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Biomedical subjects

A Kuksis

Publications and source records attributed to A Kuksis.

At least 145 records · Page 8Linked to original sources
PubMed1982

Differential uptake of cholesterol and plant sterols by rat erythrocytes in vitro.

The in vitro uptake of radioactively labeled cholesterol and the plant sterol beta-sitosterol has been examined in rat erythrocytes. From mixed micellar solutions containing egg yolk phospholipid and sodium taurocholate, the erythrocytes showed a nonlinear uptake of the two sterols. The uptake leveled off after about 45 min with the attainment of a 1:1 total sterol-to-phospholipid ratio within the cell membrane, as determined on a mass basis. From solutions containing egg yolk phospholipid, or purified egg yolk phosphatidylcholine, a preference for cholesterol over the plant sterol was observed, increasing with time from a cholesterol/beta-sitosterol uptake ratio of unity (the media ratio) to a maximum of 2 after a 60-min incubation. Correction of the data for nonspecifically bound sterol increased the ratio to a maximum of 5 at the 30-min time point. The increase in the cholesterol/beta-sitosterol uptake ratio with time, following an initial nonspecific association, showed that penetration of the plasma membrane by the sterol was required for the selectivity to be expressed. The presence of lysophosphatidylcholine or bovine serum albumin did not exert any noticeable influence over the extent or selectivity of absorption. Replacement of the egg yolk phospholipid with synthetic dipalmitoyl-phosphatidylcholine led to a loss of the sterol selectivity. No evidence was found to support a selective extraction of sterol from the erythrocyte membrane to account for the observed effects, nor was there any sign of a mass accumulation of phospholipid during the incubation. It is suggested that the media phospholipid influences the membrane permeability toward cholesterol and beta-sitosterol.

Animals↗
PubMed1982

Studies on the substrate specificity of purified human milk lipoprotein lipase.

The fatty acid specificity of purified human milk lipoprotein lipase was studied using the C18 to C54 (total acyl carbon number) saturated and the C54 mono-, di- and triunsaturated monoacid triacylglycerols. Kinetic determinations indicated that the medium-chain triacylglycerols were better substrates than long- or very short-chain saturated triacylglycerols. The unsaturated triacylglycerols were hydrolyzed at rates comparable to that of tricaprylin with triolein having the highest rate of hydrolysis of the unsaturated species tested. The enzyme attacked the primary ester bond much more readily than the secondary ester bond. The purified human milk lipoprotein lipase showed a preferential stereospecific lipolysis of the sn-1-position of the triacylglycerol molecule.

Chromatography, Gas↗
PubMed1982

Expansion of phospholipid pool size of rat intestinal villus cells during fat absorption.

The effect of fat absorption upon the phospholipid pool size of the intestinal mucosal cells was determined in rats receiving fatty emulsions as a bolus by stomach tube or as multiple meals in the form of fat-laden laboratory chow. The phospholipid content of the mucosal scrapings and of the isolated villus cells was determined 3 to 34 h after the meals and was compared with the phospholipid content of cells from similar animals receiving water alone or 10% sucrose in water. It was shown that continuously fed animals averaged 5-10% and single meal fed animals up to 40% higher phospholipid content in their mucosal cells than the corresponding controls, when compared per milligram cell protein. The expansion of the phospholipid pool involved all phospholipid classes and correlated well with the phospholipid composition of prechylomicrons and of microsomal membranes, which undergo a significant proliferation during fat absorption. The apparent lower expansion of the phospholipid pool in the continuously fed animals correlated with the lower triacylglycerol content of the lumen and of the cells at these times.

Animals↗
PubMed1982

Resolution of diacylglycerol moieties of natural glycerophospholipids by gas-liquid chromatography on polar capillary columns.

A rapid and practical method has been developed for the gas-liquid chromatographic determination of the sn-1,2-diacylglycerol moieties of natural glycerophospholipids using polar wall-coated open tubular columns. The method gives complete resolution and quantitative estimates for all species according to molecular weight and degree of unsaturation, including stearoyl docosahexaenoylglycerol and related polyunsaturates. For this purpose the sn-1,2-diacylglycerols are obtained from the glycerophospholipids by hydrolysis with phospholipase C and are converted into the trimethylsilyl or tertiary-butyldimethylsilyl ethers. The silyl ethers are separated by gas-liquid chromatography on the capillary glass columns coated with a polar cyanopropylsiloxane polymer, in the temperature range 175-250 degrees C, using hydrogen as the carrier gas. Practical applications of the method are illustrated by analyses of the sn-1,2-diacylglycerol moieties of the phosphatidylcholines of soybean phosphatides, egg yolk, and rat liver. The method of analysis is applicable to other classes of glycerophospholipids and the total time requirements for the analysis of any one phospholipid class are comparable to those for a fatty acid analysis.

Chemical Phenomena↗
PubMed1981

Effect of core composition and particle size of lipid emulsions on apolipoprotein transfer of plasma lipoproteins in vivo.

Lipid emulsions corresponding in size and surface composition to chylomicron and low density lipoprotein but differing in lipid core composition were injected into rats a tail vein cannula and 10 min later blood was withdrawn via the abdominal aorta. Plasma lipoproteins were isolated by ultracentrifugation and the lipid and apolipoprotein composition was determined by gas-liquid chromatography and gel electrophoresis, respectively. It was found that apolipoprotein C peptides were transferred to Sf greater than 400 particles of large diameter regardless of whether their cores contained cholesteryl esters or triacylglycerols, but not to Sf greater than 400 particles of small diameter. Apolipoprotein A-I transferred to both large and small diameter cholesteryl ester core particles but only to larger diameter triacylglycerol core particles. In contrast, apolipoprotein E became associated with small and large diameter particles regardless of core composition. In addition, all Sf greater than 400 fractions contained significant amounts of apopeptides with molecular weights greater than 45 000, which did not correspond to the common apolipoproteins. This is the first study to prove systematically the effects of lipid composition and particle size on apolipoprotein transfer in vivo.

Animals↗
PubMed1981

Effect of saturated and unsaturated fat diets on molecular species of phosphatidylcholine and sphingomyelin of human plasma lipoproteins.

Four healthy 21-23-year-old males with normal lipoprotein patterns and plasma lipid concentrations were subjected voluntarily to two diets of 5 weeks duration each: I, highly saturated fat diet; II, highly polyunsaturated fat diet. The VLDL, LDL and HDL3 fractions were isolated by conventional ultracentrifugation from each subject on the high fat diets and the molecular species of the component phosphatidylcholines and sphingomyelins were identified and quantitated by GC-MS of the t-butyldimethylsilyl ethers of the corresponding diacylglycerols and ceramides. It was shown that the diet markedly and rather evenly affected the molecular species of the phosphatidylcholines of all lipoprotein classes. However, the changes in the corresponding major molecular species were reciprocal in nature and were consistent with a demonstrated relative resistance to alterations in surface fluidity. In contrast, the dietary fat had only a minor effect on the composition of the sphingomyelins, and did not alter the characteristic differential distribution of the molecular species among the low and high density lipoprotein classes. These results, which were free of the uncertainties introduced by analyses of derived fatty acid and which were obtained on samples isolated from the same subjects, clearly demonstrate that a complete equilibration of the molecular species of the phospholipids is not attained amont the plasma lipoprotein classes even in the fasting state. The possible physico-chemical and metabolic basis of these observations is briefly discussed.

Adult↗
PubMed1981

Synthesis and release of lipids and lipoproteins by isolated rat jejunal enterocytes in the presence of sodium taurocholate.

Isolated rat jejunal villus and crypt cells prepared by differential scraping and hyaluronidase dispersion were used in the presence of 8 mM sodium taurocholate to study the incorporation of sn-[3H]glycerol-2-monooleate, [1-14C]palmitate, [1-14C]acetate, L-[4,5(n)-3H]leucine and D-[1-14C]glucosamine into cellular and medium lipids and proteins, respectively. The villus cells were capable of an apparently normal biosynthesis of triacylglycerols and phospholipids, as well as of proteins and glycoproteins despite an altered dye permeability and increased release of cytosolic and membrane enzymes. About 20-30% of the newly formed triacylglycerols and about 35% of the newly formed phospholipids were secreted into the medium and were recovered as triacylglycerol-rich particles. Labelled proteins and glycoproteins were also recovered from this fraction. The crypt cells synthesized about one-half as much triacylglycerol and phospholipid as did the villus cells, but secreted little or no labelled lipid into the postincubation medium. The release into the medium of triacylglycerols synthesized by the villus cells was blocked by a pretreatment of the isolated cells with the microtubule disruptors, nocodazole, colchicine and colcemid; by the amino sugar, D-galactosamine; by the inhibitors of protein synthesis, puromycin and cycloheximide, and by the inhibitor of the biosynthesis of phosphatidylcholine, chlorocholine. These results indicate that the secretion of labelled lipids, proteins and glycoproteins by the upper villus enterocytes in the presence of sodium taurocholate is not entirely due to cell breakage and spillage of contents. It is concluded that incubations of isolated villus cells of rat jejunum with mixed micellar solutions containing 8 mM taurocholate are compatible with an apparently normal biosynthesis and secretion of triacylglycerol-rich particles.

Alkaline Phosphatase↗
PubMed1981

Lipid class and molecular species interrelationships among plasma lipoproteins of normolipemic subjects.

As evidence of lipoprotein interconversion and/or equilibration, a gas-liquid chromatographic (GLC) examination was made of the lipid class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of seven male and four female normolipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by GLC of the intact cholesterol and glycerol esters and of ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and an increasing phospholipid and cholesteryl ester content when progressing from the very low (VLDL), to the low (LDL2) and high (HDL) density lipoproteins, as already established by conventional analyses. However, the LDL2 contained about a two times higher proportion of total phospholipids as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher molecular weight species than the sphingomyelins of either VLDL or LDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding fractions of lipoproteins. In general, the lipid class and molecular species distribution is incompatible with the hypothesis which postulates VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin:cholesterol acyltransferase. The significant differences noted in the lipid class and molecular species distribution suggest that the true transformations of the lipoproteins are much more complex and may also involve cholesteryl ester-triacylglycerol, triacylglycerol and phosphatidylcholine exchanges via appropriate carrier plasma proteins, as well as possible phase separation of lipids during the removal of the excess surface material from the VLDL remnants, as already demonstrated in in vitro experiments. It is concluded that a direct GLC analysis of the neutral and polar lipid components of plasma lipoprotein classes provides important evidence of lipoprotein interrelationships which may be utilized to test existing and new hypotheses of lipoprotein interconversion.

Adult↗
PubMed1981

Differential distribution of sphingomyelins among plasma lipoprotein classes.

Individual and pooled samples of plasma from normolipemic and hyperlipemic subjects were separated into very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein fractions (HDL2 and HDL3) by conventional ultracentrifugation and total lipid extracts prepared by standard methods. The composition of the molecular species of the sphingomyelins in each lipoprotein class was determined by packed column and capillary gas-liquid chromatography of the trimethylsilyl (TMS) and t-butyldimethylsilyl (t-BDMS) ethers and by gas chromatography - mass spectrometry of t-BDMS ethers of ceramides derived by phospholipase C hydrolysis of the corresponding parent compounds. It was demonstrated that the molecular weight of the species of the sphingomyelins increases with the density of the lipoprotein fraction in normolipemic subjects, and that this increase is due to an increase in the chain length of the fatty acids in the ceramide molecules. In contrast, patients with type IV hyperlipoproteinemia possessed similar species in the LDL and HDL fractions, while maintaining normal differences between HDL and VLDL. Type III patients possessed normal HDL and VLDL differences, but had variable LDL. Type II patients had ceramide profiles for VLDL, LDL, and HDL fractions that were very similar to those of normals. The differential distribution of the molecular species of the sphingomyelins is rationalized on the basis of a lateral phase separation of the short and long chain sphingomyelins during the shedding of the excess VLDL or chylomicron surface material and a subsequent preferential transformation of the long chain species into HDL. The LDL sphingomyelins in type III hyperlipemia are variable and approximate either the VLDL or HDL composition.

Ceramides↗
PubMed1981

Modulation of acylglycerol synthetase activities of rat intestinal mucosa by homogenization and sonication.

Diacylglycerol and triacylglycerol synthesis was compared in intact villus cells and in their homogenates and sonicates using 2-monoacylglycerols and free fatty acids as substrates along with appropriate cofactors. It was found that simple pestle homogenization of cells led to an apparent loss of 80-90% in the triacylglycerol and 40-fold increase in the diacylglycerol synthetase activity. Sonication of the homogenate resulted in reestablishing the normal activity of cellular triacylglycerol synthesis (12 +/- 1 nmol . mg protein-1 . h-1) but the diacylglycerol biosynthesis further increased (to a maximum of 70 +/- 5 nmol . mg protein-1 . h-1). Preincubation with trypsin and postincubation treatment with pancreatic lipase indicated that different enzymes are involved in diacylglycerol and triacylglycerol biosynthesis from 2-monoacylglycerols and that these enzymes become segregated during homogenization. It is concluded that a triacylglycerol synthetase system utilizing 2-monoacylglycerols can be reassembled upon sonication.

Acyltransferases↗
PubMed1980

Comparative determination of plasma phospholipids by automated gas--liquid chromatographic and manual colorimetric phosphorus methods.

Plasma samples obtained during a prevalence study of hyperlipemia in a free-living urban population were analyzed for phosphatidylcholine, sphingomyelin and lysophosphatidylcholine content by automated high-temperature gas--liquid chromatographic (GLC) and manual colorimetric phosphorus (thin-layer chromatographic, TLC) methods. The GLC estimates were obtained from a quantitative analysis of the diacylglycerol, ceramide and monoacylglycerol moieties released from the parent phospholipids by digestion with phospholipase C, while the TLC estimates were derived by manual colorimetric phosphorus analyses of the individual phospholipid classes resolved by TLC. On samples analyzed over a two-year period the methods gave excellent correlation for the total phospholipids (r = 0.98), phosphatidylcholine (r = 0.98) and sphingomyelin (r = 0.90), but resulted in a poor agreement for lysophosphatidylcholine (r = 0.69). Comparable results were obtained for estimates of these phospholipids in plasma very low density, low density and high density lipoproteins. The between-method coefficient of variation ranged from 3 to 5% for phosphatidylcholine and from 5 to 10% for sphingomyelin. The relative error for the estimates of lysophosphatidylcholine ranged from 10 to 25%, and was due to the inclusion in the GLC estimates of a variable proportion of plasma free monoacylglycerols. Other differences between the two methods are due to various analytical errors and biases inherent in the two techniques. The within-day, within GLC, relative error averaged 1% for phosphatidylcholine, 3% for sphingomyelin and 5% for lysophosphatidylcholine. The apparent high precision and accuracy of the GLC method recommend it as an alternative to conventional direct methods of phospholipid analyses based on TLC isolation of lipid classes and colorimetric measurements of their phosphorus content. The GLC analyses of the plasma phospholipids are particularly convenient in conjunction with GLC measurements of plasma cholesterol and triacylglycerols, where a smaller throughput of samples is not a limitation and where both total amount and relative proportion of the lipids are of interest.

Autoanalysis↗
PubMed1980

Analysis and structure determination of unsaturated 5 beta - cholanoic acids.

Unsaturated cholanoic acids are known to arise as artifacts of chemical transformation processes and during storage and high-temperature gas liquid chromatography (GLC) of various derivatives of saturated bile acids. Nevertheless, there is evidence for their natural occurrence and isolation under conditions where artifactual formation of unsaturated bile acids would be unlikely. Since structural identification of such compounds is often complicated by a lack of knowledge of their analytical properties, a representative series of monounsaturated cholanoic acids with double bonds in rings A, B and C were prepared by POCl3 and ZnCL2 dehydration of saturated bile acids with selectively blocked hydroxyl functions. The cholenoic acids were indistinguishable from their saturated analogs by thin layer chromatography (TLC) on plain silica gel, but those compounds with sterically exposed double bonds were resolved by AgNO3-TLC, using chloroform/methanol solvent systems. The synthetic 5 beta-cholenoic acids obeyed the general rules of GLC mobility based on the overall shape of the molecule and the number and configuration of the functional groups. Constant retention factors attributable to the double bond were observed for all of the double bond types on several GLC phases, and theoretical retention times could be calculated for combinations of double bonds and functional groups not specifically represented among the synthetic standards. With gas chromatography-mass spectrometry (GC-MS), the unsaturated bile acids gave several characteristic fragments, which, in conjunction with the chromatographic properties of the parent compounds, permitted an unambiguous distinction among different unsaturated acids, and between unsaturated and saturated bile acids of the same number and configuration of functional groups. For complete structural identification of saturated and unsaturated bile acids, capillary GC-MS represents the ideal state of the art, but the less expensive combination of AgNO3-TLC and GLC also can yield much useful information concerning the structure of natural and synthetic 5 beta-cholenoic acids. This study emphasizes the need for special precautions in the isolation and derivatization of bile acids intended for studies of unsaturated components.

Bile Acids and Salts↗
PubMed1980

Fat metabolism in heavy exercise.

1. To investigate differences between the metabolic effects of light and heavy exercise, five healthy males (mean maximal oxygen intake 3.92 litres/min) exercised for 40 min at 36% maximum power (light work) and 70% maximum power (heavy work) on separate days, after an overnight fast. 2. A steady state was achieved in both studies between 20 and 40 min in: oxygen intake (1.42 and 2.64 litres/min respectively); respiratory exchange ratio (0.89 and 1.01); plasma lactate concentration (1.78 and 9.94 mmol/l). 3. Plasma palmitate turnover rate (14C) was unchanged from resting values in light work but was decreased by 40% (from 104 +/- 16 to 63 +/- 8 micronml/min) in heavy work. Heavy work was associated with falls in the plasma concentrations of all free fatty acids measured: palmitic acid (C16:0), oleic acid (C18:1), stearic acid (C18:0), linoleic acid (C18:2) and palmitoleic acid (C16:1). 4. In contrast to th fall in palmitate turnover the increase in plasma glycerol was greater in heavy exercise (0.054-0.229 mmol/l) than in light exercise (0.053-0.094 mmol/l), suggesting that lipolysis was occurring which did not lead to influx of free fatty acids into plasma. 5. In light exercise fat metabolism may be controlled to favour adipose tissue lipolysis and extraction of free fatty acids by muscle from the circulation, whereas in heavy exercise adipose tissue lipolysis is inhibited and hydrolysis of muscle triglycerides may play a more important part. 6. The finding of a high respiratory exchange ratio may not exclude the use of fat as a major fuel source in exercise associated with lactate production.

Adult↗
PubMed1980

Uptake and transport of sterols by isolated villus cells of rat jejunum.

Cellular uptake and transport of cholesterol and beta-sitosterol was studied in rat jejunal villus cells isolated by hyaluronidase dispersion. Sterol absorption was measured from micellar solutions using a Millipore filtration technique. In actively metabolizing cells both cholesterol and beta-sitosterol exhibited rapid initial uptake curves which leveled off after 10 min, corresponding approximately to a 1:1 sterol:phospholipid molar ratio within the microsomal membranes of the cell, which suggested a physicochemical end point. Using various proportions of cholesterol and beta-sitosterol in the incubation medium, it was shown that each sterol interfered with the absorption of the other, the one present in the highest concentration showing the relatively higher uptake. Mixtures of equimolar proportions were taken up by the cells in a ratio that slightly favoured beta-sitosterol, but significantly more cholesterol than beta-sitosterol was found to be transferred to the microsomes. The limited mass discrimination between cholesterol and beta-sitosterol seen in the isolated cells is in contrast to the preferential absorption of cholesterol in vivo. These studies demonstrate that the molecular basis for the differential transport of cholesterol and beta-sitosterol in the lymph observed in vivo must be sought at a higher level of physiological activity of the cells (e.g., chylomicron formation and secretion) than that achieved in the present experiments.

Animals↗
PubMed1980

Stereochemical substrate requirements of lecithin:cholesterol acyltransferase and its inhibition by enantiomeric lysolecithins.

The suitability of 1-sn-phosphorylcholine- and 2-sn-phosphorylcholine-diacylglycerols as substrates for lecithin:cholesterol acyltransferase (LCAT) was assayed under standardized conditions using sonicated liposomes of radioactive phospholipids. The results were compared with those obtained for the corresponding molecular species of the natural 3-sn-phosphorylcholinediacylglycerol. It was found that with the 2-sn-phosphorylcholinediacylglycerol, LCAT had only 16% of the activity that it showed for 3-sn-phosphorylcholinediacylglycerol, while its activity with 1-sn-phosphorylcholinediacyglycerol as the substrate was not significant. It is concluded that LCAT possesses highly specific steric and positional requirements for its phosphatidylcholine substrates and that, like phospholipase A2, it has a significant activity with the 2-sn-phosphorylcholinedicacylglycerol. It wa also shown that the activity of LCAT is inhibited by both enantiomers of lysophosphatidylcholine as well as Triton X-100. In all instances the inhibition was reversed by the addition of lipid-free albumin. It is concluded, therefore, that the inhibition of LCAT by lysophosphatidylcholine is a detergent rather than an end-product inhibition phenomenon.

Glycerylphosphorylcholine↗