Dysfunctional mitochondrial bioenergetics and oxidative stress in Akita(+/Ins2)-derived ß-cells.

Insulin release from pancreatic ß-cells plays a critical role in blood glucose homeostasis, and ß-cell dysfunction leads to the development of diabetes mellitus. In cases of monogenic type 1 diabetes mellitus (T1DM) that involve mutations in the insulin gene, we hypothesized that misfolding of insulin could result in endoplasmic reticulum (ER) stress, oxidant production, and mitochondrial damage. To address this, we used the Akita(+/Ins2) T1DM model in which misfolding of the insulin 2 gene leads to ER stress-mediated ß-cell death and thapsigargin to induce ER stress in two different ß-cell lines and in intact mouse islets. Using transformed pancreatic ß-cell lines generated from wild-type Ins2(+/+) (WT) and Akita(+/Ins2) mice, we evaluated cellular bioenergetics, oxidative stress, mitochondrial protein levels, and autophagic flux to determine whether changes in these processes contribute to ß-cell dysfunction. In addition, we induced ER stress pharmacologically using thapsigargin in WT ß-cells, INS-1 cells, and intact mouse islets to examine the effects of ER stress on mitochondrial function. Our data reveal that Akita(+/Ins2)-derived ß-cells have increased mitochondrial dysfunction, oxidant production, mtDNA damage, and alterations in mitochondrial protein levels that are not corrected by autophagy. Together, these findings suggest that deterioration in mitochondrial function due to an oxidative environment and ER stress contributes to ß-cell dysfunction and could contribute to T1DM in which mutations in insulin occur.

Microbicidal activity of vascular peroxidase 1 in human plasma via generation of hypochlorous acid.

Members of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H2O2 and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H2O2/Cl⁻ system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H2O2; killing is inhibited by peroxidase inhibitors and by the H2O2 scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements.

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.

Intravenous and oral pharmacokinetic study of BCX-1777, a novel purine nucleoside phosphorylase transition-state inhibitor. In vivo effects on blood 2'-deoxyguanosine in primates.

Administration of BCX-1777 to primates results in a rapid elevation of plasma 2'-deoxyguanosine (up to 0.4 microg/ml, 1.5 microM). Maximum 2'-deoxyguanosine C(max), 0.4 microg/ml, was achieved with the lowest IV dose of BCX-1777 and increasing the IV dose of BCX-1777 did not increase the 2'-deoxyguanosine C(max). However, plasma 2'-deoxyguanosine remained elevated longer as the dose of BCX-1777 increased. In contrast, increases in the oral dose of BCX-1777 did increase the plasma C(max) of 2'-deoxyguanosine. This was in spite of the observation that overall oral bioavailability of BCX-1777 was only 8.2%. This suggests that the BCX-1777 was absorbed slowly producing a sustained low concentration of BCX-1777, resulting in prolonged plasma concentrations of 2'-deoxyguanosine. After IV dosing, the BCX-1777 was cleared relatively quickly and the plasma 2'-deoxyguanosine tracked slightly behind the BCX-1777. IV administration of 5 mg/kg of BCX-1777 twice daily maintains the plasma 2'-deoxyguanosine concentrations at around 0.3 microg/ml (1.1 microM). These data indicate that oral and IV administration of BCX-1777 induce a rapid rise in 2'-deoxyguanosine and that oral dosing at 8.8 and 17.6 mg/kg are at least equivalent to 4.4 mg/kg IV in effecting the accumulation of 2'-deoxyguanosine. Finally, 2'-deoxyguanosine plasma concentration was maintained longer in the three highest oral doses in comparison to all IV doses.

Inulin effects on bioavailability of soy isoflavones and their calcium absorption enhancing ability.

The effect of inulin on isoflavone absorption and the effect of isoflavones and synergy with inulin on calcium absorption in rats was investigated. Rats (n = 48) were divided into three groups and fed inulin (50 mg/g), isoflavone (8 mg/g) or inulin + isoflavone (50 mg/g + 8 mg/g) diets for 21 days. After a 2-h fast, rats were given (45)Ca orally or intraperitoneally, together with 25 mg of calcium as calcium acetate. Blood and femurs were collected 4 days later. Sera were analyzed for isoflavones using HPLC-MS, femurs for (45)Ca by beta-scintillation counting, and total femoral calcium by atomic absorption spectroscopy. Both groups fed isoflavones had similar and significantly higher weight-adjusted total femoral calcium content compared to the inulin-fed group (p < 0.0001). (45)Ca absorption was significantly higher (p < 0.01) when isoflavones were added to the diet, and serum equol was significantly lower (p < 0.01) when inulin was added to the diet containing isoflavones. We conclude that isoflavones enhance calcium absorption, without synergy from inulin, and that inulin decreases equol production.

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method.

A rapid 2 min liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1-5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm x 2.0 mm i.d.) column at 50 degrees C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC-MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.

Identification of isoflavone glycosides in Pueraria lobata cultures by tandem mass spectrometry.

Isoflavones in the methanolic extracts of kudzu (Pueraria lobata) callus, suspension and root cultures were compared in order to develop an experimental system in which puerarin (daidzein 8-C-glucoside) and other isoflavones could be synthesised in vitro. Quantitative variation of puerarin and other known isoflavones was estimated in kudzu culture extracts using HPLC-UV. The highest and lowest amounts of puerarin (14.56 and 0.33 mg/g) were present in in vitro root cultures and leaf tissue-derived callus cultures, respectively. A total of 48 isoflavone metabolites were detected in extracts of kudzu root cultures by HPLC-MS/MS, and the structures of 33 of them were tentatively assigned. Amongst these, 12 isoflavone C-glycosides were identified. Hydroxyderivatives of puerarin in several isomeric forms were detected, some of which have not been previously reported in kudzu root. The molecular weights, interpretation of characteristic fragment ions obtained from HPLC-MS/MS and comparison with reported data allowed the putative identification of the isoflavone metabolites.