JPH059071B2 - - Google Patents
Info
- Publication number
- JPH059071B2 JPH059071B2 JP4914984A JP4914984A JPH059071B2 JP H059071 B2 JPH059071 B2 JP H059071B2 JP 4914984 A JP4914984 A JP 4914984A JP 4914984 A JP4914984 A JP 4914984A JP H059071 B2 JPH059071 B2 JP H059071B2
- Authority
- JP
- Japan
- Prior art keywords
- reagent
- gtp
- glutamyl
- measuring
- cyclohexylamino
- 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
- 239000003153 chemical reaction reagent Substances 0.000 claims description 27
- 101000856500 Bacillus subtilis subsp. natto Glutathione hydrolase proenzyme Proteins 0.000 claims description 15
- 238000005259 measurement Methods 0.000 claims description 10
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 claims description 8
- PJWWRFATQTVXHA-UHFFFAOYSA-N Cyclohexylaminopropanesulfonic acid Chemical compound OS(=O)(=O)CCCNC1CCCCC1 PJWWRFATQTVXHA-UHFFFAOYSA-N 0.000 claims description 6
- 210000001124 body fluid Anatomy 0.000 claims description 5
- 239000010839 body fluid Substances 0.000 claims description 5
- 229940043257 glycylglycine Drugs 0.000 claims description 5
- 108010008488 Glycylglycine Proteins 0.000 claims description 4
- 239000003381 stabilizer Substances 0.000 claims description 3
- 238000006276 transfer reaction Methods 0.000 claims description 3
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 claims description 2
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 claims description 2
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims description 2
- 239000000758 substrate Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- NKTOLZVEWDHZMU-UHFFFAOYSA-N 2,5-xylenol Chemical compound CC1=CC=C(C)C(O)=C1 NKTOLZVEWDHZMU-UHFFFAOYSA-N 0.000 description 2
- PHWAJJWKNLWZGJ-UHFFFAOYSA-N 3,5-dibromo-4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC(Br)=C(O)C(Br)=C1 PHWAJJWKNLWZGJ-UHFFFAOYSA-N 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 125000002642 gamma-glutamyl group Chemical group 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- WMZTYIRRBCGARG-VIFPVBQESA-N (2s)-2-azaniumyl-5-(4-nitroanilino)-5-oxopentanoate Chemical compound OC(=O)[C@@H](N)CCC(=O)NC1=CC=C([N+]([O-])=O)C=C1 WMZTYIRRBCGARG-VIFPVBQESA-N 0.000 description 1
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical compound NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 description 1
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-M periodate Chemical compound [O-]I(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-M 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- -1 xylenol metaperiodate Chemical class 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は安定した呈色反応を示し、正確な判定
値を得ることのできる体液中のγ−グルタミルト
ランスペプチダーゼ（以下「γ−GTP」と表わ
す）測定用試薬に関する。
γ−GTPは生体内に存在する転移酵素のひと
つで、γ−グルタミルペプチドを加水分解すると
同時に、γ−グルタミル基を他のペプチドやＬ−
アミノ酸に転移させる反応を触媒する作用がある
が、種々の肝疾患により血清中のγ−GTP活性
が上昇するので、肝疾患の診断を中心とする臨床
検査ではγ−GTP活性の測定は必須の項目とな
つている。
現在、体液中のγ−GTP活性を測定する方法
としては、基質としてγ−グルタミル−Ｐ−ニト
ロアニリドあるいはγ−グルタミル−３−カルボ
キシ−４−ヒドロキシアニリド等の合成基質を用
いて、γ−GTPによる転移反応の結果反応する
Ｐ−ニトロアニリン又は４−ヒドロキシアニリン
等を直接あるいはメタ過ヨウ素酸の存在下でフエ
ノール類等のカプラーと反応させてその呈色を光
学的に測定する方法が行われているが、この際、
メタ過ヨウ素酸塩等の酸化剤を使用し、塩基性下
で発色させることにより、合成基質に加水分解を
生ぜしめ、測定値に大きな誤差を与える原因とな
つている。
従来はこの点に関して各種の緩衝剤などを安定
剤として添加して合成基質の加水分解を抑制する
方法が試みられてきたが、いずれも呈色反応を阻
害したり、濁りを発生させたりして満足すべき結
果は得られなかつた。
本発明者らは、かかる問題について種々検討し
た結果、基質としてγ−グルタミル−３，5′−ジ
ブロム−４−ヒドロキシアニリド（以下「γ−
Glu−DBHA」と表わす）を用いる下記反応式に
よつて示されるγ−GTPの測定に於て、
γ−Glu−DBHA＋グリシルグリシンγ−GTP
―――→
γ−Glu−グリシルグリシン＋DBHA
DBHA＋ｐ−キシレノールメタ過ヨウ素酸Na
―――――――――→
キノン色素
３−シクロヘキシルアミノ−１−プロパンスルホ
ン酸が有効な安定化作用を示し、該化合物を加え
ることによつて、極めて測定誤差の少ない結果が
得られることを見出した。
すなわち本発明は、γ−Glu−DBHAとグリシ
ルグリシンとの転移反応を利用して体液中のγ−
GTPを測定するγ−GTP測定用試薬において、
安定化剤として３−シクロヘキシルアミノ−１−
プロパンスルホン酸を含有することを特徴とする
γ−GTP測定用試薬に関する。
本発明のγ−GTP測定用試薬における３−シ
クロヘキシルアミノ−１−プロパンスルホン酸の
含有量は、呈色反応時の濃度が10〜500mmol／
、好ましくは50〜100mmol／となるような
量がよい。
次に本発明試薬の実施例およびそれを使用した
体液中のγ−GTP測定例を説明する。
実施例 １
γ−Glu−DBHA 5mmol／
グリシルグリシン 100mmol／
トリトンＸ−100 0.1W／Ｖ％
３−シクロヘキシルアミノ−１−プロパンス
ルホン酸 80mmol／
PH＝8.5
実施例 ２
γ−Glu−DBHA 3mmol／
グリシルグリシン 100mmol／
トリトンＸ−100 0.1W／Ｖ％
３−シクロヘキシルアミノ−１−プロパンス
ルホン酸 50mmol／
PH＝8.5
測定例
体液中のγ−GTP活性を実施例１の試薬を用
いて以下に示すように測定を行なつた。なお効果
を比較するために、実施例１の試薬より３−シク
ロヘキシルアミノ−１−プロパンスルホン酸を除
いたものを対照試薬として使用して同様に測定を
行なつた。
１ 試薬
(1) 試薬Ａ（実施例１の試薬）
(2) 試薬Ｂ（対照試薬、試薬Ａより３−シクロ
ヘキシルアミノ−１−プロパンスルホン酸を除い
たもの）
(3) 試薬Ｃ（呈色液）
ｐ−キシレノール 20mmol／
メタ過ヨウ素酸ナトリウム ５ 〃
水酸化ナトリウム 170 〃
２ 操作および結果
精製水20μおよび血清20μのそれぞれに２
mlの試薬Ａを加え、37℃で15分間加温し、次に２
mlの試薬Ｃを加えて室温に放置した。試薬Ｃを加
えてから10分後および20分後の吸光度をそれぞれ
610nmで測定した。
また、試薬Ａの代りに試薬Ｂを用いて全く同じ
操作を行なつた。
測定の結果は次表に示すとおりである。
The present invention relates to a reagent for measuring γ-glutamyl transpeptidase (hereinafter referred to as "γ-GTP") in body fluids that exhibits a stable color reaction and is capable of obtaining accurate determination values. γ-GTP is one of the transferases that exist in living organisms, and it hydrolyzes γ-glutamyl peptide and at the same time transfers the γ-glutamyl group to other peptides or L-glutamyl peptides.
It has the effect of catalyzing the reaction of transfer to amino acids, but since γ-GTP activity in serum increases with various liver diseases, measurement of γ-GTP activity is essential in clinical tests mainly for the diagnosis of liver diseases. It has become an item. Currently, the method for measuring γ-GTP activity in body fluids uses synthetic substrates such as γ-glutamyl-P-nitroanilide or γ-glutamyl-3-carboxy-4-hydroxyanilide as a substrate. A method is used in which P-nitroaniline or 4-hydroxyaniline, which reacts as a result of the transfer reaction, is reacted with a coupler such as a phenol directly or in the presence of metaperiodic acid, and the resulting coloration is optically measured. However, in this case,
The use of an oxidizing agent such as metaperiodate to develop color under basic conditions causes hydrolysis of the synthetic substrate, causing large errors in measured values. In this regard, attempts have been made in the past to suppress the hydrolysis of synthetic substrates by adding various buffers and the like as stabilizers, but these methods either inhibit the color reaction or cause turbidity. No satisfactory results were obtained. As a result of various studies on this problem, the present inventors found that γ-glutamyl-3,5'-dibromo-4-hydroxyanilide (hereinafter referred to as "γ-glutamyl-3,5'-dibromo-4-hydroxyanilide") was used as a substrate.
In the measurement of γ-GTP shown by the following reaction formula using γ-Glu-DBHA+glycylglycine γ-GTP ---→ γ-Glu-glycylglycine+DBHA DBHA+p - Sodium xylenol metaperiodate ――――――――→ The quinone dye 3-cyclohexylamino-1-propanesulfonic acid exhibits an effective stabilizing effect, and the addition of this compound significantly reduces measurement errors. It was found that results with less That is, the present invention utilizes the transfer reaction between γ-Glu-DBHA and glycylglycine to reduce γ-Glu-DBHA in body fluids.
In the γ-GTP measurement reagent for measuring GTP,
3-cyclohexylamino-1- as a stabilizer
The present invention relates to a reagent for measuring γ-GTP characterized by containing propanesulfonic acid. The content of 3-cyclohexylamino-1-propanesulfonic acid in the reagent for measuring γ-GTP of the present invention is such that the concentration during the color reaction is 10 to 500 mmol/
, preferably in an amount of 50 to 100 mmol/. Next, examples of the reagent of the present invention and examples of measuring γ-GTP in body fluids using the same will be described. Example 1 γ-Glu-DBHA 5 mmol/Glycylglycine 100 mmol/Triton Silglycine 100mmol/Triton Measurements were carried out. In order to compare the effects, the same measurements were carried out using the reagent of Example 1 except that 3-cyclohexylamino-1-propanesulfonic acid was removed as a control reagent. 1 Reagents (1) Reagent A (reagent of Example 1) (2) Reagent B (control reagent, reagent A with 3-cyclohexylamino-1-propanesulfonic acid removed) (3) Reagent C (coloring solution) ) p-xylenol 20 mmol/sodium metaperiodate 5 〃 Sodium hydroxide 170 〃 2 Procedures and results 2 for each of purified water 20μ and serum 20μ
Add ml of reagent A, warm at 37°C for 15 minutes, then add 2 ml of reagent A.
ml of Reagent C was added and allowed to stand at room temperature. Absorbance 10 minutes and 20 minutes after adding reagent C, respectively.
Measured at 610nm. Moreover, the exact same operation was performed using reagent B instead of reagent A. The measurement results are shown in the table below.
【表】
数値は吸光度を示す。
上記の測定結果から明らかなように、本発明の
試薬を用いた場合には、10分後と20分後の吸光度
差が殆どなく、呈色の安定性が非常によいことが
わかる。対照試薬の場合には10分後から20分後に
至る間に吸光度が増しており、呈色の安定性がな
いことを示している。
以上説明したとおり、本発明の試薬は３−シク
ロヘキシルアミノ−１−プロパンスルホン酸を添
加したことによつて試薬成分である合成基質の加
水分解を抑え、それによつて呈色安定性を得るこ
とができるので、本発明試薬を使用することによ
りγ−GTP測定を正確に行なうことができる。[Table] Values indicate absorbance.
As is clear from the above measurement results, when the reagent of the present invention is used, there is almost no difference in absorbance after 10 minutes and after 20 minutes, indicating that the stability of color development is very good. In the case of the control reagent, the absorbance increased from 10 minutes to 20 minutes, indicating that the color development was not stable. As explained above, the reagent of the present invention suppresses hydrolysis of the synthetic substrate, which is a reagent component, by adding 3-cyclohexylamino-1-propanesulfonic acid, thereby achieving color stability. Therefore, by using the reagent of the present invention, γ-GTP measurement can be performed accurately.
Claims (1)
ドロキシアニリドとグリシルグリシンとの転移反
応を利用して体液中のγ−グルタミルトランスペ
プチダーゼ（γ−GTP）を測定するγ−GTP測
定用試薬において、安定化剤として３−シクロヘ
キシルアミノ−１−プロパンスルホン酸を含有す
ることを特徴とするγ−GTP測定用試薬。1 In a γ-GTP measurement reagent for measuring γ-glutamyl transpeptidase (γ-GTP) in body fluids using the transfer reaction between γ-glutamyl-3,5-dibrom-4-hydroxyanilide and glycylglycine , a reagent for measuring γ-GTP, comprising 3-cyclohexylamino-1-propanesulfonic acid as a stabilizer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4914984A JPS60196200A (en) | 1984-03-16 | 1984-03-16 | γ-GTP measurement reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4914984A JPS60196200A (en) | 1984-03-16 | 1984-03-16 | γ-GTP measurement reagent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60196200A JPS60196200A (en) | 1985-10-04 |
| JPH059071B2 true JPH059071B2 (en) | 1993-02-03 |
Family
ID=12823035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4914984A Granted JPS60196200A (en) | 1984-03-16 | 1984-03-16 | γ-GTP measurement reagent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60196200A (en) |
-
1984
- 1984-03-16 JP JP4914984A patent/JPS60196200A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60196200A (en) | 1985-10-04 |
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