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PubMed1976

Nanosecond time-resolved emission spectroscopy of a fluorescence probe adsorbed to L-alpha-egg lecithin vesicles.

Nanosecond time-resolved emission spectra (TRES) are fluorescence emission spectra obtained at discrete times during the fluorescence decay. The complete data-set obtainable is a surface representing the intensity at all wavelengths and times during the emission decay time. When 2-p-toluidinonaphthalene-6-sulfonate (2,6 p-TNS) is adsorbed to egg lecithin vesicles, an excited-state reaction associated with energetic changes of the emitting species occurs on the nanosecond time scale. Convolution of the fluorescence decay with the excitation response introduces an artifact in the time-dependent spectra. A precedure is described by which this artifact can be eliminated. The data for the generation of time-resolved emission spectra are obtained with a computer-interfaced instrument based on the single-photon counting method.

Binding Sites↗
PubMed1977

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.

Chemical Phenomena↗
PubMed1979

Liquid diffraction analysis of the model membrane system. Egg lecithin + myelin protein (N-2).

The scattering from the model membrane system, egg lecithin + myelin protein (N-2), has been analyzed by liquid diffraction methods. It is found that by manipulating the protein-lipid ratio, the scattering domains of the protein and lipid can be identified. The multilayer contribution can also be identified by its position and concentration behavior in both the intensity pattern and its Fourier transform. When the multilayer and protein components are subtracted, the phospholipid scattering and an interaction term are left: these two can be resolved by a reasonable assessment of their relative magnitude in the boundary region where they overlap. The interaction term can then be used to determine the most probably position of the protein in the membrane. The deconvoluted protein and lipid transforms can then be combined in the proper way to obtain the electron density profiles. The resolution of the interaction term is not yet complete, but a method for accomplishing this is discussed.

Egg Yolk↗
PubMed1979

The model membrane system. Egg lecithin + myelin protein (N-2). Effect of solvent density variation on the X-ray scattering.

The electron density contrast method has been applied to the membrane system egg lecithin + myelin protein (N-2). The case treated here is for a low protein concentration. As theory predicts, the scattering from the different regions of the membrane (protein, hydrocarbon, and polar head group regions) are modulated differently by changing the contrast. It is then possible to separate out the electron pair correlation functions for the different regions, and from these to determine the membrane election density distribution for an external electron density p0 = 0.

Egg Yolk↗
PubMed1981

NMR study of synthetic lecithin bilayers in the vicinity of the gel-liquid--crystal transition.

1H, 2H, and 31P NMR methods have been employed in the study of dimyristoyl lecithin bilayers hydrated with D2O in the gel (L beta'), intermediate (P beta') and liquid-crystalline (L alpha) phases. For D2O/lipid molar ratios, n, in the range 7 less than or equal to n less than or equal to 11 discontinuities are observed in the deuterium NMR splittings at both main and pretransitions. A partial phase diagram based on NMR and differential scanning calorimetry data is presented. 1H NMR dipolar splittings are observed for macroscopically oriented samples in all three phases. Changes in the 1H splittings are correlated with 2H and 31P data and interpreted to show that the chain tilt in the gel phase undergoes a discontinuous change on transition to the intermediate phase, which brings the chain axes closer to the bilayer normal. An estimate of chain tilt in the gel phase is made on the basis of NMR data and found to be approximately 23 degrees for a sample with n = 11 at 18 degrees C.

Crystallization↗
PubMed1981

Osmotic shrinkage of giant egg-lecithin vesicles.

Osmotic shrinkage of giant egg-lecithin vesicles was observed by phase-contrast microscopy. The vesicles remained or became spherical when shrinking. Small and thick-walled vesicles formed visible fingers attached to the sphere. The water permeability of the single bilayer was found to be 41 micrometers/s. A variety of observations indicate that osmosis induces a parallel lipid flow between the monolayers of the bilayer, leading to a strong positive spontaneous curvature. They also suggest the formation of mostly submicroscopic daughter vesicles. The estimated coupling constant, 2 . 10(-6) mol/mol, is large enough to be biologically significant.

Lipid Bilayers↗
PubMed1983

A 1H and 13C NMR study of motional changes of dipalmitoyl lecithin associated with the pretransition.

Motional changes of the dipalmitoyl lecithin molecule associated with the pretransition in multibilayers are investigated by proton-enhanced 13C-NMR and proton spin-locking experiments. The nitrogen-bound methyl groups of the polar head exhibit faster motion and more disorder in the intermediate phase compared with the gel phase. Although little or no change occurs in the hydrocarbon chain order at the pretransition, the corresponding motional correlation time changes by one order of magnitude. This is consistent with a model involving rotational motion of the hydrocarbon chains about their long axes: in the gel phase the motion is such that neighboring chains make an oscillating disrotatory motion, while in contrast, in the intermediate phase a quasi-free chain rotation takes place. Earlier contradicting results of Davies, J., 1979, Biophys. J., 27:339-358, and ourselves, Trahms, L., and E. Boroske, 1979, Biochim, Biophys. Acta. 552:189-193, are explained by this model.

Kinetics↗
PubMed1984

Photoelectric signals generated by bovine rod outer segment disk membranes attached to a lecithin bilayer.

Purified bovine rod outer segment disk membranes were attached to a lecithin bilayer membrane. After photoexcitation with a 500-nm flash delivered by a dye laser, a negative photovoltage was observed on the bilayer under normal ionic strengths (100 mM KCl), which had a rise phase of 1-3 ms at 20 degrees C. The photoresponse was obviously due to bleaching of rhodopsin as it decreased for successive flashes of light. It originated most probably during the metarhodopsin-I metarhodopsin-II (meta-I-II) transition of rhodopsin because it was pH dependent at 2 degrees C but not at 20 degrees C. At 10 mM KCl, i.e., under hypotonic conditions, a positive photovoltage with slower kinetics than at high salt was observed. As the disk membranes were merely attached to the bilayer membrane, the photovoltage was apparently due to a light-induced transmembrane potential change in the disk membranes. Possible electrogenic mechanisms underlying the photosignal will be discussed.

Animals↗
PubMed1987

Partial specific volumes of lipid and water in mixtures of egg lecithin and water.

We describe in this paper measurements of the partial specific volumes of lipid (upsilon L) and water (upsilon W) in mechanical mixtures of egg yolk lecithin and water over a range of hydrations (5-55 wt% water) that includes the region at which excess water appears. upsilon L and upsilon W are found to be very nearly 1 cm3/g over the entire range. The water activities of the mixtures were also determined and found to be the same as for lipid deposited as oriented multilayers on solid substrates.

Liposomes↗
PubMed1993

Measurement of chain tilt angle in fully hydrated bilayers of gel phase lecithins.

The tilt angle theta tilt of the hydrocarbon chains has been determined for fully hydrated gel phase of a series of saturated lecithins. Oriented samples were prepared on glass substrates and hydrated with supersaturated water vapor. Evidence for full hydration was the same intensity pattern of the low angle lamellar peaks and the same lamellar repeat D as unoriented multilamellar vesicles. Tilting the sample permitted observation of all the wide angle arcs necessary to verify the theoretical diffraction pattern corresponding to tilting of the chains towards nearest neighbors. The length of the scattering unit corresponds to two hydrocarbon chains, requiring each bilayer to scatter coherently rather than each monolayer. For DPPC, theta tilt was determined to be 32.0 +/- 0.5 degrees at 19 degrees C, slightly larger than previous direct determinations and considerably smaller than the value required by recent gravimetric measurements. This new value allows more accurate determinations of a variety of structural parameters, such as area per lipid molecule, A = 47.2 +/- 0.5 A2, and number of water molecules of hydration, nw = 11.8 +/- 0.7. As the chain length n of the lipids was increased from 16 to 20 carbons, the parameters A and nw remained constant, suggesting that the headgroup packing is at its excluded volume limit for this range. However, theta tilt increased by 3 degrees and the chain area Ac decreased by 0.5 A2. This behavior is explained in terms of a competition between a bulk free energy term and a finite or end effect term.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2003

2,3,7,8-tetrachlorodibenzo-p-dioxin induces lecithin: retinol acyltransferase transcription in the rat kidney.

Vitamin A (retinoids) has an essential role in development and throughout life of humans and animals. Consequently, effects of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on retinoid metabolism may be contributory to its toxicity. This study was performed to clarify the mechanism behind dioxin-induced retinyl ester formation in the rat kidney. In addition we investigated the possible role of CYP1A1 in dioxin-induced all-trans-retinoic acid (atRA) formation. Male Sprague-Dawley rats were exposed to a single oral dose of TCDD in a combined dose-response and time-course study, with doses ranging from 0.1 to 100 microg/kg bw and time points from 1 to 28 days. Levels of atRA and the expression of two potentially retinoic acid (RA)-controlled proteins critically involved in retinoid storage regulation, lecithin: retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP I), were analyzed in liver and kidney. The expression and activity of cytochrome P4501A1 (assayed as ethoxyresorufin-O-deethylase activity) was assessed to gain insight into its potential role in RA synthesis. There was a significant increase in LRAT mRNA expression in the kidney, whereas no such increase could be observed in the liver, despite significantly increased atRA levels in both tissues. This suggests a tissue-specific regulation of LRAT by TCDD that may be dependent on other factors than atRA. Neither CRBP I mRNA nor protein levels were altered by TCDD. The time-course relationship between CYP1A1 activity and atRA levels in liver and kidney does not exclude a role of CYP1A1 in TCDD-induced RA synthesis. The observed altered regulation of the retinoid-metabolizing enzyme LRAT, together with the low doses and short time required by TCDD to change tissue RA levels, suggest that enzymes involved in retinoid metabolism are specific and/or direct targets of TCDD.

Acyltransferases↗
PubMed1997

Higher high density lipoprotein cholesterol associated with moderate alcohol consumption is not related to altered plasma lecithin:cholesterol acyltransferase and lipid transfer protein activity levels.

Lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are important factors involved in HDL metabolism. Altered plasma activity levels of these factors could play a role in the increase in high density lipoprotein (HDL) cholesterol associated with moderate alcohol consumption. We measured plasma LCAT, CETP and PLTP activities with exogenous substrate assays, as well as lipoproteins and HDL lipids in 6 alcohol-abstaining men, 18 matched men who used < or = 1 and 18 men who used > or = 1 alcohol-containing drinks per day. Plasma cholesterol and triglycerides were similar in the three groups. HDL total cholesterol, HDL cholesteryl ester, HDL free cholesterol and HDL triglycerides were higher in the alcohol drinkers compared to the abstainers (all P < 0.05). No differences in plasma LCAT, CETP and PLTP activity levels were observed between the three groups. Analysis of covariance also demonstrated that the use of alcohol was associated with higher HDL cholesterol (P < 0.04), whereas plasma LCAT, CETP and PLTP activity levels were not related to alcohol consumption. Furthermore, HDL cholesteryl ester was positively associated with LCAT activity (P < 0.001), PLTP activity (P < 0.01) and alcohol intake (P < 0.04) and negatively with plasma triglycerides (P < 0.001) and CETP activity (P < 0.03); indicating that alcohol influenced HDL cholesteryl ester independently from these biochemical parameters. The higher HDL cholesterol associated with moderate alcohol consumption is, therefore, unlikely to be caused by and effect on plasma LCAT, CETP or PLTP activity levels.

Adolescent↗
PubMed2001

Lecithin:cholesterol acyl transferase G30S: association with atherosclerosis, hypoalphalipoproteinemia and reduced in vivo enzyme activity.

OBJECTIVES: A 69 yr old male was referred for assessment of a very low plasma HDL cholesterol and apolipoprotein AI concentration. At age 65, he had undergone triple vessel coronary bypass graft surgery. He had a strong family history of early coronary heart disease. We analyzed the molecular basis of his clinical and biochemical abnormalities. DESIGN AND METHODS: We used DNA sequencing to determine whether mutations in LCAT were present. We also evaluated plasma biochemistry and LCAT activity. RESULTS: DNA sequencing revealed that the patient was a heterozygote for the G30S mutation in the gene encoding lecithin:cholesteol acyl transferase (LCAT). His plasma was found to have half-normal LCAT activity. CONCLUSIONS: The findings in this patient suggest that rare dysfunctional mutations in candidate genes, such as LCAT, can contribute to the spectrum of patients ascertained because of low HDL cholesterol.

Aged↗
PubMed1997

Reduced lecithin: cholesterol acyltransferase (LCAT) and Na+, K+, ATPase activity in diabetic patients.

OBJECTIVE: To investigate the plasma concentrations of lecithin:cholesterol acyltransferase (LCAT EC 2.3.1.43) and erythrocyte membrane Na+, K+, ATPase and the correlation of these parameters in diabetes mellitus. DESIGN AND METHODS: Na+, K+, ATPase was measured with spectrophotometric method and LCAT with radioactive method in 19 patients with insulin-dependent diabetes mellitus (IDDM), in 20 with non-insulin-dependent diabetes mellitus (NIDDM) and in 20 healthy volunteers as the control group. RESULTS: Compared with the control group, plasma LCAT concentrations were found to be decreased in both of the patient groups (p < 0.01 for both). Erythrocyte membrane Na+, K+, ATPase activities were higher in the controls than both in the NIDDM and IDDM groups (p < 0.01 and p < 0.001, respectively). There were significant correlations between LCAT and Na+, K+, ATPase in IDDM (r = 0.82, p < 0.001) and in NIDDM (r = 0.74, p < 0.001). In order to investigate the effect of cholesterol (C) and lysophosphatidylcholine (LPC) on Na+, K+, ATPase activity, this enzyme's activity was determined in erythrocyte membranes obtained from diabetic subjects after in vitro incubation with increasing concentrations of LPC and C (2-10 microM). Enzymatic activity was significantly reduced by in vitro C at increasing concentrations but significantly increased by in vitro LPC at increasing concentrations. CONCLUSIONS: From these data, it is to be concluded that the decrease in Na+, K+, ATPase activity in diabetes might be due to decreased LCAT concentrations and that may explain the development of atherosclerosis in diabetics.

Adult↗
PubMed2000

Low plasma lecithin:cholesterol acyltransferase and lipid transfer protein activities in growth hormone deficient and acromegalic men: role in altered high density lipoproteins.

Growth hormone (GH) deficiency and acromegaly may be associated with increased cardiovascular risk. Little is known about alterations in high density lipoproteins (HDL) in these conditions. Lecithin:cholesterol acyl transferase (LCAT) has the ability to esterify free cholesterol (FC) in HDL. Cholesteryl ester transfer protein (CETP) is able to transfer cholesteryl esters (CE) from HDL to very low and low density lipoproteins (VLDL and LDL). During phospholipid transfer protein (PLTP)-mediated HDL remodelling, small pre beta-HDL particles are generated which serve as acceptors for cellular cholesterol and provide the initial LCAT-substrate. We documented plasma lipids, LCAT, CETP and PLTP activity levels as well as plasma cholesterol esterification (EST) and cholesteryl ester transfer (CET) in 12 adult men with acquired GH deficiency, 12 acromegalic men and 24 healthy male subjects. All GH deficient and acromegalic patients received conventional hormonal replacement therapy if necessary. VLDL + LDL cholesterol and plasma triglycerides were higher in GH deficient (P < 0.01 and P < 0.05) and acromegalic patients (P < 0.05 and P < 0.01) than in healthy subjects. HDL cholesterol and HDL CE were lower (P < 0.05 for both) and the HDL FC/CE ratio was higher (P < 0.01) in these patient groups compared to healthy subjects. Plasma LCAT, CETP and PLTP activity levels were lower in acromegalic patients (P < 0.01 for all) and CETP activity was lower in GH deficient patients (P < 0.01) compared to healthy subjects. Plasma EST and CET were decreased in both acromegalic (P < 0.01 for both) and GH deficient patients (P < 0.05 for both). Multiple regression analysis demonstrated independent negative relationships of plasma insulin-like growth factor I with plasma LCAT (P = 0.0001), CETP (P = 0.009) and PLTP activity levels (P = 0.021). Plasma LCAT (P = 0.0001) and CETP activity (P = 0.0001) were also negatively associated with (substitution therapy for) adrenal insufficiency. In conclusion, GH deficient and acromegalic patients show abnormalities in HDL, consistent with impaired LCAT action. Decreases in plasma EST and CET in such patients, as well as a low PLTP activity in acromegaly suggest that reverse cholesterol transport may be impaired, contributing to increased cardiovascular risk.

Acromegaly↗
PubMed2001

Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion products. Based on these results, we propose that cigarette smoke may contribute to atherogenesis by inhibiting the activities of plasma PAF-AH and LCAT, but the nature of this inhibition differs for the enzymes.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2002

Changes in plasma lecithin: cholesterol acyltransferase activity, HDL(2), HDL(3) amounts and compositions in patients with chronic renal failure after different times of hemodialysis.

The aim of this study was to investigate the effect of hemodialysis duration on HDL(2) and HDL(3) compositions and lecithin:cholesterol acyltransferase (LCAT) activity in 58 patients on acetate hemodialysis using cuprophane membrane, after different periods of dialysis. Patients were divided into three groups-GI <1 year, GII 1-5 years, GIII 5-13 years of dialysis and were compared with 22 controls. Increase by 34% of triacylglycerols (TG) was noted in GI and by 36% in GII versus GI. Hypertriglyceridemia was correlated with hemodialysis duration (HD) (r=0.75, P<0.05). The LCAT activity decrease by 25% was noted in GII versus GI (P<0.05) and by 45% in GIII versus GI (P<0.01), this activity was negligible in 33% of GII and 39% of GIII. LCAT activity was negatively correlated with HD (r=-0.80, P<0.001). Hemodialysis duration did not influence HDL(3) and HDL(2) amounts, HDL(3)-phospholipids (PL), HDL(3) and HDL(2)-cholesteryl esters (CE) and HDL(2)-apolipoproteins. However, an increase by 56% in HDL(2)-PL was noted in GIII versus GI and GII (P<0.01). HDL(2)-unesterified cholesterol (UC) were 2-fold higher in GIII than GII and GI (P<0.01). HDL(2)-TG were 2.2-fold higher in GII and 2.4-fold in GIII than GI (P<0.001). HDL(3)-apolipoproteins were 1.5-fold and 1.8-fold lower in GI than GII (P<0.05) and GIII (P<0.01), respectively. An increase in HDL(3)-UC by 20% in GII and 33% in GIII versus GI (P<0.01) was noted. HDL(3)-TG increase by 66% was noted in GII and GIII versus GI (P<0.05). We conclude, that the long-term hemodialysis does not act on lipid anomalies following CRF. Alterations in HDL composition contribute to the reduced efficacy of reverse cholesterol transport, which is a risk factor for cardiovascular disease.

Adult↗
PubMed2002

Analysis of apolipoprotein A-I, lecithin:cholesterol acyltransferase and glucocerebrosidase genes in hypoalphalipoproteinemia.

Hypoalphalipoproteinemia (HALP) is a dyslipidemia characterized by low HDL-cholesterol (HDL-C) levels with important genetic contribution. However, no common genetic mutations have been found to be associated with this disorder. We screened the promoter and coding sequence of apolipoprotein (apo) A-I and lecithin:cholesterol acyltransferase (LCAT) genes and the 5' apo C-III region by SSCP and heteroduplex analysis, and DNA sequencing in 66 unrelated subjects with recurrent low HDL-C levels. We also analyzed the N370S and L444P variants, in the glucocerebrosidase (GBA) gene by restriction fragment analysis. Three mutations in the apo A-I gene (L144R, W108R, g.1833C>T) and 3 mutations in the LCAT gene (S208T, I178T, IVS3-23C>A) were detected, in six heterozygous subjects. In addition, a novel polymorphic site in LCAT gene (g.4886C>T) has been identified. Allelic frequencies of polymorphisms g.(-636)C>A, g.(-625)G>A, g.(-620)T>del, g.(-479C>T and g.(-452)T>C, located upstream of the apo C-III gene, were in normal range, and no other mutation was found in this region. Two HALP subjects were found to carry the N370S mutation at GBA locus. In conclusion, 12% of HALP subjects were found to carry mutations in apo A-I, LCAT, or GBA genes, which could explain this phenotype. Our results confirm the molecular, genetic and phenotypic heterogeneity of HALP.

Adult↗