JPS603838B2 - Method for measuring cholinesterase activity - Google Patents
Method for measuring cholinesterase activityInfo
- Publication number
- JPS603838B2 JPS603838B2 JP5170781A JP5170781A JPS603838B2 JP S603838 B2 JPS603838 B2 JP S603838B2 JP 5170781 A JP5170781 A JP 5170781A JP 5170781 A JP5170781 A JP 5170781A JP S603838 B2 JPS603838 B2 JP S603838B2
- Authority
- JP
- Japan
- Prior art keywords
- cholinesterase
- measuring
- activity
- hydroxybenzoylcholine
- nadph
- 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
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
本発明はコリンェステラーゼの活性測定法に関し、式を
有するヒドロキシベンゾイルコリンを合成基質として用
い、ヒドロキシベンゾイルコリン、ｍーヒドロキシ安息
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、
コリンヱステラーゼを含有する液体に混合し、コリンェ
ステラーゼの作用によってヒドロキシベンゾイルコリン
から生成されるｍ−ヒドロキシ安息香酸を、ＮＡＤＰＨ
の存在下でｍーヒドロキシ安息香酸水酸イ技酵素の作用
によって、ジヒドロキシ安息香酸に変化させ、その際に
、消費されるＮＡＤＰＨの量を、ＮＡＤＰＨの吸光度の
減少を分光法によって測定することによって求め、その
値からコリンェステラーゼ活性を測定することからなる
ものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring the activity of cholinesterase, using hydroxybenzoylcholine having the formula as a synthetic substrate, hydroxybenzoylcholine, m-hydroxybenzoic acid hydroxylase and NADPH (nicotinamide adenine dinucleotide). A solution containing phosphoric acid (reduced form),
When mixed with a liquid containing cholinesterase, m-hydroxybenzoic acid produced from hydroxybenzoylcholine by the action of cholinesterase is converted into
m-hydroxybenzoic acid is converted into dihydroxybenzoic acid by the action of a hydroxyl enzyme in the presence of hydroxybenzoic acid, and the amount of NADPH consumed at this time is determined by measuring the decrease in the absorbance of NADPH by spectroscopy. The method consists of measuring cholinesterase activity from the value.
コリンエステラーゼ（Ｃｈｏｌｉｎｅｓｔｅｒａｓｅ）
には、赤血球、神経組織などに存在するコリンェステラ
ーゼ１と、血清、隣臓などに存在するコリンェステラー
ゼロとがあり、コリンエステラーゼはアシル化コリンを
コリンと酸（アシル）に分解する。Cholinesterase
There are two types of cholinesterase: cholinesterase 1, which is present in red blood cells and nervous tissue, and cholinesterase zero, which is present in serum and neighboring organs.Cholinesterase breaks down acylated choline into choline and acid (acyl).
生体において低コリンェステラーゼ血症は、肝疾患や貧
血などの疾患にみられ、高ェステラーゼ血症は、ネフロ
ーゼや糖尿病、神経系障害の疾患にみられる。コリンェ
ステラーゼの血中でのレベルを知ることは、上記のよう
な疾患を診断する場合に有益である。従ってコリンェス
テラーゼの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、従釆その測定法としては、ｍアセチ
ルコリンを基質とし、分解によるｐＨの変化を測定する
高橋、柴田らの方法、【２ーベンゾィルコリンを基質と
して用い、遊離するコリンをコリンオキシダーゼによっ
て酸化し、これにより遊離する日２０２を、ベルオキシ
ダーゼによってフェノールおよび４ーアミノアンチピリ
ンと反応せしめ、生成するキノンィミン色素を比色定量
する方法が知られているが、上記‘１）の方法は複雑な
操作を必要とし、■の方法は、血清中に含有されている
いろいろの物質の影響を受けて精度に欠けるものである
。In living organisms, hypocholinesteraseemia is observed in diseases such as liver disease and anemia, and hypercholinesteraseemia is observed in diseases such as nephrosis, diabetes, and nervous system disorders. Knowing the level of cholinesterase in the blood is useful when diagnosing the above-mentioned diseases. Therefore, measuring the activity of cholinesterase is physiologically or clinically meaningful, and a secondary method for measuring it is the method of Takahashi and Shibata et al., which uses m-acetylcholine as a substrate and measures the change in pH due to decomposition. , [Using 2-benzoylcholine as a substrate, the liberated choline is oxidized by choline oxidase, and the choline thus liberated is reacted with phenol and 4-aminoantipyrine by peroxidase, and the resulting quinonimine pigment is colorimetrically measured. Methods for quantifying it are known, but method 1) above requires complicated operations, and method ① lacks accuracy because it is affected by various substances contained in serum. .
本発明は上記のような欠点を排除することを目的として
開発されたものであって、本発明の方法によれば、少量
の試料を用いて短時間に、試料中に含有されているコリ
ンェステラーゼの活性を測定することができる。The present invention was developed with the aim of eliminating the above-mentioned drawbacks. According to the method of the present invention, the choline contained in the sample can be removed in a short time using a small amount of sample. Sterase activity can be measured.
本発明に用いるｍーヒドロキシベンゾィルコリンの合成
法を下記に示す。The method for synthesizing m-hydroxybenzoylcholine used in the present invention is shown below.
ｍーヒドロキシ安息香酸５のと塩化コリン５夕を加え、
１洲の硫酸５のとを加えて、２曲時間加熱還流した後、
反応混合物をｐＨ４．０に調整し、不落物を炉別する。Add m-hydroxybenzoic acid and choline chloride,
After adding 5 parts of sulfuric acid and heating under reflux for 2 hours,
The reaction mixture was adjusted to pH 4.0, and the impurities were filtered out.
炉液を減圧下で濃縮し、シリカゲルクロマトグラフィー
によって分離、精製し、ｍ−ヒドロキシベンゾィルコリ
ンを得る。下記に、実施例として、血清中に含有されて
いるコリンェステラーゼの活性を測定した場合を示す。The furnace solution is concentrated under reduced pressure, separated and purified by silica gel chromatography to obtain m-hydroxybenzoylcholine. Below, as an example, a case where the activity of cholinesterase contained in serum was measured is shown.
ｍ−ヒドロキシベンゾイルコリン２のＭ、ｍ−ヒドロキ
シ安息香酸水酸化酵素３Ｕ、ＮＡＤＰＨＯ．２のＭ、リ
ン酸１００のＭを含有する溶液（ｐＨ７．５）１叫に対
して、血清５ｒ夕を加え、温度３７０において、波長３
４触れの光を用い、吸光度の減少を測定した。M of m-hydroxybenzoylcholine 2, m-hydroxybenzoic acid hydroxylase 3U, NADPHO. To one solution (pH 7.5) containing 2 M of phosphoric acid and 100 M of phosphoric acid, 5 ml of serum was added, and at a temperature of 370 ml, a wavelength of 3 was added.
The decrease in absorbance was measured using 4 exposures of light.
測定はタイムラグを５秒とし、反応時間を５分とした。
コリンェステラーゼの活性単位Ｕは下記式によって求め
ることができる。Ｕ＝３４仇ｍの激職鰍妙×分子屍轍が
肌志豚Ｘ織Ｘ瓜５ｍｉｎご＝６．２２×１ぴ
３０回測定を行なった結果は、次に示す通りであつた。In the measurement, the time lag was 5 seconds and the reaction time was 5 minutes.
The activity unit U of cholinesterase can be determined by the following formula. The results of 30 measurements of U = 34 meters of Gekisho Animyo x Molecule Corpse = 6.22 x 1 per 5 min were as shown below.
Claims (1)
安息香酸水酸化酵素およびＮＡＤＰＨ（ニチコンアミド
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、コリンエステラーゼを含有する液体に混合し、消費
されるＮＡＤＰＨの吸光度を、分光法によって測定する
段階を含むことを特徴とするコリンエステラーゼの活性
測定法。[Claims] 1. A solution containing hydroxybenzoylcholine, m-hydroxybenzoic acid hydroxylase and NADPH (reduced form of nichiconamide adenine dinucleotide phosphate) having the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ A method for measuring the activity of cholinesterase, which comprises the steps of: mixing the cholinesterase in a liquid containing the cholinesterase; and measuring the absorbance of consumed NADPH by spectroscopy.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5170781A JPS603838B2 (en) | 1981-04-08 | 1981-04-08 | Method for measuring cholinesterase activity |
| EP82300965A EP0060059B1 (en) | 1981-03-04 | 1982-02-25 | Method for determining the activity of cholinesterase and diagnostic solution for use therein |
| DE8282300965T DE3265517D1 (en) | 1981-03-04 | 1982-02-25 | Method for determining the activity of cholinesterase and diagnostic solution for use therein |
| ES510091A ES8303701A1 (en) | 1981-03-04 | 1982-03-03 | Method for determining the activity of cholinesterase and diagnostic solution for use therein. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5170781A JPS603838B2 (en) | 1981-04-08 | 1981-04-08 | Method for measuring cholinesterase activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS57166999A JPS57166999A (en) | 1982-10-14 |
| JPS603838B2 true JPS603838B2 (en) | 1985-01-30 |
Family
ID=12894362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5170781A Expired JPS603838B2 (en) | 1981-03-04 | 1981-04-08 | Method for measuring cholinesterase activity |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS603838B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60238000A (en) * | 1984-05-10 | 1985-11-26 | Nitto Boseki Co Ltd | Novel method of cholinesterase activity measurement |
-
1981
- 1981-04-08 JP JP5170781A patent/JPS603838B2/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| JPS57166999A (en) | 1982-10-14 |
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