基于超高效液相色谱法快速测定短链脂肪酸方法的建立

建立了采用超高效液相色谱法全面快速分析粪便与发酵液中短链脂肪酸的方法。以InertSustain AQ-C18色谱柱对粪便与发酵液中短链脂肪酸进行分离,流速为0.5 mL/min,波长为210 nm,流动相为20 mmol/L NaH_2PO_4(pH 2.2)与乙腈进行梯度洗脱。结果表明:该方法能在10 min内快速分离11种短链脂肪酸,加标培养基中各短链脂肪酸回收率为95.15%～107.01%,精密度为0.58%～7.33%,加标粪便样本中各短链脂肪酸回收率为98.43%～105.14%,精密度为1.13%～5.20%。该方法可用于粪便、发酵液中短链脂肪酸的检测,具有快速、简单及全面等优点。     

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),异丁酸与异戊酸水平差异无统计学意义(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,P0.000 1),与丙酸呈负相关(r=-0.358 1,P=0.006 2),与丁酸呈负相关(r=-0.421 7,P=0.001 1),与戊酸呈负相关(r=-0.326 8,P=0.042 3),与异丁酸、异戊酸无相关性。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。     

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。     

园艺植物GC-MS分析中进样方式的优化

园艺植物挥发性成分的气质联用分析选用顶空进样器进样可实现自动化,并极大地提高定性定量分析的可操作性。选择具有代表性的精油、樟树叶和花椒种子作为样品,探索顶空进样器进样的最佳炉温。结果显示,精油气质检测的峰值出现在50℃炉温;樟树叶和花椒峰值出现在70、80℃。试验认为以有机溶剂为主的液体样品,顶空进样器炉温选择50℃为宜;对园艺植物含水量较大的固体样品,则以70℃为宜。     

运动、肠道菌群代谢物——短链脂肪酸与骨骼肌代谢调控

寄生于人体的肠道菌群是一个高度动态化和个体化的复杂生态系统,受遗传、环境、饮食、年龄和运动等因素的影响,并通过其产生的代谢物与机体众多组织器官产生广泛的应答效应。短链脂肪酸(short chain fatty acid, SCFA)主要是由位于盲肠和结肠内的菌群以膳食纤维为底物发酵产生,其被吸收进入肠系膜上下静脉,随后汇入门静脉至肝。部分短链脂肪酸被肝作为糖异生和脂质合成的底物,剩余的短链脂肪酸以游离脂肪酸的形式经肝静脉进入外周循环。研究发现,运动可使产生SCFA的肠道菌群组分的丰度提高和参与调控SCFA生成的相关基因表达增加,使肠道中短链脂肪酸含量增加。由短链脂肪酸刺激结肠内分泌细胞合成分泌的胰高血糖素样肽1(glucagon like peptide-1, GLP-1)可促使胰岛B细胞合成分泌胰岛素,进而调节骨骼肌的葡萄糖摄取与糖原合成。此外,短链脂肪酸通过提高骨骼肌胰岛素受体底物1(insulin receptor substrate 1,IRS1)基因转录起始位点附近的组蛋白乙酰化水平,增强骨骼肌的胰岛素敏感性。同时,短链脂肪酸通过激活腺苷酸活化蛋白质激酶(AMP-activated protein kinase, AMPK)促进骨骼肌的脂肪酸摄取、脂肪分解和线粒体生物发生,抑制脂肪合成。本文就肠道菌群代谢物——短链脂肪酸概述、运动对产生短链脂肪酸的肠道菌群的影响和运动介导肠道菌群代谢物——短链脂肪酸对骨骼肌代谢调控机制的最新研究进展进行综述,为骨骼肌运动适应的新机制研究提供理论依据。     

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。     

苦瓜全粉和苦瓜乙醇提取物健康功效的体外评估

本研究的目的是运用体外模型评估苦瓜全粉和苦瓜乙醇提取物的健康功效。苦瓜全粉和苦瓜乙醇提取物以7.5 mg/mL和15 mg/mL的剂量与新鲜粪便样品进行体外厌氧培养。通过454焦磷酸测序技术分析粪便菌群组成的变化,同时测定发酵上清中短链脂肪酸的含量以及对Caco-2单层细胞跨膜电阻的影响。结果显示,苦瓜全粉(15 mg/mL)可以明显地扶植Faecalibacterium属细菌,同时抑制Bilophila和Desulfovibrio属细菌的增殖;同等剂量的苦瓜全粉发酵上清中短链脂肪酸的含量明显高于苦瓜乙醇提取物发酵上清;源自苦瓜乙醇提取物(15 mg/mL)的发酵上清对Caco-2单层细胞跨膜电阻的增强作用要大于苦瓜全粉(15 mg/mL)。本研究探究了体外苦瓜全粉和苦瓜乙醇提取物不同的生理功效,为以后苦瓜制品的选择提供了初步证据。     

气相色谱法测定猪结肠内容物中短链脂肪酸含量

目的:为了准确测定猪结肠内容物中蛋白质代谢物短链脂肪酸含量以研究猪结肠蛋白质代谢情况,本文拟利用气相色谱建立一种更准确快速分析猪结肠内容物中短链脂肪酸含量的方法。方法:实验选用体重相近的21日龄断奶的杜长大三元杂交仔猪,单栏饲养。试猪麻醉后放血致死,迅速剖开腹腔,收集结肠末端内容物,保存于-80℃。收集到的结肠末端内容物用偏磷酸预处理,利用毛细管气相色谱法程序升温测定猪结肠内容物中短链脂肪酸含量。结果:结肠内容物经偏磷酸预处理3 h后,在程序升温、0.8 m L/min载气流量的气相色谱条件下,内容物中乙酸、丙酸、异丁酸、丁酸、异戊酸和戊酸得到有效分离,其回收率为93%~113%,相对标准偏差为0.46%~0.70%,变异系数小于1%。结论:此方法具有操作简便、快速、准确的优点,是测定动物肠道内容物中短链脂肪酸较为理想的方法。     

白族成年人肠道菌群多样性研究

【目的】通过研究云南白族成年人肠道菌群群落结构,探索肠道菌群结构与云南白族人健康长寿的相互关系。【方法】以43份采自云南省昆明市(Urban)和大理白族自治州洱源县农村(Rural)成年白族志愿者的粪便样品为研究对象,通过种属特异性聚合酶链式反应(定性PCR)和基于聚合酶链式反应的变性梯度凝胶电泳(PCR-DGGE)对其中的优势菌群进行了分析,并测定样品中短链脂肪酸(SCFAs)含量。【结果】短链脂肪酸(SCFAs)含量测定结果显示,Rural和Urban志愿者的短链脂肪酸含量差异不显著;定性PCR结果显示Rural志愿者肠道内的乳杆菌属和双歧杆菌属的多样性明显区别于Urban志愿者;依据泳道轨迹光密度的不同对16S rRNA基因V3区PCR-DGGE图谱进行基于非加权平均距离聚类分析(Unweighted pair-group method with arithmetic means,UPGMA),显示两组样品按照组别明显地聚为两簇,多样性指数分析也呈现Rural志愿者肠道菌群多样性大于Urban的趋势;进一步对Rural谱带回收克隆测序,并构建系统发育树图,结果显示样品中双歧杆菌属、肠杆菌属和肠球菌属具有较高的多样性,是Rural健康成年志愿者肠道中的优势菌群。【结论】综合上述分析,昆明城市志愿者和大理白族自治州洱源县农村志愿者肠道菌群群落结构呈现区分趋势,多样性差异不显著。该研究为后续探索宿主肠道菌群与健康长寿的相互关系提供了数据基础。     

乳杆菌胞外多糖对人体粪便菌群及其产生短链脂肪酸的影响

目的在体外利用7名志愿者的新鲜粪便作为来源,选择菊粉作为对照,研究干酪乳杆菌LC2W与鼠李糖乳杆菌Y37胞外多糖对人体肠道菌群的影响。方法利用PCR-DGGE(变性梯度凝胶电泳)方法分析样品中菌群组成,采用高效液相色谱的方法分析短链脂肪酸组成。结果与菊粉相比,粪便菌群在添加乳杆菌胞外多糖培养后,可以促进短链脂肪酸的生成,保持肠道菌群的多样性。结论干酪乳杆菌LC2W与鼠李糖乳杆菌Y37的胞外多糖在体外有调节粪便菌群的作用。     

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography–mass spectrometry (GC–MS) analysis. A one-step derivatization using 100 μL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations of all targeted compounds showed good intra- and inter-day (within 7 days) precision (<10 %), and good stability (<20 %) within 4 days at room temperature (23–25 °C), or 7 days when stored at ?20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistical analysis of the concentrations of these targeted metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.     

Effects of Tripalmitin and Safflower Oil on the Content of Short-chain Fatty Acids in the Cecum and on the Fecal Excretion of Lipids in Rats

The effects of feeding of saturated and unsaturated fatty acids on the contents of short-chain fatty acids (SCFAs) derived from bacterial fermentation in the colon were examined in rats. The concentration of lipids in the feces was also measured. Tripalmitin or safflower oil at the concentration of 10% (w/w) in the diet with 5% (w/w) corn oil were used as the source of saturated or unsaturated fatty acids, respectively.     

The first synthetic agonists of FFA2: Discovery and SAR of phenylacetamides as allosteric modulators

Free fatty acid receptor 2 (FFA2) is a G-protein coupled receptor for which only short-chain fatty acids (SCFAs) have been reported as endogenous ligands. We describe the discovery and optimization of phenylacetamides as allosteric agonists of FFA2. These novel ligands can suppress adipocyte lipolysis in vitro and reduce plasma FFA levels in vivo, suggesting that these allosteric modulators can serve as pharmacological tools for exploring the potential function of FFA2 in various disease conditions.     

Application of liquid chromatography–mass spectrometry to measure short chain fatty acids in blood

A new liquid chromatography–mass spectrometry method is described to determine concentrations of the short chain fatty acids acetic acid, propionic acid and butyric acid (SCFAs) in human blood plasma. The method is based on reversed phase chromatography followed by post-column neutralization of the mobile phase with ammonia and a consecutive measurement of the SCFAs ammonia adducts using negative electro spray ionization. Sample preparation involved simple organic acid deproteinization, resulting in 100% recovery. SCFAs eluted baseline separated within a 25 min run cycle. A linear response was obtained in the range between 0 and 250 μmol/l (R² ranged from 0.997 to 0.9999). The limit of detection ranged from 0.05 μmol/l for propionic and butyric acid and 0.1 μmol/l for acetic acid. The method was tested by analyzing plasma of arterial blood, from portal vein and hepatic vein blood from patients undergoing a pylorus-preserving pancreaticoduodenectomy. As expected, the highest SCFA concentrations were found in portal plasma, hepatic vein levels were in between, while arterial concentrations were lowest. This newly developed method is suitable to determine SCFA concentrations in human plasma samples.     

Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers

Metabolic acidosis can result from accumulation of organic acids in the blood due to anaerobic metabolism or intestinal bacterial fermentation of undigested substrate under certain conditions. These conditions include short-bowel syndrome, grain overfeeding of ruminants and, as recently reported, severe gastroenteritis. Measuring fermentation products such as short-chain fatty acids (SCFAs) and lactic acid in various biological samples is integral to the diagnosis of bacterial overgrowth. Stereospecific measurement of D- and L-lactic acid is necessary for confirmation of the origin and nature of metabolic acidosis. In this paper, methods for the separation of SCFAs and lactic acid are reviewed. Analysis of the organic acids involved in carbohydrate metabolism has been achieved by enzymatic methods, gas chromatography, high-performance liquid chromatography and capillary electrophoresis. Sample preparation techniques developed for these analytes are also discussed.     

A quantitative headspace–solid-phase microextraction–gas chromatography–flame ionization detector method to analyze short chain free fatty acids in rat feces

This study sought to develop and validate a quantitative method to analyze short chain free fatty acids (SCFAs) in rat feces by solid-phase microextraction and gas chromatography (SPME–GC) using the salt mixture ammonium sulfate and sodium dihydrogen phosphate as salting out agent. Conditioning and extraction time, linearity, limits of detection and quantification, repeatability, and recovery were evaluated. The proposed method allows quantification with improved sensitivity as compared with other methods exploiting SPME–GC. The method has been applied to analyze rat fecal samples, quantifying acetic, propionic, isobutyric, butyric, isopentanoic, pentanoic, and hexanoic acids.     

Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters.

A protocol was developed for the analysis of volatile short chain fatty acids in microsamples of feces, short chain fatty acid (SCFA) extraction was from fecal samples using ethanol incorporating n-hexanoic acid as an internal standard. The SCFAs were converted to pentafluorobenzyl esters with alpha-2,3,4,5,6-pentafluorotoluene and analyzed on a gas-liquid chromatograph equipped with an electron capture detector. One hundred milligrams of sample was routinely used but analysis could be carried out on 20 mg of sample.     

Evaluation of intestinal microbiota,short-chain fatty acids,and immunoglobulin a in diversion colitis

It is reported that an increase in aerobic bacteria, a lack of short-chain fatty acids (SCFAs), and immune disorders in the diverted colon are major causes of diversion colitis. However, the precise pathogenesis of this condition remains unclear. The aim of the present study was to examine the microbiota, intestinal SCFAs, and immunoglobulin A (IgA) in the diverted colon. Eight patients underwent operative procedures for colostomies. We assessed the diverted colon using endoscopy and obtained intestinal samples from the diverted colon and oral colon in these patients. We analyzed the microbiota and SCFAs of the intestinal samples. The bacterial communities were investigated using a 16S rRNA gene sequencing method. The microbiota demonstrated a change in the proportion of some species, especially Lactobacillus, which significantly decreased in the diverted colon at the genus level. We also showed that intestinal SCFA values were significantly decreased in the diverted colon. Furthermore, intestinal IgA levels were significantly increased in the diverted colon. This study was the first to show that intestinal SCFAs were significantly decreased and intestinal IgA was significantly increased in the diverted colon. Our data suggest that SCFAs affect the microbiota and may play an immunological role in diversion colitis.     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.     

Structure-dependent and receptor-independent increase in osmotic fragility of rat erythrocytes by short-chain fatty acids

We examined short-chain fatty acids (SCFAs) with 1 (C1) to 5 (C5) carbon atoms for osmotic fragility (OF) in isolated red blood cells (RBCs) in rats. The RBCs were used as prototypical plasma membrane model. The dense packed RBC was incubated in a phosphate-NaCl buffer solution containing each SCFA at 0 to 100 mM. The RBC suspensions were transferred into the OF test tubes containing NaCl from 0.2 to 0.9%. The hemoglobin concentration was determined and the EC50 in hemolysis was calculated. The OF in RBCs was dose-dependently increased by exposure to SCFAs, except for C1, with an increasing number of carbon atoms. Branched-chain fatty acids (isomers of C4 and C5) have a smaller effect on OF than straight-chain fatty acids (C4 and C5). The SCFA-induced increases in OF were not affected by pretreatment of RBCs with trypsin. The response of the RBC membrane to SCFAs depends on their concentration, carbon chain length and chain structure (straight or branched). The SCFAs probably disturb the lipid bilayer of the RBC membrane and result in a decrease in osmotic resistance. The plasma membrane in rat RBCs could respond to the structure of the SCFAs in detail by using the OF as an indicator.