Enantioselective multidimensional gas chromatography–mass spectrometry in the analysis of urinary organic acids

Enantioselective multidimensional gas chromatography–mass spectrometry is a valuable tool for the enantioselective analysis of compounds from complex matrices. Samples are separated initially on a precolumn and selected substances then transferred on-line to a main-column coated with suitable chiral stationary phase for enantioselective analysis with subsequent mass selective detection. In this paper the method is used as an adjunct to urinary organic acid analysis to provide information in patients with suspected inborn errors of metabolism. Lactic acid, α-hydroxyisocaproic acid, 3-phenyllactic acid and 3-(4-hydroxyphenyl)-lactic acid are separated in a single run. In addition, the enantioselective analysis of pyroglutamic acid is presented. d-Enantiomers of amino acids and α-hydroxycarboxylic acids derived from amino acids, point to a bacterial origin whereas the l-enantiomer is predominantly of endogenous origin. Therefore the enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes.     

Deuterium-labeled Phaseic Acid and Dihydrophaseic Acids for Internal Standards

The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8′-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7′-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8′-hydroxyabscisic acid. 7′-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7′-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.     

Deuterium-labeled phaseic acid and dihydrophaseic acids for internal standards

The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8'-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7'-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8'-hydroxyabscisic acid. 7'-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7'-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.     

A new approach for sample preparation of protein hydrolyzates for amino acid analysis

Sample preparations of protein hydrolyzates for amino acid analysis by ion-exchange chromatography has been accomplished without the removal of hydrochloric acid which was used for the hydrolysis. The technique involves partial neutralization of the available hydrochloric acid after hydrolysis with a solution which neutralizes and dilutes the sample hydrolyzate at the same time. The resulting sample solution which is employed for amino acid analysis produces an amino acid chromatogram having the same elution times and resolution as compared to a mixture of amino acids prepared in pH 2.2 sodium citrate buffer. Experimental data is also presented which shows that the amount of available hydrochloric acid in the final sample solution employed for amino acid analysis can affect both the resolution and elution time of many of the amino acids found in a protein hydrolyzate.     

The simultaneous analysis of clofibric acid and probenecid and the direct analysis of clofibric acid glucuronide by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic method for the simultaneous quantitative analysis of clofibric acid and probenecid in plasma and urine and for the direct analysis of clofibric acid glucuronide in plasma is described. Both methods involve direct injection of deproteinised body fluids. Concentrations of as low as 10 mg/l of clofibric acid and probenecid and 1.5 mg/l of clofibric acid glucuronide can be measured by the analysis. The coefficient of variance for these methods ranges from 1–7%.     

ANT1, an aromatic and neutral amino acid transporter in Arabidopsis

A new amino acid transporter was identified from the Arabidopsis expressed sequence tag cDNAs by expressing the cDNA in a yeast amino acid transport mutant. Transport analysis of the expressed protein in yeast showed that it transports aromatic and neutral amino acids, as well as arginine. This transporter (ANT1, aromatic and neutral transporter) also transports indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The cDNA is 1.6 kb in length with an open reading frame that codes for a protein with 432 amino acids and a calculated molecular mass of 50 kD. Hydropathy analysis showed ANT1 is an integral membrane protein with 11 putative membrane-spanning domains. Southern analysis and a BLAST search of the Arabidopsis genome database suggests that ANT1 is part of a small gene family containing at least five members. Phylogenetic comparisons with other known amino acid transporters in plants suggests that ANT1 represents a new class of amino acid transporter. RNA gel-blot analysis showed that this transporter is expressed in all organs with highest abundance in flowers and cauline leaves.     

The poly-alpha- and -beta-1,4-glucuronic acid moiety of teichuronopeptide from the cell wall of the alkalophilic Bacillus strain C-125.

Teichuronopeptide is a structural component of the cell wall of alkalophilic Bacillus strain C-125 and is a complex composed of polyglutamate and polyglucuronate. A structural analysis of the polyglucuronic acid moiety was carried out. Periodate oxidation and Smith degradation of the moiety, and enzymic analysis after reduction of glucuronic acid to glucose, revealed that glucuronic acid bound together with alternately alpha- and beta-1,4-linkages.     

76株薄壳山核桃实生单株的果实品质差异及综合评价

对南京地区种植的76株薄壳山核桃〔Carya illinoinensis(Wangenh.)K.Koch〕实生单株的8项果实性状和种仁中5种脂肪酸含量的差异以及各指标的相关性进行分析,并筛选出单项性状优良的单株;在此基础上,结合主成分分析结果,对供试76株单株的果实品质进行综合评价.结果显示:8项果实性状(包括坚果质量、种仁鲜质量、坚果壳厚度、坚果纵径、坚果横径、果形指数、出仁率和含油率)以及种仁中5种脂肪酸(包括棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸)的含量均有较大变异,变异系数为663%~2899%,其中,出仁率的变异系数最小,亚麻酸含量的变异系数最大,8项果实性状的变异系数总体上小于5种脂肪酸含量的变异系数.部分果实性状间极显著或显著正相关,少数果实性状间极显著或显著负相关,而5种脂肪酸含量间均极显著正相关,但脂肪酸含量与果实性状间总体上无显著相关性.主成分分析结果表明:在8项果实性状中,与坚果大小有关的性状较为重要;而5种脂肪酸含量均同等重要.对果实性状和脂肪酸含量的主成分分析结果显示:第1主成分的决定指标包括含油率以及棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸的含量,第2主成分的决定指标包括坚果质量、种仁鲜质量、坚果壳厚度和坚果横径,说明薄壳山核桃果实评价的首要指标为种仁中脂肪酸含量,其次为果实大小.在利用坚果质量、种仁鲜质量、坚果壳厚度、出仁率、含油率、果形指数、油酸含量和不饱和脂肪酸含量8个单项指标优选单株的基础上,结合主成分分析结果,71号单株的综合得分较高,其种仁鲜质量、含油率以及油酸和不饱和脂肪酸的含量均较高,可作为优良品种选育的首选候选单株.根据上述研究结果,建议在薄壳山核桃不同育种目标的优良品种选育工作中,将主成分分析法作为单项性状筛选法的有效补充方法.     

Circadian Rhythm of Gastric Acid Secretion in Active Duodenal Ulcer: Chronobiological Statistical Characteristics and Comparison of Acid Secretory and Plasma Gastrin Patterns with Healthy Subjects and Post-Vagotomy and Pyloroplasty Patients

Twenty-one male patients with active duodenal ulcer underwent hourly 24-hr gastric acid collections under controlled, calorically deprived conditions. The 24-hr hourly acid secretory output for the group displayed a statistically significant (p < 0.001) rhythm, with peak rates occurring during the evening hours and low rates during the early morning hours, by population-mean cosinor statistical analysis. Population-mean cosinor analysis also verified the occurrence of a significant (p=0.034) circadian rhythm in unstimulated acid secretion in a group (N=14) of healthy male subjects similarly studied and reported previously. In contrast, population-mean cosinor analysis confirmed the absence of any detectable circadian rhythm in unstimulated acid secretion in a group (N=17) of post-vagotomy and pyloroplasty patients studied 2-11 years after surgery. Population-mean cosinor analysis of 4-hr plasma gastrin determinations, obtained in all groups during the 24-hr gastric acid collection, revealed an absence of any detectable circadian rhythm in plasma gastrin. This latter finding is compatible with the interpretation that the circadian rhythm of unstimulated gastric acid secretion, observed in the clinically healthy and active ulcer groups, is unrelated to changes in plasma gastrin levels. The employment of quantitative chronobiological inferential statistical techniques is important to the analysis of any time-dependent measurement in gastrointestinal function, of which gastric acidity is one example.     

Circadian rhythm of gastric acid secretion in active duodenal ulcer: chronobiological statistical characteristics and comparison of acid secretory and plasma gastrin patterns with healthy subjects and postvagotomy and pyloroplasty patients

Twenty-one male patients with active duodenal ulcer underwent hourly 24-hr gastric acid collections under controlled, calorically deprived conditions. The 24-hr hourly acid secretory output for the group displayed a statistically significant (p less than 0.001) rhythm, with peak rates occurring during the evening hours and low rates during the early morning hours, by population-mean cosinor statistical analysis. Population-mean cosinor analysis also verified the occurrence of a significant (p = 0.034) circadian rhythm in unstimulated acid secretion in a group (N = 14) of healthy male subjects similarly studied and reported previously. In contrast, population-mean cosinor analysis confirmed the absence of any detectable circadian rhythm in unstimulated acid secretion in a group (N = 17) of postvagotomy and pyloroplasty patients studied 2-11 years after surgery. Population-mean cosinor analysis of 4-hr plasma gastrin determinations, obtained in all groups during the 24-hr gastric acid collection, revealed an absence of any detectable circadian rhythm in plasma gastrin. This latter finding is compatible with the interpretation that the circadian rhythm of unstimulated gastric acid secretion, observed in the clinically healthy and active ulcer groups, is unrelated to changes in plasma gastrin levels. The employment of quantitative chronobiological inferential statistical techniques is important to the analysis of any time-dependent measurement in gastrointestinal function, of which gastric acidity is one example.     

Methylmalonic acid,a biochemical hallmark of methylmalonic acidurias but no inhibitor of mitochondrial respiratory chain

Methylmalonic acidurias are biochemically characterized by an accumulation of methylmalonic acid and alternative metabolites. An impairment of energy metabolism plays a key role in the pathophysiology of this disease, resulting in neurodegeneration of the basal ganglia and renal failure. It has become the subject of intense debates whether methylmalonic acid is the major toxin, inhibiting respiratory chain complex II. To elucidate whether methylmalonic acid is a respiratory chain inhibitor, we used spectrophotometric analysis of complex II activity in submitochondrial particles from bovine heart, radiometric analysis of 14C-labeled substrates (pyruvate, malate, succinate), and analysis of ATP production in muscle from mice. Methylmalonic acid revealed no direct effects on the respiratory chain function, i.e. on single electron transferring complexes I-IV, ATPase, and mitochondrial transporters. However, we identified a variety of variables that must be carefully controlled to avoid an artificial inhibition of complex II activity. In summary, the study verifies our hypothesis that methylmalonic acid is not the major toxic metabolite in methylmalonic acidurias. Inhibition of respiratory chain and tricarboxylic acid cycle is most likely induced by synergistically acting alternative metabolites, in particular 2-methylcitric acid, malonic acid, and propionyl-CoA.     

Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair with a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic plasma deproteinized by trichloroacetic acid, and after washing with water, they are eluted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to high-performance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hplc systems, fluorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis.     

Analysis of strongly acidic amino acids by the conventional amino acid analyzer: application to determination of protein-bound cysteine and glutathione

A rapid analysis method of strongly acidic amino acids and related compounds by a simple modification of an existing amino acid analyzer is presented. In this method, an anion-exchanger column (2.6 X 150 mm) packed with Hitachi 3013-N resin was developed with 0.2 M citric acid. Complete separation of phosphothreonine, phosphoserine, phosphotyrosine, cysteic acid, homocysteic acid, and glutathionesulfonic acid was achieved within 35 min, with no regeneration of the column being required. Tyrosine-O-sulfate was analyzed by the same column using 2 M sodium acetate buffer, pH 5.5. Performic acid oxidation of a variety of proteins and direct analysis of the products by this system successfully detected cysteine, homocysteine, and/or glutathione bound to proteins through disulfide bonds. This suggest the potential use of the method for analysis of the states of protein thiol groups, especially those of clinically significant mutant proteins where mutation of arginine to cysteine is rather frequently recognized.     

FIA for on-line monitoring of important lactic acid fermentation variables

An automated system with semi on-line monitoring of glucose, lactic acid, protein, and optical density during lactic acid fermentations, is set up to study the dynamics of lactic acid bacteria. The analyzers for glucose, lactic acid, and protein are based on flow injection analysis (FIA). The system consists of a laboratory fermentor with a continuous withdrawal system and an analysis system where glucose, lactic acid, and protein concentration are measured together with the optical density of the fermentor sample. The system is controlled by a personal computer.The system response is fast, and it yields a large number of reliable and precise analytical data, whoch is of great importance for mathematical model building. Some premliminary results are shown.     

Internally standardized amino acid analysis for determining peptide/carrier protein coupling ratio

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.     

High-pressure liquid chromatography of neurotoxic phenylphosphonothioate esters and related compounds

It has been suggested that perfluorooctanoic acid occurs in human plasma; however, no method of analysis for this compound in biological samples has been published to date. A method is presented for the analysis of perfluorooctanoic acid in plasma, urine, and liver tissue based on conversion of the acid to its methyl ester followed by separation and quantitation by gas chromatography.     

Higher reactivity of apolipoprotein B-100 and alpha-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction

Radical reaction of low-density lipoprotein (LDL) is a key step in atherogenesis and causes both a decrease in the sialic acid moiety and modification of apolipoprotein B-100 (apoB). Although apoB modification (cross-link and fragmentation) increases in atherosclerosis, the change in apoB-bound sialic acid in atherosclerosis is controversial. To elucidate the physiological implications of desialylation of LDL by radical reaction, the reactivity of sialic acid of LDL was compared with that of apoB, which underwent facile fragmentation in radical reactions. ApoB was determined by immunoblot analysis with anti-apoB antiserum, and the sialic acid moiety was measured by blot analysis with a biotin-bound lectin [biotin-SSA from Japanese elderberry (Sambucus sieboldiana)] specific to sialic acid. When human LDL was oxidized with Cu(2+) at 37 degrees C, apoB and apoB-attached sialic acid decreased simultaneously. Comparison of the staining bands with anti-apoB and with biotin-SSA shows that sialic acid moieties still remain on fragmented apoB proteins, indicating that the decrease in sialic acid is much slower than that of apoB fragmentation. In addition, human plasma was oxidized with 400 microM of Cu(2+) at 37 degrees C. Similar analysis indicates that the decrease in sialic acid attached to apoB also results from the fragmentation of apoB. This study indicates that the fragmentation of apoB proceeds at a much faster rate than the decrease in sialic acid content when a free radical reaction is induced in isolated LDL as well as in plasma LDL exposed to Cu(2+)-induced oxidative stress. On the basis of these results, the modification of apoB is much more sensitive than the decrease in sialic acid as an indicator of oxidative stress.     

Identification of iodotyrosines and some iodothyronines as dimethylaminoazobenzenethiohydantoin derivatives. Application to the sequencing of iodoamino acid containing peptides

A method has been developed for the gas chromatographic analysis of lipoic acid in biological samples. The lipoic acid is released from the samples by acid hydrolysis in the presence of the internal standards 1,2-dithiolane-3-butyric acid and/or 1,2-dithiolane-3-caproic acid. After hydrolysis, the lipoic acid and the internal standards are extracted from the hydrolysate and converted into the S,S-dibenzylmethyl esters. Gas chromatographic analysis of this mixture completely separates each of the homolog derivatives from the lipoic acid derivative and allows for the quantitation of the lipoic acid in the sample. Samples containing more than ～50 ng of lipoic acid can be easily assayed. Results are presented that show that the lipoic acid content of Escherichia coli depends on the carbon source used for its growth.     

Determination of the Cysteine and Cystine Content of Proteins by Amino Acid Analysis: Application to the Characterization of Disulfide-Coupled Folding Intermediates

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.