JPS5935594B2 - Angiotensin converting enzyme activity measurement method - Google Patents
Angiotensin converting enzyme activity measurement methodInfo
- Publication number
- JPS5935594B2 JPS5935594B2 JP11176180A JP11176180A JPS5935594B2 JP S5935594 B2 JPS5935594 B2 JP S5935594B2 JP 11176180 A JP11176180 A JP 11176180A JP 11176180 A JP11176180 A JP 11176180A JP S5935594 B2 JPS5935594 B2 JP S5935594B2
- Authority
- JP
- Japan
- Prior art keywords
- converting enzyme
- angiotensin converting
- activity
- measurement method
- enzyme activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明はアンジオテンシン変換酵素の活性を測定する方
法に関し、下記式で示される合成基質−ヒドロキシヒブ
リルーＬ−ヒスチジルーＬ−ロイシンとヒプリカーゼと
ｐ−ヒドロキシ安息香酸水酸化酵素を用いることを特徴
とするアンジオテンシン変換酵素の活性測定法に関する
。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the activity of angiotensin converting enzyme, which comprises a synthetic substrate represented by the following formula - hydroxyhybril-L-histidyl-L-leucine, hypercase, and p-hydroxybenzoic acid hydroxylase. The present invention relates to a method for measuring the activity of angiotensin converting enzyme, which is characterized by using the following.
アンジオテンシン変換酵素（以下ＡＣＥと略記する）は
、生体内においてアンジオテンシンＩに作用し、Ｃ−末
端のジペプチドすなわちＬ−ヒスチジル一Ｌ−ロイシン
を遊離せしめて、血圧上昇作用のあるアンジオテンシン
を生成する酵素である。Angiotensin converting enzyme (hereinafter abbreviated as ACE) is an enzyme that acts on angiotensin I in vivo, liberates the C-terminal dipeptide, namely L-histidyl-L-leucine, and produces angiotensin, which has the effect of increasing blood pressure. be.
この酵素はレニン・アンジオテンシン系あるいはキニン
・カリクレーン系と関連して生体内で重要な役割をして
いるが、この酵素の血中でのレベルによつてサルコイド
ーシスの診断をすることができる。従つて上記ＡＣＥの
酵素活性を測定することは生理的あるいは臨床的に有意
義であり、従来その測定法として、（１）放射性同位元
素を用いる方法、（２）螢光を用いる方法、（３）液体
クロマトグラフイ一による方法などが提案されているが
、これらの方法は特殊な装置を必要とするために一般的
な方法として用いられていない。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.
また上記の方法のほかに、カツシユマンらの提案した方
法があり、この方法では合成基質ヒプリル一Ｌ＝ヒスチ
ジル一Ｌ−ロイシンを用い、これをＡＣＥを含有してい
る試料（例えば血清、体液など）と所定時間反応せしめ
た後、反応停止剤例えば塩酸を添加し、先成したヒプリ
ル酸を酢酸エチルで抽出し、酢酸エチルを蒸発して乾固
した後、蒸留水に溶解し、紫外部において吸光度を測定
してヒプリル酸の濃度を求め、これから計算によつてＡ
ＣＥの活性を求める。本発明は上記の方法と異なり、Ａ
ＣＥの活性を※ε簡単に測定する方法を提供することを
目的として開発されたものであつて、本発明の測定法の
原理は次の通りである。In addition to the above method, there is a method proposed by Katsyuman et al., which uses the synthetic substrate hypril-L-histidyl-L-leucine, which is used in 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. The concentration of hiprlic acid is determined by measuring A, 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 *ε, and the principle of the measuring method of the present invention is as follows.
血清、体液などＡＣＥを含有する液体に、既述した式で
示されるｐ−ヒドロキシヒプリル一Ｌーヒスチジル一Ｌ
−ロイシンを加えると、ｐ−ヒドロキシヒプリル酸とＬ
−ヒスチジル一Ｌ−ロイシンを生ずる。In liquids containing ACE such as serum and body fluids, p-hydroxyhypril-L-histidyl-L expressed by the formula described above is added.
-When leucine is added, p-hydroxyhyprilic acid and L
- produces histidyl-L-leucine.
これにヒプリカーゼを加えると、ｐ−ヒドロキシ安息香
酸とグリシンを生ずる。ｐ−ヒドロキシ安息香酸はｐ−
ヒドロキシ安息香酸水酸化およびＮＡＤＰＨ（ニコチン
アミドアデニンージヌクレオチドリン酸還元型）の存在
下で、３・４−ジヒドロキシ安息香酸とＮＡＤＰ＋とを
生ずる。消費するＮＡＤＰＨの吸光度の減少を測定する
ことによつて、上記ＡＣＥの活性を知ることができる。
従つて上記のｐ−ヒドロキシヒプリル一Ｌ−ヒスチジル
一Ｌ−ロイシン、ヒプリカーゼ、ｐ−ヒドロキシ安息香
酸水酸化酵素及びＮＡＤＰＨを含有する溶液を試薬とし
、これにＡＣＥを含有する液体例えば血清を混合し、Ｎ
ＡＤＰＨの吸光度の減少を分光法によつて測定し、ＮＡ
ＤＰＨの消費量を求め、ＡＣＥの活性を算定することが
できる。When hiplicase is added to this, p-hydroxybenzoic acid and glycine are produced. p-hydroxybenzoic acid is p-
In the presence of hydroxybenzoic acid hydroxylation and NADPH (nicotinamide adenine dinucleotide phosphate reduced form), 3,4-dihydroxybenzoic acid and NADP+ are generated. 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-histidyl-L-leucine, hypercase, p-hydroxybenzoic acid hydroxylase and NADPH is used as a reagent, and a liquid containing ACE, such as serum, is mixed therewith. , N
The decrease in absorbance of ADPH was measured by spectroscopy and
By determining the consumption amount of DPH, the activity of ACE can be calculated.
またこの他に高感度の活性測定法としてＮＡＤＰＨの螢
光を測定する方法を用いることもできる。In addition, a method of measuring NADPH fluorescence can also be used as a highly sensitive activity measuring method.
このように本発明の方法によれば、少量の試料を用いて
短時間で測定することができ、高感度で、極めて簡単に
ＡＣＥの活性を測定することができる。本発明の方法の
原理を式をもつて示すと次のとおりである。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.
上述したように（３）で消費されるＮＡＤＰＨの吸光度
の減少を、紫外部（３４０ｎｍ）においてまたはけい光
を用いて測定する。The decrease in absorbance of NADPH consumed in (3) as described above is measured in the ultraviolet (340 nm) or using fluorescence.
下記に本発明の実施例を示す。Examples of the present invention are shown below.
例
ｐ−ヒドロキシヒプリル一Ｌ−ヒスチジル一Ｌロイシン
５ｍＭ１ヒプリカーゼ１Ｕ，．ｐ−ヒドロキシ安息香酸
水酸化酵素３Ｕ．ＮＡＤＰＨ０．２ｍ１，ホウ酸０．１
ＭおよびＮａＣｌＯ．ｌＭを含有する試薬溶液（ＰＨ８
．３）０．２５ｍＩ！に対して、血清０．０５ｍ１およ
び希釈用精製水０．０４９ｍ１を加えて混合し、温度３
７℃で波長３４０ｎｍの光を用い、吸光度の減少を測定
した。Example: p-hydroxyhypril - L-histidyl - L-leucine 5mM 1 hiplicase 1U, . p-hydroxybenzoic acid hydroxylase 3U. NADPH0.2ml, boric acid 0.1
M and NaClO. Reagent solution containing 1M (PH8
．． 3) 0.25mI! 0.05 ml of serum and 0.049 ml of purified water for dilution were added and mixed, and the mixture was heated to a temperature of 3.
The decrease in absorbance was measured using light with a wavelength of 340 nm at 7°C.
測定は遠心方式による自動分析機を用い、タイムラグを
５秒、反応時間を１０分とした。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.
ＡＣＥの活性単位ＭＵは次式によつて求めた。分子吸光
係数（４）＝６．２２×１０３
２０回測定を行なつた結果は次に示すとおりであつた。The activity unit MU of ACE was determined by the following formula. Molecular extinction coefficient (4) = 6.22 x 103 The results of 20 measurements were as shown below.
活性単位ＭＵ−１５．０±０．４０（ＮｍＯｌノ媛／Ｗ
ｌｍ）変動係数ＣＶ＝２．７％本発明の方法と比較する
ために、上記例において用いた血清と同じ血清を用い、
次のようにしてカツシユマン方法によつて測定を行ない
、下記に示す結果を得た。Activity unit MU-15.0±0.40 (NmOl no Hime/W
lm) Coefficient of variation CV = 2.7% For comparison with the method of the present invention, the same serum as used in the above example was used,
Measurements were carried out by the Katschmann method as follows, and the results shown below were obtained.
ヒプリル一Ｌ−ヒスチジル一Ｌ−ロイシンを含むリン酸
緩衝液（ＰＨ−８．３）０．２５ｍ１に血清０，１ｄを
加えて混合し、３７℃において６０分間反応せしめた後
、塩酸を加えて反応を停止させ、反応生成物であるヒプ
リル酸を抽出するため酢酸エチル１．５７ｆ１１を加え
てふりまぜ、上清を採取し、溶媒を除去した後、所定の
精製水で復元し、比色計によつて波長２２８ｎｍの紫外
線の吸光度を測定し、ヒプリル酸の生成量を求め、ＡＣ
Ｅの活性単位ＭＵを計算した。Add 0.1 d of serum to 0.25 ml of phosphate buffer (PH-8.3) containing hipryl-L-histidyl-L-leucine, mix, react at 37°C for 60 minutes, and then add hydrochloric acid. To stop the reaction and extract the reaction product, hyperric acid, 1.57ml of ethyl acetate was added and mixed, the supernatant was collected, the solvent was removed, the mixture was reconstituted with the specified purified water, and the colorimeter was measured. The absorbance of ultraviolet rays with a wavelength of 228 nm was measured by AC, and the amount of hypolytic acid produced was determined.
The activity unit MU of E was calculated.
２０回測定した結果は次の通りであつた。The results of 20 measurements were as follows.
活性単位ＭＵ＝１５．８±０．９３（ＮｍＯｌ／ｍｌ／
７！１ｊｔ）変動係数ＣＶ＝５．８９％上記結果から判
るように、本発明の方法はカツシユマンの方法に較べて
はるかに簡単であるが活性単位においては、カツシユマ
ンの方法の場合とほぼ同じ値が得られ、変動係数におい
てはカツシユマンの方法の場合に比してその値が小さく
、再現性においてすぐれている。Activity unit MU=15.8±0.93 (NmOl/ml/
7!1jt) Coefficient of variation CV = 5.89% 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, it has almost the same value as Katsuyuman's method. The coefficient of variation is smaller than that of Katschmann's method, and the reproducibility is excellent.
Claims (1)
−Ｌ−ヒスチジル−Ｌ−ロイシン、ヒプリカーゼ、ｐ−
ヒドロキシ安息香酸水酸化酵素およびＮＡＤＰＨ（ニコ
チンアミド アデニン ジヌクレオチド リン酸還元型
）を含有する溶液を、アンジオテンシン変換酵素を含有
する液体に混合し、消費されるＮＡＤＰＨの吸光度を分
光法によつて測定する段階を含むことを特徴とするアン
ジオテンシン変換酵素の活性測定法。 ▲数式、化学式、表等があります▼[Scope of Claims] 1 Synthetic substrate p-hydroxyhypril-L-histidyl-L-leucine, hypercase, p-
A solution containing hydroxybenzoic acid hydroxylase and NADPH (nicotinamide adenine dinucleotide phosphate reduced form) is mixed with a liquid containing angiotensin converting enzyme, and the absorbance of consumed NADPH is measured by spectroscopy. A method for measuring the activity of angiotensin converting enzyme, comprising steps. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11176180A JPS5935594B2 (en) | 1980-08-15 | 1980-08-15 | Angiotensin converting enzyme activity measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11176180A JPS5935594B2 (en) | 1980-08-15 | 1980-08-15 | Angiotensin converting enzyme activity measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5736997A JPS5736997A (en) | 1982-02-27 |
| JPS5935594B2 true JPS5935594B2 (en) | 1984-08-29 |
Family
ID=14569514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11176180A Expired JPS5935594B2 (en) | 1980-08-15 | 1980-08-15 | Angiotensin converting enzyme activity measurement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5935594B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108181460B (en) * | 2017-12-22 | 2020-08-07 | 苏州博源医疗科技有限公司 | Dipeptide derivative for detecting angiotensin I and preparation method and application thereof |
-
1980
- 1980-08-15 JP JP11176180A patent/JPS5935594B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5736997A (en) | 1982-02-27 |
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