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.     

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.     

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%).     

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%)。     

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.     

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。     

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.     

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.     

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...     

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.     

一株功能性乳酸菌对胃肠道环境耐受能力的测定

目的对1株具有降胆固醇功能的乳酸菌进行胃肠道环境耐受能力的测定。方法分别测定菌株对胃内酸性和胃蛋白酶的耐受能力以及对小肠内胆盐和胰蛋白酶的耐受能力。结果该菌株对胃肠道有良好的耐受能力。结论本研究为后期菌株在动物及人体内发挥其功能性奠定了实验基础。     

Detection of cholesteryl ester hydroperoxide isomers using gas chromatography-mass spectrometry combined with thin-layer chromatography blotting

Oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. During the oxidation of LDL, cholesteryl esters, the major lipid components in LDL, are oxidized to cholesteryl ester hydroperoxides (CEOOH). The isomers of CEOOH may reflect the reactive species that initiate the peroxidation reaction. In the current study, a novel analytical method for the determination of CEOOH isomers, especially cholesteryl linoleate hydroperoxide isomers, was developed using the combination of two chromatographic techniques: (i) thin-layer chromatography blotting with diphenyl-1-pyrenylphosphine (DPPP) fluorescent detection (DPPP-TLC blotting) and (ii) gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). CEOOH was applied to DPPP-TLC blotting, the obtained DPPP-derived fluorescent spots containing cholesteryl ester hydroxides were extracted and derivatized (hydrogenation, transmethylation, and trimethylsilylation), and the formed methyl ester/trimethylsilylether derivatives of hydroxyoctadecenoic acid were then analyzed by GC-EI-MS. The CEOOH isomers were determined by selected ion monitoring of isomer-specific fragment ions originated from the alpha-cleavage of the trimethylsilyloxyl group. Using these two chromatographic techniques, we were able to detect isomeric CEOOH in the oxidized human LDL. Our results indicated that GC-EI-MS analysis combined with DPPP-TLC blot is a specific method for analyzing cholesteryl ester hydroperoxide isomers in biological samples such as oxidized LDL.     

Concentrations of Tryptoline and Methtryptoline in Rat Brain

Combined gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry have been used to identify and quantify tryptoline, methtryptoline, 5-hydroxytryptoline, and 5-hydroxymethtryptoline as their heptafluorobutyryl derivatives in extracts of rat brain. Tryptoline and methtryptoline were identified on the basis of their retention times and mass spectral characteristics: they were reliably detected in brain tissue extracts without interference from artifactual formation; their whole brain concentrations ranged between 0.2 and 3 ng/g; and they had a similar neuroanatomical distribution, with the highest concentrations in the cerebellum and the cortex. Smaller quantities of 5-hydroxytryptoline and 5-hydroxymethtryptoline were also identified on the basis of their retention times and mass spectral characteristics. However, the significance of this finding is unclear, because these two compounds were accompanied by larger quantities of their tetradeuterated analogues formed from tetradeuterated-5-hydroxytryptamine added at the time of tissue homogenization; this result suggests that formation of 5-hydroxytryptoline and 5-hydroxymethtryptoline occurred during tissue homogenization, sample preparation, or both.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Quantitative gas chromatography/mass spectrometry determination of C-mannosylation of tryptophan residues in glycoproteins

C-mannosylation of Trp residue is one of the most recently discovered types of glycosylation, but the identification of these mannosylated residues in proteins is rather tedious. In a previous paper, it was reported that the complete analysis of all constituents of glycoproteins (sialic acids, monosaccharides, and amino acids) could be determined on the same sample in three different steps of gas chromatography/mass spectrometry of heptafluorobutyrate derivatives. It was observed that during the acid-catalyzed methanolysis step used for liberation of monosaccharide from classical O- and N-glycans, Trp and His were quantitatively transformed by the addition of a methanol molecule on their indole and imidazole groups, respectively. These derivatives were stable to acid hydrolysis used for the liberation of amino acids. Since monosaccharide derivatives were also stabilized as heptafluorobutyrate derivatives of O-methyl-glycosides, it was suggested that C-mannosides of Trp residues could quantitatively be recovered. Based on the analyses of standard compounds, peptides and RNase 2 from human urine, we report that C((2))-mannosylated Trp could be quantitatively recovered and identified during the step of amino acid analysis. Analyses of different samples indicated that this type of glycosylation is absent in bacteria and yeasts.     

Formation of 2-hydroxy-4-methylpentanoic acid from l-leucine by Clostridium butyricum

Abstract Five Clostridium butyricum strains of different origin were grown in trypticase-yeast extract-hemin medium with or without d-glucose (TGYH or TYH medium, respectively) and in a synthetic basal medium with d-glucose (BMG medium). 2-Hydroxy-4-methylpentanoic acid was detected by gas chromatography-mass spectrometry (GC-MS) for the five strains whether grown in TGYH or TYH medium (270 or 170 μM, respectively). In BMG medium supplemented with l-leucine (10 mM), the concentration of this metabolite was strongly increased (2.8 mM versus 10 μM in the control). After culture in TGYH or TYH medium supplemented with l-( methyl -²H₃)leucine, 2-hydroxy-4-([²H₃]methyl)pentanoic acid was detected by GC-MS. This observation demonstrates that C. butyricum is able to convert l-leucine into the corresponding 2-hydroxy acid and opens a new aspect in the study of C. butyricum metabolism.