New technique to quantify phospolipids in disordered phase in lung surfactant by electron spin resonance.

In pulmonary surfactant (PS) the coexistence of the ordered (Lo) and disordered (Ld) lipid phases would be essential for an optimal activity. Electron spin resonance (ESR) of PS labeled with 5-doxil stearic acid shows two spectral components called S and W. S/W ratio could be understood as the ratio between the probe population in Lo and in Ld. Although the specificity of S/W as an indicator of Lo/Ld has not yet been demonstrated, S/W has been used qualitatively to study changes in Lo/Ld. The goal of this paper is to stablish the correlation between S/W parameter and the amount of lipids in Ld (%Ld) measured by the ESR TEMPO technique described in our previous work. S/W and %Ld were measured in different PS samples under different experimental conditions. The results showed an inverse correlation between S/W and %Ld (r = -0.983; p < 0.001). We demonstrated that the S/W is sensible to the changes of Lo/Ld and can be used to quantify the %Ld.

Budesonide associated with exogenous pulmonary surfactant in a novel formulation to improve the delivery to the lung.

Lung delivery for glucocorticoids (GCs) is very low and depends on the system used. Exogenous pulmonary surfactant (EPS) is a promising tool to transporting GCs efficiently to the airways. We developed a new formulation of EPS with Budesonide (BUD) incorporated into EPS membranes (EPS-BUD) to improve lung delivery of BUD. We evaluated the biodistribution and pharmacokinetic of the transported BUD by intra-tracheal instillation of EPS-BUD in healthy rats. Aqueous suspension of Budesonide was used as control. Budesonide and its esters present in trachea, kidneys and lungs were determined by HPLC. The delivery of BUD in lung for EPS-BUD group was 75 % of total instilled and only 35 % for the control group. BUD was rapidly internalized in pneumocytes and a high proportion of Budesonide esters and persistent concentrations of active free BUD were found for up to 6 h after instillation. The new EPS-BUD formulation developed significantly improves the deposition and increases the permanence of BUD in lung.

Determining the fluid ordered and disordered phases in a pulmonary surfactant by electron spin resonance technique.

Pulmonary surfactant main function is to reduce surface tension at alveolar interface. Two lipids phases coexist in surfactant membranes: a liquid-ordered (Lo) and a liquid-disordered (Ld) phases. This coexistence of phases would be crucial for the surfactant activity. Until now, the proportion of phases was determined qualitatively. We design an electronic spin resonance technique to quantify the lipid fraction in Ld phase. An exogenous pulmonary surfactant (EPS) with or without extra Cho was labeled with 5-doxil stearic acid to estimate the membrane fluidity and with TEMPO to determine the PL in Ld phase. A unique equation was established for the calculation of PL in Ld phase with an error of less than 3%. TEMPO partition coefficient was (0.78 ±â€¯0.03). Cholesterol added to EPS did not modify this coefficient. The equation is valid for different batches of surfactant regardless of the cholesterol content. The proposed method is simple, precise and allows evaluating changes in lateral structure that could affect surfactant biophysical properties.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling.

Increased protein tyrosine phosphorylation and the appearance of a phosphorylated protein of 32 kD (p32) are reported among the capacitation-like changes in cryopreserved boar sperm. Egg yolk freezing extenders are composed by two fractions: insoluble granules and soluble plasma, which contains the low density lipoproteins (LDL) proposed as responsible for the egg yolk cryoprotective action. The aim of this work was to analyze the effects of complete egg yolk and its insoluble, soluble and LDL fractions on boar sperm quality and protein tyrosine phosphorylation after the first stage of a standard cryopreservation protocol. Semen samples in Androstar® Plus diluent were centrifuged and resuspended in the different egg yolk extenders. Temperature was decreased from 17 °C to 5 °C and sperm quality, protein tyrosine phosphorylation and protein pattern were analyzed. Results showed that complete egg yolk as well as soluble and LDL egg yolk fractions maintained sperm quality after temperature decrease. Cooling without any lipid component or in the presence of the insoluble fraction, significantly reduced sperm motility. About sperm protein tyrosine phosphorylation analysis, the p32 band appeared before treatments or after cooling in Androstar® Plus diluent. Complete egg yolk and its insoluble fraction interfered with sperm tyrosine phosphorylation even after cells were extensively washed. Analysis of extenders revealed a high amount of tyrosine phosphorylated proteins in the insoluble fraction, which may have co-precipitate with sperm in experiments. Samples submitted to temperature decrease from 17 °C to 5 °C in the presence of soluble and LDL egg yolk fractions in Androstar® Plus diluent did not show any change in the p32 band associated with sperm capacitation. However, a tyrosine-phosphorylated protein of 33 kD present in clarified egg yolk was also observed in sperm treated with this extender. Protein transference from plasma and LDL egg yolk extenders was also observed in sperm protein profile. Results suggested that soluble and LDL fractions might have a protective action preventing sperm protein tyrosine phosphorylation during cooling from 17 °C to 5 °C. Further studies are needed to expand the knowledge of the LDL protection mechanism as well as to determine the possible benefits of clarified egg yolk in freezing protocols.

Developing an exogenous pulmonary surfactant-glucocorticoids association: Effect of corticoid concentration on the biophysical properties of the surfactant.

Glucocorticoids (GCs) are used to treat lung disease. GCs incorporated in an exogenous pulmonary surfactant (EPS) could be an alternative management to improve drug delivery avoiding side effects. In the development of these pharmaceutical products, it is important to know the maximum amount of GC that can be incorporated and if increasing quantities of GCs alter EPS biophysical properties. Formulations containing EPS and beclomethasone, budesonide or fluticasone were analyzed (PL 10mg/ml; GC 1-2mg/ml). The microstructure was evaluated by electron paramagnetic resonance spectroscopy, GCs incorporated were determined by UV absorption and polarized light microscopy and surfactant activity with pulsating bubble surfactometer. We found that GCs have a ceiling of incorporation of around 10wt%, and that the GC not incorporated remains as crystals in the aqueous phase without altering the biophysical properties of the surfactant. This fact is important, because the greater the proportion of GC that EPS can carry, the better the efficiency of this pulmonary GC system.

Analysis of the structure and surfactant activity of novel formulations containing exogenous pulmonary surfactant and glucocorticoids.

Exogenous pulmonary surfactant (EPS) could be used as carrier of glucocorticoids (GCs) in therapy for respiratory diseases. We formulated novel combination drug products containing bovine EPS and one GC (10wt%): beclomethasone (Be), budesonide (Bu) or fluticasone (Flu), and studied the GCs action on the surface activity and biophysical properties of EPS. Subtype ratio was evaluated by phospholipid determination; surface tension (ST) with a pulsating bubble surfactometer and conformational changes by Electron Spin Resonance (ESR). GCs were incorporated into EPS in more than 80%. None of them generated disaggregation of surfactant, only Bu was found in the light subtype. Bu and Be caused minimal changes in fluidity on polar region of bilayers, but these changes were not enough to inactivate the surfactant. Flu did not significantly alter any biophysical properties or surface activity. These novel combination EPS-GC products might be a promising strategy in the therapy of pulmonary diseases as the incorporation of the GCs tested did not cause detrimental effects on EPS functionality.

Effect of serum lipoproteins and cholesterol on an exogenous pulmonary surfactant. ESR analysis of structural changes and their relation with surfactant activity.

The study of structural changes in the surfactant may help to understand the mechanisms by which the surfactant is inactivated by serum. Here, we compared the in vitro effects of serum, albumin, lipoproteins (VLDL, LDL, HDL) and cholesterol on the dynamic and structural properties of surfactant suspensions by electronic spin resonance and surface tension measurements. Our results showed that albumin seems to be responsible for macrostructure disaggregation and increased rigidity in the hydrophobic region, but it did not affect surfactant activity. Fluidity in the polar area seems to be critical for proper physiological activity, and the changes induced by serum observed in this area would be generated by HDL or cholesterol, but the amount of cholesterol transferred by serum is not significant. Statistical analysis showed that surfactant activity correlated with the fluidity in the polar area but not with that in the hydrophobic region. We obtained strong evidence that among all the serum components tested, HDL is the one that causes the structural changes that compromise surfactant performance.

Effect of serum proteins on an exogenous pulmonary surfactant: ESR analysis of structural changes and their relation with surfactant activity.

The study of the structural changes in surfactant microviscosity and bilayer organization would help to understand the mechanisms by which surfactant could be inactivated by serum components. The in vitro effects of human serum, albumin and gamma-globulin on dynamic and structural properties of surfactant suspensions and their heavy fractions were evaluated by electronic spin resonance and surface tension measurements. Our results showed that albumin and serum modified the aggregation state, transforming the active into inactive subtype, but only serum decreased the fluidity in the polar region and inactivated surfactant. In contrast, albumin and gamma-globulin generated a greater proportion of fluid-like disordered phase, without loss of surface activity. Statistical analysis showed that surface activity correlated with the fluidity in the polar area but not with that in the hydrophobic region. We concluded that one or more serum components different from albumin or gamma-globulin cause a structural change in the surfactant bilayer, increasing the rigidity in the polar area, which would be critical for proper physiological activity.

Influence of serum protein and albumin addition on the structure and activity of an exogenous pulmonary surfactant.

The comparative analysis of the deleterious action of albumin and total serum proteins (SP) might help to understand the nature of the interaction surfactant--SP. This study evaluated the effects of serum proteins and albumin on bulk shear viscosity, surface tension, surface area reduction, and the ratio between the light and heavy subtypes of surfactant suspensions. Our results showed a correlation between the bulk viscosity and aggregation degree of surfactant suspensions. The addition of albumin or SP induced the transformation from the heavy to the light subtype, reducing the viscosity. SP caused disaggregation and inactivation, whereas albumin caused only disaggregation without loss of surface activity. When SP were removed, the heavy fraction obtained recovered its surface activity. We conclude that the disaggregation may not be the primary cause for the loss of surface activity. Surfactant inactivation by a serum component, different from albumin, would be probably due to a physical interaction, a phenomenon that is reversed when SP are removed.

Antioxidant effects of water- and lipid-soluble nitroxide radicals in liposomes.

Liposomes are today useful tools in different fields of science and technology. A lack of stability due to lipid peroxidation is the main problem in the extension of the use of these formulations. Recent investigative works have reported the protective effects of stable nitroxide radicals against oxidative processes in different media and under different stress conditions. Our group has focused its attention on the natural aging of liposomes and the protection provided by the water- and lipid-soluble nitroxide radicals 2,2,6,6-tetramethylpiperdine-1-oxyl (TEMPO) and doxylstearic acids (5-DSA, 12-DSA, and 16-DSA), respectively. Unilamellar liposomes were incubated under air atmosphere at 37 degrees C, both in the absence and in the presence of these radicals. Conjugated dienes, lipid hydroperoxides, TBARS, membrane fluidity, and nitroxide ESR signal intensity were followed as a function of time. Our results demonstrated that doxylstearic acids were more efficient than TEMPO in retarding lipid peroxidation at all the concentrations tested. The inhibition percentages, depending on the total nitroxide concentration, were not proportional to the lipid-water partition coefficient. Furthermore, time-course ESR signals showed a slower decrease for doxylstearic acids than for TEMPO. No significant differences were found among 5-DSA, 12-DSA, and 16-DSA. We concluded that the nitroxide radical efficiency as antioxidant directly depends on both nitroxide concentration and lipophilicity.