Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Quantification of apoC-II and apoC-III of human very low density lipoproteins by analytical isoelectric focusing.

ApoC-II and apoC-III of human very low density lipoproteins (VLDL) have been quantified by analytical isoelectric focusing (IEF) between pH 4 and 6 in polyacrylamide gels containing 8 M urea. The isoelectric point of apoC-III0 is pH 4.93; apoC-II, pH 4.78; apoC-III1, pH 4.72, and apoC-III2, pH 4.54. ApoC-I is not found in the pH range between pH 4 and 6. Two minor peptides, apoC-IV and apoC-V, with isoelectric points of pH 4.61 and 4.44, respectively, are apoproteins not previously identified. The sensitivity (5--40 microgram) and reproducibility (+/- 8%) of this method allow quantitative analysis of apoC-II and apoC-III distribution in VLDL.     

Isolation and partial characterization of an arginine-rich apolipoprotein from human plasma very-low-density lipoproteins: apolipoprotein E.

A water-insoluble apoprotein was isolated from apo-VLDL by column chromatography on Sephadex G-200 in sodium dodecylsulfate followed by preparative polyacrylamide gel electrophoresis in a discontinous sodium dodecylsulfate system, or by preparative electrophoresis alone. The protein was similar in amino acid composition to the "arginine-rich protein" reported by Shore and Shore. It represented about 10% of the total protein mass of VLDL. The apoprotein showed one single band with an apparent Mr of 39000 in sodium dodecylsulfate gel electrophoresis, and was homogeneous in gel electrophoresis at pH 8.9 In 8M urea. Immunochemical studies also showed homogeneity of this protein, and antisera prepared against it did not react with any other of the well known apolipoproteins, but did react with VLDL and apo-VLDL preparations. Analytical isoelectric focusing in 8M urea resulted in a heterogeneous banding pattern showing three major polypeptides with pI values of 5.5, 5.6 and 5.75. Thus this apolipoprotein clearly differs from the apo-B and apo-C polypeptides of VLDL as well as from apoproteins A and D in its molecular weight, amino acid composition, focusing behavior and immunochemical properties.     

Identification and metabolic characteristics of an apolipoprotein C-II variant isolated from a hypertriglyceridemic subject

The very low density lipoprotein (VLDL) apolipoproteins from a Type IV hypertriglyceridemic Caucasian subject (plasma TG: 645 mg/dl) and his brother (plasma TG: 328 mg/dl) were separated by isoelectric focusing gel electrophoresis (IEF) and found to contain two isoforms of apoC-II, identified by immunoblot. These corresponded to normal apoC-II-1 (isoelectric point: pI 4.88) and a variant isoform (apoC-II-v, pI 4.74). The pI of C-II-v was not altered by neuraminidase treatment, indicating that it was not sialylated. The concentration of total immunoreactive C-II in VLDL was elevated (18 mg/dl vs normal; 5.0 +/- 2 mg/dl) but similar to that in other Type IV subjects. In VLDL, which contained 90% of the plasma immunoreactive apoC-II, the ratio (by IEF) of C-II-1:C-II-v was 2:1, whereas in high density lipoproteins (HDL) the ratio was 1:1. VLDL apoB turnover was measured after the pulse injection of 125I-labeled VLDL. VLDL apoB kinetic parameters for the proband and four Type IV subjects were similar: production rate, 28 mg/kg per day versus 30 mg/kg per day; fractional catabolic rate, 1.62.day-1 versus 1.96.day-1; and pool size, 17 mg/kg versus 18 mg/kg. The decline in VLDL triglyceride (TG) after the infusion of heparin (9,000 IU over 4 h) was also similar to that observed in Type IV subjects. In VLDL, the fractional catabolic rates of apoC-II-1 and C-II-v were similar (C-II-1: 0.31.day-1, C-II-v: 0.29.day-1) whereas in HDL, although similar to each other, the rates were greater than in VLDL (C-II-1: 0.48.day-1, C-II-v: 0.44.day-1). VLDL and HDL from the proband were normal in their ability to activate bovine skim milk lipase, compared to Type IV VLDL and HDL without C-II-v. Purified apoC-II-1 and apoC-II-v activated the milk lipase to a similar extent (at 1 microgram of C-II; C-II-1: 34 units/h, C-II-v: 35 units/h). Thus, apoC-II-v is a newly recognized isoform of apoC-II-1. It remains to be determined whether this mutation plays a role in the genesis of hypertriglyceridemia.     

Changes in the concentration of plasma lipoproteins and apoproteins following the administration of Triton WR 1339 to rats.

Changes in whole plasma and lipoprotien apoprotein concentrations were determined after a single injection of Triton WR 1339 into rats. Concentrations of apoproteins A-I (an activator of lecithin:cholesterol acyl transferase), arginine-rich apoprotein (ARP), and B apoprotein were measured by electroimmunoassay. The content of C-II apoprotein (an activaor of lipoprotein lipase) was estimated by the ability of plasma and lipoprotein fractions to promote hydrolysis of triglyceride in the presence of cow's milk lipase and also by isoelectric focusing on polyacrylamide gels. Apoproteins C-II and A-I were rapidly removed from high density lipoprotein (HDL) after Triton treatment and were recovered in the d 1.21 g/ml infranate fraction. A-I was then totally cleared from the plasma within 10--20 hr after injection. Arginine-rich apoprotein was removed from HDL and also partially cleared from the plasma. The rise in very low density lipoprotein (vldl) apoprotein that followed the removal of apoproteins from HDL was mostly antributed to the B apoprotein, although corresponding smaller increases were observed in VLDL ARP and C apoproteins. The triglyceride:cholesterol, triglyceride:protein, and B:C apoprotein ratios of VLDL more closely resembled nascent rather than plasma VLDL 10 hr after Triton injection. These studies suggest that the detergent may achieve its hyperlipidemic effct by disrupting HDL and thus removing the A-I and C-II proteins from a normal activating environment compirsing VLDL, HDL, and the enzymes. The possible involvement of intact HDL in VLDL catabolism is discussed in relation to other recent reports which also suggest that abnormalities of the VLDL-LDL system may be due to the absence of normal HDL.     

Separation and isolation of human apolipoproteins C-II, C-III0, C-III1, and C-III2 by chromatofocusing on the Fast Protein Liquid Chromatography system

Chromatofocusing, which separates proteins based on differences in isoelectric point, has been used on the Fast Protein Liquid Chromatography (FPLC) system (Pharmacia) to separate the C apolipoproteins from human very low density lipoproteins (VLDL). Using a Mono P column (Pharmacia), a pH gradient between pH 6.2 and pH 4.0 was generated using buffers containing 6 M urea, at a flow rate of 0.5 ml/min. Typically, runs took approximately 45 min. Chromatofocusing of delipidated whole VLDL produced sharp, well-resolved peaks for the C apolipoproteins. However, as determined by analytical isoelectric focusing (IEF), the apolipoprotein E isoforms were not separated from apoC-II, and they contaminated the other apoC species to a variable extent. In addition, apoC-II was not resolved from apoC-III0. Preliminary precipitation of VLDL with acetone prior to delipidation removed both apolipoproteins E and B. Using a start buffer of 25 mM histidine, pH 6.2, and a 1:30 dilution of the polybuffer exchanger (eluting buffer), apoC-II, C-III0, C-III1, and C-III2 were well resolved in run-times of approximately 60 min. The C apoproteins proved to be pure by analytical IEF and immunoassay with monospecific antisera against apoC-II and C-III. Recovery was over 90% of the protein chromatographed. In addition, a variant of apoC-II present in VLDL of a hypertriglyceridemic subject was clearly resolved from the other C apolipoproteins. This technique is superior to conventional methodology in terms of its time saving and high resolution. The application of this technique to the study of C apolipoprotein variants and C apolipoprotein specific radioactivity determinations is possible.     

Protein staining and pH gradient determination on the same gel in isoelectric focusing.

The apparent isoelectric point of a component focused on polyacrylamide gels is normally estimated by extrapolating a pH gradient determined on one gel to another gel which has been stained for protein in order to locate the position of the component (1). The pH gradient is determined by slicing the gel transversely and reading the pH of the eluate after soaking the segments for 1–2 hr in a small amount of degassed water. It is assumed that the gradients in both gels are identical. Alternatively, an antimony microelectrode has been used to measure pH gradients directly in unsectioned gels (2). Similar techniques have been applied to polyacrylamide gel slabs and are reviewed by Vesterberg (3). Righetti and Drysdale (4) have recently reviewed these and other aspects of isoelectric focusing in gels.I report here a very precise method for the determination of a protein “isoelectric point” that can be accomplished with a single gel. The technique is demonstrated with yeast phosphoglycerate kinase and the very low density lipoprotein (VLDL) fraction from human plasma.     

Comparative molecular properties of swine and human very low density lipoproteins-apoproteins E and C

1. By means of 2-dimensional gradient-gel electrophoresis, the very low density lipoproteins (VLDL) apoproteins E and C profiles from human and swine plasma were studied. 2. The molecular properties (isoelectric point and molecular weight) of the VLDL apoproteins and their isoforms were determined and showed many similarities between species. 3. It also appears evident that a previously unrecognized apoprotein (C-III) and several associated isoforms may exist in swine; however, it's mobility on 2-dimensional gradient gels is very similar to Apo C-II.     

Apolipoprotein E3-Leiden. A new variant of human apolipoprotein E associated with familial type III hyperlipoproteinemia

Summary A variant of apolipoprotein E, denoted apo E3-Leiden, has been identified in a 41-year-old male suffering from type III hyperlipoproteinemia with xanthomatosis. Apo E3-Leiden focus in the E3 position. In contrast with normal apo E3, apo E3-Leiden is defective in binding to the low density lipoprotein (LDL) receptor and does not contain cysteine as evaluated by cysteamine treatment of very low density lipoprotein followed by isoelectric focusing and conventional protein staining and by amino acid analysis. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, apo E3-Leiden displays an electrophoretic mobility intermediate to that of normal apo E3 and apo E2 (Arg₁₅₈Cys). The mother and four siblings of the proband also have apo E3-Leiden and hyperlipoproteinemia type III; three of them with xanthomatosis. Two siblings do not show apo E3-Leiden in their VLDL fraction and do not have hyperlipoproteinemia type III. In the VLDL fractions of all affected family members only the presence of apo E3-Leiden could be detected after cysteamine treatment and isoelectric focusing followed by conventional protein staining. However, isoelectric focusing of cysteaminetreated sera followed by immunoblotting, using anti-apo E antiserum as first antiserum, demonstrates the presence of low amounts of normal apo E3 in addition to apo E3-Leiden in serum of the affected family members. These results indicate that all affected family members are heterozygotes E3/E3-Leiden and suggest that in this family type III hyperlipoproteinemia is transmitted as a dominant trait.     

Fast protein chromatofocusing of human very-low-density lipoproteins

Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Chemical and physical properties.

Subfractions of CLDL (VLDL), Sf 100-400; CLDL2, Sf 60--100; VLDL3, Sf 20--60) and LDL (LDL), Sf 12--20; LDL2, Sf 6--12; LDL3, Sf 3--6) were isolated from the plasma of three normal, three type III and four type IV hyperlipoproteinemic subjects. In the type IV group, all VLDL subspecies were of normal composition but were increased in concentration in the order VLDL1 greater than VLDL2 greater than VLDL3. In the same subjects, although LDL2 was lowered and LDL3 increased, the total plasma LDL concentration was normal. All VLDL subfractions were elevated in the type III group, but in this case VLDL3 predominated. These subfractions were enriched in cholesteryl esters and depleted in triglyceride. In the LDL density range there was a shift of mass towards the least dense fraction, LDL1, which was of normal composition. EPR studies of the VLDL and LDL subfractions in a type IV subject demonstrated a decrease in fluidity with increasing density. The major change occurred between VLDL3 and LDL1 and was attributed to a substantial alteration in the cholesteryl ester : triglyceride ratio in the particle. A similar argument was used to explain thction in normal or type IV subjects. Particle diameters, determined by laser light-scattering spectroscopy were in good agreement with the values obtained by electron microscopy. This study provides a baseline for the examination of the relationship between the physical and metabolic properties of VLDL and LDL subfractions in type III and IV hyperlipoproteinemia.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.

Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.     

Very low density lipoprotein. Removal of Apolipoproteins C-II and C-III-1 during lipolysis in vitro.

In this study we have investigated the effects of very low density lipoprotein (VLDL) lipolysis on the removal of radiolabeled apolipoprotein C-II and apolipoprotein C-III-1 from in vitro lipolyzed lipoproteins. Lipolysis was carried out in vitro using lipoprotein lipase purified from bovine milk, and mixtures with or without plasma. Lipoproteins were isolated by ultracentrifugation and by gel filtration. Labeled apo-C-II and apo-C-III-1 distributed among plasma lipoproteins, predominantly VLDL and high density lipoprotein (HDL). Lipolysis induced transfer of apo-C-II and apo-C-III-1 from VLDL to HDL. The transfer was proportional to the extent of triglyceride hydrolysis, and similar for the two apoproteins. The apo-C-II/apo-C-III-1 radioactivity ratio did not change in either VLDL or the fraction of d greater than 1.006 g/ml during the progression of the lipolytic process. Similar observations were recorded while using plasma-devoid lipolytic systems. Gel filtration of incubation mixtures, on 6% agarose, revealed that the removal of labeled apo-C molecules from VLDL is not a consequence of either centrifugation or high salt concentration. These results suggest that there is no preferential removal of apo-C-II or apo-C-III-1 from lipolyzed VLDL particles. They further indicate that the ratio of apo-C-II to apo-C-III-1 does not regulate the extent of lipolysis of different VLDL particles, at least in VLDL isolated from normolipidemic humans.     

Catabolism of very low density lipoproteins in the rabbit. Effect of changing composition and pool size.

To determine the metabolic mechanism of hypercholesterolemia in rabbits produced by feeding cholesterol-rich diets, control and hypercholesterolemic rabbits were injected with I-labelled very low density lipoproteins (VLDL, d 1.006 g/ml) from control and/or hypercholesterolemic donors. Apolipoprotein B in VLDL decayed biphasically. The first phase occurred much more rapid than the second. 95% of the VLDL apolipoprotein B was catabolized via the first phase (t1/2 = 0.55 +/- 0.19 h) in normal rabbit with the immediate appearance of this radioactivity in intermediate density lipoproteins (IDL, d 1.006-1.025 g/ml) and low density lipoproteins (LDL, d 1.025-1.063 g/ml). The apolipoproteins C and E at the same time were transferred to high density lipoproteins where they decayed biphasically. The apolipoprotein B from hypercholesterolemic VLDL in the normal recipient disappeared at a similar rate as from normal VLDL via phase I; however, it was incompletely converted to IDL and LDL. Apolipoprotein B from normal VLDL in cholesterol-fed rabbits disappeared at a normal rate via phase I, but only 82% was catabolized by this phase. Hypercholesterolemic VLDL injected into the hypercholesterolemic recipient was less rapidly catabolized via phase I (T1/2 = 2.5 +/- 0.89 H) and only a small fraction was converted to IDL and LDL.     

Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals.

Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.     

Comparative studies of the physical state of the lipid phase of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoproteins (VLDL) were prepared from the serum of rabbits at various stages of hypercholesterolemia (95--1665 mg cholesterol/100 ml of serum). The most notable chemical change in hypercholesterolemic (hc) VLDL was the greatly increased content of cholesteryl esters and the greatly decreased content of triglycerides, compared to normal (n) VLDL. Structurally, the lipid region of n VLDL possessed a much lower microviscosity than did hc VLDL, when analyzed by fluorescence polarization and pyrene eximer methods. The microviscosity of the redispersed n VLDL lipid extract was considerably greater than the observed in n VLDL; but less than that of hc VLDL. Incorporation of pyrene into the lipid region of n VLDL and hc VLDL allowed assessment of various properties of the surface and hydrocarbon regions of these lipoproteins. Only slight differences were found in the pyrene monomer 3 : 1 fluorescence emission peak ratios, and in the rate constant for quenching of pyrene by O2. However, the quenching rate constant of pyrene by I- and iodoheptane were different for each lipoprotein.     

Cholesterol is required for secretion of very-low-density lipoprotein by rat liver.

To study potential effects of hepatic cholesterol concentration on secretion of very-low-density lipoprotein (VLDL) by the liver, male rats were fed on unsupplemented chow, chow with lovastatin (0.1%), or chow with lovastatin (0.1%) and cholesterol (0.1%) for 1 week. Livers were isolated from these animals and perfused in vitro, with a medium containing [2-14C]acetate, bovine serum albumin and glucose in Krebs-Henseleit buffer, and with an oleate-albumin complex. With lovastatin feeding, the hepatic concentrations of cholesteryl esters and triacylglycerols before perfusion were decreased, although free cholesterol was unchanged. However, hepatic secretion of all the VLDL lipids was decreased dramatically by treatment with lovastatin. Although total secretion of VLDL triacylglycerol, phospholipid, cholesterol and cholesteryl esters was decreased, the decrease in triacylglycerol was greater than that in free cholesterol or cholesteryl esters, resulting in secretion of a VLDL particle enriched in sterols relative to triacylglycerol. In separate studies, the uptake of VLDL by livers from control animals or animals treated with lovastatin was measured. Uptake of VLDL was estimated by disappearance of VLDL labelled with [1-14C]oleate in the triacylglycerol moiety, and was observed to be similar in both groups. During perfusion, triacylglycerol accumulated to a greater extent in livers from lovastatin-fed rats than in control animals. The depressed output of VLDL triacylglycerols and the increase in triacylglycerol in the livers from lovastatin-treated animals was indicative of a limitation in the rate of VLDL secretion. Addition of cholesterol (either free cholesterol or human low-density lipoprotein) to the medium perfusing livers from lovastatin-fed rats, or addition of cholesterol to the diet of lovastatin-fed rats, increased the hepatic concentration of cholesteryl esters and the output of VLDL lipids. The concentration of cholesteryl esters in the liver was correlated with the secretion of VLDL by the liver. These data suggest that cholesterol is an obligate component of the VLDL required for its secretion. It is additionally suggested that cholesteryl esters are in rapid equilibrium with a small pool of free cholesterol which comprises a putative metabolic pool available and necessary for the formation and secretion of the VLDL. Furthermore, the specific radioactivity (d.p.m./mumol) of the secreted VLDL free cholesterol was much greater than that of hepatic free cholesterol, suggesting that the putative hepatic metabolic pool is only a minor fraction of total hepatic free cholesterol.