JPH0233360B2 - TANJUSANNOTEIRYOHO - Google Patents
TANJUSANNOTEIRYOHOInfo
- Publication number
- JPH0233360B2 JPH0233360B2 JP16280082A JP16280082A JPH0233360B2 JP H0233360 B2 JPH0233360 B2 JP H0233360B2 JP 16280082 A JP16280082 A JP 16280082A JP 16280082 A JP16280082 A JP 16280082A JP H0233360 B2 JPH0233360 B2 JP H0233360B2
- Authority
- JP
- Japan
- Prior art keywords
- acid
- bile acids
- internal standard
- bile
- sample
- 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
- 239000003613 bile acid Substances 0.000 claims description 46
- 239000000126 substance Substances 0.000 claims description 22
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 17
- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 16
- 239000004380 Cholic acid Substances 0.000 claims description 16
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 claims description 16
- 229960002471 cholic acid Drugs 0.000 claims description 16
- 235000019416 cholic acid Nutrition 0.000 claims description 16
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 claims description 15
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 claims description 14
- 229960001661 ursodiol Drugs 0.000 claims description 14
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- 230000002378 acidificating effect Effects 0.000 claims description 10
- 235000001014 amino acid Nutrition 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 9
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 8
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- 235000013922 glutamic acid Nutrition 0.000 claims description 6
- 239000004220 glutamic acid Substances 0.000 claims description 6
- 239000012472 biological sample Substances 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 2
- 235000003704 aspartic acid Nutrition 0.000 claims description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- 238000004581 coalescence Methods 0.000 claims 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 239000000523 sample Substances 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 238000000926 separation method Methods 0.000 description 12
- 210000002966 serum Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 9
- 239000003480 eluent Substances 0.000 description 9
- 239000012488 sample solution Substances 0.000 description 8
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD 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](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229960002989 glutamic acid Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229950006238 nadide Drugs 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000000356 contaminant Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102100036504 Dehydrogenase/reductase SDR family member 9 Human genes 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 101000928746 Homo sapiens Dehydrogenase/reductase SDR family member 9 Proteins 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- SCPADBBISMMJAW-UHFFFAOYSA-N (10S)-3c.17t-Dihydroxy-10r.13c-dimethyl-17c-((R)-1-hydroxy-aethyl)-(5tH.8cH.9tH.14tH)-hexadecahydro-1H-cyclopenta[a]phenanthren Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(O)C)(O)C1(C)CC2 SCPADBBISMMJAW-UHFFFAOYSA-N 0.000 description 2
- SCPADBBISMMJAW-UHHUKTEYSA-N 5beta-Pregnane-3alpha,17alpha,20alpha-triol Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@@]([C@@H](O)C)(O)[C@@]2(C)CC1 SCPADBBISMMJAW-UHHUKTEYSA-N 0.000 description 2
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000012295 chemical reaction liquid Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 125000003010 ionic group Chemical group 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 229920002126 Acrylic acid copolymer Polymers 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 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 1
- 239000004471 Glycine Substances 0.000 description 1
- SMEROWZSTRWXGI-UHFFFAOYSA-N Lithocholsaeure Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 SMEROWZSTRWXGI-UHFFFAOYSA-N 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical class [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 231100000354 acute hepatitis Toxicity 0.000 description 1
- -1 amino acid ester Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 235000012501 ammonium carbonate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 1
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- QYWRBLSFXQKWQA-UHFFFAOYSA-N diazanium;acetonitrile;carbonate Chemical compound [NH4+].[NH4+].CC#N.[O-]C([O-])=O QYWRBLSFXQKWQA-UHFFFAOYSA-N 0.000 description 1
- WSEQLMQNPBNMSL-FJXQXJEOSA-N diethyl (2s)-2-aminopentanedioate;hydron;chloride Chemical compound Cl.CCOC(=O)CC[C@H](N)C(=O)OCC WSEQLMQNPBNMSL-FJXQXJEOSA-N 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229940049906 glutamate Drugs 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- SMEROWZSTRWXGI-HVATVPOCSA-N lithocholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 SMEROWZSTRWXGI-HVATVPOCSA-N 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 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 1
- 125000002347 octyl 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])[H] 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- YLLIGHVCTUPGEH-UHFFFAOYSA-M potassium;ethanol;hydroxide Chemical compound [OH-].[K+].CCO YLLIGHVCTUPGEH-UHFFFAOYSA-M 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000377 silicon dioxide 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
- 238000003756 stirring Methods 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 本発明は胆汁酸の定量法に関する。[Detailed description of the invention] The present invention relates to a method for quantifying bile acids.
従来、肝機能の検査の一つとして血液中に微量
に含まれる胆汁酸の定量が行なわれる。 BACKGROUND ART Conventionally, one of the tests for liver function is the determination of trace amounts of bile acids contained in blood.
血液中には、胆汁酸としてコール酸、デオキシ
コール酸、ケノデオキシコール酸、ウルソデオキ
シコール酸、リトコール酸が夫々遊離体、グリシ
ン抱合体、タウリン抱合体の形で含まれており、
これらの15種類の各胆汁酸を分離定量すること
が、肝胆道系疾患の病態解析並びに鑑別診断に有
用である。 Blood contains bile acids such as cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid in the form of free form, glycine conjugate, and taurine conjugate, respectively.
Separating and quantifying each of these 15 types of bile acids is useful for pathological analysis and differential diagnosis of hepatobiliary diseases.
このため従来、高速液体クロマトグラフイーを
用いて分離カラム内に溶離液と混合された血清等
の生体試料を注入して胆汁酸を各胆汁酸成分に分
離し、次いでニコチン酸アミドアデニンジヌクレ
オチド（以下NAD+と略す）を加えた反応液を供
給し、酵素3α―ヒドロキシステロイドデヒドロ
ゲナーゼ（以下3α―HSDと略す）が固定された
担体が充填された固定化酵素カラムに通して、分
離された胆汁酸とNAD+とを反応させ、螢光物質
を生成させて螢光検出器により検出し、得られた
結果に基づいて生体試料中の15種類の各胆汁酸の
定量を行つている。 To this end, conventionally, high performance liquid chromatography was used to separate bile acids into each bile acid component by injecting a biological sample such as serum mixed with an eluent into a separation column, and then nicotinamide adenine dinucleotide ( A reaction solution containing NAD The acid and NAD + are reacted to produce a fluorescent substance, which is detected by a fluorescence detector.Based on the obtained results, each of the 15 types of bile acids in biological samples is quantified.
しかしながら生体試料は液体クロマトグラフイ
ーにかける前に分離阻害物質を除去する目的で前
処理操作にかけられるので、この際に試料中の胆
汁酸が損失を受けるおそれがあり、検出結果に影
響を及ぼすので、既知の第３成分を内部標準物質
として加えておくことが行なわれている。 However, since biological samples are subjected to pretreatment to remove separation-inhibiting substances before being subjected to liquid chromatography, bile acids in the sample may be lost during this process, which may affect the detection results. , a known third component is added as an internal standard substance.
かゝる内部標準物質として従来はプレグナント
リオールが使用されてきた。しかしながらプレグ
ナントリオールは分離用カラムでの保持時間が全
ての胆汁酸より長時間で分析に時間を要し、又検
出時のピークも幅が広くなり、これを基準ピーク
とした場合に誤差が大きくなる欠点があつた。 Conventionally, pregnanetriol has been used as such an internal standard substance. However, the retention time of pregnanetriol in a separation column is longer than that of all bile acids, so it takes time to analyze, and the detection peak also has a broad width, which has the disadvantage of increasing errors when used as a reference peak. It was hot.
本発明はかゝる欠点を解消することを目的とし
てなされたものであり、内部標準物質としてすべ
ての胆汁酸よりも短かい保持時間を有するが、溶
媒その他の混在物よりは遅く溶出される性質を有
し、又検出に当つて鋭いピークを現出しうるもの
を見出すことにより完成した発明であり、短時間
での胆汁酸の定量が可能でしかも誤差の少ない、
胆汁酸の定量法を提供するものである。本発明の
要旨は、液体クロマトグラフイーによつて生体試
料中の胆汁酸を各胆汁酸成分に分離し、固定化酵
素を入れたカラムに導入し、カラム内で固定化酵
素と接触させて胆汁酸とこれと反応しうる物質と
の反応を生じさせ、反応生成物を検出器で測定す
ることにより胆汁酸を定量する方法において、内
部標準物質としてコール酸又はウルソデオキシコ
ール酸の酸性アミノ酸抱合体を用いることを特徴
とする、胆汁酸の定量法に存する。本発明におい
て内部標準物質を設計するに当り、逆相系分離用
カラム内での保持時間を短かくするためにイオン
性基を導入し親水性を高めることとした。また酵
素に対する反応性を胆汁酸に近いものとするため
に、胆汁酸の誘導体を用いることとした。そして
これに適合する内部標準物質としてコール酸又は
ウルソデオキシコール酸の酸性アミノ酸抱合体を
見出した。 The present invention was made with the aim of eliminating such drawbacks, and uses bile acids as an internal standard substance that has a retention time shorter than that of all bile acids, but has the property of eluting more slowly than solvents and other contaminants. This invention was completed by finding a method that can produce sharp peaks during detection, which enables bile acids to be quantified in a short time and with little error.
Provides a method for quantifying bile acids. The gist of the present invention is to separate bile acids in a biological sample into each bile acid component by liquid chromatography, introduce the bile acids into a column containing an immobilized enzyme, and bring the bile acids into contact with the immobilized enzyme within the column. In a method for quantifying bile acids by causing a reaction between an acid and a substance capable of reacting with the acid and measuring the reaction product with a detector, an acidic amino acid conjugate of cholic acid or ursodeoxycholic acid is used as an internal standard substance. A method for quantifying bile acids, characterized by using. In designing an internal standard substance in the present invention, an ionic group was introduced to increase hydrophilicity in order to shorten the retention time in a column for reversed-phase separation. In addition, we decided to use a bile acid derivative in order to make the reactivity toward enzymes similar to that of bile acids. We have also discovered an acidic amino acid conjugate of cholic acid or ursodeoxycholic acid as an internal standard substance that meets this requirement.
コール酸又はウルソデオキシコール酸の酸性ア
ミノ酸抱合体は希アルカリ性での加水分解に抵抗
性のあるアミド結合により化学結合されているも
のであり、イオン性基としてはカルボキシル基が
用いられる。又、酸性アミノ酸としては、グルタ
ミン酸、アスパラギン酸のようなカルボキシル基
がアミノ基よりも多いものが使用される。 The acidic amino acid conjugate of cholic acid or ursodeoxycholic acid is chemically bonded through an amide bond that is resistant to hydrolysis in dilute alkaline conditions, and a carboxyl group is used as the ionic group. Further, as the acidic amino acid, those having more carboxyl groups than amino groups, such as glutamic acid and aspartic acid, are used.
コール酸又はウルソデオキシコール酸の酸性ア
ミノ酸抱合体を合成するには、例えばコール酸又
はウルソデオキシコール酸と酸性アミノ酸エステ
ルの塩酸塩をＮ，N′―ジクロヘキシルカルボジ
イミドの存在下に撹拌して反応させ、更に水酸化
カリウム溶液により加水分解を行えばよい。 To synthesize an acidic amino acid conjugate of cholic acid or ursodeoxycholic acid, for example, the hydrochloride of cholic acid or ursodeoxycholic acid and an acidic amino acid ester are stirred and reacted in the presence of N,N'-dichlorohexylcarbodiimide. and further hydrolysis using a potassium hydroxide solution.
上記の内部標準物質を用いて胆汁酸の定量を行
う場合は、例えば第１図に示すような装置を用い
る。 When quantifying bile acids using the above-mentioned internal standard substance, for example, an apparatus as shown in FIG. 1 is used.
１，２は溶離液槽であり、溶離液槽１における
溶離液としては例えば炭酸アンモニウム―アセト
ニトリル混合液が使用され、溶離液２としてはア
セトニトリルが使用され、アセトニトリルの添加
にはグラジエントがかけられ、徐々にアセトニト
リルの含有量が増加するものとされる。４はこの
ためのグラジエントプログラマーである。 Reference numerals 1 and 2 are eluent tanks, and the eluent in eluent tank 1 is, for example, an ammonium carbonate-acetonitrile mixture, and the eluent 2 is acetonitrile, and a gradient is applied to the addition of acetonitrile. It is assumed that the content of acetonitrile gradually increases. 4 is a gradient programmer for this purpose.
５は試料注入器である。試料液としては、前処
理における血中胆汁酸の損失を補正する方法とし
て、血清試料に一定量のコール酸あるいはウルソ
デオキシコール酸の酸性アミノ酸抱合体を内部標
準物質として加えた後、前処理を行い、抽出した
ものが、使用される。該試料液は試料注入器５よ
り注入されて溶離液と混合される。又、前処理に
おける血中胆汁酸の損失を補正する必要のない場
合は、内部標準物質の一定量を前処理した血清試
料に添加した試料液が使用されてもよい。試料液
は流路３から分離用カラム６に導かれ、胆汁酸の
分離がなされる。分離用カラム６には、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。 5 is a sample injector. As a sample solution, as a method to correct the loss of blood bile acids during pretreatment, a certain amount of cholic acid or an acidic amino acid conjugate of ursodeoxycholic acid was added to the serum sample as an internal standard substance, and then pretreatment was performed. The extracted material will be used. The sample liquid is injected from the sample injector 5 and mixed with the eluent. Alternatively, if there is no need to correct the loss of blood bile acids during pretreatment, a sample solution in which a certain amount of an internal standard substance is added to the pretreated serum sample may be used. The sample liquid is led from the flow path 3 to the separation column 6, where bile acids are separated. The separation column 6 is filled with particles of, for example, porous silica into which octadecyl groups have been introduced, divinylbenzene-(meth)acrylic acid copolymer, or the like.
分離用カラム６において高速液体クロマトグラ
フイーにより胆汁酸が各胆汁酸成分に分離され
る。上記分離用カラム６から流出した分離液に
NAD+を含む反応液を反応液槽１０から供給し、
これを3α―HSDが充填されている固定化酵素カ
ラム７に導き、上記3α―HSDの作用により各胆
汁酸成分とNAD+とを順次反応させ、該反応によ
り螢光物質を生成させる。 In the separation column 6, the bile acids are separated into each bile acid component by high performance liquid chromatography. The separated liquid flowing out from the separation column 6
Supplying a reaction solution containing NAD + from the reaction solution tank 10,
This is introduced into the immobilized enzyme column 7 packed with 3α-HSD, and each bile acid component is reacted with NAD + in sequence by the action of the 3α-HSD, and a fluorescent substance is produced by this reaction.
この螢光物質の生成量は試料液中に含まれる胆
汁酸の量に依存するので螢光光度計８においてこ
れを検出しその結果を記録計９により記録する。 Since the amount of this fluorescent substance produced depends on the amount of bile acid contained in the sample solution, it is detected by a fluorophotometer 8 and the result is recorded by a recorder 9.
このようにしてまず、試料液中の蛋白質等の混
在物のピークが、次いで内部標準物質であるコー
ル酸またはウルソデオキシコール酸の酸性アミノ
酸抱合体によるピーク、各胆汁酸によるピークが
順次記録される。 In this way, first, the peaks of contaminants such as proteins in the sample solution are recorded, then the peaks due to the acidic amino acid conjugates of cholic acid or ursodeoxycholic acid, which are internal standard substances, and the peaks due to each bile acid are sequentially recorded. .
そしてこのようにして得られた結果に基づいて
各胆汁酸の量を定量することができる。 Based on the results thus obtained, the amount of each bile acid can be determined.
本発明によれば、内部標準物質としてコール酸
またはウルソデオキシコール酸の酸性アミノ酸抱
合体を用いることにより、分離用カラムでの保持
時間が各胆汁酸より短かいものとなり、各胆汁酸
の分析時間を短縮することができる。又検出時の
ピークが鋭いものとなり、定量誤差を少なくする
ことができる。 According to the present invention, by using an acidic amino acid conjugate of cholic acid or ursodeoxycholic acid as an internal standard substance, the retention time in the separation column is shorter than that of each bile acid, and the analysis time of each bile acid is can be shortened. Moreover, the peak at the time of detection becomes sharp, and quantitative errors can be reduced.
実施例 １
コール酸のグルタミン酸抱合体を次のようにし
て合成した。Example 1 A glutamate conjugate of cholic acid was synthesized as follows.
Ｌ―グルタミン酸ジエチルエステル塩酸塩
（2.4ｇ）、コール酸（4.1ｇ）のテトラヒドロフラ
ン溶液（100ml）にトリエチルアミン（1.4ml）を
加え、続いてＮ，N′―ジシクロヘキシルカルボ
ジイミド（2.1ｇ）を加え、室温で撹拌し、反応
させた。生成した不溶物を別し、液を濃縮
し、塩化メチレン（100ml）に溶解した。飽和炭
酸水素ナトリウム溶液で洗浄した後、溶媒を留去
し、続いて、３％水酸化カリウム―エタノール溶
液（100ml）にとかして加水分解した。室温にて
撹拌後、溶媒を留去し、残渣を少量の水に溶解
し、氷冷しながら塩酸を滴下しPH４に調整した。
冷蔵庫に静置し、析出する結晶を取した。この
際の収量は4.3ｇ（収率80％）であつた。この結
晶を減圧下に乾燥したものを内部標準として用い
た。 Triethylamine (1.4 ml) was added to a tetrahydrofuran solution (100 ml) of L-glutamic acid diethyl ester hydrochloride (2.4 g) and cholic acid (4.1 g), followed by N,N'-dicyclohexylcarbodiimide (2.1 g), and the mixture was heated to room temperature. The mixture was stirred and reacted. The resulting insoluble matter was separated, the liquid was concentrated, and dissolved in methylene chloride (100 ml). After washing with saturated sodium bicarbonate solution, the solvent was distilled off, followed by hydrolysis by dissolving in 3% potassium hydroxide-ethanol solution (100 ml). After stirring at room temperature, the solvent was distilled off, the residue was dissolved in a small amount of water, and the pH was adjusted to 4 by adding hydrochloric acid dropwise while cooling with ice.
The mixture was left to stand in the refrigerator, and precipitated crystals were collected. The yield at this time was 4.3 g (yield 80%). This crystal was dried under reduced pressure and used as an internal standard.
上述の内部標準物質（例えばコール酸のグルタ
ミン酸抱合体）20mgをエタノール（100ml）に溶
解し、この溶液を試料血清１mlに10μ添加し、
この添加血清0.5mlにエタノール（2.5ml）を加
え、85℃の水浴中で１分間撹拌した後、これを遠
心分離し、上澄を採取した。 Dissolve 20 mg of the above-mentioned internal standard substance (e.g. glutamic acid conjugate of cholic acid) in ethanol (100 ml), add 10μ of this solution to 1 ml of sample serum,
Ethanol (2.5 ml) was added to 0.5 ml of this added serum, stirred for 1 minute in a water bath at 85°C, and then centrifuged to collect the supernatant.
残渣にエタノール（2.5ml）を加え上記操作を
くり返し、得られた上澄を蒸発乾固し、これにメ
タノール100μを加え溶解し、この10μを試料
注入口５より注入した。 Ethanol (2.5 ml) was added to the residue and the above operation was repeated, the resulting supernatant was evaporated to dryness, 100 μm of methanol was added and dissolved, and this 10 μm was injected through the sample injection port 5.
溶離液槽１から0.3重量％の炭酸アンモニウム
20容量％とアセトニトリル80容量％の混合液をポ
ンプ１１により供給し、又溶離液槽２からアセト
ニトリルを初期値０容量％から１容量％／分のグ
ラジエントを有するようにポンプ１２により供給
し流量を0.5ml／分として試料と混合し、得られ
た試料液を分離用カラム６に導いた。分離用カラ
ム６としては、オクチル基をその表面に化学的に
結合させた粒径10μｍ前後の球状シリカゲルが充
填された内径3.9mm、長さ30cmのものを使用し、
試料液中の各胆汁酸を分離した。分離用カラム６
から出てきた各成分は反応液槽１０からポンプ１
３によつて供給される反応液と混合され固定化酵
素カラム７に導入された。 0.3% by weight ammonium carbonate from eluent tank 1
A mixed solution of 20% by volume and 80% by volume of acetonitrile was supplied by the pump 11, and acetonitrile was supplied from the eluent tank 2 by the pump 12 so as to have a gradient from the initial value of 0% by volume to 1% by volume/min, and the flow rate was adjusted. The mixture was mixed with the sample at a rate of 0.5 ml/min, and the resulting sample solution was introduced into the separation column 6. As the separation column 6, a column with an inner diameter of 3.9 mm and a length of 30 cm is used, which is filled with spherical silica gel having a particle size of approximately 10 μm and has octyl groups chemically bonded to its surface.
Each bile acid in the sample solution was separated. Separation column 6
Each component coming out from the reaction liquid tank 10 is pumped into the pump 1.
3 and introduced into the immobilized enzyme column 7.
反応液は0.3ｍＭのβ―NAD+、10ｍＭのリン
酸緩衝液（PH8.0）、１ｍＭのEDTA、0.05重量％
の２―メルカプトエタノールからなり、供給量は
0.5ml／分とした。 The reaction solution was 0.3mM β-NAD + , 10mM phosphate buffer (PH8.0), 1mM EDTA, 0.05% by weight.
It consists of 2-mercaptoethanol, and the supply amount is
The rate was 0.5ml/min.
固定化酵素カラム７としては、3α―HSD100ユ
ニツトを２c.c.の粒状セルロースに臭化シアン法で
固定化し、内径4.6mm、長さ50cmのカラムに充填
したものを用いた。 As the immobilized enzyme column 7, 100 units of 3α-HSD were immobilized on 2 c.c. of granular cellulose by the cyanogen bromide method, and the resulting column was packed into a column having an inner diameter of 4.6 mm and a length of 50 cm.
試料液中の胆汁酸成分は、酵素により３―ケト
体に酸化され、同時に反応液中のβ―NAD+は還
元されて螢光を有するNADHになり、螢光光度
計８によつてその濃度が測定された。螢光光度計
８は励起波長350mm、螢光波長450mmの条件で測定
された。螢光光度計８による測定結果を記録計９
により記録した。 The bile acid component in the sample solution is oxidized to the 3-keto form by the enzyme, and at the same time, β-NAD + in the reaction solution is reduced to fluorescent NADH, and its concentration is measured by the fluorophotometer 8. was measured. The fluorescence photometer 8 was used for measurement under the conditions of an excitation wavelength of 350 mm and a fluorescence wavelength of 450 mm. The measurement result by the fluorescence photometer 8 is recorded by the recorder 9.
Recorded by.
第２図は急性肝炎患者の血清試料を用いて測定
した例を示すものであり、内部標準であるコール
酸のグルタミン酸抱合体によるピークPBは、血
清試料中の蛋白質等の混在物によるピークPA及
び胆汁酸によるピークPCとよく分離された。 Figure 2 shows an example of measurement using a serum sample from an acute hepatitis patient. The peak P B due to the glutamic acid conjugate of cholic acid, which is an internal standard, is different from the peak P B due to contaminants such as proteins in the serum sample. It was well separated from the peak P C caused by A and bile acids.
実施例 ２
ウルソデオキシコール酸のグルタミン酸抱合体
20mgを内部標準物質としてエタノール100mlに溶
解した。この溶液を慢性肝炎患者の試料血清１ml
当り5μ量添加した。このように内部標準物質
の添加された試料血清を、疎水性ゲルを充填した
前処理カラムに添加し、続いて蒸溜水５mlで洗浄
した後、無水エタノール10mlで溶離した。更にエ
タノールを蒸発乾固した後、残渣をメタノール
100μに溶解した。Example 2 Glutamic acid conjugate of ursodeoxycholic acid
20 mg was dissolved in 100 ml of ethanol as an internal standard substance. Add 1ml of sample serum from a chronic hepatitis patient to this solution.
5μ amount was added per portion. The sample serum to which the internal standard had been added in this manner was added to a pretreatment column packed with hydrophobic gel, washed with 5 ml of distilled water, and then eluted with 10 ml of absolute ethanol. After further evaporating the ethanol to dryness, the residue was dissolved in methanol.
Dissolved in 100μ.
このようにして処理された試料液を用いて実施
例１と同様にして胆汁酸の定量を行つた。 Bile acids were quantified in the same manner as in Example 1 using the sample solution treated in this manner.
第３図にその結果を示す。内部標準であるウル
ソデオキシコール酸のグルタミン酸抱合体による
ピークPBは、血清試料中の蛋白質等の混在物に
よるピークPA及び胆汁酸によるピークPCとよく
分離された。 Figure 3 shows the results. The peak P B due to the glutamic acid conjugate of ursodeoxycholic acid, which is an internal standard, was well separated from the peak P A due to contaminants such as proteins in the serum sample and the peak P C due to bile acids.
第１図は本発明胆汁酸の定量法に使用する装置
の例を示す説明図、第２図は実施例１におけるク
ロマトグラム、第３図は実施例２におけるクロマ
トグラムである。
符号の説明、１，２…溶離液槽、４…グラジエ
ントプログラマー、５…試料注入口、６…分離用
カラム、７…固定化酵素カラム、８…螢光光度
計、９…記録計、１０…反応液槽、１１，１２，
１３…ポンプ。
FIG. 1 is an explanatory diagram showing an example of an apparatus used in the method for quantifying bile acids of the present invention, FIG. 2 is a chromatogram in Example 1, and FIG. 3 is a chromatogram in Example 2. Explanation of symbols, 1, 2... Eluent tank, 4... Gradient programmer, 5... Sample injection port, 6... Separation column, 7... Immobilized enzyme column, 8... Fluorescence photometer, 9... Recorder, 10... Reaction liquid tank, 11, 12,
13...Pump.
Claims (1)
の胆汁酸を各胆汁酸成分に分離し、固定化酵素を
入れたカラムに導入し、カラム内で固定化酵素と
接触させて胆汁酸とこれと反応しうる物質との反
応を生じさせ、反応生成物を検出器で測定するこ
とにより胆汁酸を定量する方法において、内部標
準物質としてコール酸又はウルソデオキシコール
酸の酸性アミノ酸抱合体を用いることを特徴とす
る、胆汁酸の定量法。 ２ 内部標準物質がコール酸又はウルソデオキシ
コール酸のグルタミン酸抱合体である、特許請求
の範囲第１項記載の胆汁酸の定量法。 ３ 内部標準物質がコール酸又はウルソデオキシ
コール酸のアスパラギン酸抱合体である、特許請
求の範囲第１項記載の胆汁酸の定量法。[Claims] 1. Bile acids in a biological sample are separated into each bile acid component by liquid chromatography, introduced into a column containing an immobilized enzyme, and brought into contact with the immobilized enzyme within the column. In a method for quantifying bile acids by causing a reaction between bile acids and a substance that can react with the bile acids and measuring the reaction product with a detector, an acidic amino acid conjugate of cholic acid or ursodeoxycholic acid is used as an internal standard substance. A method for quantifying bile acids, characterized by using coalescence. 2. The method for quantifying bile acids according to claim 1, wherein the internal standard substance is a glutamic acid conjugate of cholic acid or ursodeoxycholic acid. 3. The method for quantifying bile acids according to claim 1, wherein the internal standard substance is an aspartic acid conjugate of cholic acid or ursodeoxycholic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16280082A JPH0233360B2 (en) | 1982-09-17 | 1982-09-17 | TANJUSANNOTEIRYOHO |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16280082A JPH0233360B2 (en) | 1982-09-17 | 1982-09-17 | TANJUSANNOTEIRYOHO |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5951349A JPS5951349A (en) | 1984-03-24 |
| JPH0233360B2 true JPH0233360B2 (en) | 1990-07-26 |
Family
ID=15761449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16280082A Expired - Lifetime JPH0233360B2 (en) | 1982-09-17 | 1982-09-17 | TANJUSANNOTEIRYOHO |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0233360B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799126A (en) * | 2014-07-25 | 2019-05-24 | 立佳有限公司 | Dilute the analysis method of biological specimen ingredient |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0716439B2 (en) * | 1986-12-16 | 1995-03-01 | 積水化学工業株式会社 | Bile acid fractionation quantification method |
| JPH0339096A (en) * | 1989-07-04 | 1991-02-20 | Sekisui Chem Co Ltd | High-performance liquid chromatography utilizing immobilized enzyme |
| JP2771915B2 (en) * | 1991-09-11 | 1998-07-02 | 花王株式会社 | Simultaneous automatic analysis system for various compounds with absorption in the ultraviolet and visible regions |
| CN103852554B (en) * | 2012-11-30 | 2015-11-18 | 山东金诃药物研究开发有限公司 | A kind of Tibetan medicinal composition whin disappears the quality determining method of preparation of itching |
| CN106841492A (en) * | 2017-03-30 | 2017-06-13 | 杭州佰辰医学检验所有限公司 | Five kinds of methods of sequestered bile acid in high performance liquid chromatography tandem mass spectrum detection serum |
-
1982
- 1982-09-17 JP JP16280082A patent/JPH0233360B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109799126A (en) * | 2014-07-25 | 2019-05-24 | 立佳有限公司 | Dilute the analysis method of biological specimen ingredient |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5951349A (en) | 1984-03-24 |
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