Phospholipids concentration

Upon the spontaneous rearrangement of anhydrous phospholipids in the presence of water into a hydrated bilayer structure, a portion of the aqueous phase is entrapped within a continuous, closed bilayer structure. By this process water-soluble compounds are passively entrapped in liposomes. The efficiency of encapsulation varies and depends, for example, on the method of preparation of liposomes and the phospholipid concentration during preparation. Different parameters can be used to describe the encapsulation efficiency ... 

Kakiuchi et al. [75] used the capacitance measurements to study the adsorption of dilauroylphosphatidylcholine at the ideally polarized water-nitrobenzene interface, as an alternative approach to the surface tension measurements for the same system [51]. In the potential range, where the aqueous phase had a negative potential with respect to the nitrobenzene phase, the interfacial capacity was found to decrease with the increasing phospholipid concentration in the organic solvent phase (Fig. 11). The saturated mono-layer in the liquid-expanded state was formed at the phospholipid concentration exceeding 20 /amol dm, with an area of 0.73 nm occupied by a single molecule. The adsorption was described by the Frumkin isotherm. 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

Briefly, liposomes (10mM) were incubated for 30minutes at 37°C for egg phosphatidylcholine (EPC) and at 60°C for HSPC-based liposomes with 50 X 10 dpm of methylamine (1 x 10 dpm/mole). At the end of incubation an aliquot of this mixture was passed down a Sephadex G-50 minispin column equilibrated in 10 mM histidine-sucrose buffer 10%, pH 6.7 buffer. Liposomes were eluted at the column void volume and separated from the unencapsulated methylamine. The concentration of liposomes in the original liposomal dispersion and in the void volume fraction was determined from the organic phosphorus (phospholipid) concentration (see section Lipid Quantification and Chemical Stability above) (10,49,53). 

X = ratio between [ C]-methylamine radioactivity (dpm) and phospholipids concentration (mM) in the original liposome dispersion after the incubation and before Sephadex G-50 separation. 

In the case of membrane incorporation, the trapping efficiency depends on the lipophilicity of the material, phospholipid concentration and phase behavior of the membrane. For compounds covalently bound to the membrane, the number of binding sites available is the upper limit of the loading factor. 

The phospholipid concentration in a (proteo)liposome suspension can be determined by phosphorus analysis (73) and the protein concentration by automated amino acid analysis or by a calibrated colorimetric protein assay... 

Curstedt T, Hafinan M, Robertson B, et al. 1983. Rabbit lung after long-term exposure to low nickel dust combustion. I. Effects on phospholipid concentration and surfactant activity. Environ Res 30 89-94. 

Cholesterol metabolism. Hydrogenated oil, administered orally to hamsters at a dose of 20% of diet for 4 weeks, induced hypercholesterolemia. Oil feeding had no effect on cholesterol synthesis but markedly inhibited cholesterol esterification in both the liver and the intestine. The diet-induced hypercholesterolemia was strongly correlated with an increase in acyl-CoA/cholesterol acyltransferase activity. The hypercholesterolemia increased aortic uptake of cholesterol and hence acyl-CoA/cholesterol acyltransferase activity " Coconut fat, administered orally to rabbits with partial ileal bypass, produced a significant increase of serum total cholesterol and phospholipids concentrations. The effect on semm lipids of the type of fat was similar in control and partial ileal bypass rabbits A Coconut—a main source of energy for two... 

Schouten, J. A., A. C. Beynen, C. Mulder, and H. F. Hoitsma. The effect of dietary saturated fat versus polyunsaturated fat on serum cholesterol and phospholipid concentrations in rabbits with partial ileal bypass. ZErnahrung-swiss 1984 23(2) 136-142. 

The phospholipid content of milk and milk products is given in Table 4.5 (Kurtz 1974). Total phospholipid is usually determined by measuring the lipid phosphorus content of the product and multiplying by 26 (AOCS 1975). As the total milk lipid increases in a milk product, so does the phospholipid concentration. However, the ratio of phospholipid to total lipid varies greatly. Referring to Table 4.5 skim milk contains the smallest concentration of phospholipid but the highest ratio of phospholipid to total lipid. The opposite relationship is seen in cream and butter. 

Phospholipids are most actively employed in the creation of somatic and generative production. Gonad maturation is accompanied by a sharp increase in the phospholipid concentration of the liver and blood (Shchepkin, 1972), indicating enhanced synthesis and transport into the gonads. The phospholipid concentration is greater in the gonads than in somatic tissues. 

We found the 31p-NMR chemical shift of monomeric dihexanoyl PC increases upon the addition of the nonionic detergent Triton X-100. This phenomenon was used to quantitate the solubilization of this phospholipid by the detergent micelles as a function of detergent concentration using a simple phase separation model ( 5). Similar studies were carried out on dibutyryl PC. At a phospholipid concentration of 7 mM and 56 mM detergent, 85% of the dihexanoyl PC, but only 3% of the dibutyryl PC was incorporated into the micelles. 

For a large range of phospholipid concentrations, the broadening linearly depends on the lipid concentration. The slope of such plots is proportional to the affinity of the drug molecules or molecular substructures to the phospholipid. It can be taken as a measure of the degree of interaction. 

Fig. 5.13 Phosphatidylserine (PS) dependence of Ca2+-stimulated PKC activity as a function of palmitoyl- (Po), oleoylphosphatidylserine POPS concentration in the POPS/PE LUVs. Enzyme activity measured relative to 100% POPS. Total phospholipid concentration in the incubation...

Phospholipid concentration Phospholipid composition Cholesterol concentration Drug concentration Chemical stability PH... 

During the surge in interest in the phosphatidylinositol derivatives, Takai et al. (1979) noted that an unsaturated diacylglycerol diminished the Ca2+ and phospholipid concentration required for complete activation of a Ca2+-activated, phospholipid-dependent protein kinase (now known as protein kinase C). In this latter system, phosphatidylserine was most active (as the required phospholipid), with phosphatidylethanolamine and phosphatidyl inositol much less effective and phosphatidylcholine without any activity. Probably the enzyme was activated by an amphipathic molecule bearing a net negative charge, and any available cellular phospholipid mixture with the requisite surface change served this purpose. 

A CBF/NBF transition at pHcr can be demonstrated with films from lyso PC. Isoelectric points at the solution/air interface (see Section 3.3.2) and pHcr were found. The phospholipid concentration was specially chosen, Cs > Cr-, which is a necessary condition of NBF formation (see Section 3.4.3). 

Fig. 11.4 shows separately curve 1 from Fig. 11.3 which is the dependence of W on the DPPC concentrations in the AF. The W(C) curves allow to determine the threshold concentration C i.e. the minimum phospholipid concentration at which there is a 100% probability of observation of black films (see Eq. (3.130)). At concentrations lower than C, NBFs are no more observed, since W sharply decreases to zero (films rupture). At concentrations higher than C, (W = 1), NBFs always form. Special studies with phospholipid analysis of amniotic fluid indicate that of all phospholipids in the AF, it is the DPPC that stabilises the foam bilayers. This analysis gives grounds to conclude that the concentration of each phospholipid (except DPPC) in the native AF is of an order lower than the corresponding... 

The linear dependence between the threshold dilution and the initial total phospholipid concentration (respectively, DPPC) found allows to determine the threshold dilution for a 100% probability for formation of NBF instead of Ct. Fig. 11.5 shows that if a sample dilution of 3.1 times is applied, then it is possible to detect almost all cases with a developed RDS. Therefore, the threshold dilution of 3.1 times allows to distinguish the mature from immature AF samples which gives a good reason to employ it in diagnosing of RDS, and respectively, to estimate the lung surfactant deficiency. Hence, the formation of black foam films from AF samples taken at different gestation weeks and diluted 3.1 times, indicates that there is no risk of RDS, while film rupture predicts an eventual RDS development. 

Fig. 11.12. Dependence of probability Ws for black spot formation on adsorption time at phospholipid concentration of (A) 65 pg cm 3, (B) 130 pg cm 3 and (C) 170 pg cm 3 black spot formation (W, = 1) by IN and SU required about 10 min at each concentration EX required adsorption times of about 40 min at the lowest concentration (A) and about 12 min at higher concentrations black films were formed only by IN and EX at the highest concentration below C, when adsorption times were increased to longer lhan 30 min for IN and longer than 40 min for EX (arrows in (C)) films of SU always ruptured t = 22°C.

The lipid film is then hydrated and solubilised in 5 mM TES Buffer, pH 7.0. Depending on experimental requirements, the final phospholipid concentration is usually taken between 0.1 and 20 mg/mL (see Note 12). 

If desired, the precise phospholipid concentration can be calculated by means of a phosphate determination (see Subheading 3.5.1). 

The concentration of the lipophilic drugs NOAC, ara-C-NOAC, 5-FdU-NOAC or ETC-NOAC in the liposomes can be varied from 1 mg/mL to about 10 mg/mL, depending on the concentration required for biological activity (e.g. based on corresponding ICg -values), the phospholipid concentration, the lipid composition and the method of liposome preparation. The concentrations of incorporated drugs can be determined by reverse phase FFPLC (38). 

The phospholipid concentration of the liposomes is determined using a phospholipid assay kit (Wako Chemicals, Germany) according to the instructions of the manufacturer. 

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