小鼠粪便中短链脂肪酸提取与检测方法的建立及应用

目的:建立小鼠粪便中三种短链脂肪酸(乙酸、丙酸、丁酸)的检测方法及其应用。方法:使用水提法和乙醚萃取法提取小鼠粪便中的短链脂肪酸(SCFA)通过高效液相色谱(HPLC)评价两种方法的提取效果,并建立短链脂肪酸的HPLC定量检测方法并进行方法学考察,利用上述建立的体系评价新琼寡糖对小鼠短链脂肪酸合成的影响。结果:确定了SCFA的乙醚萃取方法和基于HPLC的定量检测方法,被测组分浓度与其峰面积呈良好线性关系,各组分测定的相对标准偏差在1.31%~2.61%之间,平均加标回收率为86.6%~105.8%之间;对喂饲新琼寡糖小鼠粪便中的SCFA检测显示,较对照组乙酸和丙酸含量提高了1倍,丁酸含量提高了0.45倍。结论:建立了小鼠粪便中SCFA的提取和定量检测方法,并利用该方法证明了新琼寡糖能促进小鼠短链脂肪酸的合成。     

野牦牛和家牦牛粪便菌群与短链脂肪酸关系的研究

短链脂肪酸(SCFA)是反刍动物吸收饲草、饲料中营养物质的重要形式。肠道菌群能够降解食物生成SCFA并影响其比例。本文通过16S r DNA测序和气相色谱质谱联用仪,分别测定了野牦牛(Bos mutus)和家牦牛(Bos grunniens)粪便菌群组成及SCFA含量,通过比较分析两种牦牛肠道菌群与SCFA的关系,筛选出野牦牛肠道中与SCFA高浓度有正相关关系的菌群。结果显示,野牦牛粪便菌群主要有厚壁菌门(Firmicutes)(66. 47%)、拟杆菌门(Bacteroidetes)(26. 00%)和变形菌门(Proteobacteria)(3. 48%),主要的科有瘤胃球菌科(Ruminococcaceae)(55. 18%)、拟杆菌科(Bacteroidaceae)(8. 75%)和毛螺菌科(Lachnospiraceae)(7. 57%),家牦牛的菌群结构和组成与野牦牛相似。野牦牛粪便中SCFA以乙酸和丙酸为主,乙酸、丙酸、异丁酸、正丁酸和正戊酸的含量均显著高于家牦牛(P <0. 01)。Spearman相关分析显示,野牦牛粪便菌群中紫单胞菌科(Porphyromonadaceae)、拟杆菌科(Bacteroidaceae)、普雷沃氏菌科(Paraprevotellaceae)、理研菌科(Rikenellaceae)和韦荣球菌科(Erysipelotrichaceae)与SCFA具有较强相关性(r> 0. 4),而家牦牛仅有弱相关性(r <0. 3)。说明牦牛后肠道具有丰富的能够促进SCFA生成的益生菌群,进而提高食物的转化效率。     

肠道中短链脂肪酸与过敏性疾病关系的研究进展

短链脂肪酸(short-chain fatty acid,SCFA)是肠道菌群代谢产物中最主要的标志物之一.肠道中不同种属类型的细菌产生的SCFA的种类和数量各不相同,通过检测肠道中SCFA的变化,可以反应肠道中肠道菌群的变化.过敏性疾病患儿在早期与正常儿童肠道中的肠道菌群有明显差异,故SCFA的种类和数量也表现出明显的差异.有研究显示过敏性疾病患儿肠道中丙酸、异丁酸、丁酸、异戊酸、戊酸的水平较正常儿童低,而乙酸和异己酸的水平则较高.开展这方面的研究,对于探索肠道菌群及其代谢产物与过敏性疾病发病机制的关系有十分重要的意义.     

加味芪榔方对药物依赖性便秘患者粪便短链脂肪酸的影响CSCD

目的 观察加味芪榔方对药物依赖性便秘患者粪便短链脂肪酸的影响及其可能机制。方法 将我院160例药物依赖性便秘患者随机分为治疗组和对照组,各80例,分别予以加味芪榔方+乳果糖口服液模拟剂,乳果糖口服液+加味芪榔方模拟剂,疗程均为8周。记录治疗前后患者便秘主要症状积分、中医证候积分、粪便短链脂肪酸水平及复发情况。结果 治疗组患者FAS(full analysis set,全分析集)及PPS(per protocol set,符合方案集)的总有效率分别为91.14%和93.51%,均高于对照组的73.33%和76.39%(均P<0.000 1)。治疗后,治疗组患者便秘主要症状总积分、中医证候总积分均较治疗前显著降低,且优于对照组(t=-6.113 6、-7.035 2,均P<0.000 1)。治疗后,治疗组患者粪便总短链脂肪酸水平较治疗前升高,且高于对照组(t=7.024 6,P<0.000 1)。随访至第2周和第4周时,对照组复发率均高于治疗组(χ^(2)=59.645 2,P<0.000 1;χ^(2)=17.064 5,P=0.000 4)。结论 加味芪榔方治疗药物依赖性便秘的疗效显著,患者复发率低,可能与其能调节粪便中短链脂肪酸水平和改善肠道动力有关。     

正常糖耐量个体粪菌液对db/db小鼠粪便短链脂肪酸的影响

通过气相色谱法检测经哈萨克族正常糖耐量人粪菌移植后的2型糖尿病db/db小鼠粪便中短链脂肪酸的含量,探讨肠道菌群代谢产物与糖尿病的关系。

db/db小鼠分为模型组（db/db+PBS组）和哈萨克族正常糖耐量人粪菌液干预组（db/db+KNGT组）,db/m小鼠为空白对照组（db/m+PBS组）,每组9只,干预10周。分别在干预0周、6周和10周后检测小鼠空腹血糖、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、总胆固醇和三酰甘油的水平,并收集各组小鼠粪便,运用气相色谱技术检测干预0周、6周和10周后小鼠粪便中短链脂肪酸的含量。

在哈萨克族正常糖耐量人粪菌液干预6周和10周后,与db/db+PBS组相比,db/db+KNGT组小鼠粪样中乙酸、丁酸的含量显著增加（F_乙酸 = 3.210,F_丁酸 = 8.530,均P＜0.05）。

哈萨克族正常糖耐量人粪菌液移植后小鼠粪样中菌群代谢产物乙酸和丁酸水平增加,从而改善了db/db小鼠的糖脂代谢紊乱。

     

短链脂肪酸对肠道动力影响的研究进展

人体的肠道不仅仅是消化吸收场所,也是大量微生物生存的家园.肠道作为人体最大的储菌库,其多种生理功能离不开复杂多变的肠道菌群和菌群代谢物(如短链脂肪酸等)的参与.短链脂肪酸是肠道菌群发酵膳食纤维产生的一类重要的信号分子,研究发现短链脂肪酸除参与维持人体肠道黏膜免疫屏障、调节体液及电解质平衡以及为肠上皮细胞提供能量外,...     

短链脂肪酸调控人髓母细胞瘤UW228 3细胞增殖、凋亡和侵袭作用的研究

目的 研究菌群代谢产物短链脂肪酸——丙酸、丁酸对人髓母细胞瘤UW228 3细胞增殖、凋亡和侵袭的影响。 方法 分别用10 μmol/L丙酸和5 μmol/L丁酸处理UW228 3细胞,通过HE染色观察细胞形态,MTT法测定细胞活力,流式细胞术检测细胞凋亡,细胞划痕检测细胞侵袭,PCR和Western Blot检测凋亡相关基因和蛋白的表达。 结果 10 μmol/L丙酸和5 μmol/L丁酸能够有效抑制UW228 3细胞增殖能力,增加细胞凋亡率,并显著抑制UW228 3细胞侵袭能力,提高Caspase 3基因以及蛋白表达,降低c Myc、Bcl 2、Survivin基因以及蛋白的表达。 结论 菌群代谢产物丙酸、丁酸能够抑制人髓母细胞瘤UW228 3细胞增殖和侵袭,并促进细胞凋亡,具有治疗髓母细胞瘤的潜在价值。     

基于气相色谱法分析T2DM患者粪便中SCFAs水平与HbA1c的相关性

目的 研究2型糖尿病患者粪便中6种短链脂肪酸水平与糖化血红蛋白的相关性。 方法 采用气相色谱法检测粪便中短链脂肪酸的含量,并对方法学进行考察。选取2018年在本院查体的2型糖尿病患者57例,根据糖化血红蛋白水平将其分为高糖化组(糖化血红蛋白>7.0,28例)与低糖化组(糖化血红蛋白≤7.0,29例)。应用气相色谱法检测两组患者粪便中6种短链脂肪酸水平,并分析6种短链脂肪酸水平与糖化血红蛋白的相关性。 结果 低糖化组患者粪便中乙酸、丙酸、丁酸、戊酸水平显著高于高糖化组(均P0.05)。Pearson相关性分析结果表明:在2型糖尿病患者中,糖化血红蛋白与粪便中乙酸呈负相关(r=-0.540 1,P结论 2型糖尿病患者粪便中短链脂肪酸水平与糖化血红蛋白存在一定的相关性,短链脂肪酸水平的下降可能是影响血糖控制不佳的主要原因。     

佐剂性关节炎大鼠肠道G蛋白耦联受体43、短链脂肪酸的表达及相关性分析CSCD

目的 观察佐剂性关节炎（AA）大鼠肠道短链脂肪酸（SCFAs）、G蛋白耦联受体43(GPR43)的变化并分析二者之间的相关性。方法 Wistar雄性大鼠被随机分为正常组（NC组）、模型组（AA组）,每组8只。模型组大鼠采用弗氏完全佐剂（CFA）注射法复制AA模型。复制成功后观察各组大鼠关节肿胀度（JSD）、关节炎指数（AI）的变化;采用透射电镜观察各组大鼠踝关节滑膜细胞结构的改变;使用蛋白质印迹法检测各组大鼠结肠组织中GPR43的水平;应用液相色谱—质谱联用（LC-MS/MS）检测粪便中菌群代谢物SCFAs水平。结果 与NC组相比,AA组大鼠足趾部红肿明显,JSD(t’=15.046,P<0.001)、AI评分（t’=23.910,P<0.001）均显著升高,滑膜病理损伤明显,线粒体形态评分升高（z=2.023,P<0.050）;AA组结肠组织GPR43相对表达量降低（t=3.182,P<0.050）,粪便丁酸（t’=4.798,P<0.010）、己酸（t=4.123,P<0.010）水平显著降低,其余SCFAs差异无统计学意义。结肠组织GPR43水平与大鼠粪便丁酸（r=0.817,P<0.050）、己酸（r=0.789,P<0.050）存在线性相关关系。结论 AA大鼠粪便丁酸、己酸水平及结肠组织GPR43相对表达量均下降,且结肠组织GPR43水平与粪便丁酸、己酸水平呈正相关。     

饲养方式对藏猪结肠消化酶活性、菌群结构及短链脂肪酸含量的影响

【目的】为探究饲养方式对藏猪结肠消化酶活性、菌群结构和短链脂肪酸含量的影响。【方法】研究分别选取5头相同月龄的放养藏猪和舍饲藏猪。屠宰采集结肠粪便样品,分别利用酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)试剂盒、高通量测序技术和气相色谱仪测定放养藏猪和舍饲藏猪结肠消化酶活性、菌群结构和短链脂肪酸含量。【结果】同一月龄下,放养藏猪的日增重显著低于舍饲藏猪(P<0.05)。放养藏猪结肠中纤维素酶和半纤维素酶的活性均显著高于舍饲藏猪(P<0.05);2种饲养方式藏猪结肠的6种α多样性指数均无显著差异(P>0.05),且主成分分析(principal component analysis, PCA)得到放养藏猪和舍饲藏猪结肠菌群存在一定的相似性。在门和科分类水平上,相较于舍饲藏猪,放养藏猪结肠中疣微菌门、黄杆菌科、月形单胞菌科、浮霉状菌科和伊格尔兹氏菌科的相对丰度显著升高,而链球菌科、韦荣氏球菌科、假单胞菌科、红环菌科、红螺菌科、乳杆菌科、理研菌科和巴斯德氏菌科的相对丰度显著降低(P<0.05);在属和种分类水平上,...     

Detection of microbial-derived fatty acids in carious dentin by gas chromatography and gas chromatography-mass spectrometry

The aim of the present study was to analyze the fatty acid content of carious and sound human dentin. Gas chromatography and gas chromatography-mass spectrometry revealed the presence of fatty acids of C₁₀-C₁₈ size in the carious dentin, whereas fatty acids of C₁₆ size were present in minute amounts in three samples of the corresponding sound dentine controls. No fatty acids were detected in the other sound dentin control samples. The source of fatty acids was considered to be microorganisms invading the dentin during the progression of the caries lesion. The presence of bacterial fatty acids in carious dentin may serve as a marker for the pathological process and thus contribute to the understanding of the mechanisms involved.     

Analyses of short-chain fatty acids and exhaled breath volatiles in dietary intervention trials for metabolic diseases

As an alternative to pharmacological treatment to diseases, lifestyle interventions, such as dietary changes and physical activities, can help maintain healthy metabolic conditions. Recently, the emerging analyses of volatile organic compounds (VOCs) from breath and short-chain fatty acids (SCFAs) from plasma/feces have been considered as useful tools for the diagnosis and mechanistic understanding of metabolic diseases. Furthermore, diet-induced changes of SCFAs in individuals with diagnosed metabolic abnormalities have been correlated with the composition changes of the gut microbiome. More interestingly, the analysis of exhaled breath (breathomics) has gained attention as a useful technique to measure the human VOC profile altered as a result of dietary interventions. In this mini-review, we examined recent clinical trials that performed promising dietary interventions, SCFAs analysis in plasma/feces, and VOC profile analysis in exhaling breath to understand the relationship between dietary intervention and metabolic health.     

Determination of metaldehyde in human serum by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A rapid headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method has been developed for the determination of metaldehyde in human serum samples. Metaldehyde is extensively used as a molluscicide for the control of slugs and snails, and cases of metaldehyde poisoning have been reported. Metaldehyde was headspace-extracted on a polydimethylsiloxane (PDMS) fiber at 70 degrees C for 25 min, desorbed, and analyzed rapidly by GC-MS. The method was validated for limit of detection (LOD), linearity, precision, and recovery. Although the recovery of the sample was very low, the method itself was rapid with a low detection limit of 0.25 microg/ml, R.S.D. value 12.6%, and linearity range 0.5-25.0 microg/ml (r(2)=0.999). The results demonstrated that the SPME-GC-MS method for the analysis of metaldehyde is simple, rapid, solvent-free, and does not require any pre-analysis conversions.     

Quantification of clotiazepam in human plasma by gas chromatography-mass spectrometry

An analytical procedure was developed and validated for the quantification of clotiazepam in human plasma. After subjecting plasma samples to solid-phase extraction, the extract was evaporated and the residue re-constituted. An aliquot of the mixture was injected onto a gas chromatography-mass spectrometry system. The detector response was linear for clotiazepam concentrations in the range of 5-200 ng/ml. Intra- and inter-day precision for the assay over the concentration range was below 13.1 and 13.5%, and the accuracy ranged between 99.0-107.9% and 92.4-101.3%, respectively. The drug was found to be stable under various processing conditions used. The method is applicable to human pharmacokinetic studies of clotiazepam.     

Analysis of testosterone and dehydroepiandrosterone in saliva by gas chromatography-mass spectrometry

Testosterone and 3 beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) have been identified in human parotid fluid and saliva by gas chromatography-mass spectrometry/selected ion monitoring analyses of the t-butyldimethylsilyl ether and methyl oxime, t-butyldimethylsilyl ether derivatives. High specificity of analysis has been achieved by the use of high mass spectrometric resolution or by the monitoring of metastable peaks. Quantitative analyses indicate concentrations of both unconjugated testosterone and unconjugated dehydroepiandrosterone in the range 200-800 pmol/l in the saliva and parotid fluid of the normal males examined. These represent 1.5-7.5% of the concentrations of the steroids in blood plasma taken from the same subjects.     

Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.     

Rapid and sensitive determination of nalmefene in human plasma by gas chromatography-mass spectrometry

A rapid gas chromatography-mass spectrometric method for the determination of nalmefene in human plasma is described. The procedure involves protein precipitation, extraction with ethanol-chloroform mixture and derivatization with pentafluropropionic anhydride. The deuterated analog of nalmefene, 6beta-naltrexol-d(7), was used as the internal standard. Quantitation was achieved on a HP-1 column (12 mx0.2 mm I.D.) with negative chemical ionization (NCI) using methane:ammonia (95:5) as the reagent gas. The standard curves were fitted using a quadratic equation with the curve encompassing a range of 0.5 to 200 ng/ml, and the intra- and inter-assay variations for three different nalmefene levels were less than 10% throughout. The limit of quantitation was found to be 0.5 ng/ml. The method described is highly specific and reproducible, and could also be applied for the determination of naltrexone and 6beta-naltrexol. Application of the method to actual human plasma samples is demonstrated.