Abstract
Serum alkaline phosphatase (Al-P) is separated into 6 active bands by agar-gel electrophoresis1, 2). Among these bands, Al-P4 (the 4th band from the cathode) has been found in the trimester of pregnancy, as well as in some cases of malignant tumor and other advanced diseases. All of the Al-P4 of such different origins are similar to placental Al-P in their enzymological properties and are shown to be stable to heat, being not inactivated even at 65°. This investigation is to find out any difference among these Al-P4 of different origins, by means of the radioimmunoassay method for placental Al-P.
Method:
Purification of placental Al-P and preparation of its antiserum were carried out in accordance with the previous reports3, 4), and iodination of the purified placental Al-P by Hunter-Greenwood method 5)(TABLE I). By using these materials, the radioimmunoassay (RI) method for placental Al-P was devised in accordance with double antibody method with anti-rabbit gama;-globulin serum (TABLE II).
Serum Al-P was separated by agar-gel electrophoresis 1) and determined by modified Kind-King method and heat-stable Al-P was measured after heating the sera at 65° for 10 minutes.
Results and Discussion:
1) The standard curve obtained by the RI method can be applied in detecting more than 1 ng of purified placental Al-P, which can be satisfactorily available within the range of 5 to 25 ng (Fig. 1).
2) Although the sera shown in TABLE III were tested, no reaction were seen in Al-P1, Al-P2, Al-P3. and Al-P5 by this RI method. However, dose dependence curve obtained on the heat-stable serum Al-P by this method was quite similar to that of placental Al-P. Besides, no difference was found among the heat-stable ones in pregnancy and patients with malignant tumor and non-malignant tumor (Fig 1). On the other hand, Fishman et al. reported that Regan isoenzyme originated from cancer tissue was similar to placental Al-P from the viewpoints of enzymology and of immunology6, 7). They also noticed that the heat-stable Al-P appearing in non-cancer patients showed no cross reactions to placental Al-P immunologically. Therefore, our results mentioned above are different therefrom: the heat-stable Al-P in non-malignant patients also shows the same pattern as placental Al-P immunologically.
Although another report using the electrophretic method shows a cross reaction between intestinal Al-P and placental Al-P antiserum, no reaction was revealed in both types of Al-P by this RI method.
3) Correlation between the amount of Al-P by using RI method (y) and activity of heat-stable Al-P (x) is as follows: in serum in pregnancy, y=1.99x-1.28 (r: 0.92) and non-pregnancy, y =1.96x-0.43 (r: 0.89)(Fig. 2). This means that the heat-stable Al-P in pregnancy and that in non-pregnancy are almost the same each other immunologically. For the purified placental Al-P, 33 ng of it was equivalent to 10 King-Armstrong (K-A) units, but 20ng was for the serum Al-P. The enzymatic activity of heat-stable serum Al-P was higher than that of purified placental Al-P in comparison with the same amount of Al-P by RI method. Ratio of the amount of Al-P by RI method to the activity of heat-stable Al-P remained unchanged in the following materials, i. e. water extract of placenta, crude placental Al-P by Morton's method and purified placental Al-P adding serum. Therefore, the decreased enzymatic activity of placental Al-P compared with serum heat-stable Al-P was not due to the damage caused in the course of its purification or the lack of activators. Further investigation will be needed in this mechanism.
Heat-stable Al-P can be determined for more than 0.5 K-A units which is equivalent to 1 ng/ml by RI method. The RI method is sensitive to the same degree as the enzymatic method, but is much more excellent as to specificity.
