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Amniotic fluid lecithin and sphingomyelin areas after extraction and chromatography are rendered permanently visible by the use of bromthymol blue dye buffered to a pH of 11-3.
The severity of the erosive and ulcerative lesions of the squamous gastric mucosa in 10 standardbred racehorses in training was classified according to a standard scoring system. Each horse was then treated orally for 30 days with 50 g/100 kg bodyweight daily of a pectin-lecithin complex mixed into the feed. At the end of the period of treatment, the gastric lesions were re-evaluated gastroscopically and the scores were compared with those assigned at the previous evaluation. In three of the horses the gastric ulcerations had healed completely, and in six others the lesions had improved significantly.
A sulfonium analog of lecithin has been reported to replace the ubiquitous phosphatidyl choline in a non-photosynthetic diatom, Nitzschia alba. The structure of this sulfonium analog has now been established by chemical synthesis using the following methods: (i) condensation of sn-3-phosphatidic acid (dimyristoyl-, dipalmitoyl-, distearoyl-, distearoyl-, and dioleoyl-) with sulfocholine chloride in the presence of triisopropylbenzenesulfonylchloride in chloroform-pyridine (9:1); and (ii) phosphorylation of 1,2-dipalmitoyl-sn-glycerol with monophenylphosphoryl-dichloridate followed by a reaction with sulfocholine in the presence of pyridine and finally removal of the blocking phenyl group by catalytic hydrogenolysis. The desired synthetic products were obtained in overall yields of 50-70% and 11% for methods (i) and (ii), respectively, and were characterized by elemental analyses; infrared spectroscopy, nuclear magnetic resonance spectrometry, and mass spectrometry; optical rotation; and thin-layer chromatography mobilities. Comparison of the synthetic analogs with the natural sulfolecithin showed them to be identical, except for the nature of the fatty acid chains, thus establishing the natural product as sn-3-phosphatidyl sulfocholine.
Lecithin:cholesterol acyltransferase (LCAT) has been purified from human plasma by sequential preparative ultracentrifugation, ion-exchange chromatography on DEAE-Sephacel, and affinity chromatography on HDL-Sepharose and on wheat germ agglutinin-Sepharose. After the final step, which included preparative electrophoresis or alternatively, chromatography on hydroxylapatite, a purification of about 24 000-fold was obtained. The LCAT preparation was pure according to alkaline polyacrylamide and SDS-polyacrylamide gel electrophoresis and did not react against antisera to apo AI, AII, and D. The LCAT preparation obtained by preparative electrophoresis was stimulated by Cu2+, Ni2+, Co2+, and Zn2+ at both stages of the reaction, phospholipase reaction and cholesterol esterification. This stimulatory effect was abolished by EDTA.
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.
The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis, 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E1%1cm) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Recombinant high density lipoprotein (rHDL) particles were prepared from purified lipids and human apoproteins, and the ability of these complexes to act as substrates for purified lecithin:cholesterol acyltransferase (LCAT) was determined. Increasing the triacylglycerol content relative to cholesteryl ester in rHDL markedly decreased the maximum catalytic potential of LCAT. Kinetic analysis showed that the Vmax of the LCAT reaction was significantly and negatively correlated to the triacylglycerol content. The apparent Km was not directly affected by relative neutral lipid content, but was significantly related to protein and surface lipid content as well as to particle size. These results suggest that while particulate size may regulate the interaction between LCAT and HDL, the relative neutral lipid content of the particle may play a major role in regulating the catalytic potential of the enzyme, particularly with HDL from hypertriglyceridemic patients.
Lecithin-cholesterol acetyltransferase (LCAT) is involved in the synthesis of plasma cholesteryl esters and is pivotal in the maturation of plasma high-density lipoprotein (HDL) and conversion of HDL3 to HDL2. In nephrotic syndrome (NS), the ratio of HDL2 to HDL3 is low even though the total concentration of HDL is generally normal. We hypothesize that the reduced HDL2/HDL3 ratio in NS is due to urinary losses of LCAT, leading to plasma LCAT deficiency. To test this hypothesis, Sprague-Dawley rats were randomized to NS (given 130 mg puromycin aminonucleoside on day 1 and 60 mg ip on day 14) or control groups and were studied on day 30. To dissect the effect of proteinuria from hypoalbuminemia, a group of Nagase rats with inherited hypoalbuminemia was included. Hepatic LCAT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA abundance and plasma and urine LCAT activity were measured. The NS group showed a fourfold rise in serum cholesterol and triglycerides, a fivefold rise in free cholesterol, and a fourfold fall in the HDL-to-total cholesterol ratio. Despite severe hypoalbuminemia, the Nagase rats showed only a mild elevation of serum cholesterol and triglycerides with a normal serum free cholesterol and HDL-to-total cholesterol ratio. The NS group exhibited a normal hepatic LCAT-to-GAPDH mRNA ratio, a marked reduction in plasma LCAT activity, and a significant increase in urinary LCAT excretion. LCAT/GAPDH mRNA and plasma and urine LCAT were normal in Nagase rats. Thus NS led to heavy urinary losses and reduced plasma concentration of LCAT, despite normal hepatic LCAT mRNA abundance. However, hypoalbuminemia, per se, without proteinuria as seen in the Nagase rats had no effect on plasma LCAT or the HDL-to-total cholesterol ratio. Therefore, proteinuria, not hypoalbuminemia, causes LCAT deficiency and a depressed HDL-to-total cholesterol ratio in NS.
We have previously demonstrated that the lungs of fetuses of alloxan-diabetic rabbits are functionally immature when compared with controls. In this study we have examined pulmonary lavage fluid from fetuses of diabetic and control does for the presence of a surfactant inhibitor, cholesterol content, and phosphatidylcholine (lecithin) fatty acid content. When lavage fluids from fetuses of control and diabetic does were mixed and examined on the surface balance, the measured surfactant content corresponded to the arithmetic average of the surfactant content of the individual fluids, thus ruling out the presence of an excess of a surfactant inhibitor. We were able to demonstrate inhibition of surfactant function when exogenous cholesterol was added to lavage samples, but cholesterol content of lavage fluid from fetuses of diabetic and control does was not significantly different. Finally, the fatty acid composition of surfactant phosphatidylcholine was not different between the two groups. These studies indicate that mechanisms other than the presence of a surfactant inhibitor or altered fatty acid composition must explain the functional immaturity seen in offspring of the diabetic pregnancy.
PURPOSE: Retinoids, which include vitamin A (retinol; ROL) and its derivatives, have been investigated in the treatment of bladder cancer. We have shown that expression of the enzyme lecithin:ROL acyltransferase (LRAT), which converts ROL to retinyl esters, is reduced in several human cancers. Here we evaluated expression of LRAT protein and mRNA in normal and malignant bladder tissue specimens from human patients. We also examined the effect of retinoids on LRAT expression in bladder cancer cell lines. EXPERIMENTAL DESIGN: We evaluated 49 bladder cancer specimens for LRAT protein expression using immunohistochemistry with affinity-purified antibodies to human LRAT. LRAT mRNA expression was assessed using reverse transcription-PCR in bladder specimens from an additional 16 patients. We examined the effect of retinoic acid and ROL on LRAT mRNA expression in five human bladder cancer cell lines. RESULTS: LRAT protein was detected throughout the nonneoplastic bladder epithelium in all of the specimens. In bladder tumors, LRAT protein expression was reduced compared with the nonneoplastic epithelium or was completely absent in 7 of 32 (21.9%) superficial tumors versus 16 of 17 (94.1%) invasive tumors (P < 0.001). All of the non-neoplastic bladder specimens tested (11 of 11) showed LRAT mRNA expression, compared with 5 of 8 (62%) superficial tumors and 0 of 5 (0%) invasive tumors (P = 0.001). Three of five human bladder cancer cell lines expressed LRAT mRNA independent of retinoid exposure, whereas in two cell lines LRAT mRNA expression was induced by retinoid treatment. CONCLUSIONS: We report a significant reduction in LRAT expression in bladder cancer. Moreover, we demonstrate an inverse correlation of LRAT mRNA and protein expression with increasing tumor stage. These data suggest that loss of LRAT expression is associated with invasive bladder cancer.
Dietary fats are known to influence the fatty acid profile of plasma lipids, including phospholipids which are substrates of lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43), an important enzyme in lipoprotein metabolism. We tested whether the dietary fatty acid profile has an effect on LCAT activity in an animal model. Rats were conditioned to eat two meals per day, which were enriched in either palmitic, oleic or linoleic acids, for 10 weeks. Serum was isolated from blood samples taken prior to the meal. The LCAT activity was determined in two ways: (1) by measuring serum cholesterol esterification rates, which are an estimate of LCAT action on endogenous lipoproteins, and (2) by measuring serum LCAT activity levels with excess exogenous substrates, an estimate of LCAT mass. Animals receiving the linoleic acid diet had lower serum concentrations of unesterified cholesterol and triglycerides, if compared with animals fed oleic acid or palmitic acid diets (p < 0.05). Serum LCAT activity levels (measured with excess exogenous substrates) were not different, but both the absolute and fractional rates of cholesterol esterification were highest on the linoleic acid rich diet (p < 0.01), showing that LCAT action on endogenous lipoproteins is improved. No differences were found in serum apolipoprotein B and A-IV concentrations between the dietary groups. Apolipoprotein A-I levels were lowest in the palmitic acid group (oleic and linoleic > palmitic; p < 0.05), and apolipoprotein E levels were highest in the palmitic acid group (palmitic > oleic and linoleic; p < 0.05). It is concluded that a linoleic acid rich diet may cause increased metabolism of serum cholesterol by LCAT in rats. This effect is not due to elevated serum concentrations of LCAT or of its apolipoprotein activators, but most likely to changes in the chemical composition of endogenous lipoprotein substrates. It remains to be established whether the serum cholesterol esterification rates measured in vitro are related to in vivo rates of reverse cholesterol transport.
We studied the effects of 40 and 80 mg/day of tacrine on patients with probable Alzheimer's disease (AD) in an 8-week, randomized, double-blind, placebo-controlled crossover trial with an enriched-population design. In the initial dose titration phase, an intent-to-treat analysis showed significantly more improvement with 80 mg/day of tacrine than placebo. In the subsequent crossover trial that included only 'responders', no significant improvement was observed with tacrine, whether or not it was given with lecithin. We found that individualized dose titration and enrichment strategies were not helpful and had the effect of reducing the power of the study. In the dose titration phase of this study we found that more impaired subjects were as likely to improve as those who were less impaired, suggesting that tacrine should be further investigated in more severely demented AD patients.
The electroencephalographic (EEG) and neuropsychological effects of single, oral doses or piracetam (800-6,400 mg) and of its association with lecithin (4,800 mg and 25 g, respectively) were investigated in two groups of healthy volunteers in placebo-controlled studies. The EEG was quantified by power spectral analysis; both parametric and nonparametric procedures were applied to process the EEG and neuropsychological data statistically. The drug plasma concentration was assessed (gas chromatography) in concomitance with the EEG and neuropsychological measurements. Piracetam was found to elicit systematic EEG effects, namely a decrease in the low-frequency components and an increase in the power of the 8.5- to 12.0-Hz and of the fast-frequency components. The EEG modifications were restricted to the anterior scalp areas; its detection by means of parametric statistics was conditioned by the relevant individual variability and was inferred resting upon a number of criteria (indication at nonparametric tests, topography, consistency across subjects, replicability, etc). The correlation between EEG changes and drug plasma concentration was ambiguous, and the compound was not found to be effective on any of the neuropsychological variables considered. The study exemplifies some of the methodological problems in quantitative pharmaco-EEG and emphasizes the relevance of topography and replication.
Plasma lipoprotein changes were evaluated in 65 type II patients undergoing sequential 4-week dietary treatments with: (I) standard low-lipid diet; (II) low-lipid diet with total replacement of animal proteins with textured soy proteins containing 6% of lecithin (L-TVP); (III) standard low-lipid diet; (IV) low-lipid diet with a 50% substitution of animal proteins with L-TVP. Total cholesterolemia was significantly reduced in both periods of L-TVP administration: -18.6% during phase II (total replacement) and -13.2% during phase IV (partial replacement). High-density lipoprotein (HDL) cholesterol levels tended to increase during L-TVP administration. However, only patients in the mid- and low tertiles for HDL cholesterolemia showed a significant increase of HDL levels during L-TVP. This 'normalizing' activity of L-TVP on plasma lipoproteins, even when administered as a partial dietary substituent, may be of clinical interest for subgroups of patients at high vascular risk.
We analyzed the effects of different types of dietary fat on the lecithin-cholesterol acyltransferase (LCAT) activity in 20 healthy women aged 26-49 years, consuming 6-week diets containing 54% of the calories as carbohydrates, 16% as protein and 30% as fat. The tested fats were successively: low erucic acid rapeseed (LEAR oil), sunflower oil, peanut oil and milk fats (butter and cream). The fractional and molar rates of LCAT were higher after sunflower and peanut oil diets and decreased significantly after LEAR oil and milk fat diets. The LCAT activity was independent of the P/S ratio of the diet, but positively correlated with the percentage of linoleic acid in serum phospholipids and cholesteryl esters, and negatively correlated with the percentage of oleic acid in the same fractions. Our results showed no relation between LCAT activity and the concentration of plasma cholesterol or triglycerides, no change in serum cholesteryl ester/total cholesterol ratio and confirmed that the distribution of high density lipoprotein subfractions is due to prerequisites other than LCAT.
Lecithin cholesterol acyltransferase (LCAT) and lysolecithin acyltransferase (LAT) activities in plasma were significantly lower in undialyzed than in dialyzed uremic patients and controls. Both LCAT and LAT activities were normal in the maintenance dialysis group. The free/total cholesterol ratio was significantly increased in the undialyzed patients only. This ratio and LCAT were inversely related in the dialyzed group, unrelated in the undialyzed patients, and positively correlated in controls. Unlike non-uremic populations with hypertriglyceridemia in which LCAT is increased, no correlation was found between plasma triglyceride and LCAT in the uremic population studied. Our results show that both LCAT and LAT activities are reduced in undialyzed chronic uremic patients but are not significantly different from control levels in patients treated with chronic hemodialysis. These results indicate that the changes in LCAT activity contrast with those described in lipoprotein composition and metabolism which do not appear to be affected by chronic dialysis therapy.
Serum apolipoproteins A (Apo-A) and B (Apo-B) and lecithin: cholesterol acyl transferase (LCAT) activities and 24-hour urinary cholesterol levels were estimated in 25 nephrotic children before and during steroid treatment with 4 weeks of daily prednisolone followed by another 4 weeks of alternate-day prednisolone. The patients with untreated nephrotic syndrome (NS) showed significant decrease in serum Apo-A and LCAT activities associated with significant increase in serum Apo-B and urinary cholesterol levels compared to healthy controls (n = 25). Serum Apo-A levels correlated directly and Apo-B levels inversely with the serum albumin concentrations. After a transient elevation, the serum Apo-A level returned to control range by 8 weeks of treatment accompanied by a gradual increase in serum LCAT activity and decrease in urinary cholesterol excretion. Though, the serum Apo-B level was decreased with treatment, it was still significantly high compared to the controls.
A bubble method was used to record dynamic area/surface tension diagrams of mixed lecithin-cholesterol films prepared at different weight ratios. The lecithin/cholesterol weight ratios were: 1:2, 1:1, 4:1, 6:1, 8:1 and 12:1. The minimum surface tension is about 20 dyn/cm for all weight ratios at amplitudes of 75, 25, 12.5, 6 and 3% area oscillation. The maximum surface tension depends on both the amplitude of area oscillation and the lecithin/cholesterol weight ratio. The maximum surface tension increases with increasing amplitude. At the amplitudes 25, 12.5, 6 and 3% the maximum surface tension is significant higher at the weight ratios 1:2 and 1:1 compared to the other ratios. In addition, equilibrium surface tensions during interruptions of the oscillation at maximum and minimum area were recorded. The equilibrium surface tension increases with increasing amplitude up to 25% area oscillation. A static hysteresis seems to remain. Some consequences regarding the pulmonary respiratory mechanics are discussed.