Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions

We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (B?ttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.     

The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid, an endogenous neurotoxin. Correlation of effect with structure

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.     

Physicochemical properties of low-density lipoproteins of normal human plasma. Evidence for the occurrence of lipoprotein B in associated and free forms

1. Low-density (d 1.006-1.063g/ml) lipoproteins from normal human plasma were separated by differential preparative ultracentrifugation into six subfractions. Each low-density (LD) lipoprotein subfraction contained lipoprotein B as the major and lipoproteins A and C as the minor lipoprotein families. 2. Three lipoprotein B subfractions (LP-B), LP-B-III (d 1.019-1.030g/ml), LP-B-IV (d 1.030-1.040g/ml) and LP-B-V (d 1.040-1.053g/ml) were prepared from the corresponding LD lipoprotein subfractions by immunoprecipitating small amounts of lipoproteins A and C. 3. Determination of hydrodynamic properties indicated that LD lipoproteins consisted of three molecular segments characterized by a stepwise change in the molecular weight: LDL-I and LDL-II subfractions (d 1.006-1.019g/ml) with an average mol.wt. of 4.75x10(6), LDL-III (d 1.019-1.030g/ml) with a mol.wt. of 3.99x10(6), and LDL-IV, LDL-V and LDL-VI (d 1.030-1.063g/ml) with a mol.wt. of 2.85x10(6). 4. All three lipoprotein B subfractions had an average mol.wt. of 3.16x10(6). 5. The LDL-I and LDL-II subfractions consisted of lipoprotein B and lipoprotein C families which were present in the form of an association complex. This was isolated from serum by immunoprecipitation with antibodies to lipoprotein B. The complex had a mol.wt. of 4.35x10(6). 6. The results indicate a fundamental difference between the LD lipoprotein subfractions with d 1.006-1.019g/ml and those subfractions with d 1.030-1.063g/ml. In the former, lipoprotein B occurs as a part of an association complex, whereas in the latter it occurs as a separate entity.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

A comparison of the composition and catabolism in vitro of porcine very low density lipoprotein subfractions prepared by gel exclusion and heparin-affinity chromatography

A comparison of the subfractions prepared from porcine plasma very low density lipoproteins by gel exclusion and heparin-Sepharose affinity chromatography revealed that the smallest and largest particles had the highest affinity for the glycosaminoglycan and had the highest ratio of apolipoprotein E to apolipoprotein CII. When the rates of triglyceride hydrolysis catalysed by lipoprotein lipase were compared for the subfractions the results were consistent with the view that apolipoprotein E may play a role in facilitating the catabolism of very low density lipoprotein triglyceride in the presence of glycosaminoglycan.     

Chromato-focussing of plasma low density proteins

A few alternatives of the binding of healthy patients plasma low density lipoproteins (LDL) with anion exchanger PBE-94 were revealed. In the first case the main part of LDL did not bind to the gel and the isoelectric points of the minor subfractions were 5.8 and 5.3, and pI 4.1. In the second case about half of lipoproteins did not bind to the gel, and the isoelectric points of subfractions were 5.7 and 5.0; and pI 4.1. In the third case when all lipoproteins bound to the PBE-94, there were much more subfractions and their isoelectric points were 6.2, 5.8, 5.2, 4.9, 4.5 and pI 4.1. All LDL of the patients with ischemic heart disease bound to anion exchanger, and the part of subfraction with pI 4.1 was three or five times as great as the one of the healthy person. Increasing of the LDL subfraction with pI 4.1 was observed at prolonged keeping of the LDL obtained from the healthy person plasma. LDL isoelectric point distribution of the persons with carcinoma uterine cervix did not differ from the one of the healthy persons. Acetylation and hexanol modification resulted in the isoelectric point shift from 5.7 to 4.6 and to 4.3 in the case of LDL subfraction to be obtained preparatively using the chromatofocusing.     

Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits.

Feeding rabbits 500 mg of cholesterol daily for 4 to 15 days greatly increased the concentration of esterified cholesterol in lipoproteins of d less than 1.006 g/ml. The origin of hypercholesterolemic very low density lipoproteins was investigated by monitoring the degradation of labeled lymph chyomicrons administered to normal and cholesterol-fed rabbits. Chylomicrons were labeled in vivo by feeding either 1) [3H]cholesterol and [14C]oleic acid or 2) [14C]cholesterol and [3H]retinyl acetate. After intravenous injection of labeled chylomicrons to recipient rabbits, [14C]triglyceride hydrolysis was equally rapid in normal and cholesterol-fed animals. Normal rabbits rapidly removed from plasma both labeled cholesteryl and retinyl esters, whereas cholesterol-fed rabbits retained nearly 50% of doubly labeled remnants in plasma 25 min after chylomicron injection. Ultracentrifugal separation of plasma into subfractions of very low density lipoproteins showed that chylomicron remnants in cholesterol-fed animals are found among all subclasses of very low density lipoproteins. Analysis of cholesteryl ester specific activity-time curves for the very low density lipoproteins subfraction from hypercholesterolemic plasma showed that nearly all esterified cholesterol in large very low density lipoproteins and approximately 30% of esterified cholesterol in small very low density lipoproteins was derived from chylomicron degradation. Apparently, nearly two-thirds of the esterified cholesterol in total very low density lipoproteins from moderately hypercholesterolemic rabbits is of dietary origin.     

Separation and characterization of plasma lipoproteins of rhesus monkeys (Macaca mulatta).

A group of 14 adult male rhesus monkeys was maintained on a low cholesterol-high fat diet. Periodically, animals were fasted and blood samples were taken for characterization of the plasma lipoproteins. Complete separation of individual plasma lipoprotein classes was not achieved by traditional sequential ultracentrifugation techniques. Rather, initial separation of lipoprotein classes according to size was effected and density centrifugation was used subsequently for further separation. At least six lipoprotein fractions were identified, each of which was unique as defined by the properties of size, density (d), and electrophoretic mobility. These lipoprotein fractions were characterized by determination of chemical compositions and apoprotein patterns. The lipoproteins present in highest concentration in these monkeys were designated as region IV lipoproteins. This fraction had alpha-migration on agarose electrophoresis, 1.063 < d < 1.225, and the size, composition, and apoprotein pattern characteristic of HDL. No fewer than three fractions were identified with densities that overlapped the 1.019 < d < 1.063 range. Of these, the fraction designated as region III lipoproteins was present in highest concentration, had beta-migration by agarose electrophoresis, a predominant B apoprotein, and a chemical composition and size characteristic of LDL. Two larger subfractions, identified as region II lipoproteins, were separated from each other at a density of 1.050 g/ml. Agarose electrophoresis showed that the fraction with d < 1.050 had a migration intermediate between beta and pre-beta. The chemical composition and apoprotein pattern were consistent with the possibility that these lipoproteins were remnants of VLDL catabolism. The fraction with d > 1.050, had pre-beta mobility and a size and composition similar to the Lp(a) lipoprotein in plasma of human beings. At least two VLDL subfractions, identified as region I and IIa lipoproteins, were found although both were present in very low concentrations. Region I lipoproteins were larger and contained relatively more cholesteryl ester and more of the apoproteins that migrated with the mobility of apo-B and arg-rich apoprotein in SDS-polyacrylamide gel electrophoresis. Some of the region I lipoproteins were beta-migrating by agarose electrophoresis. These results suggested the possibility that a beta-migrating VLDL was present in these normal animals.     

Anion-exchange high-performance liquid chromatography assays of plasma lipoproteins and modified low-density lipoproteins using a ProtEx-DEAE column

Previously [Anal. Biochem., 232 (1995) 163–171], we reported a high-performance liquid chromatography (HPLC) assay method for human plasma lipoproteins using a diethylaminoethyl (DEAE)-glucomannan column, which is not commercially available. In this study, HPLC assay methods for lipoproteins in plasma samples of human and experimental animals, and modified low-density lipoproteins (LDLs) of rabbits have been developed using a commercially available anion-exchange ProtEx-DEAE column. For the assays of plasma lipoproteins, the method includes complete separation of high-density lipoproteins, LDLs and very low-density lipoproteins within 20 min using stepwise elution, and determination by post-column reaction with an enzymatic cholesterol reagent as the total cholesterol (TC) level. Similarly, mild oxidative and artificially oxidised LDLs were separated into their subfractions using stepwise elution, and determined based on the TC level. The methods using the DEAE-glucomannan and ProtEx-DEAE columns were cross-validated. There was an excellent correlation between the two methods. The obtained results reveal that the anion-exchange HPLC method using the ProtEx-DEAE column could be useful for the assays of plasma lipoproteins and modified LDLs.     

Thermal behavior of cores of human serum triglyceride-rich lipoproteins: a study of induced circular dichroism of beta-carotene

Induced circular dichroism (CD) of beta-carotene has been used to study the physical state in the cores of three classes of triglyceride-rich lipoproteins from human serum: intermediate-density lipoproteins (IDL) (1.006 less than d less than 1.019 g/mL) and subfractions of the d less than 1.006 g/mL lipoproteins of beta and pre-beta electrophoretic mobility. Effects on the physical state in the cores attributable to the ratio of triglycerides to cholesteryl esters and particle diameters were assessed by comparing the temperature-dependent CD spectra of beta-carotene with those of low-density lipoproteins (LDL). Lipoproteins were prepared from serum by sequential ultracentrifugation after the donors were given supplemental dietary beta-carotene (60 mg/day) for 2 weeks. The beta- and pre-beta-migrating d less than 1.006 g/mL lipoproteins were separated by starch block electrophoresis and were then individually separated into subfractions by agarose gel filtration chromatography. Between 7 and 30 degrees C, four subfractions of the beta-migrating d less than 1.006 g/mL lipoproteins and IDL exhibited reversible, temperature-dependent induced CD of beta-carotene, with contours similar to those of LDL but with smaller magnitudes and much broader transitions of the CD bands than those of LDL. In contrast, subfractions of the pre-beta-migrating d less than 1.006 g/mL lipoproteins showed no detectable induced CD of beta-carotene. These results show that the cores of triglyceride-rich lipoproteins can exist in some ordered state between 7 and 30 degrees C if they have a relatively low ratio of triglycerides to cholesteryl esters (mass ratio less than 1.6) and relatively small particle diameter (less than 60 nm).     

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (～85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained ～18% of the activity) and the d>1.21 g/ml fraction (which contained ～32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039–1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.     

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.     

Effects of reconstituted HDL on charge-based LDL subfractions as characterized by capillary isotachophoresis

Modified LDL in human plasma including small, dense LDL (sdLDL) and oxidized LDL carries a more negative charge than unmodified LDL and is atherogenic. We examined the effects of apolipoprotein A-I (apoA-I)/POPC discs on charge-based LDL subfractions as determined by capillary isotachophoresis (cITP). Three normal healthy subjects and seven patients with metabolic disorders were included in the study. LDL in human plasma was separated into two major subfractions, fast- and slow-migrating LDL (fLDL and sLDL), by cITP. Normal LDL was characterized by low fLDL, and mildly oxidized LDL in vitro and mildly modified LDL in human plasma were characterized by increased fLDL. Moderately oxidized LDL in vitro and moderately modified LDL in a patient with hypertriglyceridemia and HDL deficiency were characterized by both increased fLDL and a new LDL subfraction with a faster mobility than fLDL [very-fast-migrating LDL as determined by cITP (vfLDL)]. cITP LDL subfractions with faster electrophoretic mobility (fLDL vs. sLDL, vfLDL vs. fLDL) were associated with an increased content of sdLDL. Incubation of a plasma fraction with d>1.019 g/ml (depleted of triglyceride-rich lipoproteins) in the presence of apoA-I/POPC discs at 37 degrees C greatly decreased vfLDL and fLDL but increased sLDL. Incubation of whole plasma from patients with an altered distribution of cITP LDL subfractions in the presence of apoA-I/POPC discs also greatly decreased fLDL but increased sLDL. ApoA-I/POPC discs decreased the cITP fLDL level, the free cholesterol concentration, and platelet-activating factor acetylhydrolase activity in the sdLDL subclasses (d=1.040-1.063 g/ml) and increased the size of LDL. ApoA-I/POPC discs reduced charge-modified LDL in human plasma by remodeling cITP fLDL into sLDL subfractions.     

Effect of ethanol on lipoprotein metabolism in human and animal blood

The amount and composition of human and animal blood plasma lipoproteins have been studied as affected by low temperature and high doses of ethanol. Results are presented. A conclusion about antiatherogenic properties of low doses of ethanol based on the data of its effect on the system of blood lipoproteins cannot be considered completely proved. Reliable interpretation of the results of the ethanol effect on lipoproteins and atherogenesis processes needs the further study of mechanisms of the ethanol effect on the exchange and modification of the basic classes of lipoproteins and their subfractions, many their peculiarities being not studied yet.     

Physico-chemical and functional characteristics of a subfraction of low-density lipoproteins isolated by ion-exchange column chromatography

Fractionation of human blood plasma low density lipoproteins (LDL) was performed by ion-exchange chromatography, using a linear NaCl gradient. It was shown that the binding of LDL subfractions eluted with a low ionic strength buffer (i.e., containing the particles with a lower negative charge) to B, E-receptors of fibroblasts was more effective than that of subfractions eluted with a high ionic strength buffer (i.e., containing the particles with a higher negative charge). The LDL particles with a lower negative charge had lower values of flotation coefficients (according to analytical ultracentrifugation data), smaller dimensions (according to gradient gel electrophoresis data) and a lower phospholipid/protein ratio (w/w). The experimental results suggest that LDL subfractions having different electrical parameters of the particle surface also differ in other physicochemical properties and seem to play a different role in atherogenesis.     

Immunoaffinity isolation of apolipoprotein E-containing lipoproteins

Discrete apolipoprotein E-containing lipoproteins can be identified when EDTA plasma is fractionated on columns of 4% agarose. The present study has demonstrated, by physical and metabolic criteria, that these apolipoprotein E-containing lipoprotein subclasses may be further isolated by immunoaffinity chromatography. Whole plasma was first bound to an anti-apolipoprotein E immunoadsorbent prior to gel filtration on 4% agarose. After elution from the affinity column and dialysis, the bound fraction was chromatographed on 4% agarose. Discrete subfractions of apolipoprotein E could be demonstrated within elution volumes similar to those observed in the original plasma. When whole plasma was first submitted to gel filtration and the apolipoprotein E-containing lipoproteins of either intermediate- or of high-density lipoprotein (HDL) size were subsequently bound to anti-apolipoprotein E columns, the bound eluted fractions maintained their size and physical properties as shown by electron microscopy and by rechromatography on columns of 4% agarose. The metabolic integrity of apolipoprotein E-containing very-low-density lipoproteins (VLDL) was examined by coinjection into a cynomolgus monkey of 125I-labeled apolipoprotein E-rich and 131I-labeled apolipoprotein E-deficient human VLDL which had been separated by immunoaffinity chromatography. The plasma specific activity time curves of the apolipoprotein B in VLDL, intermediate-density (IDL) and low-density (LDL) lipoproteins demonstrated rates of decay and precursor-product relationships similar to those obtained after injection of whole labeled VLDL, supporting the metabolic integrity of VLDL isolated by immunoaffinity chromatography.     

Evidence of increased secretion of apolipoprotein B-48-containing lipoproteins in subjects with type 2 diabetes

Patients with type 2 diabetes have high levels of triglyceride-rich lipoproteins (TRLs), including apolipoprotein B-48 (apoB-48)-containing TRLs of intestinal origin, but the mechanism leading to overaccumulation of these lipoproteins remains to be fully elucidated. Therefore, the objective of this study was to examine the in vivo kinetics of TRL apoB-48 and VLDL, intermediate density lipoprotein (IDL), and LDL apoB-100 in type 2 diabetic subjects (n = 11) and nondiabetic controls (n = 13) using a primed-constant infusion of l-[5,5,5-D(3)]leucine for 12 h in the fed state. Diabetic subjects had significantly higher fasting glycemia, higher fasting insulinemia, higher plasma triglyceride, and lower HDL-cholesterol levels than controls. Compared with controls, diabetic subjects had increased TRL apoB-48, VLDL apoB-100, and IDL apoB-100 pool sizes as a result of increased production rates (PRs) and reduced fractional catabolic rates of these lipoprotein subfractions. Furthermore, multiple linear regression analyses revealed that the diabetic/control status was an independent predictor of TRL apoB-48 PR and represented nearly 35% of its variance. These results suggest that the overaccumulation of TRLs seen in patients with type 2 diabetes is attributable to increased PRs of both intestinally derived apoB-48-containing lipoproteins and TRL apoB-100 of hepatic origin and to decreased catabolism of these subfractions.     

Determination of the fraction composition of blood lipoproteins by the small-angle X-ray scattering technique.

A new method for analyzing the fraction composition of blood lipoproteins (LP) was developed based on the small-angle X-ray scattering (SAXS) technique. The method allows quantitative determination of the contents of basic LP fractions (high-density LP, low-density LP, very low-density LP and their subfractions) in the blood plasma or serum. The results of LP analysis by the new method were compared with electron microscopy, ultracentrifugation and gel electrophoresis data. The results obtained by SAXS correlated with those obtained by traditional methods. The new method for the determination of the LP fraction composition in the blood is rapid (1-1.5 h), uses only one reagent (e.g., sucrose) and features a high accuracy and resolution up to LP subfractions. A total of 0.05 ml of the blood plasma or serum is required for an assay. The assays can be carried out in purified preparations or in the blood plasma or serum. The method developed can be used in clinical practice for diagnostics and in scientific research.     

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.