High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

A rapid method for the quantitation of cholesterol in each lipoprotein fraction has been developed which utilizes high-performance aqueous gel permeation chromatography followed by enzymatic reaction using reaction-type high-performance chromatography.Cholesterol in serum lipoproteins eluted from the column could be sensitively and selectively detected by the absorbance at 550 nm following the enzymatic reaction. The sensitivity of the detection for cholesterol measured by A₅₅₀ was compared with that for protein measured by A₂₅₀ using the standard lipoprotein fractions: low-density lipoprotein (LDL) and high-density lipoproteins (HDL₂ and HDL₃). The effects of changing the flow-rate and lengthening the column on the resolution of LDL and HDL were examined. Analyses of serum protein and cholesterol were performed with this method for human and animal subjects.     

Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with perchlorate ion-containing eluent

We have developed a high-performance liquid chromatography (HPLC) method for measurement of cholesterol in the major classes of serum lipoproteins, i.e., HDL, LDL, IDL, VLDL, and chylomicrons. Lipoproteins in serum were separated on a column containing diethylaminoethyl-ligand nonporous polymer-based gel by elution with a step gradient of sodium perchlorate concentration, and detected by post-column reaction with a reagent containing cholesterol esterase and cholesterol oxidase. The within-day assay and between-day assay coefficients of variation for cholesterol concentration in lipoproteins were in the ranges of 0.9-6.4% and 1.1-11.9%, respectively. The correlation coefficients between the values of HDL, LDL, IDL, VLDL, and chylomicron cholesterol measured by the HPLC method and those estimated by an ultracentrifugation method were 0.892, 0.921, 0.840, 0.930, and 0.873, respectively. Values of remnant-like particle cholesterol measured by an immunoseparation technique (Japan Immunoresearch Laboratories, Japan) were significantly correlated with VLDL and chylomicron cholesterol values measured by the HPLC method (r = 0.883 and r = 0.729, respectively).This rapid and accurate HPLC method was successfully applied to the analysis of plasma lipoproteins of patients with hyperlipidemia.     

Characterization of plasma lipoproteins separated and purified by agarose-column chromatography

1. A simple method for isolation of individual human plasma lipoprotein classes is presented. In this technique, lipoproteins are removed from plasma at d1.225 by ultracentrifugation, after which they are separated and purified by agarose-column chromatography. 2. Three major classes are obtained after agarose-column chromatography. Separation between classes is excellent; more than 95% of the lipoproteins eluted from the column are recovered in the form of a purified lipoprotein class. 3. Each lipoprotein class was characterized immunologically, chemically, electrophoretically and by electron microscopy. A comparison of the properties of the column-isolated lipoproteins was made with very-low-density lipoproteins, low-density lipoproteins, and high-density lipoproteins separated by sequential ultracentrifugation at densities of 1.006, 1.063 and 1.21 respectively. 4. By each criterion, peak-I lipoproteins from the agarose column are the same as very-low-density lipoproteins, peak-II lipoproteins are the same as low-density lipoproteins, and peak-III lipoproteins are the same as high-density lipoproteins. Thus the lipoprotein classes isolated by both methods are similar if not identical. 5. The agarose-column separation technique offers the advantage of a two- to three-fold saving in time. In addition, the column-elution pattern serves as a recording of the size distribution of lipoproteins in plasma. 6. The most complete characterization is reported for human plasma lipoproteins. The results with rhesus-monkey and rabbit lipoproteins were identical.     

Simultaneous measurement of phytosterols (campesterol and β-sitosterol) and 7-ketocholesterol in human lipoproteins by capillary column gas chromatography

7-Ketocholesterol (a major cholesterol oxidation product) and phytosterols are important indicators of lipoprotein oxidation and lipoprotein metabolism respectively. We describe a simple, sensitive and reproducible method for the simultaneous measurement of these sterols in human lipoprotein samples by capillary column gas liquid chromatography. The method is suitable for clinical studies as small quantities of lipoprotein are required. Sterols are analysed after extraction from lipoprotein samples obtained by sequential flotation ultracentrifugation. The method involves briefly: extraction from lipoprotein samples using chloroform-methanol, saponification of sterol esters using cold potassium hydroxide, purification and derivatisation to trimethylsilyl ethers using BSTFA and 1% TMCS. Oxidation is prevented by drying under nitrogen and the use of powerful antioxidants. Separation is achieved using a DB-1 capillary column and a two-stage temperature ramp from 180–250°C and detection using FID. The identity of sterols can be 3onfirmed by GC-MS. Phytosterol and 7-ketocholesterol are present at low concentration in all the major lipoproteins. Using [3,4-¹³C]cholesterol and GC-MS we present evidence that cholesterol oxidation does not occur during the processing of lipoproteins using this technique.     

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.     

Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein particle size. For analytical purposes, each scan was divided into 12 fractions, representing 12 particle size ranges. The relationship between the estimated cholesterol concentrations in the summed GGE/densitometric fractions corresponding to very low-density lipoproteins (VLDL) + low-density lipoproteins (LDL) and those corresponding to high-density lipoproteins (HDL) and concentrations measured by the heparin-Mn2+ precipitation/enzymatic procedure was linear over a broad range. However, a systematic overestimation of HDL cholesterol concentration and an underestimation of VLDL + LDL cholesterol concentration was apparent. Therefore, correction factors were developed for adjusting the estimates of VLDL + LDL and HDL cholesterol concentrations obtained by the GGE/densitometric method. This analytical method is rapid, repeatable, economical, and useful for genetic and dietary research in which cholesterol concentrations in multiple particle size ranges of lipoproteins must be measured in large numbers of samples. It also is adaptable to immunoblotting procedures for detecting the distribution of specific apolipoproteins among the size-resolved lipoproteins.     

Automatic gas chromatographic determination of the high-density-lipoprotein cholesterol and total cholesterol in serum

A new analytical method that combines on-line precipitation-filtration, enzymatic hydrolysis, extraction and gas chromatography was developed for the determination of total cholesterol and high-density-lipoprotein cholesterol in human serum. Very-low-density lipoprotein, intermediate-density lipoprotein and low-density lipoprotein are precipitated with sodium phosphotungstate and magnesium chloride; then, the serum is continuously filtered and unprecipitated high-density-lipoprotein cholesterol is enzymatically hydrolyzed and finally determined as cholesterol by gas chromatography. Total cholesterol is also determined by direct introduction of the serum into the proposed system. The proposed method was validated by analyzing a lipid control serum with certified contents of high-density-lipoprotein cholesterol and total cholesterol. The results obtained were consistent with the certified contents.     

Rapid transformation of [3H]cholesteryl ester in rat high-density lipoprotein: in vivo and in vitro studies

[24,25-3H]Cholesteryl ester-labeled rat high-density and low-density lipoproteins were administered to recipient rats. Following death of the rats, a major portion of the radioactivity in administered [3H]cholesteryl ester-high-density lipoprotein rapidly appeared in less dense [3H]cholesteryl ester-lipoproteins and was isolated with the low-density lipoprotein fraction. The specific activity of the esterified cholesterol in the product lipoproteins found with the low-density lipoproteins exceeded that of the precursor high-density lipoproteins. In vitro, the addition of [3H]cholesteryl ester-high-density lipoprotein to plasma resulted in a five- to six-fold increase in radioactivity recovered in the low-density lipoprotein. These results demonstrate that, under a variety of experimental conditions, isolated high-density lipoprotein particles (both in vitro and in vivo) tend to become larger and less dense. Rapid changes in the density of lipoproteins labeled with [3H]cholesteryl ester must be considered when interpreting physiologic studies using this label.     

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.     

Serum lipoproteins of normal and cholesterol-fed rats

The density distribution of lipoproteins in rats fed chow or chow containing 1% cholesterol and 10% olive oil was studied. Lipoprotein fractions were prepared in the ultra-centrifuge between narrow density bands within the density range of 1.006-1.21 and were analyzed by chemical, electrophoretic, and immunological methods. In serum from normal rats there were three major lipoprotein fractions, with densities less than 1.006, 1.030-1.063, and 1.063-1.21. Almost no lipoprotein was found between d 1.006 and 1.030. Most of the low density lipoprotein appeared between a density of 1.04 and 1.05. In the density range 1.05-1.07, small amounts of both low density and high density lipoprotein were found. Feeding a diet high in cholesterol resulted in a marked increase in the concentration of lipoproteins of density less than 1.006, and a new lipoprotein fraction appeared between d 1.006 and 1.030; this fraction contained immunologically demonstrable low density and high density lipoproteins. In addition, there was a decrease in the high density lipoprotein fraction between d 1.070 and 1.21.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Fractionation of human low density lipoprotein by column chromatography.

We have devised a method to fractionate low density lipoprotein (LDL) into subspecies by means of column chromatography. DEAE-agarose columns, 2.6 X 60 cm, were loaded with LDL (25-45 mg LDL protein) and eluted with a 0.045-0.13 M NaCl gradient. The LDL eluted over a volume of 900 ml. Specific portions of the eluted LDL, reapplied to a column identical with the original, reelute at about the same point. Altering the NaCl concentration of the elution fluid changed the elution volume. The cholesterol-protein ratio of the LDL subfractions was progressively lower in fractions eluting at higher NaCl concentrations. These results indicate the LDL is not a homogenous lipoprotein species but consists of subfractions which differ in at least charge and cholesterol content.     

Evaluation of gel chromatography for plasma lipoprotein fractionation

The fractionation of lipoproteins of normal and hyperlipidemic subjects on a column of 2% agarose was compared with ultracentrifugation and paper electrophoresis procedures. The following results were obtained. (a) Plasma lipoproteins were eluted successively from the column in the four overlapping peaks of chylomicrons, very low density lipoproteins, low density lipoproteins, and high density lipoproteins. (b) Very low density lipoproteins and high density lipoproteins (d > 1.063, containing nonlipoprotein proteins) showed continuous progressive changes in lipid composition as these fractions emerged, while low density lipoproteins showed a relatively constant lipid composition. (c) A discontinuous transition of lipid composition was observed when consecutive ultracentrifugal fractions were placed on the column. (d) The "trail" of pre-beta lipoprotein seen on paper electrophoresis was shown to consist of particles whose molecular sizes range between chylomicrons and pre-beta lipoproteins. A reverse relationship was observed between electrophoretic mobilities of "trail" components and their particle size. (e) Gel with an agarose content of 2% seemed to fractionate chylomicrons and very low density lipoproteins more effectively than other lipoprotein classes.     

Isolation and partial characterization of high-density lipoprotein HDL1 from rat plasma by gradient centrifugation.

The lipoproteins isolated from rat plasma by flotation in the density range 1.019-1.063 g/ml were further characterized. Using rate zonal ultracentrifugation, we isolated two lipoproteins in almost equal proportions from this density range. Similar isolations may be accomplished with density gradients in a swinging-bucket rotor. On isopycnic-density-gradient ultracentrifugation one component banded at rho = 1.031 g/ml and the other at rho = 1.054 g/ml. More that 98% of the apoprotein of the lighter component was B protein, and hence this particle is LD (low-density) lipoprotein. Of the apoproteins of the rho = 1.054 g/ml particles, designated lipoprotein HDL1, over 60% was arginine-rich peptide, and the remainder was A-I, A-IV and C peptides. The molecular weight of these lipoproteins determined by agarose column chromatography was 2.36 x 10(6) for LD lipoprotein and 1.30 x 10(6) for lipoprotein HDL1. On electron microscopy the radius of LD lipoprotein was 14.0 nm and that of lipoprotein HDL1 was 10.0 nm, in contrast with molecular radii of 10.4 nm and 8.4 nm respectively determined from the gel-permeation-chromatography data. The lipid and phospholipid composition of both particles was determined. Lipoprotein HDL1 was notable for both the concentration of its esterified cholesterol, which was similar to that of LD lipoprotein, and the low triacylglycerol content, resembling that of HD lipoprotein. The possible origin of lipoprotein HDL1 is discussed.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

Studies on the isolation and partial characterization of apolipoprotein D and lipoprotein D of human plasma.

This report describes further studies on the characterization of apolipoprotein D (ApoD), a recently recognized human plasma apolipoprotein, and presents results on the isolation and distribution of its lipoprotein form, lipoprotein D (LP-D). ApoD, isolated by a procedure combining hydroxylapatite and Sephadex G-100 column chromatography, migrated on 7% polyacrylamide gel as a single band with a mobility intermediate between those of A-II and C-II polypeptides. On double diffusion and immunoelectrophoresis, ApoD reacted only with antiserum to ApoD. It was characterized by the presence of all common amino acids including half-cystine. The amino terminal acid was blocked. Carbohydrate analysis demonstrated that ApoD is a glycoprotein with glucose, mannose, galactose, glucosamine, and sialic acid accounting for 18% of the dry weight of ApoD. The estimated molecular weight of ApoD IS 22 100. ApoD occurs in the serum as a lipoprotein which was isolated from high density lipoproteins3 by two different chromatographic procedures. In the first procedure, high density lipoproteins3 were treated with neuraminidase and chromatographed on concanavlin A. The retained fraction containing LP-D was purified by hydroxylapatite column chromatography. Alternatively, LP-D was isolated by a procedure combining chromatography of high density lipoproteins3 or whole serum on an immunosorber containing antibodies to ApoD, and hydroxylapatite column chromatography. LP-D displayed a single, symmetrical boundary in the analytical ultracentrifuge and a single band on 7% polyacrylamide gel electrophoresis. When injected into rabbits it produced antisera that reacted only with ApoD. On immunoelectrophoresis LP-D had a mobility different from that of lipoprotein A (LP-A). A direct immunological comparison of LP-D and LP-A showed a reaction of nonidentity. LP-D consists of 65-75% protein and 25-35% lipid. The lipid moiety contains cholesterol, cholesterol ester, triglyceride, and phospholipid. The phospholipid. composition is characterized by a relative high content of lysolecithin and sphingomyelin and a relatively low content of lecithin. We have concluded from these studies that ApoD is a unique apolipoprotein that exists in the form of a distinct lipoprotein family with a macromolecular distribution extending from very low density lipoproteins into very high density lipoproteins, but with a maximum concentration in high density lipoproteins3 and a minimum concentration in high density lipoproteins.     

NMR studies of pig low- and high-density serum lipoproteins. Molecular motions and morphology.

1. NMR spectra of porcine high- and low density lipoproteins (d 1.120--1.210 and 1.019--1.070, respectively) and their extracted lipids were obtained as functions of temperature, frequency and solution viscosity, and from solutions to which paramagnetic species had been added. 2. About one-third of the N(CH3)3 groups in low-density lipoproteins are so immobile that they do not give a sharp resonance at any temperature up to 65 degrees C, unless the particles are disrupted with sodium dodecylsulphate. Most of the protein residues also undergo little segmental motion. 3. A marked restriction of motion of acyl chain terminal CH3 groups suggests that chain interdigitation occurs in low-density lipoprotein. Apart from this, there is a general ordering of the lipids without a decrease in the rate of rotation about bonds, suggesting that the protein organizes the lipids by controlling the molecular packing rather than by direct strong interactions. The lipids are more ordered in low-density than in high-density lipoprotein. 4. All phospholipids with mobile N(CH3)3 groups are at the particle surfaces, in patches separated by protein. In low-density lipoprotein the patches are raised proud of the protein, whereas in high-density lipoproteins the protein and lipid polar groups are coplanar. 5. The high-density lipoprotein results are consistent with literature models for the structure. The low-density lipoprotein results suggest a new model, which is basically a trilayer. The centre consists of a monolayer of phospholipid with tightly-packed polar groups in contact with a protein core. The outer monolayer of phospholipid contains the rest (most) of the protein; the central layer contains the neutral lipid (cholesterol esters and triglycerides), interdigitated into both the inner and outer monolayers. Unesterified cholesterol is distributed through all three layers.     

Quantitative detection method of triglycerides in serum lipoproteins and serum-free glycerol by high-performance liquid chromatography

We have developed a simple and reliable method for quantitative detection of triglycerides (TG) in serum lipoproteins and serum-free glycerol (FG) by high-performance liquid chromatography (HPLC). After separation of serum constituents using a new gel-permeation column (TSK gel Lipopropak XL, Tosoh) and a new eluent (TSK eluent LP-2, Tosoh), TG and FG were detected by on-line reaction using a modified reagent which contained glycerol kinase, glycerol-3-phosphate oxidase and lipoprotein lipase. HPLC patterns showed five peaks corresponding to chylomicrons, very-low-density, low-density, high-density lipoproteins and FG. Absolute concentrations of TG in each lipoprotein fraction and serum FG were calculated from the corresponding peak areas using standard FG as a calibrator. Due to its very high sensitivity of peak detection, this method has become desirable for the analyses of lipoproteins of very low concentrations such as in cell culture systems. This technique will contribute to a better understanding of lipoprotein TG and serum FG distribution in human and nonhuman subjects.     

Physicochemical transfer of [3H]cholesterol from plasma lipoproteins to cultured human fibroblasts.

The transfer of free cholesterol from [3H]cholesterol-labelled plasma lipoproteins to cultured human lung fibroblasts was studied in a serum-free medium. The uptake of [3H]cholesterol depended upon time of incubation, concentration of lipoprotein in the medium, and temperature. Modified (reduced and methylated) low-density lipoprotein (LDL), which did not enter the cells by the receptor pathway, gave a somewhat lower transfer rate than unmodified LDL, but if the transfer values for native LDL were corrected for the receptor-mediated uptake of cholesterol the difference was eliminated. The initial rates of transfer of [3H]cholesterol from LDL and high-density lipoprotein (HDL) were of the same order of magnitude (0.67 +/- 0.05 and 0.75 +/- 0.06 nmol of cholesterol/h per mg of cell protein, respectively) while that from very-low-density lipoprotein (VLDL) was much lower (0.23 +/- 0.02 nmol of cholesterol/h per mg) (means +/- S.D., n = 5). The activation energy for transfer of cholesterol from reduced, methylated LDL to fibroblasts was determined to be 57.5 kJ/mol. If albumin was added to the incubation medium the transfer of [3H]cholesterol was enhanced, while that of [14C]dipalmitoyl phosphatidylcholine was decreased compared with the protein-free system. The results demonstrate that, in spite of its low water solubility, free cholesterol can move from lipoproteins to cellular membranes, probably by aqueous diffusion. We propose that physicochemical transfer of free cholesterol may be a significant mechanism for net uptake of the sterol into the artery during atherogenesis.     

Influence of contraceptive pill and menstrual cycle on serum lipids and high-density lipoprotein cholesterol concentrations.

The fluctuations of serum lipid and lipoprotein concentrations within one cycle were studied both in women using and not using oral contraceptives. High-density lipoprotein cholesterol decreased significantly from 1.47 mmol/l (57 mg/100 ml) to 1.30 mmol/l (50 mg/100 ml) during one contraceptive cycle in eight women and rose again to the initial value during the pill-free days. The mean concentration of total cholesterol also fell significantly as a result of the decrease of high-density lipoprotein cholesterol and of a not significant decrease of low-density lipoprotein cholesterol. The mean serum triglyceride concentration did not change significantly. The fluctuations in the concentration of serum lipids and lipoproteins in 10 women not using oral contraceptives were smaller than in the women using oral contraceptives and no significant changes in the concentrations were found during one cycle. Thus, high-density lipoprotein cholesterol concentration decreases during each contraceptive cycle. The time of blood sampling during the cycle is, therefore, of vital importance in interpreting the effect of oral contraceptives on high-density lipoprotein cholesterol. In women not using oral contraceptives blood can be sampled on random days during the cycle.