HEMOLYMPH CHEMISTRY REFERENCE RANGES OF THE CHILEAN ROSE TARANTULA GRAMMOSTOLA ROSEA (WALKENAER, 1837) USING THE VETSCAN BIOCHEMISTRY ANALYZER BASED ON IFCC-CLSI C28-A3.

The use of invertebrate hemolymph chemistry analysis has the potential to become a major diagnostic tool. The goal of this study was to generate statistically sound hemolymph reference ranges from healthy tarantulas. Hemolymph was drawn from wild caught, acclimatized, and apparently healthy female Chilean rose tarantulas Grammostola rosea (Walkenaer, 1837) ( n = 43) using a modified technique. Hemolymph samples were separately analyzed using the Avian-Reptilian Profile Plus reagent rotor for VetScan® for the following chemistries: aspartate aminotransferase, bile acids, creatine kinase, uric acid, glucose, total calcium, phosphorus, total protein, albumin, potassium, and sodium. With this method the authors were able to establish statistically sound reference ranges for aspartate aminotransferase, creatine kinase, glucose, phosphorus, and total protein. Further in situ studies will determine the practical usability of these values in the evaluation of tarantula health.

Human mesenchymal stem cell-derived iron oxide exosomes allow targeted ablation of tumor cells via magnetic hyperthermia.

Magnetic hyperthermia, or the heating of tissues using magnetic materials, is a promising approach for treating cancer. We found that human mesenchymal stem cells (MSCs) isolated from various tissues and MSCs expressing the yeast cytosine deaminase∷uracil phosphoribosyl transferase suicide fusion gene (yCD∷UPRT) can be labeled with Venofer, an iron oxide carbohydrate nanoparticle. Venofer labeling did not affect cell proliferation or the ability to home to tumors. All Venofer-labeled MSCs released exosomes that contained iron oxide. Furthermore, these exosomes were efficiently endocytosed by tumor cells. Exosomes from Venofer-labeled MSCs expressing the yCD∷UPRT gene in the presence of the prodrug 5-fluorocytosine inhibited tumor growth in a dose-dependent fashion. The treated tumor cells were also effectively ablated following induction of hyperthermia using an external alternating magnetic field. Cumulatively, we found that magnetic nanoparticles packaged into MSC exosomes are efficiently endocytosed by tumor cells, facilitating targeted tumor cell ablation via magnetically induced hyperthermia.

Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves.

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

Ferrous iron content of intravenous iron formulations.

The observed biological differences in safety and efficacy of intravenous (IV) iron formulations are attributable to physicochemical differences. In addition to differences in carbohydrate shell, polarographic signatures due to ferric iron [Fe(III)] and ferrous iron [Fe(II)] differ among IV iron formulations. Intravenous iron contains Fe(II) and releases labile iron in the circulation. Fe(II) generates toxic free radicals and reactive oxygen species and binds to bacterial siderophores and other in vivo sequestering agents. To evaluate whether differences in Fe(II) content may account for some observed biological differences between IV iron formulations, samples from multiple lots of various IV iron formulations were dissolved in 12 M concentrated HCl to dissociate and release all iron and then diluted with water to achieve 0.1 M HCl concentration. Fe(II) was then directly measured using ferrozine reagent and ultraviolet spectroscopy at 562 nm. Total iron content was measured by adding an excess of ascorbic acid to reduce Fe(III) to Fe(II), and Fe(II) was then measured by ferrozine assay. The Fe(II) concentration as a proportion of total iron content [Fe(III) + Fe(II)] in different lots of IV iron formulations was as follows: iron gluconate, 1.4 and 1.8 %; ferumoxytol, 0.26 %; ferric carboxymaltose, 1.4 %; iron dextran, 0.8 %; and iron sucrose, 10.2, 15.5, and 11.0 % (average, 12.2 %). The average Fe(II) content in iron sucrose was, therefore, ≥7.5-fold higher than in the other IV iron formulations. Further studies are needed to investigate the relationship between Fe(II) content and increased risk of oxidative stress and infections with iron sucrose.

In Vitro Release Kinetics and Transferrin Saturation Study of Intravenous Iron Sucrose Entrapped in Poly(ethylene glycol)-Assisted Silica Xerogel.

The presence of labile iron fractions in intravenous iron supplements compromises their safety. Poly(ethylene glycol) (PEG)-assisted silica xerogel was evaluated as a potential drug carrier for iron sucrose with the purpose of limiting labile iron available for in vitro uptake by transferrin. The drug entrapped xerogels were synthesized by the sol-gel process with varying amounts of PEG. In vitro release studies were conducted in simulated body fluid (SBF) at 37 ± 0.02 °C (pH 7.4). The results indicated that the cumulative release percentage increased with the increase in the amount of PEG in the matrix. The biphasic release profile followed first-order kinetics for the first 6 h and Higuchi model for the remaining time (up to 168 h). The sample showing highest percentage of cumulative release (the xerogel with 16 % PEG) was used for in vitro transferrin saturation studies in contrast with the plain drug. The xerogel formulation exhibited 7.25 ± 0.4 % transferrin saturation in 180 min as compared to 12.89 ± 0.2 % for the raw drug. These results indicate that encapsulation of iron sucrose in PEG-assisted silica xerogel and subsequent sustained release from the matrix can improve the safety of the drug when presence of labile iron is a major concern.

Glucaric acids from Leonurus japonicus.

Three new glucaric acids, namely 2-feruloyl-4-syringoyl or 5-feruloyl-3-syringoyl glucaric acid (1), 2-syringoyl-4-feruloyl or 5-syringoyl-3-feruloyl glucaric acid (2), and 3-feruloyl-4-syringoyl or 4-feruloyl-3-syringoyl glucaric acid (3), were isolated from Leonurus japonicus Houtt. Their structures were elucidated by detailed spectroscopic means including UV, IR, HR-ESI-MS, 1D and 2D NMR data spectra. The bioactive assays of compounds 1-3 against hepatoprotection activity were determined. The result suggested that compound 2 exhibited a moderate hepatoprotection activity and the cell survival rate was 74% (10(-5)mol/L), using bicyclol (survival rate: 66%, 10(-5)mol/L) as a positive control. Furthermore, compounds 1-3 were evaluated cytotoxic activities in vitro using HCT-8, Bel-7402, BGC-823, A-549, and A2780 model and the results exhibited no obvious cytotoxicity activity.

Overestimation of flavonoid aglycones as a result of the ex vivo deconjugation of glucuronides by the tissue ß-glucuronidase.

Flavonoid glucuronides are the main circulating metabolites of flavonoids in humans and animals. There has been a growing interest in the biological function of glucuronides. In order to differentiate biological activity and to assess efficacy it is essential to accurately determine the levels of flavonoid aglycone and metabolic conjugate in vivo. Many organs and body fluids of humans and animals exhibit ß-glucuronidase against flavonoid glucuronides. Studies have shown that ß-glucuronidase within the tissues hydrolyzes glucuronides to their aglycones during the tissue extraction, leading to artificially higher reported tissue levels of aglycone than actual in vivo concentrations. The aims of this study were to estimate the extent by which the aglycones were overestimated and to investigate the use of saccharo-1,4-lactone, a ß-glucuronidase inhibitor, to block the ex vivo hydrolysis of flavonoid glucuronides. Our data demonstrate that in mouse liver tissues and human tumor xenografts levels of quercetin and methylated quercetin aglycones could be over-estimated by 7-fold. The inhibition of deconjugation of quercetin and baicalein glucuronides by saccharo-1,4-lactone is dose-dependent. The amount of saccharo-1,4-lactone used to produce optimal inhibition of the enzyme activity is in the range of 15-24µmol per gram of liver tissue. The use of ß-glucuronidase inhibitor blocks the ex vivo deconjugation resulting in an accurate estimation of tissue levels of aglycone and conjugate. Our study described here can be extended to other animal models and human studies with different types of substrates of ß-glucuronidase.

A comparative study of antioxidative activity of calcium-D-glucarate, sodium-D-gluconate and D-glucono-1,4-lactone in a human blood platelet model.

D-glucono-1,4-lactone, sodium D-gluconate and calcium D-glucarate are non-toxic glucose derivatives occurring naturally in fruits and vegetables. Calcium D-glucarate is promoted as an orally bioavailability dietary supplement with potential chemopreventive activity without adverse effects. Despite many commercial applications in pharmaceutical and food industries the potential activity mechanisms of glucarate and gluconate are not clear. The purpose of this study was to investigate and compare the effects of these compounds on blood platelets under oxidative stress conditions and to examine their role in thrombin-induced platelet activation. Platelet activation is essential in haemostasis, tumor progression and allergic and non-allergic inflammation, where reactive oxygen species are involved. The antiplatelet and antioxidative activity was studied in vitro by measuring levels of specific oxidative stress markers: thiobarbituric acid reactive substances, superoxide anion, carbonyl groups, 3-nitrotyrosine, protein and low molecular weight thiols. All tested compounds significantly inhibited thrombin-induced arachidonic peroxidation, O2⁻ⁱ production and also platelet protein oxidation/nitration induced by peroxynitrite, which is a strong oxidant formed intravascularly in vivo. Carbonyl group generation, thiol oxidation and nitrotyrosine formation were significantly decreased in the presence of glucose derivatives. The obtained results demonstrate that tested compounds may be helpful in the prevention of excessive platelet activation through the antioxidant mechanisms. Comparative studies indicate the predominant preventive activity of sodium D-gluconate. In general, the consumption of apples or apple juice as well as oranges, grapefruit and cruciferous vegetables, sources of large amounts of tested derivatives, have beneficial effects on platelets under oxidative stress.

In vitro evaluation of the chemoprotective action mechanisms of leontopodic acid against aflatoxin B1 and deoxynivalenol-induced cell damage.

Several in vitro studies showed that free radical scavengers possess chemopreventive properties against mycotoxin-induced cell damage which are at least partially associated with the induction of phase II detoxifying enzymes and antioxidant enzymes like glutathione S-transferase (GST) and glutathione peroxidase (GPx). The aim of this project was to study the chemopreventive effects of leontopodic acid (LA), a potent natural occurring free radical scavenger isolated from the aerial parts of Leontopodium alpinum. Different mycotoxins were evaluated in two different cell lines on the basis of their specific toxicity: aflatoxin B1 (AFB1) on HepG2 cells and deoxynivalenol (DON) on U937 cells. Cell viability and reactive oxygen species concentration were determined, and the effects of pre-treatment with LA on these parameters were investigated together with the GST and GPx activity as well as the concentration of reduced glutathione. The results show that LA protects U937 cells from DON-induced cell damage but not HepG2 cells from AFB1. Moreover LA is able to enhance GPx activity in U937, but not GST activity in HepG2. We hypothesize that the increase in detoxifying enzymes is probably the main mechanism of antioxidant mediated chemoprevention.