Phospholipids ionization

As a practical example, when a mouse brain section to which 2,5-dihydroxybenzoic acid (DHB) has been applied as a matrix is subjected directly to MS in positive ion-detection conditions, strong peaks which are mainly derived from phospholipids were observed in mass region of 700 < m/z < 900 [8] on the other hand, signals derived from proteins, meanwhile, are scarcely detected at m/z > 3000 [9].This is because phospholipids ionize much more efficiently than proteins, and they, on the other hand, suppress protein/peptide ionization. Therefore, for detecting/imaging proteins and peptides, removal of such lipids improves the sensitivity for proteins analysis. To this end, tissue sections should be rinsed with organic solvent, to remove lipids from tissue samples [9-11]. 

In the case of prothrombin and related clotting factors, interruption of the vitamin K cycle leads to the production of nonfunctional, undercarboxylated proteins, which are duly exported from hepatocytes into blood (Thijssen 1995). They are nonfunctional because there is a requirement for the additional carboxyl residues in the clotting process. Ionized carboxyl groups can establish links with negatively charged sites on neighboring phospholipid molecules of cell surfaces via calcium bridges. 

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 I term is of particular relevance since, in anisotropic media such as liposomes and artiflcial membranes in chromatographic processes, ionic charges are located on the polar head of phospholipids (see Section 12.1.2) and thus able to form ionic bonds with ionized solutes, which are therefore forced to remain in the nonaqueous phase in certain preferred orientations. Conversely, in isotropic systems, the charges fluctuate in the organic phase and, in general, there are no preferred orientations for the solute. Given this difference in the I term (but also the variation in polar contributions, less evident but nevertheless present), it becomes clear that log P in anisotropic systems could be very different from the value obtained in isotropic systems. 

C. N. Partitioning of ionizing molecules between aqueous buffers and phospholipid vesicles. J. Pharm. Sci. 1995, 84, 1180-1183. 

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). 

The same approach derived for weak bases can also be applied to the uptake of simple weak acids, and to the transbilayer transport of acidic lipids, such as fatty acids and some phospholipids. We consider uptake of a simple weak acid into an LUV with basic interior. Let [AH], ([A ]o) and [AH] ([A ] ) refer to the concentrations of the neutral (ionized) form(s) of the weak acid on the outside and inside of the vesicle, respectively. Then, the total external and internal concentrations of the weak acid can be written as... 

Section 3.3.4 pointed out that cosolvents alter aqueous ionization constants as the dielectric constant of the mixture decreases, acids appear to have higher pKa values and bases appear (to a lesser extent than acids) to have lower values. A lower dielectric constant implies that the force between charged species increases, according to Coulomb s law. The equilibrium reaction in Eq. (3.1) is shifted to the left in a decreased dielectric medium, which is the same as saying that pKa increases. Numerous studies indicate that the dielectric constant in the region of the polar head groups of phospholipids is 32, the same as the value of methanol. [381,446-453] Table 5.2 summarizes many of the results. 

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. 

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). 

Phospholipids are amphiphilic substances i.e. their molecules contain both hydrophilic and hydrophobic groups. Above a certain concentration level, amphiphilic substances with one ionized or polar and one strongly hydrophobic group (e.g. the dodecylsulphate or cetyltrimethylammonium ions) form micelles in solution these are, as a rule, spherical structures with hydrophilic groups on the surface and the inside filled with the hydrophobic parts of the molecules (usually long alkyl chains directed radially into the centre of the sphere). Amphiphilic substances with two hydrophobic groups have a tendency to form bilayer films under suitable conditions, with hydrophobic chains facing one another. Various methods of preparation of these bilayer lipid membranes (BLMs) are demonstrated in Fig. 6.10. 

Ayanoglu, E. Wegmann, A. Pilet, O. Marbury, G. D. Hass, J. R. Djerassi, C. Mass spectrometry of phospholipids—some applications of desorption chemical ionization and fast atom bombardment. /. Am. Chem. Soc. 1984, 106, 5246-5251. 

Black, G. E. Snyder, A. P. Heroux, K. S. Chemotaxonomic differentiation between the Bacillus cereus group and Bacillus subtilis by phospholipid extracts analyzed with electrospray ionization tandem mass spectrometry. J. Microbiol. Meth. 1997, 28,187-199. 

Fang, J. Barcelona, M. J. Structural determination and quantitative analysis of bacterial phospholipids using liquid chromatography electrospray ionization mass spectrometry./. Microbiol. Meth. 1998,33,23-35. 

Smith, P. B. W. Snyder, A. P. Harden, C. S. Characterization of bacterial phospholipids by electrospray ionization tandem mass spectrometry. Anal. Chem. 1995, 67,1824-1830. 

Fig. 10.5 Schematic diagrams a micelle consisting of ionized fatty acid molecules, a phospholipid bilayer and the vesicle bilayer of a liposome...

The tissue sections must be washed with organic solvents when the detection targets include peptides and proteins. Washing with organic solvents promotes the ionization of peptides and proteins mainly by removing phospholipids from the sections.14 Washing also flushes out salts that could interfere with the crystallization of the matrix. 

The cross-validated r2 values for these improved models were 0.627 and 0.776, respectively. The models implied that the brain favors cationic compounds over phospholipids membranes. Although log iam offered no advantage in these models over log P(Xt in predicting log BB ratios of the drugs (in particular because log P0ct can be calculated with sufficient accuracy), it appeared to provide a better model than log Poet for the membrane distribution of the ionized compounds. 

Provided a better model for membrane distribution of ionized compounds and concluded that brain favors cationic compounds over phospholipid membranes. 

Kakela R, Somerharju P, Tyynela J. 2003. Analysis of phospholipids molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography-electrospray ionization mass spectrometry. J Neurochem 84 1051. 

Schiller, J. Suss, R. Amhold, J. Fuchs, B. Lessig, J. Muller, M. Petkovic, M. Spalteholz, H. Zschomig, O. Arnold, K. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research. Prog. Lipid Res. 2004, 43, 449-488. 

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