Thermodynamic and infrared analyses of the interaction of chlorpromazine with phospholipid monolayers

An investigation has been made of the interaction between chlorpromazine (CPZ) and monolayers of 1,2-dipalmitoyl-sn-3-glycerophosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-3-glycero[phospho-rac-(1-glycerol)] (DPPG), both at the air/water interface and in transferred Langmuir-Blodgett films. The Gibbs free energy, DeltaG, and the compressibility modulus (C(S)(-1)), obtained from the surface pressure isotherms, indicated changes in the in-plane interactions of CPZ/DPPG mixed monolayers, with positive values of DeltaG. The arrangement of CPZ in the zwitterionic DPPC monolayers causes a weaker interaction in CPZ/DPPC mixed monolayers, with the DeltaG fluctuating around zero. IR measurements in transferred monolayers showed that CPZ did not affect the conformational order of the acyl chains, its effects being limited to the bands corresponding to the headgroups. Furthermore, since no shift was observed for the acyl chain bands, the phase transition induced by CPZ is not a liquid expanded (LE) to liquid condensed (LC) transition, as the latter is associated with chain ordering. Taken together, the IR and compressibility results demonstrate that the effect from CPZ cannot be correlated with temperature changes in the subphase for pure monolayers, in contrast to models proposed by other authors.     

A method for assessing and maintaining the reproducibility of mass spectrometric analyses of complex samples

Direct injection mass spectrometric analysis of biological samples is potentially an attractive approach to the discovery of diagnostic patterns for specific pathophysiological conditions because of its speed and simplicity. Despite the possible benefits offered by such a method, its extensive application has been limited so far by several factors, including the inadequate reproducibility of the analytical results. We describe a method for monitoring and optimizing the performance of mass spectrometers used for biomarker discovery studies, based on the analysis of patterns of standardized spectral features. The method was successfully applied to maintaining spectral reproducibility during a multi‐day analysis of hundreds of serum samples despite an ion source failure, which necessitated minor maintenance. The monitoring method allowed the early detection of that failure and the restoration of the spectral profiles after the system was restarted. Copyright © 2009 John Wiley & Sons, Ltd.     

Improvement of reproducibility of trace analysis by spark-source mass-spectrometry

Summary Mechanisms responsible for poor reproducibility of analysis by spark-source mass-spectroscopy are discussed. A new scheme for the output cascade of a radiofrequency generator has been developed. It allows for stabilizing both the charge and impurity mass-spectrum composition at changing the width of the interelectrode gap over a wide range. The causes of transient irreproducibility in mass-spectrometric analysis have been studied and traced to temporal variation in the discharge-circuit parameters. Methods of eliminating this irreproducibility are proposed.

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Verbesserung der Reproduzierbarkeit der funkenmassenspektrometrischen Spurenanalyse

Zusammenfassung Die Ursachen der schlechten Reproduzierbarkeit der mit Hilfe der Fun-kenmassenspektrometrie durchgeführten Spurenanalyse wurden erörtert. Ein neues Schema der Ausgangsstufe des Hochfrequenzgenerators wurde ausgearbeitet. Dies ermöglicht die Konstanthaltung sowohl der Ladung wie der Zusammensetzung des Massenspektrums auch bei weitgehender Veränderung des Elektroden-Zwischenraumes. Die Ursachen der vorübergehenden Nicht-Reproduzierbarkeit massenspektrometrischer Analysen wurden untersucht. Diese wird von der zeitlichen änderung der Ladungsparameter verursacht. Vorschläge für die Behebung dieser Nicht-Reproduzierbarkeit wurden gemacht.

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Presented at the 8th International Microchemical Symposium, Graz, August 25–30, 1980.     

Structural and morphological transition of long-chain phospholipid vesicles induced by mixing with short-chain phospholipid

Effects of a short-chain phospholipid, dihexanoylphosphatidylcholine (DHPC), on the structure and morphology of membrane assemblies of a long-chain phospholipid, dimyristoylphosphatidylcholine (DMPC), were examined by fluorescence spectroscopy, differential scanning calorimetry (DSC), and cryogenic transmission electron microscopy (cryo-TEM). It was found by fluorescence measurements that DHPC affects on the gel and liquid crystalline state of DMPC vesicle membranes in different ways. Further, the result of DSC suggested that, along the transition process from DMPC vesicle to DMPC–DHPC mixed micelle, there are at least three different concentration regions which are characterized by the individual variation pattern of the transition temperature and enthalpy change. The cryo-TEM micrographs demonstrated the formation of thread-like assemblies in the second region and the coexistence of the assemblies and spherical micelles in the third region. Thus, it was concluded that the structural transition from DMPC vesicle to DMPC–DHPC mixed micelle could occur in a stepwise manner through the formation of the thread-like assembly, which cannot be described by the three-stage model of vesicle to micelle transition.     

Synthesis of novel phospholipid polymers by polycondensation

To synthesize novel phospholipid polymers by polycondensation, 1,3‐dichloroisopropyl phosphorylcholine (DCPC) was synthesized as a new monomer. The DCPC was reacted with 1,4‐dibromobutane in the presence of potassium carbonate, and polycarbonates having phosphorylcholine group were obtained with changing in the mole fraction of DCPC unit. The number averaged molecular weight (Mn) of the polycarbonate determined by gel‐permeation chromatography was in the range between 4×10³–1×10⁴ g·mol^–1. The polycarbonate containing 30 mol‐% of DCPC units was dissolved in water. The surface tension measurement of an aqueous solution containing the polycarbonate indicated the formation of a polymer associate when the concentration of the polycarbonate was higher than 5.0×10^–3 g/dL.     

The synthesis of an amine-bearing polymerizable phospholipid

Exchange of the natural glycerol phospholipid backbone with an artificial 1,3-diaminopropanol backbone led to the first synthetic 1,3-diaminophospholipid. The amines in the polar head group region were reacted to acrylamides giving a UV polymerizable phospholipid. Preliminary experiments demonstrate that formulated vesicles can be polymerized into large aggregates of vesicles.     

Solubilization of unilamellar phospholipid bilayers by nonionic surfactants

The mechanisms governing the solubilization of neutral or electrically charged unilamellar liposomes by a series of octylphenol polyethoxylated surfactants (average of ethylene oxide units between 8.5 and 20.0) were investigated. Solubilization was detected as a decrease in light-scattering of liposome suspensions. To this end, in accordance with the nomenclature adopted by Lichtenberg, three parameters were considered as corresponding to the effective surfactant/lipid molar ratios (Re) at which the surfactant (a) saturated the liposomesRe _sat; (b) resulted in a 50% solubilization of vesiclesRe _50% and (c) led to a total solubilization of liposomesRe _sol. These parameters, corresponded to theRe at which light scattering starts to decrease, reaches 50% of the original value and shows no further decrease.It is noteworthy that theRe _sat parameter decreases as the EO contents or the surfactant critical micellar concentration (CMC) increases. However, theRe _50% and theRe _sol parameters show the lowest values for the surfactant with 12.5 EO units in its molecular structure regardless of the electrical charge of the lipid bilayers. As a consequence, these last parameters are not linearly correlated with the CMC of these surfactants. The CMC values of the surfactant/lipid systems at 0.5 mM lipid concentration corresponded in all cases to the surfactant concentration at which liposomes were saturated by surfactants (S _sat).     

Solubilization kinetics of phospholipid vesicles by sodium taurocholate

The solubilization kinetics of phospholipid vesicles, about 100 nm in diameter and composed of egg phosphatidylcholine (EPC) and EPC/cholesterol in molar ratio 7/3, by sodium taurocholate (TC) used as a model bile salt were investigated by monitoring the turbidity at 500 nm and by quasielastic light scattering (QELS). The solubilization process was found to be dependent on the rate of TC addition. Although the solubilization profiles were identical whatever the rate of TC addition, an increase in the amount of TC needed to solubilize phosphatidylcholine liposomes was observed at higher rates. These results suggest that at low TC concentrations the permeability of the membrane to taurocholate is the rate-limiting step of the solubilization. In the case of cholesterol-containing vesicles, the effect of the rate of addition of TC was observed only at the solubilization characteristic points, called B and C, corresponding to a sharp decrease in the turbidity. This suggests that cholesterol greatly reduces the permeability of the membrane. In addition, the kinetic process was found to be independent of the micellar concentration of the detergent added to the aqueous medium, indicating that the solubilization of liposomes by TC was independent of the initial state of aggregation of the detergent. The calculated values of lipid/TC aggregates and of the partition coefficient show that the kinetic effect observed at high TC concentrations prior to complete solubilization might also be due to the diffusion of the detergent into the membranes. This gives rise to the differences in composition of the aggregates as a consequence of the variation in the rate of TC addition. In addition, QELS scattered intensity variations confirm the presence of a kinetic process for the solubilization of liposomes by TC. In conclusion, our results suggest that solubilization of lipid vesicles by TC is governed by kinetic parameters that might be controlled by liposome membrane permeability at low TC concentrations and by the lateral diffusion of the detergent into aggregates at higher TC concentrations.     

Penetration of mercury-adsorbed phospholipid monolayers by polynuclear aromatic hydrocarbons

The penetration of phospholipid monolayers (dioleoyl lecithin) adsorbed on mercury by polynuclear aromatic hydrocarbons (PAH) is described. The PAH studied were anthracene, phenanthrene, pyrene, benzo[a]anthracene, fluoranthene and perylene. The penetration is monitored by measuring the differential capacitance of the monolayer; the uptake of PAH causes a potential shift (up to ?0.25 V) in the cathodic capacitance peaks. This occurs without displacement of the lipid from the mercury. The differential capacitance is measured by out-of-phase (90°) a.c. voltammetry and rapid cyclic voltammetry. The PAH permeate the mercury-adsorbed lipid layers from dilute aqueous solution; the order of affinity is benzo[a]anthracene > fluoranthrene = pyrene > anthracene = phenanthrene. The rates of penetration vary for the different compounds and depend on their water solubility.     

Pore formation in phospholipid bilayers by amphiphilic cavitands

Five new cavitands were prepared that have four pendant n-undecyl chains and "headgroups" connected by 2-carbon spacers. The headgroups were ~OCH(2)CONH-Ala-OCH(3), 1; ~OCH(2)CONH-Phe-OCH(3), 2; ~OCH(2)CONH-Ala-OH, 3; ~OCH(2)CONH-Phe-OH, 4; and ~OCH(2)CONHCH(2)CH(2)-thyminyl, 5. Pore formation by each cavitand was studied by use of the planar bilayer conductance experiment. All five compounds were found to form pores in asolectin bialyer membranes. Compounds 1-3 behaved in a generally similar fashion and exhibited open-close dynamics. Compounds 4 and 5 formed pores more rapidly, were more dynamic, and led more quickly to membrane rupture. Differences in the ion transport activity are rationalized in terms of structure and aggregate cavitand assemblies.     

Enzymatic lithography of phospholipid bilayer films by stereoselective hydrolysis

The stereoselective phospholipase A2-catalyzed hydrolysis of patterned phospholipid bilayers consisting of the l- and d-isomers of alpha-dilauroylphosphatidylcholine (DLPC) and alpha-dipalmitoylphosphatidylcholine (DPPC) is reported. The stereochemically directed enzyme lithography demonstrated herein allows the parallel modification of large surface areas and constitutes a potentially useful method to structure biomimetic films, given the stereospecific action of many enzymes.     

银杏磷脂复合物复合率的测定

为制定银杏磷脂复合物的制备工艺的评价标准．以高效液相色谱法测定复合物中黄酮醇苷的含量,计算复合率,以此为评估标准,用正交设计试验优化了制备工艺．确定了高效液相色谱法测定复合物复合率的方法,并对复合物中的银杏内酯进行了鉴别。     

Improving the reproducibility of quantitative gas-liquid chromatography by modifying the injection system

Summary In order to increase accuracy of split ratio measurements by interruption of the septum purge gas stream a gas chromatograph (GLC) sample injection system was modified by the introduction of a rapid-action gate-valve in the appropriate gas line. The effects of this modification are described. It is shown to improve the reproducibility of injected amounts in multiple analysis and thus the accuracy of quantitative determinations.     

The orientational ordering of benzyl alcohol in phospholipid bilayers

The α-methylene segment of benzyl alcohol has been deuterated at one site and the proton—deuterium dipole spin interaction measured for this compound dissolved in bilayers of dimyristoylphosphatidylcholine. These data, when combined with measured deuterium quadrupole interactions at other sites, allow for a more complete understanding of the orientational ordering and conformation of benzyl alcohol in the bilayers. Models for the rotary motion of the ring are discussed.     

On-line slurry analyses by californium-252

In chemical processing technology on-line activation methods gain an increasing importance for process monitoring and control. In the paper presented a method is described according to which the different fluorspar contents at various strategic points of a flotation plant are to be determined through neutron activation by 100 μg²⁵² californium. For that purpose, a continuous analytical system for on-stream process control of slurries was designed and constructed. This SUSAC compact facility allows continuous application of the method on an industrial scale.     

Fluorescence quenching of anthracene by indole derivatives in phospholipid bilayers

The quenching of anthracene fluorescence by indole, 1,2-dimethylindole (DMI), tryptophan (Trp) and indole 3-acetic acid (IAA) in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers was investigated. A very efficient quenching of the anthracene fluorescence in the lipid membrane is observed. Stern-Volmer plots are linear for DMI but present a downward curvature for the other quenchers. This was interpreted as an indication of the presence of an inaccessible fraction of anthracene molecules. By a modified Stern-Volmer analysis the fraction accessible to the quenchers and the quenching constant were determined. The changes in the fluorescence emission spectrum of indole and DMI have been used to calculate the partition constants of these probes into the membranes, and bimolecular quenching rate constants were determined in terms of the local concentration of quencher in the lipid bilayer. The rate constants are lower than those in homogeneous solvents, which may be ascribed to a higher viscosity of the bilayer. No changes in the emission spectra of Trp and IAA are observed in the presence of vesicles, indicating that these probes locate preferentially in the aqueous phase, or in close proximity to the vesicular external interface in a medium resembling pure water. In these cases quenching rate constants were determined in terms of the analytical concentration. In the quenching by DMI a new, red shifted, emission band appears; it is similar to that observed in non-polar solvents and it is ascribable to an exciplex emission. The exciplex band is absent in the quenching by IAA and Trp and only very weakly present when the quencher is indole. From the position of the maximum of the exciplex emission, a relatively high local polarity could be estimated for the region of the bilayer where the quenching reaction takes place.     

The effect of numerical error on the reproducibility of molecular geometry optimizations

Geometry optimization is one of the most often applied techniques in computational drug discovery. Although geometry optimization routines are generally deterministic, the minimization trajectories can be extremely sensitive to initial conditions, especially in case of larger systems such as proteins. Simple manipulations such as coordinate transformations (translations and rotations), file saving and retrieving, and hydrogen addition can introduce small variations ( approximately 0.001 A) in the starting coordinates which can drastically affect the minimization trajectory. With large systems, optimized geometry differences of up to 1 A RMSD and final energy differences of several kcal/mol can be observed when using many commercially available software packages. Differences in computer platforms can also lead to differences in minimization trajectories. Here we demonstrate how routine structure manipulations can introduce small variations in atomic coordinates, which upon geometry optimization, can give rise to unexpectedly large differences in optimized geometries and final energies. We also show how the same minimizations run on different computer platforms can also lead to different results. The implications of these findings on routine computational chemistry procedures are discussed.