JPH022600B2 - - Google Patents
Info
- Publication number
- JPH022600B2 JPH022600B2 JP14955282A JP14955282A JPH022600B2 JP H022600 B2 JPH022600 B2 JP H022600B2 JP 14955282 A JP14955282 A JP 14955282A JP 14955282 A JP14955282 A JP 14955282A JP H022600 B2 JPH022600 B2 JP H022600B2
- Authority
- JP
- Japan
- Prior art keywords
- noradrenaline
- dopamine
- adrenaline
- catecholamines
- converted
- 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
Links
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 72
- UCTWMZQNUQWSLP-UHFFFAOYSA-N adrenaline Chemical compound CNCC(O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-UHFFFAOYSA-N 0.000 claims description 51
- SFLSHLFXELFNJZ-UHFFFAOYSA-N norepinephrine Natural products NCC(O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-UHFFFAOYSA-N 0.000 claims description 44
- 229960002748 norepinephrine Drugs 0.000 claims description 44
- SFLSHLFXELFNJZ-QMMMGPOBSA-N (-)-norepinephrine Chemical compound NC[C@H](O)C1=CC=C(O)C(O)=C1 SFLSHLFXELFNJZ-QMMMGPOBSA-N 0.000 claims description 41
- 229960003638 dopamine Drugs 0.000 claims description 36
- 150000003943 catecholamines Chemical class 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 18
- 238000000926 separation method Methods 0.000 claims description 10
- PSTISBTWOZSQID-UHFFFAOYSA-N 1h-indole-2,3,4,5-tetrol Chemical compound C1=C(O)C(O)=C2C(O)=C(O)NC2=C1 PSTISBTWOZSQID-UHFFFAOYSA-N 0.000 claims description 9
- 238000004811 liquid chromatography Methods 0.000 claims description 7
- 108010015720 Dopamine beta-Hydroxylase Proteins 0.000 claims description 6
- 102100033156 Dopamine beta-hydroxylase Human genes 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 description 18
- 108090000790 Enzymes Proteins 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000003480 eluent Substances 0.000 description 9
- 238000006911 enzymatic reaction Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 7
- 239000012472 biological sample Substances 0.000 description 6
- -1 potassium ferricyanide Chemical compound 0.000 description 6
- 235000010323 ascorbic acid Nutrition 0.000 description 5
- 239000011668 ascorbic acid Substances 0.000 description 5
- 229960005070 ascorbic acid Drugs 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 235000011121 sodium hydroxide Nutrition 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000008363 phosphate buffer Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- JHFAEUICJHBVHB-UHFFFAOYSA-N 1h-indol-2-ol Chemical class C1=CC=C2NC(O)=CC2=C1 JHFAEUICJHBVHB-UHFFFAOYSA-N 0.000 description 2
- 229920002126 Acrylic acid copolymer Polymers 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- MRDDZASGYAFJTG-UHFFFAOYSA-N 2-ethenyl-3-phenylpenta-2,4-dienoic acid Chemical compound OC(=O)C(C=C)=C(C=C)C1=CC=CC=C1 MRDDZASGYAFJTG-UHFFFAOYSA-N 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) 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-J 0.000 description 1
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 1
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 239000001744 Sodium fumarate Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- MSJMDZAOKORVFC-SEPHDYHBSA-L disodium fumarate Chemical compound [Na+].[Na+].[O-]C(=O)\C=C\C([O-])=O MSJMDZAOKORVFC-SEPHDYHBSA-L 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910003002 lithium salt Inorganic materials 0.000 description 1
- 159000000002 lithium salts Chemical class 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 description 1
- 235000010378 sodium ascorbate Nutrition 0.000 description 1
- 229960005055 sodium ascorbate Drugs 0.000 description 1
- 229940005573 sodium fumarate Drugs 0.000 description 1
- 235000019294 sodium fumarate Nutrition 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9406—Neurotransmitters
Description
【発明の詳細な説明】
本発明は、カテコールアミンの定量法に関し、
血液、尿等の生体試料液中に極めて微量に含有さ
れるカテコールアミンを液体クロマトグラフイー
を利用して分離し、ノルアドレナリン、アドレナ
リン、ドーパミンの３成分を定量する方法に関す
る。
従来生体試料液中に含有されるカテコールアミ
ンの定量法として、高速液体クロマトグラフイー
で、ノルアドレナリン、アドレナリン、ドーパミ
ンの３成分を分画し、これらの各成分の電気化学
的性質を利用して検出する方法（VMD法）と、
高速液体クロマトグラフイーでノルアドレナリ
ン、アドレナリン、ドーパミンの３成分に分画
し、次いで蛍光物質に変換した後、その蛍光を検
知し定量する方法（THI法）の二法が実用化さ
れている。
しかしながらVMD法では、生体試料液中の電
気化学的性質を有する全ての成分を同感度で検出
するため、カテコールアミンを検出するためには
かなり繁雑な前処理を必要とする欠点があり、又
血中カテコールアミンはその濃度が血清１ml中に
10-12ｇ（pg）程度であつて極めて微量であるた
め、アドレナリン、ドーパミンの検出が困難であ
つた。又THI法では感度的にVMD法より優れ、
血中カテコールアミンも、ノルアドレナリン、ア
ドレナリンの検出が可能である。しかしドーパミ
ンはTHI物質への変換効率が悪く、しかも測定
波長がノルアドレナリン、アドレナリンとは異な
るため検出できないという欠点があつた。
本発明は上記の欠点を解消し、生体試料液中の
ノルアドレナリン、アドレナリン、ドーパミンの
各成分を液体クロマトグラフイーにより分画し、
ドーパミンを選択的にノルアドレナリンに変換
し、更に蛍光を発現する誘導体に変換して蛍光を
検知し、定量する方法を提供することを目的とす
る。
本発明の要旨は、カテコールアミンを含有する
試料液を液体クロマトグラフイーにおける分離カ
ラムに導入して、ノルアドレナリン、アドレナリ
ン、ドーパミンの３成分を分離し、これにドーパ
ミン−β−水酸化酵素を作用させてドーパミンを
選択的にノルアドレナリンに変換し、次いでノル
アドレナリン、アドレナリン、ドーパミンから変
換されたノルアドレナリンにテトラヒドロキシイ
ンドールを接触させ、これにより得られた夫々の
誘導体の蛍光を検出し、これに基づいてノルアド
レナリン、アドレナリン及びドーパミンの３成分
を定量することを特徴とする、カテコールアミン
の定量法に存する。
次に本発明カテコールアミンの定量法について
更に詳細に説明する。
生体液中のカテコールアミンは血清中には数
pg／ml〜数百pg／ml、尿中には数ng／ml〜数百
ng／ml程度にごく微量含有されている。この生
体液を試料とし、その中に含有されるカテコール
アミンを定量するために液中クロマトグラフイー
を用いる。液体クロマトグラフイーにかけられる
生体試料は、カテコールアミンの分画を阻害する
成分を予め除去するための前処理操作にかけられ
る。液体クロマトグラフイー工程を第１図に示
す。１は溶離液槽であり、例えばリン酸緩衝液が
使用され、定流量ポンプ２により送液される。３
は試料注入器であり、カテコールアミンを含有す
る試料液が供給されて溶離液と混合される。４は
分離用カラムであり、例えばオクタデシル基を導
入した多孔性シリカ、ジビニルベンゼン−（メタ）
アクリル酸共重合体等の粒子が充填されている。
溶離液のPH値、イオン強度、イオンの種類、流
速は分離用カラム４の分離能に応じて適宜調整さ
れる。
溶離液と混り合つた前記試料液は分離用カラム
４を通過することにより、ノルアドレナリン、ア
ドレナリン、ドーパミンの３成分に順次分離され
る。
ところでドーパミンは、カテコールアミンの分
画の常法とされてきたVMD法やTHI法では感度
よく検出されない。そこで本発明においては、ド
ーパミンβ−水酸化酵素を作用させる。５は酵素
溶液槽であり、定流量ポンプ６によつてドーパミ
ンβ−水酸化酵素溶液が供給される。前記酵素は
ドーパミンを選択的にノルアドレナリンに変換す
る酵素であり、ノルアドレナリン、アドレナリ
ン、ドーパミンの順に分離されたカテコールアミ
ンは酵素反応によつてドーパミンがノルアドレナ
リンに変換され、ノルアドレナリン、アドレナリ
ン、ドーパミンから変換されたノルアドレナリン
となる。
前記酵素溶液は、ドーパミンβ−水酸化酵素の
他に、例えば緩衝液、助酵素としてのアスコルビ
ン酸、酵素活性を安定化するためのカタラーゼ、
アデノシントリフオスフエートとグルコースデヒ
ドロゲナーゼの混合物等を構成成分とする。緩衝
液としては、例えばリン酸、酢酸、クエン酸等の
酸とそのナトリウム塩、カリウム塩、リチウム塩
等よりなるものが使用される。又、酵素活性は−
SH基を有する化合物によつて活性が低下するの
で、−SH基を有する化合物を失活させるためにＮ
−エチルマレイミド、硫酸銅等を構成成分とす
る。又、ドーパミンからノルアドレナリンへの変
換効率を高めるためフマル酸等のジカルボン酸を
構成成分とするのが好適である。
前記酵素はその酵素反応の好適PH値は4.8〜5.7
であり、更に好適には5.2〜5.4であり、最適には
5.3である。
このような酵素反応に適したPH値とするために
緩衝液のPH値、イオン強度を調整する必要があ
る。緩衝液のPH値、イオン強度は分離カラム４を
通過後の溶離液のPH値、イオン強度、流速に応じ
て調整するのが好ましい。酵素反応によりドーパ
ミンをノルアドレナリンに変換するには、前記酵
素溶液を酵素溶液槽５から定流量ポンプ６で送給
し、反応槽７で反応させるか、又は反応槽７にか
えて前記酵素を固定化した担体が充填されている
カラムを設置し、緩衝液によりPHを調整しカラム
中で酵素反応を行なわせるのが好適である。反応
槽７としては、例えば反応コイルを設置しコイル
を流通するノルアドレナリン、アドレナリン、ド
ーパミンに分離されたカテコールアミンと溶離液
との混合成分に前記酵素溶液を供給し、ドーパミ
ンと前記酵素との選択的反応を生じさせ、ドーパ
ミンをノルアドレナリンに変換する。
反応槽７又はカラムから出たノルアドレナリ
ン、アドレナリン、ドーパミンから変換されたノ
ルアドレナリンにテトラヒドロキシインドールを
接触させて、これにより得られた夫々の誘導体の
蛍光を検出する。テトラヒドロキシインドール誘
導体に変換するには、フエリシアン化カリウム水
溶液、アスコルビン酸水溶液、水酸化ナトリウム
水溶液が供給される。これらの各水溶液は、ノル
アドレナリン、アドレナリン、ドーパミンから変
換されたノルアドレナリンの夫々と反応し、テト
ラヒドロキシインドール誘導体を生成する。
９はフエリシアン化カリウム水溶液槽、１０は
アスコルビン酸水溶液槽、１１はカセイソーダ水
溶液槽であり、これらの各槽から定流量３連ポン
プ８により夫々の水溶液が反応槽１２に供給され
る。これらの３液と反応してノルアドレナリンの
テトラヒドロキシインドール誘導体、アドレナリ
ンのテトラヒドロキシインドール誘導体、ドーパ
ミンから変換されたノルアドレナリンのテトラヒ
ドロキシインドール誘導体に変換される。
夫々の誘導体は蛍光性物質であり、蛍光検出器
１３により検出することができる。１４は記録計
であり、蛍光検出部１３による検出結果は記録計
で記録される。そしてこの検出結果に基づいて、
ノルアドレナリン、アドレナリン、ドーパミンか
ら変換されたノルアドレナリンのヒドロキシイン
ドール誘導体の量を知ることができ、これにより
知り得た量から生体試料中に元々含有されていた
ノルアドレナリン、アドレナリン、ドーパミンを
定量とすることができる。
本発明によれば試料中に微量にしか含有されな
いカテコールアミンにおける、アドレナリン、ノ
ルアドレナリン、ドーパミンの３成分を同時にか
つ高感度に測定することができる。又測定に際し
て一つの装置で測定を行うことができ、測定時間
も短縮される等の利点が存する。
実施例 １
健常人５検体の血液５mlを真空採血管に採取
し、30分間室温放置後、3000r・ｐ・ｍで５分間
遠心分離し、血清２mlを得た。
次いでこの血清を活性アルミナにより前処理
し、液体クロマトグラフイーによるカテコールア
ミンの分離を阻害する成分を除去し、カテコール
アミンを含有する血清試料を得た。
溶離液槽１から0.1モルリン酸緩衝液を定流量
ポンプ２で0.8ml／分の流速で供給し、試料注入
器３からカテコールアミンを含有する血清試料を
注入し、前記リン酸緩衝液と混合した。分離用カ
ラム４としては、ジビニルベンゼン−アクリル酸
共重合体粒子を充填した内径５mm、長さ30cmのス
テンレスカラムを使用した。
酵素溶液槽５から定流量ポンプ６により0.2
ml／分の流速で酵素溶液を供給した。酵素溶液の
組成はドーパミンβ−水酸化酵素５単位／200ml、
PH6.0の0.5モルリン酸緩衝液、40ミリモルのＮ−
エチルマレイミド、１重量％のカタラーゼ、10ミ
リモルのフマル酸ナトリウム、10ミリモルのアス
コルビン酸ナトリウム、5μモルの硫酸銅から成
るものとした。酵素反応槽７は0.5mmの内径を有
し、10mの長さを有するコイルを使用し、37℃で
酵素反応を生じさせた。酵素反応によりドーパミ
ンはノルアドレナリンに変換された。
反応槽７を出たノルアドレナリン、アドレナリ
ン、ドーパミンから変換されたノルアドレナリン
に、フエリシアン化カリウム水溶液、アスコルビ
ン酸水溶液、水酸化ナトリウム水溶液を夫々接触
させて、夫々のテトラヒドロキシインドール誘導
体を生成させた。
フエリシアン化カリウム水溶液槽９は0.05重量
％のフエリシアン化カリウム、0.2モルのリン酸
一カリウム、0.4モルのリン酸二カリウムを組成
とし、アルコルピン酸水溶液槽１０は0.2重量％
の濃度に調整し、カセイソーダ水溶液は６規定の
濃度に調整し、夫々0.2ml／分の流速で定流量３
連ポンプ８により供給した。反応槽１２の温度は
60℃に保持した。
これらの各水溶液との反応によりノルアドレナ
リン、アドレナリン、ドーパミンから変換された
ノルアドレナリンの夫々のヒドロキシインドール
誘導体が生成された。夫々の誘導体を蛍光検出器
１３で検出し、記録計１４により記録した。P1.
P2.P3は夫々ノルアドレナリン、アドレナリン、
ドーパミンから変換されたノルアドレナリンの誘
導体のピークである。
このようにして記録計１４により記録された
夫々の誘導体のクロマトグラムを第２図に示す。
このようにして得られた結果に基づいて、標準カ
テコールアミンを用いた検量線、及びプール血清
に標準カテコールアミンを添加し、前処理操作を
施して得た回収率より５検体夫々のカテコールア
ミンの血中濃度が表１の通り得られた。
【表】DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying catecholamines,
The present invention relates to a method for separating extremely small amounts of catecholamines contained in biological sample fluids such as blood and urine using liquid chromatography, and quantifying the three components of noradrenaline, adrenaline, and dopamine. Conventionally, as a method for quantifying catecholamines contained in biological sample fluids, three components, noradrenaline, adrenaline, and dopamine, are fractionated using high-performance liquid chromatography and detected using the electrochemical properties of each of these components. method (VMD method) and
Two methods have been put into practical use: fractionating it into three components, noradrenaline, adrenaline, and dopamine, using high-performance liquid chromatography, then converting it into a fluorescent substance, and then detecting and quantifying the fluorescence (THI method). However, since the VMD method detects all components with electrochemical properties in a biological sample with the same sensitivity, it has the disadvantage that it requires quite complicated pretreatment to detect catecholamines. The concentration of catecholamines in 1ml of serum is
It was difficult to detect adrenaline and dopamine because the amount was extremely small, about 10 -12 g (pg). In addition, the THI method is superior to the VMD method in sensitivity,
Blood catecholamines such as noradrenaline and adrenaline can also be detected. However, dopamine has the disadvantage that it cannot be detected because it has a low conversion efficiency into THI substances and the measurement wavelength is different from that of noradrenaline and adrenaline. The present invention solves the above-mentioned drawbacks, and fractionates the components of noradrenaline, adrenaline, and dopamine in a biological sample liquid by liquid chromatography.
The object of the present invention is to provide a method for selectively converting dopamine into noradrenaline and further converting it into a derivative that expresses fluorescence, detecting the fluorescence, and quantifying the fluorescence. The gist of the present invention is to introduce a sample solution containing catecholamines into a separation column in liquid chromatography to separate three components, noradrenaline, adrenaline, and dopamine, and to allow dopamine-β-hydroxylase to act on the three components. Dopamine is selectively converted to noradrenaline, and then tetrahydroxyindole is contacted with noradrenaline, adrenaline, and noradrenaline converted from dopamine, and the fluorescence of each derivative obtained is detected, and based on this, noradrenaline and adrenaline are converted. The present invention relates to a method for quantifying catecholamines, which is characterized by quantifying the three components of dopamine and dopamine. Next, the method for quantifying catecholamines of the present invention will be explained in more detail. Catecholamines in biological fluids are few in serum.
pg/ml to several hundred pg/ml, several ng/ml to several hundred in urine
It is present in very small amounts, around ng/ml. This biological fluid is used as a sample, and submerged chromatography is used to quantify the catecholamines contained therein. A biological sample to be subjected to liquid chromatography is subjected to a pretreatment operation to remove components that inhibit the fractionation of catecholamines. The liquid chromatography process is shown in FIG. Reference numeral 1 denotes an eluent tank in which, for example, a phosphate buffer is used, and the eluent is fed by a constant flow pump 2. 3
is a sample injector into which a sample solution containing catecholamines is supplied and mixed with the eluent. 4 is a separation column, for example, porous silica with an octadecyl group introduced, divinylbenzene-(meth)
Filled with particles such as acrylic acid copolymer. The pH value, ionic strength, type of ion, and flow rate of the eluent are adjusted as appropriate depending on the separation ability of the separation column 4. The sample liquid mixed with the eluent passes through the separation column 4 and is sequentially separated into three components: noradrenaline, adrenaline, and dopamine. By the way, dopamine cannot be detected with good sensitivity using the VMD method and THI method, which are conventional methods for fractionating catecholamines. Therefore, in the present invention, dopamine β-hydroxylase is used. 5 is an enzyme solution tank, into which a dopamine β-hydroxylase solution is supplied by a constant flow pump 6. The enzyme is an enzyme that selectively converts dopamine into noradrenaline, and the catecholamines separated in the order of noradrenaline, adrenaline, and dopamine are converted into noradrenaline through an enzymatic reaction, and noradrenaline is converted from noradrenaline, adrenaline, and dopamine. becomes. In addition to dopamine β-hydroxylase, the enzyme solution contains, for example, a buffer, ascorbic acid as a coenzyme, catalase to stabilize enzyme activity,
Its constituent components include a mixture of adenosine triphosphate and glucose dehydrogenase. As the buffer solution, for example, one composed of acids such as phosphoric acid, acetic acid, and citric acid, and their sodium salts, potassium salts, and lithium salts is used. Also, the enzyme activity is -
Since the activity is lowered by compounds having a SH group, N
- Consists of ethylmaleimide, copper sulfate, etc. Further, in order to increase the conversion efficiency from dopamine to noradrenaline, it is preferable to use a dicarboxylic acid such as fumaric acid as a constituent component. The preferred pH value for the enzyme reaction is 4.8 to 5.7.
, more preferably 5.2 to 5.4, optimally
It is 5.3. It is necessary to adjust the pH value and ionic strength of the buffer solution to obtain a pH value suitable for such enzymatic reactions. The PH value and ionic strength of the buffer solution are preferably adjusted according to the PH value, ionic strength, and flow rate of the eluent after passing through the separation column 4. In order to convert dopamine into noradrenaline by an enzymatic reaction, the enzyme solution is fed from the enzyme solution tank 5 with a constant flow pump 6 and reacted in the reaction tank 7, or the enzyme is immobilized in the reaction tank 7. It is preferable to install a column packed with a carrier prepared by the reaction system, adjust the pH with a buffer solution, and carry out the enzyme reaction in the column. As the reaction tank 7, for example, a reaction coil is installed, and the enzyme solution is supplied to a mixed component of an eluent and catecholamines separated into noradrenaline, adrenaline, and dopamine flowing through the coil, and selective reaction between dopamine and the enzyme is performed. and converts dopamine into noradrenaline. Tetrahydroxyindole is brought into contact with noradrenaline, adrenaline, and noradrenaline converted from dopamine released from the reaction tank 7 or the column, and the fluorescence of each derivative obtained thereby is detected. For conversion into a tetrahydroxyindole derivative, a potassium ferricyanide aqueous solution, an ascorbic acid aqueous solution, and a sodium hydroxide aqueous solution are supplied. Each of these aqueous solutions reacts with noradrenaline, adrenaline, and noradrenaline converted from dopamine, respectively, to produce tetrahydroxyindole derivatives. 9 is a potassium ferricyanide aqueous solution tank, 10 is an ascorbic acid aqueous solution tank, and 11 is a caustic soda aqueous solution tank, and the respective aqueous solutions are supplied from these tanks to the reaction tank 12 by a constant flow triple pump 8. It reacts with these three liquids and is converted into a tetrahydroxyindole derivative of noradrenaline, a tetrahydroxyindole derivative of adrenaline, and a tetrahydroxyindole derivative of noradrenaline converted from dopamine. Each derivative is a fluorescent substance and can be detected by a fluorescence detector 13. 14 is a recorder, and the detection result by the fluorescence detection section 13 is recorded by the recorder. And based on this detection result,
It is possible to know the amount of hydroxyindole derivatives of noradrenaline converted from noradrenaline, adrenaline, and dopamine, and from this, it is possible to quantify the amount of noradrenaline, adrenaline, and dopamine originally contained in the biological sample. . According to the present invention, three components of catecholamines, adrenaline, noradrenaline, and dopamine, which are contained only in trace amounts in a sample, can be measured simultaneously and with high sensitivity. Further, there are advantages such as being able to perform measurements with one device and shortening the measurement time. Example 1 5 ml of blood from 5 healthy subjects was collected into vacuum blood collection tubes, left at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 5 minutes to obtain 2 ml of serum. Next, this serum was pretreated with activated alumina to remove components that inhibit the separation of catecholamines by liquid chromatography, and a serum sample containing catecholamines was obtained. A 0.1M phosphate buffer was supplied from the eluent tank 1 at a flow rate of 0.8 ml/min using a constant flow pump 2, and a serum sample containing catecholamine was injected from the sample injector 3 and mixed with the phosphate buffer. As the separation column 4, a stainless steel column with an inner diameter of 5 mm and a length of 30 cm filled with divinylbenzene-acrylic acid copolymer particles was used. 0.2 by constant flow pump 6 from enzyme solution tank 5
The enzyme solution was fed at a flow rate of ml/min. The composition of the enzyme solution is dopamine β-hydroxylase 5 units/200ml;
0.5 molar phosphate buffer at pH 6.0, 40 mmol N-
It consisted of ethylmaleimide, 1% by weight catalase, 10 mmol sodium fumarate, 10 mmol sodium ascorbate, 5 μmol copper sulfate. The enzyme reaction tank 7 used a coil having an inner diameter of 0.5 mm and a length of 10 m, and the enzyme reaction was caused at 37°C. Dopamine was converted to noradrenaline by an enzymatic reaction. The noradrenaline, adrenaline, and noradrenaline converted from dopamine leaving the reaction tank 7 were brought into contact with a potassium ferricyanide aqueous solution, an ascorbic acid aqueous solution, and a sodium hydroxide aqueous solution, respectively, to produce respective tetrahydroxyindole derivatives. The potassium ferricyanide aqueous solution tank 9 has a composition of 0.05% by weight of potassium ferricyanide, 0.2 mol of monopotassium phosphate, and 0.4 mol of dipotassium phosphate, and the alcoholic acid aqueous solution tank 10 has a composition of 0.2% by weight.
The caustic soda aqueous solution was adjusted to a concentration of 6 normal, and a constant flow rate of 3 was applied at a flow rate of 0.2 ml/min.
It was supplied by a continuous pump 8. The temperature of the reaction tank 12 is
It was maintained at 60°C. Reactions with these aqueous solutions produced respective hydroxyindole derivatives of noradrenaline, adrenaline, and noradrenaline converted from dopamine. Each derivative was detected by a fluorescence detector 13 and recorded by a recorder 14. P1 .
P 2 .P 3 are noradrenaline, adrenaline, and
This is the peak of the derivative of noradrenaline converted from dopamine. The chromatograms of each derivative recorded by the recorder 14 in this manner are shown in FIG.
Based on the results obtained in this way, the concentration of catecholamines in the blood of each of the five samples was calculated from the calibration curve using standard catecholamines and the recovery rate obtained by adding standard catecholamines to pooled serum and performing pretreatment operations. were obtained as shown in Table 1. 【table】
第１図は本発明方法における実施態様の一例を
示す説明図、第２図は実施例において得られたク
ロマトグラムを示している。
符号の説明、１……溶離液槽、２，６……定流
量ポンプ、３……試料注入器、４……分離用カラ
ム、５……酵素溶液槽、７，１２……反応槽、８
……定流量３連ポンプ、９……フエリシアン化カ
リウム水溶液槽、１０……アスコルビン酸水溶液
槽、１１……カセイソーダ水溶液槽、１３……蛍
光検出器、１４……記録計。
FIG. 1 is an explanatory diagram showing an example of an embodiment of the method of the present invention, and FIG. 2 shows a chromatogram obtained in an example. Explanation of symbols, 1... Eluent tank, 2, 6... Constant flow pump, 3... Sample injector, 4... Separation column, 5... Enzyme solution tank, 7, 12... Reaction tank, 8
... Constant flow triple pump, 9 ... Potassium ferricyanide aqueous solution tank, 10 ... Ascorbic acid aqueous solution tank, 11 ... Caustic soda aqueous solution tank, 13 ... Fluorescence detector, 14 ... Recorder.
Claims (1)
ロマトグラフイーにおける分離カラムに導入し
て、ノルアドレナリン、アドレナリン、ドーパミ
ンの３成分を分離し、これにドーパミン−β−水
酸化酵素を作用させてドーパミンを選択的にノル
アドレナリンに変換し、次いでノルアドレナリ
ン、アドレナリン、ドーパミンから変換されたノ
ルアドレナリンにテトラヒドロキシインドールを
接触させ、これにより得られた夫々の誘導体の蛍
光を検出し、これに基づいてノルアドレナリン、
アドレナリン及びドーパミンの３成分を定量する
ことを特徴とする、カテコールアミンの定量法。1 A sample solution containing catecholamines is introduced into a separation column in liquid chromatography to separate three components, noradrenaline, adrenaline, and dopamine, and dopamine-β-hydroxylase is applied to this to selectively release dopamine. Noradrenaline is converted into noradrenaline, and then tetrahydroxyindole is contacted with noradrenaline converted from noradrenaline, adrenaline, and dopamine, and the fluorescence of each derivative obtained is detected. Based on this, noradrenaline,
A method for quantifying catecholamines, which is characterized by quantifying three components: adrenaline and dopamine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14955282A JPS5938651A (en) | 1982-08-27 | 1982-08-27 | Quantitative analysis of catecholamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14955282A JPS5938651A (en) | 1982-08-27 | 1982-08-27 | Quantitative analysis of catecholamine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5938651A JPS5938651A (en) | 1984-03-02 |
| JPH022600B2 true JPH022600B2 (en) | 1990-01-18 |
Family
ID=15477652
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14955282A Granted JPS5938651A (en) | 1982-08-27 | 1982-08-27 | Quantitative analysis of catecholamine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5938651A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60205262A (en) * | 1984-03-29 | 1985-10-16 | Doujin Kagaku Kenkyusho:Kk | Assay of catecholamine using 1,2-diphenylethylenediamine |
| JPH0820426B2 (en) * | 1989-10-20 | 1996-03-04 | 株式会社日立製作所 | Liquid chromatograph analyzer and pre-label reaction treatment method |
| JP2768079B2 (en) * | 1991-08-29 | 1998-06-25 | 和光純薬工業株式会社 | High precision analysis method |
| JP4517422B2 (en) | 1999-11-24 | 2010-08-04 | 株式会社ニコン | Afocal zoom lens and microscope equipped with the lens |
| JP4488283B2 (en) | 2003-09-17 | 2010-06-23 | オリンパス株式会社 | Afocal zoom lens for microscope |
-
1982
- 1982-08-27 JP JP14955282A patent/JPS5938651A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5938651A (en) | 1984-03-02 |
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