JPH01202297A - Fractional determination of adenosine deaminase isozyme - Google Patents
Fractional determination of adenosine deaminase isozymeInfo
- Publication number
- JPH01202297A JPH01202297A JP2824188A JP2824188A JPH01202297A JP H01202297 A JPH01202297 A JP H01202297A JP 2824188 A JP2824188 A JP 2824188A JP 2824188 A JP2824188 A JP 2824188A JP H01202297 A JPH01202297 A JP H01202297A
- Authority
- JP
- Japan
- Prior art keywords
- isozyme
- adenosine deaminase
- adenosine
- ada
- substrate
- 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.)
- Granted
Links
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 title claims abstract description 49
- 102000055025 Adenosine deaminases Human genes 0.000 title claims abstract description 49
- 108010044467 Isoenzymes Proteins 0.000 title claims abstract description 31
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 32
- 239000000758 substrate Substances 0.000 claims abstract description 23
- IOSAAWHGJUZBOG-UHFFFAOYSA-N 3-(6-amino-9h-purin-9-yl)nonan-2-ol Chemical compound N1=CN=C2N(C(C(C)O)CCCCCC)C=NC2=C1N IOSAAWHGJUZBOG-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 16
- 229960005305 adenosine Drugs 0.000 claims abstract description 16
- 239000003112 inhibitor Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 49
- 230000000694 effects Effects 0.000 abstract description 25
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 108090000790 Enzymes Proteins 0.000 abstract description 10
- 102100025976 Adenosine deaminase 2 Human genes 0.000 description 12
- 101000720051 Homo sapiens Adenosine deaminase 2 Proteins 0.000 description 12
- 101000657352 Homo sapiens Transcriptional adapter 2-alpha Proteins 0.000 description 12
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- 238000000691 measurement method Methods 0.000 description 9
- 238000005259 measurement Methods 0.000 description 7
- 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 description 7
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 4
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 4
- PTKRUDMLGIIORX-ITGWJZMWSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2r,3s,4r,5r)-5-(3-carbamoyl-4h-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate;cyclohexanamine Chemical compound NC1CCCCC1.NC1CCCCC1.NC1CCCCC1.NC1CCCCC1.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](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 PTKRUDMLGIIORX-ITGWJZMWSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical compound OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 description 3
- 101000780338 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) Adenosine deaminase 1 Proteins 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- 102100036664 Adenosine deaminase Human genes 0.000 description 2
- 208000009746 Adult T-Cell Leukemia-Lymphoma Diseases 0.000 description 2
- 206010001413 Adult T-cell lymphoma/leukaemia Diseases 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 101000929495 Homo sapiens Adenosine deaminase Proteins 0.000 description 2
- 101000625338 Homo sapiens Transcriptional adapter 1 Proteins 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- 208000019423 liver disease Diseases 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- UVCLNKKMPGHLGP-UHFFFAOYSA-N 3-(6-amino-7h-purin-2-yl)nonan-2-ol Chemical compound CCCCCCC(C(C)O)C1=NC(N)=C2NC=NC2=N1 UVCLNKKMPGHLGP-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 229920002271 DEAE-Sepharose Polymers 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 description 1
- 102000006746 NADH Dehydrogenase Human genes 0.000 description 1
- 108010086428 NADH Dehydrogenase Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 102100033220 Xanthine oxidase Human genes 0.000 description 1
- 108010093894 Xanthine oxidase Proteins 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 description 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000007882 cirrhosis Effects 0.000 description 1
- 208000019425 cirrhosis of liver Diseases 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 229960003966 nicotinamide Drugs 0.000 description 1
- 235000005152 nicotinamide Nutrition 0.000 description 1
- 239000011570 nicotinamide Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 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 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【産業上の利用分野１
本発明は、アデノシンデアミナーゼアイソザイム■と■
とを簡単に分別定量する方法に関する。
【従来の技術１
（背景）
アデノシンデアミナーゼ（ＡＤＡ）は、アデノシンをイ
ノシンとアンモニアとに分解する酵素であって、生物の
核酸代謝に関与しており、牌臓、肝臓、赤血球等に高濃
度に存在している。このアデノシンデアミナーゼは、遺
伝子の異なる主分子量３５，０００ダルトンのアイソザ
イム■（以下“ＡＤＡ 、 ”とも略す）と主分子量１
００，０００ダルトンのアイソザイム■（以下“ＡＤＡ
２”とも略す）の二種のアイソザイムに区別できること
が知られている。
ところで、本酵素の臨床的意義については、従来から、
各種悪性腫瘍、肝疾患、炎症性疾患時等における活性値
の変動が注目されてきたが、近来に至り、本酵素の活性
をアイソザイムのレベルで精査したときの個々のアイソ
ザイムレベルの高低と、各種疾患との関連性が頓に注視
されるようになってきた。例えば倉田ら（（臨床病理）
３２巻、８７５頁、１９８４）は、健康成人及び肝炎、
肝硬変などの肝疾患、悪性腫瘍の患者の血清がらゲル濾
過法（分子篩クロマトグラフィー）によりアイソザイム
を分離した後、各アイソザイムの活性を測定した。その
結果、下表−１記載の通り、これら各種疾患との関連が
示唆されている。
表−１
（次頁へ続く）
（以下余白）
表−１（続き）
＊ Ｖｏ ： ＡＤＡＩと結合プロティン（ｃｐ）との
結合体が複数結合したもの。
大型： ＡＤＡ、とｃｐの結合体。
中間型： ＡＤＡ２
小型： ＡＤＡ。
なお、より近来では、後天性免疫不全症候群（エイズ）
又は成人Ｔ細胞白血病リンパ腫（ＡＴＬ）患者血清中に
おけるＡＤＡ 、とＡＤＡ２との関連も指摘されている
。
【従来の技術とその問題点１
ところで、既存のＡＤＡアイソザイムの測定方法として
は、大別して以下三種の方法が知られている。
■ 基質に対する反応性の違いを利用する方法（ａ＞二
種類の基質を用いる方法
（ｂ）二種類の基質濃度を用いる方法
（ｅ）阻害剤を使用する方法。
■ 分子量、等電点の違いを利用する方法。
（ａ＞セファデックスＧ−２００によるゲル濾過法（ｂ
） ＤＥＡＥセファロースＣＬ−６Ｂによるイオン交換
クロマトグラフィー
（ｃ）高速液体クロマトグラフィー
（ｄ）電気泳動法
■ 抗原性の違いを利用する方法
（ａ）酵素免疫測定法
（ｂ）放射免疫測定法
以上の各方法の中、■の抗原性の相違を利用する方法は
、キット化に時間とコストがかかるので実用化迄に至っ
ていない。特に（ｂ）の放射免疫測定法は、同位元素の
放射能を利用する関係で、設備的にも大きな制約を受け
る。
■のクロマトグラフィーや電気泳動による方法は、アイ
ソザイムを個別的に分離して各フラクションの活性を測
定するので、精密ではあるが操作が繁雑であって、時間
とコストがかかるため、自動分析用には不向きである。
従って、現状では■の方法が最も実用化し易い方法であ
ろうと考えられるが、現実には簡単で、しかも多量検体
の処理に適した測定手段は未だ確立していない。
【発明が解決しようとする課題】
以上の事情に鑑み、本発明は、多検体処理に適したアデ
ノシンデアミナーゼアイソザイムの簡便かつ正確な分別
定量法の提供を目的とする。[Industrial Application Field 1] The present invention relates to adenosine deaminase isozymes ■ and ■.
This paper relates to a method for easily separating and quantifying [Conventional technology 1 (Background) Adenosine deaminase (ADA) is an enzyme that decomposes adenosine into inosine and ammonia, and is involved in nucleic acid metabolism in living organisms, and is found in high concentrations in the spleen, liver, red blood cells, etc. Existing. This adenosine deaminase is an isozyme with a main molecular weight of 35,000 daltons (hereinafter also abbreviated as "ADA") and a main molecular weight of 1
00,000 dalton isozyme■ (hereinafter referred to as “ADA
It is known that the enzyme can be distinguished into two types of isozymes:
Changes in activity values during various malignant tumors, liver diseases, inflammatory diseases, etc. have been attracting attention, but recently, when the activity of this enzyme was examined at the isozyme level, it was found that the levels of individual isozymes and various The relationship between this and other diseases has been attracting close attention. For example, Kurata et al. ((clinical pathology)
32, p. 875, 1984), healthy adults and hepatitis,
After isozymes were separated by gel filtration (molecular sieve chromatography) from serum samples from patients with liver diseases such as cirrhosis or malignant tumors, the activity of each isozyme was measured. As a result, as shown in Table 1 below, a relationship with these various diseases has been suggested. Table 1 (Continued on next page) (Margins below) Table 1 (Continued) *Vo: Multiple conjugates of ADAI and binding protein (cp). Large: A combination of ADA and cp. Intermediate: ADA2 Small: ADA. In addition, more recently, acquired immunodeficiency syndrome (AIDS)
It has also been pointed out that there is a relationship between ADA and ADA2 in the serum of adult T-cell leukemia-lymphoma (ATL) patients. [Prior art and its problems 1] By the way, as existing methods for measuring ADA isozyme, the following three types of methods are known. ■ Method using differences in reactivity to substrates (a> Method using two types of substrates (b) Method using two types of substrate concentrations (e) Method using inhibitors. ■ Differences in molecular weight and isoelectric point (a>Gel filtration method using Sephadex G-200 (b)
) Ion exchange chromatography using DEAE Sepharose CL-6B (c) High performance liquid chromatography (d) Electrophoresis■ Methods that utilize differences in antigenicity (a) Enzyme immunoassay (b) Radioimmunoassay Each of the above Among the methods, method (2) that utilizes the difference in antigenicity has not been put into practical use because it takes time and cost to prepare a kit. In particular, the radioimmunoassay method (b) uses the radioactivity of isotopes, and is therefore subject to major restrictions in terms of equipment. Methods using chromatography and electrophoresis (2) separate isozymes individually and measure the activity of each fraction, so although they are accurate, they are complicated, time-consuming, and costly, so they are not suitable for automatic analysis. is not suitable. Therefore, at present, method (2) is considered to be the easiest method to put into practical use, but in reality, a measurement method that is simple and suitable for processing a large amount of specimen has not yet been established. [Problems to be Solved by the Invention] In view of the above circumstances, an object of the present invention is to provide a simple and accurate method for differentially quantifying adenosine deaminase isozyme, which is suitable for processing multiple samples.
（概要）
本発明に係るアデノシンデアミナーゼアイソザイムの分
別定量法は、以上の目的を達成せんがため、アデノシン
を基質としてアデノシンデアミナーゼを定−量するに当
たり、アデノシンデアミナーゼアイソザイム■の阻害剤
として、０．１ ｍＭ〜０．３ ｍＭのエリスロ−９−
（２−ヒドロキシ−３−ノニル）−アデニンを選択し、
基質濃度６ｎ＋）４〜２０ｍＭにおいて、上記阻害剤を
併用しない全酵素活性値（ａ）から該阻害剤を併用した
ときの酵素活性値（ｂ）を控除してアデノシンデアミナ
ーゼアイソザイム■の値（ａ−ｂ）及びアデノシンデア
ミナーゼアイソザイムＩＩの値（ｂ）を夫々求めること
を特徴とする。
（原理）
本発明は、主としてＡＤＡ １を選択的に阻害する阻害
剤、エリスロ−９−（２−ヒドロキシ−３−ノニル）−
アデニン（ＥＨＮＡ）を用るＡＤＡ １とＡＤＡ２へ
の分別定量である。即ち、特定濃度範囲内の基質（検体
）にＥ ＨＮ Ａを添加しないで測定した全酵素活性値
から、特定濃度範囲内のＥＨＮＡを添加してＡＤＡ Ｉ
活性を阻害することにより得られるＡＤＡ２活性値を控
除して得られる差値を以ってＡＤＡ １活性値を求める
方法である。
（発明の経緯）
ＥＨＮＡがＡＤＡ １を選択的に阻害することは、既に
Ｒａｔｅｃｈら（Ｅｎｚｙｍｅ ２６，７４−８４．１
９８１）により報告されている。しかし、ＥＨＮＡによ
る阻害は必ずしもＡＤＡ、に対してのみ特異的である訳
ではなく、ＡＤＡ２に対してもある程度阻害作用を及ぼ
すから、本方法を臨床検査に応用するときの鍵となる基
質濃度と阻害作用の関連、 ＡＤＡ２に対する阻害剤の
影響の程度及び測定結果の再現性など、本方法を実用化
するのに必要な要件については未知であった。
そこで本発明者は、基質濃度とＥＨＮＡの阻害作用との
関連、殊にこれを臨床現場で使用されているアデノシン
デアミナーゼ測定法に準用したときの測定条件につき検
討した結果、ここに本発明に到達した。
（測定条件）
アデノシンは緩衝液に溶けにくいので、アデノシン濃度
の上限は２０ｍＭが限界であり、逆に濃度が低過ぎると
反応速度が非実用的なレベル迄低下するため、実用的な
濃度の下限は６ｍＭである。
次にこれら基質濃度下における阻害剤ＥＨＮＡの濃度を
検討すると、第１図に示されるように、アデノシン６ｍ
ＭのときＥＨＮＡの濃度が帆１ｍ１４てあればＡＤＡ、
は完全に阻害され、ＡＤＡ２は殆ど影響を受けない（同
図Ａ）。同様にアデノシン２０ｍＭのときのＡＤＡ、と
ＡＤＡ２の阻害割合を検討してみると、ＥＨＮＡが０．
３１のときＡＤＡ、は完全に阻害されるが、ＡＤＡ２は
殆ど影響を受けない（同図Ｂ〉。
そこで、３５検体のＡＤＡ２のみを精製した試料の活性
と、同じ試料に０．１ｎ＋Ｍ Ｅ ＨＮ Ａを添加して
測定した活性との相関を検討すると、相関係数０．９６
２６が得られ、０．１ｍＭ Ｅ ＨＮ Ａの添加により
ＡＤＡｌがほぼ１００％阻害されていることが確認でき
たく第２図）。
さらに、既知ＡＤＡ測定法である■ＧＬＤＨ−ＮＡＤＨ
法、■ＧＬＤＨ−ＮＡＤＰＨ法及び■ＸＯＤ−ＰＮＰ法
（下注参照）における相関関係を検討したところ以下の
値が得られ、いずれも極めて良好な相関を示すことが判
明した。それ故、本発明測定法は、何れの測定方式にも
応用できることが明らかである（第３図〜第５図参照）
。
ｌＪＬ 糺ｆＬＩＬ
■と■ ０．９９２■と■
０．９９２■と■ ０
．９９３（各測定法の詳細）
以下、上記ＧＬＤＨ−ＮＡＤＨ法、ＧＬＤＨ−ＮＡＤＰ
Ｈ法及びχ０Ｄ−ＰＮＰ法の各詳細につき記述する。
＋１） Ｇ Ｌ Ｄ Ｈ−Ｎ Ａ Ｄ Ｈ法（ａ）原
理
アデノシンは、アデノシンデアミナーゼの作用によって
イノシンとアンモニアになる。このアンモニアは、ニコ
チンアミド補酵素ＮＡＤＨとグルタミン酸デヒドロゲナ
ーゼ（ＧＬＤＨ）の存在下でα−ケトグルタル酸と反応
してグルタミン酸を生じる。その際、アンモニアと等モ
ルのＮＡＤＨが消費される。このＮＡＤＨの減少速度を
測定してＡＤＡ活性を求める。
（ｂ）操作
■ 試薬（２５０回用）
○基質液（アデノシン、α−Ｋ Ｇ Ａ ２０ｍ Ｘ○
酵素剤（ＧＬＤＨ，ＮＡＤＨ，ＬＤＨ凍結乾燥品２０ｄ
Ｘ５）
■ 試薬の調製
酵素剤１本分を基質液２０−中に溶かし、基質Ｍ衝液と
する。
（以下余白）
■ 測定法（用手法）
ＡＤＡ活性（Ｕ／Ｌ）＝キルた４Ｎへ”Ｘ ２１ Ｘ
１０６＊ ＮＡＤＨの分子吸光係数
（２１Ｇ Ｌ Ｄ Ｈ−Ｎ Ａ Ｄ Ｐ Ｈ法（ａ）原
理
アデノシンからＡＤＡの作用で生成したアンモニアを、
α−ケトグルタル酸とＮＡＤＰＨの存在下でＧＬＤＨと
共役させ、ＮＡＤＰＨの減少速度を波長３４０ｎｍにお
ける吸光度変化として測定する。
↓
グルタミン酸
（ｂ）試薬
（以下余白）
（Ｃ）測定法（用手法）
↓
充分攪拌後、３７℃で２分間加温
↓
↓
充分攪拌後、３７℃、３４０ｎｍにおける３〜５分後の
吸光度変化を測定して１分間当たりの吸光度減少速度を
求め、下式によりＡＤＡ活性を計算。
（以下余白）
明細書の浄書く内容に変更なし）
＋３） Ｘ ＯＤ −Ｐ Ｎ Ｐ法
（ａ）原理
ＡＤＡにより生成したイノシンをプリンヌクレオシドフ
ォスホリラーゼ（ＰＮＰ）によりキサンチンとして、テ
トラゾリウム塩、例えばニトロブルーテトラゾ１）ラム
（ＮＢＴ）の存在下にキサンチンオキシダーゼと共役さ
せ、生じたホルマザンの吸光度を測定する。
（ｂ）試薬
■標準液（０，２ｍＭヒポキサンチン溶液）＝２．７２
２ｎ＋ｇのヒポキサンチンを蒸留水に溶かし、１００
Ｆｄとする。
■基質・発色緩衝液；アデノシン１６８．４ｍｇに蒸留
水５０ ｍＱを加え、加温、溶解させる。冷後、０．５
Ｍリン酸Ｍ街液（ｐＨ７，０）４２ ｄを加え、これに
アジ化ナトリウム１０．５Ｔｆｉｇ、ツイーン２０４２
０ｍｇ 、 Ｎ Ｂ Ｔ ２５．７６ｍｇ、 Ｐ Ｎ
Ｐ ５２．５Ｕ及びＸＯＤ ２．６２５Ｕを添加し
、蒸留水を加えて全量を１００−とする。
（ｃ）測定法
下表−２記載の通りである。
↓ ３７℃、１０分間加熱(Summary) In order to achieve the above-mentioned purpose, the method for differentially quantifying adenosine deaminase isozyme according to the present invention is to quantify adenosine deaminase using adenosine as a substrate. mM to 0.3 mM erythro-9-
(2-hydroxy-3-nonyl)-adenine,
At a substrate concentration of 6n+) 4 to 20 mM, the value of adenosine deaminase isozyme ■ (a- b) and the value (b) of adenosine deaminase isozyme II, respectively. (Principle) The present invention mainly relates to an inhibitor that selectively inhibits ADA 1, erythro-9-(2-hydroxy-3-nonyl)-
This is a differential determination of ADA 1 and ADA2 using adenine (EHNA). That is, from the total enzyme activity value measured without adding EHNA to a substrate (sample) within a specific concentration range, to the ADA I by adding EHNA within a specific concentration range.
This is a method of determining the ADA1 activity value using the difference value obtained by subtracting the ADA2 activity value obtained by inhibiting the activity. (Background of the invention) It has already been reported by Ratech et al. (Enzyme 26, 74-84.1) that EHNA selectively inhibits ADA 1.
981). However, inhibition by EHNA is not necessarily specific only to ADA, and also exerts an inhibitory effect on ADA2 to some extent, so substrate concentration and inhibition are the key factors when applying this method to clinical tests. The requirements necessary to put this method into practical use, such as the relationship between effects, the extent of the inhibitor's influence on ADA2, and the reproducibility of measurement results, were unknown. Therefore, the present inventor investigated the relationship between the substrate concentration and the inhibitory effect of EHNA, and in particular, the measurement conditions when applying this to the adenosine deaminase measurement method used in clinical practice, and as a result, the present invention has been arrived at. did. (Measurement conditions) Adenosine is difficult to dissolve in buffer solutions, so the upper limit for the adenosine concentration is 20mM.On the other hand, if the concentration is too low, the reaction rate will drop to an impractical level, so the lower limit for practical concentration is is 6mM. Next, when considering the concentration of the inhibitor EHNA under these substrate concentrations, as shown in Figure 1, adenosine 6m
When M, if the concentration of EHNA is 1m14, then ADA,
was completely inhibited, and ADA2 was hardly affected (Figure A). Similarly, when examining the inhibition ratio of ADA and ADA2 at 20mM adenosine, it was found that EHNA was 0.
31, ADA is completely inhibited, but ADA2 is hardly affected (Figure B). Therefore, the activity of the 35 samples purified only of ADA2 and the same sample with 0.1n+M E HN A When examining the correlation with the activity measured by adding , the correlation coefficient was 0.96.
26 was obtained, confirming that ADAl was almost 100% inhibited by the addition of 0.1 mM EHNA (Figure 2). In addition, ■GLDH-NADH, which is a known ADA measurement method.
The following values were obtained by examining the correlations in the GLDH-NADPH method, (1) GLDH-NADPH method, and (2) XOD-PNP method (see note below), and it was found that all of them showed extremely good correlations. Therefore, it is clear that the measurement method of the present invention can be applied to any measurement method (see Figures 3 to 5).
. lJL 纺fLIL ■and■ 0.992■and■
0.992 ■ and ■ 0
．． 993 (Details of each measurement method) Below, the above GLDH-NADH method, GLDH-NADP
The details of the H method and the χ0D-PNP method will be described. +1) GLDH-NADH method (a) Principle Adenosine becomes inosine and ammonia by the action of adenosine deaminase. This ammonia reacts with α-ketoglutarate to produce glutamic acid in the presence of nicotinamide coenzyme NADH and glutamate dehydrogenase (GLDH). At this time, ammonia and equimolar NADH are consumed. The rate of decrease in NADH is measured to determine ADA activity. (b) Operation■ Reagent (for 250 times) ○Substrate solution (adenosine, α-K GA 20m X○
Enzyme agent (GLDH, NADH, LDH freeze-dried product 20d
X5) ① Preparation of reagent Dissolve one bottle of enzyme preparation in substrate solution 20-20 and use it as substrate M solution. (Left below) ■Measurement method (technique) ADA activity (U/L) = Killed 4N”X 21
106* Molecular extinction coefficient of NADH (21G LDH-NADPH method (a) Principle Ammonia produced from adenosine by the action of ADA,
It is conjugated with GLDH in the presence of α-ketoglutaric acid and NADPH, and the rate of decrease in NADPH is measured as a change in absorbance at a wavelength of 340 nm. ↓ Glutamic acid (b) reagent (blank below) (C) Measurement method (manual method) ↓ After thorough stirring, heat at 37℃ for 2 minutes ↓ ↓ After thorough stirring, absorbance change after 3 to 5 minutes at 37℃ and 340 nm Measure the rate of decrease in absorbance per minute, and calculate the ADA activity using the formula below. (Left below) No changes to the text in the specification) +3) It is conjugated with xanthine oxidase in the presence of blue tetrazo1)ram (NBT), and the absorbance of the resulting formazan is measured. (b) Reagent ■Standard solution (0.2mM hypoxanthine solution) = 2.72
Dissolve 2n+g of hypoxanthine in distilled water and add 100
Let it be Fd. (2) Substrate/coloring buffer: Add 50 mQ of distilled water to 168.4 mg of adenosine and dissolve by heating. After cooling, 0.5
Add 42 d of M phosphoric acid M street solution (pH 7,0), and add 10.5 Tfig of sodium azide and Tween 2042 to this.
0mg, N B T 25.76 mg, P N
Add 52.5 U of P and 2.625 U of XOD, and add distilled water to bring the total volume to 100-. (c) Measurement method As described in Table 2 below. ↓ Heat at 37℃ for 10 minutes
本発明は、エリスロ−９−（２−ヒドロキシ−３−ノニ
ル）−アデニン（ＥＨＮＡ）が一定の濃度範囲内におい
て、一定基質濃度範囲内のアデノシンに対するＡＤＡｌ
の作用を選択的に阻害するという自然法則の発見に基づ
き、これをＡＤＡアイソザイムの分別定量に利用しよう
とするものである。従ってこの方法は、測定に要する手
数及び時間を短縮、軽減できることの他に、特に現在広
く実用化されている自動分析手段への適用が容易である
点で、ＡＤＡアイソザイム活性測定の臨床的応用を実現
できるという効果を奏する。The present invention provides that erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) is an ADAl for adenosine within a certain substrate concentration range within a certain concentration range.
Based on the discovery of a natural law that selectively inhibits the action of ADA isozymes, we aim to utilize this for the differential quantification of ADA isozymes. Therefore, this method not only shortens and reduces the number of steps and time required for measurement, but also facilitates the clinical application of ADA isozyme activity measurement, especially in that it is easy to apply to automatic analysis methods that are currently widely used. It has the effect of being achievable.
以下、実施例により発明実施の態様を説明するが、例示
は当然説明用のものであって、発明思想の限界又は制限
を意味するものではない。
（以下余白）
実施例１
２５のヒト血清検体につき、出願人の製造、発売に係る
前記性（１）項記載のＧＬＤＨ−ＮＡＤＨ法用試薬（＜
ＡＤオートマルホ）商標）を用い、基質アデノシン濃度
６ｍＭにおいて、ＥＨＮＡを加えないときの全ＡＤＡ活
性値から、同じ＜ ０．１ｍＭのＥＨＮＡを加えた場合
の活性値を控除してＡＤＡ １とＡＤＡ２の値を求めた
。結果は下表−３の通りであった。
表−３
表−３（続き）Hereinafter, embodiments of the invention will be explained with reference to Examples, but the examples are, of course, for illustrative purposes and do not imply any limitations or restrictions on the idea of the invention. (Left below) Example 1 For 25 human serum samples, the reagent for the GLDH-NADH method (<
Using AD Automaruho (Trademark), at a substrate adenosine concentration of 6mM, the activity value when EHNA was added at the same <0.1mM was subtracted from the total ADA activity value when EHNA was not added, and the values of ADA1 and ADA2 were calculated. I asked for The results were as shown in Table 3 below. Table-3 Table-3 (continued)
以上、説明した通り、本発明は、ＡＤＡアイソザイムの
簡便かつ正確な定量法を提供しうろことを通じて、各種
疾病の診断及び治療の向上に寄与しうる。As described above, the present invention can contribute to improving the diagnosis and treatment of various diseases by providing a simple and accurate method for quantifying ADA isozymes.
第１図は、異なった基質濃度下におけるエリスロ−９−
（２−ヒドロキシ−３−ノニル）−アデニンのＡＤＡア
イソザイムに対する阻害作用を示すグラフ（Ａ：基質濃
度６ｍＭ、Ｂ：同２０ｍＭ） 、第２図は、ゲル濾過法
により精製されたＡＤＡ２活性と発明測定法によるＡＤ
Ａ２活性との相関を示すグラフ、第３図〜第５図は、夫
々ＡＤＡ活性の測定に関し、ＮＡＤＨ法とＮＡＤＰＨ法
、ＮＡＤＰＨ法とＸＯＤ−ＰＮＰ法及びＮＡＤＨ法とＸ
ＯＤ−ＰＮＰ法との相関を示すグラフである。
ＡＤＡ活・避ＣＡ）
０ ８ ８ ８ °３ｏ
８
Ｃす
ｏＪ７′ルｉ７１！孟ＦＭ）ＡＤＡ 馳１１（ｍＵ／ｍ
ｌ） ０（Ｙ） （’Ｊ
″第５図
二手続祁１′Ｘ１ミマ！）
昭和６３年 ６月 ６日
１、事件の表示
昭和６３年特許願第２８２４１号
２ 発明の名称−。
ア
アデノシンアミナーゼアイソザイムの分別定量法３、補
正をする者
事件との関係 特許出願人
住 所 大阪市大淀区中津１丁目６番２４号名 称 マ
ルポ株式会社
代表者 高木 二部
４、代理人■５３２
住 所 大阪府大阪市淀川区東三国１−３２−１２電話
０６−３９５−２７１４ ／（Ｘｌ−３９１−６７１２
；０６−３９７−１００７（ＦＡＸ＞
７、補正の対象
（１）明細書の浄書（内容に変更ない
（２）代理権を証明する書面
ａ 補正の内容
（１）別紙の通り明細書の１５頁を提出する。
（２１別紙の通り委任状を補充する。
９、添１；を書類の目録
（１）明ａ書（１５頁） １通Figure 1 shows erythro-9-9 under different substrate concentrations.
A graph showing the inhibitory effect of (2-hydroxy-3-nonyl)-adenine on ADA isozyme (A: substrate concentration 6mM, B: substrate concentration 20mM), Figure 2 shows ADA2 activity purified by gel filtration method and the invention measurement. AD by law
Graphs showing the correlation with A2 activity, Figures 3 to 5, respectively relate to the measurement of ADA activity by the NADH method and the NADPH method, the NADPH method and the XOD-PNP method, and the NADH method and the XOD-PNP method.
It is a graph showing the correlation with the OD-PNP method. ADA active/evasive CA) 0 8 8 8 °3o
8 CsoJ7'le i71! Meng FM) ADA Hase 11 (mU/m
l) 0(Y) ('J
``Figure 5 2 Procedures 1' Relationship with the case of the person making the amendment Patent Applicant Address 1-6-24 Nakatsu, Oyodo-ku, Osaka Name Name: Marupo Co., Ltd. Representative Takagi Nibe 4, Agent 532 Address Higashimikuni, Yodogawa-ku, Osaka-shi, Osaka Prefecture 1-32-12 Telephone 06-395-2714 / (Xl-391-6712
;06-397-1007 (FAX> 7. Subject of amendment (1) Engraving of the specification (no change in content) (2) Document certifying authority of agency a Contents of amendment (1) Page 15 of the specification as attached (Supplement the power of attorney as shown in Attachment 21. 9. Attachment 1; List of documents (1) Letter A (15 pages) 1 copy
Claims (1)
定量するに当たり、アデノシンデアミナーゼアイソザイ
ム I の阻害剤として、０．１ｍＭ〜０．３ｍＭのエリ
スロ−９−（２−ヒドロキシ−３−ノニル）−アデニン
を選択し、基質濃度６ｍＭ〜２０ｍＭにおいて、上記阻
害剤を併用しない全酵素活性値（ａ）から該阻害剤を併
用したときの酵素活性値（ｂ）を控除してアデノシンデ
アミナーゼアイソザイム I の値（ａ−ｂ）及びアデノ
シンデアミナーゼアイソザイムIIの値（ｂ）を夫々求め
ることを特徴とするアデノシンデアミナーゼアイソザイ
ムの分別定量法。 ２ アデノシンデアミナーゼアイソザイムの定量法とし
てＧＬＤＨ−ＮＡＤＨ法、ＧＬＤＨ−ＮＡＤＰＨ法及び
ＸＯＤ−ＰＮＰ法から選ばれた方法が使用される特許請
求の範囲第１項記載のアデノシンデアミナーゼアイソザ
イムの分別定量法。[Claims] 1. In quantifying adenosine deaminase using adenosine as a substrate, 0.1 to 0.3 mM of erythro-9-(2-hydroxy-3-nonyl)-adenine is used as an inhibitor of adenosine deaminase isoenzyme I. At a substrate concentration of 6mM to 20mM, the value of adenosine deaminase isozyme I ( 1. A method for differentially quantifying adenosine deaminase isozyme, which comprises determining the values of a-b) and adenosine deaminase isozyme II (b), respectively. 2. The method for differentially quantifying adenosine deaminase isozyme according to claim 1, wherein a method selected from the GLDH-NADH method, GLDH-NADPH method, and XOD-PNP method is used as the method for quantifying adenosine deaminase isozyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2824188A JPH0650992B2 (en) | 1988-02-09 | 1988-02-09 | Fractional quantification method of adenosine deaminase isozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2824188A JPH0650992B2 (en) | 1988-02-09 | 1988-02-09 | Fractional quantification method of adenosine deaminase isozyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01202297A true JPH01202297A (en) | 1989-08-15 |
| JPH0650992B2 JPH0650992B2 (en) | 1994-07-06 |
Family
ID=12243091
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2824188A Expired - Lifetime JPH0650992B2 (en) | 1988-02-09 | 1988-02-09 | Fractional quantification method of adenosine deaminase isozyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0650992B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2530569A (en) * | 2014-09-25 | 2016-03-30 | Osama Aldirbashi | Methods of detecting adenosine deaminase deficiency |
-
1988
- 1988-02-09 JP JP2824188A patent/JPH0650992B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2530569A (en) * | 2014-09-25 | 2016-03-30 | Osama Aldirbashi | Methods of detecting adenosine deaminase deficiency |
| EP3204487A4 (en) * | 2014-09-25 | 2018-09-05 | Children's Hospital of Eastern Ontario Research Institute Inc. | Methods of detecting adenosine deaminase deficiency |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0650992B2 (en) | 1994-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Skrede et al. | Bile acids measured in serum during fasting as a test for liver disease. | |
| Roda et al. | Bioluminescence measurement of primary bile acids using immobilized 7 alpha-hydroxysteroid dehydrogenase: application to serum bile acids | |
| Washtien et al. | Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil | |
| Kasidas et al. | A new enzymatic method for the determination of glycollate in urine and plasma | |
| Jelikić-Stankov et al. | Determination of uric acid in human serum by an enzymatic method using N-methyl-N-(4-aminophenyl)-3-methoxyaniline reagent | |
| Nazar et al. | An improved microfluorometric enzymatic assay for the determination of ammonia | |
| Atkins et al. | Plasma concentrations of histamine measured by radioenzymatic assay: effects of histaminase incubations | |
| JPS6357040B2 (en) | ||
| Shamma'a et al. | Serum xanthine oxidase: a sensitive test of acute liver injury | |
| CN109112181A (en) | A kind of 5`- activity of 5 '-nucleotidase measuring method and 5`- nucleotidase detection kit | |
| JPH01202297A (en) | Fractional determination of adenosine deaminase isozyme | |
| Kato et al. | Improved assay for alcohol dehydrogenase activity in serum by centrifugal analysis. | |
| CN111808918B (en) | Kit for determining 5' -nucleotidase | |
| Härkönen et al. | Enzymatic techniques in steroid assay | |
| JPS61502443A (en) | Novel reagents and their use | |
| JPH02276595A (en) | Method for measuring bile acid in high sensitivity and composition for measuring bile acid | |
| TW593681B (en) | Quantitative determination method of mannose and reagent therefor | |
| Hyldgaard-Jensen et al. | A study of the determination of lactic dehydrogenase isoenzymes by means of agar gel and starch-agar gel zymograms | |
| Singkamani et al. | A direct assay for pyridoxal 5′-phosphate using pig heart apo-aspartate transaminase | |
| JPH06153991A (en) | Phosphatase analysis reagent | |
| Chrostek et al. | Serum class I and II alcohol dehydrogenase activity during the course of viral hepatitis | |
| JP3493411B2 (en) | Reagent composition for potassium ion measurement and test piece | |
| Ravid et al. | Interrelation between salvage of purine nucleotides and protein synthesis in rat heart cells | |
| Henley et al. | Mitochondrial oxidase activities and pyridine nucleotide concentrations of normal human liver | |
| JPS6024199A (en) | Determination of substance relating to disease |