JPH0218840B2 - - Google Patents
Info
- Publication number
- JPH0218840B2 JPH0218840B2 JP56143924A JP14392481A JPH0218840B2 JP H0218840 B2 JPH0218840 B2 JP H0218840B2 JP 56143924 A JP56143924 A JP 56143924A JP 14392481 A JP14392481 A JP 14392481A JP H0218840 B2 JPH0218840 B2 JP H0218840B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- hydrogen peroxide
- peroxidase
- buffer
- compound
- 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
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 31
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- 102000003992 Peroxidases Human genes 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 14
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 13
- 239000000126 substance Substances 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000003086 colorant Substances 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims 1
- 238000000034 method Methods 0.000 description 11
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 9
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 9
- 229940116269 uric acid Drugs 0.000 description 9
- 238000005259 measurement Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 238000011088 calibration curve Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 108010092464 Urate Oxidase Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000013060 biological fluid Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 238000009833 condensation Methods 0.000 description 3
- 230000005494 condensation Effects 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 108010000659 Choline oxidase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- -1 aniline compound Chemical class 0.000 description 2
- 235000012000 cholesterol Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000003925 fat Substances 0.000 description 2
- 235000021588 free fatty acids Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 125000000542 sulfonic acid group Chemical group 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- PHOLIFLKGONSGY-CSKARUKUSA-N (e)-(3-methyl-1,3-benzothiazol-2-ylidene)hydrazine Chemical compound C1=CC=C2S\C(=N\N)N(C)C2=C1 PHOLIFLKGONSGY-CSKARUKUSA-N 0.000 description 1
- BOTGCZBEERTTDQ-UHFFFAOYSA-N 4-Methoxy-1-naphthol Chemical compound C1=CC=C2C(OC)=CC=C(O)C2=C1 BOTGCZBEERTTDQ-UHFFFAOYSA-N 0.000 description 1
- 102000004539 Acyl-CoA Oxidase Human genes 0.000 description 1
- 108020001558 Acyl-CoA oxidase Proteins 0.000 description 1
- HOPVGFKDVOOCHD-UHFFFAOYSA-N Benzoylcholine Chemical compound C[N+](C)(C)CCOC(=O)C1=CC=CC=C1 HOPVGFKDVOOCHD-UHFFFAOYSA-N 0.000 description 1
- 108010089254 Cholesterol oxidase Proteins 0.000 description 1
- 102000003914 Cholinesterases Human genes 0.000 description 1
- 108090000322 Cholinesterases Proteins 0.000 description 1
- 102000005870 Coenzyme A Ligases Human genes 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 102000057621 Glycerol kinases Human genes 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- WZKXBGJNNCGHIC-UHFFFAOYSA-N Leucomalachite green Chemical compound C1=CC(N(C)C)=CC=C1C(C=1C=CC(=CC=1)N(C)C)C1=CC=CC=C1 WZKXBGJNNCGHIC-UHFFFAOYSA-N 0.000 description 1
- 102000017055 Lipoprotein Lipase Human genes 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 108010011449 Long-chain-fatty-acid-CoA ligase Proteins 0.000 description 1
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N N-phenyl amine Natural products NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 1
- 102000011420 Phospholipase D Human genes 0.000 description 1
- 108090000553 Phospholipase D Proteins 0.000 description 1
- 101710163410 Probable glycerol kinase Proteins 0.000 description 1
- IFTGQCOAUCEONK-UHFFFAOYSA-N Reduced 2,6-Dichlorophenolindophenol Chemical compound C1=CC(O)=CC=C1NC1=CC(Cl)=C(O)C(Cl)=C1 IFTGQCOAUCEONK-UHFFFAOYSA-N 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229940048961 cholinesterase Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 108010046301 glucose peroxidase Proteins 0.000 description 1
- 108010054790 glycerol-3-phosphate oxidase Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000000852 hydrogen donor Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000008362 succinate buffer Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
Description
本発明は過酸化水素定量用試薬にかんする。
過酸化水素の検出および定量は、従来から種々
の分野でさまざまな方法で行われているが、臨床
検査の分野において、その重要性は、ここ数年来
とみに増している。すなわち生体液試料中の成分
に酵素を作用させ、生成する過酸化水素を測定
し、当該成分を定量する方法が広く行われてい
る。このことは、生体液中の微量成分であるグル
コース、コレステロール、尿酸、リン脂質、中性
脂肪、遊離脂肪酸等の測定を容易にし、臨床診断
等に利用することで、その有効性が十分に立証さ
れている。
このような過酸化水素の検定及び定量法に用い
られている試薬は、過酸化水素をパーオキシダー
ゼまたはパーオキシダーゼよう作用を有する物質
の存在下で反応させ、発色あるいは発螢光に導く
ことが可能な水素供与体を含むもので、それらに
ついては数多くの開発がなされている。大別する
と、
(1) 酸化還元色素を用いる方法で、還元型の色素
を酸化し発色させて測定する方法。例えば、還
元型２，６−ジクロルフエノールインドフエノ
ール、ロイコマラカイトグリーン等がある。
(2) 酸化縮合による発色を測定する方法。例え
ば、４−メトキシナフトールを用いそれ自身の
酸化縮合による発色を測定する方法、またデベ
ロツパーとして、４−アミノアンチピリンまた
は３−メチル−２−ベンゾチアゾロンヒドラゾ
ンを用い、カツプラーとしてアニリン系化合物
あるいはフエノール系化合物を用い両者の間で
酸化縮合を行わせ、その際の発色を測定する方
法がある。
しかし、これらの方法は、いずれも完全なもの
ではなく、特に生体液中成分の測定においては、
感度、安定性、測定波長、水溶性、毒性の点でい
ずれかの欠点を持つている。
本発明者等はこれらの欠点を除くべく種々研究
を重ねた結果、パーオキシダーゼの存在下で高感
度、長波長で測定可能な、さらに発色の安定性が
よく、しかも水溶性である発色剤を合成した。こ
れにより分散剤等を用いることなく均一な水溶液
の調製が可能となり、しかも酵素活性に対し影響
のない再現性の良い試薬を開発し、本発明を完成
した。
本発明に従つて、次の一般式で表わされる化合
物、あるいはその塩を発色剤として含有すること
を特徴とする液体中の過酸化水素定量用試薬：
（ここでR1は−Ｈ、−OCH3、−CH3
R2は−CH3，−C2H5
R3は（−CH2−）oSO3Hまたは（−CH2−）oCOOHで
ｎ＝１〜３
を表わす）が提供される。
本発明における発色剤化合物は一般式に示す通
り、スルホン酸基または、カルボキシル基を有す
るトリアリールイミダゾール誘導体であるが、こ
れに類似のトリアリールイミダゾール染料前駆体
が公開特許公報昭53−26188に開示されている。
しかし、当該公報から明らかな通り当該トリアリ
ールイミダゾール染料前駆体にはスルホン酸基ま
たはカルボキシル基を有する化合物は全く含まれ
ていない。しかも、当該公報に記載の化合物は水
に対して不溶または難溶性物質であり、水溶性の
具体的物質名は示されていない。
一般的に過酸化水素の存在または生成する試料
は水溶液の状態で反応させる方法をとるのが大部
分であり、その定量用試薬も取扱い上水溶液が好
ましい。また液体と固体を反応させる方法より、
液体と液体を混合し反応させる方法がより迅速で
しかも再現性が良好であるといえるし、本発明者
等が水溶性を重視した所以である。
本発明の化合物は極めて水溶性がよく、さらに
過酸化水素と反応して生成する色素の極大吸収波
長が610nmであるので、測定に際し短波長に吸収
のある物質、例えばビリルビン、ヘモグロビン等
の影響を受けない。また、その分子吸光係数が４
〜６万cm-1／mol-1／であるので微量成分の定
量には効果的である。
本発明の化合物は一般に周知の次の方法で合成
することができる。
例えば、
の合成工程を経て得られた。
次に、本発明化合物の水溶性について、類似の
トリアリールイミダゾール染料前駆物質の一部と
比較した試験結果を表−１に示す。
The present invention relates to a reagent for quantifying hydrogen peroxide. Detection and quantification of hydrogen peroxide has been conventionally carried out in various fields using various methods, but its importance in the field of clinical testing has increased significantly over the past few years. That is, a widely used method is to apply an enzyme to a component in a biological fluid sample, measure the generated hydrogen peroxide, and quantify the component. This makes it easy to measure trace components such as glucose, cholesterol, uric acid, phospholipids, neutral fats, and free fatty acids in biological fluids, and its effectiveness has been fully demonstrated by using it for clinical diagnosis. has been done. The reagents used in such hydrogen peroxide assay and quantitative methods are capable of reacting hydrogen peroxide in the presence of peroxidase or a substance that acts like peroxidase, leading to color development or fluorescence. contains hydrogen donors, and many developments have been made regarding them. Broadly divided: (1) A method using a redox dye, in which the reduced dye is oxidized to develop color for measurement. Examples include reduced 2,6-dichlorophenol indophenol and leucomalachite green. (2) A method of measuring color development due to oxidative condensation. For example, 4-methoxynaphthol is used to measure color development due to its own oxidative condensation, and 4-aminoantipyrine or 3-methyl-2-benzothiazolone hydrazone is used as a developer, and an aniline compound or phenol compound is used as a coupler. There is a method of performing oxidative condensation between the two using a compound and measuring the color development at that time. However, none of these methods is perfect, especially when it comes to measuring components in biological fluids.
They have some drawbacks in terms of sensitivity, stability, measurement wavelength, water solubility, and toxicity. As a result of various studies to eliminate these drawbacks, the present inventors have developed a color former that can be measured with high sensitivity and long wavelength in the presence of peroxidase, has good color stability, and is water-soluble. Synthesized. As a result, a uniform aqueous solution can be prepared without using a dispersant or the like, and a reagent with good reproducibility that does not affect enzyme activity has been developed and the present invention has been completed. According to the present invention, a reagent for quantifying hydrogen peroxide in a liquid, which is characterized by containing a compound represented by the following general formula or a salt thereof as a coloring agent: (Here, R 1 is -H, -OCH 3 , -CH 3 R 2 is -CH 3 , -C 2 H 5 R 3 is (-CH 2 -) o SO 3 H or (-CH 2 -) o COOH (representing n=1 to 3) are provided. As shown in the general formula, the color former compound in the present invention is a triarylimidazole derivative having a sulfonic acid group or a carboxyl group, and a triarylimidazole dye precursor similar to this is disclosed in Japanese Patent Application Publication No. 1986-26188. has been done.
However, as is clear from the publication, the triarylimidazole dye precursor does not contain any compound having a sulfonic acid group or a carboxyl group. Furthermore, the compounds described in the publication are insoluble or poorly soluble substances in water, and the names of specific water-soluble substances are not indicated. In general, most samples in which hydrogen peroxide is present or produced are reacted in the form of an aqueous solution, and the reagent for quantitative determination is preferably an aqueous solution for handling reasons. Also, from the method of reacting liquid and solid,
It can be said that the method of mixing and reacting liquids is faster and has better reproducibility, and this is why the inventors placed importance on water solubility. The compound of the present invention has extremely good water solubility, and the maximum absorption wavelength of the dye produced by reacting with hydrogen peroxide is 610 nm. Therefore, during measurement, the influence of substances that absorb at short wavelengths, such as bilirubin and hemoglobin, can be avoided. I don't accept it. Also, its molecular extinction coefficient is 4
Since it is ~60,000 cm -1 /mol -1 /, it is effective for quantifying trace components. The compounds of the present invention can be synthesized by the following generally known methods. for example, It was obtained through the synthesis process of Next, Table 1 shows the test results of the water solubility of the compound of the present invention compared with some similar triarylimidazole dye precursors.
【表】【table】
【表】
表−１より明らかなように、２，３，４，５，
６，７は水溶性であり、その反応生成物の色調も
安定であつた。過酸化水素の検出又は定量に際
し、本発明化合物を用いる場合の緩衝液のPHは約
６ないし8.5の範囲内が適当で、例えば、リン酸
塩緩衝液、ホウ酸塩緩衝液、クエン酸塩緩衝液、
コハク酸塩緩衝液、トリス緩衝液、グリシン緩衝
液、トリエタノールアミン緩衝液、マクイルバイ
ン緩衝液、グツド緩衝液等を用いることができ
る。
グルコースを測定する場合は、グルコースオキ
シダーゼとパーオキシダーゼを作用させ、PH６〜
７のリン酸塩緩衝液中で行うことが好ましい。
コレステロールを測定する場合はコレステロー
ルオキシダーゼとパーオキシダーゼを作用させ、
PH６〜７のリン酸塩緩衝液中で行うことが好まし
い。
リン脂質を測定する場合は、ホスフオリパーゼ
Ｄとコリンオキシダーゼおよびパーオキシダーゼ
を作用させ、PH８のトリス緩衝液中で行うことが
好ましい。
中性脂肪を測定する場合はリポプロテインリパ
ーゼとグリセロキナーゼとＬ−α−グリセロリン
酸オキシダーゼ、およびパーオキシダーゼを作用
させPH７のグツド緩衝液中で行うことが好まし
い。
遊離脂肪酸を測定する場合は、アシルCoAシ
ンセターゼとアシルCoAオキシダーゼ、および
パーオキシダーゼを作用させPH６〜８のグツド緩
衝液中で行うことが好ましい。
尿酸を測定する場合はウリカーゼとパーオキシ
ダーゼを作用させ、PH６〜７のホウ酸塩緩衝液中
で行うことが好ましい。
また、コリンエステラーゼ活性も、ベンゾイル
コリンを基質とし、コリンオキシダーゼとパーオ
キシダーゼを作用させて測定することが可能であ
る。
次に実施例により本発明を説明する。
実施例 １
過酸化水素の定量：
(1) 試薬：70m mol／ホウ酸塩緩衝液（PH６）
にパーオキシダーゼを0.5U／ml、本発明化合
物（No.４（表−１参照）50μmol／になるよう
に調製した。
(2) 試料液：市販過酸化水素水を用い2.0，1.6，
1.4，0.8，0.4m mol／になるように水で希釈
調製した。
(3) 操作：試薬3.0mlと試料液のそれぞれを20μ
加え、37℃で５分間加温し、波長610nmの吸光
度を測定した。
(4) 結果：第１図から明からなように過酸化水素
の検量線は良好な直線性を示した。また呈色の
安定性も十分であつた。
実施例 ２
尿酸の定量：
(1) 原理：尿酸にウリカーゼを作用させ定量的に
生成する過酸化水素をパーオキシダーゼと本発
明化合物を用いて測定しその値から尿酸を定量
する。
(2) 試薬：70m mol／ホウ酸塩緩衝液（PH６）
にウリカーゼ0.2U／ml、パーオキシダーゼ
0.5U／ml、本発明化合物No.４ 50μmol／の
濃度になるよう調製した。
(3) 操作：試薬3.0mlに血清20μを加え、37℃で
15分間加温し、波長610nmの吸光度を測定し
た。
別に作製した検量線を用いて尿酸を定量し
た。
(4) 検量線の作製：尿酸を水に溶解し、1.5，
1.2，0.9，0.6，0.3m mol／になるよう尿酸
試料液を調製した。
これを用い血清の場合と同様に操作し夫々の吸
光度を測定し第２図の検量線を得た。これは良好
な直線性を示した。また試薬は使用上十分な保存
安定性のあることが確認された。[Table] As is clear from Table-1, 2, 3, 4, 5,
6 and 7 were water-soluble, and the color tone of the reaction product was also stable. When detecting or quantifying hydrogen peroxide, the pH of the buffer solution when using the compound of the present invention is suitably within the range of about 6 to 8.5, such as phosphate buffer, borate buffer, citrate buffer. liquid,
Succinate buffer, Tris buffer, glycine buffer, triethanolamine buffer, McIlvain buffer, Gud buffer, etc. can be used. When measuring glucose, use glucose oxidase and peroxidase to
Preferably, the reaction is carried out in a phosphate buffer of No. 7. When measuring cholesterol, cholesterol oxidase and peroxidase are used to
Preferably, the reaction is carried out in a phosphate buffer with a pH of 6 to 7. When measuring phospholipids, it is preferable to allow phospholipase D to interact with choline oxidase and peroxidase in a Tris buffer at pH 8. When measuring neutral fats, it is preferable to carry out the measurement in a pH7 buffer solution with the action of lipoprotein lipase, glycerokinase, L-α-glycerophosphate oxidase, and peroxidase. When measuring free fatty acids, it is preferable to carry out the measurement in a neutral buffer solution having a pH of 6 to 8 with acyl-CoA synthetase, acyl-CoA oxidase, and peroxidase acting on it. When measuring uric acid, it is preferable to allow uricase and peroxidase to interact and perform the measurement in a borate buffer solution with a pH of 6 to 7. Cholinesterase activity can also be measured by using benzoylcholine as a substrate and allowing choline oxidase and peroxidase to act. Next, the present invention will be explained with reference to examples. Example 1 Quantification of hydrogen peroxide: (1) Reagent: 70mmol/borate buffer (PH6)
Peroxidase was prepared at 0.5 U/ml and the compound of the present invention (No. 4 (see Table-1)) at 50 μmol/ml. (2) Sample solution: Commercially available hydrogen peroxide solution was used at 2.0, 1.6,
It was diluted with water to give a concentration of 1.4, 0.8, and 0.4 m mol/. (3) Operation: Add 3.0ml of reagent and 20μ of sample solution each.
In addition, the mixture was heated at 37°C for 5 minutes, and the absorbance at a wavelength of 610 nm was measured. (4) Results: As is clear from Figure 1, the calibration curve for hydrogen peroxide showed good linearity. Furthermore, the stability of color development was also sufficient. Example 2 Quantification of uric acid: (1) Principle: Uricase is reacted with uric acid to quantitatively produce hydrogen peroxide, which is measured using peroxidase and the compound of the present invention, and uric acid is quantified from the value. (2) Reagent: 70mmol/borate buffer (PH6)
uricase 0.2U/ml, peroxidase
The concentration was adjusted to 0.5 U/ml and 50 μmol/ml of Compound No. 4 of the present invention. (3) Procedure: Add 20μ of serum to 3.0ml of reagent and incubate at 37℃.
After heating for 15 minutes, absorbance at a wavelength of 610 nm was measured. Uric acid was quantified using a separately prepared calibration curve. (4) Preparation of calibration curve: Dissolve uric acid in water,
Uric acid sample solutions were prepared to have concentrations of 1.2, 0.9, 0.6, and 0.3m mol/. This was used in the same manner as in the case of serum, and the respective absorbances were measured to obtain the calibration curve shown in Figure 2. It showed good linearity. It was also confirmed that the reagent had sufficient storage stability for use.
第１図は本発明の試薬を用いる過酸化水素測定
の検量線を示し、第２図は本発明の試薬を用いる
尿酸測定の検量線を示す。
FIG. 1 shows a calibration curve for hydrogen peroxide measurement using the reagent of the invention, and FIG. 2 shows a calibration curve for uric acid measurement using the reagent of the invention.
Claims (1)
塩を発色剤として含有することを特徴とする液体
中の過酸化水素定量用試薬。 （ここでR1は−Ｈ，−OCH3，−CH3 R2は−CH3，−C2H5 R3は（−CH2−）oSO3Hまたは （−CH2−）oCOOHでｎ＝１〜３ を表わす） ２ 物質に作用し、最終的に過酸化水素を生成す
る一連の反応にあずかる全ての試薬成分、パーオ
キシダーゼまたはパーオキシダーゼよう作用を有
する物質および特許請求の範囲第１項に記載の化
合物からなる当該物質の定量用試薬。[Scope of Claims] 1. A reagent for quantifying hydrogen peroxide in a liquid, characterized by containing a compound represented by the following general formula or a salt thereof as a coloring agent. (Here, R 1 is -H, -OCH 3 , -CH 3 R 2 is -CH 3 , -C 2 H 5 R 3 is (-CH 2 -) o SO 3 H or (-CH 2 -) o COOH n = 1 to 3) 2 All reagent components that act on a substance and participate in a series of reactions that ultimately produce hydrogen peroxide, peroxidase or a substance that has a peroxidase-like action, and claims No. A reagent for quantitative determination of the substance comprising the compound described in item 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14392481A JPS5845557A (en) | 1981-09-14 | 1981-09-14 | Reagent for quantitative determination of hydrogen peroxide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14392481A JPS5845557A (en) | 1981-09-14 | 1981-09-14 | Reagent for quantitative determination of hydrogen peroxide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5845557A JPS5845557A (en) | 1983-03-16 |
| JPH0218840B2 true JPH0218840B2 (en) | 1990-04-26 |
Family
ID=15350258
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14392481A Granted JPS5845557A (en) | 1981-09-14 | 1981-09-14 | Reagent for quantitative determination of hydrogen peroxide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5845557A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59193352A (en) * | 1983-04-18 | 1984-11-01 | Fuji Photo Film Co Ltd | Reagent for analysis, method of analysis and element of multiple chemical analysis |
| JPS614960A (en) * | 1984-06-19 | 1986-01-10 | Fuji Photo Film Co Ltd | Analyzing reagent, analyzing method and multilayered chemical analyzing element |
| US5024935A (en) * | 1987-12-18 | 1991-06-18 | Eastman Kodak Company | Dye-providing composition, diagnostic test kit and their use in method for ligand determination using peroxidase labeled-receptor |
| US5047318A (en) * | 1988-06-13 | 1991-09-10 | Eastman Kodak Company | Imidazole leuco dye composition containing 4'-hydroxyacetanilide, diagnostic kit and method using same |
| US5372931A (en) * | 1992-12-22 | 1994-12-13 | Eastman Kodak Company | Use of 4'-hydroxy- and 4'-alkoxy-substituted electron transfer agents in compositions, elements, test kits and analytical methods |
| AU697785B2 (en) | 1994-07-19 | 1998-10-15 | Johnson & Johnson Clinical Diagnostics, Inc. | Analytical element,composition and method using modified apo-horseradish peroxidase |
| JP3657535B2 (en) | 2001-05-29 | 2005-06-08 | 株式会社日本トリム | Hydrogen radical detection method and quantitative analysis method |
| JP5265257B2 (en) | 2008-06-30 | 2013-08-14 | 富士フイルム株式会社 | Antibodies that recognize dog CRP and human CRP |
| JP5292270B2 (en) | 2009-12-21 | 2013-09-18 | 富士フイルム株式会社 | Dry analytical element for dog CRP measurement |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5326188A (en) * | 1976-08-09 | 1978-03-10 | Eastman Kodak Co | Composition for detecting hydrogen peroxide |
| JPS5642599A (en) * | 1979-09-13 | 1981-04-20 | Wako Pure Chem Ind Ltd | Novel method of hydrogen peroxide determination |
-
1981
- 1981-09-14 JP JP14392481A patent/JPS5845557A/en active Granted
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5326188A (en) * | 1976-08-09 | 1978-03-10 | Eastman Kodak Co | Composition for detecting hydrogen peroxide |
| JPS5642599A (en) * | 1979-09-13 | 1981-04-20 | Wako Pure Chem Ind Ltd | Novel method of hydrogen peroxide determination |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5845557A (en) | 1983-03-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3342077B2 (en) | Improved oxidative coupling dyes for spectrophotometric determination of analytes | |
| EP0330517B1 (en) | Method, system and devices for the assay and detection of biochemical molecules | |
| RU2052819C1 (en) | Method of assay of electrone-saturated aromatic amine which is a marker of analyte content in biological fluid, and an agent for its realization | |
| US6121050A (en) | Analyte detection systems | |
| KR101282539B1 (en) | Method of multiquantification for cholesterol of low-density lipoprotein | |
| JPS6129462B2 (en) | ||
| EP0223977B1 (en) | Colour developing method in clinical examinations | |
| EP0243066B1 (en) | Element and method for the determination of creatinine or creatine | |
| CN104245952B (en) | The determination method of material in blood sample | |
| JPH0218840B2 (en) | ||
| JPS61138163A (en) | Color developing coupler and use to measurement of hydrogen peroxide or other substance to be analyzed | |
| JPS5819279B2 (en) | Method and reagent for measuring NAD(P)H or salicylate | |
| EP0797774B1 (en) | Multilayer analytical element | |
| JPS59162899A (en) | Method and composition for determination of beta-hydroxybutyric acid | |
| JP3065692B2 (en) | Naphthotriazolium salt | |
| EP0420893B1 (en) | Reducible indicators containing pyrogallol and use thereof | |
| JPH0372280B2 (en) | ||
| JPH04252197A (en) | Assay method for catechol and its product and factor thereof | |
| JPS5847498A (en) | Measurement of component of living body solution | |
| JPH0244396B2 (en) | BIRIRUBINTEIRYOHO | |
| JPH06237794A (en) | Determination of 1,5-anhydroglucitol | |
| JPH1118798A (en) | Determination of cholesterol and reagent therefor | |
| JPS62291567A (en) | Usage of methine dye to analysis of hydrogen peroxide or specimen forming said hydrogen peroxide | |
| JPS60241899A (en) | Determination of hydrogen peroxide | |
| JPS5847499A (en) | Measurement of component of living body solution |