JPH0586198B2 - - Google Patents
Info
- Publication number
- JPH0586198B2 JPH0586198B2 JP8885285A JP8885285A JPH0586198B2 JP H0586198 B2 JPH0586198 B2 JP H0586198B2 JP 8885285 A JP8885285 A JP 8885285A JP 8885285 A JP8885285 A JP 8885285A JP H0586198 B2 JPH0586198 B2 JP H0586198B2
- Authority
- JP
- Japan
- Prior art keywords
- reaction
- acid
- pyruvate
- icdh
- salts
- 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
- 238000006243 chemical reaction Methods 0.000 claims description 24
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 claims description 16
- 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 16
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 16
- 101100072035 Haloferax volcanii (strain ATCC 29605 / DSM 3757 / JCM 8879 / NBRC 14742 / NCIMB 2012 / VKM B-1768 / DS2) icd gene Proteins 0.000 claims description 14
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 12
- 229940076788 pyruvate Drugs 0.000 claims description 12
- 239000002738 chelating agent Substances 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 229940107700 pyruvic acid Drugs 0.000 claims description 8
- 239000007795 chemical reaction product Substances 0.000 claims description 5
- ODBLHEXUDAPZAU-UHFFFAOYSA-N isocitric acid Chemical compound OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 4
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 4
- 229910001437 manganese ion Inorganic materials 0.000 claims description 4
- 229910021645 metal ion Inorganic materials 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- 150000003839 salts Chemical class 0.000 description 19
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 13
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 7
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 4
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 102000004882 Lipase Human genes 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 150000003626 triacylglycerols Chemical class 0.000 description 3
- 101710098398 Probable alanine aminotransferase, mitochondrial Proteins 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- VASZYFIKPKYGNC-UHFFFAOYSA-N 2-[[2-[bis(carboxymethyl)amino]cyclohexyl]-(carboxymethyl)amino]acetic acid;hydrate Chemical compound O.OC(=O)CN(CC(O)=O)C1CCCCC1N(CC(O)=O)CC(O)=O VASZYFIKPKYGNC-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- IWTIBPIVCKUAHK-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)amino]propanoic acid Chemical compound OC(=O)CCN(CCC(O)=O)CCC(O)=O IWTIBPIVCKUAHK-UHFFFAOYSA-N 0.000 description 1
- FTEDXVNDVHYDQW-UHFFFAOYSA-N BAPTA Chemical compound OC(=O)CN(CC(O)=O)C1=CC=CC=C1OCCOC1=CC=CC=C1N(CC(O)=O)CC(O)=O FTEDXVNDVHYDQW-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 102000057621 Glycerol kinases Human genes 0.000 description 1
- 108700016170 Glycerol kinases Proteins 0.000 description 1
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 description 1
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 1
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 1
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 1
- JYXGIOKAKDAARW-UHFFFAOYSA-N N-(2-hydroxyethyl)iminodiacetic acid Chemical compound OCCN(CC(O)=O)CC(O)=O JYXGIOKAKDAARW-UHFFFAOYSA-N 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- BAECOWNUKCLBPZ-HIUWNOOHSA-N Triolein Natural products O([C@H](OCC(=O)CCCCCCC/C=C\CCCCCCCC)COC(=O)CCCCCCC/C=C\CCCCCCCC)C(=O)CCCCCCC/C=C\CCCCCCCC BAECOWNUKCLBPZ-HIUWNOOHSA-N 0.000 description 1
- PHYFQTYBJUILEZ-UHFFFAOYSA-N Trioleoylglycerol Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCCCCCCCC)COC(=O)CCCCCCCC=CCCCCCCCC PHYFQTYBJUILEZ-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- YDONNITUKPKTIG-UHFFFAOYSA-N [Nitrilotris(methylene)]trisphosphonic acid Chemical compound OP(O)(=O)CN(CP(O)(O)=O)CP(O)(O)=O YDONNITUKPKTIG-UHFFFAOYSA-N 0.000 description 1
- MKBUQYWFFBCMFG-UHFFFAOYSA-N acetic acid propane-1,1-diamine Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.CCC(N)N MKBUQYWFFBCMFG-UHFFFAOYSA-N 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 229910001431 copper ion Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- NFDRPXJGHKJRLJ-UHFFFAOYSA-N edtmp Chemical compound OP(O)(=O)CN(CP(O)(O)=O)CCN(CP(O)(O)=O)CP(O)(O)=O NFDRPXJGHKJRLJ-UHFFFAOYSA-N 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- ZZGUZQXLSHSYMH-UHFFFAOYSA-N ethane-1,2-diamine;propanoic acid Chemical compound NCCN.CCC(O)=O.CCC(O)=O ZZGUZQXLSHSYMH-UHFFFAOYSA-N 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- IFQUWYZCAGRUJN-UHFFFAOYSA-N ethylenediaminediacetic acid Chemical compound OC(=O)CNCCNCC(O)=O IFQUWYZCAGRUJN-UHFFFAOYSA-N 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- -1 iron ions Chemical class 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
- 229960003330 pentetic acid Drugs 0.000 description 1
- 229930029653 phosphoenolpyruvate Natural products 0.000 description 1
- DTBNBXWJWCWCIK-UHFFFAOYSA-N phosphoenolpyruvic acid Chemical compound OC(=O)C(=C)OP(O)(O)=O DTBNBXWJWCWCIK-UHFFFAOYSA-N 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229910001432 tin ion Inorganic materials 0.000 description 1
- PHYFQTYBJUILEZ-IUPFWZBJSA-N triolein Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(OC(=O)CCCCCCC\C=C/CCCCCCCC)COC(=O)CCCCCCC\C=C/CCCCCCCC PHYFQTYBJUILEZ-IUPFWZBJSA-N 0.000 description 1
- 229940117972 triolein Drugs 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明はピルビン酸を反応生成物とする生体物
質の定量方法に関するものである。
一般に、血液などの検体中のトリグリセライド
を測定したり、酵素例えばGOT（グルタミン酸オ
キザロ酢酸トランスアミナーゼ）やGPT（グルタ
ミン酸ピルビン酸トランスアミナーゼ）、の活性
を測定することは普通に行われている。
そして、トリグリセライドの検出や各種酵素反
応においては最終段階でピルビン酸を生成させ、
生成したピルビン酸をLDH（乳酸脱水素酵素）に
よつて乳酸に変換し、この際NADH→NAD+の
共役反応によつて減少するNADHの量を340nm
で測定し定量していた。
しかし、この反応系では必ずピルビン酸を生成
するために、そもそも検体中に存在するピルビン
酸や遊離のグリセロールが測定値に含まれてしま
つて、正確な定量を困難にしていた。
そこで、検体中に存在するピルビン酸や遊離の
グリセロールを前処理で、LDHによつて乳酸に
変換させてしまえば問題はなくなるのである。
NAD+→NADHの逆反応でNADHに戻す必要が
あり、この際イソクエン酸を基質としてiCDH
（イソクエン酸脱水素酵素）とマグネシウムイオ
ン又はマンガンイオンなどの金属イオンによつて
共役反応を生起させることができる。この反応系
は、次の式（）に示される。
The present invention relates to a method for quantifying biological substances using pyruvate as a reaction product. In general, it is common practice to measure triglycerides in samples such as blood, and to measure the activity of enzymes such as GOT (glutamate oxaloacetate transaminase) and GPT (glutamate pyruvate transaminase). In triglyceride detection and various enzymatic reactions, pyruvic acid is produced in the final step.
The generated pyruvate is converted to lactic acid by LDH (lactate dehydrogenase), and at this time, the amount of NADH reduced by the coupling reaction of NADH → NAD + is measured at 340 nm.
It was measured and quantified. However, since this reaction system always produces pyruvic acid, the measured values include pyruvic acid and free glycerol that are present in the sample, making accurate quantification difficult. Therefore, if the pyruvic acid and free glycerol present in the sample are pretreated and converted to lactic acid by LDH, the problem will disappear.
It is necessary to convert NAD + → NADH back to NADH by the reverse reaction, and at this time, iCDH is converted using isocitrate as a substrate.
(isocitrate dehydrogenase) and metal ions such as magnesium ions or manganese ions can cause a conjugation reaction. This reaction system is shown in the following formula ().
【化】
式（）に示されるように、検体中のピルビン
酸の消費とトリグリセライドからもたらされたピ
ルビン酸の測定は同じ共役反応によつて行うこと
ができるのであるが、検体中のピルビン酸や遊離
グリセロールの消費が完了し、NAD+→NADH
の反応が完全に停止されてはじめてトリグリセラ
イドからもたらされたピルビン酸の正確な定量が
行なえるのである。
そこで、問題となるのは、式（）における
NADHNAD+においてNAD+→NADHの反応
をいかにして完全に停止させるかであつた。従来
NAD+→NADHの反応のみを完全に停止させる
ことは知られていなかつた。
本発明者らは、上述の式（）におけるイソク
エン酸iCDH→αKGの反応のみを完全に停止させる
方法を求めて鋭意研究したところ、ATP又は／
及びキレート剤の添加によつて、イソクエン酸
iCDH→α−KGの反応を完全に停止させることに
成功したのである。
本発明は、検体にLDH、NADH、イソクエン
酸、マグネシウムイオンまたはマンガンイオンな
どの金属イオンおよびiCDHを添加混合し、混合
液中にすでに存在するピルビン酸や遊離のグリセ
ロールを消費せしめ、次いでATP又は／及びキ
レート剤を添加し、iCDH反応を停止し、これと
同時もしくはしかる後反応生成物としてピルビン
酸を生成せしめる酵素もしくは酵素群を添加し
て、生成するピルビン酸を定量することを特徴と
するピルビン酸を反応生成物とする生体物質の定
量方法である。
ここで、金属イオンとはマグネシウムイオン、
マンガンイオン、鉄イオン、銅イオン、亜鉛イオ
ン、スズイオン、カルシウムイオンなどを云う
が、これらのイオン種に制限されることはない。
また、キレート剤とはEDTAおよびその塩、
１，２−ビス（Ｏ−アミノフエノキシ）エタン−
Ｎ，Ｎ，N′，N′−四酢酸およびその塩、トラン
ス−１，２−シクロヘキサンジアミン−Ｎ，Ｎ，
N′，N′−四酢酸およびその塩、ジヒドロキシエ
チルグリシンおよびその塩、１，３−ジアミノプ
ロパノール−Ｎ，Ｎ，N′，N′−四酢酸およびそ
の塩、ジエチレントリアミン五酢酸およびその
塩、エチレンジアミンジオルトヒドロキシフエニ
ル酢酸およびその塩、エチレンジアミン二酢酸お
よびその塩、エチレンジアミン二プロピオン酸お
よびその塩、ヒドロキシエチルエチレンジアミン
三酢酸およびその塩、エチレンジアミンテトラキ
ス（メチレンホスホン酸）およびその塩、グリコ
ールエーテルジアミン四酢酸およびその塩、ヒド
ロキシエチルイミノ二酢酸およびその塩、イミノ
二酢酸およびその塩、ジアミノプロパン四酢酸お
よびその塩、ニトリロ三酢酸およびその塩、ニト
リロ三プロピオン酸およびその塩、ニトリロトリ
ス（メチレンホスホン酸）およびその塩、トリエ
チレンテトラミン六酢酸およびその塩などを云う
が、これらのキレート剤に制限されることはな
い。[C] As shown in formula (), the consumption of pyruvate in the sample and the measurement of pyruvate derived from triglyceride can be performed by the same coupled reaction; and consumption of free glycerol is completed, NAD + →NADH
Accurate quantification of pyruvate derived from triglycerides is only possible when the reaction is completely stopped. Therefore, the problem is that in formula ()
The challenge was how to completely stop the NAD + →NADH reaction in NADHNAD + . Conventional
It was not known to completely stop only the NAD + →NADH reaction. The present inventors conducted extensive research in search of a method to completely stop only the reaction of isocitrate iCDH → αKG in the above formula (), and found that ATP or /
and by addition of chelating agents, isocitric acid
They succeeded in completely stopping the iCDH→α-KG reaction. In the present invention, LDH, NADH, isocitrate, metal ions such as magnesium ions or manganese ions, and iCDH are added and mixed to a sample, pyruvic acid and free glycerol already present in the mixed solution are consumed, and then ATP or/ and a chelating agent to stop the iCDH reaction, and at the same time or after that, an enzyme or enzyme group that produces pyruvate as a reaction product is added, and the produced pyruvate is quantified. This is a method for quantifying biological substances that uses acid as a reaction product. Here, the metal ions are magnesium ions,
Examples include manganese ions, iron ions, copper ions, zinc ions, tin ions, calcium ions, etc., but the ion species are not limited to these. In addition, chelating agents include EDTA and its salts,
1,2-bis(O-aminophenoxy)ethane-
N,N,N',N'-tetraacetic acid and its salts, trans-1,2-cyclohexanediamine-N,N,
N',N'-tetraacetic acid and its salts, dihydroxyethylglycine and its salts, 1,3-diaminopropanol-N,N,N',N'-tetraacetic acid and its salts, diethylenetriaminepentaacetic acid and its salts, ethylenediamine Diorthohydroxyphenylacetic acid and its salts, ethylenediaminediacetic acid and its salts, ethylenediaminedipropionic acid and its salts, hydroxyethylethylenediaminetriacetic acid and its salts, ethylenediaminetetrakis (methylenephosphonic acid) and its salts, glycol ether diaminetetraacetic acid and its salts, hydroxyethyliminodiacetic acid and its salts, iminodiacetic acid and its salts, diaminopropanetetraacetic acid and its salts, nitrilotriacetic acid and its salts, nitrilotripropionic acid and its salts, nitrilotris (methylenephosphonic acid) and salts thereof, triethylenetetraminehexaacetic acid and salts thereof, but are not limited to these chelating agents.
【化】
本発明はATP又は／及びキレート剤の添加に
よつて上記式（）→式（）への変化を行なわ
せるものである。即ち、検体中のピルビン酸又
は／及び遊離のグリセロールの完全消費を式
（）で行わせ、完全消費ののち反応系にATP又
は／及びキレート剤を添加し、NAD+→NADH
の反応を停止させるものである。
iCDHの反応を停止させた後は、ATP又は／及
びキレート剤の添加と同時もしくはその後で検体
中にピルビン酸を生成せしめる酵素又は酵素群を
添加し、生成したピルビン酸から乳酸への共役反
応としてNADH→NAD+の反応にともなう
340nm吸光度の減少によつて物質を定量するもの
である。
ピルビン酸を生成せしめる酵素群の反応として
は次の式（）が示される。[Image Omitted] The present invention allows the change from the above formula () to formula () by adding ATP or/and a chelating agent. That is, complete consumption of pyruvic acid and/or free glycerol in the sample is carried out using the formula (), and after complete consumption, ATP or/and a chelating agent is added to the reaction system, and NAD + →NADH
It stops the reaction. After stopping the iCDH reaction, an enzyme or enzyme group that generates pyruvate is added to the sample at the same time as or after the addition of ATP and/or a chelating agent, and a coupling reaction from the generated pyruvate to lactic acid is performed. Along with the reaction of NADH → NAD +
The substance is quantified by the decrease in absorbance at 340 nm. The following equation () is shown as the reaction of the enzyme group that produces pyruvic acid.
【化】
即ち、リパーゼ、グリセロールキナーゼ、
ATP、ホスホエノールピルビン酸及びピルビン
酸キナーゼの添加によつてトリグリセライドが測
定でき、また、α−KG、アラニンの添加でGTP
の活性が測定できるものである。
本発明においては、検体中の被検物を分解し、
NADH→NAD+の反応によつてNADHを消費し
て正確な被検物の定量を行うものである。
本発明において用いる、ATP又は／及びキレ
ート剤によるiCDH反応の停止は、反応を停止し
たそのままの媒質でNADH→NAD+の反応を用
い各種反応が行える点できわめて有用である。
反応系に対するATPの添加量は15mM以上で
あればよい。第１図はiCDH活性におよぼすATP
濃度の影響をみた図であるが、ATP濃度が
15mM以上でiCDHは完全に活性を失つているの
が分る。
また、反応系に対するキレート剤、例えば
EDTAの添加量は10mM以上であればよい。第２
図はiCDH活性におよぼすEDTA濃度の影響をみ
た図であるが、EDTA濃度が10mM以上でiCDH
は完全に活性を失つているのが分る。
次に本発明の実施例を示す。
実施例 １
ATP 1mM
ホスホエノールピルビン酸 5mM
NADH 0.2mM
イソクエン酸 5mM
KCl 100mM
MgCl2 3mM
グリセロキナーゼ 20u／ml
ピルビン酸キナーゼ 10u／ml
LDH 10u／ml
iCDH 5u／ml
を含有する0.1Mトリス塩酸（PH8.0）2.88mlに
200mMグリセロールを含む様々な濃度に調整し
たトリグリセライド（TG）含有検体（Ａ＝100
mg／dl、Ｂ＝300mg／dl、Ｃ＝750mg／dl）10ml添
加した。それぞれ37℃で10分間保温した後、
ATP、リパーゼ濃度がそれぞれ20mM、100u／
mlになるように、ATP、リパーゼ混液を100μl添
加し、分光光度計により37℃での340nmにおける
吸光度の減少を測定した。
ΔE；Ａ＝0.021、Ｂ＝0.069、Ｃ＝0.179、であ
つた。
これを次式により計算した結果、検体中にすで
に存在していたグリセロールは完全に消去され、
引きつづき測定されるトリグリセライドの定量に
影響なく、検体中のトリグリセライド含量が定量
された。
トリグリセライド量
mg／dl＝ΔE／6.2×3.00／0.01×885／1000×100
ΔE＝NADHの減少による吸光度の変化
6.2＝NADHの1mMの吸光度
3.00＝全反応液量（ml）
0.01＝検体量（ml）
885＝トリオレインの分子量[chemical] Namely, lipase, glycerol kinase,
Triglycerides can be measured by adding ATP, phosphoenolpyruvate and pyruvate kinase, and GTP can be measured by adding α-KG and alanine.
activity can be measured. In the present invention, the analyte in the specimen is decomposed,
Accurate quantification of the analyte is performed by consuming NADH through the NADH → NAD + reaction. Termination of the iCDH reaction using ATP or/and a chelating agent used in the present invention is extremely useful in that various reactions can be carried out using the reaction of NADH→NAD + in the same medium in which the reaction has been terminated. The amount of ATP added to the reaction system may be 15 mM or more. Figure 1 shows the effect of ATP on iCDH activity.
This is a diagram looking at the effect of concentration, and the ATP concentration
It can be seen that iCDH completely loses its activity at 15mM or higher. Also, chelating agents for the reaction system, e.g.
The amount of EDTA added may be 10 mM or more. Second
The figure shows the effect of EDTA concentration on iCDH activity.
It can be seen that it has completely lost its activity. Next, examples of the present invention will be shown. Example 1 0.1M Tris-HCl (PH8 . 0) to 2.88ml
Triglyceride (TG) containing samples adjusted to various concentrations including 200mM glycerol (A = 100
mg/dl, B=300mg/dl, C=750mg/dl) 10ml was added. After incubating each at 37℃ for 10 minutes,
ATP and lipase concentrations are 20mM and 100u/
100 μl of the ATP/lipase mixture was added to the solution, and the decrease in absorbance at 340 nm at 37° C. was measured using a spectrophotometer. ΔE; A=0.021, B=0.069, C=0.179. As a result of calculating this using the following formula, the glycerol that was already present in the sample was completely erased.
The triglyceride content in the sample was determined without affecting the subsequent determination of triglyceride. Triglyceride amount mg/dl = ΔE/6.2 x 3.00/0.01 x 885/1000 x 100 ΔE = Change in absorbance due to decrease in NADH 6.2 = Absorbance of 1mM of NADH 3.00 = Total reaction volume (ml) 0.01 = Sample amount (ml) ) 885 = molecular weight of triolein
第１図はiCDH活性におよぼすATP濃度の影響
をみた図で、第２図はiCDH活性におよぼす
EDTA濃度の影響をみた図である。
Figure 1 shows the effect of ATP concentration on iCDH activity, and Figure 2 shows the effect of ATP concentration on iCDH activity.
It is a diagram showing the influence of EDTA concentration.
Claims (1)
ネシウムイオンまたはマンガンイオンなどの金属
イオンおよびiCDHを添加混合し、混合液中にす
でに存在するピルビン酸を消費せしめ、次いで
ATP又は／及びキレート剤を添加し、iCDH反応
を停止し、これと同時もしくはしかる後反応生成
物としてピルビン酸を生成せしめる酵素もしくは
酵素群を添加して、生成するピルビン酸を測定す
ることを特徴とするピルビン酸を反応生成物とす
る生体物質の定量方法。1 Add and mix LDH, NADH, isocitrate, metal ions such as magnesium ions or manganese ions, and iCDH to the sample to consume the pyruvic acid already present in the mixed solution, and then
It is characterized by adding ATP or/and a chelating agent to stop the iCDH reaction, and simultaneously or afterwards adding an enzyme or enzyme group that produces pyruvate as a reaction product, and measuring the produced pyruvate. A method for quantifying biological substances whose reaction product is pyruvate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8885285A JPS61247968A (en) | 1985-04-26 | 1985-04-26 | Quantitatively determining method for vital material with pyruvic acid as resulted product of reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8885285A JPS61247968A (en) | 1985-04-26 | 1985-04-26 | Quantitatively determining method for vital material with pyruvic acid as resulted product of reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61247968A JPS61247968A (en) | 1986-11-05 |
| JPH0586198B2 true JPH0586198B2 (en) | 1993-12-10 |
Family
ID=13954514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8885285A Granted JPS61247968A (en) | 1985-04-26 | 1985-04-26 | Quantitatively determining method for vital material with pyruvic acid as resulted product of reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61247968A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06102040B2 (en) * | 1985-07-11 | 1994-12-14 | オリエンタル酵母工業株式会社 | Quantitative determination of dorigeride |
-
1985
- 1985-04-26 JP JP8885285A patent/JPS61247968A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS61247968A (en) | 1986-11-05 |
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