RESUMEN

Human plasma lipoproteins or human whole plasma have been incubated in vitro with canine hepatic lipase (HL) and bovine milk lipoprotein lipase (LPL) to determine the effects of lipases on the particle size distribution of HDL. Confirming previous reports, HL preferentially hydrolysed high density lipoprotein (HDL) triacylglycerol while LPL hydrolysed predominantly very low density lipoprotein (VLDL) triacylglycerol; however, neither lipase altered HDL particle size unless both VLDL and cholesteryl ester transfer protein (CETP) were present. Under these conditions HL promoted marked reduction in HDL particle size in a process dependent on the concentration of VLDL triacylglycerol while LPL was virtually without effect. When both LPL and HL were included in the same incubation, however, LPL prevented the effects of HL. These results are consistent with a proposition that HL has a direct effect on HDL particle size in a process which is dependent on concurrent lipid transfers between HDL and VLDL and that LPL reduces the effect of HL by reducing the concentration of VLDL triacylglycerol.

Asunto(s)

RESUMEN

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.

Asunto(s)

RESUMEN

We have examined the effect of added apolipoprotein A-I (apoA-I) on the changes in high density lipoprotein (HDL) particle size that occur when human plasma is incubated in vitro. In the absence of added apoA-I, incubation of plasma at 37 degrees C resulted in a dramatic increase in HDL particle size. When these incubations contained an inhibitor of LCAT, an additional population of smaller HDL particles was formed. These changes in particle size were even more pronounced when the incubations were supplemented with an artificial triglyceride emulsion, Intralipid. All these changes in HDL particle size were markedly inhibited when incubations were supplemented with apoA-I. Even when the amount of added apoA-I was as little as 4.5% of the endogenous apolipoprotein there was an obvious inhibition of the changes in HDL particle size. The presence of added apoA-I sufficient to increase the plasma concentration by 18% virtually abolished the changes in HDL particle size. This effect did not relate to an inhibition of cholesterol esterification, nor did it appear to depend on an incorporation of the added apoA-I into the HDL.

Asunto(s)

RESUMEN

Regulation of plasma cholesterol transport is to a large extent a function of factors that regulate plasma cholesterol esterification and the transfers of cholesteryl esters between plasma lipoprotein fractions. Plasma cholesterol esterification is catalysed by the action of lecithin: cholesterol acyltransferase on lipids on the surface of HDL, while the transfers of cholesteryl esters require activity of a specific lipid transfer protein. Esterification of the cholesterol on the surface of HDL generates a concentration gradient down which unesterified cholesterol moves from tissues into the plasma. Once within the plasma and esterified, the newly formed cholesteryl esters are incorporated initially into the core of HDL particles before being redistributed to other classes of lipoproteins. The end result of these processes of esterification and transfer is that most of the cholesterol in human plasma is accommodated within the core of LDL, where its transport is a function of the highly regulated uptake by tissues of intact LDL particles. The capacity of HDL to act as substrates for lecithin: cholesterol acyltransferase varies inversely with HDL particle size. Thus, factors such as the concentration of triglyceride-rich lipoproteins and activities of the lipid transfer protein, hepatic lipase, lipoprotein lipase and the HDL conversion protein, which are known to influence HDL particle size, may also be important as regulators of plasma cholesterol esterification.

Asunto(s)

RESUMEN

Human plasma contains a number of proteins that promote movement of lipids between lipoprotein fractions. One of these proteins, designated lipid transfer protein, is known to promote bidirectional transfers of cholesteryl esters, triglyceride, and phospholipids between all plasma lipoprotein fractions. This report briefly reviews the role of lipid transfer protein in plasma cholesterol transport and in the regulation of the particle size distribution of high-density lipoproteins. Studies are described that show that the small particle size of high-density lipoproteins in human subjects with hypertriglyceridemia is a result of the combined actions of lipid transfer protein and hepatic lipase.

Asunto(s)

RESUMEN

These studies examined the proposition that the small particle size of HDL3 in the plasma of hypertriglyceridemic subjects is the consequence of the sequential actions of lipid transfer protein and hepatic lipase on HDL. Incubation of unmodified total HDL or HDL3 in the presence of hepatic lipase resulted in a depletion of phospholipid, but little change in the size of the particles. On the other hand, HDL3 that had first been depleted of cholesteryl ester and enriched with triglyceride and phospholipid, during prior incubation with Intralipid and a source of lipid transfer protein, were much more susceptible to the action of hepatic lipase. When these modified HDL3 were incubated with hepatic lipase there was a depletion of the triglyceride and phospholipid content and a conversion into much smaller particles the same size as those predominant in hypertriglyceridemic subjects. These very small particles were derived from a population of modified particles that were larger than the original HDL3 and were within the size range of HDL2. It is proposed, therefore, that in the plasma of hypertriglyceridemic subjects there exists a dynamic balance between the formation of enlarged triglyceride-rich HDL and a secondary conversion of these particles by hepatic lipase to form populations of very small HDL.

Asunto(s)

RESUMEN

Subpopulations of lipoproteins within the high density lipoprotein subfraction 3 (HDL3) have been isolated from human plasma and characterized in terms of their chemical composition and particle size. Since HDL3 are the major substrates for the esterification of plasma cholesterol, and thus play a central role in the transport of cholesterol through the plasma, these studies also examined the relative capacities of different HDL3 subpopulations to interact with lecithin: cholesterol acyltransferase. Substrate reactivity of a given preparation of lipoproteins was defined in terms of the Vmax of the cholesterol esterification reaction in incubations containing a fixed amount of human lipoprotein-free plasma as a source of the enzyme. Substrate reactivity was found to correlate inversely with the radius of HDL3 particles. This inverse relationship between particle size and substrate reactivity was independent of the particle content of cholesteryl ester.

Asunto(s)

Asunto(s)

RESUMEN

Polyacrylamide gradient gel electrophoresis has been used to examine the particle size distribution of high density lipoproteins (HDL) in human subjects with a wide range of plasma triglyceride concentrations. In studies of groups of both male and female subjects, it was confirmed that the concentration of HDL cholesterol decreases with increasing plasma triglyceride concentration. The HDL fraction from subjects with elevated concentrations of plasma triglyceride was depleted of cholesteryl ester and enriched in triglyceride. It was also confirmed that the proportion of HDL subfraction 2 (HDL2) declines as the plasma triglyceride increases. A new finding was that there were also significant changes in the size of particles in HDL subfraction 3 (HDL3). At low concentrations of plasma triglyceride the predominant subpopulation of HDL3 comprised particles of mean radius 4.3 nm. As the triglyceride concentration increased, however, there was a progressive appearance of HDL3 particles of radius 3.9 nm; in plasma samples with the highest concentrations of triglyceride there was an almost complete disappearance of the 4.3-nm particles, with the population of 3.9-nm particles now predominant.

Asunto(s)

RESUMEN

The effect of lipid transfers on the structure and composition of high density lipoproteins (HDL) has been studied in vitro in incubations that contained the lipoprotein-free fraction of human plasma as a source of lipid transfer protein. These incubations did not contain lecithin:cholesterol acyltransferase activity and were not supplemented with lipoprotein lipase. Incubations were performed at 37 degrees C for 6 hr in both the presence and absence of either added very low density lipoproteins (VLDL) or the artificial triglyceride emulsion, Intralipid. Incubation in the absence of added VLDL or Intralipid had little or no effect on the HDL. By contrast, incubation in the presence of either VLDL or Intralipid resulted in marked changes in the HDL. The effect of incubation with VLDL was qualitatively similar to that of Intralipid; both resulted in obvious transfers of lipid and changes in the density, particle size, and composition of HDL. Incubation of the plasma fraction of density 1.006-1.21 g/ml, total HDL, or HDL3 with either VLDL or Intralipid resulted in the following: 1) a depletion of the cholesteryl ester and free cholesterol content and an increase in the triglyceride content of both HDL2 and HDL3; 2) a decrease in density and an increase in particle size of the HDL3 to form a population of HDL2-like particles; and 3) the formation of a discrete population of very small lipoproteins with a density greater than that of the parent HDL3. The newly formed lipoproteins had a mean particle radius of 3.7-3.8 nm and consisted mainly of protein, predominantly apolipoprotein A-I and phospholipid.

Asunto(s)

RESUMEN

Previous studies have shown that high concentrations of very low density lipoproteins (VLDL) stimulate the formation of cholesteryl esters in human plasma, possibly by acting as recipients of cholesteryl esters transferred from high density lipoproteins (HDL). To gain further insight into this phenomenon, experiments were performed to determine whether the triacylglycerol emulsion Intralipid, which acts as an artificial recipient of HDL cholesteryl esters, has an effect on cholesterol esterification similar to that of VLDL. Intralipid, in contrast to VLDL, is devoid of apolipoproteins which may stimulate cholesterol esterification. Human plasma, which had previously been depleted of VLDL, was pre-incubated in the presence or absence of Intralipid. After pre-incubation, the Intralipid was removed and rates of cholesterol esterification were measured in subsequent incubations of the Intralipid-depleted fractions. The presence of Intralipid during the pre-incubation had a marked stimulatory effect on cholesterol esterification, comparable to that of VLDL in earlier studies. The pre-incubation with Intralipid also markedly reduced the cholesteryl ester content, and increased the triacylglycerol content, of the HDL. These changes in composition coincided with two changes in the elution profile of HDL after density-gradient ultracentrifugation which were (i) a reduction in the density of particles in the HDL3 subfraction, which virtually disappeared as an identifiable peak, and (ii) the emergence of a discrete population of very dense lipoproteins consisting primarily of protein and phospholipid. Since all these changes were related to redistributions of lipids, the results highlight the importance of lipid transfers in the regulation of plasma cholesterol esterification.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

A recent observation that lecithin: cholesterol acyltransferase (EC 2.3.1.43) interacts with both low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in human plasma is in apparent conflict with an earlier finding that the purified enzyme, while highly reactive with isolated HDL, was only minimally reactive with LDL. There is evidence, however, that lecithin: cholesterol acyltransferase may exist physiologically as a component of a complex with other proteins and that studies with the isolated enzyme may therefore provide misleading results. Consequently, interactions of the enzyme with isolated human lipoproteins have been re-examined in incubations containing lecithin: cholesterol acyltransferase as a component of human lipoprotein-free plasma in which a physiologically active complex of the enzyme with other proteins may have been preserved. In this system there was a ready esterification of the free cholesterol associated with both LDL and HDL-subfraction 3 (HDL3) in reactions that obeyed typical enzyme-saturation kinetics. For a given preparation of lipoprotein-free plasma the Vmax values with LDL and with HDL3 were virtually identical. The apparent Km for free cholesterol associated with HDL3 was 5.6 X 10(-5) M, while for that associated with LDL it was 4.1 X 10(-4) M. This implied that, in terms of free cholesterol concentration, the affinity of HDL3 for lecithin: cholesterol acyltransferase was about 7-times greater than that of LDL. When expressed in terms of lipoprotein particle concentration, however, it was apparent that the affinity of LDL for the enzyme was considerably greater than that of HDL3. When the lipoprotein fractions were equated in terms of lipoprotein surface area, the apparent affinities of the two fractions for the enzyme were found to be comparable.

Asunto(s)

RESUMEN

The role of esterified cholesterol transfers in mediating the known effect of very low density lipoproteins (VLDL) on plasma cholesterol esterification has been examined. Advantage has been taken of the fact that pig plasma, in contrast to human plasma, is deficient in activity of the protein that transfers esterified cholesterol between plasma lipoproteins. The experimental protocol consisted of a pre-incubation in which VLDL-deficient pig plasma or isolated pig lipoproteins were incubated with added VLDL, in the presence or absence of an exogenous source of the esterified cholesterol transfer protein. After pre-incubation, any VLDL that may have been present was removed and rates of cholesterol esterification measured in subsequent incubations of the recovered VLDL-depleted fractions. It was, thus, possible to assess the effect of VLDL and/or transfer protein activity on the capacity of other lipoproteins to act as substrates for cholesterol esterification. In the absence of transfer protein activity, the presence of VLDL in pre-incubations had no effect on the subsequently measured rate of cholesterol esterification. However, when pig plasma was supplemented with human lipoprotein-free plasma (a source of esterified cholesterol transfer protein activity) the presence of VLDL in pre-incubations resulted in both a reduction in the esterified cholesterol content of the re-isolated VLDL-deficient fraction and a markedly enhanced rate of cholesterol esterification in subsequent incubations of this fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

RESUMEN

It has been shown previously that there are two pathways by which the esterified cholesterol formed in human plasma in the reaction catalysed by lecithin: cholesterol acyltransferase may be delivered to very low density lipoproteins (VLDL) and low density lipoproteins (LDL): (a) an indirect pathway in which esterified cholesterol which was incorporated initially into high density lipoproteins (HDL) is transferred subsequently to VLDL and LDL in a process mediated by an esterified cholesterol transfer/exchange protein and (b) a direct pathway in which a small proportion of the esterified cholesterol formed in the lecithin:cholesterol acyltransferase reaction is delivered to VLDL and LDL directly from its site of synthesis via a pathway which bypasses the bulk HDL fraction. These present studies have been designed to examine the incorporation of esterified cholesterol into VLDL relative to that into LDL via each of these two pathways. It has been found that a delivery of esterified cholesterol from HDL to VLDL and LDL via the indirect pathway has a marked preference for VLDL over LDL; equating the concentrations of esterified cholesterol in the two fractions revealed an incorporation into VLDL which was 7-11 times greater than that into LDL. By contrast, delivery via the direct pathway showed a marginal preference for LDL over VLDL.

Asunto(s)

RESUMEN

In vitro incubations of human or pig plasma containing a tracer amount of [3H]cholesterol have been performed to determine which lipoprotein fractions are the immediate recipients of the esterified cholesterol formed in the reaction catalysed by lecithin: cholesterol acyltransferase. In pig plasma, which is deficient in activity of the protein which promotes transfer of esterified cholesterol between different lipoprotein fractions, 87-90% of the lecithin: cholesterol acyltransferase-derived esterified cholesterol was incorporated into the high density lipoprotein (HDL) fraction. In human plasma there was an initial recovery of more than 80% in HDL, although the proportion recovered in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) became progressively greater with increasing duration of incubation, consistent with a transfer from an HDL -esterified cholesterol pool of increasing specific activity. Nevertheless, as in the pig plasma incubations, there was evidence that some 10-15% of the esterified cholesterol formed in the lecithin: cholesterol acyltransferase reaction was incorporated directly into human VLDL and LDL. In quantitative terms, however, it was found that most of the esterified cholesterol delivered to human VLDL and LDL was the result of transfers from HDL rather than as a direct incorporation from its site of synthesis.

Asunto(s)

Asunto(s)

RESUMEN

Exchanges of esterified cholesterol between human high density lipoproteins (HDL) and very low density lipoproteins (VLDL) and between VLDL and low density lipoproteins (LDL) have been fitted to a mathematical model previously developed to describe exchanges between human HDL and LDL. In all cases the fit of the model predicted exchanges to those measured experimentally was extremely good, thus greatly increasing confidence in the validity of the model. The model assumes that esterified cholesterol exchanges are achieved by means of a transfer protein which interacts with lipoprotein particles from which it picks up and deposits esterified cholesterol. The values generated for the model constants indicated that, given equal concentrations of esterified cholesterol in each fraction, the relative probability that the transfer protein will pick up a molecule of esterified cholesterol in HDL vs. VLDL vs. LDL is in the ratio 28.9:4.65:1. According to the model the transfer protein may 'bind' to lipoproteins. The model predicts that, at physiological lipoprotein concentrations, the proportion of transfer protein bound to HDL will be more than double that which is unbound to lipoprotein and that bound to VLDL will be about one tenth that unbound. The model was unable to detect evidence of the transfer protein binding to LDL.

Asunto(s)

RESUMEN

Rates of esterification of plasma cholesterol have been compared in two groups of human subjects with widely differing concentrations of very low density lipoproteins (VLDL). Group 1 consisted of subjects with fasting plasma triacylglycerol concentrations over 2.0 mM and group 2 consisted of subjects with concentrations under 1.6 mM. The rate of esterified cholesterol production in incubations of whole plasma from group 1 subjects was much greater than that from group 2 subjects. A clear difference between the rates of esterification was also evident when either total lipoproteins, total high density lipoproteins (HDL) or the HDL subfraction, HDL3, from the two types of subjects were incubated in the presence of a common source of lecithin:cholesterol acyltransferase. Since these findings appeared to reflect fundamental differences within the HDL3 subfractions, which may have been modified by prior exposure to the high concentrations of VLDL in group 1 subjects, VLDL-deficient plasma and the plasma fraction of d greater than 1.125 g/ml (containing HDL3) from hypotriglyceridaemic subjects were preincubated at 37 degrees C with an excess of added VLDL in the presence of a reversal of the inhibition of lecithin:cholesterol acyltransferase, the capacity of the original fractions to esterify cholesterol had been markedly increased. These studies, therefore, show that the lecithin:cholesterol acyltransferase substrate capacity of particles within the HDL3 subfraction is enhanced by exposure to VLDL and that this enhancement is not dependent on the continued presence of VLDL during the actual esterification reaction.

Asunto(s)

RESUMEN

Different methods of incorporating esterified [3H]cholesterol into human high density lipoproteins (HDL) have been assessed in terms of their influence on the subsequent rate at which esterified [3H]cholesterol was transferred from HDL to other plasma lipoproteins in 37 degrees C incubations containing tracer amounts of the labelled preparations added to aliquots of a common pool of unlabelled human plasma. Two basic methods were used to label the HDL esterified cholesterol: (a) by the action of lecithin: cholesterol acyltransferase in incubations of plasma containing exogenous [3H]cholesterol and (b) by exchange in incubations of plasma containing preparations of prelabelled LDL. The exchange labelling approach was also used to examine the effects of various HDL compositional changes on the subsequent behaviour of its esterified [3H]cholesterol. It was found that the source of the label, per se, whether from the esterification of exogenous [3H]cholesterol or from exchange with labeled LDL, had no influence on the subsequent behaviour of HDL esterified [3H]cholesterol. However, modification of the HDL composition during the labelling process had profound effects. For example, in preparations in which the esterified cholesterol content of the labelled HDL was increased by the action of lecithin: cholesterol acyltransferase, there was a reduced rate of transfer of esterified [3H]cholesterol out of the HDL fraction in subsequent incubations of the labelled HDL with fresh, unlabelled plasma. Conversely, when the esterified cholesterol content of the labelled HDL had been decreased by pre-incubation with Intralipid or very low density lipoproteins (VLDL), the subsequent rate of transfer of esterified [3H]cholesterol out of the HDL fraction was increased markedly. It has been concluded that to obtain a biologically valid, labelled tracer of human HDL esterified cholesterol, the labelling should be achieved with minimal modification to the HDL composition. This means labelling by exchange in incubations in which lecithin: cholesterol acyltransferase is inhibited in plasma from which VLDL has been removed.

Asunto(s)