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Biomedical subjects

A Kuksis

Publications and source records attributed to A Kuksis.

At least 127 records · Page 7Linked to original sources
PubMed1984

Effect of intravenous infusion of Intralipid, cholesterol, and plant sterols on hepatic cholesterogenesis.

Male Wistar rats were infused intravenously with various amounts (2-20 mL/24 h) of Intralipid or Intralipid containing cholesterol or plant sterols (5-100 mg/24h), and hepatic cholesterogenesis was monitored by measuring the incorporation of [1-14C]acetate into the nonsaponifiable sterols of liver slices. It was observed that the infusion of Intralipid alone resulted in a hypercholesterolemia that varied with the amount of Intralipid administered and that it was accompanied by up to a threefold increase in hepatic cholesterogenesis. Inclusion of cholesterol in the Intralipid at 5 mg/mL prevented the increase in hepatic cholesterol biosynthesis, while an inhibition of up to 95% of control synthesis was achieved when a total of 33 mg of cholesterol in 20 mL Intralipid was infused over a 24-h period. It is concluded that the feedback regulation of cholesterol biosynthesis is operative even when the entry of cholesterol bypasses the intestine and the lipoprotein synthesis taking place there and that Intralipid is a suitable medium for the intravenous introduction of a large mass of metabolically active cholesterol molecules. Similar infusions of mixed plant sterols failed to prevent the activation or inhibition of cholesterol biosynthesis, but the administration of much larger doses of plant sterols (100 mg) brought about a partial inhibition of hepatic cholesterogenesis. It is concluded that the presence of an alkyl group in the side chain prevents the plant sterols from an effective interaction with the critical sites of the feedback regulatory system of cholesterogenesis. The effects of the larger doses of plant sterols were attributed to the displacement of increasing amounts of free cholesterol from the vascular tissues, which resulted in an effective elevation of plasma cholesterol levels. The infusion of either cholesterol or plant sterols over the 24-h period did not appear to have a consistent effect upon the composition or secretion of biliary bile acids.

Acetates↗
PubMed1984

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

A sensitive and practical procedure has been developed for the determination of the sn-1-alk-1-enyl-2-acylglycerol moieties of natural glycerophospholipids using capillary gas-liquid chromatography with flame ionization detection. The method gives excellent resolution and quantitative estimates for molecular species according to carbon number and degree of unsaturation. The diradylglycerols are released from the glycerophospholipids by hydrolysis with phospholipase C and the alk-1-enylacylglycerols are resolved by thin-layer chromatography. Either the trimethylsilyl or the tertiary-butyldimethylsilyl ethers are then resolved at 250 degrees C by gas-liquid chromatography on a glass capillary column wall-coated with a polar cyanopropylsiloxane polymer using hydrogen as the carrier gas. The method has been applied to the analysis of the alk-1-enylacyl- and diacyl-glycerol moieties of the phosphatidylethanolamines of rat heart and kidney.

Animals↗
PubMed1984

Quantitative and positional analysis of fatty acids.

As a result of steady advances made in the analytical methodology of fatty acids and glycerolipids, it is now possible to identify, quantitate, and in many instances to isolate individual molecular species of fatty acids and their glyceryl esters. Essentially complete resolution of most natural fatty acids is now possible by GLC on polar capillary columns, which are commercially available for routine application. Combinations of GLC with mass spectrometry provide identification of many unknown acids but are not necessary for work with most known acids. Likewise, GLC on polar capillary columns can provide essentially complete resolution of the diacyl-, alkylacyl-, and alkenylacyl-glycerols. Although separations of the lower molecular weight triacylglycerols are also possible, the resolution of long chain triacylglycerols would appear to be best accomplished by HPLC. Thus, HPLC on microparticulate reversed-phase columns has yielded complete resolution of the critical pairs, triplets, and quadruplets of natural long chain triacylglycerols. Provided sufficiently large fractions can be collected, a stereospecific analysis of the individual triacylglycerols or their simple mixtures should yield both positional placement and molecular association of the component fatty acids from which exact structures of the component triacylglycerols could be reconstructed. A complete separation of intact natural glycerophospholipids has also been recently demonstrated by means of HPLC, and it is possible that this technique will completely revolutionize the identification and separation of the molecular species of glycerophospholipids, although quantitation remains a problem for the time being. Also the separations have not yet been extended to the alkylacyl- and alkenylacyl-glycerophospholipids. Since the various fractions arising from HPLC may be collected, it should be possible to obtain meaningful positional analyses of fatty acids by means of specific enzymic hydrolyses. The high purity of the fractions should allow a reliable estimation of the proportions of the reverse isomers and of fully saturated triacylglycerols, which are not resolved by the chromatographic techniques. The small fractions available from HPLC will place increased importance on the microtechniques of enzymic hydrolyses and fatty acid and acylglycerol analyses. The essentially complete analyses of the molecular species of the glycerolipids made up of the common fatty acids will greatly simplify the structural analyses of membrane and lipoprotein lipids, especially if combined with automated operation and computerized data processing.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1983

Simultaneous quantitation of Krebs cycle and related acids by mass fragmentography.

Methods are described for simultaneous quantitation of Krebs cycle and related acids by gas chromatography--mass spectrometry using deuterium-labelled acids and n-butyl-d9-esters of the organic acids as internal standards. Using sulphuric acid as esterification catalyst, only lactic, succinic, fumaric, malic, maleic and citric acids were found to be stable to hydrogen exchange and could be used as reference standards in the deuterated form. In contrast, pyruvic, oxalacetic, alpha-ketoglutaric and malonic acids were found to exchange their deuterium readily and could not be employed for this purpose. All the acids could be quantitated using n-butyl-d9-esters of reference organic acids as internal standards, following a separate preparation of the n-butyl derivatives of the unknown acids. The method is suitable for routine analysis of organic acids at the picogram level in perchloric acid extracts of tissues.

Adipates↗
PubMed1983

Studies on the substrate specificity of purified human milk bile salt-activated lipase.

The fatty acid specificity of the bile salt-activated lipase purified from human milk was studied using C12 to C54 (total acyl carbon) saturated and the C54 unsaturated triacylglycerols. Kinetic studies indicated that the short chain triacylglycerols were hydrolyzed more readily than the long chain triacylglycerols, and that the long chain unsaturated triacylglycerols were attacked more readily than the long chain saturated triacylglycerols. This fatty acid specificity was also apparent intramolecularly, both short chain and unsaturated fatty acids being released at higher rates than the saturated long chain acids. The enzyme possessed neither positional specificity nor stereospecificity as indicated by the nearly simultaneous appearance of the sn-1,2-, sn-2,3-, and sn-1,3-dioleoylglycerols from trioleoylglycerol. The hydrolyses of these three ester bonds were approximately at their anticipated chemical reactivities. Synthetic rac-1-monooleoylglycerols were hydrolyzed about 2 times faster than the sn-2-monooleoylglycerols. It is concluded that the bile salt-activated lipase may possess a special potential for a rapid release of short chain and polyunsaturated fatty acids from dietary triacylglycerols in the intestinal lumen of infants.

Bile Acids and Salts↗
PubMed1983

Differential transfer of C and E apolipoproteins from very-low-density lipoprotein to lysophosphatidylcholine-phosphatidylcholine micelles.

Rat plasma VLDL was incubated with lysoPC/PC micelles consisting of 45-90 mol% lysoPC at micelle/VLDL phospholipid ratios of 0.33-6. Following incubation, the VLDL and micellar particles were reisolated by ultracentrifugation and the lipid and apolipoprotein composition determined by high-temperature gas chromatography and polyacrylamide gel electrophoresis, respectively. Lysophosphatidylcholine was found to equilibrate between the very-low-density lipoproteins and micellar fraction without transfer of phosphatidylcholine or sphingomyelin between these fractions. This produced reisolated VLDL and micellar particles with nearly identical lysoPC/PC ratios. Only apolipoprotein E transferred to reisolated micellar fractions with less than 35 mol% lysoPC. The C apolipoproteins were also transferred to the micellar fraction when the reisolated micelles contained more than 35 mol% lysoPC. It is concluded that apolipoproteins E and C can bind to HDL-size micellar particles of appropriate composition. The differential transfer of apolipoproteins E and C indicates that fundamental differences exist between these apolipoproteins in their interaction with lipid interfaces.

Animals↗
PubMed1983

Strategy of glycerolipid separation and quantitation by complementary analytical techniques. Plenary lecture.

Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable fo complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas--liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas--liquid chromatography with both liquid and gas--liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.

Animals↗
PubMed1983

Influence of divalent cations on rat apolipoprotein transfer to synthetic lipoproteinlike lipid emulsions in vitro.

Synthetic lipid emulsions of chylomicron (TG1) and low density lipoprotein (TG2) diameter, consisting of triacylglycerol and phosphatidylcholine, were incubated with rat serum, whole blood, plasma, and postheparin plasma in the presence or absence of divalent cations. The lipid and apoprotein composition of the reisolated Sf greater than 400 particles was examined by high temperature gas chromatography and sodium dodecyl sulfate - glycerol polyacrylamide gel electrophoresis, respectively. It was found that apoproteins C, E, H, and A-I were associated with TG1 after incubation with whole blood + EDTA, EDTA-plasma, or EDTA-postheparin plasma. Significant amounts of unidentified apoproteins with molecular weights greater than 45 000 were also associated with these particles. Apoprotein E, apoprotein A-I, and a 51 000 dalton apoprotein were associated with the Sf greater than 400 fraction of TG2 recovered after incubation under the same conditions. The apoprotein profiles were indistinguishable from those previously reported for the corresponding Sf greater than 400 fraction recovered after intravenous infusion of TG1 and TG2. In the presence of Ca2+ and Mg2+ either in serum or in plasma and postheparin plasma, two additional proteins were recovered in the Sf greater than 400 fractions of TG1 and TG2. These apoproteins were of molecular weight 28 000 and 48 000 and had not been previously reported as components of plasma lipoproteins.

Animals↗
PubMed1983

Further evidence for enhanced phospholipid synthesis by rat jejunal villus cells during fat absorption.

The effect of fat absorption on the phospholipid turnover of rat intestinal mucosa was determined in animals receiving single fatty meals by stomach tube or multiple meals in the form of corn-oil-soaked laboratory chow diet. The specific activity and relative specific activity of the total phospholipids and of individual phospholipid classes were measured in the isolated jejunal villus cells of fasting and fat-fed animals following an injection of radioactive inorganic phosphate 0.5-31 h prior to sacrifice, which was scheduled to coincide with the peak of fat absorption (2.5-3 h after the last meal). It was shown that the relative specific activity of the fat-absorbing cells increased by about 33% when the samples were taken 0.5 h after intravenous injection of radioactive phosphate. Samples taken 11 and 31 h after the introduction of the radioactive phosphate showed about 16% decrease in the relative specific activity of the phospholipids of the fat-absorbing cells when compared with the fasting controls. These changes in the relative specific activity of the total phospholipids included all phospholipid classes and corresponded to the recently described expansion of the cellular phospholipid pool owing partly to increased de novo synthesis of the membrane phospholipids. The present results are consistent with the known biochemical and physiological changes taking place in the mucosal cells during fat absorption and transport and find support in various less direct biochemical and morphometric measurements.

Animals↗
PubMed1983

Analysis of triacylglycerols by reversed-phase high pressure liquid chromatography with direct liquid inlet mass spectrometry.

Natural triacylglycerols were resolved by high pressure liquid chromatography on Supelcosil LC-18 columns using a gradient of 30-90% propionitrile in acetonitrile as eluting solvent. The effluent was admitted to a quadrupole mass spectrometer via a direct liquid inlet interface. The mass spectra of the solutes were recorded in the chemical ionization mode. The triacylglycerol elution profile was obtained from the total ion current. Individual molecular species of the triacylglycerols were identified from the (MH)+ and the (MH--RCOOH)+ ions. The reversed-phase system allowed the separation of triacylglycerols on the basis of both carbon number and double-bond number, as well as of certain critical pairs of triplets of triacylglycerols with the same partition number. The method is applicable to the determination of triacylglycerol composition of both plant and animal fats. The minimum amount of sample which is required is about 50 ng in the scanning mode. However, 100 times more material must be injected in the liquid chromatograph because only 1/100th of the effluent is being injected into the mass spectrometer.

Arachis↗
PubMed1983

Critical role of ring structure in the differential uptake of cholesterol and plant sterols by membrane preparations in vitro.

To determine the role of the ring structure in the differential absorption of sterols, we have used rat jejunal brush border vesicles and erythrocytes to examine the uptake of cholesterol, campesterol, and sitosterol following successive chemical degradations of rings A and B. The cell and membrane preparations were incubated with the sterols and sterol analogues (about 30 micromolar each) dissolved in 7 mM sodium taurocholate and 0.6 mM egg phospholipid. The uptake of the analogues was analyzed by high performance liquid chromatography and capillary gas--liquid chromatography. In both membrane preparations, the uptake of the 7-dehydroanalogues of cholesterol, campesterol, and sitosterol was linear with time. 7-Dehydrocholesterol was absorbed 4-5 times faster than 7-dehydrositosterol by both preparations. The uptake of the campesterol analogue was intermediate between that of the analogues of cholesterol and sitosterol at all time points. Following conversion of the 7-dehydrosterols to their calciferol derivatives, the 27-carbon sterols were absorbed only 1.9 and 1.4 times faster than those of the 29-carbon sterols by the erythrocyte and brush border membranes, respectively. A similar degree of selectivity was expressed in the erythrocytes during the uptake of a steroid series possessing keto-4-ene ring system. Complete oxidation of the calciferol derivatives to the des-AB-8-ones resulted in a total loss of discrimination among the various side-chain homologues during absorption from micellar solutions. It is concluded that the selective absorption of animal and plant sterols depends upon the presence of a ring system having the bulk of the cholestane nucleus, although not necessarily a rigid or planar one containing a hydroxyl group.

Absorption↗
PubMed1983

Uptake of 7-dehydro derivatives of cholesterol, campesterol, and beta-sitosterol by rat erythrocytes, jejunal villus cells, and brush border membranes.

We have determined the effect of the side chain on the uptake of sterols from micellar solutions by isolated rat jejunal villus cells, brush border membranes, and erythrocytes. From an equimolar mixture of 7-dehydrosterols, the uptake decreased with an increasing number of carbon atoms at C24 of the sterol side chain in a manner identical to that observed for the parent-delta 5-sterols. The brush border and erythrocyte membranes were found to absorb 4-5 times more 7-dehydrocholesterol than 7-dehydro-beta-sitosterol over a 60-min period of incubation. A somewhat lower specificity of sterol uptake by the villus cells was attributed to the exposure of large areas of the basolateral membrane, which apparently was less able to discriminate between sterols. The higher sterol selectivity was associated with higher membrane organization anticipated from the higher free cholesterol/phospholipid and protein/phospholipid ratios of the brush border and red blood cell membrane. On a mass basis the villus cells and brush borders absorbed 30-60 times more sterol than the erythrocytes. Assuming that the delta 5,7-sterols accurately represent the absorption of the delta 5 parent sterols, it is suggested that the 3- to 5-fold excess of absorbed cholesterol over beta-sitosterol that is typically found in the jejunal wall of the rat following feeding of radioactive sterols arises from an inability of beta-sitosterol to enter the brush border membrane as easily as cholesterol.

Animals↗
PubMed1982

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects.

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type IIi and Type IV hyperlipemic 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 gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL1, and LDL2 of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL nd HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.

Adult↗
PubMed1982

Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells.

The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction.

Acyltransferases↗