Quantitative determination of amino acids in tissue cells by high performance liquid chromatography

Amino acids derivatized with o-phthaldialdehyde were separated by high performance liquid chromatography on a reverse phase C8 column with a ternary gradient. Multilevel calibration permits the analytical quantitation within the range of 50 to 3500 pMol of the individual amino acids in one run. The reproducibility of the analysis in consecutive runs is ± 3%. HeLa cells grown in suspension cultures were harvested by either centrifugation in the cold or by centrifugation through dibutylphthalate. Amino acids were extracted with methanol-water. Cells not exposed to aqueous media prior to extraction show an up to 3 fold higher level of amino acids in the intracellular pool.     

Quantitative determination of free intracellular alpha-keto acids in neutrophils

For the first time, a procedure is described for the quantitative analysis of free alpha-keto acid content in human neutrophils (PMNs) relative to single cell number by reversed-phase fluorescence high-performance liquid chromatography. The procedure is minimally invasive and is unsurpassed in the quality of PMN separation, ease of sample preparation as well as sample stability. This method can satisfy the rigorous demands for an ultra-sensitive, comprehensive and rapid intracellular alpha-keto acid analysis in particularly for the surveillance of severe diseases as well as cellular or organ dysfunction.     

Gas chromatographic determination of free amino acids and amino acids attached to transfer RNA in biological samples

A method was developed to analyze quantitatively free amino acids and amino acids attached to transfer RNA (tRNA) in tissue samples by gas chromatography. Free amino acids were purified by ion-exchange chromatography after deproteinization. Total cellular aminoacyl-tRNA was extracted from rabbit reticulocytes and liver by a modified phenol extraction method under conditions which were designed to prevent deacylation of the attached amino acids. After deacylation and separation from tRNA by pressure ultrafiltration, eighteen amino acids were determined by gas chromatography as their N-heptafluorobutyryl isobutyl derivatives.     

Comparative study of various extraction methods for the quantitative determination of free amino acids from brain tissue

Seven published extraction methods for the quantitative determination of the amino acids of biological fluids and tissues were applied to brain tissue and the experimental data analyzed statistically.     

Chemiluminescence determination of hydroperoxides following radiolysis and photolysis of free amino acids

Hydroperoxides were determined in selected amino acids using three free radical generating systems by a sensitive (50 pmol limit of detection) and specific high performance liquid chromatography (HPLC)/chemiluminescence method. UVB and gamma radiation produced significant hydroperoxide formation, particularly in the aromatic amino acids tyrosine and tryptophan. Hydroperoxide yield was found to be dependent on both amino acid and irradiation source. Generation of hydrogen peroxide as a by-product of irradiation caused interference with chemiluminescence detection demonstrating the need for catalase addition. Hydroperoxides were not detectable following metal-catalysed H₂O₂ breakdown. We suggest that metal ions could interfere with the detection of hydroperoxides by causing preferential decomposition.     

Dynamics of free amino acids in rat brain tissue during hypothermia

A comparative study is conducted for the effect of one-, three- and six-hour artificial deep (20-19 degrees C) hypothermia on the content of free amino acids in the blood serum, tissue, nuclei and mitochondria of the rat brain. It is found out that the content is the highest in the blood serum after a three-hour cooling. In the brain tissue the amount of amino acids lowers, especially under conditions of a six-hour hypothermia. In nuclei a three-hour effect of hypothermia decreases sharply the content of free amino acids and the six-hour one increases the amount of most of them. Under hypothermia the content of nearly all amino acids in the brain mitochondria is higher than in the intact animals.