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The Effect of pH on Monoglyceride-Caseinate Mixed Monolayers at the Air-Water Interface.

The surface pressure (pi) area (A) isotherms and Brewster angle microscopy (BAM) of monoglyceride-caseinate mixed films spread on buffered water at pH 5 and 7 and at 20 degrees C were determined as a function of the mass fraction (X) of monoglyceride (monopalmitin or monoolein) in the mixture. The structural characteristics, miscibility, and morphology of monoglyceride-caseinate mixed films are very dependent on surface pressure and monolayer composition. The monolayer structure was more expanded as the pH and the monoglyceride concentration in the mixture were increased. From the concentration and surface pressure dependence on excess area, free energy, and collapse pressure, it was deduced that, at a macroscopic level, monoglyceride (either monopalmitin or monoolein) and caseinate form a practically immiscible monolayer at the air-water interface. The BAM images and the evolution with the surface pressure of the relative reflectivity of BAM images give complementary information on the interactions and structural characteristics of monoglyceride-caseinate mixed monolayers, which at a microscopic level corroborated in part the conclusions derived from the pi-A isotherm at a macroscopic level. Over the overall range of existence of the mixed film the monolayer presents some heterogeneity due to the fact that domains of monoglyceride (especially of monopalmitin) and spots of collapsed caseinate residues are present during the monolayer compression-expansion cycle, giving relative intensity peaks with high relative film thickness. At higher pi, after the caseinate collapse, characteristic squeezing-out phenomenon was observed. At the monoglyceride monolayer collapse the mixed film is practically dominated by the presence of monoglyceride. The prevalence of monoglyceride in the interface increases with the amount of monoglyceride in the mixture and at higher pi. However, some degree of interactions exists between monoglyceride and caseinate in the mixed film and these interactions are more pronounced as the monolayer is compressed at the highest surface pressures. Copyright 2001 Academic Press.

Journal Article↗

Penetration of beta-lactoglobulin into monoglyceride monolayers. dynamics, interactions, and topography of mixed films.

In this work we have analyzed the penetration of betalactoglobulin into a monoglyceride monolayer (monopalmitin or monoolein) spread at the air-water interface and its effects on the structural, dilatational, and topographical characteristics of mixed films. Dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology have been used, maintaining the temperature constant at 20 degrees C and the pH and ionic strength at 7 and 0.05 M, respectively. The initial surface pressure (mN/m) of the spread monoglyceride monolayer (pii(MONOGLYCERIDE)) at 10, 20, and the collapse point is the variable studied. Beta-lactoglobulin can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-lactoglobulin into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, requires monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of monoglyceride penetrated by beta-lactoglobulin mixed monolayers are essentially dominated by the presence of monoglyceride (either monopalmitin or monoolein) in the mixed film. In fact, monoglyceride molecules have the capacity to re-enter the monolayer after expansion and recompression of the mixed monolayer. Thus, monoglyceride molecular loss by collapse and/or desorption is reversible. The topography of the monolayer under dynamic conditions corroborates these conclusions.

Air↗

Thermodynamic and dynamic characteristics of monoglyceride monolayers penetrated by beta-casein.

In this work, we have analyzed the dynamics of the penetration of beta-casein into monoglyceride monolayers (monopalmitin and monoolein) and the structural, dilatational, and topographical characteristics of mixed films formed by monoglyceride penetrated by beta-casein. Different complementary experimental techniques [dynamic tensiometry, surface film balance, Brewster angle microscopy (BAM), and surface dilatational rheology] have been used, maintaining the temperature constant at 20 degrees C and the pH at 7. The surface pressure of the monoglyceride monolayer at the beginning of the penetration process (at pi(i)MP and pi(i)MO for monopalmitin and monoolein, respectively) was the variable studied. beta-Casein can penetrate into a spread monoglyceride monolayer at every surface pressure. The penetration of beta-casein into the monoglyceride monolayer with a more condensed structure, at the collapse point of the monoglyceride, is a complex process that is facilitated by monoglyceride molecular loss by collapse and/or desorption. However, the structural, topographical, and dilatational characteristics of the monoglyceride penetrated by beta-casein mixed monolayers are essentially dominated by the presence of the monoglyceride (either monopalmitin or monoolein) in the mixed film.

Animals↗

The specificity of monoglyceride-protein interactions and mechanism of the protein induced L(beta) to coagel phase transition.

This study aims at gaining insight into the specificity and molecular mechanism of monoglyceride-protein interactions. We used beta-lactoglobulin (beta-LG) and lysozyme as model proteins and both monostearoylglycerol and monopalmitoylglycerol as defined gel phase monoglycerides. The monoglycerides were used in different combinations with the two negatively charged amphiphiles dicetylphosphate and distearylphosphate. The interactions were characterized using the monolayer technique, isothermal titration calorimetry, (2)H-nuclear magnetic resonance (NMR) using deuterium labelled monoglycerides and freeze fracture electron microscopy (EM). Our results show that lysozyme inserts efficiently into all monolayers tested, including pure monoglyceride layers. The insertion of beta-LG depends on the lipid composition of the monolayer and is promoted when the acylchains of the negatively charged amphiphile are shorter than that of the monoglyceride. The binding parameters found for the interaction of beta-LG and lysozyme with monoglyceride bilayers were generally similar. Moreover, in all cases a large exothermic binding enthalpy was observed which was found to depend on the nature of the monoglycerides but not of the proteins. (2)H-NMR and freeze fracture EM showed that this large enthalpy results from a protein mediated catalysis of the monoglyceride L(beta) to coagel phase transition. The mechanism of this phase transition consists of two steps, an initial protein mediated vesicle aggregation step which is followed by stacking and probably fusion of the bilayers.

Freeze Fracturing↗

The Effect of monoglycerides on structural and topographical characteristics of adsorbed beta-casein films at the air-water interface.

The effect of monoglycerides (monopalmitin and monoolein) on the structural and topographical characteristics of beta-casein adsorbed film at the air-water interface has been analyzed by means of surface pressure (pi)-area (A) isotherms and Brewster angle microscopy (BAM). At surface pressures lower than that for the beta-casein collapse (pi(c)(beta-casein)), attractive interactions between beta-casein and monoglycerides were observed. At higher surface pressures, the collapsed beta-casein is partially displaced from the interface by monoglycerides. However, beta-casein displacement by monoglycerides is not quantitative at the monoglyceride concentrations studied in this work. From the results derived from these experiments, we have concluded that interactions, miscibility, and displacement of proteins by monoglycerides in adsorbed mixed monolayers at the air-water interface depend on the particular protein-monoglyceride system, the interactions between film-forming components being higher for adsorbed than for spread films. The adsorbed films are more segregated than spread films, and both collapsed protein domains and monoglyceride domains in adsorbed films are smaller than for spread films.

Adsorption↗

The production of a bactericidal monoglyceride in staphylococcal abscesses.

The treatment of abscess homogenates with calcium ionophores stimulated the production of a bactericidal lipid with properties indistinguishable from those of a previously unidentified bactericidal lipid that had been detected in staphylococcal abscesses. The lipid was identified as a monoglyceride by thin layer chromatography. It resembled the unidentified lipid in that it had a high specific activity, exhibited differential activity, was inhibited by Staphylococcus aureus delta toxin, lecithin and Ca++, and its activity was reduced by oxidation. Stimulation of monoglyceride production by calcium ionophore requires the joint presence of components from the sedimented and supernatant fractions of abscess homogenates, and was not produced if boiled homogenate was used. The addition of verapamil interfered with the production of monoglyceride in homogenates treated with calcium ionophore. Monoglyceride was produced only in abscess homogenates and not in homogenates of other normal tissues or tissues taken from mice infected with S. aureus. Calcium ionophore could be replaced by inositol triphosphate, suggesting that monoglyceride production involved the release of calcium from intracellular stores. The 2-monoglyceride was the form originally produced in abscess homogenates, but this spontaneously isomerized to the 1-monoglyceride. The fatty-acid moiety of the monoglyceride consisted primarily of 16:0 and 16:1 fatty acids.

Abscess↗

1-arachidonyl-monoglyceride causes platelet aggregation: indirect evidence for an acylglycerol acylhydrolase involvement in the release of arachidonic acid for prostaglandin synthesis.

Phosphatidylcholine liposomes containing 1-arachidonyl-monoglyceride were found to cause aggregation of human platelets. In contrast, addition of phosphatidylcholine liposomes, 1-arachidonyl-monoglyceride, or phosphatidylcholine liposomes containing I-oleoyl-monoglyceride to a similar platelet preparation had no effect. Aggregation stimulated by 1-arachidonyl-monoglyceride was inhibited by 100 microM aspirin or 1 microM indomethacin, suggesting that the arachidonic acid is first released by; a platelet acylglycerol acylhydrolase and then converted to PGG2 and thromboxane A2 which initiate the platelet aggregation. Changes in platelet morphology in response to 1-arachidonyl-monoglyceride were similar to those reported previously to occur following stimulation of platelets by arachidonic acid or PGG2 providing further support for this concept. EDTA inhibited aggregation of platelets but no shape change or granule centralization in response to 1-arach-idonyl-monoglyceride. PGE1 and theophylline inhibited both aggregation and morphological changes. These results with inhibitors are similar to the effects of these inhibitors on PGG2 and provide further evidence for similarity between the action of 1-arachidonyl-monoglyceride and PGG2. The results provide important evidence to support the concept that an acylglycerol acylhydrolase may be involved in arachidonic acid release and platelet aggregation.

Arachidonic Acids↗

Monoglyceride hydrolase activities of rat plasma and platelets. Their properties and roles in the activity of lipoprotein lipase.

Rat plasma contains monoglyceride hydrolase activities against both 1(3)- and 2-monoglycerides. These activities are present as lipoprotein complexes recovered by either density flotation or by agarose gel chromatography with plasma high density lipoprotein. However neither activity is complexed with the major apoproteins (apo-A-I, apo-E) of this lipoprotein class. 2-Monoglyceride hydrolase (but not 1(3)-monoglyceride hydrolase) activity associates with the triglyceride-rich lipoprotein class. The two activities are also noncompetitive with respect to substrate, and differ in pH- and cofactor-dependence and sensitivity to inhibition by diethyl p-nitrophenyl phosphate. Rat platelets also contain both 1(3)- and 2-monoglyceride hydrolase activities. These differ in reactivity with antiesterase and after solubilization and electrophoretic migration with each other and with the corresponding plasma activities. Studies with the isolated perfused rat heart suggest that a major role in the catabolism of 2-monoglyceride generated from lipoprotein lipase activity at the coronary bed is played by the plasma 2-monoglyceride hydrolase activity.

Animals↗

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences.

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.

Adsorption↗

Microstructural investigation of monoglyceride-water coagel systems by NMR and CryoSEM.

Monoglyceride coagels consist of a network of plate-like crystals and are formed from a swollen gel state (alpha-gel). In order to resolve the transition mechanism, coagels were prepared with monoglycerides that differ in fatty acid composition (monomyristate and palmitate/stearate, respectively). Rheology provided information on kinetics of coagel formation and the strength of the resulting crystal network. From NMR measurements, the surface-to-volume ratio, tortuosity, and dimensionality of the network were obtained. These findings were in line with qualitative and quantitative structural information obtained from CryoSEM. As a model for the behaviour of non-monoglyceride species, the dynamics of (perdeuterated) palmitic acid was monitored in both alpha-gels and coagels. The experimental data support a two-stage mechanism. In the first stage, two-dimensional separation of D- and L-isomers in the monoglyceride bilayers of the alpha-gel occurs. This process depends primarily on lateral diffusion rate of the monoglycerides. Palmitic acid can be accommodated in the alpha-gel bilayer, but in the coagels it is separated into relative mobile and mechanically weak junction zones between the crystal plates. In the second stage of coagel formation, the crystal plates also grow in the third dimension. Both monoglyceride type and concentration determine the kinetics of this process.

Cryoelectron Microscopy↗

Proton transfer in gramicidin channels is modulated by the thickness of monoglyceride bilayers.

The thickness of monoglyceride planar bilayers has significant effects on the transfer of protons in both native gramicidin A (gA) and in covalently linked SS- and RR-dioxolane-linked gA proteins. Planar bilayers with various thicknesses were formed from an appropriate combination of monoglyceride with various fatty acid lengths and solvent. Bilayer thicknesses ranged from 25 A (monoolein in squalene) to 54 A (monoeicosenoin in decane). Single-channel conductances to protons (g(H)) were measured in the concentration range of 10-5000 mM HCl. In native gA as well as in RR channels, the shape of the log(g(H))-log([H(+)]) relationships was nonlinear and remained basically unaltered in monoglyceride bilayers with various thicknesses. For both native gA and RR channels, g(H) values were systematically and significantly larger in thin than in thick bilayers. By contrast, the shape of the log(g(H))-log([H(+)]) relationships in the SS channel was linear (with a slope considerably smaller than 1) in thick (>37 A) bilayers. However, in thin (<37 A) bilayers these plots became nonlinear and g(H) values approached those obtained in native gA channels. The linearization of the log-log plots in the SS channel in thick bilayers is a consequence of a dramatic increase (instead of a decrease as in native gA and RR channels) of g(H) in these bilayers in [H(+)] <1 M. The gating characteristics of the various gA channels as a function of bilayer thickness followed the same pattern as described previously. It was noticed, however, that in the thickest monoglyceride bilayer used in this study, both the SS- and RR-dioxolane-linked channels opened in a mode of bursting activity instead of remaining in the open state as in thin bilayers. It is proposed that the thickness of monoglyceride bilayers modulates proton transfer in native gA channels by a combination of factors including the access resistances of channels to H(+), and fluctuations in both the structure of the lipid bilayer and in the distance between gA monomers. The differential effects of relatively thick monoglyceride bilayers on proton transfer in both dioxolane-linked gA channels must relate to distinct interactions between the bilayers and the SS and RR dioxolanes.

Electric Capacitance↗

Structural and topographical characteristics of adsorbed WPI and monoglyceride mixed monolayers at the air-water interface.

In this work we have analyzed the structural and topographical characteristics of mixed monolayers formed by an adsorbed whey protein isolate (WPI) and a spread monoglyceride monolayer (monopalmitin or monoolein) on the previously adsorbed protein film. Measurements of the surface pressure (pi)-area (A) isotherm were obtained at 20 degrees C and at pH 7 for protein-adsorbed films from water in a Wilhelmy-type film balance. Since the surface concentration (1/A) is actually unknown for the adsorbed monolayer, the values were derived by assuming that the A values for adsorbed and spread monolayers were equal at the collapse point of the mixed film. The pi-A isotherm deduced for adsorbed WPI monolayer in this work is practically the same as that obtained directly by spreading. For WPI-monoglyceride mixed films, the pi-A isotherms for adsorbed and spread monolayers at pi higher than the equilibrium surface pressure of WPI are practically coincident, a phenomenon which may be attributed to the protein displacement by the monoglyceride from the interface. At lower surface pressures, WPI and monoglyceride coexist at the interface and the adsorbed and spread pi-A isotherms (i.e., the monolayer structure of the mixed films) are different. Monopalmitin has a higher capacity than monoolein for the displacement of protein from the air-water interface. However, some degree of interactions exists between proteins and monoglycerides and these interactions are higher for adsorbed than for spread films. The topography of the monolayer corroborates these conclusions.

Adsorption↗

Structural, topographical, and shear characteristics of milk protein and monoglyceride monolayers spread at the air-water interface.

In this contribution we are concerned with the study of structure, topography, and surface rheological characteristics under shear conditions of monoglyceride (monopalmitin and monoolein) and milk protein (beta-casein, kappa-casein, caseinate, and WPI) spread monolayers at the air-water interface. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (Brewster angle microscopy: BAM) techniques have been applied in this study to pure emulsifiers (proteins and monoglycerides) spread at the air-water interface. To study the shear characteristics of spread films, a homemade canal viscometer was used. The experiments have demonstrated the sensitivity of the surface shear viscosity (eta(s)) of protein and monoglyceride films at the air-water interface, as a function of surface pressure (or surface density). The surface shear viscosity was higher for proteins than for monoglycerides. In addition, eta(s) was higher for the globular WPI than for disordered beta-casein and caseinate due to the strong forces acting on spread globular proteins. This technique makes it possible to distinguish between beta-casein and caseinate spread films, with the higher eta(s) values for the later due to the presence of kappa-casein. The eta(s) value varies greatly with the surface pressure (or surface density). In general, the greater the surface pressure, the greater the values of eta(s). Finally, the eta(s) value is also sensitive to the monolayer structure, as was observed for monoglycerides with a rich structural polymorphism (i.e., monopalmitin).

Air↗

Rheologic and NMR characterization of monoglyceride-based formulations.

This paper describes the production and characterization of semi-solid formulations based on monoglycerides from canola oil and water as drug-delivery systems. In order to obtain new formulations with different characteristics in terms of viscosity, bioadhesiveness, and solubilization capacity, a third component was added to the monoglyceride-water system. Nine excipients were tested, namely soy oil, isopropylmyristate, isopropylpalmitate, tripalmitin, tristearin, glyceryl monostearate, glycerol, propylene glycol, and ethanol. In particular, the effect of each excipient on the viscosity and stability of the formulation was investigated. It was found that ethanol dramatically influenced the viscosity of the monoglyceride-water system, resulting in the formation of stable forms. In addition, ethanol suitably could be used for the solubilization of water-insoluble lipophilic drugs. This promising ternary system was characterized by microscopic, NMR spectroscopic, and rheologic techniques. (1)H and (13)C NMR studies were made of Myverol to verify the molecular structure and isomer distributions of this commercial monoacylglycerol mixture. The microstructure of an isotropic solution consisting of Myverol [1.8% (w/w)], ethanol (42.9%), and water (55.3%) was studied by the multi-component self-diffusion NMR method. From the self-diffusion coefficient (D) of the monoglycerides (8.8 x 10-11 m(2)/s), an "apparent spherical hydrodynamic radius" of ca. 2.48 nm was calculated for the micellar aggregate. A nearly spherical shape is consistent with these values since the extended hydrocarbon chain of the longest monoglyceride (17 carbons) is ca. 2.2 nm long. The D's of water and ethanol reveal they do not associate (no attractive nonbonding interactions) appreciably with the fatty acid micelles.

Biocompatible Materials↗

Absorption enhancement of rectally infused cefoxitin by medium chain monoglycerides in conscious rats.

The enhancing effect of the medium chain monoglycerides glyceryl-1-monoctanoate (GMO), glyceryl-1-monodecanoate (GMD), and glyceryl-1-monododecanoate (GMDD) on rectal absorption of the cephalosporin antibiotic cefoxitin [(6R,7S)-3-hydroxymethyl)-7-methoxy-8-oxo-7-[2-(2-thienyl)acetamido]-5- thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid carbamate (ester)] was investigated in unanesthetized rats. Rectal infusion of 3 mg of cefoxitin sodium without monoglyceride resulted in a mean bioavailability of 31 +/- 18% and a mean residence time (MRT) of 134 +/- 44 min. Coadministration with 53% (w/w) GMO significantly enhanced cefoxitin absorption, resulting in a mean bioavailability of 84 +/- 11% and a mean MRT of 75 +/- 8 min. In a lower concentration, GMD (13% w/w) also significantly promoted cefoxitin bioavailability to 68 +/- 14% and reduced MRT to 70 +/- 11 min. With GMDD only, a trend of increasing bioavailability with increasing monoglyceride concentration was observed, which may be explained by its limited aqueous solubility. Concerning the action of GMO and GMD, the longer monoglyceride is, in terms of effective concentration, more potent in enhancing the extent and rate of cefoxitin absorption. However, a further increase in chain length results in a loss of effect, indicating that the effect of monoglycerides on drug absorption may be determined by their intrinsic absorption enhancing action and solubility.

Administration, Rectal↗

Shear characteristics, miscibility, and topography of sodium caseinate-monoglyceride mixed films at the air-water interface.

In this contribution, we are concerned with the study of structure, topography, and surface rheological characteristics (under shear conditions) of mixed sodium caseinate and monoglycerides (monopalmitin and monoolein) at the air/water interface. Combined surface chemistry (surface film balance and surface shear rheometry) and microscopy (Brewster angle microscopy, BAM) techniques have been applied in this study to mixtures of insoluble lipids and sodium caseinate spread at the air-water interface. At a macroscopic level, sodium caseinate and monoglycerides form an heterogeneous and practically immiscible monolayer at the air-water interface. The images from BAM show segregated protein and monoglyceride domains that have different topography. At surface pressures higher than that for the sodium caseinate collapse, this protein is displaced from the interface by monoglycerides. These results and those derived from interfacial shear rheology (at a macroscopic level) appear to support the idea that immiscibility and heterogeneity of these emulsifiers at the interface have important repercussions on the shear characteristics of the mixed films, with the alternating flow of segregated monoglyceride domains (of low surface shear viscosity, etas) and protein domains (of high etas) across the canal.

Air↗

Overfeeding fat as monoglyceride or triglyceride: effect on appetite, nutrient balance and the subsequent day's energy intake.

OBJECTIVE: To examine the effect of overfeeding isoenergetic diets enriched in 1-monoglyceride or triglyceride on nutrient oxidation and appetite throughout the day that it was given and the subsequent day's food and energy intake. DESIGN: Six men [mean (s.d.) weight; 76.89 (7.00) kg, height; 1.77 (0.05) m, age; 26.4 (6.0) y], were each studied twice in a 3 d protocol. On day 1 (maintenance day) they were fed a medium fat (MF) maintenance diet (MF: 40% fat, 47% carbohydrate and 13% protein by energy) calculated at 1.6 x resting metabolic rate (RMR). Subjects entered the calorimeter at 06.30 on day 2 for 49.5 h. On day 2 (manipulation day) subjects consumed a MF diet at 1.6 x RMR with an additional 0.45 x RMR as either 1-monoglyceride or triglyceride. On day 3 (outcome day), subjects had ad libitum access to isoenergetic, isoenergetically dense MF (40 :47: 13, 550 kJ/ 100 g) foods. Subjective hunger and satiety were tracked hourly, during waking hours throughout days 1-3. RESULTS: There was no significant effect of diet on nutrient oxidation or balance either during day 2 (manipulation day) or day 3 (outcome day), fat oxidation was similar on both diets. Subjective hunger was not affected by diet on either day with mean values of 34.3 and 35.0 mm (SED 5.2) on manipulation day (day 2) and outcome day (day 3), 35.3 and 40.8 mm (SED 5.2) on the 1 -monoglyceride or triglyceride diets respectively. Day 3 food and energy intake were unaffected by the previous day's dietary treatment, with mean intakes of 15.9 and 15.6 MJ (SED 1.07) on the 1-monoglyceride or triglyceride treatments, respectively. CONCLUSIONS: This study suggests that when 1-monoglyceride is covertly incorporated into a diet at unusually high levels, it behaves in a manner that is very similar to triglyceride, in its effects on appetite, feeding behaviour and net nutrient balance.

Adult↗