Fundamental Studies for the Clinical Application of High Voltage Paper Electrophoresis
On the quantitative analysis of the amino acids
1959 Volume 6 Issue 1 Pages 9-24
Details
1959 Volume 6 Issue 1 Pages 9-24
1) In order to use the high-voltage paper electrophoresis of amino acids as a quantitative method, several fundamental experiments were carried out by the pure crystal of amino acid, the hydrolysate of ovalbumin and human serum.
2) Electrophoresis were performed at a potential gradient of 100 to 150v/cm and in acetic acidpyridine buffer (pH3.5).
3) Amino acid spectra which were colorized by ninhydrin reagent were altered to a series of peaks by densitometer (Filter 550mμ).
4) The relative migrativities of various amino acids, which were evaluated with respect to the position of glutamic acid, showed a good reproducibility and were constant independently of the periods of electrophoresis (12 to 60min).
5) The standard curves of 15 kinds of amino acids were obtained. As a result, in a region of 3 to 5γ of applying amount of amino acids, the straight proportionalities were found between the color yields and applying amount of various amino acids.
6) The ratio of color yields of various amino acids against that of glutamic acid were evaluated from the slopes of standard curves.
7) The quantitative determinations of amino acids were thus possible by correcting the peak areas obtained by densitometry by using these ratios of color yields of amino acids. This was verified by the electrophoretic analysis of the artificial mixture of amine acids and the hydrolysate of ovalbumin.
8) The serum (1.0ml) was deproteinized by adding methanol-acetone (3:1) mixture and supernatant was concentrated to 0.1ml in water-bath and 0.01ml of that was used for the electrophoresis.
9) In the analysis of pherograms, the ratio of each peak area for the sum of all peaks obtained by densitometry was determined. No ratios of color yields was used.
10) Although the amino acids patterns of five normal sera hardly showed any individual difference, the pathological states (carcinoma of the stomach and Banti's syndrome) gave various different patterns.
11) It was convinced that this method is very useful for the clinical application of amino acid anlyasis of human blood serum.
