependió significativamente de APO-AI/B-100 (r=0.73, p<0.001), APO-B-100 (r+0.60, p<0.001), LDL/HDL (r=0.51, p<0.001) y CT (r=0.50, p=<0.001). En la regresión múltiple la presencia de EC dependió de la relación APO-AI/B-100 (r=0.73) y del colesterol total (r=0.50). Conclusiones : el índice APO-AI/B-100 mejoró de manera importante la correlación con la presencia de EC con respecto a las demás variables, no así la medición de APO-AI y B-100 por separado. Esta misma relación no discriminó la ausencia de EC de la EC leve, pero si la ausencia de EC de la EC moderada y severa.

Hepatic production of very low density lipoprotein, catabolism of low density lipoprotein, biliary lipid secretion, and bile salt synthesis in rats fed a bean (Phaseolus vulgaris) diet.

Rats fed a bean diet develop a significant hypocholesterolemia. The catabolism of low density lipoprotein (LDL; d 1.019-1.063 g/ml) was studied in vivo and in vitro in the isolated perfused liver of rats fed either a casein or a bean diet. The clearance of LDL was significantly increased by 100% from 0.38 +/- 0.04 to 0.63 +/- 0.04 ml/h x 100 g body wt in vivo in the bean-fed rat. Similarly, the clearance of homologous and heterologous (human) LDL was also increased by 100% in the isolated perfused liver of bean-fed animals. Spleen, kidney, and hepatic cholesterogenesis was increased by 150% in these animals. Bile salt synthesis was increased from 1.54 +/- 0.02 to 2.84 +/- 0.09 nmol/min x g liver wt (P < 0.02) and biliary cholesterol output by 200% from 0.81 +/- 0.03 to 2.18 +/- 0.04 nmol/min x g (P < 0.02) in the isolated perfused liver of rats fed a bean diet. These results explained the depletion of hepatic cholesterol and were consistent with the LDL turnover studies, suggesting that apoB/E receptor activity was increased in these animals. ApoB and triglyceride secretion in the d < 1.060 g/ml lipoprotein fraction of liver perfusates remained normal in the bean-fed rats. In contrast, total sinusoidal cholesterol output isolated in the d < 1.060 g/ml fraction significantly decreased by 100% after 90 min of perfusion. Cholesterol output in the d > 1.060 g/ml lipoprotein fraction was unmodified by the bean diet. These data demonstrate that key metabolic pathways of hepatic cholesterol are modified in the bean-fed rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol absorption, elimination, and synthesis related to LDL kinetics during varying fat intake in men with different apoprotein E phenotypes.

Cholesterol absorption, fecal elimination, and synthesis and low density lipoprotein (LDL) metabolism were measured in 29 middle-aged men while on their normal diet and a diet low in fat and cholesterol, and the obtained values were related to apoprotein (apo) E phenotypes. Basal cholesterol absorption efficiency was positively related to production rate (PR) for LDL apo B and negatively to cholesterol synthesis (measured by fecal steroids and dietary cholesterol), which in turn was negatively associated with the LDL level and positively with the fractional removal (FCR) of LDL apo B. The apo E subscript (e.g., E2/2 = 1, E2/3 = 2, etc.) was positively associated with cholesterol absorption and the LDL apo B and cholesterol levels and negatively with cholesterol synthesis and FCR for LDL apo B. Effective bile acid and cholesterol synthesis, fecal elimination of cholesterol, removal of LDL apo B, and low cholesterol absorption characterized men with the epsilon 2 allele. Reduction of dietary fat and cholesterol intakes lowered LDL cholesterol levels and cholesterol absorption but increased cholesterol synthesis proportionally to the apo E subscript; the FCR and PR for LDL apo B were significantly increased and decreased, respectively. The decrease in absorption was related to enhanced removal of LDL apo B and synthesis of cholesterol. During the modified diet, cholesterol metabolism was poorly related to LDL, apo E phenotypes, and LDL apo B kinetics. A positive correlation of cholesterol absorption with dietary fat intake in combined studies suggests that a dietary fat reduction-associated decrease in LDL cholesterol is at least partly caused by reduced cholesterol absorption.(ABSTRACT TRUNCATED AT 250 WORDS)