Lipoic acid nanoforms based on phosphatidylcholine: production and characteristics.

We describe the dynamics of lipoic acid (LA) alone, incorporated in liposomes and as a part of nanoemulsions. Mass spectrometry shows that LA in water forms aggregates of two or three molecules in the form of a negatively charged ion and a neutral molecule. Phosphatidylcholine (PC)-based nanoforms of LA as liposomes and nanoemulsions with a particle size equal to 145 nm are characterized by a high degree of incorporation of LA into the nanoparticles and long-term stability during storage at room temperature. Dynamic light scattering (DLS) gives the polydispersity index of the nanoforms (> 0.3), characterizing the homogeneity of the obtained nanodispersions. We found that such emulsions can significantly (5 ×) increase the concentration of LA in the aqueous phase (5-7 mg/mL) when compared with an aqueous solution of LA (1 mg/mL) and by 40% when compared with PC liposomes (4 mg/mL). Moreover, the inclusion of LA in liposomes and nanoemulsions from PC did not change the neutral ζ-potential characteristic of PC nanoforms. CryoTEM established that the structural organization of the liposomes practically did not differ from nanoemulsions and both nanoforms contained both multilayer and single-layer vesicles. When studying the release kinetics of LA from phosphatidylcholine nanoforms, we found that at 22 h, 45-55% of LA was released from nanoparticles, but that at the initial stage of the process LA was slowly released from the nanoemulsions and rapidly from the liposomes. Conductance measurements indicate that LA delivered in all the three forms increase membrane permeability, though this result is most marked with the LA in PC liposomes.

[The profile of plasma non-esterified fatty acids in children with different terms of type 1 diabetes mellitus].

Composition and quantitative content of non-esterified fatty acids (NEFA) were investigated in plasma samples of healthy children (12) and children with type 1 diabetes mellitus (DM1) (31) by gas chromatography (GC) after preliminary NEFA solid-phase extraction from plasma lipids. There was a significant (p<0.001) 1.6-fold increase in the total level of NEFA regardless of the disease duration. In the group of DM1 children with the disease period less than 1 year there was an increase in the arachidonic acid (20:4) content (30%) and the oleic acid trans-isomer (18:1) content (82%), and also a decrease in the docosahexaenoic acid (22:6 n3) content (26% ) and the docosapentaenoic acids (22:5 n-6) content (60%). In the group of DM1 children with prolonged course of this disease the altered NEFA levels returned to the normal level.

Antibacterial Effects of Liposomes Containing Phospholipid Cardiolipin and Fluoroquinolone Levofloxacin on Mycobacterium tuberculosis with Extensive Drug Resistance.

The effects of liposomes containing phospholipid cardiolipin without antibiotic and loaded with levofloxacin on the growth of Mycobacterium tuberculosis with extensive drug resistance were studied in vitro. Liposomes consisting of cardiolipin alone in a concentration of 335 µM completely suppressed the growth of M. tuberculosis. In order to reduce the minimum inhibitory concentration of cardiolipin, complex liposome preparation consisting of phosphatidylcholin/cholesterol/cardiolipin and loaded with levofloxacin was prepared. Due to this, the cardiolipin concentration was reduced to 33.5 µM (50 µg/ml) and concentration of levofloxacin - to 2 µg/ml.

[Non-esterified fatty acids of blood serum in type 1 diabetic women during late pregnancy].

Using gas chromatography a comparative study of the range and content of individual non-esterified fatty acids in serum of patients with diabetes mellitus type 1 in the third trimester of pregnancy, and healthy pregnant and non-pregnant women has been carried out. In groups of pregnant women there was activation of lipid metabolism, confirmed by corresponding changes in serum biochemical parameters, as well as in the content of non-esterified fatty acids. Intergroup differences in the non-esterified fatty acids were not found. However, there were significant differences between the examined groups in the quantitative content of non-esterified fatty acids.

Autoantibodies to neuron-specific proteins S100, GFAP, MBP and NGF in the serum of rats with streptozotocin-induced diabetes.

The appearance of autoantibodies to neuronal proteins (S100, GFAP, MBP, and NGF) in rat serum was analyzed by ELISA on days 5, 10, 17, 25, and 32 after streptozotocin injection. Simultaneously, blood glucose and insulin autoantibodies were assayed. Serum glucose level increased on the next day after streptozotocin injection and the level of autoantibodies to insulin significantly increased on day 5 indicating the development of diabetes. The levels of antibodies to specific neuronal proteins (S100, GFAP, MBP, and NGF) also increased at this term. It is concluded that diabetes with streptozotocin is associated with damage to the blood-brain barrier.

[The proinsulin level in the blood of children with type 1 diabetes mellitus of various duration].

Proinsulin content was measured in the serum of 82 children (aged from 3 to 14 years) with type 1 diabetes mellitus of various duration. Three groups of patients characterized by low (54%), normal (42%) and high (4%) levels of this prohormone were recognized. No dependence the proinsulin level on the disease term was found. The serum proinsulin level may be used as a parameter specifying the pathogenesis of type 1 diabetes mellitus.

[Distal polyneuropathy in children with diabetes mellitus type 1].

To identify the association between distal peripheral neuropathy (DPN) and type 1 diabetes mellitus (T1D), 62 children and adolescents with different duration of T1D have been studied using electromyography (EMG). Subclinical symptoms of DPN occur with equal frequency in 74% of children with short duration of T1D (group 1) and in 72% of children with long duration of disease. In both groups, the highest frequency of disturbances of the neurophysiological parameters was found in the sural sensory nerve and the lowest frequency in the median motor nerve. Correlations between HbAlc levels and compound muscle action potential were revealed only in tibial and peroneal nerves and only in the early stages of T1D. However, this dependence disappears with increasing duration of the disease.

[Erythrocyte lipid composition at different stages of type 1 diabetes in children].

Complete profiles of phospholipid and ceramide molecular species from erythrocyte lipid extracts of children without carbohydrate metabolism disorders, and children with type 1 diabetes were compared by means of high performance liquid chromatography/mass spectrometry. For the first time a statistically significant increase (p<0.05) of lysophosphatidylcholine content in two groups of diabetic children with different duration of the disease (less than one year and more than one year) was found. Statistically significant changes in other lipid classes were not observed. The dependence of the content of phosphatidylcholine and phosphatidylethanolamine molecular species containing arachidonic acid residue (20:4) on the duration of the disease was found. The observed shift in lipid metabolism suggests of phospholipase A2 and chronic inflammatory process at different stages of diabetes mellitus, in cells (erythrocytes), which aer not involved in the immune response.

Role of lipid components in formation and reactivation of Mycobacterium smegmatis "nonculturable" cells.

We have found that transition of actively dividing Mycobacterium smegmatis cells into the dormant "nonculturable" state is accompanied by increase in the protein/lipid ratio and disappearance of one of the main lipid components of the mycobacterial cells, trehalose monomycolate. In this case, oleic acid is accumulated in the culture medium due to its secretion by the mycobacterial cells. Addition of lipids of different classes to "nonculturable" M. smegmatis cells induces their resuscitation. The lipid reactivating effect is evidently caused by the presence of fatty acids in their composition, because free fatty acids also exhibited reactivation effect. Oleic acid in concentration of 0.05-3 µg/ml exhibited maximal effect, and that allows us to draw a conclusion concerning its signal role in the transition of dormant cells into active state.

Biochemical and morphological changes in dormant ("Nonculturable") Mycobacterium smegmatis cells.

Biochemical and morphological changes have been studied during transition of Mycobacterium smegmatis cells into their dormant ("nonculturable") state. A significant fraction of the population of irreversibly "nonculturable" (NC) cells has a thicker cell wall, condensed cytoplasm, and a decreased number of ribosomes. The lipid contents in the NC cells are lower than in the metabolically active cells, with a relatively decreased amount of phospholipid and neutral lipid. Free mycolic acids, which are abundant in metabolically active cells, were not found in the NC cells. The NC forms are also characterized by decreased respiratory activity on endogenous substrates; however, the respiratory chain enzymes retain their activities in the isolated membranes. Activities of the Krebs cycle and glyoxylate cycle enzymes are markedly decreased. Despite a significant decrease in metabolic activity, NC cells possess membrane potential that seems to provide for reversibility of the NC state of mycobacteria, i.e. their capability of reactivating.

[An approach to the identification of the phospholipid molecular species in human erythrocytes using HPLC with mass-spectrometric detection].

A modified RP-HPLC-MS approach has been proposed for a single run separation and identification of the molecular species of different phospholipid classes in a complex extract. This approach has been applied to the analysis of glycero- and sphingolipid composition of human erythrocytes and a number of ceramide fractions have been identified; these fractions was missed in previous studies employing similar methods. The fine experimental design leads to the decrease in the number of procedures needed for a complete phospholipid profiling of the sample.

[Measurement of the endogenous alloxane in human blood].

To determine the endogenous alloxane content in blood of healthy donors we stabilized it by rapid lowering of pH in the sample to 2.0. Alloxane was then allowed to form a colored product in a reaction with o-phenylenediamine and its content was measured by spectrophotometry using an internal calibration curve. The experiment showed that the concentration of alloxane in blood of most of the healthy donors varied from 41 to 265 micromol/l. However, a small group of healthy people was had their blood alloxane level greatly exceeding the above-mentioned values.

Effects of liposomes of different lipid composition on in vitro growth of Mycobacterium tuberculosis H37Rv.

The growth of M. tuberculosis H37RV in culture medium was studied after addition of liposomes from different lipids (phosphatidylcholine, cardiolipin, and glycosphyngolipids). Addition of phosphatidylcholine into culture medium did not modify the growth and multiplication of mycobacteria. Addition of glycosphyngolipids and their mixture with phosphatidylcholine partially inhibited the growth. Addition of cardiolipin inhibited the growth of mycobacteria and even suppressed it, depending on the dose. Presumably, high concentrations of cardiolipin added into the culture medium, can transfer the mycobacteria into an uncultivable state.

[Liposomal formulations of protein proteinase inhibitors: preparation and specific activity].

Here we investigated encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and between the bilayers. The biological activity (antitrypsin and antichymotrypsin) assay of the protein entrapped in liposomes showed its active sites were spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI confirmed this hypothesis and suggested that this shielding is reversible. The liposomes stability in three various media-artificial gastric juice and intestinal fluids was also examined. The liposomes prepared seem to be promising formulations for BBI and BPTI delivery.

[Determination of the distribution coefficient of rifabutin by the fluorescence study in the system liposome-water].

The distribution coefficient (Kd) of tuberculostatic rifabutin in a liposome/water system at pH 6.4 and 7.4 has been determined by the fluorescence quenching method. Large unilamellar vesicles composed of phosphatidylcholine alone or its combination with cholesterol or cardiolipin were used. The fluorescent probe anthrylphosphatidylcholine, which contains the anthryl moiety in the hydrophobic part, was incorporated into large unilamellar vesicles. The Kd values calculated with the use of dynamic quenching theory (Stern-Volmer model), were comparable for phosphatidylcholine and its combination with cholesterol at both pH. The value of lgKd (24-2.6) demonstrates the hydrophobicity of the rifabutin molecule. After the introduction of negatively charged cholesterol, Kd increases more than tenfold at pH 6.4. At pH 7.4, a second phosphate group of cholesterol undergoes ionization, and Kd of rifabutin gains an additional increase. The results obtained demonstrate a strong influence of electrostatic forces on rifabutin-model membranes interaction.

[The effect of liposomes on the growth and sensitivity of Mycobacterium smegmatis to isoniazide].

The effects of the liposome form of isoniazide (IN) and liposomes without IN on the growth of Mycobacterium smegmatis were studied. Fluorescent assay demonstrated that the fraction of liposomes that interacted with M. smegmatis amounted to 1-3%. It was shown that the IN efficiency in a liposomal form decreased depending on the liposome composition and concentration as compared with the IN in water solution. A preincubation of mycobacteria with liposomes led to a decrease in their sensitivity to IN. An analogous effect was observed when incubating M. smegmatis with oleic acid. It was postulated that the relative resistance of M. smegmatis to the antibiotic when using lipids as a carbon substrate appeared due to a change in the agent's metabolism and should be taken into account when testing in vitro the liposomal forms of antibiotics.

[Role of carbon substrates of culture media on Mycobacterium smegmatis susceptibility to antitubercular agents].

Influence of medium composition on Mycobacterium smegmatis growth and susceptibility to antituberculosis drugs (ATD)--isoniazid and rifabutin--was studied. It was shown that addition of phospholipids (PL) in form of liposomes to meat peptone broth resulted in activation of M. smegmatis growth and decrease of its susceptibility to ATD. Growth characteristics of M. smegmatis and its susceptibility to ATD were studied using variants of modified on source of carbon synthetic medium Sauton as growth substrate. It was revealed that presence of acetate or PL in growth medium results in significant decrease of M. smegmatis susceptibility to isoniazid and rifabutin. It was suggested that this phenomenon is determined by activation of glyoxylate cycle by PL and fatty acids, which, in its turn, can stimulate expression of a number of proteins, including cell membrane pumps excreting antibiotics out of microbial cell.

[Evaluation of the efficiency of activity of the liposomal form of isoniazid against different types of mycobacteria in vitro].

M. avium and three M. tuberculosis strains were used as an example to study the bacteriostatic activity of the liposomal form of isoniazid in the liquid nutrient medium by serial dilutions. Unlike isoniazid in solution, its liposomal form was found to suppress the multi plication of all study mycobacterial samples and diminishes their viability, but at different concentrations. The liposomes that did not contain the drug exerted an inhibitory effect only on the M. tuberculosis strains H37Rv and Erdman. It is suggested that the change in isoniazid sensitivity results from the effect of liposomal phospholipids on some mycobacterial enzymes. Electron microscopy indicated that the liposomal form of isoniazid is able to penetrate into the alveolar macrophageal cytoplasm of guinea pigs just 30 min after incubation.