Phospholipids assays

How well do the compositional values for the individual lipid components and protein and carbohydrate values predicted by Table II agree with experimental values For this comparison, we have selected the experimental values for fractionated LDL published by Chapman et al. (1988). Before these values could be compared, it was necessary to make three adjustments to the experimental values. These workers employed an enzymatic phospholipid assay (Takayama et al., 1977) specific for phosphocholine-containing lipids, which included phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine, making up about 93% of LDL lipids (Skipski etal., 1967). Therefore, the weight of phospholipid has been increased by a divisor of 0.93 in calculating the adjusted experimental values listed in Table 111. Second, Chapman and co-workers... 

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

PhilHppe M, Acker D, Torday J, Schiff I, Frigoletto FD. The effects of vaginal contamination on two pulmonary phospholipid assays. J Reprod Med 1982 27 283-6. 

Phospholipids assay Polar lipids were determined using colorimetric method according to [12]. After acidic hyckolysis, phosphorus was complexed with ammonium molybdate, leading to the formation of a chromogen which absorbed at 820 nm. Standard... 

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. 

Liu, A. et al.. Validated LC/MS/MS assay for curcumin and tetrahydrocurcumin in rat plasma and application to pharmacokinetic study of phospholipid complex of curcumin, J. Pharm. Biomed. Anal, 40, 720, 2006. 

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.]. 

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... 

Faller and Wohnsland [509,554] developed the PAMPA assay using phospholipid-free hexadecane, supported on 10-pm thick polycarbonate filters(20% porosity,... 

Sugano et al. [561,562] explored the lipid model containing several different phospholipids, closely resembling the mixture found in reconstituted brush border lipids [433,566] and demonstrated dramatically improved property predictions. The best-performing lipid composition consisted of a 3% wt/vol lipid solution in 1,7-octadiene (lipid consisting of 33% wt/wt cholesterol, 27% PC, 27% PE, 7% PS, 7% PI). The donor and acceptor compartments were adjusted in the pH interval between 5.0 and 7.4 [562]. With such a mixture, membrane retention is expected to be extensive when lipophilic drugs are assayed. The use of 1,7-octadiene in the assay was noted to require special safety precautions. 

Four neutral lipid models were explored at pH 7.4 (1) 2% wt/vol DOPC in dode-cane, (2) olive oil, (3) octanol, and (4) dodecane. Table 7.5 lists the effective permeabilities Pe, standard deviations (SDs), and membrane retentions of the 32 probe molecules (Table 7.4). The units of Pe and SD are 10 6 cm/s. Retentions are expressed as mole percentages. Figure 7.22a is a plot of log Pe versus log Kd (octanol-water apparent partition coefficients, pH 7.4) for filters loaded with 2% wt/vol DOPC in dodecane (model 1.0, hlled-circle symbols) and with phospholipid-free dodecane (model 4.0, open-circle symbols). The dashed line in the plot was calculated assuming a UWL permeability (see Section 7.7.6) Pu, 16 x 10-6 cm/s (a typical value in an unstirred 96-well microtiter plate assay), and Pe of 0.8 x 10-6 cm/s... 

Highly insoluble molecules are in part transported in the GIT by partitioning into the mixed micelles injected into the lumen from the biliary duct in the duodenum (Fig. 2.3). Mixed micelles consist of a 4 1 mixture of bile salts and phospholipids (Fig. 7.13). In contrast, at the point of absorption in the BBB, highly insoluble molecules are transported by serum proteins. This distinction is expected to be important in in vitro assay modeling. The use of simulated intestinal fluids is appealing. 

This book is written for the practicing pharmaceutical scientist involved in absorption-distribution-metabolism-excretion (ADME) measurements who needs to communicate with medicinal chemists persuasively, so that newly synthesized molecules will be more drug-like. ADME is all about a day in the life of a drug molecule (absorption, distribution, metabolism, and excretion). Specifically, this book attempts to describe the state of the art in measurement of ionization constants (p Ka), oil-water partition coefficients (log PI log D), solubility, and permeability (artificial phospholipid membrane barriers). Permeability is covered in considerable detail, based on a newly developed methodology known as parallel artificial membrane permeability assay (PAMPA). 

Faller and Wohnsland [18, 19] developed the PAMPA assay using phospholipid-free hexadecane, supported on 10 pm-thick polycarbonate filters, and were able to demonstrate interesting predictions. Their PAMPA method appeared to be a satisfactory substitute for obtaining alkane/water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. Apparently, membrane retention was not measured. 

Hendrickson, H. S., Hendrickson, E. K., Johnson, I. D. and Farber, S. A. (1999). Intramolecularly quenched BODIPY-labeled phospholipid analogs in phospholipase A(2) and platelet-activating factor acetylhydrolase assays and in vivo fluorescence imaging. Anal. Biochem. 276, 27-35. 

Silvius, J.R., Leventis, R., Brown, P.M., and Zuchermann, M. (1987) Novel fluorescent phospholipids for assays of lipid mixing between membranes. Biochemistry 26, 4279-4287. 

A variety of methods have been developed to study exocytosis. Neurotransmitter and hormone release can be measured by the electrical effects of released neurotransmitter or hormone on postsynaptic membrane receptors, such as the neuromuscular junction (NMJ see below), and directly by biochemical assay. Another direct measure of exocytosis is the increase in membrane area due to the incorporation of the secretory granule or vesicle membrane into the plasma membrane. This can be measured by increases in membrane capacitance (Cm). Cm is directly proportional to membrane area and is defined as Cm = QAJV, where Cm is the membrane capacitance in farads (F), Q is the charge across the membrane in coulombs (C), V is voltage (V) and Am is the area of the plasma membrane (cm2). The specific capacitance, Q/V, is the amount of charge that must be deposited across 1 cm2 of membrane to change the potential by IV. The specific capacitance, mainly determined by the thickness and dielectric constant of the phospholipid bilayer membrane, is approximately 1 pF/cm2 for intracellular organelles and the plasma membrane. Therefore, the increase in plasma membrane area due to exocytosis is proportional to the increase in Cm. 

Prior to being able to study the function and mechanism of an enzyme, it is essential that suitable assays be available to monitor enzyme activity toward different substrates and to determine the kinetic parameters kcat and Km for the reactions. A brief overview of the known assays for the evaluation of PLCB(. activity is thus appropriate. The ideal assay for a phospholipase C would utilize a phospholipid substrate, not an analogue with a modified headgroup or side chains. Such an assay should be sensitive to minimize the quantities of enzyme and substrates that would be required, and it should be convenient to implement so that analyses may be readily performed. 

Several other methods, including a 31P NMR [39,40] and radiometric assay [28, 41] have also been used to monitor the PLCBc catalyzed hydrolysis of phospholipids. The assays that are currently available for PLCBc are compared in Table 1. 

Recently, a series of soluble phospholipids with n-hexanoyl side chains and a variety of headgroups were assayed as substrates for PLCBc [37]. The results (Table 4) indicate that both l,2-dihexanoyl-s -glycero-3-phosphocholine (C6PC) and l,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE) are processed with similar catalytic efficiencies (kcaXIKm) by the enzyme, while 1,2-dihe-... 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Wohnsland and Faller ([175] performed measurements using a thin (9-10 //in) supported, phospholipid-free hexadecane layer. To validate their model, they used 32 well-characterized chemically diverse compounds. The permeability values obtained with their model could be correlated with known human absorption values if the maximum permeability obtained at different pH was taken into account. However, several disadvantages are related to this method. For hydrophilic drugs, hexadecane by itself has an increased barrier function in comparison with membranes. In addition, the hexadecane layers are not very stable, which makes this assay difficult to apply as a routine screening method. The advantage of this PAMPA setup is that it appears to be a satisfactory substitute for obtaining alkane-water partition coefficients, which are usually very difficult to measure directly, due to the poor solubility of drug molecules in alkanes. 

The appearance of anionic phospholipids, particularly phosphatidylserine, on the cell siuface activates prothrombinase complex culminating in the formation of thrombin (Bevers et al., 1982 Connor et al., 1989). The assay can be performed with pure coagulation proteins and specific chromogenic substtates to produce a very sensitive test to detect the appearance of phosphatidylserine on ceU siufaces. Nevertheless, it has been shown that changes in the disposition of phosphatidylethanolamine and sphingomyelin may interfere with the ability of phosphatidylserine-containing membranes to activate prothrombinase (Smeets et al., 1996). 

McIntyre, J.C. and Sleight, R.G., 1991, Fluorescence assay for phospholipid membrane asymmetry. Biochemistry, 30 11819-11827. 

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