Biomedical subjects

P E Fielding

Publications and source records attributed to P E Fielding.

At least 37 records · Page 2Linked to original sources

PubMed1993

Quantitation of pre beta-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid.

Both receptor-mediated and diffusional processes have been proposed as mechanisms for the efflux of cellular cholesterol to plasma. The depletion of a minor high-density lipoprotein subfraction (pre beta-1-HDL) from plasma by incubation was associated with a proportional reduction in up to 58% of cholesterol and lecithin efflux from cultured fibroblasts. Pre beta-HDL-dependent efflux was blocked by protease pretreatment of the cells, while residual ("nonspecific") efflux was protease-insensitive. The whole of cholesterol efflux from blood erythrocytes was both pre beta-1-HDL-and protease-independent. These data suggest that two distinct pathways contribute to total efflux from fibroblast monolayers; one of these is directly proportional to plasma pre beta-1-HDL concentration and may involve a cell-surface protein.

Cells, Cultured↗

PubMed1992

Regulation of the concentration of pre beta high-density lipoprotein in normal plasma by cell membranes and lecithin-cholesterol acyltransferase activity.

A minor fraction of plasma high-density lipoprotein (pre beta-1 HDL) has been shown to promote cholesterol efflux from peripheral cell membranes [Castro, G. R., & Fielding, C. J. (1988) Biochemistry 27, 25-29]. When isolated native plasma is incubated at 37 degrees C, this fraction is specifically decreased. On the other hand, the level of plasma pre beta-1 HDL is fully protected in the presence of even very low levels of fibroblasts, vascular smooth muscle cells, or macrophages. Blood cells were completely inactive in maintaining plasma pre beta-1 HDL levels in the absence of peripheral cells, even at the relatively high levels present in whole blood. The loss of pre beta-1 observed in isolated plasma was dependent upon lecithin-cholesterol acyltransferase (LCAT) activity. These data suggest that reverse cholesterol transport catalyzed by pre beta-1 HDL, and subsequent LCAT-mediated cholesterol esterification, is directly dependent upon the interaction between this HDL species and competent peripheral cells.

Cell Membrane↗

PubMed1991

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase.

The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters.

Carrier Proteins↗

PubMed1990

Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma.

The transfer of [3H]cholesterol in low-density lipoprotein (LDL) to different high-density lipoprotein (HDL) species in native human plasma was determined by using nondenaturing two-dimensional electrophoresis. Transfer from LDL had a t1/2 at 37 degrees C of 51 +/- 8 min and an activation energy of 18.0 kCal mol-1. There was unexpected specificity among HDL species as acceptors of LDL-derived labeled cholesterol. The largest fraction of the major alpha-migrating class (HDL2b) was the major initial acceptor of LDL-derived cholesterol. Kinetic analysis indicated a rapid secondary transfer from HDL2b to smaller alpha HDL (particularly HDL3) driven enzymatically by the lecithin-cholesterol acyltransferase reaction. Rates of transfer among alpha HDL were most rapid from the largest alpha HDL fraction (HDL2b), suggesting possible protein-mediated facilitation. Simultaneous measurements of the transport of LDL-derived and cell-derived isotopic cholesterol indicated that the former preferably utilized the alpha HDL pathway, with little label in pre-beta HDL. The same experiments confirmed earlier data [Castro, G.R., & Fielding, C.J. (1988) Biochemistry 27, 25-29] that cell-derived cholesterol is preferentially channeled through pre-beta HDL. We suggest that the functional heterogeneity of HDL demonstrated here includes the ability to independently process cell- and LDL-derived free cholesterol.

Cholesterol↗

PubMed1990

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques.

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.

Cells, Cultured↗

PubMed1989

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding.

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.

Apolipoproteins E↗

PubMed1989

Nascent very low density lipoproteins from rat hepatocytic Golgi fractions are enriched in phosphatidylethanolamine.

The phospholipid composition of nascent very low density lipoproteins (VLDL) of rat hepatocytic Golgi fractions differs greatly from that of plasma VLDL. The phospholipids of nascent VLDL contain about four times more phosphatidylethanolamine (PE) than plasma VLDL, whereas plasma VLDL contain considerably more sphingomyelin. Thus, the ratio of PE to sphingomyelin differs by a factor of about 12 between nascent Golgi VLDL and circulating plasma VLDL. It is evident from these data that the PE/sphingomyelin ratio of VLDL can be used to estimate endosomal contamination of hepatocytic Golgi fractions.

Animals↗

PubMed1989

Cutaneous xanthoma in association with paraproteinemia in the absence of hyperlipidemia.

In the present report we describe a patient with multiple myeloma and long-standing paraproteinemia who developed xanthoma in the absence of an elevation in plasma cholesterol or triglyceride concentrations. Studies demonstrated that our patient's monoclonal IgG antibody interacted with apoprotein B-100. The LDL-antibody complex isolated from our patient did not affect the degradation of LDL by human fibroblasts, indicating that while IgG derived from our patient interacted with LDL it did not alter the metabolism of this lipoprotein by the LDL receptor pathway. Since the LDL receptor pathway is the major route of LDL metabolism, this probably explains why our patient was not hyperlipidemic. In contrast to an absence of effect on the LDL receptor, our patient's LDL-antibody complex stimulated cholesterol esterification within macrophages indicating the uptake and degradation of the LDL-antibody complex. The LDL-antibody complex inhibited the degradation of acetyl LDL by macrophages (scavenger pathway), demonstrating that our patient's LDL-antibody complex was recognized as a modified LDL. Moreover, mixing Ig from our patient with normal LDL also resulted in the normal LDL increasing the esterification of cholesterol by macrophages. One can hypothesize that our patient's monoclonal IgG-LDL complex interacted with the macrophage scavenger receptor, thereby resulting in the occurrence of xanthoma in the absence of hyperlipidemia.

Acetylation↗

PubMed1989

Chronic dietary fat and cholesterol inhibit the normal postprandial stimulation of plasma cholesterol metabolism.

The response of parameters of plasma cholesterol metabolism was studied in baboons adapted either to a low-fat, low-cholesterol diet or a high-fat, high-cholesterol diet. Animals adapted to the low-fat diet responded to a single low-fat or high-fat meal, as do normal humans, by a stimulation of cholesterol transport from blood cells to plasma, a stimulation of esterification of cholesterol, and a stimulation of cholesteryl ester transfer to very low and low density lipoproteins. While fasting rates of esterification and transfer increased as a result of diet-induced hypercholesterolemia, the postprandial response was reversed, so that postprandial metabolism was characterized by a movement of cholesterol from plasma to blood cells, an inhibition of cholesterol esterification, and a net transfer of cholesteryl esters from VLDL and LDL to HDL. These data indicate that the effects of postprandial lipemia on plasma cholesterol metabolism critically depend upon fasting plasma cholesterol levels.

Animals↗

PubMed1988

A change in apolipoprotein B expression is required for the binding of apolipoprotein E to very low density lipoprotein.

Factors affecting the association of apolipoprotein E (apoE) with human plasma very low density lipoprotein (VLDL) were investigated in experiments in which the lipid content of the lipoprotein was modified either by lipid transfer in the absence of lipolysis or through the action of lipoprotein lipase. In both cases, lipoprotein particles initially containing no apoE (VLDL-E), isolated by heparin affinity chromatography, were modified until they had the same lipid composition as native apoE-containing VLDL (VLDL+E) from the same plasma. Transfer-modified lipoproteins, unlike native VLDL+E, did not bind apoE or interact with heparin. In contrast, VLDL-E, whose lipid composition was modified to the same extent by lipase, bound apoE and bound to heparin under the same conditions as native VLDL+E. A structural protein (apolipoprotein B) epitope characteristic of VLDL+E was expressed during lipolysis prior to ApoE or heparin binding. The data suggest that the reaction of apoE with VLDL-E is a two-step reaction. The appearance of apoB is modified during lipolysis, with expression of a major heparin-binding site. The modified VLDL then becomes competent to bind apoE. The lipid composition of VLDL appears not to be a major factor in the ability of VLDL to bind apoE or to bind to heparin.

Animals↗

PubMed1986

An apo-E-free very low density lipoprotein enriched in phosphatidylethanolamine in human plasma.

Normal human plasma contains a fraction of very low density lipoprotein (VLDL) which, unlike most VLDL, contains no apolipoprotein E and, unlike apo-E-containing VLDL, is enriched in phosphatidylethanolamine. This fraction made up 0.28 +/- 0.09 of total VLDL triglyceride. Interconversion of the two isolated VLDL fractions was not detected during incubation (2 h, 37 degrees C) and they may represent the physical forms of apo-B corresponding to distinct metabolic pathways in plasma.

Apolipoproteins B↗

PubMed1986

Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2.

The human hepatoma cell line Hep-G2 has been shown to express the major enzymes of intra- and extracellular cholesterol metabolism. These include lecithin:cholesterol acyltransferase, acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-7 alpha-hydroxylase. Regulatory mechanisms that have been described in other hepatic systems also appear to be active in Hep-G2 cells: perturbations of cholesterol and triglyceride metabolism affected the enzyme activities and the accumulation of specific apolipoproteins in the culture media. The results indicate that studies of Hep-G2 cells may provide useful information for the elucidation of mechanisms of regulation of human hepatocyte cholesterol, lipoprotein, and biliary metabolism.

Apolipoproteins↗

PubMed1986

Distribution of apolipoprotein E in the plasma of insulin-dependent and noninsulin-dependent diabetics and its relation to cholesterol net transport.

Noninsulin-dependent diabetics, whose plasma contained no detectable beta-VLDL (very low density lipoprotein), had a proportion (0.23 +/- 0.04) of plasma apolipoprotein E in the form of an abnormal lipoprotein not recognized by antibodies to apoB-100 from LDL (low density lipoprotein) or apoA-I from HDL (high density lipoprotein). This lipoprotein, abnormally rich in free cholesterol and apoE, had a calculated particle density within the low density lipoprotein range. It competed with LDL at the apoB,E receptor of normal fibroblasts and stimulated cholesteryl ester accumulation in mouse peritoneal macrophages. However, it did not compete with the binding of labeled rabbit beta-VLDL to macrophages. A much lower proportion of apoE (0.04 +/- 0.03) was in this form in the plasma of patients with insulin-dependent diabetes who had a comparable degree of hyperglycemia. The diabetic lipoprotein was absent in normoglycemic control subjects. The net transport of cholesterol from cell membranes to the plasma of noninsulin-dependent diabetics (and to a lesser extent, insulin-dependent diabetics) was inhibited relative to control values, and the magnitude of this inhibition was well correlated with the concentration of the abnormal lipoprotein of diabetes in plasma (r = 0.66 and 0.75, respectively). These findings suggest that diabetic plasma contains an abnormal and novel low density lipoprotein that mediates the abnormal cholesterol transport characteristic of human diabetes mellitus.

Apolipoproteins↗

PubMed1984

Increased free cholesterol in plasma low and very low density lipoproteins in non-insulin-dependent diabetes mellitus: its role in the inhibition of cholesteryl ester transfer.

Recombination of low and very low density lipoproteins (VLDL and LDL) from normal subjects with plasma from patients with non-insulin-dependent diabetes mellitus significantly increased the reduced rate of transfer of cholesteryl ester to these lipoproteins, which is characteristic of diabetic plasma, whereas diabetic VLDL and LDL reduced cholesteryl ester transfer rates in normal plasma. VLDL and LDL from diabetic plasma had an increased ratio of free cholesterol to phospholipid compared to normal, and unlike normal VLDL and LDL spontaneously lost free cholesterol to high density lipoprotein. These data suggest that the block to cholesteryl ester transfer to these lipoproteins in non-insulin-dependent diabetes is mediated by their increased free cholesterol content and may be related to the increased risk of these patients for developing atherosclerosis.

Cholesterol↗

PubMed1984

Lecithin:cholesterol acyltransferase and cholesteryl ester transfer activity from the isolated perfused rabbit liver.

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester (CE) transfer activity accumulate linearly in the recirculated medium of the isolated perfused rabbit liver. The appearance of both activities in the perfusate was blocked by the addition of 10 microM colchicine, indicating that these two proteins are synthesized and secreted by the liver. CE transfer activity catalyzed the net transport of cholesteryl ester from high density lipoprotein (donor) to very low and low density lipoproteins (acceptor), both in the perfusate medium and in whole rabbit blood plasma. The activity of LCAT in the perfusate was dependent on the presence of the major protein of the high density lipoprotein class, apoA-I. The similar properties of LCAT and CE transfer activity in rabbit liver perfusate and plasma, compared to these same activities in human blood, suggest that the rabbit is an appropriate model for the study of the cholesterol transport system in man.

Animals↗

PubMed1983

Cholesterol net transport, esterification, and transfer in human hyperlipidemic plasma.

Cholesterol esterification, cholesteryl ester transfer between lipoproteins, and cholesterol transport between lipoproteins and cultured cells have been measured in the plasma of 22 patients with primary hyperlipidemia and 10 normolipidemic subjects. In hyperbetalipoproteinemia, increase in plasma low density lipoprotein levels was associated with a reduction of cholesteryl ester transfer rates, and with a reversal of the normal direction of sterol transport between fibroblasts and their plasma culture medium. Instead of net transport from cells to medium there was a net uptake of sterol from plasma by the cells, despite a level of plasma lecithin/cholesterol acyltransferase activity that was within the normal range. In dysbetalipoproteinemia, esterification rates were increased above normal levels, but cholesteryl ester transfer was reduced and the direction of sterol transport between the cells and plasma medium was reversed, as in the hyperbetalipoproteinemic group. In hypertriglyceridemia, those subjects with cardiovascular disease showed a metabolic pattern similar to the hyperbetalipoproteinemic group. The subjects in this group without symptoms of cardiovascular disease showed a normal direction of sterol transport, normal or raised rates of cholesteryl ester transfer between lipoproteins, and an increased rate of sterol esterification in plasma that decreased towards normal levels as plasma triglyceride levels decreased. Despite their quite distinct metabolic patterns there was no consistent difference between the two hypertriglyceridemic groups in triglyceride or cholesterol levels, very low density lipoprotein composition, or electrophoretic or isoelectric focussing patterns. All hypertriglyceridemic subjects with documented cardiovascular disease showed reversed cell-plasma sterol transport and all subjects without such disease showed a normal direction of cell-plasma sterol transport. The results of this study indicate major and reproducible abnormalities in plasma cholesterol metabolism in several groups of subjects with genetically distinct hyperlipidemias, who are at risk for atherosclerotic vascular disease. The possible predictive value of sterol metabolic measurements in the analysis of cardiovascular disease is discussed.

Adult↗

PubMed1982

Promotion of sterol efflux and net transport by apolipoprotein E in lecithin:cholesterol acyltransferase deficiency.

In the plasma of 4 subjects homozygous for deficiency of lecithin:cholesterol acyltransferase, the level of many apolipoproteins (apo A-I, apo A-II, apo B, apo D) was greatly relative to normal, while that of apo E is increased 5-fold. The lipoprotein complex containing lecithin:cholesterol acyltransferase with apo A-I and apo D in normal plasma is completely absent. The major part of apo E is unassociated with other apolipoproteins. The apoprotein-dependence of sterol efflux and net transport from human skin fibroblasts into plasma was determined by immunoaffinity chromatography. In normal plasma the major component of efflux of sterol radioactivity from labeled fibroblasts was dependent upon unassociated apo A-I. In LCAT-deficient plasma, apoprotein-dependent efflux was largely a function of unassociated apo E. When fibroblasts were incubated with fibrinogen-free unfractionated LCAT-deficient plasma, there was no spontaneous net transport of sterol either into or from the cells, indicating that efflux and influx rates were in balance. When apo E was removed by affinity chromatography, there was net transport from plasma to cells. These findings suggest a novel metabolic role for apo E in the promotion of sterol transport uncoupled to LCAT-activity.

Apolipoproteins↗

PubMed1982

Cholesterol transport between cells and body fluids. Role of plasma lipoproteins and the plasma cholesterol esterification system.

The manner in which cells retain their sterol content is reviewed. Although most of what is known at the molecular level has been defived from studies in continuous cell culture, the findings appear to be broadly applicable to conditions in vivo. The main impetus to this research has come from the potential direct relevance of cell sterol balance to lipid disorders.

Animals↗