Physical stability of sonicated arsonoliposomes: effect of calcium ions.

The physical stability of sonicated arsonoliposomes in the absence and presence of Ca(2+) ions is evaluated. Cholesterol-containing arsonoliposomes composed of arsonolipids [having different acyl chains (C(12)-C(18))], or mixtures of arsonolipids with phospholipids (phosphatidylcholine or distearoyl-phosphatidylcholine) were prepared, and physical stability was evaluated in the absence and presence of CaCl(2), by vesicle dispersions turbidity measurements and cryo-electron microscopy morphological assessment. In some cases, vesicle zeta-potential was measured, under identical conditions. Results demonstrate that self-aggregation of the vesicles studied is low and influenced by the acyl chain length of the arsonolipid used, whereas calcium-induced aggregation is higher, correlating well with the decreased values of vesicle zeta-potential in the presence of Ca(2+) ions (weaker electrostatic repulsion). Acyl chain length of arsonolipids used has a significant quantitative effect on Ca(2+)-induced vesicle aggregation mainly for arsonoliposomes that contain phospholipids (mixed), compared with the vesicles that consist of plain arsonolipids (significant effect only for initial aggregation at time 0). Another difference between plain and mixed arsonoliposomes is that for mixed arsonoliposomes Ca(2+)-induced increases in turbidity are irreversible by ethylenediaminotetraacetic acid, suggesting that vesicle fusion is taking place. This was confirmed by cryo-electron microscopy observations. Finally, when phosphatidylcholine is replaced by distearoyl-phosphatidylcholine, arsonoliposomes are more stable in terms of self-aggregation, but in the presence of calcium, the turbidity and morphology results are similar.

The effect of pH on the electrophoretic behaviour of a new class of liposomes: arsonoliposomes.

Herein we report the effect of pH on the surface charge of a new class of liposomes: arsonoliposomes. Plain or mixed arsonoliposomes with cholesterol (Chol) and distearoyl-phosphatidylcholine (DSPC) in 1:1 molar ratio were prepared with lauryl-(C12), myristoyl-(C14) and palmitoyl-(C16) acyl side chain arsonolipids. The one step hydration method was used for vesicle preparation and zeta potential measurements were performed in the pH range from 3 to 9. The results revealed that these lipids hold a negative surface charge at all pH values investigated. The presence of cholesterol in 1:1 molar ratio results in higher zeta potential compared with plain arsonoliposomes with the exception of palmitoyl-(C16) acyl chain arsonolipids in neutral and slightly basic pH values. Oppositely, the DSPC (1:1 molar ratio) containing arsonoliposomes had lower values of zeta potential compared with plain arsonoliposomes. Concluding, the experimental results reveal that zeta potential of arsonoliposomes is indeed modified when the vesicles are incubated in environments with different acidity. In most cases these changes are in accordance with the ionization pattern of the arsonolipid headgroup, while some peculiar deviations may be connected with the known difference in the structure between some of the vesicle types studied.

Capability of arsonolipids to transport divalent cations: a Pressman cell study.

The ability of the newly synthesized arsonolipids (2,3-diacyloxyprophlarsonic acids) to transport cations was studied using the Pressman cell. Experimental results demonstrate that arsonolipids are much more efficient carriers of Ca(2+) and Mg(2+) than natural phosphatidic acid in the Pressman cell experiments. The ability of arsonolipids to transfer Ca(2+) is affected by the lipid side chain length in the order: C(12)>>C(14) approximately C(16). Ca(2+) is transferred faster than Mg(2+), suggesting that the latter is more tightly bound to the arsonolipids. The transfer kinetic curves are parabolic for C(12), while initially linear with a tendency to reach a steady state for C(14) and C(16), when the pH in the donor compartment was 8.3. The transport kinetics for both ions studied were best fitted by an equation derived from saturation kinetics that apply in reversible chemical reactions. The ion transfer rates increased as the pH in the donor compartment decreased.

Preparation and properties of arsonolipid containing liposomes.

Arsonolipids are analogs of phosphonolipids which have a chemically versatile head group. In preliminary cell culture studies, liposomes composed solely of arsonolipids or of phosholipid-arsonolipid mixtures, demonstrate a specific toxicity against cancer cells (Gortzi et al., unpublished results). The possibility of using such formulations as an alternative of arsenic trioxide with or without combination of other cytostatic agents (encapsulated in their aqueous interior) prompted the investigation of their physicochemical characteristics. Herein we compared the characteristics of arsonolipid containing vesicles with different lipid compositions. Experimental results and morphological observations reveal that non-sonicated formulations have different structures and stability (when both membrane integrity and aggregation are taken into account) depending on the acyl chain length of the arsonolipid. When phospholipids and especially cholesterol are included in their membranes almost all arsonolipids studied produce more stable vesicles. An interesting aspect of these arsonolipid containing vesicles is also their negative surface charge, which may be modulated by mixing phospholipids with arsonolipids. Sonicated vesicles have smaller sizes and profoundly higher stability, especially when containing cholesterol and phosphatidylcholine mixed with arsonolipids. The only exception is that of the arsonolipid with the C(12) acyl chain which was observed to produce long tubes which break down to cubes by sonication. In conclusion, these initial studies demonstrate that sonicated vesicles composed of arsonolipid and phospholipid mixtures mixed with cholesterol posses the stability required to be used as an arsonolipid delivery system. In addition, although cryo-electron microscopy demonstrated that the sonicated vesicles are elliptical in shape, their encapsulation efficiency is not significantly lower than sonicated phospholipid liposomes. Thereby, these vesicles may be also used for the delivery of other drug molecules which can be sufficiently retained in their aqueous interior.

Kinetic basis for the substrate specificity during hydrolysis of phospholipids by secreted phospholipase A2.

Kinetics of hydrolysis of aqueous dispersions of arsono-, sulfo-, phosphono- and phospholipids by phospholipase A2 from pig pancreas are characterized in terms of interfacial rate and equilibrium parameters. The enzyme with or without calcium binds with high affinity to the aqueous dispersions of the four classes of anionic lipids and shows the same general kinetic behavior. The rate of hydrolysis of anionic substrates does not show an anomalous change at the critical micelle concentration because the enzyme is present in aggregates even when bulk of the substrate is dispersed as a solitary monomer. Apparent affinities of the enzyme for the interface of different anionic lipids are virtually the same. Also, affinities of these substrates for the active site of the enzyme at the interface are comparable. However, a significant change in the catalytic turnover rate is seen as the sn-3 phosphodiester group is modified; the apparent maximum rate at saturating bulk substrate concentration, V(M)app values, increase in the order: homo- and arsonolipids < sulfo- < phosphono- < phospholipids. Not only the basis for the sn-2 enantiomeric selectivity but also the decrease in the rate of hydrolysis with the increasing chain length is due to a decrease in the value of V(M)app. Results show that even when the bulk concentration of anionic phospholipid is below cmc, hydrolysis occurs in aggregates of enzyme and substrate where the chemical step of the turnover cycle remains rate-limiting, which provides a basis for the assumption that V(M)app is directly related to Kcat. The fact that Kcat depends on the nature of the head group (phosphate, phosphonate, sulfate, arsonate) implies that the head group plays a critical role in the rate-limiting chemical step of the catalytic cycle, possibly during the decomposition of the tetrahedral intermediate. The significance of these results for the microscopic steady-state condition for hydrolysis at the micellar interface, mechanism of esterolysis by phospholipase A2, and inhibitor design are discussed.

Carbonic Anhydrase Interaction With Lipothioars Enites: A Novel Class of Isozymes I and II Inhibitors.

The interaction of carbonic anhydrase (CA) isozymes I and II with a series of As(III) derivatives, dialkyl and diaryl rac-2,3-dimyristoyloxypropyldithioarsonites, was investigated kinetically and spectrophotometrically, utilizing the native and Co(II)-substituted enzymes. Depending on the substitution pattern at the -As(SR)(2) moiety of the investigated derivatives, inactive compounds were found for R = phenyl or naphthyl, and active ones for derivatives containing carboxyl groups (R = CH(2)COOH, cysteinyl and glutathionyl). Together with the arsonolipids previously investigated, the active compounds of this series - the "lipothioarsenites"- constitute a novel class of CA inhibitors that bind to the metal ion within the enzyme active site, as proved by changes in the electronic spectra of adducts of such inhibitors with Co(II)CA.

Selenium in human milk and dietary selenium intake by Greeks.

Fluorimetric determination of selenium in colostrum, transitional and mature human milk gave the following concentrations (mean and standard deviation): 41 +/- 16, 23 +/- 6 and 17 +/- 3 ng Se ml-1, respectively. The ranges for each kind of milk, especially for mature milk, were narrow. For all cases studied, the Se concentration in milk decreased with lactation time, reaching a plateau, at 17 ng Se ml-1, after 20 days. It is estimated that breast-fed-only babies in Greece receive approximately 5-11 micrograms Se day-1 up to 6 months of age. From consumed food data it was estimated that adult Greeks receive 100 +/- 6 micrograms Se day-1, in close agreement with our previously determined value of 110 micrograms Se day-1 estimated from food disappearance data.

Selenium status of cancer patients in Greece.

The Se status of 177 Greek cancer patients has been assessed by fluorimetric analysis of the Se concentration in whole blood, urine (morning) and hair. Patients either with newly diagnosed cancers or with metastases or who are undergoing therapy have statistically significant less blood, urine and hair Se than age- and sex-matched healthy controls. There is a strong and significant correlation between blood and urine or hair Se in the newly diagnosed cancers. In cancers of systems involving absorption, metabolism and excretion of Se, and in female breast cancer, the Se levels in blood, urine and hair are significantly lower than in the controls. In cancers of the respiratory and haematologic systems, blood and urine Se are normal, while hair Se is significantly lower than in controls. The results are compared with literature data; conflicting data may be due to different dietary habits of the various populations. Some cancerous tissues, such as those of the peptic system, accumulate Se compared with their adjacent, apparently healthy tissues, but others, of the urinary system for example, do not. The reason(s) for this behaviour, however, is not known.

The nutritional selenium status of healthy Greeks.

The nutritional selenium status of apparently healthy Greeks has been assessed by measuring fluorimetrically the selenium content of whole blood, morning urine, hair and finger nails. The means and standard deviations were 165 +/- 33, 25 +/- 7 ng Se ml-1, 416 +/- 86, and 536 +/- 91 ng Se g-1, respectively. No significant difference was found between the selenium content of whole blood, hair and finger nails, but, for morning urine, there was a significant difference between males and females. The young and the elderly have less selenium in these biological materials than other Greeks. Whole blood selenium correlates significantly with morning urine, hair, and finger nail selenium, as does hair and nail selenium of male, female and male + female Greeks. The results are compared with those in the literature and possible explanations for the observations are presented. It is concluded that the selenium status of Greeks is satisfactory.

The regional distribution of selenium in Greek cereals.

The selenium content of hard and soft wheat, barley, oats, rye and corn grown in approximately 100 different locations of Greece has been determined fluorimetrically. The mean values +/- SD for these cereal types were 0.29 +/- 0.19, 0.21 +/- 0.12, 0.16 +/- 0.10, 0.14 +/- 0.10, 0.19 +/- 0.10 and 0.12 +/- 0.08 ppm Se (dry weight basis), respectively. Based on data for selenium in corn from 96 different locations, a geobotanic map of Greece for the selenium in soil available for uptake by plants was prepared. Macedonia, West Epirus, south-east Thessaly, north-east Sterea Hellas and the Aegean Islands produce corn deficient or low in selenium, but only sporadic selenium-deficiency diseases in animals have been observed in many of these areas, probably because the farm animals are given mixed food or they are free to graze. No areas with toxic levels of selenium in soil were found.

Total selenium concentration in tap and bottled drinking water and coastal waters of Greece.

The total selenium concentration in various waters from all over Greece was determined fluorimetrically. The concentration in most of the drinking water, either from the tap or bottled, was within the range 100-200 ng l-1 and showed no great seasonal fluctuations. Most Greeks receive 0.2-0.4 micrograms selenium per day from drinking water. Coastal surface water contains 100-250 ng Se l-1, while higher concentrations are found at locations regarded as polluted.

Phosphatidylcholesterol bilayers. A model for phospholipid-cholesterol interaction.

Aqueous dispersions of monovalent and divalent cations salts of O-(1,2-dipalmitosyl-sn-glycero-3-phosphoryl)cholesterol form multilamellar vesicles as shown by freeze-fracture electron microscopy, by electron micrographs of the negatively stained liposomes, and by swelling curves of liposomes in hypo-osmotic medium. Differential scanning calorimetry reveals that aqueous dispersions of divalent metal salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol undergo a characteristic thermotropic phase transition with a relatively large cooperative unit (n greater than 250 for the calcium salt). In contrast, monovalent cation salts of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol do not show a thermotropic phase transition under comparable conditions. The molecular area of 0-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol in a monolayer is the same in the presence and absence of Ca2+, and is virtually equal to the area of an equimolar mixture of dipalmitoyl phosphatidic acid and cholesterol. To account for the novel state induced by Ca2+, on aqueous dispersions of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol (i.e., bilayer organization and highly cooperative phase transition), a linear array model is proposed in which Ca2+ bridges adjacent arrays of O-(1,2-dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol molecules, thus freezing the acyl chains in their normal state. One of the main corollaries of the model is that the cooperative unit for a thermotropic phase transition is essentially one-dimensional, rather than a two-dimensional matrix. O-(1,2-Dipalmitoyl-sn-glycero-3-phosphoryl)cholesterol is proposed as an orientationally and conformationally restricted analog of glycerophospholipid plus cholesterol in bilayers.

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects.

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.