Determination of erythrocyte amino acids by gas chromatography

Erythrocyte amino acid levels were determined, by gas chromatography, in a group of 34 normal human adults. No significant sex or age correlations were noted.A method for the quantitative gas chromatographic analysis of free amino acids in erythrocytes is described. Following hemolysis and deproteinization the amino acids were isolated on a cation-exchange resin. Glutathione was removed from the amino acid mixture by adsorption on an anion-exchange resin. Following conversion to their N-acetyl-n-propyl esters, 19 amino acids were separated and quantitated by gas chromatography on a single column in 18 min. Typical reproducibility data indicate that a coefficient of variation of 2–5% is attainable.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

A modified technique for the quantitative analysis of amino acids by gas chromatography using heptafluorobutyric n-propyl derivatives.

The quantitative analysis of amino adids by gas chromatography as their heptafluorobutyric n-propyl derivatives has been examined and a modified technique developed which has been found to give improved results. A single propylation step has been adopted using 6 m HCl n-propanol at 150°C for 3.5 min, followed by a 12-min acylation step at the same temperature. Variability of response for methionine and histidine has been overcome in most cases by the introduction of an antioxidant at the acylation stage. An improved column performance on the previously used nonpolar OV silicone phase has been obtained by using a mildly polar OV phase.     

Analysis of the amino acid 3-methylhistidine by gas-liquid chromatography

A method for the quantitative separation of 3-methylhistidine from other amino acids, by gas-liquid chromatography, has been developed. This method gives complete resolution of the N-heptafluorobutyryl isobutyl esters of 20 amino acids with the use of a single column packed with 3% SE-30 on $\text{100}\text{120}$ mesh Gas Chrom Q. Using this method the 3-methylhistidine content of urine and meat has been determined.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that some straight-chained α-amino acids are converted by yeast to the alcohols with correspondingly longer carbon chains in the alcoholic fermentation contrary to F. Ehrlich’s scheme, i.e., isobutyl alcohol from alanine and active amyl alcohol from α-amino-n-butyric acid or threonine.

In this report, we confirmed this fact in the alcoholic fermentation of many aliphatic amino acids by 2 yeast strains using gas chromatography. Moreover, n-propyl alcohol was proved to come from α-amino-n-butyric acid or threonine. Small quantities of n-propyl, isobutyl, active amyl and isoamyl alcohols were found in all the fermented solutions. There was some difference in the composition of higher alcohols of the alcoholic solutions fermented by different yeasts.     

Mass-spectrometric evidence for quinonoid-lysine coupling products in cigar protein

On subjecting hydrolysates of hydrogenated cigar protein to derivatization and preparative GLC, two fractions were obtained which had volatilities and mass spectra consistent with an origin from oxidative-condensation products of chlorogenic acid with lysine residues. The mass-spectral evidence was chiefly from high-resolution mass spectrometry of the heaviest fragment ions; there was also high-resolution comparison of the lighter fragment ions with those from relevant model compounds. New compounds prepared were the hydantoin from 6-(1-trans-octahydroquinol-2-onyl)-DL-norleucine and the heptafluorobutyrylated n-propyl esters of four other less-common amino acids. Details of the mass spectra are provided in a Supplementary Publication 4 or have been sent to the Mass Spectrometry Data Centre.     

Quantitative analysis of oxepinac in human plasma,urine and saliva by gas chromatography—mass fragmentography

A sensitive and specific method is described for the quantitative analysis of 6,11-dihydro-11-oxo-dibenz[b,e]oxepin-3-acetic acid (oxepinac) in human plasma, urine and saliva. Oxepinac and internal standard are extracted from acidified plasma, urine or saliva, converted to the corresponding n-propyl esters and analysed by gas chromatography—mass fragmentography using selected ion monitoring. The method is accurate and precise over the range 100 μg/ml to 1.0 ng/ml. The method has been applied to the analysis of plasma, urine and saliva from healthy volunteers receiving therapeutic doses of oxepinac.     

Antimalarial activity of 10-alkyl/aryl esters and -aminoethylethers of artemisinin

A series of n-alkyl/aryl esters were synthesized and their in vitro antiplasmodial activity was measured alongside that of previously synthesized aminoethylethers of artemisinin ozonides against various strains of Plasmodium falciparum. The cytotoxicity against human cell lines was also assessed. The esters were synthesized in a one-step reaction by derivatization on carbon C-10 of dihydroartemisinin. Both classes were active against both the 3D7 and K1 strains of P. falciparum, with all compounds being significantly more potent than artemether against both strains. The majority of compounds possessed potency either comparable or more than artesunate with a high degree of selectivity towards the parasitic cells. The 10α-n-propyl 11 and 10α-benzyl 18 esters were the most potent of all synthesized ozonides, possessing a moderate (∼3-fold) and significant (22- and 12-fold, respectively) potency increases against the 3D7 and K1 strains, respectively, in comparison with artesunate.     

Sterols and fatty acids from three species of mangrove

The hydrolysis of steryl esters on thin-layer chromatographic plates by porcine pancreatic lipase is described. The sterols and fatty acids produced were separated on the same plate, recovered, and analysed by gas-liquid chromatography for their compositions. Synthetic cholesteryl esters containing various saturated and unsaturated fatty acids and synthetic steryl oleates with various sterols were lipolysed along with steryl esters of Acanthus ilicifolius, Bruguiera gymnorhiza and Rhizophora mucronata mangrove leaves. The major sterol was sitosterol which was accompanied by cholesterol, campesterol, stigmasterol and 28-isofucosterol. In addition, stigmast-7-en-3β-ol was present in R. mucronata leaves. The component fatty acids found in all three species were 16:0, 18:0, 18:1, 18:2 and 18:3. The relative proportions of the sterols and fatty acids were significantly different from the chemotaxonomic standpoint. The results obtained by carrying out plate lipolysis for 45 min at 40° compared well with those produced by conventional chemical hydrolysis.     

Formation of volatile alcohols and esters from aldehydes in strawberries

The aldehydes acetaldehyde, propanal, 2-methylpropanal, butanal, 3-methylbutanal, pentanal and hexanal, were reduced to their corresponding alcohols by incubation with strawberry fruit. The alcohols formed were then converted to their acetate, propionate, n-butyrate, isovalerate and n-caproate esters during the incubation with strawberry fruit. Simultaneous reaction of isobutyric acid, n-valeric acid and isocaproic acid with aldehyde and strawberry fruit resulted in the formation of esters of these acids. In all seven alcohols and 54 esters were produced by means of incubation of aldehydes and volatile fatty acids with strawberry fruit.     

The interaction of amino acids with o-phthaldialdehyde: A kinetic study and spectrophotometric assay of the reaction product

A detailed study has been made of the kinetics of interaction between amino acids and esters of amino acids and o-phthaldialdehyde in the presence of mercaptoethanol. The reaction products have been characterized. A spectrophotometric method for quantitative analysis of all amino acids, except proline and hydroxyproline, has been developed. The possibility of determination of amino acid esters in mixtures containing free amino acids has been demonstrated. It is noted that determination of glycine and histidine with the help of o-phthaldialdehyde has certain specificities associated with faster, compared to other amino acids, degradation of their derivatives. Optimal conditions for quantitative analysis of amino acids in solutions of higher than 10^?5m concentration are recommended. The reproducibility of the determination was ±2%.     

Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination

Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-l-cysteine, d-phenylglycine and S-aminoethyl-l-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.     

Plasma-free amino acid profiling of cervical cancer and cervical intraepithelial neoplasia patients and its application for early detection

In this study, plasma-free amino acid profiles were used to investigate pre-cancerous cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) metabolic signatures in plasma. Additionally, the diagnostic potential of these profiles was assessed, as well as their ability to provide novel insight into CSCC metabolism and systemic effects. Plasma samples from CIN patients (n = 26), CSCC patients (n = 22), and a control healthy group (n = 35) were analyzed by high-performance liquid chromatography, and their spectral profiles were subjected to the t test for statistical significance. Potential metabolic biomarkers were identified using database comparisons that examine the significance of metabolites. Compared with healthy controls, patients with CIN and CSCC demonstrated lower levels of plasma amino acids; plasma levels of arginine and threonine were increased in CIN patients but were decreased in cervical cancer patients. Additionally, the levels of a larger group of amino acids (aspartate, glutamate, asparagine, serine, glycine, histidine, taurine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine) were gradually reduced from CIN to invasive cancer. These findings suggest that plasma-free amino acid profiling has great potential for improving cancer screening and diagnosis and for understanding disease pathogenesis. Plasma-free amino acid profiles may have the potential be used to determine cancer diagnoses in the early stage from a single blood sample and may enhance our understanding of its mechanisms.     

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2–50 μg of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88–108%. Analytical results for serum amino acids from normal subjects are presented.     

Long chain esters of Virola species

The esters of n-fatty acids and ω-hydroxy n-fatty acids of β-sitosterol, D-glucose and ferulic acid (trans and cis) as well as β-sitosterol, fatty acids and β-sitosteryl-β-D-glucoside were isolated from three Virola species and identified by optical data and chemical reactions. A novel series of acidic esters derived from C₂₂C₂₉ ω-hydroxy fatty acids and cis- and trans-ferulic acid is reported for the first time. These compounds also occurred as the corresponding diester 1-monoglycerides whereas the ω-hydroxy acids themselves were also present as the corresponding glucosyl esters.     

High-performance liquid chromatography of side-chain-protected amino acid phenylthiohydantoins

Eighteen side-chain-protected amino acids, routinely employed in solid-phase peptide synthesis, were derivatized to their phenylthiohydantoins (PTH) by one cycle of the Edman degradation. All of these side-chain-protected PTH amino acids elute, with almost-baseline resolution, in less than 18 min by high-performance liquid chromatography, utilizing a biphasic gradient of acetonitrile in 0.01 n sodium acetate, pH 4.5, or a linear gradient of 0 to 100% acetonitrile with the exception of the coelution of a O-benzyl-threonine and carbobenzoxy-lysine phenylthiohydantoin amino acids. The derivatized amino acids were subjected to reverse-phase chromatography on a Zorbax ODS column and monitored at 254 nm. None of the PTH amino acids coelute with side-chain-protected PTH amino acid counterparts, although PTH-tosyl-histidine undergoes deprotection to PTH-histidine in the Edman degradation. A protected decapeptide attached to a chloromethylated polystyrene resin was degraded on a solid-phase sequencer in 16 h. The PTH amino acids resulting from the automated Edman degradation on the decapeptide were fully resolved and quantified in less than 3 h demonstrating that automated high-performance liquid chromatography can keep pace with both the automated sequencer and synthesizer which requires minimally 2–3 h for attachment of each residue to the growing peptide chain.     

Amino-acid biosynthesis in the cotyledons of Sinapis alba L. in darkness and far-red light studied by deuterium labelling and mass spectrometry

The incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba L. was studied by gas chromatography-mass spectrometry and was similar, both qualitatively and quantitatively, after incubation of the seedlings in darkness or far-red light. The results support studies which show that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is synthesised de novo, rather than activated, in response to far-red light.Abbreviations GC-MS Gas chromatography-mass spectrometry - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - HFB n-propyl heptafluorobutyryl n-propyl     

Glycosyl esters of amino acids : Part IV. Synthesis of 1-O-(acylaminoacyl)-2,3,4,6-tetra-O-benzyl-D-glucopyranoses by the imidazole-promoted active ester and dicyclohexylcarbodiimide methods

1-O-(Acylaminoacyl)-2,3,4,6-tetra-O-benzyl-D-glucopyranoses were prepared in high yields by two routes involving direct participation of imidazole in the ester linkage formation; namely, (a) the accelerated active-ester method, and (b) the imidazole-promoted dicyclohexylcarbodiimide condensation. The compounds were synthesized from 2,3,4,6-tetra-O-benzyl-α-D-glucopyranose and pentachlorophenyl esters of optically active benzyloxycarbonyl- and tert-butyloxycarbonyl-amino acids in method (a) or benzyloxycarbonyl- and acetyl-amino acids in method (b). By both methods, anomeric mixtures of D-glucosyl esters were obtained; they were resolved by column chromatography and the α and β anomers were fully characterized. The retention of configuration of the amino acid moiety was determined from optical rotations of acylamino acid methyl esters formed from D-glucosyl esters with methanolic sodium methoxide. With benzyloxycarbonyl and tert-butyloxycarbonyl protecting groups, a high degree of retention of optical activity was established in both methods-method (a) being slightly superior.     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

The Formation of Higher Alcohols in the Fermentation of Amino Acids by Yeast

We have found that in the alcoholic fermentation of amino acids by yeast isobutyl alcohol is produced from alanine and n-propyl and active amyl alcohols are formed from α-amino-n-butyric acid or threonine contrary to the F. Ehrlich’s scheme. These results suggest the close relationship among the formation of these higher alcohols and biosynthesis of valine from alanine and biosynthesis of isoleucine from α-amino-n-butyric acid or threonine.

In this report, we studied the formation of n-propyl alcohol and active amyl alcohol from α-amino-n-butyric acid using washed yeast cells.