Phospholipids measurement

The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

Grazing incidence excitation of a fluorescent probe in a phospholipid monolayer can also be used to indicate order. The collective tilt of the molecules in a domain inferred from such measurements is indicative of long-range orientational order [222]. 

In an extensive SFA study of protein receptor-ligand interactions, Leckband and co-workers [114] showed the importance of electrostatic, dispersion, steric, and hydrophobic forces in mediating the strong streptavidin-biotin interaction. Israelachvili and co-workers [66, 115] have measured the Hamaker constant for the dispersion interaction between phospholipid bilayers and find A = 7.5 1.5 X 10 erg in water. 

Phospholipid molecules form bilayer films or membranes about 5 nm in thickness as illustrated in Fig. XV-10. Vesicles or liposomes are closed bilayer shells in the 100-1000-nm size range formed on sonication of bilayer forming amphiphiles. Vesicles find use as controlled release and delivery vehicles in cosmetic lotions, agrochemicals, and, potentially, drugs. The advances in cryoelec-tron microscopy (see Section VIII-2A) in recent years have aided their characterization [70-72]. Additional light and x-ray scattering measurements reveal bilayer thickness and phase transitions [70, 71]. Differential thermal analysis... 

Activated partial thromboplastin time (aPTT) is a coagulation assay, which measures the time for plasma to clot upon activation by a particulate substance (e.g., kaolin) in the presence of negatively charged phospholipids. 

The increase in Ca is initiated rapidly and begins to recover after 1 min. The order of potency correlates fairly well with the solubilities of these compounds in organic solvents (37) and their abilities to accumulate in phospholipid vesicles (38), i.e., 6>y>a>p, but not with their insecticidal activity (y 6>a p 39). At these concentrations, crystals of p-, a-, and y-HCH were evident in the cell suspensions when we made simultaneous measurements of the right-angle light scatter, indicating that the order of aqueous solubilities is 6>y>a>p. However, stimulation by 6-HCH at concentrations below its aqueous solubility limit shows a typical dose dependency of the response (Figure 10). 

Rand, R. P. (1981). Interacting phospholipid bilayers Measured forces and induced structural changes, Ann. Rev. Biophys. Bioeng.. 10. 277-314. 

There is also inside-outside (transverse) asymmetry of the phospholipids. The choline-containing phospholipids (phosphatidylcholine and sphingomyelin) are located mainly in the outer molecular layer the aminophospholipids (phosphatidylserine and phos-phatidylethanolamine) are preferentially located in the inner leaflet. Obviously, if this asymmetry is to exist at all, there must be limited transverse mobility (flip-flop) of the membrane phospholipids. In fact, phospholipids in synthetic bilayers exhibit an extraordinarily slow rate of flip-flop the half-life of the asymmetry can be measured in several weeks. However, when certain membrane proteins such as the erythrocyte protein gly-cophorin are inserted artificially into synthetic bilayers, the frequency of phospholipid flip-flop may increase as much as 100-fold. 

The popular applications of the adsorption potential measurements are those dealing with the surface potential changes at the water/air and water/hydrocarbon interface when a monolayer film is formed by an adsorbed substance. " " " Phospholipid monolayers, for instance, formed at such interfaces have been extensively used to study the surface properties of the monolayers. These are expected to represent, to some extent, the surface properties of bilayers and biological as well as various artificial membranes. An interest in a number of applications of ordered thin organic films (e.g., Langmuir and Blodgett layers) dominated research on the insoluble monolayer during the past decade. 

Immunoassays have been based on the potentiometric measurement of marker ions such tetrapentylammonium ion (TPA ) that are loaded in phospholipid liposomes Complement mediated immunolysis of these loaded vesicles is caused... 

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

The lag-phase measurement at 234 nm of the development of conjugated dienes on copper-stimulated LDL oxidation is used to define the oxidation resistance of different LDL samples (Esterbauer et al., 1992). During the lag phase, the antioxidants in LDL (vitamin E, carotenoids, ubiquinol-10) are consumed in a distinct sequence with a-tocopherol as the first followed by 7-tocopherol, thereafter the carotenoids cryptoxanthin, lycopene and finally /3-carotene. a-Tocopherol is the most prominent antioxidant of LDL (6.4 1.8 mol/mol LDL), whereas the concentration of the others 7-tocopherol, /3-carotene, lycopene, cryptoxanthin, zea-xanthin, lutein and phytofluene is only 1/10 to 1/300 of a-tocopherol. Since the tocopherols reside in the outer layer of the LDL molecule, protecting the monolayer of phospholipids and the carotenoids are in the inner core protecting the cholesterylesters, and the progression of oxidation is likely to occur from the aqueous interface inwards, it seems reasonable to assign to a-tocopherol the rank of the front-line antioxidant. In vivo, the LDL will also interact with the plasma water-soluble antioxidants in the circulation, not in the artery wall, as mentioned above. 

Koryta et al. [48] first stressed the relevance of adsorbed phospholipid monolayers at the ITIES for clarification of biological membrane phenomena. Girault and Schiffrin [49] first attempted to characterize quantitatively the monolayers of phosphatidylcholine and phos-phatidylethanolamine at the ideally polarized water-1,2-dichloroethane interface with electrocapillary measurements. The results obtained indicate the importance of the surface pH in the ionization of the amino group of phosphatidylethanolamine. Kakiuchi et al. [50] used the video-image method to study the conditions for obtaining electrocapillary curves of the dilauroylphosphatidylcholine monolayer formed on the ideally polarized water-nitrobenzene interface. This phospholipid was found to lower markedly the surface tension by forming a stable monolayer when the interface was polarized so that the aqueous phase had a negative potential with respect to the nitrobenzene phase [50,51] (cf. Fig. 5). 

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. 

Capacitance measurements of phospholipid monolayers at the ITIES have been proposed as a suitable tool for studying the enzyme activity under the precise control of the electrical state of the monolayer [81]. Kinetics of hydrolysis of phosphatidylcholine... 

Dynamic surface tension has also been measured by quasielastic light scattering (QELS) from interfacial capillary waves [30]. It was shown that QELS gives the same result for the surface tension as the traditional Wilhelmy plate method down to the molecular area of 70 A. QELS has recently utilized in the study of adsorption dynamics of phospholipids on water-1,2-DCE, water-nitrobenzene and water-tetrachloromethane interfaces [31]. This technique is still in its infancy in liquid-liquid systems and its true power is to be shown in the near future. 

Activated partial thromboplastin time aPTT is performed by adding calcium phospholipids and kaolin to citrated blood and measures the time required for a fibrin clot to form. In this manner, aPTT measures the activity of intrinsic and common pathways. Prolongation of aPTT may be due to a deficiency or inhibitor for factors II, V, VIII, IX, X, XI, and XII. It also may be due to heparin, direct thrombin inhibitors, vitamin K deficiency, liver disease, or lupus anticoagulant. 

Antiphospholipid antibodies include lupus anticoagulants (LAs) and anticardi-olipin (aCL) antibodies. Lupus anticoagulants are immunoglobulins that are characterized by their ability to inhibit phospholipid-dependent coagulation assays. In contrast, aCL antibodies are measured in an enzyme-linked immunosorbent assay... 

A method where phospholipids are entrapped in the pores of resin beads, in the forms of multilamellar vesicles, has been described [313-319,376]. In some ways, the idea is similar to that of IAM chromatography, even though the resin is modified differently. The retention indices correlate very well with the partition coefficients measured in liposome-water systems (described below). 

---