Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.     

双歧杆菌三联活菌散对儿童轮状病毒性肠炎患者肠道菌群及肠黏膜通透性的影响

目的 观察双歧杆菌三联活菌散对儿童轮状病毒性肠炎（RVE）患者肠道菌群和肠黏膜通透性的影响。方法 选择2016年1月至2017年8月于丽水市中心医院儿科门诊治疗的RVE患儿94例,随机分为观察组和对照组各47例。两组患儿均予饮食调整、补液及纠正电解质紊乱和酸碱失衡等治疗。观察组患儿加用双歧杆菌三联活菌散1.0 g/次,3次/d,温水冲服。观察两组患儿治疗前后肠道菌群（双歧杆菌、乳杆菌和大肠埃希菌）数量和肠黏膜通透性指标[内毒素（ETX）和D-乳酸]的变化,并比较其临床疗效。结果 治疗72 h后,观察组患儿双歧杆菌和乳杆菌数量明显上升,大肠埃希菌数量明显下降（P0.05）。治疗后观察组患儿双歧杆菌、乳杆菌数量较对照组更高,大肠埃希菌数量较对照组更低（P<0.05）。两组患儿治疗后血清ETX和D-乳酸水平均明显下降（P<0.05）,且观察组下降幅度更大（P<0.05）;同时观察组患儿总有效率高于对照组（χ2=4.11,P<0.05）。结论 双歧杆菌三联活菌散治疗儿童RVE的疗效确切,其作用机制可能与其能增加双歧杆菌和乳杆菌等有益菌的数量,减少大肠埃希菌等致病菌的数量,改善患儿肠道微生态状况;并与其能降低肠黏膜通透性和减少肠液的分泌渗出相关。     

柔肝固肠方改善慢性酒精性肝损伤大鼠肠道通透性的机制研究

目的：从肠道紧密连接及黏附连接角度探讨柔肝固肠方改善慢性酒精性肝损伤大鼠肠道通透性的作用机制。方法：Lieber-DeCarli酒精液体饲料饲养6周诱导大鼠慢性酒精性肝损伤模型。30只SD雄性大鼠,随机分无酒精液体饲料对照组（对照组,n＝10）和酒精液体饲料造模组（n＝20）,造模第4周将造模组大鼠随机分模型组（n＝10）,柔肝固肠方组（n＝10）,并开始灌胃给柔肝固肠方或蒸馏水直至6周末,取材前3．5 h各组以10 mg／kg的内毒素（LPS）灌胃。取材后检测：1）血清AST、ALT活性变化;2）通过门脉血浆内毒素含量测定判断小肠通透性变化;3）小肠组织电镜超微结构观察;4）小肠紧密连接蛋白ZO-1、Occludin蛋白与mRNA表达变化;5）小肠黏附连接蛋白β-Catenin和E-Cadherin蛋白表达。结果：1）柔肝固肠方可显著降低模型大鼠显著升高的AST水平（P＜0．05）;2）柔肝固肠方可降低模型大鼠显著升高的小肠通透性,柔肝固肠方组血浆内毒素含量显著低于模型组;3）电镜结果显示柔肝固肠方可显著改善Lieber-DeCarli酒精饲料喂养大鼠小肠黏膜表面微绒毛局灶性减少、变短、稀疏,排列不规则,同微绒毛相连的细胞终末网变性模糊等病理改变;4）柔肝固肠方可显著升高酒精饲料喂养肝损伤大鼠小肠组织紧密连接蛋白ZO-1、Occludin蛋白与mRNA表达及黏附连接蛋白β-Catenin和E-Cadherin蛋白表达。结论：柔肝固肠方通过改善肠上皮紧密连接及黏附连接改善慢性酒精性肝损伤大鼠肠道通透性改变。     

非酒精性脂肪性肝炎患者肠道菌群失调与肠道通透性及血清内毒素的相关性研究

目的 探讨非酒精性脂肪性肝炎患者肠道菌群失调与肠道通透性及血清内毒素的相关性.方法 选择肠道菌群中具有代表性的细菌共8种进行培养.研究对象为健康成人(A组)和非酒精性脂肪性肝炎(B组)各30例,计数两组肠道菌群中8种细菌的数量,检测所有被研究者的血清内毒素、二胺氧化酶(DAO)、D-乳酸及TNF-α的浓度,比较两组细菌数量和血清指标的变化,并进行相关分析.结果 与A组比较,B组双歧杆菌、乳杆菌和拟杆菌的菌落数显著减少(P＜0.01或P＜0.05),而肠球菌、肠杆菌的菌落数则有显著增加(P＜0.01或P＜0.05);酵母真菌、葡萄球菌、梭菌菌落数没有发生显著改变(P＞0.05);血清内毒素、DAO、D-乳酸、TNF-α水平显著增高(P＜0.01);相关分析显示肠杆菌与内毒素、DAO、D-乳酸相关(r=0.644,P＜0.001;r=0.415,P=0.023;r =0.383,P=0.037);血清内毒素和DAO、D-乳酸、TNF-α显著相关(r=0.485,P=0.007;r=0.477,P=0.008;r=0.490,P=0.006);TNF-α则与DAO、D-乳酸相关(r=0.426,P =0.019;r =0.440,P=0.015).结论 非酒精性脂肪性肝炎患者存在肠道菌群失调、肠道通透性增高及肠源性内毒素血症,肠杆菌的过度生长与肠源性内毒素血症及肠道通透性密切相关.     

Gas chromatographic method using photoionization detection for the determination of breath pentane

Lipid peroxidation is thought to be an important event in the pathogenesis of atherosclerosis. It has been suggested that pentane, which can be formed during the oxidation of ω-6 fatty acids, is a marker of lipid peroxidation. Previous studies have reported elevated breath pentane and serum markers of lipid peroxidation in smokers. However, chromatographic separation of pentane from isoprene in virtually all of these studies was incomplete and the methods used did not resolve pentane into its isomers, n-pentane and isopentane. Additionally, most current methods are complicated, requiring trapping and concentrating steps to obtain adequate sensitivity prior to hydrocarbon analysis. The purpose of the current study was to develop a gas chromatographic system to analyze breath pentane, that addressses the above technical problems and that would provide a simple in vivo method for measuring lipid. n-Pentane and isopentane standards were easily separated from isoprene with a Al₂O₃/KCl capillary column contained in a portable gas chromatograph equipped with a photoionization detector. The analysis of repeated measures showed a low coefficient of variation for measurements of n-pentane (10%) and isopentane (9%). We measured breath pentane in 27 subjects (15 smokers, 12 non-smokers). There were no significant difference between the baseline and 4 week interval measurements of n-pentane for smokers both before and after cigarette smoking. The within-subject variability data showed that the assay is highly reproducible for both low and high pentane levels in smokers. Smokers were found to have higher levels of both n-pentane and isopentane than non-smokers (P<0.001). In addition, smokers had further significant elevation of pentane levels 10 min after smoking (P<0.001), which returned to baseline by 1 h. These studies demonstrate that measurement of breath pentane, using a gas chromatograph with a photoionization detector, is simple and reproducible. Additionally, these results suggest that pentane elevation associated with smoking is secondary to the oxidant effects of cigarette smoke and an important temporal relationship exists between cigarette smoking and breath sample analysis.     

The intestinal barrier in lepidopteran larvae: permeability of the peritrophic membrane and of the midgut epithelium to two biologically active peptides

Endogenous peptide regulators of insect physiology and development are presently being considered as potential biopesticides, but their efficacy by oral delivery cannot be easily anticipated because of the limited information on how the insect gut barrier handles these kind of molecules. We investigated, in Bombyx mori larvae, the permeability properties of the two components of the intestinal barrier, the peritrophic membrane (PM) and the midgut epithelium, separately isolated and perfused in conventional Ussing chambers. The PM discriminated compounds of different dimensions but was easily crossed by two small peptides recently proposed as bioinsecticides, the neuropeptide proctolin and Aedes aegypti Trypsin Modulating Oostatic Factor (Aea-TMOF), although their flux values indicated that the permeability was highly affected by their steric conformation. To date, there is very little functional data available on how peptides cross the insect intestinal epithelium, but it has been speculated that peptides could reach the haemocoel through the paracellular pathway. We characterized the permeability properties of this route to a number of organic molecules, showing that B. mori septate junction was highly selective to both the dimension and the charge of the permeant compound. Confocal images of whole-mount midguts incubated with rhodamine(rh)-proctolin or fluorescein isothiocyanate (FITC)-Aea-TMOF added to the mucosal side of the epithelium, revealed that rh-proctolin did not enter the cell and crossed the midgut only by the paracellular pathway, while FITC-Aea-TMOF did cross the cell apical membrane, permeating also through the transcellular route.     

口炎清颗粒对小鼠肠道菌群失衡的调节作用

目的探讨口炎清颗粒对肠道菌群失衡的调节作用。方法小鼠随机分为6组：正常对照组、重度失调组、自然恢复组、口炎清低剂量组、口炎清高剂量组和大豆低聚糖对照组。正常对照组小鼠灌服蒸馏水,其他组以头孢曲松钠终浓度为8 g/（kg.d）的剂量,连续灌胃8 d,建立小鼠菌群失衡模型,自然恢复组在失衡模型建立后不使用任何药物,自然恢复7 d;口炎清低剂量组、高剂量组、大豆低聚糖对照组分别灌服相应剂量的药物7 d,观察各组小鼠盲肠指数和肠道内双歧杆菌、类杆菌、韦荣球菌、乳酸杆菌、消化球菌、优杆菌、葡萄球菌、肠球菌、大肠埃希菌、链球菌和酵母菌11种菌群变化。结果与菌群失衡组比较,口炎清组与大豆低聚糖组盲肠指数均显著下降（P〈0.05）,呈恢复趋势。自然恢复组肠道菌群种类和数量变化不明显,口炎清组厌氧菌重新出现,尤其是低剂量组双歧杆菌、优杆菌和类杆菌的出现预示着肠道菌群正在逐渐恢复正常。结论一定浓度的口炎清可以明显促进菌群失衡小鼠盲肠大小的恢复,对小鼠肠道菌群有调节作用,具有类似大豆低聚糖的益生元功效。     

新型肠壁通透性标志物用于评价炎症性疾病的研究进展

肠道菌群及其代谢产物失衡可能通过增加肠壁通透性（intestinal permeability,IP）影响宿主免疫反应。研究发现一些新型IP标志物不但能反映肠道局部功能性或机械性损伤程度,还能反映全身炎症反应。而炎症反应是炎症性疾病发生的基础,其严重程度是炎症性疾病进展的关键。最新研究表明,再生胰岛衍生蛋白3α（Reg3α）不但可用于诊断评价炎性肠病,还可用于评估消化道肿瘤的进展、分期、治疗效果及预后,而且还能反映酒精性肝病患者、肝细胞癌患者及HIV感染者的全身免疫反应状态。小肠脂肪酸结合蛋白（I-FABP）能用于评估新生儿坏死性肠炎和急性胰腺炎的进展与预后。而血浆瓜氨酸（CIT）对肠上皮细胞的功能性损伤敏感。本文综述了与局部或全身炎症反应相关的IP标志物用于评价炎症性疾病的研究进展以及这些标志物的临床应用价值与局限性,这些标志物包括Reg3α、I-FABP、CIT、D-乳酸、肠黏膜紧密连接蛋白及血清二胺氧化酶等。

     

浅析肠道菌群干预治疗癫痫的潜在应用前景

肠道菌群是存在于人体内的庞大而复杂的微生物群落,菌群的变化会影响大脑的生理、行为和认知功能,而大脑可以通过免疫、内分泌和神经通路等途径调节神经生理行为,与很多神经和精神疾病(如癫痫、脱髓鞘疾病、阿尔茨海默症、自闭症等)的发生发展有关。近年来诸多证据表明自身免疫机制在癫痫进展中发挥重要作用,肠道菌群可通过调节免疫反应来影响疾病的进展。本文从肠道菌群与大脑间关系的认知、相互间功能影响及与癫痫的关系等方面,探讨通过肠道菌群干预治疗癫痫的机制与潜在应用前景,希望为菌群干预治疗与预防一些神经及精神疾病提供参考。     

Gas chromatographic method with mass-selective detection for the determination of 2-isopropoxyphenol in human urine

Human metabolism of the insecticide propoxur yields 2-isopropoxyphenol (IPP) which is excreted conjugated in urine. In this publication a sensitive and selective analytical method is described which permits the determination of IPP as a suitable parameter for biomonitoring. The clean-up of the hydrolysed urine samples consisted of steam distillation and solid-phase extraction using a reversed-phase column. IPP and the internal standard 2-ethoxyphenol were converted to their pentafluorobenzyl ethers. Excess of the derivatisation reagent was removed using deactivated silica gel. Separation and quantitative analysis was carried out by capillary gas chromatography and mass selective detection. Coefficients of variation were below 5% for concentrations from 6 to 300 μg/l. The detection limit was 0.5 μg/l. The method was checked by analysing six urine samples from pest controllers after indoor application of propoxur. The IPP concentrations ranged from 45 to 306 μg/g creatinine. IPP was not detected in urine specimens from 10 non-exposed persons. The sensitivity of the developed method permits the detection of latent exposure to propoxur.     

New sensible method to quantize the intestinal absorption of receptor ligands

In recent years, special attention has been paid to the A₃ adenosine receptor (A₃AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A₃AR agonist reported so far, named AR 170 (N⁶-methyl-2-phenylethynyl-5′-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A₃AR could presuppose good topical anti-inflammatory effects of this compound.     

暗黑鳃金龟甲幼虫肠道细菌分离及鉴定

目的从微生态学角度研究暗黑鳃金龟甲幼虫营养生理活动,探讨其肠道菌群的构成,为其资源开发及生物防治提供理论依据。方法按传统分离方法,从暗黑鳃金龟甲幼虫肠道环境中分离纯化获得10个细菌菌株,对其菌体形态、染色反应、培养性状、生理生化反应进行了系统研究。结果研究结果表明,上述10个细菌菌株分别属于鲁氏耶尔森菌（Yersinia．ruckeri）、侧胞芽胞杆菌（Bacilluslaterosporus）、坚强芽胞杆菌（Bacillus．firmus）、放线杆菌属（Actinobacillus）、飞虫杀雄菌（Arsenophonusllasoniae）、不动杆菌属（Acinetobacter）、气单胞菌Aeromonas）、沙门菌属（Sal—moneUa）、短芽胞杆菌（Bacillusbrevis）、变形菌属（Proteus）。结论通过对暗黑鳃金龟甲幼虫肠道细菌的鉴定,其肠道细菌在培养性状、生理性状、生理生化测定等方面存在较多差异。     

A method for estimating free Ca within human red blood cells,with an application to the study of their Ca-dependent K permeability

Summary Murphy, Coll, Rich and Williamson (J. Biol. Chem. 255:6600–6608, 1980) described a null-point method for estimating intracellular free Ca in liver cells. They used digitonin to lyse the cells in solutions of varying Ca concentration. This method has been adapted for use with human red cells. The values found are about 0.4 m Ca in fresh cells, and from 0.4 to 0.7 m Ca in blood-bank cells, at pH 7.2 and 37°C. These are likely to be overestimates, and the errors and limitations of the method are discussed. Red cells may be loaded with Ca by metabolic depletion in Ca-containing solutions. Such cells have an elevated K permeability, and the relationships between free Ca, total Ca and K permeability were investigated, using⁸⁶Rb as a tracer for K.⁸⁶Rb flux studies show that the affinity of the K channel for Ca is the same in cells as in resealed ghosts where intracellular Ca can be controlled with Ca buffers, but the rate of tracer equilibration is 3–6 times faster in ghosts than in cells.     

肠道菌群影响黏膜屏障结构与功能的研究进展

肠道黏膜屏障具有防止致病性抗原侵入、维护肠道健康的功能。而肠道菌群是肠道黏膜屏障的重要构成部分,肠道菌群失调会导致肠道黏膜屏障的损伤,引起炎性肠病、肠易激综合征及肝、肾等多种疾病的发生发展。因此,本文从肠道黏膜的结构与功能及肠道菌群对其的影响等方面归纳总结肠道菌群对屏障系统的调控作用,从调节肠道微生态平衡、促进黏液分泌、影响紧密连接和肠道上皮通透性、激发肠黏膜免疫、调控肠上皮凋亡、影响肠上皮DNA稳定性及产生特殊代谢产物等方面阐述其作用机制,为临床胃肠道疾病及其并发症的治疗提供新的思路和方法。     

试论肠菌与中草药联合和发酵防治疾病

【摘 要】 本文阐述了肠道原始固有菌群（肠菌）与天然药用植物（中草药）联合应用和用肠菌发酵中草药的可行性及应用前景。     

Study on the intestinal permeability of lamivudine using Caco-2 cells monolayer and Single-pass intestinal perfusion

BackgroundThe aim of this work is to investigate the intestinal permeability of lamivudine and explore its absorption mechanism.MethodCaco-2 cells monolayer and single-pass intestinal perfusion (SPIP) were selected for the investigation of lamivudine under different conditions, such as different concentration, absorption time, bidirectional transportation, and transportation with efflux transporters inhibitor. The concentration of lamivudine both in Caco-2 cells monolayer samples and SPIP samples was detected by HPLC-UV. Then the permeability parameters were calculated.ResultsThe established HPLC-UV method reach the requirements for detection. There is no statistically difference between absorption parameters of lamivudine both in Caco-2 cells monolayer and SPIP (P > 0.05) under different dose groups. After transportation with efflux transporters inhibitor, the efflux rate of lamivudine in three dose groups was significantly decreased from 2.67, 2.59 and 2.59 to 1.78, 1.61, and 1.81 respectively. Lamivudine exhibits an absorption mechanism of passive diffusion.ConclusionThe absorption of lamivudine may be related to efflux transporters. In addition, lamivudine is a moderate-permeability drug in Biopharmaceutics Classification System.     

Sensitive high-performance liquid chromatographic method for profiling phytoestrogens using coulometric electrode array detection: application to plasma analysis.

An HPLC method for profiling 13 phytoestrogens and their metabolites using coulometric electrode array detection was developed. Sensitivity of the method was slightly less than that of our GC-MS method, but significantly higher compared to the HPLC methods using diode-array or UV detection. Detection limits varied from 3.4 (secoisolariciresinol) to 40.3 (genistin) pg on column. Signal linearities ranged from the detection limits to 61 ng on column. Resolution values for the peak pairs varied from 1.1 (O-desmethylangolensin-anhydrosecoisolariciresinol) to 16 (daidzin-genistin). Intra- and interassay retention time variations were negligible and detector response variation was eliminated by frequent calibration. Chromatographic method was applied to plasma analyses and 6 of the 13 compounds were detected. Method accuracy for those six analytes varied from 69% (enterodiol) to 118% (genistein). Intraassay precision CVs ranged from 1.5% (enterolactone, 12.4 nmol/liter) to 14% (genistein, 245 nmol/liter) and interassay precision CVs ranged from 9.9% (daidzein, 67.4 nmol/liter) to 44% (enterodiol, 1.20 nmol/liter).     

口炎清颗粒调整菌群失调有效组分的研究

目的 探讨口炎清组分玄参对于小鼠肠道菌群的调节作用.方法 应用抗生素头孢曲松钠灌胃的方法建立小鼠肠道菌群失衡模型.利用观察称量等方法观察小鼠临床表征的变化,以及盲肠指数和脾指数的变化.利用活菌计数法分析肠道优势菌群的变化趋势.结果 (1)菌群失衡组盲肠指数增高(P＜0.05);自然恢复组盲肠指数变化不明显;玄参流膏与大豆低聚糖回灌组盲肠指数均显著下降(P＜0.05),呈恢复趋势.(2)菌群失衡组脾脏指数有下降趋势,自然恢复组脾脏指数无明显变化;玄参流膏组与大豆低聚糖回灌组脾脏指数均有所上升,呈恢复趋势.(3)菌群失衡组厌氧菌被抑制,需氧球菌大量繁殖.自然恢复组菌群种类和数量变化不明显,玄参组厌氧菌重新出现,尤其是双歧杆菌、乳杆菌、韦荣球菌和类杆菌的出现预示着菌群正在逐渐恢复正常.结论 玄参流膏可以促进菌群失衡小鼠盲肠和脾脏大小的恢复,可以调节小鼠肠道菌群失衡,具有类似大豆低聚糖的益生元功效.