JP4217815B2 - Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose - Google Patents
Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose Download PDFInfo
- Publication number
- JP4217815B2 JP4217815B2 JP11401499A JP11401499A JP4217815B2 JP 4217815 B2 JP4217815 B2 JP 4217815B2 JP 11401499 A JP11401499 A JP 11401499A JP 11401499 A JP11401499 A JP 11401499A JP 4217815 B2 JP4217815 B2 JP 4217815B2
- Authority
- JP
- Japan
- Prior art keywords
- glucose
- concentration
- dehydrogenase
- quantifying
- reagent
- 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 - Fee Related
Links
- 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 title claims description 68
- 239000008103 glucose Substances 0.000 title claims description 68
- 238000000034 method Methods 0.000 title claims description 38
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 title claims description 32
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 23
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 26
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 13
- 239000004327 boric acid Substances 0.000 claims description 13
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical group NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 claims description 12
- 229950006238 nadide Drugs 0.000 claims description 11
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 8
- 229940124186 Dehydrogenase inhibitor Drugs 0.000 claims description 5
- 239000005515 coenzyme Substances 0.000 claims description 5
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 4
- 229930024421 Adenine Natural products 0.000 claims description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH 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](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 229960000643 adenine Drugs 0.000 claims description 2
- 229960003966 nicotinamide Drugs 0.000 claims description 2
- 235000005152 nicotinamide Nutrition 0.000 claims description 2
- 239000011570 nicotinamide Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 claims 1
- 239000003112 inhibitor Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 description 11
- 210000002700 urine Anatomy 0.000 description 11
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 229940088598 enzyme Drugs 0.000 description 10
- 238000011002 quantification Methods 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 238000000691 measurement method Methods 0.000 description 8
- 239000012086 standard solution Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 7
- 239000012895 dilution Substances 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- IZXGZAJMDLJLMF-UHFFFAOYSA-N methylaminomethanol Chemical compound CNCO IZXGZAJMDLJLMF-UHFFFAOYSA-N 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 229910021538 borax Inorganic materials 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 206010012601 diabetes mellitus Diseases 0.000 description 4
- 235000010339 sodium tetraborate Nutrition 0.000 description 4
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 208000017169 kidney disease Diseases 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 2
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 2
- 108010015776 Glucose oxidase Proteins 0.000 description 2
- 239000004366 Glucose oxidase Substances 0.000 description 2
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 2
- 102000005548 Hexokinase Human genes 0.000 description 2
- 108700040460 Hexokinases Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 229940116332 glucose oxidase Drugs 0.000 description 2
- 235000019420 glucose oxidase Nutrition 0.000 description 2
- 108010046301 glucose peroxidase Proteins 0.000 description 2
- 238000012886 linear function Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000012190 activator Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- SCPOPPZYKMBXRM-UHFFFAOYSA-N boric acid;2-methylaniline Chemical compound OB(O)O.CC1=CC=CC=C1N SCPOPPZYKMBXRM-UHFFFAOYSA-N 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000002562 urinalysis Methods 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【０００１】
【発明の属する技術分野】
本発明は、グルコースの定量方法、特に、臨床検査分野における例えば検尿などの試料中のグルコース濃度を、酵素グルコースデヒドロゲナーゼを用いて反応速度論に基づいて定量する方法、並びに、その方法の使用に適する測定用試薬に関する。
より詳しくは、本発明は、グルコースを含有する試料にグルコースデヒドロゲナーゼを作用させる際、グルコースデヒドロゲナーゼの拮抗阻害剤としてホウ酸もしくはその塩を使用し、グルコースデヒドロゲナーゼと基質（グルコース）との親和力を小さくして見かけのＫｍ値を大きくすることにより、試料中のグルコース濃度が高くとも、そのグルコースを正確に定量することができるグルコース濃度の測定方法に関する。
【０００２】
【従来の技術】
血清および尿などの体液中のグルコース濃度は各種の疾患または病態を反映していることから、ヒトの健康診断、特に成人病として発症率の高い糖尿病の鑑別診断においては、グルコース濃度の検査は、必須の検査項目となっている。
ヒトの尿中のグルコース濃度の基準値は、およそ２０ないし４０ｍｇ／ｄＬの範囲であり、測定値がその範囲に該当するとき、そのヒトは健常であると判断される。しかるに、重篤な糖尿病とか腎障害などを患っている患者にあっては、大量のグルコースが尿中に排泄されるため、尿中のグルコース濃度は約１０ｇ／ｄＬの非常に高濃度にまで及ぶ。
グルコース濃度の測定法としては、従来より化学法と酵素法が知られている。化学法としては、還元法（Ｓｏｍｏｇｙｉ−Ｎｅｌｓｏｎ法）とか、酸化的縮合法（ｏ−トルイジンホウ酸法）などが挙げられる。しかし、これらの方法は、還元物質の影響を受ける、またグルコースに対する特異性が低いなどの欠点があるため、一般に、化学法よりも酵素法がより優れていると判断されている。
酵素法としては、従来、グルコースオキシダーゼ・ペルオキシダーゼ系を用いた測定法並びにヘキソキナーゼ・グルコース６−リン酸デヒドロゲナーゼ系を用いた測定法が主に使用されている［Ｈａｎｓ Ｕ．Ｂ．：Ｍｅｔｈｏｄｓ ｏｆＥｎｚｙｍａｔｉｃ Ａｎａｌｙｓｉｓ，ｖｏｌ．３，１１９６−１２２７（１９７４）など参照］。
【０００３】
【発明が解決しようとする課題】
しかし、これら両測定法は、ともに、複数の酵素を使用するものであって、その反応系が複雑であり、簡便な測定法とは言い難い。その上、グルコースオキシダーゼ・ペルオキシダーゼ系を用いた測定法にあっては、過酸化水素の呈色系が測定液中の共存物質の影響を受けやすく、とりわけ溶存酸素の濃度が全反応を律速するものとなるとき、グルコース濃度の測定が困難になる。他方、ヘキソキナーゼ・グルコース６−リン酸デヒドロゲナーゼ系を用いた測定法にあっては、反応終末端において残存する還元型ニコチンアミドアデニンジヌクレオチド（以下、ＮＡＤＨと表わすこともある。）もしくは還元型ニコチンアミドアデニンジヌクレオチドホスフェート（以下、ＮＡＤＰＨと表わすこともある。）の紫外部吸収を測定する方法であるため、試料中の種々の共存物質のうち、紫外部に吸収域をもつものの影響を受ける。その上、ＮＡＤＨもしくはＮＡＤＰＨの濃度については、光学測定器の測定可能な吸光度限界によって、その測定可能な濃度範囲が一定の範囲に制限されるため、この測定法によるグルコース濃度の測定可能な濃度範囲は、低い濃度レベルの狭い範囲に限られるという欠点がある。
【０００４】
また、酵素グルコースデヒドロゲナーゼを用いたヒト血清中のグルコースの定量法も、有効な測定法のひとつとして提案されている。正常ヒト血清中のグルコース濃度は６５〜１０５ｍｇ／ｄＬ（３．６５．８ｍＭ）程度であるのに対して、グルコースデヒドロゲナーゼのグルコースに対するＫｍ値は数ｍＭ程度と小さいものであるため、グルコースデヒドロゲナーゼを用いたグルコース定量においては、終末点法が適当な測定法として従来採用されていた。
しかしながら、ヒト尿中のグルコース濃度については、その基準値は２０〜４０ｍｇ／ｄＬ程度であるが、重篤な糖尿病もしくは腎障害などを患う患者にあっては、尿中のグルコース濃度が大変高い濃度に及ぶため、そのような患者の尿試料（つまり高濃度グルコース試料）を測定するときには、試料を希釈して、グルコース濃度を低下させる必要があった。しかし、この希釈操作は煩雑なものであり、しかも、希釈の精度および回数が誤差範囲として、最終的な測定値に与える影響も大きく、無視し得ないものである。
【０００５】
本発明は、上述した従来事情を考慮してなされたものであって、その課題とするところは、とくに臨床領域におけるグルコースの定量において、測定可能な濃度範囲が広く、たとえ高濃度の尿検体であっても希釈せずに測定が可能であり、汎用する自動分析機への適用性に優れたグルコースの定量方法、並びに、その方法に好適に使用されるグルコース定量用試薬を提供することにある。
本発明のその他の課題は、特許請求の範囲を含む明細書の記載並びに図面を参照することにより、理解される。
【０００６】
【課題を解決するための手段】
一般に、酵素の反応速度論に基づいて、その酵素の基質となる物質を定量する場合には、酵素の基質に対するＫｍ値が測定系における基質濃度よりも充分により大きいことが必須の条件となる。
しかるに、上述した、グルコースデヒドロゲナーゼを用いたグルコースの定量法にあっては、グルコースデヒドロゲナーゼのグルコースに対するＫｍ値が低すぎるため、そのＫｍ値を適当な高い値に調節することが必要とされる。Ｋｍ値を高い値に調節する手段として、当該酵素に対する拮抗阻害剤を反応系に添加する方法がある。この方法を採択すれば、見かけのＫｍ値が定量可能な高い値にまで上昇し、グルコース濃度の測定範囲を高濃度域にまで広げることができ、よって、尿検体も希釈しないで測定することが可能になる。
本発明者らは、上記の技術的課題を解決するべく鋭意研究した結果、グルコースデヒドロゲナーゼの拮抗阻害剤としてホウ酸もしくはその塩を採用すると、グルコース濃度の高い尿検体であっても、希釈しないでグルコース濃度を正確に測定することができ、有効なグルコース定量法を確立することができることを見出し、本発明を完成した。
【０００７】
したがって、本発明は、より明確には、酵素グルコースデヒドロゲナーゼを用いて試料中のグルコースを定量する方法であって、グルコースを含有する試料に、グルコースデヒドロゲナーゼ阻害剤としてホウ酸もしくはその塩の存在下、補酵素としてニコチンアミドアデニンジヌクレオチド（以下、ＮＡＤと表わすこともある。）もしくはニコチンアミドアデニンジヌクレオチドホスフェート（以下、ＮＡＤＰと表わすこともある。）とともにグルコースデヒドロゲナーゼを作用させ、その際生成する還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）もしくは還元型ニコチンアミドアデニンジヌクレオチドホスフェート（ＮＡＤＰＨ）の量増加速度を測定することを特徴とする、グルコースの定量方法に関する。
また、本発明は、上記の定量方法の使用に適する試薬、つまり、酵素グルコースデヒドロゲナーゼ、補酵素ニコチンアミドアデニンジヌクレオチド（ＮＡＤ）もしくはニコチンアミドアデニンジヌクレオチドホスフェート（ＮＡＤＰ）およびグルコースデヒドロゲナーゼ阻害剤ホウ酸もしくはその塩を含有する溶液よりなる、グルコース定量用試薬にも関する。
【０００８】
【発明の実施の形態】
本発明に従う酵素グルコースデヒドロゲナーゼを用いたグルコースの定量法は、グルコースを含有する試料にグルコースデヒドロゲナーゼを作用させると、同時に、その系に共存する補酵素のＮＡＤもしくはＮＡＤＰが水素還元されてＮＡＤＨもしくはＮＡＤＰＨが生じるが、そのときのＮＡＤＨ量（濃度）もしくはＮＡＤＰＨ量（濃度）の増加速度を例えば紫外部における吸光度の時間変化値にて測定するというものである。
そして、本発明の特徴的な事項は、グルコースデヒドロゲナーゼ阻害剤としてホウ酸もしくはその塩を用いて、グルコースデヒドロゲナーゼのグルコースに対する反応を拮抗阻害させることにより、グルコースデヒドロゲナーゼのグルコースに対する見かけのＫｍ値をより大きな値に変え、高濃度のグルコースを含有する検体であっても、希釈を必要とせずに、グルコースの正確な定量を可能にした点にある。
本発明の方法の実施にあたり、酵素グルコースデヒドロゲナーゼの濃度並びに阻害剤ホウ酸もしくはその塩の濃度は、各々、試料中に含まれる基質グルコースの濃度により決定される。すなわち、高濃度のグルコースを含む検体の定量において見かけのＫｍ値を適当な基準にまで高めるべく、酵素および阻害剤の添加量はそれぞれ調節され、通常、ＮＡＤＨもしくはＮＡＤＰＨの紫外部吸収の測定に用いる吸光度計の実行感度（つまり、単位時間当たりの吸光度の変化値ΔＡｂｓ／ｍｉｎ）に基づいて、最適な条件が設定される。一例を挙げると、グルコース１ｇ／ｄＬの標準液を用い、実行感度（ΔＡｂｓ／ｍｉｎ）を得るには、グルコースデヒドロゲナーゼ０．８ｕ／ｍＬ添加のときで、阻害剤ホウ酸の添加量は１６０ｍＭ程度に調節される。
本発明の方法に使用されるＮＡＤもしくはＮＡＤＰの添加量は、試料中のグルコース量（濃度）に相応して適宜変更される。例えば、グルコース１ｇ／ｄＬの標準液を検体として測定した場合において、ＮＡＤは３ｍＭ以上含まれているのが好都合であることを本発明者は確認している。
また、本発明に係る反応系および定量用試薬に使用する緩衝液としては、酵素による定量技術において通常使用されている緩衝液であれば、いずれも利用可能である。緩衝液には、周知の各種の安定剤、賦活剤などを同時に添加することも可能である。
【０００９】
【実施例】
以下、本発明の最良の実施形態と思われる実施例を説明することにより、本発明をより明確なものにする。
【００１０】
本例は、実際の臨床検体（希釈しない尿）の代わりに、濃度既知のグルコース標準液各種を対象として酵素グルコースデヒドロゲナーゼをＮＡＤとともに作用させ、生じるＮＡＤＨの濃度を紫外部における単位時間当たりの吸光度変化値（ΔＡｂｓ／ｍｉｎ）で以って表わすことにより、グルコース濃度と酵素の反応速度との関係を調べたものである。両者の間に一次関数の関係が現われているとき、グルコース濃度の正確な測定が可能であると認められる。
−標準試料
グルコース濃度１〜１０ｇ／ｄＬの標準液１０種を作製した。これら標準液は、１ｇ／ｄＬより、１ｇ／ｄＬの等間隔で濃度が増加する１０種の試料よりなる。最もグルコース濃度が高い標準液は、１０ｇ／ｄＬの標準液である。
−試薬
次の組成より成る試薬１ａ、１ｂおよび１ｃ（阻害剤試薬）と、試薬２ａおよび２ｂ（酵素試薬）をそれぞれ作製した。
試薬１ｃ（比較）：塩化ナトリウム ５００ｍＭ
上記の各濃度に必要な量の塩化ナトリウムを１００ｍＭトリス（ヒドロキシメチルアミノメタン）緩衝液に溶解し、次いで希塩酸を添加して、最終ｐＨが８．０になるように調製した。
−測定
試薬１ａまたは１ｂと試薬２ａとを自動分析装置Ｈ７１５０形（株式会社日立製作所製）に組み入れ、反応速度法に従いグルコースの定量を行なった。すなわち、グルコースの各標準液１０μＬに３００μＬの試薬１ａまたは１ｂを添加し、３７℃で５分間加温した後、さらに１００μＬの試薬２ａを添加し、紫外部（３４０ｎｍ）における単位時間当たりの吸光度変化値（ΔＡｂｓ／ｍｉｎ）を測定した。
また、比較のため、試薬１ａ、１ｂに代えて試薬１ｃ、また試薬２ａに代えて試薬２ｂを用いて、上記と同様の操作を行ない、グルコース濃度（ｇ／ｄＬ）と紫外部（３４０ｎｍ）における単位時間（毎分）当たりの吸光度変化値（ΔＡｂｓ／ｍｉｎ）との関係を調べた。
【００１１】
−結果
測定の結果を図１にまとめて示す。図１中、○印は、阻害剤としてホウ酸を使用した場合を表わし、△印は、阻害剤としてホウ酸ナトリウムを使用した場合を表わし、そして×印は、阻害剤を使用しない場合を表わす。
図１よりわかるように、阻害剤を添加した反応系と添加しない反応系とを対比することにより、阻害剤としてホウ酸もしくはホウ酸ナトリウムを添加した反応系にあっては、グルコース濃度（ｇ／ｄＬ）と単位時間当たりの吸光度変化値（ΔＡｂｓ／ｍｉｎ）との間に、精度のよい一次関数の関係が認められる。従って、酵素グルコースデヒドロゲナーゼの拮抗阻害剤としてホウ酸もしくはホウ酸ナトリウムを用いる測定法により、１〜１０ｇ／ｄＬのグルコース濃度が高い検体であっても、希釈せずに、感度良く、グルコースの正確な定量が可能であることが判明した。
【００１２】
【発明の効果】
以上説明したように、本発明の方法によれば、とくに臨床領域においてグルコースの定量を行なうとき、グルコースの測定可能な濃度範囲が広く、高い濃度域にまで及び、たとえ高濃度グルコース検体であっても、希釈せずに正確な定量が可能であるという効果が得られる。例えば重篤な糖尿病もしくは腎障害などを患う患者の尿検体をも、希釈しないで、グルコース濃度の正確な測定を為しうる。さらに、本発明の方法は、その実施にあたって特別な装置および設備が不要であり、汎用する自動分析機への適用性にも優れているという利点を有する。
また、本発明のグルコース定量用試薬によれば、上記の本発明に係るグルコース定量方法に好適に使用することができるところの試薬組成物が提供される。
【図面の簡単な説明】
【図１】図１は、試料中のグルコース濃度と該試料について３４０ｎｍにて測定された単位時間当たりの吸光度変化値（ΔＡｂｓ／ｍｉｎ）との関係を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention is suitable for a method for quantifying glucose, in particular, a method for quantifying glucose concentration in a sample such as urinalysis in the clinical laboratory field based on reaction kinetics using the enzyme glucose dehydrogenase, and use of the method. The present invention relates to a measurement reagent.
More specifically, the present invention uses boric acid or a salt thereof as a competitive inhibitor of glucose dehydrogenase when glucose dehydrogenase is allowed to act on a sample containing glucose, thereby reducing the affinity between glucose dehydrogenase and the substrate (glucose). The present invention relates to a method for measuring glucose concentration by increasing the apparent Km value so that even if the glucose concentration in a sample is high, the glucose can be accurately quantified.
[0002]
[Prior art]
Glucose concentrations in body fluids such as serum and urine reflect various diseases or pathologies, so in human health examinations, especially in differential diagnosis of diabetes, which has a high incidence as adult diseases, the examination of glucose concentration is It is an indispensable inspection item.
The standard value of the glucose concentration in human urine is in the range of approximately 20 to 40 mg / dL, and when the measured value falls within that range, the human is judged to be healthy. However, in a patient suffering from severe diabetes or renal disorder, a large amount of glucose is excreted in the urine, so that the glucose concentration in the urine reaches a very high concentration of about 10 g / dL. .
Conventionally known chemical methods and enzymatic methods are methods for measuring glucose concentration. Examples of the chemical method include a reduction method (Somogyi-Nelson method) and an oxidative condensation method (o-toluidine boric acid method). However, these methods are disadvantageous in that they are affected by reducing substances and have low specificity for glucose, and therefore, it is generally judged that enzymatic methods are superior to chemical methods.
As the enzyme method, a measurement method using a glucose oxidase / peroxidase system and a measurement method using a hexokinase / glucose 6-phosphate dehydrogenase system are mainly used [Hans U. B. : Methods of Enzymatic Analysis, vol. 3, 1196-1227 (1974)].
[0003]
[Problems to be solved by the invention]
However, both of these measurement methods use a plurality of enzymes, and the reaction system is complicated, and it is difficult to say that these are simple measurement methods. In addition, in the measurement method using the glucose oxidase / peroxidase system, the coloration system of hydrogen peroxide is easily affected by coexisting substances in the measurement solution, and in particular, the concentration of dissolved oxygen controls the overall reaction. The glucose concentration becomes difficult to measure. On the other hand, in the measurement method using the hexokinase / glucose 6-phosphate dehydrogenase system, reduced nicotinamide adenine dinucleotide (hereinafter sometimes referred to as NADH) or reduced nicotinamide remaining at the terminal end of the reaction. Since it is a method for measuring the ultraviolet absorption of adenine dinucleotide phosphate (hereinafter sometimes referred to as NADPH), it is affected by various coexisting substances in the sample having an absorption region in the ultraviolet region. In addition, for NADH or NADPH concentration, the measurable concentration range is limited to a certain range due to the measurable absorbance limit of the optical measuring instrument. Has the disadvantage of being limited to a narrow range of low density levels.
[0004]
In addition, a method for quantifying glucose in human serum using the enzyme glucose dehydrogenase has been proposed as an effective measurement method. The glucose concentration in normal human serum is about 65 to 105 mg / dL (3.65.8 mM), whereas the Km value of glucose dehydrogenase for glucose is as small as several mM, so glucose dehydrogenase is used. In the conventional glucose determination, the end point method has been conventionally adopted as an appropriate measurement method.
However, as for the glucose concentration in human urine, the reference value is about 20 to 40 mg / dL. However, in patients suffering from severe diabetes or renal disorder, the concentration of glucose in urine is very high. Therefore, when measuring such a patient's urine sample (ie, a high concentration glucose sample), it was necessary to dilute the sample to reduce the glucose concentration. However, this dilution operation is complicated, and the accuracy and number of dilutions have a large influence on the final measurement value as an error range and cannot be ignored.
[0005]
The present invention has been made in consideration of the above-described conventional circumstances, and the problem is that, particularly in the quantitative determination of glucose in the clinical field, the measurable concentration range is wide, even with high-concentration urine samples. An object of the present invention is to provide a glucose quantification method that can be measured without diluting, and is excellent in applicability to a general-purpose automatic analyzer, and a glucose quantification reagent suitably used in the method. .
Other objects of the present invention will be understood by referring to the description of the specification including the claims and the drawings.
[0006]
[Means for Solving the Problems]
In general, when a substance serving as a substrate for an enzyme is quantified based on the reaction kinetics of the enzyme, it is essential that the Km value for the enzyme substrate is sufficiently larger than the substrate concentration in the measurement system.
However, in the above-described glucose determination method using glucose dehydrogenase, the Km value of glucose dehydrogenase with respect to glucose is too low, and therefore it is necessary to adjust the Km value to an appropriate high value. As a means for adjusting the Km value to a high value, there is a method of adding a competitive inhibitor for the enzyme to the reaction system. If this method is adopted, the apparent Km value increases to a quantifiable high value, and the measurement range of the glucose concentration can be expanded to a high concentration range. Therefore, the urine sample can be measured without dilution. It becomes possible.
As a result of intensive studies to solve the above technical problems, the present inventors have adopted boric acid or a salt thereof as a competitive inhibitor of glucose dehydrogenase. It was found that the glucose concentration can be accurately measured and an effective glucose determination method can be established, and the present invention has been completed.
[0007]
Therefore, more specifically, the present invention is a method for quantifying glucose in a sample using the enzyme glucose dehydrogenase, in the presence of boric acid or a salt thereof as a glucose dehydrogenase inhibitor in a sample containing glucose. Reduced form produced by allowing glucose dehydrogenase to act together with nicotinamide adenine dinucleotide (hereinafter also referred to as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter also referred to as NADP) as a coenzyme. The present invention relates to a method for quantifying glucose, characterized by measuring the rate of increase in the amount of nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH).
The present invention also provides reagents suitable for use in the above quantification methods, ie, enzyme glucose dehydrogenase, coenzyme nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) and glucose dehydrogenase inhibitor boric acid or The present invention also relates to a glucose quantification reagent comprising a solution containing the salt.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
In the method for quantifying glucose using the enzyme glucose dehydrogenase according to the present invention, when glucose dehydrogenase is allowed to act on a glucose-containing sample, simultaneously, NAD or NADP of the coenzyme coexisting in the system is reduced by hydrogen, and NADH or NADPH is The increase rate of the NADH amount (concentration) or NADPH amount (concentration) at that time is measured by, for example, the time change value of the absorbance in the ultraviolet region.
And the characteristic matter of the present invention is that the apparent Km value of glucose dehydrogenase with respect to glucose is increased by competitively inhibiting the reaction of glucose dehydrogenase with glucose using boric acid or a salt thereof as a glucose dehydrogenase inhibitor. In other words, even in the case of a specimen containing a high concentration of glucose, the glucose can be accurately quantified without requiring dilution.
In carrying out the method of the present invention, the concentration of the enzyme glucose dehydrogenase and the concentration of the inhibitor boric acid or salt thereof are each determined by the concentration of substrate glucose contained in the sample. That is, in order to increase the apparent Km value to an appropriate standard in the quantification of a sample containing a high concentration of glucose, the addition amounts of the enzyme and the inhibitor are adjusted, respectively, and are usually used for measuring the ultraviolet absorption of NADH or NADPH. Optimal conditions are set based on the execution sensitivity of the absorptiometer (that is, the absorbance change value ΔAbs / min per unit time). For example, in order to obtain an execution sensitivity (ΔAbs / min) using a standard solution of glucose 1 g / dL, when adding glucose dehydrogenase 0.8 u / mL, the amount of inhibitor boric acid added is about 160 mM. Adjusted.
The amount of NAD or NADP used in the method of the present invention is appropriately changed according to the amount (concentration) of glucose in the sample. For example, when the standard solution of glucose 1 g / dL is measured as a specimen, the present inventor has confirmed that NAD is conveniently contained at 3 mM or more.
In addition, as the buffer solution used in the reaction system and the quantification reagent according to the present invention, any buffer solution that is usually used in an enzyme quantification technique can be used. Various well-known stabilizers, activators, and the like can be simultaneously added to the buffer solution.
[0009]
【Example】
In the following, the present invention will be made clearer by describing examples which are considered to be the best modes of the present invention.
[0010]
In this example, instead of an actual clinical sample (undiluted urine), the enzyme glucose dehydrogenase is allowed to act with NAD on various glucose standard solutions with known concentrations, and the resulting NADH concentration changes in absorbance per unit time in the ultraviolet region. The relationship between the glucose concentration and the reaction rate of the enzyme was examined by expressing the value (ΔAbs / min). When a linear function relationship appears between the two, it is recognized that an accurate measurement of the glucose concentration is possible.
-Standard sample Ten standard solutions having a glucose concentration of 1 to 10 g / dL were prepared. These standard solutions consist of 10 samples whose concentrations increase from 1 g / dL at equal intervals of 1 g / dL. The standard solution with the highest glucose concentration is a standard solution of 10 g / dL.
-Reagents Reagents 1a, 1b and 1c (inhibitor reagents) and reagents 2a and 2b (enzyme reagents) having the following compositions were prepared, respectively.
Reagent 1c (comparison): Sodium chloride 500 mM
The amount of sodium chloride required for each of the above concentrations was dissolved in 100 mM Tris (hydroxymethylaminomethane) buffer, and then diluted hydrochloric acid was added to adjust the final pH to 8.0.
-Measurement reagent 1a or 1b and reagent 2a were incorporated into automatic analyzer H7150 (manufactured by Hitachi, Ltd.), and glucose was quantified according to the reaction rate method. That is, 300 μL of reagent 1a or 1b was added to 10 μL of each glucose standard solution, heated at 37 ° C. for 5 minutes, and then 100 μL of reagent 2a was added, and the absorbance change per unit time in the ultraviolet region (340 nm) The value (ΔAbs / min) was measured.
For comparison, the same operation as described above was performed using the reagent 1c instead of the reagents 1a and 1b and the reagent 2b instead of the reagent 2a, and the glucose concentration (g / dL) and the ultraviolet region (340 nm) were measured. The relationship with the absorbance change value (ΔAbs / min) per unit time (every minute) was examined.
[0011]
-Results of the measurement are summarized in FIG. In FIG. 1, ◯ represents the case where boric acid was used as an inhibitor, Δ represents the case where sodium borate was used as an inhibitor, and X represents the case where no inhibitor was used. .
As can be seen from FIG. 1, by comparing the reaction system to which the inhibitor is added and the reaction system to which the inhibitor is not added, in the reaction system to which boric acid or sodium borate is added as an inhibitor, the glucose concentration (g / dL) and a change in absorbance per unit time (ΔAbs / min), a highly accurate linear function relationship is recognized. Therefore, even if a sample having a high glucose concentration of 1 to 10 g / dL is measured using boric acid or sodium borate as a competitive inhibitor of the enzyme glucose dehydrogenase, it is sensitive and accurate without any dilution. It was found that quantification was possible.
[0012]
【The invention's effect】
As described above, according to the method of the present invention, particularly when glucose is quantified in the clinical region, the concentration range of glucose is wide and extends to a high concentration range, even if it is a high concentration glucose sample. However, there is an effect that accurate quantification is possible without dilution. For example, a urine sample of a patient suffering from severe diabetes or renal disorder can be accurately measured without diluting it. Furthermore, the method of the present invention does not require special equipment and equipment for its implementation, and has the advantage that it is excellent in applicability to general-purpose automatic analyzers.
Moreover, according to the glucose determination reagent of the present invention, a reagent composition that can be suitably used in the glucose determination method according to the present invention is provided.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the glucose concentration in a sample and the absorbance change value (ΔAbs / min) per unit time measured for the sample at 340 nm.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11401499A JP4217815B2 (en) | 1999-03-17 | 1999-03-17 | Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11401499A JP4217815B2 (en) | 1999-03-17 | 1999-03-17 | Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000262299A JP2000262299A (en) | 2000-09-26 |
| JP4217815B2 true JP4217815B2 (en) | 2009-02-04 |
Family
ID=14626902
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11401499A Expired - Fee Related JP4217815B2 (en) | 1999-03-17 | 1999-03-17 | Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4217815B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013051897A (en) * | 2011-09-01 | 2013-03-21 | Mitsubishi Chemical Medience Corp | Method for suppressing influence of interfering substance |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5286511B2 (en) * | 2005-03-31 | 2013-09-11 | 株式会社シノテスト | Glucose measurement method and reagent |
| JP2012211810A (en) * | 2011-03-31 | 2012-11-01 | Cci Corp | Biosensor having improved measurement accuracy |
| CN103088108A (en) * | 2012-11-16 | 2013-05-08 | 李立和 | Kit for detecting glucose by using glucose dehydrogenase method and preparation method |
| CN110726842B (en) * | 2018-07-17 | 2024-01-30 | 上海瀚联诊断科技有限公司 | Glucose quality control liquid |
-
1999
- 1999-03-17 JP JP11401499A patent/JP4217815B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013051897A (en) * | 2011-09-01 | 2013-03-21 | Mitsubishi Chemical Medience Corp | Method for suppressing influence of interfering substance |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2000262299A (en) | 2000-09-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fossati et al. | Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement. | |
| US5516700A (en) | Automated urinalysis method | |
| US4729959A (en) | Glucose reference control for glucose test strips | |
| US8883439B2 (en) | Blood component measurement method utilizing hemolyzed whole blood, and kit for the method | |
| EP0823943B1 (en) | Determination of glycated proteins | |
| Wiener | Whole blood glucose: what are we actually measuring? | |
| JP3217066B2 (en) | Compositions useful for anaerobic determination of analytes | |
| JP4217815B2 (en) | Method for quantifying glucose by glucose dehydrogenase and reagent for quantifying glucose | |
| EP0140589B1 (en) | Enzymatic determination of d-3-hydroxybutyric acid or acetoacetic acid, and reagents therefor | |
| Yao et al. | Electrochemical dehydrogenase-based homogeneous assays in whole blood | |
| JPH06253895A (en) | Measurement of total amount of ketone compounds and reagent therefor | |
| CN112255219A (en) | 1, 5-sorbitan determination kit, and preparation method and application thereof | |
| JP2955443B2 (en) | Analytical element and analyte detection method | |
| JPH0698031B2 (en) | LD-1 isozyme assay and reagents | |
| Kimata et al. | Evaluation of a new automated, enzymatic inulin assay using D-fructose dehydrogenase | |
| Kayamori et al. | Enzymatic method for assaying uric acid in serum with a new tetrazolium salt produces water-soluble formazan dye | |
| JP3760250B2 (en) | Method for quantifying sulfate-conjugated bile acids and kits thereof | |
| JP3981190B2 (en) | Cholesterol determination method and reagent for determination | |
| JP2761768B2 (en) | Method for determining NADH and method for determining bile acid using the same | |
| Alqobaisi et al. | Interference of Mannose and Galactose in Glucose Assay by the Glucose Oxidase/Peroxidase Method | |
| JP3159273B2 (en) | Sorbitol measurement method and composition thereof | |
| Ruiz et al. | Determination of plasma lipid hydroperoxides by an NADPH/NADP+ coupled enzyme reaction system. Evaluation of a method | |
| WO2006030866A1 (en) | Method of quantitative determination of uric acid | |
| JP3227486B2 (en) | Copper measurement method | |
| JP3034984B2 (en) | Highly sensitive method and composition for quantification of D-galactose |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20060228 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20060228 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20080930 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20081027 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111121 Year of fee payment: 3 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111121 Year of fee payment: 3 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121121 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121121 Year of fee payment: 4 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20131121 Year of fee payment: 5 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |