Lipid quantifying

The interest in vesicles as models for cell biomembranes has led to much work on the interactions within and between lipid layers. The primary contributions to vesicle stability and curvature include those familiar to us already, the electrostatic interactions between charged head groups (Chapter V) and the van der Waals interaction between layers (Chapter VI). An additional force due to thermal fluctuations in membranes produces a steric repulsion between membranes known as the Helfrich or undulation interaction. This force has been quantified by Sackmann and co-workers using reflection interference contrast microscopy to monitor vesicles weakly adhering to a solid substrate [78]. Membrane fluctuation forces may influence the interactions between proteins embedded in them [79]. Finally, in balance with these forces, bending elasticity helps determine shape transitions [80], interactions between inclusions [81], aggregation of membrane junctions [82], and unbinding of pinched membranes [83]. Specific interactions between membrane embedded receptors add an additional complication to biomembrane behavior. These have been stud-... 

A number of methods are available for following the oxidative behaviour of food samples. The consumption of oxygen and the ESR detection of radicals, either directly or indirectly by spin trapping, can be used to follow the initial steps during oxidation (Andersen and Skibsted, 2002). The formation of primary oxidation products, such as hydroperoxides and conjugated dienes, and secondary oxidation products (carbohydrides, carbonyl compounds and acids) in the case of lipid oxidation, can be quantified by several standard chemical and physical analytical methods (Armstrong, 1998 Horwitz, 2000). 

These assumptions were confirmed by the electrophoresis study of the washed creams. Electrophoresis of purified fat globules is a convenient method to characterize and quantify proteins adsorbed at the oil-water interface [35]. Electrophoretic data indicate that no casein, nor whey proteins, were adsorbed at the surface of raw-milk fat globule. Upon homogenization, caseins adsorbed preferentially at the lipid-water interface. In this case, bound a-lactalbumin accounted for 16% of the total interfacial proteins. Heat treatment also induced the interaction of proteins with the fat globules. The amount of bound proteins (per mg of lipids) for heated raw milk was half that for homogenized milk. 

Major determinants of membrane fluidity may be grouped within two categories [53] (1) intrinsic determinants, i.e., those quantifying the membrane composition and phase behavior, and (2) extrinsic determinants, i.e., environmental factors (Table 1). In general, any manipulation that induces an increase in the molal volume of the lipids, e.g., increase in temperature or increase in the fraction of unsaturated acyl chains, will lead to an increase in membrane fluidity. In addition, several intrinsic and extrinsic factors presented in Table 1 determine the temperature at which the lipid molecules undergo a transition from the gel state to liquid crystalline state, a transition associated with a large increase in bilayer fluidity. 

Food products can generally be considered as a mixture of many components. For example, milk, cream and cheeses are primarily a mixture of water, fat globules and macromolecules. The concentrations of the components are important parameters in the food industry for the control of production processes, quality assurance and the development of new products. NMR has been used extensively to quantify the amount of each component, and also their states [59, 60]. For example, lipid crystallization has been studied in model systems and in actual food systems [61, 62]. Callaghan et al. [63] have shown that the fat in Cheddar cheese was diffusion-restricted and was most probably associated with small droplets. Many pioneering applications of NMR and MRI in food science and processing have been reviewed in Refs. [19, 20, 59]. 

The compounds that are identifiable in the sea represent a vast array of biochemicals attributable to the life and death of marine plants and animals. They are generally grouped into six classes based on structural similarities hydrocarbons, carbohydrates, lipids, fatty acids, amino acids, and nucleic acids. Because they represent compounds that can be quantified and understood for their chemical properties and known role in biological systems, a great deal of information has been accumulated over the years about these groups and the specific compounds found within them.7... 

The system reported by Avdeef and co-workers [25-28,556-560] is an extension of the Roche approach, with several novel features described, including a way to assess membrane retention [25-28,556,557] and a way to quantify the effects of iso-pH [558] and gradient pH [559] conditions applied to ionizable molecules. A highly pure synthetic phospholipid, dioleoylphosphatidylcholine (DOPC), was initially used to coat the filters (2% wt/vol DOPC in dodecane). Other lipid mixtures were subsequently developed, and are described in detail in this chapter. 

Up to now in this chapter, we have concentrated on the measurement via electric field sensitive dyes of the transmembrane electrical potential, which by itself should produce a linear drop in the electrical potential across a membrane. However, at least through the lipid matrix of a cell membrane, the electrical potential, /, at any point does not change linearly across the membrane. Instead, it follows a complex profile (see Fig. 6). This is due to contributions other than the transmembrane electrical potential to /. The other contributions come from the surface potential and the dipole potential. Both of these can also be quantified via electric field sensitive dyes. 

The determination of F2-isoprostanes, oxidation products of arachidonic acid, has been proposed as a more reliable index of oxidative stress in vivo, overcoming many of the methodological problems associated with other markers. The isoprostanes have emerged as a most effective method of quantifying the potential of antioxidants to inhibit lipid peroxidation. However, one drawback of this method is that quantification of F2-iP requires sophisticated techniques, in particular GC/MS and HPLC/MS... 

Bors W, Michel C and Saran M. 1984. Inhibition of the bleaching of the carotenoid crocin. A rapid test for quantifying antioxidant activity. Biochim Biophys Acta Lipids Lipid Metab 796(3) 312-319. 

The initial mixture and each time point are then assayed for doxorubicin and lipid. Lipid concentrations can be quantified by the phosphate assay (see above) or by liquid scintillation counting of an appropriate radiolabel. Doxorubicin is quantified by an absorbance assay (see below). The percent uptake at any time point (e.g., t = 30 minutes) is determined by %-uptake = [(D/L), =30minutes] x 100/[(D/L) inuiai]. Doxorubicin can be assayed by both a fluorescence assay and an absorbance assay, but we find the latter to be more accurate. The standard curve consists of four to five cuvettes containing 0 to 150 nmol doxorubicin in a volume of 0.1 mL samples to be assayed are of the same volume. To each tube is added 0.9 mL of 1% (v/v) Triton X-100 (in water) solution. For saturated lipid systems such as DSPC/Chol, the tubes should be heated in a boiling water bath for 10 to 15 seconds, until the detergent turns cloudy. Samples are allowed to cool, and absorbance is read at 480 nm on a UV/Visible spectrophotometer. 

The initial mixture and each time point are then assayed for ciprofloxacin and lipid. Lipid can be quantified using the phosphate assay (64,65) or by liquid scintillation counting. Ciprofloxacin is quantified by an absorbance assay following removal of drug from lipid by a Bligh-Dyer extraction procedure (78) (see below). The percent uptake is determined as described in the section Remote Loading of Doxorubicin into DSPC/Cholesterol (55 45) Large Unilamellar Vesicle. ... 

A colorimetric estimation of inserted PEG-phospholipids was developed based on the two-phase system used to quantify phospholipids (49). The formation of a complex between the phospholipids and Fe(SCN)3 transfers the chromophore Fe(SCN)3 to the organic phase, allowing quantification of the phospholipids present in the solution. This system was applied to PEG-phospholipids (50). It is quite sensitive but obviously limited to PEG-phospholipids. The PEG-lipids, which bear no phosphate group, cannot be quantified by this method. 

Another colorimetric method of the associated PEG-lipid might be performed using the method described by Baleux (51). This assay consists of the formation of a complex between the PEG moiety and iodine forming a solution absorbing at 500 nm. This method is less sensitive than the previous one because it does not rely on a specific chemical function on the PEG, although it allows for wider type of PEG-lipid to be quantified. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

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