Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans. Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency

The extent to which lipid and apolipoprotein (apo) concentrations in tissue fluids are determined by those in plasma in normal humans is not known, as all studies to date have been performed on small numbers of subjects, often with dyslipidemia or lymphedema. Therefore, we quantified lipids, apolipoproteins, high density lipoprotein (HDL) lipids, and non-HDL lipids in prenodal leg lymph from 37 fasted ambulant healthy men. Lymph contained almost no triglycerides, but had higher concentrations of free glycerol than plasma. Unesterified cholesterol (UC), cholesteryl ester (CE), phosphatidylcholine (PC), and sphingomyelin (SPM) concentrations in whole lymph were not significantly correlated with those in plasma. HDL lipids, but not non-HDL lipids, were directly related to those in plasma. Lymph HDLs were enriched in UC. However, as the HDL cholesterol/non-HDL cholesterol ratio in lymph exceeded that in plasma, whole lymph nevertheless had a lower UC/CE ratio than plasma. Lymph also had a significantly higher SPM/PC ratio. The lymph/plasma (L/P) ratios of apolipoproteins were as follows: A-IV > A-I and A-II > C-III and E > B. Comparison with the L/P ratios of seven nonlipoprotein proteins suggested that apoA-IV was predominantly lipid free. Concentrations of apolipoproteins A-II, A-IV, C-III, and E in lymph, but not of apolipoproteins A-I or B, were positively correlated with those in plasma. The L/P ratios of apolipoproteins B, C-III, and E in two subjects with lipoprotein lipase (LPL) deficiency, and of apolipoproteins A-I and A-IV in a subject with lecithin:cholesterol acyltransferase (LCAT) deficiency, were low relative to those in normal subjects. Thus, the concentrations of lipids, apolipoproteins, and lipoproteins in human tissue fluid are determined only in part by their concentrations in plasma. Other factors, including the actions of LPL and LCAT, are at least as important.     

Secretion of adipokines by human adipose tissue in vivo: partitioning between capillary and lymphatic transport

Peptides secreted by adipose tissue (adipokines) may enter blood via capillaries or lymph. The relative importance of these pathways for a given adipokine might influence its biological effects. Because this has not been studied in any species, we measured the concentrations of seven adipokines and eight nonsecreted proteins in afferent peripheral lymph and venous plasma from 12 healthy men. Data for nonsecreted proteins were used to derive indices of microvascular permeability, which in conjunction with the molecular radii of the adipokines were used to estimate the amounts leaving the tissue via capillaries. Transport rates via lymph were estimated from the lymph adipokine concentrations and lymph flow rates and total transport (secretion) as the sum of this and capillary transport. Concentrations of nonsecreted proteins were always lower in lymph than in plasma. With the exception of adiponectin, adipokine concentrations were always higher in lymph (P < 0.01). Leptin and MCP-1 were secreted at the highest rates (means: 43 μg/h or 2.7 nmol/h and 32 μg/h or 2.4 nmol/h, respectively). IL-6 and MCP-1 secretion rates varied greatly between subjects. The proportion of an adipokine transported via lymph was directly related to its molecular radius (r(s) = +0.94, P = 0.025, n = 6), increasing from 14 to 100% as the radius increased from 1.18 (IL-8) to 3.24 nm (TNFα). We conclude that the lymph/capillary partitioning of adipokines is a function of molecular size, which may affect both their regional and systemic effects in vivo. This finding may have implications for the physiology of peptides secreted by other tissues.     

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.     

Apolipoprotein B amino acid 3611 substitution from arginine to glutamine creates the Ag (h/i) epitope: the polymorphism is not associated with differences in serum cholesterol and apolipoprotein B levels

Summary A G-to A-DNA sequence change in exon 26 of the human apolipoprotein B (apo B) gene leads to a glutamine substitution for arginine at codon 3611 of the mature apolipoprotein B100 and causes a loss of an MspI site. In 106 Finnish individuals, a complete correspondence exists between this MspI polymorphic site and the Ag (h/i) immunochemical polymorphism. Linkage disequilibrium was found between this MspI polymorphic site and the apo B XbaI and EcoRI variable sites and the Ag (a1/d) and (c/g) epitope pairs; there is apparent linkage equilibrium with the apo B PvuII variable site. Based on three population studies (samples from London, Finland and Italy), no significant association was found between this RFLP and serum cholesterol and apo B levels. These data suggest that the arginine 3611glutamine 3611 substitution has no significant effect on apo B function.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

An In-Silico Model of Lipoprotein Metabolism and Kinetics for the Evaluation of Targets and Biomarkers in the Reverse Cholesterol Transport Pathway

High-density lipoprotein (HDL) is believed to play an important role in lowering cardiovascular disease (CVD) risk by mediating the process of reverse cholesterol transport (RCT). Via RCT, excess cholesterol from peripheral tissues is carried back to the liver and hence should lead to the reduction of atherosclerotic plaques. The recent failures of HDL-cholesterol (HDL-C) raising therapies have initiated a re-examination of the link between CVD risk and the rate of RCT, and have brought into question whether all target modulations that raise HDL-C would be atheroprotective. To help address these issues, a novel in-silico model has been built to incorporate modern concepts of HDL biology, including: the geometric structure of HDL linking the core radius with the number of ApoA-I molecules on it, and the regeneration of lipid-poor ApoA-I from spherical HDL due to remodeling processes. The ODE model has been calibrated using data from the literature and validated by simulating additional experiments not used in the calibration. Using a virtual population, we show that the model provides possible explanations for a number of well-known relationships in cholesterol metabolism, including the epidemiological relationship between HDL-C and CVD risk and the correlations between some HDL-related lipoprotein markers. In particular, the model has been used to explore two HDL-C raising target modulations, Cholesteryl Ester Transfer Protein (CETP) inhibition and ATP-binding cassette transporter member 1 (ABCA1) up-regulation. It predicts that while CETP inhibition would not result in an increased RCT rate, ABCA1 up-regulation should increase both HDL-C and RCT rate. Furthermore, the model predicts the two target modulations result in distinct changes in the lipoprotein measures. Finally, the model also allows for an evaluation of two candidate biomarkers for in-vivo whole-body ABCA1 activity: the absolute concentration and the % lipid-poor ApoA-I. These findings illustrate the potential utility of the model in drug development.     

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.