JPH0738798B2 - Pretreatment method for urine samples - Google Patents
Pretreatment method for urine samplesInfo
- Publication number
- JPH0738798B2 JPH0738798B2 JP3519287A JP3519287A JPH0738798B2 JP H0738798 B2 JPH0738798 B2 JP H0738798B2 JP 3519287 A JP3519287 A JP 3519287A JP 3519287 A JP3519287 A JP 3519287A JP H0738798 B2 JPH0738798 B2 JP H0738798B2
- Authority
- JP
- Japan
- Prior art keywords
- urine sample
- polyamine
- free
- acid
- pretreatment
- 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 - Lifetime
Links
- 210000002700 urine Anatomy 0.000 title claims description 67
- 238000002203 pretreatment Methods 0.000 title claims description 5
- 229920000768 polyamine Polymers 0.000 claims description 68
- 238000000034 method Methods 0.000 claims description 39
- 230000001603 reducing effect Effects 0.000 claims description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 16
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 16
- 108010019718 putrescine oxidase Proteins 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 14
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 9
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 9
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 8
- 108010065763 Aminobutyraldehyde dehydrogenase Proteins 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 230000003301 hydrolyzing effect Effects 0.000 claims 2
- 238000005259 measurement Methods 0.000 description 12
- 239000008363 phosphate buffer Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000000975 dye Substances 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 238000010306 acid treatment Methods 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 description 5
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 238000004040 coloring Methods 0.000 description 4
- 230000001590 oxidative effect Effects 0.000 description 4
- FGEPRNXUNITOCW-UHFFFAOYSA-N 2-aminobutanal Chemical compound CCC(N)C=O FGEPRNXUNITOCW-UHFFFAOYSA-N 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 2
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102100037209 Peroxisomal N(1)-acetyl-spermine/spermidine oxidase Human genes 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- 238000003381 deacetylation reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 108010089000 polyamine oxidase Proteins 0.000 description 2
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 108010024957 Ascorbate Oxidase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- GUNURVWAJRRUAV-UHFFFAOYSA-N N(1)-acetylspermine Chemical compound CC(=O)NCCCNCCCCNCCCN GUNURVWAJRRUAV-UHFFFAOYSA-N 0.000 description 1
- FONIWJIDLJEJTL-UHFFFAOYSA-N N(8)-acetylspermidine Chemical compound CC(=O)NCCCCNCCCN FONIWJIDLJEJTL-UHFFFAOYSA-N 0.000 description 1
- KLZGKIDSEJWEDW-UHFFFAOYSA-N N-acetylputrescine Chemical compound CC(=O)NCCCCN KLZGKIDSEJWEDW-UHFFFAOYSA-N 0.000 description 1
- 102100033153 NADH-cytochrome b5 reductase 3 Human genes 0.000 description 1
- 101710173867 NADH-cytochrome b5 reductase 3 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 101710132584 Peroxidase 3 Proteins 0.000 description 1
- 239000005700 Putrescine Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- -1 accepts electrons Chemical compound 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 229940067621 aminobutyrate Drugs 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は尿検体の前処理法に関し、特に尿中のポリアミ
ンの測定において、光学的測定に妨害を与える尿検体中
の還元性成分、例えばアスコルビン酸などの除去を行
い、尿中のポリアミン、すなわち遊離ポリアミンまたは
総ポリアミンの光学的測定を可能ならしめる方法に関す
る。 臨床検査診断学においては、尿中のポリアミンの増加
は、ガンの指標として注目されているので、精度のよい
測定法を提供することは本分野の進歩に供する所が大き
い。The present invention relates to a pretreatment method for a urine sample, particularly in the measurement of urinary polyamine, removal of reducing components such as ascorbic acid in the urine sample that interferes with optical measurement, and polyamines in urine, that is, It relates to a method which allows the optical determination of free or total polyamines. In clinical laboratory diagnostics, an increase in urinary polyamine has attracted attention as an indicator of cancer, and therefore providing an accurate measurement method is largely used for progress in this field.
尿中のポリアミンの測定法は、抱合体ポリアミン（例え
ばアセチルプトレシン、アセチルスペルミジン、アセチ
ルスペルミンなどのアシルポリアミン）のアミド結合を
加水分解し、遊離ポリアミンを生成するデアセチラーゼ
（アシルポリアミンアミドヒドロラーゼ）の発見とその
実用化に関わる発明により大きく進歩をとげた（特公昭
59−7435号公報参照）。すなわち尿検体中の抱合体ポリ
アミンをデアセチラーゼにより遊離ポリアミンに変え、
尿検体中の遊離ポリアミンとともに測定する総ポリアミ
ンの測定法が新しく開発された。この方法は、尿検体中
の遊離ポリアミンまたは抱合体ポリアミンからの遊離ポ
リアミンにプトレシンオキシダーゼなどのポリアミンオ
キシダーゼを作用させ、生成する過酸化水素をペルオキ
シダーゼにより測定する方法（特公昭62−679号公報参
照）またはこれらの遊離ポリアミンにプトレシンオキシ
ダーゼなどのポリアミンオキシダーゼを作用させ、生成
するアミノブチルアルデヒドにアミノブチルアルデヒド
デヒドロゲナーゼおよびNADを作用させ、生成するNADH
を還元性発色性色素、例えばジホルマザン発色系にて測
定する方法（特願昭61−279425号）などがある。 しかし実際には尿検体中の抱合体ポリアミンをデアセチ
ラーゼにより遊離ポリアミンとしても、該遊離ポリアミ
ンを陽イオン交換体カラムにより吸着させ、還元性成分
などの他の成分と分離してから遊離ポリアミンを測定す
る方法が実用化されているにすぎない。The method for measuring polyamines in urine is based on the discovery of a deacetylase (acyl polyamine amide hydrolase) that hydrolyzes the amide bond of a conjugated polyamine (eg, an acyl polyamine such as acetylputrescine, acetylspermidine, and acetylspermine) to produce a free polyamine. Significant progress was made due to inventions related to its practical application
59-7435). That is, the conjugated polyamine in the urine sample is converted to free polyamine by deacetylase,
A new method for the determination of total polyamines with free polyamines in urine samples has been developed. This method is a method in which a polyamine oxidase such as putrescine oxidase is allowed to act on a free polyamine in a urine specimen or a free polyamine from a conjugated polyamine, and the produced hydrogen peroxide is measured by peroxidase (see JP-B-62-679). Alternatively, a polyamine oxidase such as putrescine oxidase is allowed to act on these free polyamines, and the resulting aminobutyraldehyde is allowed to act with aminobutyraldehyde dehydrogenase and NAD to produce NADH.
Is measured with a reducing color forming dye, for example, a diformazan coloring system (Japanese Patent Application No. 61-279425). However, in practice, even if the conjugated polyamine in the urine sample is made into a free polyamine by deacetylase, the free polyamine is adsorbed by a cation exchanger column and separated from other components such as a reducing component, and then the free polyamine is measured. The method has just been put to practical use.
尿中の還元性成分、例えばアスコルビン酸などはペルオ
キシダーゼによる酸化発色系を抑制し、酸化発色をしな
い場合がほとんどである。また、還元性色素、例えばジ
ホルマザン発色系は過剰な還元が生じ、検体ブランクの
高値と検体によるブランクの不同性のため、測定が実際
上出来ない。 本発明者らの知見によれば、尿中のアスコルビン酸の測
定系妨害は極めて大きいが、アスコルビン酸オキシダー
ゼにより簡単に除去される。しかしなお還元性成分が残
り、色素系を還元して妨害を与える。この還元性成分は
フェナジンメトサルフェート（PMS）やフラビン類を存
在させて、生成する過酸化水素をカタラーゼを介して除
去する方法でも極めて時間を要する。 したがって、本発明では尿中のポリアミンを他の成分と
分離するカラム操作を含まず、手軽な操作で測定におけ
る妨害物質を除去する方法を提供する。In most cases, reducing components in urine, such as ascorbic acid, suppress the oxidative coloring system caused by peroxidase and do not cause oxidative coloring. In addition, reducing dyes, for example, diformazan color-developing system, cause excessive reduction, and cannot be measured practically due to the high value of the sample blank and the disparity of the sample blank. According to the findings of the present inventors, the interference of ascorbic acid in urine with the measurement system is extremely large, but it is easily removed by ascorbate oxidase. However, the reducing component still remains, reducing the dye system and interfering. This reducing component requires phenazine methosulfate (PMS) and flavins, and the hydrogen peroxide produced is removed via catalase. Therefore, the present invention provides a method for removing an interfering substance in measurement by a simple operation without including a column operation for separating polyamine in urine from other components.
すなわち本発明は尿検体中のポリアミンを光学的に測定
する前の尿検体の処理法において、尿検体に塩酸、トリ
クロル酢酸またはポリクロル酢酸をを加えて、pH3以下
とすることにより、還元性成分を除去することを特徴と
する尿検体の前処理法である。 尿検体は一般にはpHが約５〜９であるが、本発明におい
ては、尿検体に塩酸、トリクロル酢酸またはポリクロル
酢酸をを加えて、pH3以下、望ましくはpH2以下にする。 具体的には尿検体中の遊離ポリアミンを光学的に測定す
る前の尿検体の処理法においては、尿検体に塩酸、トリ
クロル酢酸またはポリクロル酢酸をを加えて、pH3以下
とすることにより、還元性成分を除去する。 また尿検体中の総ポリアミンを光学的に測定する前の尿
検体の処理法においては、尿検体にリン酸緩衝液を加え
てpH7〜８に調整し、デアセチラーゼを加えて反応さ
せ、抱合体ポリアミンを遊離ポリアミンへ加水分解させ
た後、塩酸、トリクロル酢酸またはポリクロル酢酸をを
加えて、pH3以下とすることにより、還元性成分を除去
する。 デアセチラーゼを加えて、脱アセチル化を行う場合に
は、デアセチラーゼの作用性のよいpH7〜８になるよう
にリン酸緩衝液を加えた後、デアセチラーゼを10〜100
単位（Ｕ）/mlの範囲で加える。20U/ml以上加えた場合
は脱アセチル化反応は５分程度で完結する。反応後、酸
を加えてよく混合してpH3以下にする。ポリアミン（抱
合体ポリアミンおよび遊離ポリアミン）は尿中にもとも
と少量しか存在しないので尿検体中に加える緩衝液や酸
あるいはデセチラーゼは出来るだけ高濃度にし、尿検体
の容量が増えないようにする。 デアセチラーゼとしては、特公昭59−7435号公報に記載
されるアシルポリアミンアミドデヒドロゲナーゼなどが
例示される。 尿検体中の遊離ポリアミンを光学的に測定する方法とし
ては、遊離ポリアミンにプトレシンオキシダーゼを作用
させ、生成する過酸化水素を測定する方法、または遊離
ポリアミンにプトレシンオキシダーゼ、アミノブチルア
ルデヒドデヒドロゲナーゼおよびNAD+を作用させ、生成
するNADHを測定する方法がある。 また尿検体中の総ポリアミンを光学的に測定する方法と
しては、尿検体にデアセチラーゼを加えて反応させ、抱
合体ポリアミンを遊離ポリアミンへ加水分解させた後、
遊離ポリアミンにプトレシンオキシダーゼを作用させ、
生成する過酸化水素を測定する方法、または尿検体にデ
アセチラーゼを加えて反応させ、抱合体ポリアミンを遊
離ポリアミンへ水解させた後、遊離ポリアミンにプトレ
シンオキシダーゼ、アミノブチルアルデヒドデヒドロゲ
ナーゼおよびNAD+を作用させ、生成するNADHを測定する
方法がある。 NADHを測定する方法のひとつとしては、ジアフォラーゼ
およびホルマザン色素、MTT（３−4,5−ジメチルチアゾ
ール−２−イル）−2,5−ジフェニルテトラゾール−２
−イル）を使用する方法が挙げられる。また生成するNA
DHの波長340nmの吸光度を測定する方法もある。 アミノブチルアルデヒドデヒドロゲナーセは、NAD+とと
もに、アミノブチルアルデヒドに作用して、アミノブチ
レートとNADHを生成する。 ジアフォラーゼはNADHの作用し、電子を受け取り、NAD+
へと変換する酵素であり、いかなる起源のものでも好適
に利用できる。ホルマン色素、MTTはジアフォラーゼに
より電子を受け取り、ジホルマザンとなり発色する。 プトレシンオキシダーゼは、プトレシンに作用して、ア
ミノブチルアルデヒドと過酸化水素を生成する。生成し
た過酸化水素を測定する方法としては、ペルオキシダー
ゼ、４−アミノアンチピリン、EHSPT［Ｎ−エチル−Ｎ
−（２−ヒドロキシ−３−スルホプロピル）−ｍ−トル
イジン］などの酸化発色性色素を作用させ、発色した色
素の吸光度変化を測定する方法が例示される。That is, the present invention is a method for treating a urine sample before optically measuring polyamines in the urine sample, by adding hydrochloric acid, trichloroacetic acid or polychloroacetic acid to the urine sample, and by adjusting the pH to 3 or less, a reducing component is obtained. It is a pretreatment method for a urine sample characterized by removal. Generally, a urine sample has a pH of about 5 to 9, but in the present invention, hydrochloric acid, trichloroacetic acid or polychloroacetic acid is added to the urine sample to adjust the pH to 3 or less, preferably pH 2 or less. Specifically, in the method for treating a urine sample before optically measuring free polyamine in the urine sample, hydrochloric acid, trichloroacetic acid or polychloroacetic acid is added to the urine sample to adjust the pH to 3 or less, thereby reducing Remove the ingredients. Further, in the method for treating a urine sample before optically measuring the total polyamine in the urine sample, a phosphate buffer is added to the urine sample to adjust the pH to 7-8, and deacetylase is added to react the urine sample with the conjugated polyamine. Is hydrolyzed to a free polyamine, and hydrochloric acid, trichloroacetic acid or polychloroacetic acid is added to adjust the pH to 3 or less to remove the reducing component. When deacetylation is performed by adding deacetylase, a phosphate buffer is added so that the pH of the deacetylase having good activity is 7 to 8, and then 10 to 100 of the deacetylase is added.
Add in the range of unit (U) / ml. When 20 U / ml or more is added, the deacetylation reaction is completed in about 5 minutes. After the reaction, add acid and mix well to bring the pH to 3 or less. Since polyamines (conjugated polyamines and free polyamines) are originally present in a small amount in urine, the buffer solution, acid or deacetylase added to the urine sample should be at a concentration as high as possible to prevent the volume of the urine sample from increasing. Examples of the deacetylase include acylpolyamine amide dehydrogenase described in JP-B-59-7435. As a method for optically measuring free polyamine in a urine sample, putrescine oxidase is allowed to act on the free polyamine to measure the hydrogen peroxide produced, or free polyamine is treated with putrescine oxidase, aminobutyraldehyde dehydrogenase and NAD + . There is a method to act and measure the produced NADH. As a method for optically measuring the total polyamine in the urine sample, a deacetylase is added to the urine sample and reacted to hydrolyze the conjugated polyamine into a free polyamine,
Let putrescine oxidase act on free polyamine,
A method of measuring the hydrogen peroxide produced, or by adding a deacetylase to a urine sample and reacting it to hydrolyze the conjugated polyamine to a free polyamine, and then letting putrescine oxidase, aminobutyraldehyde dehydrogenase and NAD + act on the free polyamine, There is a method to measure the NADH produced. One of the methods for measuring NADH is diaphorase and formazan dye, MTT (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole-2.
-Yl). Also generate NA
There is also a method of measuring the absorbance of DH at a wavelength of 340 nm. Aminobutyraldehyde dehydrogenase, together with NAD + , acts on aminobutyraldehyde to produce aminobutyrate and NADH. Diaphorase acts as NADH, accepts electrons, and NAD +
It is an enzyme that converts into the enzyme and can be suitably used from any origin. The formant dye, MTT, receives electrons by diaphorase and becomes diformazane, which develops color. Putrescine oxidase acts on putrescine to produce aminobutyraldehyde and hydrogen peroxide. As a method for measuring the produced hydrogen peroxide, peroxidase, 4-aminoantipyrine, EHSPT [N-ethyl-N
Examples include a method in which an oxidative color-forming dye such as-(2-hydroxy-3-sulfopropyl) -m-toluidine] is allowed to act and the change in absorbance of the color-developed dye is measured.
本発明では尿検体に塩酸、トリクロル酢酸またはポリク
ロル酢酸を添加して、pH3以下とすることにより、ポリ
アミン（遊離ポリアミンまたは総ポリアミン）の測定に
影響を与えないで、測定の妨害となる還元性成分を消去
することができる。また本発明では尿検体中のポリアミ
ンを他の成分と分離する操作を含まず、手軽な操作で測
定における妨害を除去することができる。さらに本発明
では尿検体に酸を加えて１分を越えない時間範囲で、pH
3以下とすることにより、尿検体中のポリアミンが還元
性成分の妨害なく測定できる。In the present invention, hydrochloric acid, trichloroacetic acid or polychloroacetic acid is added to the urine sample to adjust the pH to 3 or less, thereby not affecting the measurement of polyamine (free polyamine or total polyamine) and reducing components that interfere with the measurement. Can be erased. Further, the present invention does not include the operation of separating the polyamine in the urine sample from other components, and the interference in the measurement can be removed by a simple operation. Furthermore, in the present invention, pH is added to the urine sample within a time range not exceeding 1 minute by adding acid.
By setting it to 3 or less, the polyamine in the urine sample can be measured without interfering with the reducing component.
以下に尿検体中の遊離ポリアミンを測定する場合と抱合
体を含む総ポリアミンを測定する場合について、比較例
と実施例で説明する。 比較例１ 尿検体1mlをとり、0.2mlの0.1Mリン酸緩衝液pH7.5を加
えた。デアセチラーゼ処理を行う場合は120U/mlのデア
セチラーゼを0.1Mリン酸緩衝液に加えて37℃で５分以上
反応させた。更に蒸留水0.2mlを加えて測定試料とし
た。 測定は、以下の反応組成で行った。 試液1. リン酸緩衝液、pH7.0 （含 0.1％ Triton Ｘ−100） 100 mM NAD+ １ mM アミノブチルアルデヒドデヒドロゲナーゼ 0.5 U/ml ジアフォラーゼ １ U/ml MTT 0.03％ 試液2. リン酸緩衝液、pH7.0 100mM プトレシンオキシダーゼ 5U/ml 試液１、2.7mlに0.2mlの尿検体を加え、５分間、37℃、
565nmの吸光度をトレースし、0.1mlのプトレシンオキシ
ダーゼ（試液２）を加えた。図１に反応の吸光度変化を
示す。Ａはデアセチラーゼ処理しない尿検体、Ｂはデア
セチラーゼ処理した尿検体を示す。 実施例１ 尿検体1mlをとり、0.2mlの0.1Mリン酸緩衝液、ph7.5を
加えた。デアセチラーゼ処理を行う時は120U/mlのデア
セチラーゼを0.1Mリン酸緩衝液に加えて添加した。37℃
で５分以上反応させた後、0.2mlの0.5Mトリスクロル酢
酸（TCA）を加えて測定試料とした。測定法、組成は比
較例と同じである。結果は図２に示すが反応は定常的発
色を示し、検体ブランクの増加が生じないことを示して
いる。Ａはデセチラーゼ処理なし、Ｂはデセチラーゼ処
理をした場合を示している。この方法により尿中ポリア
ミンはカラムによる分離を要せず、正確に測定される。
前処理検体を用いれば測定は１ステップで済み、自動分
析機に適用される。 実施例２ ペルオキシダーゼを用いた酸化発色について、尿検体の
酸処理による測定例を示す。前処理は実施例１に示す方
法による。 試液1. リン酸緩衝液、pH7.0 100 mM ４−アミノアンチピリン 0.04％ ペルオキシダーゼ ３ U/ml 試液2. リン酸緩衝液、pH7.0 100 mM プトレシンオキシダーゼ ５ U/ml EHSPT 0.02％ 試液１、2.7mlに0.2mlの処理検体を加え、５分、37℃、
553nmで測定し、0.1mlのプトレシンオキシダーゼ（試液
２）を加えて更に５分反応させた。図3Aは酸処理しない
場合、ＢとＣはデセチラーゼ処理をせず酸処理をした場
合、及びデアセチラーゼ処理後酸処理をした場合を示
す。 図３より、酸処理をしない検体ではほとんど発色を認め
なかった。酸処理をした試料では定常的な発色を認め
た。The case of measuring the free polyamine in the urine sample and the case of measuring the total polyamine including the conjugate will be described below in Comparative Examples and Examples. Comparative Example 1 1 ml of a urine sample was taken and 0.2 ml of 0.1 M phosphate buffer pH 7.5 was added. When performing deacetylase treatment, 120 U / ml of deacetylase was added to 0.1 M phosphate buffer and reacted at 37 ° C. for 5 minutes or more. Further, 0.2 ml of distilled water was added to prepare a measurement sample. The measurement was performed with the following reaction composition. Reagent 1. Phosphate buffer, pH 7.0 (containing 0.1% Triton X-100) 100 mM NAD + 1 mM aminobutyraldehyde dehydrogenase 0.5 U / ml diaphorase 1 U / ml MTT 0.03% Reagent 2. phosphate buffer, pH7.0 100mM putrescine oxidase 5U / ml Reagent solution 1, 2.7ml, 0.2ml urine sample was added, 5 minutes, 37 ℃,
The absorbance at 565 nm was traced, and 0.1 ml of putrescine oxidase (Test solution 2) was added. FIG. 1 shows the change in absorbance of the reaction. A indicates a urine sample not treated with deacetylase, and B indicates a urine sample treated with deacetylase. Example 1 1 ml of a urine sample was taken, and 0.2 ml of 0.1 M phosphate buffer, ph7.5 was added. When carrying out the deacetylase treatment, 120 U / ml of deacetylase was added in addition to 0.1 M phosphate buffer. 37 ° C
After reacting for 5 minutes or longer, 0.2 ml of 0.5 M trischloroacetic acid (TCA) was added to prepare a measurement sample. The measuring method and composition are the same as those of the comparative example. The results are shown in FIG. 2 and show that the reaction shows a steady color development and no increase in analyte blank occurs. A shows the case without deacetylase treatment, and B shows the case with deacetylase treatment. By this method, urinary polyamine can be accurately measured without the need for column separation.
If the pretreated sample is used, the measurement is completed in one step and applied to the automatic analyzer. Example 2 An example of measurement of oxidative coloring using peroxidase by acid treatment of a urine sample will be shown. The pretreatment is according to the method shown in Example 1. Reagent 1. Phosphate buffer, pH 7.0 100 mM 4-aminoantipyrine 0.04% Peroxidase 3 U / ml Reagent 2. Phosphate buffer, pH 7.0 100 mM putrescine oxidase 5 U / ml EHSPT 0.02% Reagent 1, 2.7 Add 0.2 ml of the treated sample to 5 ml at 37 ° C for 5 minutes.
It was measured at 553 nm, 0.1 ml of putrescine oxidase (reagent solution 2) was added, and the reaction was continued for 5 minutes. FIG. 3A shows the case without acid treatment, B and C with acid treatment without deacetylase treatment, and with acid treatment after deacetylase treatment. From FIG. 3, almost no color development was observed in the sample not treated with acid. A steady color was observed in the acid-treated sample.
図1:ホルマザン系発色による酸処理をしない尿検体での
吸光度変化 図2:ホルマザン系発色による酸処理をした尿検体での吸
光度変化 図3:ペルオキシダーゼ発色系における吸光度変化Figure 1: Absorbance change in urine sample without acid treatment due to formazan color development Figure 2: Absorbance change in urine sample after acid treatment with formazan color development Figure 3: Absorbance change in peroxidase color development system
Claims (8)
前の尿検体の処理法において、尿検体に塩酸、トリクロ
ル酢酸またはポリクロル酢酸をを加えて、pH3以下とす
ることにより、還元性成分を除去することを特徴とする
尿検体の前処理法。1. A method for treating a urine sample before optically measuring polyamines in the urine sample, by adding hydrochloric acid, trichloroacetic acid or polychloroacetic acid to the urine sample to adjust the pH to 3 or less, thereby reducing components. A method for pretreatment of a urine sample, which comprises removing
アミンであることを特徴とする特許請求の範囲第１項記
載の尿検体の前処理法。2. The method for pretreatment of a urine sample according to claim 1, wherein the polyamine is a free polyamine or a total polyamine.
する前の尿検体の処理法において、尿検体に塩酸、トリ
クロル酢酸またはポリクロル酢酸をを加えて、pH3以下
とすることにより、還元性成分を除去することを特徴と
する特許請求の範囲第２項記載の尿検体の前処理法。3. A method for treating a urine sample before optically measuring free polyamine in the urine sample, by adding hydrochloric acid, trichloroacetic acid or polychloroacetic acid to the urine sample to adjust the pH to 3 or less, the reducing property is improved. The method for pretreatment of a urine sample according to claim 2, wherein the component is removed.
る前の尿検体の処理法において、尿検体をpH7〜８に調
整し、デアセチラーゼを加えて反応させ、抱合体ポリア
ミンを遊離ポリアミンへ加水分解させた後、塩酸、トリ
クロル酢酸またはポリクロル酢酸をを加えて、pH3以下
とすることにより、還元性成分を除去することを特徴と
する特許請求の範囲第２項記載の尿検体の前処理法。4. A method for treating a urine sample before optically measuring total polyamines in the urine sample, wherein the urine sample is adjusted to pH 7 to 8 and deacetylase is added and reacted to convert the conjugated polyamine into a free polyamine. The pretreatment of the urine sample according to claim 2, characterized in that after reducing the reducing component by adding hydrochloric acid, trichloroacetic acid or polychloroacetic acid to adjust the pH to 3 or less. Law.
する方法が、遊離ポリアミンにプトレシンオキシダーゼ
を作用させ、生成する過酸化水素を測定する方法である
特許請求の範囲第３項記載の尿検体の前処理法。5. The urine according to claim 3, wherein the method for optically measuring the free polyamine in the urine sample is a method for allowing putrescine oxidase to act on the free polyamine and measuring the hydrogen peroxide produced. Sample pretreatment method.
する方法が、遊離ポリアミンにプトレシンオキシダー
ゼ、アミノブチルアルデヒドデヒドロゲナーゼおよびNA
D+を作用させ、生成するNADHを測定する方法である特許
請求の範囲第３項記載の尿検体の前処理法。6. A method for optically measuring free polyamine in a urine sample is a method in which free polyamine is putrescine oxidase, aminobutyraldehyde dehydrogenase and NA.
The method for pretreatment of a urine sample according to claim 3, which is a method of measuring NADH produced by allowing D + to act.
る方法が、尿検体にデアセチラーゼを加えて反応させ、
抱合体ポリアミンを遊離ポリアミンへ加水分解させた
後、遊離ポリアミンにプトレシンオキシダーゼを作用さ
せ、生成する過酸化水素を測定する方法である特許請求
の範囲第４項記載の尿検体の前処理法。7. A method for optically measuring total polyamines in a urine sample is the reaction of adding deacetylase to a urine sample,
The method for pretreatment of a urine sample according to claim 4, which is a method of hydrolyzing the conjugated polyamine to free polyamine, and then allowing putrescine oxidase to act on the free polyamine to measure the hydrogen peroxide produced.
る方法が、尿検体にデアセチラーゼを加えて反応させ、
抱合体ポリアミンを遊離ポリアミンへ水解させた後、遊
離ポリアミンにプトレシンオキシダーゼ、アミノブチル
アルデヒドデヒドロゲナーゼおよびNAD+を作用させ、生
成するNADHを測定する方法である特許請求の範囲第４項
記載の尿検体の前処理法。8. A method of optically measuring total polyamines in a urine sample is the reaction of adding deacetylase to a urine sample,
The method for measuring the amount of NADH produced by hydrolyzing a conjugated polyamine into free polyamine, and then allowing putrescine oxidase, aminobutyraldehyde dehydrogenase and NAD + to act on the free polyamine to measure the produced NADH. Pretreatment method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3519287A JPH0738798B2 (en) | 1987-02-17 | 1987-02-17 | Pretreatment method for urine samples |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3519287A JPH0738798B2 (en) | 1987-02-17 | 1987-02-17 | Pretreatment method for urine samples |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63202399A JPS63202399A (en) | 1988-08-22 |
| JPH0738798B2 true JPH0738798B2 (en) | 1995-05-01 |
Family
ID=12434989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3519287A Expired - Lifetime JPH0738798B2 (en) | 1987-02-17 | 1987-02-17 | Pretreatment method for urine samples |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0738798B2 (en) |
-
1987
- 1987-02-17 JP JP3519287A patent/JPH0738798B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63202399A (en) | 1988-08-22 |
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