Jung Han Kim

Agmatine reduces infarct area in a mouse model of transient focal cerebral ischemia and protects cultured neurons from ischemia-like injury

Agmatine is a primary amine formed by the decarboxylation of L-arginine synthesized in mammalian brain. In this study, we investigated the neuroprotective effect of agmatine on ischemic and ischemia-like insults. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation (OGD), a model of ischemia-like injury, and treated with agmatine before or at the start of OGD, or upon reperfusion. Neuronal death was reduced when agmatine was present during OGD, and this protection was associated with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not inducible NOS (iNOS). Protection by agmatine was also studied at the in vivo level using a model of middle cerebral artery occlusion (MCAO) in mice. Mice were subjected to 2 h MCAO. Agmatine was administered either 30 min before ischemia, at the start of MCAO, at the start of reperfusion, or 2 or 5 h into reperfusion. Agmatine markedly reduced infarct area in all treatment groups except when treatment was delayed 5 h. The number of nNOS immunopositive cells was correlated with neuroprotection. Interestingly, immunoreactivity for iNOS was reduced only when agmatine was administered before and at the onset of MCAO. Our study suggests that agmatine may be a novel therapeutic strategy to reduce cerebral ischemic injury, and may act by inhibiting the detrimental effects of nNOS.

Rapid gas chromatographic profiling and screening of biologically active amines

An efficient method is described for the simultaneous determination of 57 amines including volatile aliphatic amines, nonvolatile polyamines and catecholamines present in aqueous samples. The method is based on two-phase isobutyloxycarbonylation (isoBOC) with a pH shift. In 1.0 M phosphate buffer at pH 7.5, phenolic hydroxyl groups were allowed to react with isobutyl chloroformate in the dichloromethane phase, and subsequently pH of the aqueous phase was increased to 12.0 for the reaction of basic amino functions. The resulting N(O)-isoBOC amines were recovered by solid-phase extraction using Chromosorb P in normal phase partition mode, with subsequent tert.- butyldimethylsilylation of the remaining hydroxyl groups for gas chromatographic analysis. Using this combined procedure, linear responses were obtained in the concentration range of 0.2-12 ppm, with correlation coefficients varying from 0.945 to 0.999 for most of the amines studied except for 5-methoxytryptamine (0.864). Temperature-programmed retention index (I) sets as measured on DB-5 and DB-17 dual-capillary columns of different polarity were characteristic of each amine and thus, useful in the screening for amines by computer I matching. When applied to saliva samples, the present method allowed rapid screening for each spiked amine and unspiked polyamines such as 1,3-diaminopropane, putrescine, cadaverine and spermidine.

Gas chromatographic amino acid profiling of wine samples for pattern recognition

A rapid gas chromatographic profiling and screening method is described for the determination of free amino acids in wine samples. The amino acids in alkaline aqueous wines were allowed to react with isobutyl chloroformate. The resulting N(O,S)-isobutyloxycarbonyl amino acids were extracted by solid-phase extraction using Chromosorb P as the adsorbent and diethyl ether as the eluent after acidification, and then converted into stable tert.-butyldimethylsilyl derivatives with subsequent analysis by dual-capillary column gas chromatography and gas chromatography-mass spectrometry. Seventeen free amino acids were identified from the four wine brands studied. When the GC profiles were simplified to the corresponding amino acid retention index spectra in bar graphical form, they presented characteristic patterns for each wine brand. Stepwise discriminant analysis performed on the amino acid profiles provided star symbols characteristic of the wine brands.

Enantioseparation of chiral amino acids as the N(O,S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography

The enantioseparation of 30 racemic amino acids in a single analysis is described for the determination of their absolute configurations. Two-phase extractive ethoxycarbonyl (EOC) reaction with ethyl chloroformate present in the dichloromethane phase was performed to recover amino acids from alkaline aqueous solutions. The resulting N(O,S)-EOC amino acids extracted into an organic solvent after acidification were reacted with a chiral alcohol such as (S)-(+)-3-methylbutan-2-ol, (S)-(+)-butan-2-ol and (S)-(+)-octan-2-ol for gas chromatographic analysis on achiral dual-capillary DB-5 and DB-17 columns of different polarities. Among the chiral reagents examined, (S)-(+)-3-methylbutan-2-ol provided the best diastereomeric structures in resolving all the racemic amino acids into their enantiomeric pairs with high resolution factors (1.2-8.0). Moreover, the temperature-programmed retention index (I) values measured on the two columns were characteristic of each enantiomer. Hence simple I matching with the reference values was useful in cross-checking for chemical identification and also chiral discrimination. When the present method was applied to a fermented dairy product (Yakult), D-alanine, D-aspartic acid, D-glutamic acid and D-proline were positively detected along with their respective L-forms in addition to glycine.

Configurational analysis of chiral acids as O-trifluoroacetylated (−)-menthyl esters by achiral dual-capillary column gas chromatography

The simultaneous enantiomeric separation of 30 racemic acids including 24 hydroxy acids in a single analysis is described for the determination of their absolute configurations. It involves the conversion of each enantiomer into diastereomeric O-trifluoroacetylated (-)-menthyl ester for the direct separation by gas chromatography on achiral dual-capillary columns of different polarities, with subsequent identification and chiral discrimination by retention index (I) library matching. Among the acids studied, the enantiomers of 27 acids were discriminatively resolved on both non-polar DB-5 and the intermediate-polar DB-17 columns with resolution factors in the range of 0.7-7.7 and separation factors in the range of 1.002-1.021. Enantiomers of 3-hydroxybutyric and alpha-methoxyphenylacetic acids were partially resolved on DB-5 (resolution factor of 0.9), but not resolved on DB-17, while the baseline resolution for 3-hydroxydecanoic acid and the minimal separation on the peak top (resolution factor less than 0.7) for 2-hydroxyglutaric acid were achieved on DB-17 but not on DB-5. The temperature-programmed I values measured on both columns were characteristic of each enantiomer and thus simple I matching with the reference values was useful in cross-checking for their chemical identification and the chiral discrimination as well. When applied to a clinical urine sample, the present method allowed positive identification of endogenous (S)-lactic acid and (S)-2-hydroxybutyric acid along with (R)-3-hydroxybutyric acid.

Enantiomeric separation and discrimination of 2-hydroxy acids as O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl esters by achiral dual-capillary column gas chromatography

An efficient method is described for the simultaneous enantiomeric separation of 18 different racemic 2-hydroxy acids for the determination of their absolute configurations. It involves the conversion of each enantiomer into a diastereomeric O-trifluoroacetylated (S)-(+)-3-methyl-2-butyl ester for the direct separation by achiral dual-capillary column gas chromatography with subsequent identification and determination of its chirality by retention index (I) library matching. The enantiomers of each acid were well separated with high resolution values (R > or = 1.4) on DB-5 and DB-17 columns of different polarity. When temperature-programmed I values of 2-hydroxy acid enantiomers as their diastereomeric derivatives were measured on both columns, the I values were characteristic of each enantiomer. Simple I matching with the reference values was thus useful in cross-checking each acid enantiomer for the identification and chiral discrimination. When applied to urine samples, the present method allowed positive identification of most of the spiked 2-hydroxy acids from normal urine and for endogenous (S)-lactic acid and (S)-2-hydroxybutyric acid from a clinical urine specimen.