JPH024279B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring serum alkaline phosphatase activity, particularly in a method for measuring activity using thymol phthalein monophosphate as a substrate.
The accuracy and reliability of measurement is improved by using diethanolamine/hydrochloric acid buffer.

Alkaline phosphatase is an enzyme that hydrolyzes phosphate esters.
It is present in liver bile canaliculi, mammary glands, small intestinal cilia, osteoblasts, placenta, etc., and because its localization is in cell membranes, it is thought to be involved in active transport across membranes. Therefore, measuring serum alkaline phosphatase is useful for differential diagnosis of liver disease, biliary tract disease, bone disease, or monitoring the outcome of pregnancy, and is now widely used as a routine screening test. The basis for its measurement method has been established through reports such as the method of Shinowara et al., the Bessey-Lowry method, and the Kind-King method. For example, a method using phenolphthalein monophosphate as a substrate proposed by Babson et al.
Babson, Clin, Chem, 12, (8) 482 (1966), etc. However, these measurement methods are affected by coexisting substances in serum, such as hemoglobin, bilirubin, and turbidity, and it is often difficult to obtain accurate measurement values.

Subsequently, completely new measurement methods were proposed that improved on these methods (A.V., Roy, Clin,
Chem..16.(5)431 (1970)). The method (hereinafter referred to as Roy's method) uses thymolphthalein monophosphate as a substrate and 2-amino-2-methyl-1-propanol as a buffer component at 37°C and 10°C.
The method is based on minute-minute measurements, has better sensitivity than conventional methods, and can be measured almost unaffected by coexisting substances in serum as described above. However, when this substrate buffer is stored refrigerated and tightly sealed, its pH changes by 0.05 or more in just one month, and the blinded value in measurements increases significantly, so it is not stable enough to be applied to routine tests. There is a problem of lack of reliability.

The present invention solves the drawbacks of Roy's method described above, and uses diethanolamine instead of 2-amino-2-methyl-1-propanol as a buffer to improve the stability of the substrate and PH. , which made it possible to accurately measure alkaline phosphatase for at least 6 months.

The present invention will be explained in detail below.

According to the present invention, a diethanolamine/hydrochloric acid buffer containing thymolphthalein monophosphate as a substrate is added to a sample, the liberated thymolphthalein is colored with alkaline, and the absorbance is measured, thereby accurately determining alkaline phosphatase activity. A value is required.

The reagents used in the measurement method of the present invention include a substrate buffer containing thymolphthalein monophosphate, an alkaline reaction stop solution for stopping the reaction and developing color of liberated thymolphthalein, and a constant concentration of thymolphthalein. It consists of a standard solution containing concentration. The substrate buffer includes diethanolamine/hydrochloric acid buffer, thymolphthalein monophosphate (sodium salt, magnesium salt, ammonium salt, etc. are used from the viewpoint of solubility), and an alkaline phosphatase activator (preferably magnesium chloride, etc.). ), and a surfactant (preferably,
Brij-35, etc.), and if necessary, preservatives (e.g.
It is made by blending sodium azide, etc.)
The concentration of the substrate thymolphthalein monophosphate is
Preferably, the content is 0.15 to 0.2%, and the liquid pH is adjusted to 9.75 to 9.85 (37°C). The reaction stop solution is an aqueous solution containing sodium hydroxide and sodium carbonate, each with a concentration of about 0.1M, but about 0.5M.
By adjusting the concentration before and after high concentrations, the yellow color caused by unfree thymolphthalein monophosphate can be faded after the reaction has stopped, and the blue color of liberated thymolphthalein can be accurately reflected in the absorbance measurement. The standard solution consists of thymolphthalein and a surfactant (preferably,
Brij-35, etc.), and the appropriate concentration of thymolphthalein is 0.1 to 0.2%. Preferred composition examples of each of the above reagents are as follows.

Substrate buffer: Sodium thymolphthalein monophosphate
...3mM Brij-35 ...0.5 (W/V)% Magnesium chloride ...1mM Sodium azide ...0.1 (W/V)% 0.6M diethanolamine/hydrochloric acid buffer containing the above (PH9.80±0.05, liquid temperature 37℃).

Reaction stop solution: Sodium hydroxide …0.5M Sodium carbonate…0.5M Standard solution: Thymolphthalein…3mM Brij−35…1.5 (W/V)% 60% ethanol aqueous solution containing the above.

Comparing the stability of each reagent used in the present invention after preparation with that in Roy's method, we find that the stability of both reaction stop solutions and standard solutions at room temperature is the same, but that of the substrate It can be seen that the present invention is superior in terms of the pH of the buffer solution and the stability of the substrate, and that the assay method of the present invention is highly reliable from the standpoint of routine testing.

Substrate buffer (stored at 2-8℃) Invention: No change in PH for 12 weeks, Roy: PH decreased by 0.05 in 3 weeks.

Reaction stop solution (stored in a tightly capped container) Invention: No deterioration Roy: Standard solution without any deterioration (stored in a tightly capped container) Invention: No deterioration Roy: Only the stock solution remains unchanged (however, 2-amino-2-
If the working solution is diluted 100 times with methyl-1-propanol/hydrochloric acid buffer, thymolphthalein will precipitate during long-term storage).

Blinded blank value This invention: 10% increase in 6 months Roy: 30% increase in one month.

To explain an example of the measurement method of the present invention, first, three small test tubes are prepared, each containing a sample, a standard, and
Blinded, add 0.5 ml of substrate buffer to each
Incubate at 37°C for 5 minutes. Then add serum to each test tube,
Add standard solution and purified water 0.05ml each and mix to make sure
Incubate for 10 minutes. Thereafter, 5 ml of the reaction stop solution was added to each, mixed well, and heated at 37°C for 15 minutes, whereby thymolphthalein liberated by alkaline phosphatase developed a color. Since its color development is proportional to alkaline phosphatase activity, by measuring the absorbance of the standard and sample at 590 nm using a blind control, the activity of the sample can be determined using the following formula.

Activity of specimen = Absorbance of specimen / Absorbance of standard x Activity value of standard The measurement results obtained for 30 specimens by the above measurement are shown in FIG. The figure shows a comparison with measurement values obtained for the same specimen by the Kind-King method, which is considered to be a standard measurement method for alkaline phosphatase. As is clear from the figure, the values measured by the kind-king method are normal values (2.7~
10King-Armstrong units), all of the 19 samples measured by the method of the present invention showed normal values, and the 11 samples that showed abnormal values by the Kind-King method, although some of the values were slightly different, were measured using the method of the present invention. The measured values obtained by the method also showed similar abnormal values, and a high correlation was observed between the measured values of both methods (correlation coefficient γ =
0.9678), and its regression equation is Y=1.006X+1.237
(Y: value measured by the Kind-King method, X: value measured using the method of the present invention) was obtained.

In addition, among the samples that showed abnormal values, regarding the two samples a and b whose measured value by the present invention method and the measured value by the Kind-King method were significantly different, the measured value by the present invention method (a: 11.4 K-A unit, b: 12.0 K-A units) are close to normal values, whereas the Kind-King method measurements (a: 21.3 K-A units, b: 21.7 K-A units) are significantly high. Therefore, as a result of searching for isozymes in these two samples, it was found that there was no abnormality in the alkaline phosphatase values derived from the liver and bones in both samples, and that values derived from the small intestine were higher. In other words, the reason why the assay method of the present invention showed low values close to normal values for the above two samples is because thymolphthalein monophosphate, which is the substrate for the assay method of the present invention, is lower than phenyl phosphate, which is the substrate for the kind-king method. This is due to the low substrate specificity for alkaline phosphatase derived from the small intestine. Normally, in the measurement of alkaline phosphatase for in vitro differential diagnosis of diseases, the measurement targets are those derived from the liver and bone, so the measurement obtained by the method of the present invention has low substrate specificity for that derived from the small intestine. value is,
It can be said that this method more accurately reflects the presence or absence of an abnormality in alkaline phosphatase than the Kind-King method. Moreover, by utilizing this fact and using it in combination with the kind-king method, it is also possible to detect isozymes derived from the small intestine.

The measuring method of the present invention is more suitable for daily inspection than the conventional Roy method and can provide accurate measured values. Furthermore, it is superior for differential diagnosis compared to the Kind-King method, which is currently considered a standard method.

[Brief explanation of drawings]

FIG. 1 is a graph showing serum alkaline phosphatase measurements by the method of the present invention and the Kind-King method.

Claims (1)

[Claims] 1. Serum alkalinity, which is characterized by adding a diethanolamine/hydrochloric acid buffer containing thymolphthalein monophosphate as a substrate to a sample, causing the liberated thymolphthalein to develop color in alkaline conditions, and determining the alkaline phosphatase activity by measuring its absorbance. Method for measuring phosphatase activity.