Search PubMed⌕ Search

Biomedical subjects

A G Lacko

Publications and source records attributed to A G Lacko.

At least 37 records · Page 2Linked to original sources
PubMed1992

Physiologic role and clinical significance of reverse cholesterol transport.

Low levels of high-density lipoproteins have been consistently shown to be a major risk factor for coronary heart disease. However, the precise role of HDL in the prevention or reversal of atherosclerosis (or both) is unknown. It has been proposed that HDL functions jointly with the enzyme lecithin:cholesterol acyltransferase and the cholesteryl ester transfer protein to facilitate the movement of cholesterol from tissues to the liver. This mechanism--referred to as reverse cholesterol transport--has been shown to be an important physiologic mechanism. However, its clinical significance, though intriguing, is unclear. This article reviews recent advances concerning the components of reverse cholesterol transport and evaluates their potential significance in the early diagnosis and treatment of atherosclerosis.

Arteriosclerosis↗
PubMed1991

Age related changes in the lipoprotein substrates for the esterification of plasma cholesterol in rats.

The activity of the enzyme lecithin:cholesterol acyltransferase (LCAT) and the properties of its lipoprotein substrates have been investigated in 6- and 19-month-old Fischer-344 rats. These studies were carried out to determine the nature of the relationship between the observed hypercholesterolemia and the age-related decrease in the fractional rate of lipoprotein cholesterol esterification. The distribution of LCAT activity of plasma fractions was determined following gel chromatography and ultracentrifugation respectively. LCAT activity was found to be associated with the high density lipoprotein (HDL) fraction when rat plasma was passed through a Bio-Gel A-5 M column. Upon density gradient ultracentrifugation for 24 h it was found associated with HDL fraction; d = 1.125-1.21 g/ml. However, following prolonged ultracentrifugation (40 h), the majority of the LCAT activity was displaced into the lipoprotein-free infranatant (d greater than 1.225 g/ml). The dissociation of LCAT from its complex with HDL occurred to a smaller extent in aged rat plasma than in young rat plasma. Substrate specificity studies indicated that HDL was a considerably better substrate for LCAT than very low density lipoproteins (VLDL) in both young and aged rats. In addition, HDL from young rats was a better substrate for LCAT than the HDL from aged rats. Incubation experiments followed by the isolation of lipoproteins and the subsequent analyses of their cholesterol contents revealed that the age-related hypercholesterolemia was mainly due to an increase in the cholesterol carried by lipoprotein fractions d = 1.025 -1.07 g/ml (LDL + HDL1). These and other low density lipoproteins (d less than 1.025 g/ml) were poor substrates for LCAT. However, these lipoproteins could provide free cholesterol for esterification by first transferring it to HDL (d = 1.07-1.21). The HDL isolated from the plasma of aged rats was enriched with apolipoprotein (apo) E and these lipoprotein particles were found to be inferior substrates for LCAT. These data suggest that the decreased fractional rate of esterification observed in aged rats is due to the slower utilization of the HDL lipid substrate pool by the enzyme LCAT as a result of the accumulation of unfavorable substrates (compositionally altered HDL particles) for the LCAT reaction.

Aging↗
PubMed1990

International Symposium on Reverse Cholesterol Transport. Report on a meeting.

The purpose of this symposium was to provide a forum for the reporting of recent findings and the exchange of ideas concerning reverse cholesterol transport, an area of intense interest and some controversy. Data from epidemiological studies have consistently shown that elevated levels of high density lipoproteins (HDL) are an index of increased protection against coronary heart disease. However, the mechanism whereby HDL is involved in the prevention and/or reversal of atherosclerosis is unknown. According to one of the hypotheses, HDL acts as the primary acceptor of unesterified cholesterol from cells and functions jointly with the enzyme lecithin:cholesterol acyltransferase (LCAT) and the cholesteryl ester transfer protein (CETP) to facilitate the movement of cholesterol from peripheral tissues to the plasma and ultimately to the liver. Although this mechanism as originally proposed by Glomset is an essential physiological mechanism, the clinical significance of this hypothesis remains unsubstantiated. Key elements of knowledge are lacking that would allow the linking of cholesterol efflux from cells and tissues with specific events in HDL metabolism, particularly those that are relevant to the prevention and/or reversal of atherosclerosis. Because of the intricate nature of the interaction between the components of reverse cholesterol transport, a conference involving the leading investigators of the field, where extensive discussion of the findings and ideas is allowed, appeared highly desirable. Indeed, from the distance of nearly 4 months, feedback from the participants indicates that the meeting was highly successful and the organizers feel that all the projected goals of the symposium were accomplished.

Biological Transport↗
PubMed1989

Relationship between cholesteryl ester transfer activity and high density lipoprotein composition in hyperlipidemic patients.

Cholesteryl ester transfer from solid-phase bound HDL to endogenous plasma HDL or VLDL/LDL was determined in 50 patients with primary disorders of lipid metabolism and 27 normolipidemic subjects. Transfer to the plasma HDL pool was significantly reduced in familial hypercholesterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia and dysbetalipoproteinemia. Subfractionation of HDL revealed that the lipid transfer to HDL3 was significantly reduced in all patient groups while transfer to HDL2 was increased in those with dysbetalipoproteinemia and familial hypertriglyceridemia. Transfer to LDL and VLDL was increased only in patients with dysbetalipoproteinemia and hypoalphalipoproteinemia. Reduced transfer to HDL occurred in samples with altered HDL composition; particularly where HDL-triglyceride was significantly increased and HDL-cholesteryl esters were reduced. Transfer of cholesteryl ester to HDL3 was significantly decreased in patients with vascular disease. These findings indicate that impaired interaction of cholesteryl ester transfer protein with the HDL3 pool may contribute to the risk of coronary heart disease in patients with specific plasma lipid abnormalities.

Cholesterol Esters↗
PubMed1989

Studies on the structure of lecithin:cholesterol acyltransferase (LACT)--comparisons of the active site region and secondary structure of the human and the porcine enzymes.

1. Chemical modification of essential serine, histidine and cysteine residues of porcine LCAT were accompanied by loss of enzymatic activity. 2. Modification of cysteine with DTNB inactivated the enzyme which could not be reactivated by KCN suggesting direct involvement of the cysteine residue(s) in catalysis. 3. About half of the primary structure of the porcine enzyme was determined. 4. Respective regions of the human and porcine LCAT are highly homologous; especially, the amino-terminus and the region surrounding the DFP-labeled serine residues. 5. The observed primary structure differences represent amino acid substitutions that are projected to induce significant changes in secondary structure.

Amino Acid Sequence↗
PubMed1988

Lecithin:cholesterol acyltransferase in familial HDL deficiency (Tangier disease).

These studies were performed to investigate the relationship between the enzyme lecithin:cholesterol acyltransferase and plasma lipoproteins in Tangier disease, a condition characterized by a virtual absence of high-density lipoproteins (HDLs) and an accumulation of cholesteryl esters in peripheral tissues. Apolipoprotein A-I was nearly absent from the patient's plasma (1% of the normal levels were found). However, apolipoprotein A-I purified from the plasma of the Tangier disease patient, was found to activate both purified and the plasma enzyme. At lower apolipoprotein concentrations (up to 25 micrograms/ml), about twice the amount of Tangier apolipoprotein A-I was required to achieve a certain level of lecithin:cholesterol acyltransferase activity as compared with the activating potential of the normal apolipoprotein. Gel chromatography studies revealed that as in normal plasma, lecithin:cholesterol acyltransferase in Tangier plasma was associated with an HDL-size lipoprotein fraction. However, unlike in normal plasma, this lipoprotein complex (containing lecithin:cholesterol acyltransferase) was not removed from Tangier plasma by immunoaffinity chromatography utilizing immobilized anti-apolipoprotein A-I antibodies. Plasma incubation studies showed that free cholesterol was primarily supplied by LDL in normal plasma, whereas both LDL and VLDL donated the free cholesterol for lecithin:cholesterol acyltransferase reaction in Tangier plasma. The majority of the cholesteryl esters, generated during the incubation experiments, were transferred back to LDL in normal plasma, whereas in Tangier plasma both LDL and VLDL served as cholesteryl ester acceptors. The cholesteryl ester transfer from HDL to lower-density lipoproteins was lower in Tangier plasma as compared to this process in a normal control, suggesting that a minimal cholesteryl ester core may be required for the stability of HDL.

Apolipoprotein A-I↗
PubMed1988

Effect of dietary protein on cholesterol homeostasis in diabetic rats.

Normal and streptozotocin (STZ)-diabetic rats were studied in order to examine the effects of altering the type of dietary protein on cholesterol homeostasis. Rats were fed a non-purified or a purified diet containing either casein or soybean protein. The results obtained on the specific aspects of lipid metabolism were remarkably similar in control rats fed the non-purified (Purina Lab Chow) diet or the purified diet with the soybean protein. However, most of the findings obtained with the above two groups were different from those obtained with rats fed the purified diet containing casein. In the latter group, plasma cholesterol was elevated following a 15-day feeding period as compared to the other two dietary groups. The excess plasma cholesterol in the casein-fed group was found in two lipoprotein fractions with densities of 1.023-1.045 g/ml and 1.045-1.086 g/ml, respectively. The latter lipoprotein fraction was also enriched with apolipoprotein E. The casein-fed animals also showed a lower fractional rate of plasma cholesterol esterification and an abnormal accumulation of cholesterol in the body despite inhibition of cholesterol synthesis in the liver and in the intestines. Twelve to 15 days after the induction of diabetes, plasma cholesterol increased to a similar extent in the rats on all three diets. However, the distribution of cholesterol among the lipoprotein fractions was markedly different. The percentage of cholesterol in fractions of d less than 1.086 g/ml was increased while that carried in the fraction of d 1.086-1.161 g/ml decreased in the rats fed the nonpurified diet and the casein diet. In contrast, there was no change in the distribution of lipoprotein cholesterol between the diabetic and the control rats fed the soybean protein diet. The hepatic synthesis of cholesterol was unaltered in diabetic rats fed the nonpurified diet and the purified diet with soybean protein, but was increased 2.4-fold in diabetic rats fed casein. Intestinal cholesterol synthesis was increased in all three dietary groups. The increase was highest in the rats fed casein and lowest in rats fed soybean protein. The rate of sterol synthesis in the kidneys was not significantly affected by the diet or diabetes. In all three dietary groups diabetes led to an abnormal accumulation of cholesterol in the body. This accumulation was highest in the casein-fed rats and lowest in those fed the soybean protein diet. The cholesterol content of the kidneys was markedly increased by dietary casein.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗
PubMed1987

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency.

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.

Cholesterol↗
PubMed1987

The catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides.

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with [3H]-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Amino Acid Sequence↗
PubMed1986

Purification of biologically active apolipoproteins by chromatofocussing.

Chromatofocussing has been used to isolate homogeneous apolipoproteins (apo) from human very-low-density lipoproteins and high-density lipoproteins with protein recovery of 70%. The inclusion of sulfhydryl-reducing agent (dithiothreitol) was required during solubilization of the lipoproteins (following delipidation) to achieve reproducible elution profiles. Removal of polyvalent buffers from apoproteins was rapidly accomplished on small columns of hydroxylapatite. The biological activity of purified apo AI and apo CII was confirmed by assessment of their ability to activate lecithin:cholesterol acyltransferase or lipoprotein lipase, respectively. Functional properties of isolated apo E were assessed by in vitro interaction with the low-density lipoprotein receptor expressed by cultured fibroblasts. Apolipoproteins purified by this rapid procedure exhibit identical physical, chemical and biological properties to those purified by other, more tedious techniques.

Animals↗
PubMed1986

Isolation and characterization of lecithin-cholesterol acyltransferase from hog plasma.

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified from hog plasma by a highly efficient procedure. The final enzyme preparation was purified 30,000-fold over the starting material and was homogeneous as indicated by polyacrylamide gel electrophoreses in the presence of both SDS and urea. The purified hog lecithin-cholesterol acyltransferase had an apparent molecular weight of 66 000 on SDS-polyacrylamide gel electrophoresis and HPLC and was found to contain about 21.4% (w/w) carbohydrate-hexose, 11.3%; hexosamine, 1.9%; sialic acid, 8.2%. The amino acid composition analysis showed that hog lecithin-cholesterol acyltransferase contains four half cystines per mol; two cysteines were titrated at neutral pH with 5,5'-dithiobis(2-nitrobenzoic acid). Nearly all the phenolic groups were unavailable to the solvent at neutral pH, while they become exposed at around pH 11. Hog lecithin-cholesterol acyltransferase was found to be associated with HDL in the plasma and it prefers HDL as a substrate. The physicochemical properties of hog lecithin-cholesterol acyltransferase were generally similar to those of the human and the rat enzyme.

Amino Acids↗
PubMed1986

Age-related changes in the rate of esterification of plasma cholesterol in Fischer-344 rats.

Plasma cholesterol and triglyceride levels and selected molecular species of plasma cholesteryl esters and triglycerides were determined in 6-, 12-, 15-, 18-, 21-, and 24-month-old Fischer-344 rats. Lecithin:cholesterol acyltransferase (LCAT) activity was also determined using two independent methods utilizing endogenous and exogenous substrates. Plasma cholesterol levels increased up to 18 months of age and then plateaued. Of the plasma triglyceride molecular species investigated (C50, C52, C54 and C56), only the levels of C52 increased linearly with age. The concentration of other triglyceride molecular species did not change with age. The fractional rate of plasma cholesterol esterification showed a decreasing trend with age, whereas, the net cholesterol esterification rate showed a gradual age related increase. However, this latter parameter remained unchanged with age when the data were normalized for body weight. The cholesterol esterification rates measured using an exogenous substrate (estimating LCAT enzyme levels) showed essentially no change with age. These data indicate that changes in the levels and/or composition of lipoprotein substrate(s) for LCAT are likely causes of the observed age-related changes in the fractional rate of plasma cholesterol esterification. The net esterification rate of plasma cholesterol was significantly correlated with the plasma triglyceride levels when the animals for all age groups were treated as one experimental group.

Aging↗
PubMed1985

Age-related changes in plasma lipid levels and tissue lipoprotein lipase activities of Fischer-344 rats.

Fischer-344 rats of 6, 12, 18, 21 and 24 mth of age were investigated. The data were collected from groups of rats ranging from 5-10 rats per age group. Modest age-related increases were noted in body weight, plasma triglycerides, total cholesterol and free (unesterified) cholesterol. Much more significant changes were noted in tissue lipoprotein lipase activities where the adipose tissue lipases declined sharply (r = -0.90, P less than 0.0001; r = -0.86, P less than 0.0001) and the muscle tissue lipases decreased moderately (r = -0.86, P less than 0.0001; r = -0.58, P less than 0.0001) with age. These observed trends suggest that enzymatic parameters, specifically tissue lipoprotein lipase activities, may be considerably more accurate indicators of age-related physiological changes than levels of plasma lipids such as cholesterol and triglyceride.

Adipose Tissue↗
PubMed1985

Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma.

High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components that the control as indicated by acrylamide gel electrophoresis in the presence of urea. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.

Animals↗
PubMed1985

Detection of lecithin: cholesterol acyltransferase (LCAT) in a human hepatoma cell line.

A human hepatoma cell line (HepG-2) was probed for the presence of lecithin: cholesterol acyltransferase (LCAT) using an antiserum to human plasma LCAT. Double immunodiffusion analysis using antiserum to human plasma LCAT revealed a single precipitin line in the sonicated cell homogenate. This precipitin line showed a reaction of identity with highly purified plasma LCAT. The presence of LCAT within the hepatoma cells was also confirmed by an immunofluorescence test. In contrast, the cell culture supernate showed a weak and inconsistent precipitin line. These data suggest that HepG-2 cells synthesize LCAT but secretion of the enzyme by these cells into the culture medium may be partially or totally impaired.

Carcinoma, Hepatocellular↗
PubMed1984

Effects of detergents on the lecithin:cholesterol acyltransferase reaction.

This paper describes the effect of an ionic (sodium dodecyl sulfate; SDS) and a nonionic detergent (Triton X-100) on the substrate and enzyme components of the lecithin: cholesterol acyltransferase (LCAT) reaction. When the enzyme sources (purified or partially purified) or the respective substrates [high-density lipoproteins (HDL) or proteoliposomes] were preincubated with detergents, a consistent trend in LCAT activity was only seen when partially purified LCAT was used as the enzyme source. This trend indicated an approximately 25% increase in enzyme activity over the control when 10(-4) M SDS and 2 X 10(-3)% Triton X-100 were present in the preincubation mixtures, respectively. Those observations suggested that, during the preincubations and subsequent assays, the enzyme (in the presence of detergents) was allowed to dissociate from the endogenous substrate and subsequently interact with the exogenous substrate molecules. Additional experiments utilizing molecular-sieve chromatography with whole plasma and partially purified enzyme also showed that dissociation of LCAT/lipoprotein complexes occurred in the presence of detergent. SDS was also shown to enhance the reaction of LCAT in whole plasma with anti-LCAT antibody in an enzyme-linked immunoassay system, indicating that the detergent treatment facilitated the exposure of additional antigenic sites, perhaps via dissociation of the enzyme from plasma lipoproteins.

Animals↗