JP2739473B2 - Highly sensitive measurement method for D-lactic acid - Google Patents
Highly sensitive measurement method for D-lactic acidInfo
- Publication number
- JP2739473B2 JP2739473B2 JP33138087A JP33138087A JP2739473B2 JP 2739473 B2 JP2739473 B2 JP 2739473B2 JP 33138087 A JP33138087 A JP 33138087A JP 33138087 A JP33138087 A JP 33138087A JP 2739473 B2 JP2739473 B2 JP 2739473B2
- Authority
- JP
- Japan
- Prior art keywords
- lactic acid
- highly sensitive
- present
- pyruvate
- amount
- 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 - Fee Related
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、体液中に微量に存在するＤ−乳酸の高感度
定量法に関する。
本発明者らは体液即ち血液，尿等でヒトの健康時に微
量に存在するＤ−乳酸は糖尿病、或いはリンパ腫等の疾
病時において著しく増加することを発見した。従って本
発明のＤ−乳酸の高感度定量法はこれら疾病の診断を目
的とする臨床検査の分野で有用である。
〔従来技術〕
これまでにも体液中のＤ−乳酸をＤ−乳酸脱水素酵素
（以下Ｄ−LDHという。）を用いて測定する試みがなさ
れており〔例えばMethods in Enzymology 89巻,35〜40
（1982）〕、その反応式は以下に示される。
しかしながら、従来の測定法においてはその反応式に
おいて生成したNADHを紫外部（例えば波長340nm）にて
直接測定するか又はNADHを種々の発色剤（例えばホルマ
ザン色素）にて発色させ可視部にて測定することが試み
られてきた。
〔従来技術の問題点〕
しかしながら、このような測定法による体液中のＤ−
乳酸を定量する方法では感度が低すぎるため、極く微量
のＤ−乳酸の定量を行うためには体液の測定試料を多く
せねばならず、これでは、極少量の試料を用い、短時間
に測定することを要する臨床検査の分野において到底使
用することが不可能であった。
〔問題点を解決するための手段〕
そこで本発明者らは、微量のＤ−乳酸を感度よく定量
できる方法を見出すべく鋭意検討した結果、生成するピ
ルビン酸に蛍光化合物をカップリングさせ、カップリン
グ化合物の蛍光強度を定量することによりＤ−乳酸を感
度よく定量できる方法を見出したのである。これにより
健常人の体液中に極微量存在するＤ−乳酸を精度よく測
定することが可能になり、各種疾病により体液中のＤ−
乳酸量が増加した場合健常人の値と比較することにより
診断することが可能となった。即ち本発明は、体液にNA
D存在下、Ｄ−LDHを作用させ、ピルビン酸とNADHを生成
せしめ、次いで生成したピルビン酸と蛍光化合物とを反
応させることによってカップリング化合物を形成せしめ
た後、該カップリング化合物の蛍光強度を測定すること
により、体液中のＤ−乳酸量を求めることを特徴とする
ところのＤ−乳酸の高感度定量法である。この場合にお
いて、さらに本発明者らは、反応系にジアホラーゼとチ
オクタミドとを存在せしめ、NADHを酸化させることによ
ってＤ−LDHによるピルビン酸からＤ−乳酸生成反応即
ち逆反応を阻止し、Ｄ−乳酸からピルビン酸を生成する
反応を促進せしめたのである。
本発明において使用する体液には血液，尿等があり、
血液は通常血清、血漿等に分画したものが用いられる。
又本発明において使用するＤ−LDHは微生物起源のもの
であれ動物起源のものであれ、いずれでもよいがＬ−乳
酸に作用せずＤ−乳酸のみに特異的に作用するものが望
ましい。具体的にはスタフィロコッカス属菌から得られ
るＤ−LDH（例えば天野製薬製LDH“AMANO"）等が挙げら
れる。
本発明のピルビン酸と蛍光化合物とのカップリング反
応は塩酸酸性化で行われ、形成されたカップリング化合
物の蛍光強度を測定することによって体液中のＤ−乳酸
量が求められる。蛍光化合物として好ましいのはｏ−フ
ェニレンジアミン,4,5−ジメトキシ−1,2−ジアミノベ
ンゼン等であり、形成されるカップリング化合物として
は２−メチルキサール，ジメトキシ２−メチルキサール
等が挙げられる。
以下に本発明を試験例，実施例にて具体的に説明す
る。
試験例１ 人尿中のＤ−乳酸量とＤ−乳酸の添加回収実
験
正常な人尿0.15mlを２倍量の0.5M過塩素酸で除蛋白
し、上清を1M KOHにて中和した試料0.5mlに15nmol,30nm
ol,60nmol,90nmol量のＤ−乳酸を添加したもの及びＤ−
乳酸無添加のものを検体とした。
各検体に21mM硫酸ヒドラジン50μ,0.1Mリン酸緩衝
液（pH7.0）0.2mlを加え、37℃,30分反応させ、内因性
ピルビン酸を除去し、次いで10mM NAD 50μ,100u/ml
のＤ−LDH 50μ,26mM DL−6,8チオタミド100μ,25u
/mlのジアホラーゼ50μにて37℃,2時間反応を行い、
生じたピルビン酸を1M HCl 0.3ml,70mM o−フェニレン
ジアミンと50℃,1時間反応させることにより２−メチル
キサノールとし、このものをHPLCにて定量することによ
り検体中のＤ−乳酸量を求めた。その結果は表１に示さ
れる。
表１より明らかのように人尿中に含まれるＤ−乳酸量
は29.9nmol/0.5mlであり、又本発明の測定法によるＤ−
乳酸の人尿への添加回収は良好であった。
実施例１ 血清中のＤ−乳酸の定量法
健常人４人の食前，食後における血液を採取し、その
各血清0.15mlを２倍量の0.5M過塩素酸で除蛋白し、上清
を1M KOHにて中和した試料0.5mlに21mM硫酸ヒドラジン5
0μ,0.1Mリン酸緩衝液（pH7.0）0.2mlを加え37℃,30
分反応させて内因性ピルビン酸を除去し、次に10mM NAD
50μ,100u/mlのＤ−LDH 50μ,26mM DL−6,8チオク
タミド100μ,25u/mlのジアホラーゼ50μを添加し、
37℃,2時間反応を行い生じたピルビン酸を1M HCl 0.3m
l,70mM o−フェニレンジアミンと50℃,1時間反応させる
ことにより２−メチルキサノールとし、このものをHPLC
にて定量することにより各血清中のＤ−乳酸量を求め
た。HPLCの分析条件は次の通りである。
移動相 10mM KH2PO4（pH2.1）
（20％ CH3CNを含む）
流 速 1ml/ml
カラム UNISIL−ODS−QT 5ケ
温 度 41℃
波 長 λex341nm λem416nm
その結果は表２に示される。
表２より明らかのように食後に血清中のＤ−乳酸量が
増加することが分かる。
実施例２
ラットにストレプトゾシンを腹腔内投与し、普通食で
17日間飼育し慢性糖尿病にした後、採血を行い血清を分
取したのち実施例１の方法に準じてＤ−乳酸の定量を行
った。
その結果を表３に示す。
表３から明らかのように糖尿ラットでＤ−乳酸の値の
高い投与が得られた。
〔発明の効果〕
本発明のＤ−乳酸の高感度定量法は、体液中に微量に
存在するＤ−乳酸を正確に定量することができる。それ
故、本発明の方法は糖尿病，リンパ腫等の疾病の診断を
目的とする臨床検査の分野に使用することが可能であ
る。Description: TECHNICAL FIELD The present invention relates to a highly sensitive method for quantifying D-lactic acid, which is present in a small amount in a body fluid. The present inventors have found that D-lactic acid, which is present in a very small amount in human fluids, such as blood and urine during human health, significantly increases during diseases such as diabetes and lymphoma. Therefore, the highly sensitive method for quantifying D-lactic acid of the present invention is useful in the field of clinical tests for the purpose of diagnosing these diseases. [Prior art] Attempts have been made to measure D-lactic acid in body fluids using D-lactate dehydrogenase (hereinafter referred to as D-LDH) [for example, Methods in Enzymology Vol. 89, 35-40.
(1982)], and the reaction formula is shown below. However, in the conventional measurement method, NADH generated in the reaction formula is directly measured in the ultraviolet (for example, at a wavelength of 340 nm), or NADH is colored with various coloring agents (for example, formazan dye) and measured in the visible region. Attempts have been made to do so. [Problems of the prior art] However, D-
Since the method of quantifying lactic acid is too low in sensitivity, it is necessary to increase the number of measurement samples of body fluids in order to quantify a very small amount of D-lactic acid. It has never been possible to use it in the field of clinical tests which need to be measured. [Means for Solving the Problems] Accordingly, the present inventors have conducted intensive studies to find a method capable of quantifying a trace amount of D-lactic acid with high sensitivity. As a result, a fluorescent compound was coupled to the generated pyruvic acid, and the coupling was performed. They have found a method for quantifying D-lactic acid with high sensitivity by quantifying the fluorescence intensity of the compound. This makes it possible to accurately measure D-lactic acid, which is present in a very small amount in the body fluid of a healthy person, and to detect D-lactic acid in the body fluid due to various diseases.
When the amount of lactic acid was increased, it was possible to make a diagnosis by comparing it with the value of a healthy person. That is, the present invention relates to
In the presence of D, D-LDH is acted to generate pyruvate and NADH, and then react the generated pyruvate with a fluorescent compound to form a coupling compound.After that, the fluorescence intensity of the coupling compound is reduced. This is a highly sensitive method for determining D-lactic acid, characterized in that the amount of D-lactic acid in a body fluid is determined by measurement. In this case, the present inventors further confirmed that diaphorase and thioctamide were present in the reaction system and oxidized NADH to thereby prevent the D-LDH-forming reaction from pyruvate by D-LDH, that is, the reverse reaction. This accelerated the reaction to produce pyruvic acid from pulp. The body fluid used in the present invention includes blood, urine, etc.
Normally, blood fractionated into serum, plasma and the like is used.
The D-LDH used in the present invention may be of microbial origin or animal origin, but it is preferred that it does not act on L-lactic acid but specifically acts only on D-lactic acid. Specific examples include D-LDH (for example, LDH “AMANO” manufactured by Amano Pharmaceutical Co.) obtained from Staphylococcus sp. The coupling reaction between pyruvic acid and the fluorescent compound of the present invention is performed by acidification with hydrochloric acid, and the amount of D-lactic acid in the body fluid is determined by measuring the fluorescence intensity of the formed coupling compound. Preferred examples of the fluorescent compound include o-phenylenediamine, 4,5-dimethoxy-1,2-diaminobenzene and the like, and examples of the coupling compound formed include 2-methylxal and dimethoxy-2-methylxal. Hereinafter, the present invention will be described specifically with reference to Test Examples and Examples. Test Example 1 D-lactic acid content in human urine and addition and recovery experiment of D-lactic acid 0.15 ml of normal human urine was deproteinized with twice the volume of 0.5 M perchloric acid, and the supernatant was neutralized with 1 M KOH. 15 nmol, 30 nm in 0.5 ml sample
ol, 60 nmol, 90 nmol amount of D-lactic acid added and D-lactic acid
A sample without lactic acid was used as a sample. To each sample was added 50 μl of 21 mM hydrazine sulfate, 0.2 ml of 0.1 M phosphate buffer (pH 7.0), and reacted at 37 ° C. for 30 minutes to remove endogenous pyruvate. Then, 10 mM NAD 50 μ, 100 u / ml
D-LDH 50μ, 26mM DL-6,8 thiotamide 100μ, 25u
Reaction at 37 ° C for 2 hours with 50 μl / ml diaphorase,
The resulting pyruvic acid was reacted with 0.3 ml of 1M HCl and 70 mM o-phenylenediamine at 50 ° C. for 1 hour to give 2-methylxanol, which was quantified by HPLC to determine the amount of D-lactic acid in the sample. I asked. The results are shown in Table 1. As is clear from Table 1, the amount of D-lactic acid contained in human urine is 29.9 nmol / 0.5 ml, and the amount of D-lactic acid measured by the method of the present invention is
The recovery of lactic acid added to human urine was good. Example 1 Method for Quantifying D-Lactic Acid in Serum Blood of four healthy subjects before and after meals was collected, 0.15 ml of each serum was deproteinized with twice the amount of 0.5M perchloric acid, and the supernatant was concentrated to 1M. 21 mM hydrazine sulfate 5 in 0.5 ml of sample neutralized with KOH
Add 0.2 ml of 0μ, 0.1M phosphate buffer (pH 7.0) and add
Reaction to remove endogenous pyruvate, then 10 mM NAD
50μ, 100u / ml D-LDH 50μ, 26mM DL-6,8 thiooctamide 100μ, 25u / ml diaphorase 50μ were added,
Pyruvic acid produced by reaction at 37 ° C for 2 hours is converted into 1M HCl 0.3m
l-70 mM o-phenylenediamine to give 2-methylxanol by reacting at 50 ° C. for 1 hour,
The amount of D-lactic acid in each serum was determined by quantification. The HPLC analysis conditions are as follows. Mobile phase 10 mM KH 2 PO 4 (pH 2.1) (including 20% CH 3 CN) Flow rate 1 ml / ml Column UNISIL-ODS-QT 5 temperature 41 ° C Wavelength λ ex 341 nm λ em 416 nm As shown in FIG. As is clear from Table 2, the amount of D-lactic acid in the serum increases after eating. Example 2 Streptozocin was intraperitoneally administered to rats and given a normal diet
After breeding for 17 days with chronic diabetes, blood was collected and serum was collected, and the amount of D-lactic acid was determined according to the method of Example 1. Table 3 shows the results. As is apparent from Table 3, administration of high D-lactic acid was obtained in diabetic rats. [Effects of the Invention] The highly sensitive method for quantifying D-lactic acid of the present invention enables accurate quantification of D-lactic acid present in minute amounts in body fluids. Therefore, the method of the present invention can be used in the field of clinical tests for the purpose of diagnosing diseases such as diabetes and lymphoma.
Claims (1)
せ、ピルビン酸とNADHを生成せしめ、次いでピルビン酸
と蛍光化合物とを反応させることによって得られるカッ
プリング化合物の蛍光強度をHPLCを用いて測定すること
により体液中のＤ−乳酸量を求めることを特徴とするＤ
−乳酸の高感度定量法。 ２．反応系にジアホラーゼとチオクタミドとを存在せし
め、NADHを酸化させることによってＤ−乳酸からピルビ
ン酸の生成を促進せしめることを特徴とする特許請求の
範囲第１項記載のＤ−乳酸の高感度定量法。 ３．蛍光化合物がｏ−フェニレンジアミン又は4,5−ジ
メトキシ1,2−ジアミノベンゼンであるところの特許請
求の範囲第１項記載のＤ−乳酸の高感度定量法。 ４．カップリング化合物が２−メチルキサノール又はジ
メトキシ２−メチルキサノールであるところの特許請求
の範囲第１項記載のＤ−乳酸の高感度定量法。(57) [Claims] In the presence of NAD, D-lactate dehydrogenase is allowed to act on bodily fluids to generate pyruvate and NADH, and then the fluorescence intensity of the coupling compound obtained by reacting pyruvate with the fluorescent compound is measured using HPLC. The amount of D-lactic acid in the body fluid by calculating
-A sensitive method for the determination of lactic acid. 2. 2. A highly sensitive method for quantifying D-lactic acid according to claim 1, wherein diaphorase and thioctamide are present in the reaction system, and the production of pyruvate from D-lactic acid is promoted by oxidizing NADH. . 3. 2. The method for highly sensitive determination of D-lactic acid according to claim 1, wherein the fluorescent compound is o-phenylenediamine or 4,5-dimethoxy-1,2-diaminobenzene. 4. 2. The method for highly sensitive determination of D-lactic acid according to claim 1, wherein the coupling compound is 2-methylxanol or dimethoxy-2-methylxanol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33138087A JP2739473B2 (en) | 1987-12-26 | 1987-12-26 | Highly sensitive measurement method for D-lactic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP33138087A JP2739473B2 (en) | 1987-12-26 | 1987-12-26 | Highly sensitive measurement method for D-lactic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01171498A JPH01171498A (en) | 1989-07-06 |
| JP2739473B2 true JP2739473B2 (en) | 1998-04-15 |
Family
ID=18243040
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP33138087A Expired - Fee Related JP2739473B2 (en) | 1987-12-26 | 1987-12-26 | Highly sensitive measurement method for D-lactic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2739473B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH1033196A (en) * | 1996-07-23 | 1998-02-10 | Unitika Ltd | Test piece |
| US6030802A (en) * | 1998-05-29 | 2000-02-29 | Roche Diagnostics Corporation | Liquid reagent set for L-lactate determination |
| JP3707479B2 (en) | 2002-04-26 | 2005-10-19 | ヤマハ株式会社 | Drum head tensioning device |
| CN109342634A (en) * | 2018-12-07 | 2019-02-15 | 西北大学 | A kind of method of column front derivation-HPLC measurement DXS enzymatic activity |
-
1987
- 1987-12-26 JP JP33138087A patent/JP2739473B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01171498A (en) | 1989-07-06 |
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