IL33898A

IL33898A - Process and agent for the determination of the content of alkaline phosphatase in serum

Info

Publication number: IL33898A
Application number: IL33898A
Authority: IL
Original assignee: Merck Anlagen Gmbh
Priority date: 1969-03-04
Filing date: 1970-02-13
Publication date: 1972-12-29
Also published as: BR6915155D0; GB1273506A; DE1910803A1; BE746829A; CH535433A; NL7003093A; SE360929B; IL33898A0; FR2037607A5

Description

Process and agent for the determination of the content of alkaline phosphatase in serum T»03a nvv*%ypi?R ΠΤΧΠΒΟΙΒ η*7Τ3η nvzp1? »PSDKI T»*?rn The present invention is concerned with a process and agent for the determination of the content of alkaline phosphatase in serum.

The determination of the content of alkaline phosphatase in serum is of importance for the diagnosis and continuous control of bone diseases and tumorous diseases, as well as of gall and liver diseases.

Such an enzyme determination can be carried out by means of a large number of different processes, which differ from one another with regard to the substrate used, the concentration of the substrate, the nature of the detection of the reaction products, the amount of serum needed, the pH value, the period of incubation and the incubation temperature and the nature of the buffer employed.

In the case of one of the most important known processes, serum is incubated in a glycine-sodium hydroxide buffer at a pH of 10.5, together with £-nitro-phenyl phosphate as substrate, for 30 minutes at a temperature of 37°C. and the amount of j3-nitrophenol formed subsequently used for the calculation of the , enzyme content present. The £-nitrophenol formed can be determined photometrically. This discontinuous, so-called two-point method involves, in the same way as all other . previously known methods, various possibilities of error. Thus, for example, an enzyme inhibation occurring during the period of incubation, i.e. a non-linear course of the reaction, brought about, for example, by a displacement of the pH value, cannot be observed. This may well be one of the reasons why the Commission of Enzymes of that enzyme reactions should, whenever possible, be measured op ically-kinetically, i.e. for example, in the case of the determination of alkaline phosphates, the initial velocity of the hydrolysis of the phosphoric acid ester should be determined.

In the case of the use of j -nitrophenyl phosphate as substrate, the detection of the £-nitrophenol formed in the alkaline medium used by the action of the enzyme can be determined photometrically by direct measurement at kOO nm since the pK„ value of p-nitrophenol is 7.16.

Thus, the determination is possible by the op ical-kinetic method by mixing the buffered substrate solution with the serum to be tested, whereafter the extinction caused by the liberated p_-nitrophenol is continuously read off or is recorded with the help of a compensation recorder.

In the case of this process, the incubation is carried put in the known buffers of the conventional low molarity and thus with low buffer capacity.

A process is. also known for the determination of the content of alkaline phosphate in serum with the use of a buffered aqueous p_-nitrophenyl phosphate solution as substrate and with the help of a photometric measurement of the initial velocity of the hydrolysis of this phosphoric diethanolamine acid ester, 1 M/diethanolamine hydrochloride being used as the buffer substance* (a diethanolamine - HC1 buffer).

In the case of the use of diethanolamine hydrochloride buffer at a pH of 9.2+, the enzyme activity is, surprisingly, about 2.3 times higher than in all the previous known buffers. Due to this, it is possible to measure, with only a small amount of serum and in an extremely short Thus, this process permits the determination of the alkaline phosphate in an optical-kinetic way. An especially optimum increase of the enzyme activity is achieved, in the case of this process, when working at a pH of 9.-4. and with the use of a 1 molar buffer concentration.

An important advantage of the method consists in that it works quickly and simply and that, in particular, it gives a very good reproducibility. The other previously known methods are characterised by a very unsatisfactory reproducibility, which always leads to relatively uncertain measurement results.

We have now found, surprisingly, that the determination of the alkaline phosphate in serum proceeds espec- diethanolamine ially favourably when the/diethanolamine hydrochloride is used in a concentration of at least 1.5 and at most Z+.5 mole/litre, especially of about 2 mole/litre. Especially by the use of this relatively high buffer concentration, there is obtained a high buffer capacity which precludes the harmful action of carbon dioxide from the air and keeps the pH value constant during the whole period of the experiment.

Thus, the present invention provides a process for the determination of the content of alkaline phosphatase in' serum with the use of an aqueous solution of p-nitro- dietnano1amine phenyl phosphate as substrate buffered with/diethanolamine hydrochloride and with the help of a photometric measurement of the initial velocity of the hydrolysis of this phosphoric acid ester, which is characterised in that the diethanolamine /diethanolamine hydrochloride is used in a molar concentration of at least 1.5 and at most i+.5» especially of The present invention also provides an agent for carrying out this process, which comprises £-nitrophenyl diethanolamine phosphate as substrate buffered with diethanolamine/ hydrochloride in a molar concentration of at least 1.5 and at most 4.5, especially of about 2.

The following Examples are given for the purpose of illustrating the present invention: - Example . a) Preparation of the buffer. 210.28 g. diethanolamine (crystallisable) are dissolved in about 600 ml. double distilled water. Sub-sequently, about 160 ml. 2N hydrochloric acid are added, with stirring, using a glass stirrer. 500 mg. sodium azide are then added, together with 101.5 mg. magnesium chloride hexahydrate. The solution is made up with water to almost 1000 ml. Subsequently, the pH value is adjusted to precisely 9.8 by the dropwise addition of H hydrochloric acid and the solution then made up to precisely 1000 ml. with double distilled water. b) Preparation of the buffer substrate solution. 1000 A 1-00- mg. tablet which contains 131.5 mg. 100 disodium j>-nitrophenyl phosphate, the remainder being sodium chloride and possibly some moisture, is dissolved in 10 ml. of the above-described buffer solution. When this solution is stored at 1+°C> with the exclusion of light, it can be used for 2 days for carrying put the enzyme determination described hereinafter. c) Enzyme determination.

The buffer substrate and the serum to be tested are warmed to 25°C., the temperature being measured in the solution. 2 ml. of the buffered substrate solution are mixed with 0.02 ml. fresh serum and thoroughly mixed. After about 1 minute, the extinction is measured at 25°C. and, simultaneously, the time is ascertained with a stopwatch. The measurement is repeated every minute for 3 minutes.

The extinction measurement is carried out with, for example, a photometer which is equipped with a filter for the mercury line at i+05 nm. The layer thickness is 1 cm.

If the extinction differences Δ E/min at the beginning of the measurement are greater than 0.2 , then the determination is repeated with the serum diluted 1 : 5 with a physiological solution of sodium chloride and the result obtained multiplied by 5. 1 International Enzyme Unit (u) is the amount of enzyme which reacts with 1 μ, mole of substrate in one minute under standard conditions. 1 milli-unit (mil) = 1 /1 000 U.

The extinction difference per minute Δ E/+05 is introduced into the following calculation formula: enzyme concentration = _» Ei05 . 5kS0 mU/ml.

In this Example, 90 - 250 mU ( 25°C.)/ml. is to be regarded as the normal range.

Example 2. a) Preparation of the buffer.

The preparation of the buffer is carried out in a manner analogous to that described in Example , except that the magnesium chloride hexahydrate is used in an amount of 203.0 mg. b) Preparation of the buffered substrate solution.

A tablet which contains 39.45 mg. 00% disodium _-nitrophenyl phosphate is dissolved in 10 ml. of the buffer solution of Example 2a) in a manner analogous to that described in Example 1b). c) Enzyme determination.

The enzyme determination is carried out in a manner analogous to that described in Example 1c), except that 1 ml. of buffered substrate solution is added to 0.02 ml. serum.

The calculation of the enzyme content is carried out, in this Example, by means of the formula: enzyme concentration = ΔΕ/min . 2757 mU/ml.

When, in a variant of this Example, i+.O ml. buffered substrate solution are added to 0.02 ml. serum, .the following formula is used for the calculation of the enzyme content: enzyme concentration = ΔΣ/mln . 10865 mU/ml.

Example 3.

The preparation of the buffer is carried out in a manner analogous to that described in«Example 1a) but with the use of the following amounts of the individual components : 168.22 g. diethanolamine 128 ml. 2N hydrochloric acid 101.5 mg. magnesium chloride hexahydrate The remaining components are used in the same concentrations as in Example 1.

The preparation of the buffered substrate solution and the enzyme determination are also carried out in a manner analogous to that described in Example 1b) and c), respectively.

Example k* By way of variation of Example 1 , the buffer is . prepared from the following components: i+20.56 g. diethanolamine, without additional water 320 ml. 2N hydrochloric acid 101.5 mg. magnesium chloride hexahydrate.

The preparation of the substrate solution, the preparation of the buffered substrate mixture and the enzyme determination are carried out in a manner analogous to that described in Example 1.

Claims

1. What we claim is :- 1. An agent for carrying out the determination of the content of alkaline phosphatase in serum, which comprises /diethano1amine an aqueous solution of £-nitrophenyl phosphate containing/ diethanolamine hydrochloride in a molar concentration of at least 1.5 and at most h.5.

2. An agent according to claim 1, wherein the molar diethano1amine concentration of the /diethanolamine hydrochloride is about 2.

3. An agent according to claim 1 for carrying out the determination of the content of alkaline phosphatase in serum, substantially as hereinbefore described and exemplified. k» Process for carrying out the determination of the content of alkaline phosphatase in serum, wherein, as substrate, there is used an aqueous solution of _-nitro- diethanolamine phenyl phosphate which contains, as a buffer/ diethanolamine hydrochloride in, a molar concentration of at least 1.5 and at most

4. » the initial velocity of the hydrolysis of the £-nitrophenyl phosphate being measured photometrically,

5. Process according to claim 2+, wherein the molar diethanolamine concentration of the/ diethanolamine hydrochloride is about 2.

6. Process according to claim i+ for carrying out the determination of the content of alkaline phosphatase in serum, substantially as hereinbefore described and exemplified.