JP2000352564A - Method for determining 5-hydroxycreatinine - Google Patents
Method for determining 5-hydroxycreatinineInfo
- Publication number
- JP2000352564A JP2000352564A JP11164727A JP16472799A JP2000352564A JP 2000352564 A JP2000352564 A JP 2000352564A JP 11164727 A JP11164727 A JP 11164727A JP 16472799 A JP16472799 A JP 16472799A JP 2000352564 A JP2000352564 A JP 2000352564A
- Authority
- JP
- Japan
- Prior art keywords
- hydroxycreatinine
- methylguanidine
- methylguadinine
- quantifying
- liquid chromatography
- 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.)
- Pending
Links
- SMQPRZPBBUJEGU-UHFFFAOYSA-N 2-amino-4-hydroxy-3-methyl-4h-imidazol-5-one Chemical compound CN1C(O)C(=O)N=C1N SMQPRZPBBUJEGU-UHFFFAOYSA-N 0.000 title claims abstract description 120
- 238000000034 method Methods 0.000 title claims abstract description 55
- CHJJGSNFBQVOTG-UHFFFAOYSA-N methylguanidine Chemical compound CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 claims abstract description 63
- 238000002372 labelling Methods 0.000 claims abstract description 12
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000004327 boric acid Substances 0.000 claims abstract description 11
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 17
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000007850 fluorescent dye Substances 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- ISAOCJYIOMOJEB-UHFFFAOYSA-N benzoin Chemical compound C=1C=CC=CC=1C(O)C(=O)C1=CC=CC=C1 ISAOCJYIOMOJEB-UHFFFAOYSA-N 0.000 abstract description 4
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 abstract description 4
- 244000028419 Styrax benzoin Species 0.000 abstract description 2
- 235000000126 Styrax benzoin Nutrition 0.000 abstract description 2
- 235000008411 Sumatra benzointree Nutrition 0.000 abstract description 2
- 229960002130 benzoin Drugs 0.000 abstract description 2
- 235000019382 gum benzoic Nutrition 0.000 abstract description 2
- 238000004811 liquid chromatography Methods 0.000 abstract 3
- 230000001131 transforming effect Effects 0.000 abstract 2
- 230000003907 kidney function Effects 0.000 abstract 1
- 238000005259 measurement Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 12
- 238000011002 quantification Methods 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 10
- 210000002700 urine Anatomy 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 6
- 238000004445 quantitative analysis Methods 0.000 description 6
- 239000012086 standard solution Substances 0.000 description 6
- 210000001124 body fluid Anatomy 0.000 description 5
- 239000010839 body fluid Substances 0.000 description 5
- 238000012423 maintenance Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 208000001647 Renal Insufficiency Diseases 0.000 description 3
- 238000001215 fluorescent labelling Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 201000006370 kidney failure Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- YYVYAPXYZVYDHN-UHFFFAOYSA-N 9,10-phenanthroquinone Chemical compound C1=CC=C2C(=O)C(=O)C3=CC=CC=C3C2=C1 YYVYAPXYZVYDHN-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 208000020832 chronic kidney disease Diseases 0.000 description 2
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008085 renal dysfunction Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000011533 pre-incubation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、腎機能障害の検査法と
して有用な、高速液体クロマトグラフィーを用いた５-
ヒドロキシクレアチニンの定量法に関する。FIELD OF THE INVENTION The present invention relates to a method for detecting renal dysfunction, which is useful for high-performance liquid chromatography.
The present invention relates to a method for determining hydroxycreatinine.
【０００２】[0002]
【従来の技術】５-ヒドロキシクレアチニンは、クレア
チニンの非酵素的酸化により産成され、腎不全患者の血
中に蓄積する主要な尿毒素の一つとして知られているメ
チルグアニジンが産生される際の前駆体である。５-ヒ
ドロキシクレアチニンは健常人血清中からは検出されな
いが、腎不全患者の血清中に早期から検出され、症状の
悪化に伴い増加していることより、５-ヒドロキシクレ
アチニンの定量的な測定は患者の病態把握や腎不全の早
期発見マーカーとして重要視されている。また、クレア
チニンの非酵素的酸化には、反応性が極めて高いことで
知られるヒドロキシルラジカルが関与しており、クレア
チニンからメチルグアニジンへの変換反応の中間体であ
る５-ヒドロキシクレアチニンは、尿毒素メチルグアニ
ジンの前駆体としてだけではなく、生体内におけるヒド
ロキシルラジカル産生の指標（酸素ストレス状態の指
標）としても注目を集めている。2. Description of the Related Art 5-Hydroxycreatinine is produced by the non-enzymatic oxidation of creatinine and produces methylguanidine, which is known as one of the major uretoxins that accumulates in the blood of patients with renal failure. Is a precursor of 5-Hydroxycreatinine is not detected in the serum of healthy subjects, but is detected in the serum of patients with renal failure from an early stage and increases with worsening of the symptoms. It is regarded as an important marker for understanding the disease state of the disease and for early detection of renal failure. In addition, the non-enzymatic oxidation of creatinine involves the hydroxyl radical, which is known to have extremely high reactivity, and 5-hydroxycreatinine, which is an intermediate in the conversion reaction from creatinine to methylguanidine, is converted to urexin methyl Attention has been drawn not only as a precursor of guanidine, but also as an index of hydroxyl radical production in vivo (index of oxygen stress state).
【０００３】このように各種の重要な指標となる５-ヒ
ドロキシクレアチニンの定量法については様々な研究が
なされていた。中でも、本発明者の一人である中村らに
よる高速液体クロマトグラフィーを用いた方法（ポスト
カラムラベル法：特開平４−１６１８５４号公報及び特
開平５−１１９０３８号公報）が具体的な５-ヒドロキ
シクレアチニンの定量法として知られていた。この定量
法は、高速液体クロマトグラフィー（ＨＰＬＣ）により
５-ヒドロキシクレアチニンを分離した後に、得られた
５-ヒドロキシクレアチニンを加水分解してメチルグア
ニジンへ変換し、蛍光ラベルして定量する方法である。As described above, various studies have been made on a method for quantifying 5-hydroxycreatinine, which is an important index. Among them, a method using high-performance liquid chromatography by Nakamura et al., One of the present inventors (post column label method: JP-A-4-161854 and JP-A-5-119038), is a specific 5-hydroxycreatinine. It was known as a quantitative method. This quantification method is a method of separating 5-hydroxycreatinine by high performance liquid chromatography (HPLC), hydrolyzing the obtained 5-hydroxycreatinine to convert it to methylguanidine, and quantifying it by fluorescent labeling.
【０００４】[0004]
【発明が解決しようとする課題】しかしながら、前述の
ポストカラムラベル法による定量においては、蛍光試薬
を送液するポンプが２台余分に必要であり、大掛りなＨ
ＰＬＣシステムが必要となっていた。また高濃度のアル
カリ溶液を溶出液として使用するためＨＰＬＣシステム
の消耗が激しく、維持・管理が繁雑で、初期導入費用や
維持費用が比較的高額であった。However, in the above-described quantification by the post-column labeling method, two extra pumps for sending a fluorescent reagent are required, and a large amount of H is required.
A PLC system was needed. In addition, since a high-concentration alkaline solution is used as an eluate, the HPLC system is greatly consumed, maintenance and management are complicated, and initial introduction costs and maintenance costs are relatively high.
【０００５】[0005]
【課題を解決するための手段】そこで本発明者らは、５
-ヒドロキシクレアチニンの定量法について更に鋭意研
究を行った結果、従来技術の問題点を克服した実用的な
本発明の５-ヒドロキシクレアチニンの定量法を見出し
本発明を完成した。本発明の目的は、腎機能障害の検査
法として有用な、高速液体クロマトグラフィーを用いた
５-ヒドロキシクレアチニンの高感度且つ実用的な定量
法を提供することにある。Means for Solving the Problems Accordingly, the present inventors have proposed a method of 5
As a result of further intensive studies on the method for quantifying 5-hydroxycreatinine, the present inventors have found a practical method for quantifying 5-hydroxycreatinine of the present invention which has overcome the problems of the prior art, and have completed the present invention. An object of the present invention is to provide a highly sensitive and practical method for quantifying 5-hydroxycreatinine using high performance liquid chromatography, which is useful as a method for testing renal dysfunction.
【０００６】[0006]
【発明の実施の形態】本発明は、５-ヒドロキシクレア
チニンをメチルグアニジンに変換し、得られたメチルグ
アニジンを蛍光ラベルした後に高速液体クロマトグラフ
ィーにより測定することを特徴とする５-ヒドロキシク
レアチニンの定量法に関する。BEST MODE FOR CARRYING OUT THE INVENTION The present invention is characterized in that 5-hydroxycreatinine is converted into methylguanidine, and the obtained methylguanidine is fluorescently labeled and then measured by high performance liquid chromatography. About the law.
【０００７】以下に本発明について詳細に説明する。５
-ヒドロキシクレアチニンをメチルグアニジンに変換す
る方法としては、例えば５-ヒドロキシクレアチニンを
加水分解する方法が挙げられる。具体的には、５-ヒド
ロキシクレアチニンを含む試料をホウ酸又はホウ酸塩溶
液中で加温する方法などが挙げられる。ホウ酸又はホウ
酸塩溶液中で加温する方法を用いる場合、ホウ酸又はホ
ウ酸塩を終濃度５０〜５００ｍｍｏｌ／Ｌで５-ヒドロ
キシクレアチニンと共存させ、ｐＨ７に調整し５０℃で
０．５〜２時間加温すれば、選択的に５-ヒドロキシク
レアチニンの加水分解がおこり、クレアチニン等の他の
生体成分からのメチルグアニジンの産生を防止すること
ができるため特に好ましい。Hereinafter, the present invention will be described in detail. 5
Examples of a method for converting -hydroxycreatinine to methylguanidine include a method for hydrolyzing 5-hydroxycreatinine. Specifically, there is a method in which a sample containing 5-hydroxycreatinine is heated in a boric acid or borate solution. When using a method of heating in a boric acid or borate solution, boric acid or borate is co-existed with 5-hydroxycreatinine at a final concentration of 50 to 500 mmol / L, adjusted to pH 7 and adjusted to 50 ° C at 0.5 ° C. Heating for れ ば 2 hours is particularly preferable because 5-hydroxycreatinine is selectively hydrolyzed and the production of methylguanidine from other biological components such as creatinine can be prevented.
【０００８】メチルグアニジンを蛍光ラベルする方法と
しては、ニンヒドリン法、ベンゾイン法、フェナンスレ
ンキノン（ＰＱ）法等が挙げられる。As a method for fluorescently labeling methylguanidine, a ninhydrin method, a benzoin method, a phenanthrenequinone (PQ) method and the like can be mentioned.
【０００９】メチルグアニジンを蛍光ラベルして得られ
た反応液は、高速液体クロマトグラフィーに供する前に
ｐＨを適宜調整する。このとき、ｐＨを１〜９、好まし
くは５〜７に調整すれば、ＨＰＬＣシステムの消耗を防
ぐことが可能である。The pH of the reaction solution obtained by fluorescently labeling methylguanidine is appropriately adjusted before subjecting it to high performance liquid chromatography. At this time, if the pH is adjusted to 1 to 9, preferably 5 to 7, it is possible to prevent the consumption of the HPLC system.
【００１０】メチルグアニジンを蛍光ラベルして得られ
た反応液を高速液体クロマトグラフィーにより分離し、
メチルグアニジンの蛍光ラベル体を定量する。この場
合、分解前のメチルグアニジン量を別途に求め、全メチ
ルグアニジン量よりこの量を差し引いて補正しても良い
が、このような補正を行なわずとも、クレアチニンの酸
化物である５-ヒドロキシクレアチニンとメチルグアニ
ジンの両方を合わせて測定することは、生体内あるいは
検体の前処理中での５-ヒドロキシクレアチニンのメチ
ルグアニジンへの代謝・分解の影響を受けないため、よ
り有用な腎障害の診断指標と成り得る。The reaction solution obtained by fluorescently labeling methylguanidine is separated by high performance liquid chromatography,
Quantify the fluorescent label of methylguanidine. In this case, the amount of methylguanidine before decomposition may be separately determined and corrected by subtracting this amount from the total amount of methylguanidine, but without such correction, 5-hydroxycreatinine, which is an oxide of creatinine, may be used. Measurement of both methylguanidine and methylguanidine is a more useful diagnostic index for renal damage because it is not affected by the metabolism / decomposition of 5-hydroxycreatinine to methylguanidine in vivo or during sample pretreatment. Can be
【００１１】蛍光ラベルしたメチルグアニジンの定量に
関しては、通常の逆相系ＨＰＬＣカラム、イオン交換カ
ラム、ゲル濾過カラムなどが使用できる。また、用いる
移動相は使用するカラムに応じて適宜選択して用いるこ
とができる。検出は通常の蛍光検出器を用いることがで
き、蛍光誘導体測定に至適な励起波長、蛍光波長で測定
する。For quantitative determination of fluorescently labeled methylguanidine, a conventional reversed-phase HPLC column, ion exchange column, gel filtration column, or the like can be used. The mobile phase used can be appropriately selected and used according to the column used. An ordinary fluorescence detector can be used for the detection, and the measurement is performed at an excitation wavelength and a fluorescence wavelength which are optimal for the measurement of the fluorescent derivative.
【００１２】また、測定の対象としては、動物由来の体
液、例えば、ヒト血液、ヒト血清、ヒト尿等を挙げるこ
とができる。[0012] The object of measurement may be a body fluid derived from an animal, for example, human blood, human serum, human urine and the like.
【００１３】本発明の好ましい実施態様を以下に挙げ
る。 （１）５-ヒドロキシクレアチニンをメチルグアニジン
に変換し、蛍光ラベルした後に高速液体クロマトグラフ
ィーにより測定することを特徴とする５-ヒドロキシク
レアチニンの定量法。 （２）５-ヒドロキシクレアチニンを加水分解してメチ
ルグアニジンに変換する上記（１）に記載５-ヒドロキ
シクレアチニンの定量法。 （３）ホウ酸又はホウ酸塩を用いて加水分解する上記
（２）に記載の５-ヒドロキシクレアチニンの定量法。 （４）ホウ酸又はホウ酸塩溶液中で加温することにより
加水分解する上記（２）又は（３）に記載の５-ヒドロ
キシクレアチニンの定量法。 （５）ニンヒドリン法によりメチルグアニジンを蛍光ラ
ベルする上記（１）乃至（４）に記載の５-ヒドロキシ
クレアチニンの定量法。 （６）高速液体クロマトグラフィーを用いて測定する前
に、メチルグアニジンを蛍光ラベルして得られた反応液
のｐＨを調整する上記（１）乃至（５）に記載の５-ヒ
ドロキシクレアチニンの定量法。Preferred embodiments of the present invention are described below. (1) A method for quantifying 5-hydroxycreatinine, which comprises converting 5-hydroxycreatinine into methylguanidine, performing fluorescent labeling, and measuring the resulting product by high performance liquid chromatography. (2) The method for quantifying 5-hydroxycreatinine according to the above (1), wherein 5-hydroxycreatinine is hydrolyzed to be converted into methylguanidine. (3) The method for quantifying 5-hydroxycreatinine according to (2), wherein the method is hydrolyzed using boric acid or a borate. (4) The method for quantifying 5-hydroxycreatinine according to the above (2) or (3), which is hydrolyzed by heating in a boric acid or borate solution. (5) The method for quantifying 5-hydroxycreatinine according to any one of (1) to (4), wherein methylguanidine is fluorescently labeled by a ninhydrin method. (6) The method for quantifying 5-hydroxycreatinine according to (1) to (5) above, wherein the pH of the reaction solution obtained by fluorescently labeling methylguanidine is adjusted before measurement using high performance liquid chromatography. .
【００１４】（７）メチルグアニジンを蛍光ラベルして
得られた反応液のｐＨを１〜９に調整する上記（６）に
記載の５-ヒドロキシクレアチニンの定量法。 （８）メチルグアニジンを蛍光ラベルして得られた反応
液のｐＨを５〜７に調整する上記（６）に記載の５-ヒ
ドロキシクレアチニンの定量法。 （９）動物由来の体液中の５-ヒドロキシクレアチニン
を定量する上記（１）乃至（８）に記載の５-ヒドロキ
シクレアチニンの定量法。 （１０）体液が血清である上記（９）に記載の５-ヒド
ロキシクレアチニンの定量法。 （１１）体液が尿である上記（９）に記載の５-ヒドロ
キシクレアチニンの定量法。 （１２）体液がヒト由来のものである上記（９）乃至
（１１）に記載の５-ヒドロキシクレアチニンの定量
法。 以下に実施例を挙げて本発明を更に具体的に説明する
が、本発明はこれらによって何ら限定されるものではな
い。(7) The method for quantifying 5-hydroxycreatinine according to (6), wherein the pH of the reaction solution obtained by fluorescently labeling methylguanidine is adjusted to 1 to 9. (8) The method for quantifying 5-hydroxycreatinine according to (6), wherein the pH of the reaction solution obtained by fluorescently labeling methylguanidine is adjusted to 5 to 7. (9) The method for quantifying 5-hydroxycreatinine according to any one of (1) to (8), wherein 5-hydroxycreatinine is quantified in a body fluid derived from an animal. (10) The method for quantifying 5-hydroxycreatinine according to (9), wherein the body fluid is serum. (11) The method for quantifying 5-hydroxycreatinine according to (9), wherein the body fluid is urine. (12) The method for quantifying 5-hydroxycreatinine according to the above (9) to (11), wherein the body fluid is derived from a human. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
【００１５】[0015]
【実施例】以下の実施例において、本発明定量法の一例
をより詳細に示す。尚、以下の実験においては、高速液
体クロマトグラフィーは８８０ＰＵ ｓｅｒｉｅｓ（日
本分光（株））を使用し、インテグレーターにはＳＩＣ
Ｌａｂｃｈａｒｔ １８０を用いた。EXAMPLES In the following examples, an example of the quantification method of the present invention will be described in more detail. In the following experiments, 880 PU series (JASCO Corporation) was used for high-performance liquid chromatography, and SIC was used as an integrator.
Labchart 180 was used.
【００１６】実施例１ 実験は以下の手順に従って行った。 （１）５-ヒドロキシクレアチニン標準溶液の調製法 ５-ヒドロキシクレアチニンを秤量後、水に溶解して２
０００μｍｏｌ／Ｌ溶液を調製し、５-ヒドロキシクレ
アチニン標準原液とした。原液を水で希釈し、１０００
μｍｏｌ／Ｌ、２００μｍｏｌ／Ｌ、１００μｍｏｌ／
Ｌ、５０μｍｏｌ／Ｌ、２０μｍｏｌ／Ｌ、１０μｍｏ
ｌ／Ｌ、５μｍｏｌ／Ｌ、２μｍｏｌ／Ｌ、１μｍｏｌ
／Ｌ、０．５μｍｏｌ／Ｌ及び０．２μｍｏｌ／Ｌの５
-ヒドロキシクレアチニン標準溶液を調製した。Example 1 An experiment was performed according to the following procedure. (1) Preparation method of 5-hydroxycreatinine standard solution After weighing 5-hydroxycreatinine, dissolving it in water
A 000 μmol / L solution was prepared and used as a 5-hydroxycreatinine standard stock solution. Dilute the stock solution with water, 1000
μmol / L, 200 μmol / L, 100 μmol / L
L, 50 μmol / L, 20 μmol / L, 10 μmo
1 / L, 5 μmol / L, 2 μmol / L, 1 μmol
/ L, 0.5 μmol / L and 0.2 μmol / L
-Hydroxycreatinine standard solution was prepared.
【００１７】（２）血清及び尿の調製法 血清は早朝空腹時に採血し、４℃で遠心分離した後、上
清を凍結保存した。また、尿は２４時間尿を採取し、尿
量を記録後、凍結保存した。(2) Preparation of serum and urine Serum was collected on an empty stomach in the early morning, centrifuged at 4 ° C., and the supernatant was frozen and stored. In addition, urine was collected for 24 hours, and the amount of urine was recorded and stored frozen.
【００１８】（３）５-ヒドロキシクレアチニン標準溶
液、血清及び尿中５-ヒドロキシクレアチニンの選択的
加水分解 ５-ヒドロキシクレアチニン標準溶液、血清及び尿１２
０μＬを遠心分離（１０，０００ｒｐｍ、６０ｍｉｎ）
し、その６０μＬにホウ酸溶液２０μＬ加え、５０℃で
１時間インキュベートし、５-ヒドロキシクレアチニン
をメチルグアニジンへ変換した。ホウ酸溶液は１ｍｏｌ
／Ｌホウ酸を２Ｎ水酸化ナトリウムによりｐＨ７に調製
したものを用いた。この処理により５-ヒドロキシクレ
アチニンは完全にメチルグアニジンへ変換され、またこ
の条件下ではクレアチニンなどからメチルグアニジンが
生成することはなかった。(3) Selective hydrolysis of 5-hydroxycreatinine standard solution, serum and urine 5-hydroxycreatinine standard solution, 5-hydroxycreatinine standard solution, serum and urine
0 μL is centrifuged (10,000 rpm, 60 min)
Then, 20 μL of a boric acid solution was added to 60 μL of the mixture, and the mixture was incubated at 50 ° C. for 1 hour to convert 5-hydroxycreatinine into methylguanidine. 1 mol of boric acid solution
/ L boric acid adjusted to pH 7 with 2N sodium hydroxide was used. By this treatment, 5-hydroxycreatinine was completely converted to methylguanidine, and under this condition, methylguanidine was not produced from creatinine or the like.
【００１９】（４）加水分解液のプレカラム蛍光ラベル 反応液から６０μＬをとり、６Ｎ水酸化ナトリウムを１
０μＬ添加、撹拌し、正確に2分間室温に放置した。次
いで、４０℃の温浴中で正確に５分間プレインキュベー
トした。プレインキュベート後、１．６％（ｗ／ｖ）ニ
ンヒドリン／メチルセロソルブ溶液１０μＬを添加、撹
拌し、１８分間４０℃の温浴中でインキュベートした。
インキュベート後、６Ｎ塩酸１０μＬを添加撹拌し、４
０秒後に８０μＬをＨＰＬＣで分析した。(4) Pre-column fluorescent labeling of hydrolyzed solution Take 60 μL from the reaction solution and add 1N 6N sodium hydroxide.
0 μL was added, stirred, and left at room temperature for exactly 2 minutes. It was then pre-incubated for exactly 5 minutes in a 40 ° C. water bath. After the pre-incubation, 10 μL of a 1.6% (w / v) ninhydrin / methyl cellosolve solution was added, stirred, and incubated for 18 minutes in a 40 ° C. warm bath.
After the incubation, 10 μL of 6N hydrochloric acid was added, and the mixture was stirred.
After 0 seconds, 80 μL was analyzed by HPLC.
【００２０】（５）プレカラム蛍光ラベルした検体のＨ
ＰＬＣ測定 カラムは逆相ＯＤＳカラム（Inertsil ODS-3V I.D.4.6
x 250mm : GL science）をメインカラムとして用い、ガ
ードカラムとしてDevelosil ODS-5 I.D.4.6 x30mm（Nom
ura Chemical）を用いた。溶離液は３０％メタノール／
０．０５Ｍ酢酸ナトリウム緩衝液（ｐＨ６．０）を用い
た。流速は１．０ｍＬ／ｍｉｎとし、２５℃で測定し
た。(5) Precolumn Fluorescently labeled sample H
The PLC measurement column was a reversed-phase ODS column (Inertsil ODS-3V ID4.6
x 250mm: GL science) as main column and Develosil ODS-5 ID4.6 x 30mm (Nom
ura Chemical) was used. The eluent was 30% methanol /
A 0.05 M sodium acetate buffer (pH 6.0) was used. The flow rate was 1.0 mL / min, and the measurement was performed at 25 ° C.
【００２１】５-ヒドロキシクレアチニン標準溶液を本
発明の５-ヒドロキシクレアチニン定量法を用いて測定
した結果の一例を表１に示す。本発明の５-ヒドロキシ
クレアチニン定量法は再現性も優れ、実際の生体試料の
分析でも十分適用可能であった。また、本発明測定法の
定量性を検討したところ、本実施例の試験系の測定条件
においては、定量下限は０．５μｍｏｌ／Lであり、定
量上限は１００μｍｏｌ／Lであった。Table 1 shows an example of the results obtained by measuring the 5-hydroxycreatinine standard solution using the 5-hydroxycreatinine quantitative method of the present invention. The 5-hydroxycreatinine quantification method of the present invention has excellent reproducibility and was sufficiently applicable to the analysis of actual biological samples. Further, when the quantitativeness of the measurement method of the present invention was examined, the lower limit of quantification was 0.5 μmol / L and the upper limit of quantification was 100 μmol / L under the measurement conditions of the test system of this example.
【表１】 [Table 1]
【００２２】また、慢性腎不全患者の血清中の５-ヒド
ロキシクレアチニン量（５-ヒドロキシクレアチニンと
メチルグアニジンの両方を足した値）を本発明定量法及
びポストカラムラベル法（特開平４−１６１８５４号公
報記載の方法）で測定し、その相関について調べた結果
の一例を表２及び図１に示す。両測定法での結果は共に
非常に良い相関を示しており（相関係数ｒ＝０．９９
３）、同等の定量値を与えることが明らかとなった。Further, the amount of 5-hydroxycreatinine (the value obtained by adding both 5-hydroxycreatinine and methylguanidine) in the serum of a patient with chronic renal failure was determined by the quantitative method of the present invention and the post-column labeling method (JP-A-4-161854). Table 2 and FIG. 1 show an example of the results of the measurement according to the method described in the official gazette and the examination of the correlation. The results of both measurement methods show a very good correlation (correlation coefficient r = 0.99).
3), it was found to give equivalent quantitative values.
【表２】 [Table 2]
【００２３】[0023]
【発明の効果】上記の結果より明らかなように、本発明
の５-ヒドロキシクレアチニン定量法は再現性に優れ、
定量性についても従来のポストカラムラベル法と遜色の
ない、実際の生体試料の分析にも十分適用しうる高感度
な定量法である。また、本発明の５-ヒドロキシクレア
チニン定量法は比較的単純な操作のみで構成されている
ため、オートサンプラー等の使用により操作を機械化し
自動化することが可能であり、実用的な定量法である。
操作を機械化し自動化すれば、操作をより正確且つ精密
に行うことができ、測定のばらつきの低減につながるほ
か、終夜測定などの無人化も可能である。さらに本発明
の５-ヒドロキシクレアチニン定量法では、送液ポンプ
は１台しか必要なく、極めて単純なシステムで測定する
ことができる。また、ＨＰＬＣによる測定の前にｐＨを
適宜調整できるので、ＨＰＬＣシステムの消耗を防ぐこ
とが可能であり、従来のポストカラムラベル法に比べて
ＨＰＬＣシステムの維持・管理が行いやすく、初期導入
費用や維持費用が比較的安価な、コスト面でも優れた非
常に実用的な定量法である。As is clear from the above results, the 5-hydroxycreatinine quantification method of the present invention has excellent reproducibility,
It is a highly sensitive quantification method that is comparable to the conventional post-column label method in terms of quantification and can be sufficiently applied to the analysis of actual biological samples. Further, since the 5-hydroxycreatinine quantification method of the present invention comprises only relatively simple operations, the operation can be automated and automated by using an autosampler or the like, and is a practical quantification method. .
If the operation is mechanized and automated, the operation can be performed more accurately and precisely, which leads to a reduction in measurement variation and also enables unmanned measurement such as overnight measurement. Furthermore, in the 5-hydroxycreatinine quantitative determination method of the present invention, only one liquid sending pump is required, and measurement can be performed with a very simple system. In addition, since the pH can be appropriately adjusted before the measurement by HPLC, it is possible to prevent the consumption of the HPLC system, and the maintenance and management of the HPLC system are easier than the conventional post-column label method, and the initial introduction cost and It is a very practical quantitative method with relatively low maintenance costs and excellent cost.
【図１】図１は慢性腎不全患者の血清中の５-ヒドロキ
シクレアチニン量を本発明定量法及びポストカラムラベ
ル法で測定し、その相関について調べた結果の一例であ
る。FIG. 1 shows an example of the results of measuring the amount of 5-hydroxycreatinine in the serum of a patient with chronic renal failure by the quantitative method of the present invention and the post-column label method, and examining the correlation.
Claims (3)
ニジンに変換し、得られたメチルグアニジンを蛍光ラベ
ルした後に高速液体クロマトグラフィーを用いて測定す
ることを特徴とする５-ヒドロキシクレアチニンの定量
法。1. A method for quantifying 5-hydroxycreatinine, which comprises converting 5-hydroxycreatinine into methylguanidine, labeling the obtained methylguanidine with a fluorescent label, and measuring the resulting product using high performance liquid chromatography.
てメチルグアニジンに変換する請求項１記載の５-ヒド
ロキシクレアチニンの定量法。2. The method for quantifying 5-hydroxycreatinine according to claim 1, wherein 5-hydroxycreatinine is hydrolyzed and converted into methylguanidine.
請求項２記載の５-ヒドロキシクレアチニンの定量法。3. The method for quantifying 5-hydroxycreatinine according to claim 2, wherein the hydrolysis is carried out using boric acid or a borate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11164727A JP2000352564A (en) | 1999-06-11 | 1999-06-11 | Method for determining 5-hydroxycreatinine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11164727A JP2000352564A (en) | 1999-06-11 | 1999-06-11 | Method for determining 5-hydroxycreatinine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2000352564A true JP2000352564A (en) | 2000-12-19 |
Family
ID=15798761
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11164727A Pending JP2000352564A (en) | 1999-06-11 | 1999-06-11 | Method for determining 5-hydroxycreatinine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2000352564A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002350449A (en) * | 2001-05-30 | 2002-12-04 | Fuji Photo Film Co Ltd | Control serum for dry analytical element |
| US6696301B2 (en) | 2000-10-03 | 2004-02-24 | Nippon Zoki Pharmaceutical Co., Ltd. | Method for determination of 5-hydroxycreatinine |
-
1999
- 1999-06-11 JP JP11164727A patent/JP2000352564A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6696301B2 (en) | 2000-10-03 | 2004-02-24 | Nippon Zoki Pharmaceutical Co., Ltd. | Method for determination of 5-hydroxycreatinine |
| JP2002350449A (en) * | 2001-05-30 | 2002-12-04 | Fuji Photo Film Co Ltd | Control serum for dry analytical element |
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