Comparison of formulation characteristics of drugs and dietary supplements containing alpha-lipoic acid relevant to therapeutic efficacy.

OBJECTIVE: Healthcare professionals lack the knowledge about the impact of formulations on treatment effectiveness. This is further complicated by the existence of dietary supplements containing the same active pharmaceutical ingredients (API) as drug formulations [e.g., alpha-lipoic acid (ALA)], to which the strict formulation testing requirements do not apply. This research aimed to compare ALA-containing drugs and dietary supplements through the determination of uniformity of content, disintegration time and dissolution rates. MATERIALS AND METHODS: A total of seven different ALA formulations (5 dietary supplements, 2 drugs) were tested for uniformity of content, disintegration time and dissolution rates. All tests were performed in accordance with the 10th European Pharmacopoeia. ALA was determined spectrophotometrically. RESULTS: Uniformity of content testing revealed larger variations of ALA content in three formulations of dietary supplements. Dissolution curves generated at 50 and 100 rpm differed significantly. Testing requirements were met only by one dietary supplement at 50 rpm, and one drug and two dietary supplements at 100 rpm. Disintegration testing showed limited impact on the release kinetic of ALA, as opposed to formulation type. CONCLUSIONS: Considering the lack of regulation on dietary supplement formulations and the variable success of them conforming to pharmacopoeial requirements, it is an imperative for stricter regulations on the dietary supplements' formulations to be imposed globally.

Epidemiology of dietary supplement use in Serbia: Report from Novi Sad.

OBJECTIVES: The primary objectives were to assess the prevalence of dietary supplement (DS) use and to identify specific demographic and lifestyle characteristics of DS users from Novi Sad, Serbia as well as the most commonly used DS and reasons for their use. DESIGN: Observational, cross-sectional study. SETTING AND INTERVENTIONS: Data on demographics, lifestyle and dietary supplement use of 435 adults from Novi Sad, Serbia were collected using an online questionnaire. RESULTS: In total, 435 subjects completed the questionnaire (62.3% women). Prevalence of dietary supplement use in the sample was 42.8%. More women used DS than men (p = 0.002). Higher use of DS was reported among individuals 65+, while the young used DS less (p = 0.001), but the highest proportions of DS users was from the 45-54 age group. DS were used more among those with lower education levels (p < 0.001) and no income (p = 0.009). The highest percentages of DS users reported daily intakes of fruits and moderate physical activity, were non-smokers and social drinkers. Main reason for DS use was maintaining general health. The most commonly used DS were minerals and/or vitamins (68.8%). CONCLUSIONS: We report a high prevalence of dietary supplement use in Novi Sad. DS use was associated with being a female, being older and having minimal/average income, the latter being opposite of the usual findings. Our results warrant a more detailed examination of the association between income, DS use and healthcare availability in developing countries such as Serbia.

Distribution and characterization of cAMP-dependent protein kinase isoenzymes in bovine adrenal cells.

Subcellular localization and characterization of cAMP-kinase isoenzymes in fasciculata reticularis bovine adrenal cells has been investigated. Different subcellular fractions were purified on a Percoll gradient and characterized by marker enzymes. cAMP-kinase was located principally in cytosol and microsomes. In the low-speed particulate fractions cAMP-kinase was found associated mainly with plasma membrane but not with mitochondria. Characterization of isoenzyme patterns in subcellular fractions by conventional DEAE-cellulose chromatography and by anion-exchange HPLC gives essentially the same results. Isoenzyme I appears to be the main enzyme in cytosol whereas isoenzyme II predominates in solubilized microsome and plasma membrane enriched fraction. Photoaffinity labelling of chromatographic fractions demonstrated that HPLC separates both cAMP binding subunits. Photoaffinity labelling of the different subcellular fraction by 8-azido-[32P]cAMP confirmed the data obtained by anion-exchange chromatography. However, in microsomes this method revealed the presence of both isoenzymes and the preferential solubilization of isoenzyme II by Triton X-100. In summary, our results indicate a subcellular compartmentalization of cAMP-kinase in bovine adrenal cells with a preferential localization of isoenzyme I in cytosol and of isoenzyme II in membrane. However, the relation between the distribution and the role of each isoenzyme has so far not been documented.

Glycoside hydrolase production by an anaerobic rumen fungus Caecomyces communis.

The ruminal fungus Caecomyces communis was grown anaerobically either in a discontinuous cultivation system or in a fermentor with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The best culture conditions for glycoside hydrolase production were obtained in Lowe medium with daily fresh medium addition, whereas the Orpin medium with ruminal fluid was favourable to fungal growth and to the enzyme export process. Among glycoside hydrolases assessed in both culture fluid and cellular homogenate, beta-D-fucosidase activity was preponderant. Most studied enzymes were mainly associated with cells (from 50% to 99%). Glycoside hydrolase activities were constitutive, but their level was regulated by a carbon source. beta-D-fucosidase and beta-D-xylosidase activity production was activated by the association of glucose plus cellobiose, whereas beta-D-glucosidase activity production was stimulated by cellobiose alone. Enzyme release could be favoured by glucose alone or by Ray grass hay added to glucose plus cellobiose.

Polysaccharide hydrolase production by the rumen fungus Caecomyces communis.

The anaerobic fungus Caecomyces communis was grown in a fermentor in either a discontinuous cultivation system or in a culture system with daily withdrawal and addition of fresh medium. Lowe and Orpin media were tested. The Lowe medium was best for the stimulation of enzyme production, the Orpin medium, for the stimulation of fungal growth and enzyme release. Xylanase activity was predominant among the polysaccharide hydrolases. Most of the enzymes studied were associated with cells except when the culture medium contained glucose or Ray grass hay. Enzymatic activities were constitutive, but their level was regulated by a carbon source. Cellulase production in both the cellular and extracellular fractions and the extracellular xylanase activity were stimulated by the presence of glucose. Cell-associated xylanase activity, however, was stimulated by glucose plus cellobiose. The presence of glucose enhanced enzyme release.

Lipids of three microsporidian species and multivariate analysis of the host-parasite relationship.

Sporal lipids of 3 microsporidia, Encephalitozoon cuniculi from mammals and Glugea atherinae and Spraguea lophii from fishes, were investigated. High phospholipid levels were found (54.8-64.5% of total lipids), which is in agreement with the presence of highly developed internal membranes in microsporidian spores. Sphingomyelin was not detected in G. atherinae. Triglycerides (less than 10% of total lipids), cholesterol, and free fatty acids were identified in all species. Analysis of fatty acids from the phospholipid fraction revealed the predominance of docosahexaenoic acid (30-40% of total phospholipid fatty acids) in G. atherinae and S. lophii and oleic acid (25.8% of total phospholipid fatty acids) in E. cuniculi. The 3 microsporidia possessed a significant amount of branched-chain fatty acids (iso and anteiso forms) not found in the hosts, supporting the existence of some parasite-specific metabolic steps for these fatty acids. On the basis of phospholipid fatty acid profiles, host-parasite relationships were investigated through correspondence factorial analysis. It shows 3 distinct clusters with the first corresponding to fishes, the second to fish parasites, and the third to E. cuniculi and its host cell. These data suggest that the mammal microsporidia developing within parasitophorous vacuoles are more dependent on host cells than the fish microsporidia that induce cystlike structures.

[The humoral immune response in recent pulmonary tuberculosis (author's transl)]. / La réponse immunitaire humorale dans la tuberculose pulmonaire récente.

The average serum concentration of immunoglobulins, alpha-1-antitrypsin, haptoglobin and alpha-2-macroglobulin, calculated in 45 adults with pulmonary tuberculosis increased in a variable way for each of the six parameters studied. Tuberculosis does not induce new correlations between the parameters but modifies the intensity of existing links.

[Study of various serum proteins in lung cancer. Immunoglobulins A, G, M, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin]. / Etude de quelques protéines sériques dans le cancer du poumon. Immunoglobulines A, G, M, haptoglobine, alpha-1-antitrypsine, alpha-2-macroglobuline.

A statistical study of the plasma concentration of immunoglobulin, alpha-1-antitrypsin, haptoglobin, and alpha-2-macroglobulin in 153 patients with primary carcinoma of the lung showed a strong increase in alpha-1-antitrypsin, haptoglobin and A and C immunoglobulins, whilst alpha-2-macroglobulin increases very moderately, and IgM does not vary. The relationships between variables are also modified, thus there appears a correlation between IgA and IgG to the detriment of the IgG/IgM relationship. Furthermore the coefficient of the correlation IgG/alpha-2-macroblobulin is lower in cancer patients.

[Alpha 1-antitrypsin/lymphocyte interactions: cytofluorometry study]. / Interactions alpha-1-antitrypsine/lymphocytes: étude par cytofluorimétrie.

Purified alpha 1-antitrypsin (alpha 1AT) was previously shown to prevent primary antibody response and lymphocyte DNA synthesis. We have reported that radiolabelled alpha 1-AT could bind to human lymphocytes and inhibit surface proteolytic activity. However, the radiolabelling method brings no information on the eventual heterogeneity of alpha 1AT distribution among the population nor on the presence of alpha 1AT on untreated lymphocytes. In this report, we have investigated these two points using indirect fluorescence followed by flow cytofluorometric analysis. The presence of alpha 1AT was revealed on a variable percentage of untreated peripheral blood and tonsillar lymphocytes. The incubation of cells with additional alpha 1AT induced an increase of the percentages of fluorescent lymphocytes. This binding was specific and could be inhibited by pretreatment with the protease inhibitor tosyl-L-phenylalanine-chloromethyl-ketone (TPCK) (2 X 10(-5)M). Furthermore, TPCK and EDTA (3 mM) could displace alpha 1AT initially bound to the lymphocyte surface.

Interaction between alpha 1 antitrypsin and lymphocyte surface proteases: immunoregulatory effects.

Alpha 1 antitrypsin (alpha 1-AT) is the major plasma protease inhibitor. Radioiodinated alpha 1-AT binds to human lymphocytes. The binding is fast and reversible, and the cells can be saturated with a maximum of approximately 1.2 x 10(6) molecules of alpha 1-AT per lymphocyte. The receptor for alpha 1-AT is a surface-associated protease. Addition of alpha 1-AT completely inhibits cell surface proteolytic activity. Furthermore alpha 1-AT decreases 3H-thymidine incorporation into lymphocytes stimulated by B or T cell mitogens or by allogeneic cells. Since alpha 1-AT was shown to be produced by activated monocytes and to bind to lymphocytes, it is likely to represent a mediator of monocyte-lymphocyte interactions.

[alpha-Amylase activity in lysosomes of Aspergillus oryzae (author's transl)]. / Mise en évidence d'une activité alpha-amylasique dans les lysosomes d'Aspergillus oryzae.

The alpha-amylase of mycelial cells of Aspergillus oryzae exists in a particular form in 8000 g pellet. The lysosomal localization of acid phosphatase is confirmed by electron microscopy. The purification of lysosomes by discontinuous gradient of sucrose in D2O shows that alpha-amylase activity is bound to these particles.

Estimation of C3 levels in cerebrospinal fluid by electroimmunodiffusion.

C3 levels have been determined by the electroimmunodiffusion technique in the CSF of patients with a wide variety of pathologies. The patients were grouped on the basis of protein content and G/A ratio of the CSF as I) patients with normal meningeal permeability and apparent absence of local gamma-globulin synthesis; II) patients with increased meningeal permeability; III) patients with characteristics of MS, i.e. increase of IgG accompanied by a normal or slightly elevated protein content, Group III showed a lower level of C3 when expressed at % of the total protein and also as % of the total protein less gamma-globulins of the CSF. Other parameters of the CSF are also recorded. It was shown that only the expression of C3 concentration relative to the total protein content of the CSF produced meaningful analytical data.

Spontaneous dissociation of an IgG-3 paraprotein through disulfide interchange.

A paraprotein of the gamma3 subclass was observed to dissociate "spontaneously" under various experimental conditions, such as during chromatography on DEAE-cellulose and on Sephadex G-200 or on starch block electrophoresis. This phenomenon was accompanied by the formation of various complexes of higher molecular weight, displaying antigenic properties different from those of the original paraprotein. These changes did not occur in the presence of iodoacetamide, indicating that dissociation of the paraprotein was due to disulfide interchange(s) and not to the absence of interchain disulfide bridges.

[Optimal conditions of alpha-amylase production by Aspergillus oryzae in liquid media]. / Conditions optimales de production d'alpha-amylase en milieu liquide par Aspergillus oryzae.

The alpha-amylase secretion in a mineral culture medium containing starch and glucose follow the lysis of mycelium. This lysis seems to result from the hydrolysing action of dextranase and levulanase on cell wall. Cell lysis and amylase secretion are greatly enhanced by pH elevation of culture medium (optimal pH 8,8). In such conditions of production the amylase is not stable but can be stabilized by addition of starch. A method is described using pH and starch content modifications, which allows to obtain an amylase production three times greater than in standard culture medium.

[Inhibition of DNA biosynthesis in human lymphocytes by alpha 1-antitrypsin]. / Inhibition de la biosynthèse du DNA chez les lymphocytes humains par l'effet de l'alpha1-antitrypsine.

Incorporation of tritiated thymidine into human lymphocytes from tonsils is markedly inhibited by purified alpha1-antitrypsin-preparations. This inhibitory effect is observed in lymphocytes stimulated by a mitogen factor (phytohemagglutinin) as in non stimulated lymphocytes.

Modification of lymphocyte DNA synthesis by alpha 1-antitrypsin.

Purified human alpha 1-antitrypsin (alpha 1-AT) was shown to inhibit 3H-thymidine incorporation into mouse or human lymphocytes stimulated by various mitogens or by allogeneic cells. In the mouse, both B- and T-cell responses were affected. In the human, proliferative responses of peripheral blood lymphocytes, thymocytes and T-enriched tonsillar lymphocytes to phytohaemagglutinin were inhibited as well as that of tonsillar lymphocytes to Salmonella typhi-murium lipopolysaccharide. Spontaneous 3H-thymidine incorporation was moderately and inconstantly decreased, without evidence of altered cell viability. The inhibitory effect of alpha 1-AT appears to be related to its protease inhibitory capacity. These data bring further evidence for the role of proteolytic enzymes in the early events of lymphocyte activation, and support the hypothesis that serum inhibitors of proteases may contribute to the modulation of the immune response.

[Intracellular cyclic AMP in vesicular stomatitis or Sendaï virus infected cells (author's transl)]. / Variations des concentrations de cAMP intracellulaire de différents types de cellules après infection par le virus Sendaï ou le virus de la stomatite vésiculeuse.

In KB cells, MRC5 and adult skin fibroblasts infected by low doses of Sendaï virus, intracellular cyclic AMP levels rose and fell in the first hours following infection, then remained lower than basal level during at least 2 days in KB cells and adult skin fibroblasts. When compared to other viruses or cAMP inducers previously described, this effect appeared specific of Sendaï virus. Mechanisms and roles of cAMP variations are discussed. VSV-infected KB cells showed slightly decreased cAMP levels during the first hours following infection.

Glycoside and polysaccharide hydrolase activity of the rumen anaerobic fungus Caecomyces communis (Sphaeromonas communis SENSU ORPIN) at early and final stages of the developmental cycle.

The rumen anaerobic fungus Caecomyces communis was grown in a fermentor in Lowe medium. We studied four polysaccharide hydrolases and three glycoside hydrolases at early and final stages. We found a difference in cell association for these enzymes depending on the developmental stage. The endocellulase and beta-D-fucosidase were early synthesized, and their activities decreased at the end of the developmental cycle. On the contrary, the beta-D-glucosidase, beta-D-xylosidase and xylanase activities increased during the cycle. The avicelase and the CM-cellulase activities linked with thalli increased, whereas the extracellular activities of these enzymes decreased.

Encephalitozoon cuniculi (Microspora): characterization of a phospholipid metabolic pathway potentially linked to therapeutics.

Phospholipid metabolism of the microsporidian Encephalitozoon cuniculi, an obligate intracellular parasite, has been investigated. Labeled precursor incorporation experiments have shown that phosphatidylserine decarboxylase and phosphatidylethanolamine N-methyltransferase are more active in cells infected by E. cuniculi than in uninfected cells. In contrast, no difference was observed in the activity of Kennedy pathway's enzymes, the mammalian pathway. This suggests the occurrence in microsporidia of a bacteria- and fungi-typical pathway for phospholipid synthesis, which is supported by the identification of two genes implicated in this pathway, the cds gene encoding the key enzyme CDP-diacylglycerol synthase (E.C. 2.7.7.41) and the pss gene for CDP-alcohol phosphatidyltransferase. The pss gene could encode phosphatidylserine synthase (E.C. 2.7.8.8.), which catalyses the de novo synthesis of phosphatidylserine in bacteria and fungi. The complete CDP-diacylglycerol synthase messenger has been isolated and shows very short 5' and 3' untranslated regions. This is strong evidence for the functionality of a metabolic pathway which could be a potential target against microsporidia which infect humans.

On small genomes in eukaryotic organisms: molecular karyotypes of two microsporidian species (Protozoa) parasites of vertebrates.

Pulsed field gel electrophoresis (PFGE) was used to separate chromosome-sized DNA from two species of microsporidia of fishes. The molecular karyotype of Glugea atherinae exhibits 16 DNA bands from 420 to 2,700 kb, and that of Spraguea lophii 12 bands from 230 to 980 kb. Until now they represent respectively the largest and the smallest genomes visualized for microsporidia: 19.5 Mb for G. atherinae and 6.2 Mb for S. lophii (the smallest nuclear genome in eukaryotic organism). We have analysed separately five strains of G. atherinae (individual cysts), with this technique. The electrophoretic spectra are the same for these strains, except for the absence of the 2,380-kb band in one case. Therefore, the karyotype seems to be rather well conserved for this species.