Determination of 1-Methylimidazole-4-Acetic Acid in Brain Tissue from Rat and Mouse

The concentration of the histamine metabolite 1-methylimidazole-4-acetic acid was determined in brain tissue from rat and mouse with a gas chromatographic-mass spectrometric method. Mouse brain contained 1.7-3.2 nmol/g, depending on the strain. The concentration in cerebrum from Sprague-Dawley rats was 1.2 nmol/g, whereas cerebellum contained 0.24 nmol/g. The concentration of tele-methylhistamine in mouse brain was 1.4-2.2 nmol/g. The concentration of 1-methylimidazole-4-acetic acid in rat brain after death did not change significantly during 2 h at room temperature.     

A novel model of adenine-induced chronic kidney disease-associated gastrointestinal dysfunction in mice: The gut-kidney axis

Although constipation is a common complication of chronic kidney disease (CKD), there is no animal model that can be used to study the association between renal impairment and gastrointestinal function without interfering with the gastrointestinal tract of the model. Therefore, we determined whether adenine could induce CKD in association with gastrointestinal dysfunction. Six-week-old ICR mice were intraperitoneally injected with saline, 25, 50, or 75 mg adenine/kg body weight for 21 days. Blood urea nitrogen (BUN), plasma creatinine, and renal histopathology were evaluated. Defecation status was evaluated from defecation frequency and fecal water content. Colonic smooth muscle contraction was measured by the organ bath technique, and transepithelial electrical resistance (TEER) was measured using an Ussing chamber. In the 50 mg/kg treatment group, BUN and creatinine were significantly increased compared with control, and inflammatory cell infiltration, glomerular necrosis, tubular dilatation, and interstitial fibrosis were observed in renal tissues. Mice in this group also showed a significant decrease in defecation frequency, fecal water content, colonic motility index, and TEER. Overall, 50 mg/kg of adenine was the best dose to induce CKD with associated constipation and intestinal barrier impairment. Therefore, this adenine administration model can be recommended for CKD-associated gastrointestinal dysfunction research.     

16S rDNA高通量测序分析腹膜透析胃肠功能障碍患者肠道菌群特征

采用16S rDNA高通量测序技术分析腹膜透析（PD）胃肠功能障碍患者的肠道菌群变化特征,探讨肠道菌群在腹膜透析胃肠功能障碍中的具体作用。

收集25例PD胃肠功能正常者（PDGF组）、25例PD胃肠功能障碍者（PDGD组）和13例健康者（Normal组）的粪便样本,提取肠道菌群基因组,应用16S rDNA高通量测序技术对各组测序结果进行菌群多样性、物种组成差异和菌群功能分析。

3组的Observed species指数（H = 6.905,P = 0.032）和Shannon指数（H = 6.993,P = 0.030）差异具有统计学意义。与Normal组相比,PDGD组Shannon指数显著降低（P = 0.027）,PDGF组Observed species指数显著升高（P = 0.044）;与PDGF组相比,PDGD组Observed species和Shannon指数显著降低（P＜0.05）。PCoA结果显示3组各自聚集,区分较明显。UPGMA聚类分析结果显示3组大部分样本均在本组中聚类后再与其他组进一步合并。LEfSe分析结果显示,Normal组优势菌群包括颤螺菌目（Oscillospirales）、瘤胃菌科（Ruminococcaceae）、栖粪杆菌属（Faecalibacterium）、拟杆菌科（Bacteroidaceae）等;PDGF组优势菌群包括毛螺菌目（Lachnospirales）、毛螺菌科（Lachnospiraceae）、布劳特菌属（Blautia）;PDGD组优势菌群包括芽孢杆菌纲（Bacilli）、乳杆菌目（Lactobacillales）、链球菌属（Streptococcus）、链球菌科（Streptococcaceae）等。KEGG L1水平的功能组成集中在代谢、遗传信息处理和环境信息处理;KEGG L2水平的功能主要集中在碳水化合物、氨基酸代谢、遗传信息的复制和修复及转运、膜运输途径;与Normal组和PDGF组相比,PDGD组在碳水化合物、氨基酸代谢途径富集减少,感染性疾病、聚糖生物合成和代谢途径富集增加。

PD胃肠功能障碍患者存在肠道菌群失调,调整肠道微生态是防治PD患者出现胃肠功能障碍的潜在干预靶标。

     

Determination of N-Acetylaspartic Acid in Human Cerebrospinal Fluid by Gas Chromatography–Mass Spectrometry

N-Acetyl-L-aspartic acid was identified and determined in human cerebrospinal fluid. The concentration in lumbar fluid was about 2 nmol/ml and about 20 nmol/ml in ventricular fluid. There was no difference between healthy subjects and schizophrenic patients.     

菘蓝种子脂肪酸的GC-MS分析

目的:分析菘蓝种子中的脂肪酸。方法:分别利用索氏提取法,运用气相色谱-质谱(GC-MS)联用技术,计算机检索和人工解析对菘蓝种子中的脂肪酸进行分析和鉴定。结果:鉴定了11种脂肪酸成分。结论:菘蓝种子中脂肪酸成分主要是亚麻酸(24．72%)、芥酸(23．9%)、油酸(19．11%)和亚油酸(10．76%)。     

Quantification of D-amino acids in human urine using GC-MS and HPLC

Summary The relative amounts of free D-amino acids (D-AA) in the urine of seven healthy volunteers (age 27 to 49 years) were determined using chiral phase (Chirasil-L-Val) capillary gas chromatography in conjunction with selected ion monitoring mass spectrometry. The absolute amounts of free D-AA were determined by pre-column derivatization of the amino acids witho-phthaldialdehyde andN-isobutyryl-L-cysteine followed by high-performance liquid chromatographic separation and fluorescence detection of the isoindol derivatives formed. The following most abundant D-AA were found (highest and lowest absolute and relative amounts): D-Ser (379.8 — 30.1µMol/L; 56.5 — 19.0%), D-Ala (53.8 — 7.6µMol/L; 19.6 — 5.7%), D-Thr (5.8 — 0.25µMol/L; 3.4 — 1.0%), D-Val (3.7 — 0µMol/L; 4.2 — 0%), and D-Phe (3.5 — 0.35µMol/L; 4.8 — 1.4%).     

植物应答非生物胁迫的代谢组学研究进展

代谢组学技术是研究植物代谢的理想平台, 通过现代检测分析技术对胁迫环境下植物中代谢产物进行定性和定量分析, 可以监测其随时间变化的规律。而各种组学平台包括基因组学、转录组学及代谢组学的整合, 更是一个强有力的工具箱, 将所获得的不同组学的信息联系起来, 有利于从整体研究生物系统对基因或环境变化的响应, 如可判断代谢物的变化是从哪一个层面开始发生的, 帮助人们揭开复杂的植物胁迫应答机制。该文对近期代谢组学技术及其与蛋白质组学、基因组学技术相结合探索植物应答非生物胁迫的研究进行了综述。代谢组学的应用, 拓展了对植物耐受非生物胁迫分子机制的认识, 开展更多这方面的研究, 再通过植物代谢组学、转录组学、蛋白质组学和基因组学整合, 有助于从整体水平上把握植物胁迫应答机制。     

Bioconversion of 2-amino acids to 2-hydroxy acids by Clostridium butyricum

Analysis by gas chromatography-mass spectrometry (GC-MS) of 24-h cultures of Clostridium butyricum type strain in synthetic BMG medium supplemented with various 2-amino acids (10 mM) revealed the presence of the corresponding 2-hydroxy acids. C. butyricum was able to bioconvert l-valine, dl-norvaline, l-leucine, dl-norleucine, l-methionine and l-phenylalanine as well as unusual 2-amino acids, i.e., l-2-aminobutyric acid, l-2-amino-4-pentenoic acid, dl-2-aminooctanoic acid, and dl-2-amino-4-phenylbutanoic acid. l-Isoleucine and cycloleucine were not converted into their corresponding 2-hydroxy acids. The bioconversion rate was maximal with dl-norvaline (6.2%). Chiral GC analysis demonstrated that only d-2-hydroxy-4-methylpentanoic acid is formed from l-leucine, indicating that the bioconversion is stereospecific, with inversion of configuration. d-Leucine and d-methionine were also converted to the corresponding 2-hydroxy acids. This observation opens new aspects in the study of C. butyricum and raises questions about the amino acid metabolism by this species.     

Capture and reconstitution of G protein-coupled receptors on a biosensor surface

Surface plasmon resonance (SPR) biosensors offer a unique opportunity to study the binding activity of G protein-coupled receptors (GPCRs) in real time with minimal sample preparation. Using two chemokine receptors (CXCR4 and CCR5) as model systems, we captured the proteins from crude cell preparations onto the biosensor surface and reconstituted a lipid environment to maintain receptor activity. The conformational states of the receptors were probed using conformationally dependent antibodies, and by characterizing the binding properties of a native chemokine ligand (stromal cell-derived factor 1alpha). The results suggest that the detergent-solubilized receptors are active for ligand binding in the presence and absence of a reconstituted bilayer. There are three advantages to using this receptor-capturing approach: (1) there is no need to purify the receptor prior to immobilization on the biosensor surface, (2) the receptors are homogeneously immobilized through the capturing step, and (3) the receptors can be captured at high enough densities to allow the study of relatively low-molecular-mass ligands (2000-4000Da). We also demonstrated that the receptors are sensitive to the solubilizing conditions, which illustrates the potential for using SPR biosensors to rapidly screen solublization conditions for GPCRs.     

肠内营养联合益生菌对重症脑卒中伴胃肠功能障碍患者肠黏膜屏障的保护作用

目的 探讨肠内营养联合益生菌对重症脑卒中伴胃肠功能障碍患者肠黏膜屏障的保护作用,为该类患者的治疗提供参考.方法 选择2016年1月至2019年7月于我院住院的84例重症脑卒中伴胃肠功能障碍患者为研究对象,根据肠内营养支持时是否添加益生菌将患者分为联用组和单用组,各42例.两组患者均予控制颅内压、营养脑细胞、保护胃黏膜和...     

Regional distribution of the histamine metabolite, tele-methylimidazoleacetic acid, in rat brain: effects of pargyline and probenecid

Abstract: tele -Methylimidazoleacetic acid (t-MIAA), a major brain histamine metabolite, was measured in nine rat brain regions by a gas chromatography-mass spectrometric method that also measures the precursor amine, tele -methylhistamine (t-MH). The t-MIAA concentration of cerebellum, medulla-pons, midbrain, caudate nucleus, hypothalamus, frontal cortex, hippocampus, and thalamus varied 15-fold, hypothalamus showing the highest level (2.21 nmol/g) and cerebellum the lowest (0.15 nmol/ g). The concentrations of t-MIAA and t-MH were significantly correlated in all regions except midbrain, which had relatively more t-MIAA. Probenecid did not alter whole-brain t-MIAA levels. Treatment with pargyline, an inhibitor of monoamine oxidase, lowered the t-MIAA levels in all regions.     

GC/MS分析血浆中丁丙诺啡

目的:建立血浆中丁丙诺啡GC/MS分析方法。方法:血浆中丁丙诺啡,加入内标长春西汀,加pH 7缓冲溶液,用三氯甲烷提取,提取物经BSTFA衍生化后进行GC/MS分析。结果:方法的线性范围为2～100 g·L~(-1),检出限为1g·L~(-1)。结论:该方法灵敏度高,可用于涉毒案件血浆中丁丙诺啡的分析。     

Chemical Constituents of <i>Pedicularis longiflora</i> var. <i>tubiformis</i> (Orobanchaceae), a Common Hemiparasitic Medicinal Herb from the Qinghai Lake Basin,China

Pedicularis longiflora var. tubiformis (Orobanchaceae) is an abundant parasitic herb mainly found in the Xiaopohu wetland of the Qinghai Lake Basin in Northwestern China. The species has an important local medicinal value, and in this study, we evaluated the chemical profile of its stems, leaves and seeds using mass spectrometry. Dried samples of stems, leaves and seeds were grinded, weighted, and used for a series of extractions with an ultrasonic device at room temperature. The chemical profiles for each tissue were determined using Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid ChromatographyMass Spectrometry (LC-MS). Twenty-seven amino acids and organic acids were identified and quantified from stems, leaves and seeds. The content of amino acids detected in leaves and seeds was higher than the amount found in stems. Six flavonoids were also detected, including isoorientin, orientin, luteolin-7-O-glucoside, luteolin, apigenin and tricin. The concentrations of luteolin-7-O-glucoside, luteolin and tricin were the highest and more concentrated in leaves, while that of orientin was the lowest and mainly found in stems. Soluble monosaccharides and oligosaccharides below tetramer were also examined, and our analyses detected the presence of arabitol, fructose, galacturonic acid, glucose, glucuronic acid, inositol, sucrose, and trehalose. This is the first study to identify and quantify the main components of amino acids, organic acids, flavonoids and soluble sugars from stems, leaves and seeds of P. longiflora var. tubiformis. Eight of the amino acids detected are essential for humans, highlighting the medicinal importance of this species. Results shown here can be used as a reference case to develop future studies on the chemical constituents of Pedicularis herbs and other medicinal plants from the Tibetan region.     

Neuronal Metabolism of Branched-Chain Amino Acids: Flux Through the Aminotransferase Pathway in Synaptosomes

Abstract: The metabolism of branched-chain amino acids (BCAAs) was studied in cortical synaptosomes. With [¹⁵N]leucine (1 mM) as precursor, the cumulative appearance of ¹⁵N in [¹⁵N]glutamate and [¹⁵N]aspartate was 0.2 nmol/min/mg of protein without supplemental α-ketoglutarate and 0.3 nmol/min/mg of protein in the presence of α-ketoglutarate (0.5 mM). The BCAA aminotransferase reaction also proceeded in the “reverse” direction [α-ketoisocaproate (KIC) + glutamate → leucine + α-ketoglutarate]. This was documented by incubating synaptosomes with [¹⁵N]glutamate and measuring the formation of [¹⁵N]leucine. Without KIC in the medium, the rate of [¹⁵N]leucine production was 0.13 nmol/min/mg of protein. In the presence of 25 µM KIC the rate was 0.79 nmol/min/mg of protein and even greater (1.0 nmol/min/mg of protein) in the presence of 500 µM KIC. The reamination of KIC was two- to threefold faster with [2-¹⁵N]glutamine as precursor compared with [¹⁵N]glutamate. The ketoacid of valine, α-ketoisovalerate (KIV), was reaminated to [¹⁵N]valine at a rate comparable to that observed with respect to KIC. The BCAA transaminase mediated not only the bidirectional transfer of amino groups between leucine or valine and glutamate, but also the direct transfer of nitrogen between leucine and valine. This was ascertained in studies in which the incubation medium was supplemented with either [¹⁵N]leucine and KIV or [¹⁵N]valine and KIC (amino acids at 1 mM and ketoacids at 25 or 500 µM). The rate was faster in the direction of leucine formation at both the lower (6.1-fold) and higher (1.7-fold) KIC concentration. It is suggested that in synaptosomes the BCAA transaminase (a) functions predominantly in the direction of leucine formation and (b) maintains a constant ratio of BCAAs and ketoacids to one other.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.     

Recent progress in polar metabolite quantification in plants using liquid chromatography-mass spectrometry

Metabolite analysis or metabolomics is an impor-tant component of systems biology in the post-genomic era. Although separate liquid chromatography (LC) methods for quantification of the major classes o...     

One-pot microwave derivatization of target compounds relevant to metabolomics with comprehensive two-dimensional gas chromatography

Metabolomics has been defined as the quantitative measurement of all low molecular weight metabolites (sugars, amino acids, organic acids, fatty acids and others) in an organism's cells at a specified time under specific environmental/biological conditions. Currently, there is considerable interest in developing a single method of derivatization and separation that satisfies the needs for metabolite analysis while recognizing the many chemical classes that constitute the metabolome. Chemical derivatization considerably increases the sensitivity and specificity of gas chromatography–mass spectrometry for compounds that are polar and have derivatizable groups. Microwave-assisted derivatization (MAD) of a set of standards spanning a wide range of metabolites of interest demonstrates the potential of MAD for metabolic profiling. A final protocol of 150 W power for 90 s was selected as the derivatization condition, based upon the study of each chemical class. A study of the generation of partially derivatized components established the conditions where this could potentially be a problem; the use of greater volumes of reagent ensured this would not arise. All compounds analyzed by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry in a standard mixture showed good area ratio reproducibility against a naphthalene internal standard (RSD < 10% in all but one case). Concentrations tested ranged from 1 μg/mL to 1000 μg/mL, and the calibration curves for the standard mixtures were satisfactory with regression coefficients generally better than 0.998. The application to gas chromatography–quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry for a typical reference standard of relevance to metabolomics is demonstrated.     

Presence and Measurement of Imidazoleacetic Acid, a γ-Aminobutyric Acid Agonist, in Rat Brain and Human Cerebrospinal Fluid

Imidazoleacetic acid (IAA) was unequivocally demonstrated in rat brain, human CSF, and human plasma by a gas chromatographic-mass spectrometric method that can reliably quantify as little as 8 pmol, i.e., 1 ng. Owing to tautomerism of the imidazole ring, IAA and [15N, 15N]IAA, the internal standard, each formed two chromatographically distinct isomers after derivatization of the ring nitrogens with either ethyl chloroformate or methyl chloroformate. The isomers of n-butyl(N-ethoxycarbonyl)imidazole acetate and n-butyl(N-methoxycarbonyl)imidazole acetate were identified by analysis with methane chemical ionization and electron impact ionization of molecular and fragment ions. The levels (mean +/- SEM) of free IAA were 140 +/- 14 pmol/g and 2.7 +/- 0.2 pmol/ml in brains of untreated rats and human lumbar CSF, respectively. Mean levels of IAA in brains of anesthetized rats, perfused free of blood, did not differ significantly from mean levels of anesthetized, nonperfused controls or from untreated rats. The source or sources of IAA in brain and CSF are unknown. Because IAA is a potent agonist at gamma-aminobutyrate receptors, it merits examination as a regulator in brain.     

Norepinephrine Metabolism in Man Using Deuterium Labelling: The Conversion of 4-Hydroxy-3-Methoxyphenylglycol to 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study the disposition of peripherally administered HMPG. Five healthy men were given an intravenous pulse dose of 4.3 μmol of labelled HMPG and subsequent plasma and urine levels of endogenous and labelled HMPG as well as those of 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) were determined by gas chromatography-mass spectrometry, using selected ion detection. Approximately 40% of the injected amount of deuterium-labelled HMPG was recovered in the urine as HMMA and another 40% was eliminated as HMPG conjugates. Thus, the HMPG formed from norepinephrine either in the central or peripheral nervous system undergoes both conjugation and extensive oxidation.     

Further Studies on 4-Hydroxy-3-Methoxyphenylglycol Oxidation in Humans: Effect of Pool Expansion and Stereochemistry

The in vivo oxidation of the norepinephrine metabolite 4-hydroxy-3-methoxyphenylglycol (HMPG) to 4-hydroxy-3-methoxymandelic acid was studied in man with two different doses of deuterium-labeled HMPG and a tracer dose of [14C]HMPG. HMPG oxidation appeared to be dose-dependent with an oxidation of 62-70% for doses below or equal to 2.2 mumol. With the use of a capillary column coated with an optically active phase (Chirasil-Val) and gas chromatography mass-spectrometry the human urinary excretions of the two stereoisomers of deuterium-labelled HMPG (free + conjugates) were found to be equal.