Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples

The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency’s Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.     

碳纳米管修饰电极的制备及其对NADH的电催化氧化

采用丝网印刷技术,制备出羧基化多壁碳纳米管修饰的丝网印刷碳电极,并采用循环伏安法研究了该电极对还原型烟酰胺腺嘌呤二核苷酸(NADH)的电催化氧化性能.结果表明,与未修饰丝网印刷碳电极相比,多壁碳纳米管修饰丝网印刷碳电极显著降低了NADH的氧化峰电位,消除了反应产物对电极的污染及其它电化学反应对测量的干扰.将修饰电极与流...     

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism‐dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO₂, field potentials and changes in extracellular potassium concentration ([K⁺]_o). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus‐induced FAD, field potential, [K⁺]_o and pO₂ changes as compared to continuous illumination.     

Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a partial block of AO import in this mutant. This resulted in the accumulation of AO precursors in the cytosol where they remained separated from the putative peroxisomal AO assembly factors. In order to circumvent the peroxisomal membrane barrier, we have now studied AO assembly in a peroxisome-deficient/Rf-autotrophic double mutant (Δper1.rif1) of H. polymorpha. By sucrose density centrifugation and native gel electrophoresis, three conformations of AO were detected in crude extracts of Δper1.rif1 cells grown under Rf-limitation, namely active octameric AO and two inactive, monomeric forms. One of the latter forms lacked FAD; this form was barely detectable in extracts wild-type and Δper1 cells, but had accumulated in the cytosol of rif1 cells. The second form of monomeric AO contained FAD; this form was also present in Δper1 cells but absent/very low in wild-type and rif1 cells. In vivo only these FAD-containing monomers associate into the active, octameric protein. We conclude that in H. polymorpha FAD binding to the AO monomer is mediated by a yet unknown peroxisomal factor and represents the crucial and essential step to enable AO oligomerization; the actual octamerization and the eventual crystallization in peroxisomes most probably occurs spontaneously.     

Treatment of Chronic Kidney Disease with Extracellular Vesicles from Mesenchymal Stem Cells and CD133+ Expanded Cells: A Comparative Preclinical Analysis

Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133⁺ cells (eCD133⁺) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133⁺ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133⁺ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.     

Lutein Prevents High Fat Diet‐Induced Atherosclerosis in ApoE‐Deficient Mice by Inhibiting NADPH Oxidase and Increasing PPAR Expression

Epidemiological and experimental studies provide supportive evidence that lutein, a major carotenoid, may act as a chemopreventive agent against atherosclerosis, although the underlying molecular mechanisms are not well understood. The main aim of this study was to investigate the effects of lutein on the alleviation of atherosclerosis and its molecular mechanisms involved in oxidative stress and lipid metabolism. Male apolipoprotein E knockout mice (n = 55) were fed either a normal chow diet or a high fat diet (HFD) supplemented with or without lutein for 24 weeks. The results showed that a HFD induced atherosclerosis formation, lipid metabolism disorders and oxidative stress, but noticeable improvements were observed in the lutein treated group. Additionally, lutein supplementation reversed the decreased protein expression of aortic heme oxygenase‐1 and increased the mRNA and protein expressions of aortic nicotinamide‐adenine dinucleotide phosphate oxidase stimulated by a HFD. Furthermore, the decreased mRNA and protein expression levels of hepatic peroxisome proliferator‐activated receptor‐α, carnitine palmitoyltransferase 1A, acyl CoA oxidase 1, low density lipoprotein receptors and scavenger receptor class B type I observed in mice with atherosclerosis were markedly enhanced after treatment with lutein. Taken together, these data add new evidence supporting the anti‐atherogenic properties of lutein and describing its mechanisms of action in atherosclerosis prevention, including oxidative stress and lipid metabolism improvements.     

(R)-9-[2-(二乙氧基磷酰甲氧)丙基]腺嘌呤合成过程中催化剂的优选

目的:(R)-9-[2-(二乙氧基磷酰甲氧)丙基]腺嘌呤(3)合成过程中的催化剂优选。方法:以亚磷酸二乙酯和多聚甲醛经缩合、酯化得到对甲苯磺酸羟甲基磷酸二乙基酯(1)。另以(R)-1,2-亚丙基碳酸酯与腺嘌呤反应得到9-(2-羟丙基)腺嘌呤(2),在(1)与(2)缩合得到(3)的反应中对四种催化剂进行实验对比。结果:优选催化剂为叔丁醇锂的无水DMF溶液,降低成本和污染。结论:合成得到(3),产率提高到40.5%。     

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1–3 steps with moderate yields (10–57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.     

基于腺嘌呤核苷酸单体的光接枝生态阻燃棉织物制备及其性能

为了提高核糖核酸(RNA)单元应用于棉织物阻燃整理的耐久性以及降低生物阻燃成本,采用烯丙基溴对RNA单元衍生物5'-腺嘌呤核苷酸(AMP)进行改性制备生物阻燃单体。通过紫外光接枝法将AMP单体和AMP单体/丙烯酰胺分别接枝到纯棉织物上,制备了光接枝AMP单体和光接枝AMP单体/丙烯酰胺阻燃棉织物。对阻燃单体化学结构进行了表征。测试了纯棉织物及阻燃棉织物的阻燃性能。研究结果表明:AMP单体中成功引入不饱和双键;2种光接枝阻燃棉织物的分解温度均低于纯棉织物,阻燃棉织物的失重率降低;光接枝AMP单体/丙烯酰胺阻燃棉织物的续燃时间及阴燃时间可降低至0 s,AMP单体与丙烯酰胺之间存在一定的协效阻燃作用且可降低成本。     

Antimicrobial activity of Elettaria cardamomum: Toxicity,biochemical and histological studies

Elettaria cardamomum is one of the most broadly used spices in Moroccan gastronomy. Its antimicrobial activity against both Gram-positive and Gram-negative bacterial species was demonstrated. Likewise, its toxicity was investigated on Swiss albinos mice. Daily, mice were treated orally with 0.003 and 0.3 mg during 7 days. Plasmatic markers and antioxidant defence systems were assessed and histological alterations were evaluated. A significant increase in creatine phosphokinase level was observed. The microscopic evaluation shows that E. cardamomum induce morphological perturbation in mice’s heart. The results show also an inhibitory effect of glyceraldehyde 3-phosphate dehydrogenase and an important increase in the level of thiobarbituric acid reactive substances, succinate dehydrogenase and catalase activities. Results show that E. cardamomum induces toxicity at 0.3 mg/g mouse and affect energy metabolism and oxidative stress.