Is the probable spillage of the lung surfactant dipalmitoylphosphatidylcholine the ultimate source of diabetes type 1?

The lung surfactant dipalmitoylphosphatidylcholine (DPPC) most probably leaks into the blood, settling on the luminal aspect of blood vessels to create active hydrophobic spots (AHS). Nanobubbles are formed at these spots from dissolved gas. We hypothesized that when a large molecule in the blood comes into contact with a nanobubble at the AHS, its tertiary structure is disrupted. An epitope not previously having undergone thymus education may then prompt an autoimmune response. There are thus two independent processes which may share the blame for autoimmune disease: spillage of large molecules into the blood, and the creation of AHS. DPPC was measured in 10 diabetes type 1 patients and 10 control subjects. DPPC in the diabetic group was 4.63 ± 0.68 µg/mL, non-significantly higher than in the control group (4.23 ± 0.94 µg/mL). However, in the diabetic group, DPPC was high when the samples were taken within 1.5 years of disease onset. This is closer to the time of AHS production, which takes place ahead of the disease. Further investigation, with sampling for DPPC as soon as possible after onset of the disease, may provide additional support for our hypothesis. If proved true, this may open up considerable therapeutic potential.

Homogeneous and Heterogeneous Bilayers of Ternary Lipid Compositions Containing Equimolar Ceramide and Cholesterol.

Cell membranes have been proposed to be laterally inhomogeneous, particularly in the case of mammalian cells, due to the presence of "domains" enriched in sphingolipids and cholesterol (Chol). Among membrane sphingolipids, sphingomyelin (SM) in the cell plasma membrane is known to be degraded to ceramide (Cer) by acid sphingomyelinases under stress conditions. Since cholesterol (Chol) is abundant in the plasma membrane, the study of ternary mixtures SM:Chol:Cer is interesting from the point of view of membrane biophysics, and it might be physiologically relevant. In previous studies, we have described the homogeneous gel phase formed by phospholipid:Chol:Cer at 54:23:23 mol ratios, where phospholipid was either SM or dipalmitoylphosphatidylcholine (DPPC). We now provide new data, based on trans-parinaric acid and diphenylhexatriene fluorescence, supporting that the gel phase includes all three components in a single bilayer. The main question addressed in this paper is the stability of the ternary gel phase when bilayer composition is changed, specifically when the SM proportion is varied. To this aim, we have prepared bilayers of composition phospholipid:Chol:Cer at X:Y:Y ratios, in which phospholipid increased between 54 and 70 mol %. The N-palmitoyl derivatives of SM (pSM) and Cer (pCer) have been used. We observe that for X = 54 or 60 mol %, a gel phase is clearly predominant. However, when the proportion of phospholipid increases beyond 60 mol %, i.e., in 66:17:17 or 70:15:15 mixtures, a lateral phase separation occurs at the micrometer scale. These data can be interpreted in terms of a pCer:Chol interaction, that would predominate at the lower phospholipid concentrations. The putative pCer:Chol complexes (or nanodomains) would mix well with the phospholipid. At the higher SM concentrations pSM:pCer and pSM:Chol interactions would become more important, giving rise to the coexisting gel and liquid-ordered phases respectively. Heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicating a higher affinity of SM over DPPC for Chol or Cer. The observation that heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicates a higher affinity of SM over DPPC for Chol or Cer, and can be related to cell regulation through the sphingolipid signaling pathway.

Ovine plasma dipalmitoylphosphatidylcholine does not predict decompression bubbling.

Decompression illness (DCI) is the main risk associated with scuba diving. Some divers ("bubblers") are more sensitive to DCI than others ("non-bubblers"). We found that there are active hydrophobic spots (AHS) on the luminal aspect of ovine blood vessels, which contain the surfactant dipalmitoylphosphatidylcholine (DPPC). DPPC leaks from the lung into the plasma, settling on the blood vessel to create AHS. These are the main source of gas micronuclei from which bubbles develop after decompression. A correlation between bubbling ovine blood vessels and the animal's plasma DPPC might lead to the development of a blood test for vulnerability to DCI. Samples from ovine blood vessels were stretched on microscope slides, placed anaerobically in saline at the bottom of a Pyrex bowl, and exposed to high pressure. Automated photography was used after decompression to reveal AHS by visualising their bubble production. Phospholipids were extracted from the AHS and plasma for determination of DPPC. Bubbling was unrelated to the concentration of DPPC in the plasma (2.15 ±â€¯0.87 µg/ml). Bubble production from the AHS (n = 130) as a function of their DPPC content yielded two groups, one unrelated to DPPC and the other which demonstrated increased bubbling with elevation of DPPC. We suggest this may be related to alternate layering with hydrophobic and hydrophilic phospholipids. This study reinforces the connection between DPPC and DCI. However, a blood test for diver vulnerability to decompression stress is not recommended.

Brucine-loaded liposomes composed of HSPC and DPPC at different ratios: in vitro and in vivo evaluation.

OBJECTIVE: The objective of this study is to test the hypothesis that the phase transition temperature (T(m)), the main property of liposomes, can be easily controlled by changing the molar ratio of hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphacholine (DPPC) after drug encapsulation. MATERIALS AND METHODS: Brucine, an antitumor alkaloid, was encapsulated into the liposomes with different HSPC/DPPC compositions. The T(m)s of the brucine-loaded liposomes (BLs) were determined by differential scanning calorimetry (DSC). Then the physicochemical properties and pharmacokinetics of the BLs with different HSPC/DPPC compositions were investigated and compared. RESULTS: The results of DSC revealed that HSPC and DPPC can combine into one phase. The findings of molecular modeling study suggested that HSPC interacts with DPPC via electrostatic interaction. The molar ratio of HSPC/DPPC influenced the sizes of BLs but had little effect on the entrapment efficiency (EE). The stability of BLs was improved with the increase of the HSPC ratios, especially with the presence of plasma. Following i.v. administration, it was found that AUC values of BLs in vivo were directly related to the HSPC/DPPC ratios of BLs, namely the T(m)s of BLs. DISCUSSION: The behavior of liposomes, especially in vivo pharmacokinetic behavior, can be controlled by the modification of T(m). CONCLUSION: The characterization of BLs in vitro and in vivo had demonstrated that the Tm could be flexibly modified for liposomes composed of both HSPC and DPPC. Using HSPC/DPPC composition may be an efficient strategy to control the T(m), thus control the in vivo pharmacokinetic behavior, of BLs.

Development of an enzymatic assay for sphingomyelin with rapid and automatable performances: Analysis in healthy subjects and coronary heart disease patients.

BACKGROUND: Sphingomyelin (SM) is an important choline group-containing phospholipid and is considered to be an independent risk factor for coronary heart disease. METHODS: We have developed a specific enzymatic assay for SM measurement with rapid and automatable performances by using two-reagent system involving sphingomyelinase. We performed within-run and between-run precision, linearity test, detection limit, recovery test and interference to validate this assay. Then, we measured the serum SM concentration in 194 healthy subjects and 141 consecutive patients undergoing coronary angiography. RESULTS: The within-run and between-run coefficients of variation for SM concentrations were 1.1-1.3% and 1.0-1.2%, respectively. Quantitative measurements to a lower limit of 30 µmol/L were shown to be possible. The recoveries of the exogenously added SM to the control samples were 98.7%-101.5%. No effect was observed after the addition of some interference materials. The mean ± SD of the serum SM concentration in the 194 healthy subjects was 553.3 ± 100.1 µmol/L. We found that the SM concentration was significantly higher among an acute coronary syndrome subjects than among the healthy subjects (P<0.01) and that the serum SM concentrations were significantly correlated with the serum magnesium concentration. CONCLUSIONS: We have developed a rapid and automatable enzymatic assay for SM that enables the automatic measurement of choline-containing phospholipids. This assay may be useful for various types of biochemical and clinical research.

Apolipoprotein A-I localization and dipalmitoylphosphatidylcholine dynamics in reconstituted high density lipoproteins.

The structure and molecular dynamics of recombinant high density lipoproteins (rHDL) were studied by non-radiative energy transfer (NRET), fluorescence anisotropy and intensity measurements. The rHDL particles contained human plasma apolipoprotein (apo) A-I and dipalmitoylphosphatidylcholine (DPPC). Fluorescent cis- and trans-parinaric acids were used both as probes of molecular motion in the particle lipid phase and as acceptors in the Forster's energy transfer from apo A-I tryptophan residues to determine particle dimensions, apolipoprotein localization and lipid dynamics. The probes are sensitive to thermal wobbling (macromobility) and conformational deformations (micromobility) of phospholipid acyl chains. The experimental data fitted to various models of the particle structure are compatible with the following: (a) at T < Tt the particles appeared as lens-like discs with a radius of the lipid phase of 5 nm and a mean thickness of 4 nm, the value being more by 20% in the particle centre, the alpha-helices of about 1 nm thickness were located around the edge of the lipid core. Compared to liposomes, both macro- and micromobility of DPPC molecules in rHDL were more rapid due to a significant disorder of the boundary lipid molecules close to the apo A-I molecule. This disorder led to the increase of the specific surface area per one lipid molecule, S(o). The lipid phase can be divided into three regions: (i) zone I of the most tightly packed lipid (0-1.7 nm from the disc axis) with a S(o) value small as 0.5 nm2; (ii) intermediate zone II (from 1.7 to 4.0 nm); and (iii) boundary lipid zone III (4-5 nm) of significantly disordered lipid with a S(o) value large as 0.65 nm2. (b) at T> Tt the S(o) heterogeneity disappeared, the radius of the lipid phase did not increase significantly, not exceeding 5.2-5.4 nm, but protein-induced immobilization of lipid molecules which affected about half or more of the total lipid, became remarkable. The overall effect was the suppression of the transition amplitude in rHDL compared to liposomes. The structural inhomogeneity might underlie the function of the native plasma HDL as the key component of the transport and metabolism of plasma lipids.

Formulation of liposomes with a soybean-derived sterylglucoside mixture and cholesterol for liver targeting.

In this study, we investigated a liposomal formulation of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch) for targeting the liver in mice. We found two distribution profiles of liposomes (reverse-phase evaporation vesicles, REV) in the liver depending on the concentration of Ch and SG in vivo; Ch tends to enhance liposomes containing 10 mol% SG accumulation in the liver, but to decrease those containing 20 mol% SG, and it prolongs the circulation in blood. Dicetylphosphate did not enhance liver targeting by liposomes with SG and Ch. More DPPC/SG/Ch-liposomes (6:1:3) were significantly taken up by cultivated hepatocytes than DPPC/SG/Ch-liposomes (6:0:4), suggesting a glucose residue. The optimal composition for the maximal liver targeting was a mixture of 0.2 micron DPPC/SG/Ch-liposomes (REV) at a molar ratio of 6:1:3. This composition of liposomes was distributed 3 times greater in hepatocytes than non-parenchymal cells 1 h after intravenous injection.

In vivo turnover of 1,2-dipalmitoylphosphatidylcholine and sphingomyelin in rabbit erythrocytes.

The in vivo turnover of both 1,2-dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) in rabbit erythrocytes was studied. DPPC, either 14C-labelled in the fatty acyl chain at the 2-position of the glycerol moiety or 3H-labelled in the choline's methyl group, and [N-methyl-14C]SM (bovine) were introduced into the membrane of freshly isolated rabbit erythrocytes by using phospholipid transfer proteins. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. Analysis of blood samples shows that both labels disappear from the circulating cells with the same rate, resulting in a half-time value of about 6.4-6.6 days. This result demonstrates that the loss of DPPC from the cells is due to transfer of intact molecules to the plasma and that a deacylation process is of no or minor importance as mechanism of renewal of DPPC. Labelled sphingomyelin, introduced into the rabbit erythrocyte membrane in a similar way, disappears from the circulating red cell with a half-time value of 15.5 days. This accounts for a daily replacement of the total SM pool by 3.2%.

Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studies with poly(ethylene glycol)-coated vesicles.

Well-defined liposome systems have previously established the influence of size, surface charge lipid composition and surface ligands, on in vivo fate and behaviour of model compounds entrapped in liposomes. In the present study, preformed liposomes which quantitatively retain aqueous markers were covalenty coupled via dipalmitoylphosphatidyl-ethanolamine, to the hydrophilic polymer, monomethoxypoly(ethylene glycol) (MPEG 5000). Such liposomes retain the coating in the presence of plasma, and appear to adsorb plasma components more slowly than liposomes without the polymer, shown using an aqueous two-phase partitioning technique. MPEG-coupled liposomes were cleared from the blood circulation up to 30% more slowly than liposomes without MPEG after intravenous administration to mice, despite the unmodified liposomes being of a composition and size shown previously to favour achievement of maximum half-life. It is suggested that the polymer acts as a surface barrier to plasma factors which otherwise bind to liposomes in the blood and accelerate vesicle removal.

Characterization of temperature-dependent drug release from liposomes using electron spin resonance.

Liposome blood clearance and temperature-dependent drug release were studied using an electron spin resonance technique on whole blood samples. Temperature-sensitive liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1 by weight) were formed by reversed-phase evaporation. The liposomes were stable in the blood for greater than 2 h after iv injection. Liposome blood clearance exhibited a fast and slow component which may be due to the presence of both large and small liposomes in the preparation. The onset temperature for liposome release of the water-soluble nitroxide spin label was reduced from 40 degrees C in Hepes buffered saline to 38 degrees C in the rat blood, and the total release in the blood was approximately 25% greater than that measured in saline. These results show the influence of blood constituents on temperature-dependent drug release from liposomes.

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.