JPS5935595B2 - Angiotensin converting enzyme activity measurement method - Google Patents

Description

Detailed Description of the Invention The present invention relates to a method for measuring the activity of angiotensin converting enzyme, which is characterized in that it uses a synthetic substrate p represented by the following formula, hiplicase, and p-hydroxybenzoic acid hydroxylase. Concerning a method for measuring the activity of.

Angiotensin-converting enzyme (hereinafter abbreviated as ACE) acts on angiotensin I in vivo, and has a C-terminal dipeptide, namely L-his-hydroxyhypri-L-glycyl-L-glycine JH-CH2-C-NH
-CH2-COOH This is an enzyme that liberates tidyl-L-leucine and generates angiotensin (2), which has an effect of increasing blood pressure.

This enzyme plays an important role in the body in association with the renin-angiotensin system or the kinin-kallikrene system, and sarcoidosis can be diagnosed based on the level of this enzyme in the blood. Therefore, it is physiologically or clinically meaningful to measure the enzymatic activity of ACE, and the conventional methods for this measurement include (1) a method using radioisotopes, (2) a method using fluorescence, and (3) a method using fluorescence. Although methods using liquid chromatography have been proposed, these methods require special equipment and are therefore not commonly used.

In addition to the above method, there is a method proposed by Katsyuman et al., which uses the synthetic substrate hipryl-L-histidyl-L-leucine and transfers it to samples containing ACE (e.g. serum, body fluids, etc.). After reacting for a predetermined period of time, a reaction terminator such as hydrochloric acid is added, and the preformed hyperric acid is extracted with ethyl acetate. After evaporating the ethyl acetate to dryness, it is dissolved in distilled water, and the absorbance is measured under ultraviolet light. is measured to determine the concentration of moprilic acid, and from this, A is calculated.
Determine the activity of CE. The present invention differs from the above method in that A
It was developed with the aim of providing a method for easily measuring CE activity, and the principle of the measuring method of the present invention is as follows.

In liquids containing ACE such as serum and body fluids, p-hydroxyhypril-L-glycyl-L-
When glycine is added, p-hydroxyhyprilic acid and L-
yields glycyl-L-glycine.

Adding hipricase to this produces p-hydroxybenzoic acid and glycine. p-hydroxybenzoic acid is p-
In the presence of hydroxybenzoic acid hydroxylase and NADPH (nicotinamide adenine dinucleotide phosphate reduced form), 3,4-dihydroxybenzoic acid and NADP' are produced. The activity of the ACE can be determined by measuring the decrease in the absorbance of consumed NADPH. Therefore, the above solution containing p-hydroxyhypril-L-glycyl-L-glycine, hypercase, p-hydroxybenzoic acid hydroxylase, and NADPH is used as a reagent, and a liquid containing ACE, such as serum, is mixed therewith. ,
The decrease in absorbance of NADPH was measured by spectroscopy and
The activity of ACE can be calculated by determining the consumption amount of ADPH.

In addition, a method of measuring NADPH fluorescence can also be used as a highly sensitive activity measuring method.

As described above, according to the method of the present invention, it is possible to perform measurement in a short time using a small amount of sample, and the activity of ACE can be measured extremely easily with high sensitivity. The principle of the method of the present invention is expressed by the following formula.

The decrease in absorbance of NADPH consumed in (3) as described above is measured in the ultraviolet (340 nm) or using fluorescence.

Examples of the present invention are shown below.

Example p-hydroxyhypril-L-glycyl-L-glycine 10mM) hiplicase IU, . p-hydroxybenzoic acid hydroxylase 3U, NADPHO. 2mM, boric acid 0.
IM and NaClO. Reagent solution containing IM (PH
8.3) Add 0.05 mj of serum and 0.049 mj of purified water for dilution to 0.25 mj, mix, and heat at 37°C.
The decrease in absorbance was measured using light with a wavelength of 340 nm.

The measurement was performed using an automatic analyzer using a centrifugal method, with a time lag of 5 seconds and a reaction time of 10 minutes.

The activity unit MU of ACE was determined by the following formula. The results of 20 measurements were as shown below.

For comparison with the method of the present invention, measurements were carried out by the Katschmann method as follows using the same serum as used in the above example, and the results shown below were obtained.

0.1 serum in 0.25 ml of phosphate buffer (PH = 8.3) containing hipryl-L-histidyl-L-leucine
After adding 1.5 ml of ethyl acetate and mixing, and reacting at 37°C for 60 minutes, add hydrochloric acid to stop the reaction, add 1.5 ml of ethyl acetate to extract the reaction product, hipric acid, and mix. After removing the solvent, reconstitute with specified purified water, measure the absorbance of ultraviolet rays with a wavelength of 228 nm using a colorimeter, determine the amount of hyperlic acid produced, and
The activity unit MU of CE was calculated.

The results of 20 measurements were as follows.

As can be seen from the above results, the method of the present invention is much simpler than Katsuyuman's method, but in terms of activity units, almost the same value as Katsuyuman's method is obtained.
The coefficient of variation is smaller than that of Katschmann's method, and the reproducibility is excellent.

Claims (1)

[Scope of Claims] 1 Synthetic substrates p-hydroxyhypril-L-glycyl-L-glycine, hypercase, p-hydroxybenzoic acid hydroxylase and NADPH (nicotinamide adenine dinucleotide phosphate reduced form) represented by the following formula: )
A method for measuring the activity of angiotensin converting enzyme, comprising the steps of: mixing a solution containing the angiotensin converting enzyme with a liquid containing the angiotensin converting enzyme, and measuring the absorbance of consumed NADPH by spectrometry. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼