Abstract
Amino acids in hydrolysates of some pure proteins have recently been gas chromatographed by Gehrke et al., using n-butyl N-trifluoroacetyl derivatives. We have modified this method at the step of esterification and applied it to analysis of amino acids inplasma and needle biopsy specimen of the liver, after extracting amino acids from biologicalsample by passing through ion-exchange columns.
The procedure is shown in Figure 1: To omit methylesterification by stirring at 100°C in a sealed tube, in which amino acids are directly n-butylesterifid.
The column (1.0-1.5m×4mm i. d., glass) was packed with NPGSb or EGA for determinating amino acids other than cystine, arginine and histidine. An OV-17 column was used for analysis of the last three amino acids. The detector was hydrogen flame ionization. The condition for gas chromatography was same as Gehrke's. Quantative analysis was made by peak area by using ε-amino caproic acid as an internal standard.
Results: 1) Twenty amino acids in plasma (Figure 2) and liver were identified with satisfactory recovery. 2) Four hours were found to be sufficient for the period of esterification (Figure 3), and the yields of derivatives were more than 90 per cent of all amino acids (TABLE II). 3) NPGSb showed an excellent capacity to separate most of amino acids except glycine and isoleucine, and EGA did so except methionine and aspartic acid (EGA). 4) The concentration of most amino acids in human liver was less than100 μmoles per gram protein nitrogen and varied markedly in the injured liver. The amino acid concentration in the regenarating rat liver was significantly increased.
Because of its rapidity, accuracy and sensitivity, this method offers advantages for the determination of amino acids in minimum amount of plasma and liver tissue.
