Phytosterolemia and hypercholesterolemia in childhood.
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Child, Preschool↗
Biomedical subjects
A Kuksis
Publications and source records attributed to A Kuksis.
Acylation of lysophosphatidylcholine and glycerolphosphate and fatty acid pattern in phosphatidylcholine and -ethanolamine in microsomes of normal and dystrophic human muscle.
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were isolated from microsomes obtained from normal and dystrophic human muscle and the fatty acid (FA) pattern estimated by GLC. In PC a decrease of the fatty acids of 16:0 and 18:2 and an increase of 18:0 and 18:1 was observed. In PE the decrease measured 18:2 and the increase 18:0 and 18:1. The acylation of lysophosphatidylcholine (LPC) and glycerol-3-phosphate (G3P) was measured in a microsomal system containing exogenously added LPC or G3P and labelled palmitic and oleic acid CoA esters. The incorporation of both labelled fatty acids in LPC-forming PC is reduced in dystrophic microsomes. On the other hand the acylation of glycerolphosphate and the formation of phosphatidic acid (PA) is greater in dystrophic microsomes when compared with normal controls. Possible correlations between the shifted FA pattern and the acylation rate by dystrophic microsomes measured in vitro in the two systems are discussed.
Acylation↗
Effect of different neutral phospholipids on apolipoprotein binding by artificial lipid particles in vivo.
Soybean triacylglycerol particles, stabilized with egg yolk sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylethanolamine (PE), or PC-PE mixtures, with diameters ranging from 170 to 550 nm were prepared by sonication and isolated by ultracentrifugation. Binding of apoproteins to the lipid particles was studied in vivo using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had decreased in size and undergone minimal changes in lipid composition, ranged from 70% and 57% for SPH- and PC-stabilized particles to 14% for particles stabilized with egg yolk PC-PE mixtures. The apoprotein (apo) composition of the recovered particles showed qualitative and quantitative differences, which were affected by the number of washes during isolation. After four washes, the SPH and PC particles contained apoE, apoC-II, and apoC-III as major components and apoA-IV as minor components. In addition, all particles, except those stabilized with egg yolk PC, contained large amounts of albumin. In contrast to egg yolk PC, the dipalmitoyl PC particles bound albumin as a major component. The recovered PC-PE and PE particles were characterized by a relative decrease of apoC and greatly increased binding of albumin. The higher rate of clearance of the PE-containing particles was attributed to a relative absence of apoC-III, which is known to delay hepatic uptake of lipid particles containing it, and to a more rapid hydrolysis of PE by lipoprotein lipases. Since PE occurs as a minor and variable component of chylomicrons and plasma lipoproteins, the present observations are of physiological interest.
1,2-Dipalmitoylphosphatidylcholine↗
Effect of acidic phospholipids on apolipoprotein binding by artificial lipid particles in vivo.
Soybean triacylglycerol particles stabilized with soybean phosphatidylinositol (PI), bovine brain phosphatidylserine (PS), egg yolk phosphatidylcholine (PC) or mixtures of these acidic and neutral phospholipids were prepared with diameters ranging from 250 to 520 nm. Binding of apoproteins to the lipid particles was studied using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had undergone minimal changes in lipid composition, ranged rom 57% for the PC-stabilized emulsions to 21% for the emulsions stabilized with PS and 8% for the emulsions stabilized with PI. The apoprotein (apo) composition of the recovered particles showed characteristic qualitative and quantitative differences. The particles stabilized with PI and PS or PI-phosphatidylethanolamine contained an unknown protein of molecular weight 117,000 (43-48%) and albumin (9-13%) as major components. The apoC-II, apoC-III, apoA-I, apoE, and apoA-IV were present as minor components in ratios that were the reverse of those seen for the PC-stabilized particles, which contained these proteins as major components. The relative strength of the binding of the proteins, which was determined by washing the particles with saline under standard conditions, also showed variations among the different particles and different apoproteins. The lipid particles stabilized with the acidic phospholipids had less total apoprotein and held it less tightly than the particles stabilized with PC. It is concluded that the binding of apoproteins by lipid particles stabilized with acidic phospholipids involves hydrophobic and ionic interactions, both of which may be physiologically important.
Animals↗
Investigation of the role of micellar phospholipid in the preferential uptake of cholesterol over sitosterol by dispersed rat jejunal villus cells.
The uptake of radioactive cholesterol and sitosterol by rat jejunal villus cells was examined using mixed micellar solutions containing sodium taurocholate, equimolar mixtures of the two sterols, and a variety of phospholipid types. The addition of phospholipid to the incubation solutions reduced the cellular absorption of both sterols and gave rise to uptake kinetics that were linear with time. In the presence of egg yolk phospholipid, uptake of the sterols by villus cells occurred with a modest preference for cholesterol over sitosterol. The ratio of accumulated cholesterol/sitosterol increased from 1.0 initially to 1.23 +/- 0.04 (n = 18) after a 30-min incubation at 37 degrees C. The selectivity displayed in the villus cells increased significantly as egg phosphatidylethanolamine was added to the egg phosphatidylcholine (PC) preparation in micellar solution. It was markedly decreased when dipalmitoyl PC or the primarily saturated egg yolk sphingomyelin were incorporated into the micelles. In every case examined, phospholipid was taken up by the cells concurrently with the sterols. The selectivity between cholesterol and sitosterol was maintained when the donor species were multilamellar vesicles composed of egg PC and the sterols, but not when the donor particles were albumin-stabilized sterol dispersions or taurocholate solutions in the absence of PC. The results show that the selective absorption of cholesterol over the plant sterol occurs only in the presence of unsaturated phospholipid. The phospholipid may act by influencing the permeability of the cellular membranes to the two sterols or the rate of sterol desorption from the phospholipid-containing micellar or liposomal carriers.(ABSTRACT TRUNCATED AT 250 WORDS)
1,2-Dipalmitoylphosphatidylcholine↗
Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine.
rac-1-[1-14C]Lauroyl-2-oleylglycero-3-phospho[methyl-3H]choline and rac-1-lauroyl-2-[1-14C]oleoylglycero-3-phospho[methyl-3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D. There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.
Brassica↗
Acyl selectivity in the transfer of molecular species of phosphatidylcholines from human erythrocytes.
This report describes the molecular species composition of phosphatidylcholines (PC) transferred from human erythrocytes to acceptor vesicles composed of cholesterol and single PC species in the presence of PC-specific transfer protein from bovine liver. The compositions of the PC isolated from the vesicles were determined by capillary GLC as the diacylglycerol trimethylsilyl ethers. The cellular PC species appearing in the acceptor vesicles were enriched in unsaturated species and showed a low content of dipalmitoyl PC compared to untreated erythrocytes. This trend was independent of the composition of the PC used to construct the acceptor vesicles and it was possible to determine that the relative rates of efflux of the palmitoyl-containing phosphatidylcholines decreased in the order: palmitoyl-linoleoyl greater than palmitoyl-oleoyl greater than dipalmitoyl and in the stearoyl series, stearoyl-linoleoyl greater than stearoyl-oleoyl. No clear trend was distinguished for the influence of chain-length on the efflux, thus preventing an unambiguous assignment of the order of removal of all species from the cell membrane. Results derived for arachidonoyl-containing species were compromised by evidence for oxidation occurring during incubations at 37 degrees C. To confirm that acyl selectivity was also possible during transfer in the absence of the transfer protein, the efflux of 14C-labeled soya PC and [14C]dipalmitoyl PC from prelabeled erythrocytes was measured using plasma as the acceptor. As predicted by the chromatographic analyses, 14C-labeled soya PC effused up to 10-times faster than [14C]dipalmitoyl PC from the red cell membrane. Thus, the more rapid transfer of unsaturated PC cannot be explained entirely as a specificity of the transfer protein and is consistent with the hypothesis that intermolecular interactions involving PC molecules within the erythrocyte membrane, become weaker with increasing unsaturation. The results suggest a potential role of PC-specific transfer protein as a probe of the nature of PC interactions within biological membranes.
Androgen-Binding Protein↗
Comparative studies of triacylglycerol structure of very low density lipoproteins and chylomicrons of normolipemic subjects and patients with type II hyperlipoproteinemia.
The triacylglycerols of very low density lipoproteins (VLDL) and of chylomicrons were analyzed in the fasting and postabsorptive states from normolipemic subjects and patients with Frederickson's Type II hyperlipoproteinemia, who subsisted on free choice diets, standard diets excluding lard, or were given a breakfast enriched in lard. The VLDL and chylomicrons were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2-, sn-2-3- and sn-1,3-diacylglycerols were generated by partial Grignard degradation of the triacylglycerols and a stereospecific hydrolysis by phospholipase C of the mixed sn-1,2(2,3)-diacyl phosphatidylcholines prepared as intermediates. Representative sn-2-acylglycerols were obtained by hydrolysis with pancreatic lipase. Positional distribution of the fatty acids was established by subtracting in turn the fatty acid composition of the sn-2-position from the fatty acid composition of the sn-1,2- and sn-2,3-diacylglycerols. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gas-liquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl (t-BDMS) ethers. The molecular association of the fatty acids in the triacylglycerols was determined by 1-random 2-random 3-random calculation following experimental validation of the distribution. The results confirm a marked asymmetry in the positional distribution of the fatty acids in all triacylglycerol samples, with the palmitic acid predominantly in the sn-1-position, the unsaturated acids about equally divided between the sn-2- and sn-3-positions, and the stearic acid divided about equally between the sn-1- and sn-3-positions. The overall structure of the VLDL and chylomicron triacylglycerols from patients and control subjects was characterized by a non-correlative distribution of fatty acids under all dietary conditions.
Adult↗
Milk and plasma lipid composition in a lactating patient with type I hyperlipoproteinemia.
This report describes studies on the plasma and milk lipid composition of a patient with primary Type I hyperlipoproteinemia who had been followed through her second pregnancy. Post-partum she lactated, supplying milk for assay. It was abnormal in the low content of its total lipid and in the bizarre composition of its fatty acids. The proportion of long chain fatty acids was unusually low, and that of medium chain fatty acids unusually high. Furthermore, the fatty acids of the patient's milk differed greatly from those of her plasma triglycerides. This was in marked contrast to normal nursing mothers' milk, in which the fatty acid composition is comparable to that of plasma triglycerides. The patient's milk fatty acids were shorter in chain length and deficient in essential fatty acids. During the time of lactation, the patient remained hyperlipidemic and her post-heparin plasma had no lipolytic activity. These data and the differences between the plasma and milk fatty acids suggested that in the patient the circulating triglyceride fatty acids did not enter the mammary gland. Without preformed fatty acids entering it from plasma or adipose tissue, the lactating breast apparently synthesized fatty acids de novo. These newly synthesized fatty acids were of medium, rather than long chain length. This accounted for the abundance of medium chain length triglycerides in the patient's milk. The studies suggested that the deficit of lipoprotein lipase in Type I hyperlipoproteinemia extended to the mammary gland.
Adult↗
Rapid isolation of a triacylglycerol synthetase complex from rat intestinal mucosa.
A triacylglycerol synthetase complex made up of acyl-CoA synthetase, acyl-CoA:monoacylglycerol acyltransferase, and acyl-CoA:diacylglycerol acyltransferase has been solubilized by sodium taurocholate and isolated by chromatography on phenyl-Sepharose. For this purpose microsomes of the villus cells of rat intestinal mucosa were dissolved in 2% sodium taurocholate prepared in 1 M (NH4)2SO4 and 25 mM Tris-HCl (pH 8.5) (buffer A). After dialysis against buffer A, the sample was loaded on a phenyl-Sepharose column and the enzyme complex was eluted with 25 mM Tris-HCl (pH 8.5) (buffer B). The enzymatically active fractions were pooled and rechromatographed on a Bio-Gel A-0.5m column equilibrated with buffer B and 150 mM NaCl. The recovered triacylglycerol synthetase complex accounted for over 75% of the original enzyme activity and represented a 145-fold purification from villus cells and a 10-fold purification from microsomes. It exhibited maximal activity at pH 8.0-9.0. On the basis of Bio-Gel A-0.5m chromatography the three enzymic activities appeared as a single fraction in the molecular weight range of 350 000-375 000. The complex migrated as a single peak on high performance liquid chromatography on an ion-exchange column using a NaCl gradient. The ratio of the activities of the three enzymes remained constant during the purification. The purified enzyme complex lost about 50% of its diacylglycerol acyltransferase activity on storage for 2 weeks at -20 degrees C in presence of phenylmethylsulfonyl fluoride.
Acyltransferases↗
Deacylation of endogenously deuterated rat liver microsomal phospholipids by endogenous phospholipases.
The relative deacylation of microsomal phospholipid molecular species was reexamined. Microsomal membranes were prepared from the livers of rats injected, over a period of 20 h, with perdeuterated ethanol. The phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography of the total lipid extracts and the distribution of deuterium among the molecular species of the diacylglycerol moieties was determined by reversed-phase high pressure liquid chromatography in combination with chemical ionization mass spectrometry. Deuterium was found to be incorporated into newly formed glycerol and newly synthesized palmitic and stearic acids (4-22% replacement) which were distributed throughout the molecular species in proportion to their relative rates of turnover (10-45% replacement). Within each unsaturation class, the palmitoyl species were labelled more extensively than the corresponding stearoyl species. Following in vitro incubation of the membranes with 10 mM Ca2+ at pH 8.5, there occurred a rapid degradation of the phosphatidylethanolamines (to 60% of control values after 90 min), while the phosphatidylcholines remained essentially unaffected. The various molecular species of the phosphatidylethanolamines were degraded linearly and in proportion to their masses. The deuterium content of the phosphatidylethanolamine and phosphatidylcholine remained constant throughout the incubation. It was concluded that under the present experimental conditions all molecular species of phosphatidylethanolamine, both old and newly synthesized, are equally accessible to the endogenous phospholipase.
Animals↗
Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells.
We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate--polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol-3-phosphate.
Acyltransferases↗
Molecular species of plant phosphatidylinositol with selective cytotoxicity towards tumor cells.
The molecular species of highly purified phosphatidylinositol from soybeans were determined as an aid in the investigation of the mechanism of their reported selective cytotoxicity towards tumor cells. Unlike the animal phosphatidylinositol, which contains predominantly stearic acid in the sn-1 and arachidonic in the sn-2 position (18:0 20:4), the soybean phosphatidylinositol was found to contain mainly linoleic acid in the sn-2 position and palmitic (16:0 18:2), stearic (18:0 18:2) and linoleic (18:2 18:2) acids in the sn-1 position of its molecular species.
Antineoplastic Agents↗
An examination of the stereochemical course of acylation of 2-monoacylglycerols by rat intestinal villus cells using [2H3]palmitic acid.
The stereochemical course of acylation of 2-monoacylglycerols by rat intestinal mucosa was investigated using isolated villus cells with 2-lauroylglycerol and [2H3]palmitic acid as substrates. The newly synthesized X-1,2-diacylglycerols and triacylglycerols were recognized on the basis of the content, positional distribution and molecular association of the fatty acids by gas-liquid chromatography/mass spectrometry in combination with sterospecific analysis. It was found that the free X-1,2-[2H3]palmitoyllauroylglycerols contained 74% sn-1,2- and 26% sn-2,3-enantiomers, which were utilized for triacylglycerol formation in the same proportion. Some of the 2-monolauroylglycerol became hydrolyzed and the released lauric acid was utilized along with [2H3]palmitic acid for diacylglycerol and triacylglycerol formation via both the phosphatidic acid and the 2-monoacylglycerol pathway. The contribution of the phosphatidic acid pathway (12-16%) was determined by estimating the relative proportion of the newly synthesized triacylglycerols containing [2H3]palmitic acid in the sn-2 position. After correcting for the contribution of the phosphatidic acid pathway, the sn-1,2-/sn-2,3-enantiomer ratio in the X-1,2-[2H3]palmitoyllauroylglycerol was estimated to be 71/29. These results are consistent with those previously obtained with glycerol-labelled 2-monoacylglycerols and intact tissue or homogenates of rat intestinal mucosa. It is suggested that the 2-mono-acylglycerol transacylase, if a single enzyme, possesses a low stereospecificity, or that two enzymes exist of unequal concentration and/or activity.
Acyltransferases↗
Quantitation of natural triacylglycerols by reversed-phase liquid chromatography with direct liquid inlet mass spectrometry.
Using acetonitrile and propionitrile as eluting solvents and reagent gases the yields of both quasi-molecular and fragment ions were found to vary with the molecular weight, degree of unsaturation and positional distribution of the fatty acids in the triacylglycerol molecule, and appropriate calibration factors were necessary for accurate quantitation. In the absence of pure structural isomers and mixed acid standards, preliminary calibration factors have been determined for total ion and specific ion current responses by comparing the peak area ratios obtained by liquid chromatography-mass spectrometry with the proportions of the molecular species known to be present in randomized oils and in natural oils of known chemical composition. Although the derived factors include both chromatographic and mass spectrometric effects and are obtained with a gradient of reagent gases, they appear to be generally applicable. It was shown that positional isomers affected the yield of the (MH-RCOOH)+ ions over a 1-3-fold range of intensities, while the nature of the fatty acid affected it over a range of 1.25-fold. After suitable calibration of the relative ion responses it was possible to determine the identities and amounts of the individual molecular species in natural fats and oils.
Arachis↗
Milk fat structure of a patient with type 1 hyperlipidemia.
Structural analyses were performed on milk fat samples obtained 3-10 days postpartum from a lactating patient with primary Type 1 hyperlipidemia. The milk triacylglycerols contained 3-7% C10, 14-21% C12, 20-30% C14, 22-26% C16 and 20-30% C18 (largely oleic) acids. Gas liquid chromatographic (GLC) analyses of the X-1,3- and X-1,2-diacylglycerols on polar siloxane columns showed a markedly non-random association of acyl chains. Stereospecific analyses indicated that the short chain length fatty acids were confined essentially to the sn-3-position of the triacylglycerol molecule. Furthermore, these acids were largely absent from the phosphatidylcholines and the endogenous sn-1,2-diacylglycerols of the milk fat. It is concluded that the short chain fatty acids are incorporated into the milk triacylglycerols during the final stage of biosynthesis via the phosphatidic acid pathway, and that the overall fatty acid distribution is consistent with the 1-random 2-random 3-random hypothesis.
Animals↗
Studies of triacyglycerol structure of very low density lipoproteins of normolipemic subjects and patients with type III and type IV hyperlipoproteinemia.
The triacylglycerols of very low density lipoproteins (VLDL-TG) were analyzed in samples from normal subjects and patients with Frederickson's Type III and Type IV hyperlipoproteinemia. VLDL were obtained by conventional ultracentrifugation, and the triacylglycerols were isolated by thin-layer chromatography (TLC). Representative sn-1,2(2,3)- and sn-1,3-diacylglycerols were generated by Grignard degradation of the triacylglycerols, and were resolved by TLC on borate-treated silica gel. The molecular association of the fatty acids in the diacylglycerol moieties was determined by gas-liquid chromatography with mass spectrometry (GC/MS) of the tertiary-butyldimethylsilyl ethers. The positional distribution of the fatty acids was established by the Brockerhoff stereospecific analysis. The results showed a marked asymmetry in the distribution of the fatty acids in all samples, with the saturated acids predominantly in the sn-1-position and the unsaturated fatty acids distributed about equally between the sn-2- and sn-3-positions. In all instances, the molecular species composition of the sn-1,2-, sn-2,3- and sn-1,3-diacylglycerols was found to be similar to that calculated for 1-random 2-random 3-random distribution of triacylglycerols. There were marked differences in the quantitative composition of the molecular species of the VLDL-TG between normal subjects and patients, but these discrepancies were attributed to differences in the fatty acid composition of the samples.
Diglycerides↗
Determination of plasma total lipid profiles by capillary gas-liquid chromatography.
We have obtained improved qualitative and quantitative information about plasma lipid composition by adopting capillary gas-liquid chromatography to the determination of plasma total lipid profiles. The new procedure takes advantage of the good recoveries of high molecular weight solutes provided by short capillary columns as well as of the absence of sample fractionation during on-column injection. The major improvements in the gas-liquid chromatographic determination of plasma lipid profiles are the greatly increased carbon number resolution of the solutes and the minimal sample size requirements on the capillary columns. In many instances there is a partial resolution of the saturated and unsaturated molecules within a carbon number, which provides clues to the fatty acid composition of the molecular species. In other respects the new plasma total lipid profiles are similar to those obtained on packed columns. The plasma lipids are dephosphorylated by phospholipase C and trimethylsilylated before analysis.
Chromatography, Gas↗