Use of reversed-phase high-performance liquid chromatography for simultaneous determination of glutamine synthetase and glutamic acid decarboxylase in crude extracts

Glutamine and γ-aminobutyric acid (GABA), formed from glutamic acid in crude tissue extracts by glutamine synthetase and glutamic acid decarboxylase respectively, were separated by derivatization with dansyl chloride followed by reversed-phase high-performance liquid chromatography on an Altex Ultrasphere ODS-5 column. The mobile phase was a gradient of 100 mM potassium dihydrogen phosphate (pH 2.1) with 0–40% acetonitrile. The amounts of glutamine and GABA formed from glutamic acid were determined under different reaction conditions.     

A simple HPLC technique for accurate monitoring of mammalian cell metabolism

We have developed a simple and accurate isocratic HPLC method, without any prederivatisation, for the determination of glucose, lactate, glutamine, glutamate, pyrrolidone carboxylic acid and alanine in samples from mammalian cell cultures. The method has been successfully validated with enzyme analysis for each of the compounds. Quantification of pyrrolidone carboxylic acid makes the correction for glutamine decrease due to chemical decomposition very simple and accurate, and avoids some possibly erroneous calculations.     

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.     

Inhibition by glucose of the synthesis of proline from glutamic acid

While in the absence of glucose, proline is not a required amino acid, in the presence of glucose the growth of Micrococcus pyogenes var. aureus in amino acid medium is proportional to the concentration of proline when all other amino acids and growth factors are present in amounts adequate for optimal growth. The data presented here and the ideas prevailing in the literature indicate that glutamic acid is a precursor of proline. Glucose inhibits the conversion of glutamic acid into proline, which in turn causes failure of growth. Thus, 1 μg. and 10 μg. glucose/ ml. cause 50% and 100% inhibition, respectively, of the growth dependent on the synthesis of proline. One μg. proline antagonizes completely the inhibition in the presence of 5,000 μg. glucose/ml.One μg. glycerol, 100 μg. pyruvate, 250 μg. lactate, or 100 μg. α-glycerophosphate/ml., individually, cause from 25 to 50% inhibition of the growth dependent on the synthesis of proline from glutamic acid. Five thousand μg./ml. either of malic, succinic, fumaric, α-keto-glutaric, cis-aconitic acid, or dihydroxyacetone, or 500 μg. citric acid/ml. fails to cause inhibition.Pyrrolidone carboxylic acid was found to substitute for glutamic acid but not for proline in tests with M. pyogenes var. aureus. Also, seven proline-less mutant strains of Escherichia coli were unable to utilize pyrrolidone carboxylic acid in place of proline. No evidence was obtained to indicate that pyrrolidone carboxylic acid could serve as a direct precursor of proline.     

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5,6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HLPC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectrometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds. © 1996 Wiley-Liss, Inc.     

Hominoid evolution as judged by fibrinopeptide structures

Summary The fibrinopeptides A and B from gorilla, organgutan and siamang have been characterized, thereby completing a study of all six extant hominoids. The gorilla peptides were identical with the corresponding fibrinopeptides previously reported for human and chimpanzee. The orangutan peptide A was also identical with the human-chimpanzee-gorilla type A, but its fibrinopeptide B had two amino acid differences. The siamang A peptide differed from the others in one of its sixteen residues, but its peptide B was identical with the orangutan B. A cladogram based on the fibrinopeptide sequences of all six hominoids indicates that five amino acid replacements and one deletion can account for the evolution of present day sequences. It was also possible to deduce the amino acid sequence of the fibrinopeptides of the common ancestor of Old World monkeys and hominoids.Abbreviations Used PITC phenylisothiocyanate - DNS dimethylaminonaphthalene sulfonyl- - PCA pyrrolidone carboxylic acid - ASP aspartic acid - ASN asparagine - THR threonine - SER serine - GLU glutamic acid - GLN glutamine - GLY glycine - ALA alanine - VAL valine, ILE isoleucine - LEU leucine - PHE phenylalanine Supported by grants from the National Science Foundation (GB 7332) and the National Institutes of Health (GM-17, 702 and HE-12, 759).     

Isolation and characterization of rabbit gastrin

The heptadecapeptide form the rabbit gastrin was extracted from 16 rabbit antra and purified by a combination of DEAE Sephadex, C-18 SEP PAK cartridges, fast performance liquid chromatography (FPLC) and reverse phase high pressure liquid chromatography (HPLC) steps. After the HPLC purification, a sharp, single peak of gastrin-like immunoreactivity was detected that had the same absorption to immunoreactivity ratio as human gastrin. An amino terminal pyrrolidone carboxylic acid blocking group was removed by incubation with pyrrolidone carboxylic peptidase. The amino acid analysis, microsequence analysis and mass spectrometry all confirmed the structure of rabbit gastrin being pQGPWLQEEEEAYGWMDFamide. This sequence is identical to human gastrin-17 except for glutamine in position 6 which replaces glutamate in human gastrin. Both sulfated and unsulfated rabbit gastrin-17 were characterized by mass spectrometry.     

The single proline-glutamine substitution at position 5 enhances the potency of amyloid fibril formation of murine apo A-II

The primary structure of murine apolipoprotein A-II (apo A-II) has been determined. Apo A-II consists of a single polypeptide chain of 78 amino acid residues, of which the amino-terminus is pyrrolidone carboxylic acid. Except for residues 5 and 38, the amino acid sequence is identical to that of murine senile amyloid protein (ASSAM), which has a common antigenicity with apo A-II. Substitution of glutamine (ASSAM) for proline (apo A-II) at position 5 is distinct and may possibly be related to murine senile amyloid-ogenesis.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the chiral reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C₈ column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Nitrogen regulation of arginase in Neurospora crassa.

The final products of the arginine catabolism that can be utilized as a nitrogen source in Neurospora crassa are ammonium, glutamic acid, and glutamine. The effect of these compounds on arginase induction by arginine was studied. In wild-type strain 74-A, induction by arginine was almost completely repressed by glutamic acid plus ammonium, whereas ammonium or glutamic acid alone had only moderate effects. Arginine products of catabolism also repressed arginase induction. A mutant, ure-1, which lacks urease activity, hyperinduced its arginase with arginine as a nitrogen source. The addition of either ammonium or glutamine produced effects similar to those in the wild-type strain. The effect of ammonium on arginase induction is mediated through its conversion into glutamine. This was demonstrated in mutant am-1, which lacks L-glutamate dehydrogenase activity. In this mutant, the effect of glutamic acid was reduced, and, with ammonium, it was completely lost. The addition of glutamine or glutamic acid plus ammonium to this strain decreased by threefold the induction of arginase by arginine. Proline, a final product of arginine catabolism, competitively inhibited arginase activity. This effect and the repression of arginase by glutamine are examples of negative modulation of the first enzyme in a catabolic pathway by its final products.     

Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantitation of tBDMS derivatives using ammonia positive chemical ionization

The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.     

Pharmacokinetic studies with the lipid-regulating agent beclobrate: enantiospecific assay for beclobric acid using a new fluorescent chiral coupling component (S-FLOPA)

The major biotransformation pathway for the chiral lipid-regulating agent beclobrate is conversion to the corresponding carboxylic acid, which is then metabolized to the acyl glucuronide. An enantiospecific assay for biological material was developed that is based on chiral derivatization with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a new chiral coupling component for carboxylic acids derived from the 2-arylpropionic acid S-flunoxaprofen. Conversion of beclobric acid to the acyl chloride prior to coupling with the amine is also feasible. From plasma or urine beclobric acid was extracted into n-hexane/ethanol (9:1) at pH 4 after addition of sodium chloride. Clofibric acid was used as internal standard. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane containing trace amounts of pyridine (ambient temperature/2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:0.75, by vol) as mobile phase [flow rate, 2 ml/min; fluorescence detection, 305/355 nm; elution order of the derivatives, (-) before (+)]. Coefficients of variation were between 1.3 and 9.3% for both plasma and urine. Limit of quantification was 20-25 ng/ml for plasma based on a sample volume of 0.2 ml. Application of the assay in a pilot pharmacokinetic study showed significant differences between the kinetics of the two enantiomers. In plasma and urine, the concentrations of the dextrorotatory enantiomer exceeded those of the levorotatory enantiomer significantly.     

Hormone mediated changes of brain glutamic acid system in amino acid imbalance

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.     

Mass spectra of alpha-amino acid oxazolidinones

2-Bis-(chlorodifluoromethyl)-4-substituted-1,3-oxazolidin-5-ones, a new type of alpha-amino acid derivative for gas chromatographic separation, have been studied by low resolution mass spectrometry. These derivatives are obtained by reacting alpha-amino acids with dichlorotetrafluoroacetone. Their structure has been established or confirmed for most protein amino acids and several non-protein alpha-amino acids. The mechanisms responsible for the mass spectral pattern have been rationalized. An interesting feature of this derivatization procedure is that it distinguishes aspartic and glutamic acid from the respective amides. The structure of asparagine and glutamine derivatives has been established. A survey of oxazolidinone mass spectra has provided a list of diagnostically useful ions. Each amino acid can be identified by one or two of its most abundant fragments.     

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

Transport of Glutamate and Glutamine in the Light Leaf Spot Pathogen Pyrenopeziza brassicae

The kinetics of glutamic acid and glutamine uptake in the light leaf spot fungus, Pyrenopeziza brassicae , are biphasic. At low and high concentrations, glutamic acid and glutamine share a high-affinity and a low-affinity carrier, respectively, with K_ms of 4.0 and 4.4 μ m for uptake of glutamic acid and glutamine, respectively, by the high-affinity system, and K_m s of 580 and 560 μ m for uptake of glutamic acid and glutamine by the low-affinity system. The data suggest that glutamic acid and glutamine are taken up by a different system to that responsible for the uptake of ornithine, arginine, lysine and asparagine, and may represent system IV described in Neurospora crassa for the uptake of acidic amino acids.     

Determination of amino acids in different regions of the rat brain. Application to the acute effects of tetrahydrocannabinol (THC) and trimethyltin (TMT)

A modified HPLC method is described for the determination of amino acids [aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA)] in brain tissue utilizing precolumn derivatization with o-phthalaldehyde (OPA)-tert-butyl-thiol and electrochemical detection. A simple extraction procedure was employed and DL-homoserine used as internal standard. A neurotoxin previously shown to affect brain amino acids (trimethyltin, TMT) and a psychoactive compound hypothesized to act on these neurochemicals (delta-9-tetrahydrocannabinol, THC) were administered to adult male rats and amino acids were measured. Results revealed a gradient of distribution of most amino acids, with lowest levels posteriorly in the brain stem and increasing to the highest values in anterior cortical regions. TMT increased glutamine significantly in all brain regions examined, but increased glycine and decreased taurine only in the frontal cortex and hippocampus. No significant changes in any amino acid were found in hippocampus after THC treatment. The results establish the validity and usefulness of this HPLC method for detecting neurotoxicity-related changes in brain amino acid metabolism.     

Rapid and selective derivatizatin method for the nitrogen-sensitive detection of carboxylic acids in biological fluids prior to gas chromatographic analysis

A rapid and selective derivatization procedure is described for the pre-column labelling of carboxylic acids with a nitrogen-containing label. The carboxylic acid function is activated with 2-bromo-1-methylpyridinium iodide and the activated carboxylic acid function reacts with a primary or a secondary amine to yield an amide. With flurbiprofen as the test compound and dipropylamine as a label the acid was completely converted to the corresponding amide. The method was tested for several aliphatic, aromatic and for phenylacetic or phenylpropionic carboxylic acid dervatives, and was found to result in the complete derivatization of these compounds with a few exceptions only. The derivatization procedure is potentially useful for drug monitoring purposes, as is shown with the analysis of valproic acid and flurbiprofen in plasma.     

Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria

The enzyme complex F1-ATPase has been isolated from bovine heart mitochondria by gel filtration of the enzyme released by chloroform from sub-mitochondrial particles. The five individual subunits alpha, beta, gamma, delta and epsilon that comprise the complex have been purified from it, and their amino acid sequences determined almost entirely by direct protein sequence analysis. A single overlap in the gamma-subunit was obtained by DNA sequence analysis of a complementary DNA clone isolated from a bovine cDNA library using a mixture of 32 oligonucleotides as the hybridization probe. The alpha, beta, gamma, delta and epsilon subunits contain 509, 480, 272, 146 and 50 amino acids, respectively. Two half cystine residues are present in the alpha-subunit and one in each of the gamma- and epsilon-chains; they are absent from the beta- and delta-subunits. The stoichiometry of subunits in the complex is estimated to be alpha 3 beta 3 gamma 1 delta 1 epsilon 1 and the molecular weight of the complex is 371,135. Mild trypsinolysis of the F1-ATPase complex, which has little effect on the hydrolytic activity of the enzyme, releases peptides from the N-terminal regions of the alpha- and beta-chains only; the C-terminal regions are unaffected. Sequence analysis of the released peptides demonstrates that the N terminals of the alpha- and beta-chains are ragged. In 65% of alpha-chains, the terminus is pyrrolidone carboxylic acid; in the remainder this residue is absent and the chains commence at residue 2, i.e. lysine. In the beta-subunit a minority of chains (16%) have N-terminal glutamine, or its deamidation product, glutamic acid (6%), or the cyclized derivative, pyrrolidone carboxylic acid (5%). A further 28% commence at residue 2, alanine, and 45% at residue 3, serine. The delta-chains also are heterogeneous; in 50% of chains the N-terminal alanine residue is absent. The sequences of the alpha- and beta-chains show that they are weakly homologous, as they are in bacterial F1-ATPases. The sequence of the bovine delta-subunit of F1-ATPase shows that it is the counterpart of the bacterial epsilon-subunit. The bovine epsilon-subunit is not related to any known bacterial or chloroplast H+-ATPase subunit, nor to any other known sequence. The counterpart of the bacterial delta-subunit is bovine oligomycin sensitivity conferral protein, which helps to bind F1 to the inner mitochondrial membrane.(ABSTRACT TRUNCATED AT 400 WORDS)