JP3147953B2 - Biological substance measurement method - Google Patents
Biological substance measurement methodInfo
- Publication number
- JP3147953B2 JP3147953B2 JP30520091A JP30520091A JP3147953B2 JP 3147953 B2 JP3147953 B2 JP 3147953B2 JP 30520091 A JP30520091 A JP 30520091A JP 30520091 A JP30520091 A JP 30520091A JP 3147953 B2 JP3147953 B2 JP 3147953B2
- Authority
- JP
- Japan
- Prior art keywords
- hydroxycreatinine
- methylguanidine
- renal
- serum
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- Investigating Or Analysing Biological Materials (AREA)
Description
【０００１】[0001]
【産業上の利用分野】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に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for quantifying 5-hydroxycreatinine, which comprises measuring 5-hydroxycreatinine by converting it to methylguanidine.
【０００２】[0002]
【従来の技術】メチルグアニジンは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おり、クレ
アチニンから活性酸素により生成するものと考えられて
いる。本発明者らは腎障害におけるクレアチニン代謝に
関して研究した結果、腎障害患者の血中及び尿中から新
規な化合物、５−ヒドロキシクレアチニンを単離同定し
た。この物質は健常人の血中にはなく、腎障害患者の血
中に特有にみられ、病態の進行に伴い産生昂進が認めら
れること、また５−ヒドロキシクレアチニンは種々の実
験結果より、クレアチニンからメチルグアニジンが産生
される際のメチルグアニジンの前駆体であることを見出
した。本発明はこれらの事実に基づき、血中又は尿中に
おける５−ヒドロキシクレアチニンを測定することによ
って、腎不全、***等各種腎障害の早期診断を可能と
したものである。2. Description of the Related Art Methylguanidine is known as one of the major urinary toxins that accumulates in the blood of patients with renal failure and is considered to be produced from creatinine by active oxygen. The present inventors have studied creatinine metabolism in renal impairment, and have isolated and identified a novel compound, 5-hydroxycreatinine, from the blood and urine of patients with renal impairment. This substance is not found in the blood of healthy individuals but is found in the blood of patients with renal impairment, and its production is increased as the disease progresses. It has been found that methylguanidine is a precursor of methylguanidine when produced. Based on these facts, the present invention enables early diagnosis of various renal disorders such as renal failure and uremic disease by measuring 5-hydroxycreatinine in blood or urine.
【０００３】[0003]
【発明が解決しようとする課題】本発明の目的は、腎機
能障害の検査法として有用な５−ヒドロキシクレアチニ
ンの定量法を提供することにある。SUMMARY OF THE INVENTION An object of the present invention is to provide a method for quantifying 5-hydroxycreatinine which is useful as a method for examining renal dysfunction.
【０００４】[0004]
【課題を解決するための手段】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で
ある。SUMMARY OF THE INVENTION The present invention relates to a method for quantifying 5-hydroxycreatinine, which comprises measuring 5-hydroxycreatinine by converting it to methylguanidine.
【０００５】グアニジノ化合物の測定法としては、ニン
ヒドリン、ベンゾイン、９，１０−フェナンスレンキノ
ン（ＰＱ試薬）などを用いる蛍光分析が繁用されてい
る。５−ヒドロキシクレアチニンは分子内にグアニジノ
骨格を有しているが、これら蛍光分析用試薬との反応性
は極めて低く微量分析が容易でない。そこで、上記蛍光
試薬に対してメチルグアニジンが５−ヒドロキシクレア
チニンよりも反応性が高いことから、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て測定する本発
明定量法を見出した。As a method for measuring a guanidino compound, fluorescence analysis using ninhydrin, benzoin, 9,10-phenanthrenequinone (PQ reagent) or the like has been widely used. Although 5-hydroxycreatinine has a guanidino skeleton in the molecule, its reactivity with these reagents for fluorescence analysis is extremely low, and trace analysis is not easy. Then, since methylguanidine has higher reactivity with the above fluorescent reagent than 5-hydroxycreatinine, the present inventors have found a quantitative method of the present invention in which 5-hydroxycreatinine is converted to methylguanidine for measurement.
【０００６】以下に本発明測定法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
変換させる方法としては、一般に用いられている加水分
解等の方法が利用でき、例えば酸又はアルカリで処理す
る方法や酸又はアルカリの存在下或いは非存在下にて加
熱処理する方法などが挙げられる。また、酵素を用いて
５−ヒドロキシクレアチニンをメチルグアニジンに変換
させる方法も利用できる。Hereinafter, the measurement method of the present invention will be described in detail. As a method for converting 5-hydroxycreatinine to methylguanidine, a commonly used method such as hydrolysis can be used. For example, a method of treating with acid or alkali, or heating in the presence or absence of an acid or alkali can be used. And a method of processing. A method of converting 5-hydroxycreatinine into methylguanidine using an enzyme can also be used.
【０００７】上記メチルグアニジンへの変換処理につい
ては、陽イオン交換樹脂等による分画法や該樹脂を用い
た高速液体クロマトグラフィー（ＨＰＬＣ）などによっ
て５−ヒドロキシクレアチニンを分離した後に加水分解
処理する方法が挙げられる。また、５−ヒドロキシクレ
アチニンを分離する前に加水分解処理を行い、その試料
中の全メチルグアニジンを測定することも可能である。
この場合、分離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引いて補正してもよ
いが、このような補正を行なわずとも、腎障害の病態の
進行に伴い産生昂進が認められる５−ヒドロキシクレア
チニン及びメチルグアニジンの両方を合わせて測定する
ことも、腎機能障害の診断として有用である。加水分解
の方法や条件によっては、試料中に存在する５−ヒドロ
キシクレアチニン以外の物質からメチルグアニジンが産
生されてくる場合を考慮に入れると、５−ヒドロキシク
レアチニンのみを選択的にメチルグアニジンに変換でき
るような加水分解等の方法及び条件を適宜設定するのが
好ましい。The above-mentioned conversion to methylguanidine is carried out by fractionation using a cation exchange resin or the like, or separation of 5-hydroxycreatinine by high performance liquid chromatography (HPLC) using the resin, followed by hydrolysis. Is mentioned. It is also possible to carry out a hydrolysis treatment before separating 5-hydroxycreatinine, and to measure the total methylguanidine in the sample.
In this case, the amount of methylguanidine before separation is separately determined,
This amount may be subtracted from the total methylguanidine amount for correction, but without such correction, both 5-hydroxycreatinine and methylguanidine, whose production is increased along with the progression of the renal impairment, are combined. Measurement is also useful as a diagnosis of renal dysfunction. Depending on the method and conditions of hydrolysis, taking into account the case where methylguanidine is produced from a substance other than 5-hydroxycreatinine present in the sample, only 5-hydroxycreatinine can be selectively converted to methylguanidine. It is preferable to appropriately set the method and conditions for such hydrolysis and the like.
【０００８】５−ヒドロキシクレアチニンを変換して得
られたメチルグアニジンを測定する方法としては、ニン
ヒドリン、ベンゾイン、ＰＱ試薬等の蛍光試薬と反応さ
せる蛍光分析法、メチルグアニジンアミジノヒドラーゼ
を用いる酵素法など通常行われているメチルグアニジン
の測定法が利用できる。例えば、ＰＱ試薬を用いる方法
においては、ＰＱ試薬はアルカリ性でグアニジノ化合物
と反応するため、アルカリの存在下にて５−ヒドロキシ
クレアチニンを加水分解処理してメチルグアニジンに変
換するのが、後のＰＱ試薬との反応において有利であり
手間を省くことができる。As a method for measuring methylguanidine obtained by converting 5-hydroxycreatinine, there are a fluorescence analysis method in which a reaction is caused with a fluorescent reagent such as ninhydrin, benzoin, PQ reagent, and an enzymatic method using methylguanidine amidinohydrase. A commonly used method for measuring methylguanidine can be used. For example, in the method using a PQ reagent, the PQ reagent is alkaline and reacts with a guanidino compound, and therefore, 5-hydroxycreatinine is hydrolyzed in the presence of an alkali to convert it to methylguanidine. This is advantageous in the reaction with and can save labor.
【０００９】[0009]
実施例１． （１）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。又、尿は２４時間尿を採取し、尿量
を記録後、凍結保存した。これら検体をトリクロロ酢酸
等により除蛋白処理してから以下の測定に供した。Embodiment 1 FIG. (1) 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. The urine was collected for 24 hours, and the amount of the urine was recorded and stored frozen. These specimens were subjected to the following measurement after deproteinization treatment with trichloroacetic acid or the like.
【００１０】（２）ＨＰＬＣによる分画 クレアチニン、メチルグアニジン等のグアニジノ化合物
の測定は、ＨＰＬＣで分離した後、ＰＱ試薬と反応させ
蛍光を測定する山本らの方法に準じて行った。〔Ｊｏｕ
ｒｎａｌ ｏｆ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ，１６
２（１９７９），３２７−３４０〕カラムは陽イオン交
換樹脂（ＴＯＳＯ ＳＣＸ，ＳＨＩＭＡＺＵ ＩＳＣ−
０７／Ｓ１５０４等）を用い、溶離液の組成は東館らの
方法に準じた。〔分析化学，３３（１９８４），３６６
−３７０〕(2) Fractionation by HPLC The measurement of guanidino compounds such as creatinine and methylguanidine was carried out according to the method of Yamamoto et al., Which was separated by HPLC and reacted with a PQ reagent to measure fluorescence. [Jou
rnal of Chromatography, 16
2 (1979), 327-340] column is a cation exchange resin (TOSO SCX, SHIMAZU ISC-
07 / S1504), and the composition of the eluent was in accordance with the method of Higashidate et al. [Analytical Chemistry, 33 (1984), 366]
-370]
【００１１】（３）蛍光試薬 ポストカラムにおける蛍光試薬としては、２Ｍ水酸化ナ
トリウム水溶液及びＰＱ試薬溶液（１ｌのジメチルホル
ムアミドに５００ｍｇのＰＱを溶解して調製）を用い
た。(3) Fluorescent reagent As a fluorescent reagent in the post column, a 2M aqueous sodium hydroxide solution and a PQ reagent solution (prepared by dissolving 500 mg of PQ in 1 liter of dimethylformamide) were used.
【００１２】（４）５−ヒドロキシクレアチニンの測定
系 ５−ヒドロキシクレアチニンをメチルグアニジンに変換
して測定する定量分析システムの流路構成図を第１図に
示す。第１反応コイルには長さが５ｍ、内径が０．５ｍ
ｍのステンレス管を用い、反応器を１２５℃に設定する
ことにより、定量分析系における発色が最大となった。
蛍光検出器の励起波長は３４０ｎｍに、蛍光波長は４９
５ｎｍに設定した。(4) 5-Hydroxycreatinine Measurement System FIG. 1 shows a flow path configuration diagram of a quantitative analysis system for measuring 5-hydroxycreatinine by converting it to methylguanidine. The first reaction coil has a length of 5 m and an inner diameter of 0.5 m
The color development in the quantitative analysis system was maximized by setting the reactor at 125 ° C. using a stainless steel tube of m.
The excitation wavelength of the fluorescence detector is 340 nm and the fluorescence wavelength is 49 nm.
It was set to 5 nm.
【００１３】実施例２． （１）ホウ酸による５−ヒドロキシクレアチニンのメチ
ルグアニジンへの変換 １ｍｌの血清に１Ｍホウ酸−水酸化ナトリウム緩衝液
（ｐＨ７．０）を加え、１時間水浴（約５０℃）で加熱
した。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に変換されることが確かめられ
た。尚、この条件下ではクレアチニン等からメチルグア
ニジンが生成することはなかった。Embodiment 2 FIG. (1) Conversion of 5-hydroxycreatinine to methylguanidine with boric acid To 1 ml of serum was added 1M boric acid-sodium hydroxide buffer (pH 7.0) and heated in a water bath (about 50 ° C) for 1 hour. It was confirmed that 5-hydroxycreatinine was completely converted to methylguanidine by this treatment. Under these conditions, methylguanidine was not produced from creatinine or the like.
【００１４】（２）陽イオン交換樹脂を用いたメチルグ
アニジンの分離 血清中にはアルギニン等のグアニジノ化合物が大量に含
まれており、微量分析のためには、これらとメチルグア
ニジンを分離する必要がある。メチルグアニジンは強塩
基性物質であり陽イオン交換樹脂に強く吸着するため、
試料を添加した陽イオン交換樹脂カラムを、先ずｐＨ９
〜１０の緩衝液（例えば、０．１Ｍ〜０．２Ｍリン酸緩
衝液）で洗浄し、グアニジン以外のクアニジノ化合物を
溶出させた。その後、１Ｎ〜２Ｎ水酸化ナトリウム溶液
等の強塩基性溶液によって吸着したメチルグアニジン等
を溶出させた。(2) Separation of Methyl Guanidine Using Cation Exchange Resin Serum contains a large amount of guanidino compounds such as arginine. For trace analysis, it is necessary to separate methyl guanidine from these compounds. is there. Methylguanidine is a strongly basic substance and strongly adsorbs to cation exchange resin,
The cation exchange resin column to which the sample was added was first placed at pH 9
Washing was performed with a buffer solution (e.g., 0.1 M to 0.2 M phosphate buffer solution) to elute guanidino compounds other than guanidine. Thereafter, the adsorbed methylguanidine and the like were eluted with a strongly basic solution such as a 1N to 2N sodium hydroxide solution.
【００１５】ホウ酸処理をしなかったときのメチルグア
ニジン量との差から５−ヒドロキシクレアチニンの量を
求めることができる。またグアニジンが混在していても
それが大量で無い限り問題はない。グアニジンも尿毒素
の一つと考えられており、５−ヒドロキシクレアチニ
ン、メチルグアニジン及びグアニジンの総和を腎不全の
指標として設定することもでき、この場合は特にホウ酸
無処理時との差を求める必要はない。The amount of 5-hydroxycreatinine can be determined from the difference from the amount of methylguanidine without the boric acid treatment. Even if guanidine is mixed, there is no problem unless it is in a large amount. Guanidine is also considered to be one of the urinary toxins, and the sum of 5-hydroxycreatinine, methylguanidine and guanidine can be set as an index of renal failure. In this case, it is particularly necessary to determine the difference from the case without boric acid treatment There is no.
【００１６】（３）メチルグアニジンの定量法 血清をホウ酸による変換処理を行った後、トリクロロ酢
酸を用いて除蛋白処理した。次いで、陽イオン交換樹脂
を用いた分離操作を行い、得られた試料中のメチルグア
ニジン量を、ニンヒドリン、ベンゾイン、ＰＱ試薬等の
蛍光試薬を用いて測定した。(3) Method for Quantifying Methylguanidine Serum was subjected to conversion treatment with boric acid, followed by deproteinization treatment using trichloroacetic acid. Next, a separation operation using a cation exchange resin was performed, and the amount of methylguanidine in the obtained sample was measured using a fluorescent reagent such as ninhydrin, benzoin, and a PQ reagent.
【００１７】ａ）ニンヒドリン法 試料液２ｍｌ（１Ｎ水酸化ナトリウム溶液）に０．５％
ニンヒドリン水溶液を加え攪拌した。１０分間反応させ
た後、３９０ｎｍの励起光を用い、４９５ｎｍの蛍光を
測定した。A) Ninhydrin method 0.5% in 2 ml of a sample solution (1N sodium hydroxide solution)
A ninhydrin aqueous solution was added and stirred. After reacting for 10 minutes, fluorescence at 495 nm was measured using excitation light at 390 nm.
【００１８】ｂ）ベンゾイン法 試料液１ｍｌ（２Ｎ水酸化ナトリウム溶液）に０．５ｍ
ｌの４ｍＭベンゾイン／メチルセロソルブ溶液と０．５
ｍｌの０．１Ｍβ−メルカプトエタノール／０．２Ｍ亜
硫酸ナトリウム溶液を加え混和した後、１００℃で１０
分間加熱した。冷却してから１Ｍトリス緩衝液（ｐＨ
９．２）と４Ｎ塩酸の等量混合液を１ｍｌ加えた後、３
２７ｎｍの励起光を用い、４２７ｎｍの蛍光を測定し
た。B) Benzoin method 0.5 ml per 1 ml of sample solution (2N sodium hydroxide solution)
l of a 4 mM benzoin / methyl cellosolve solution and 0.5
ml of 0.1 M β-mercaptoethanol / 0.2 M sodium sulfite solution, and mixed.
Heated for minutes. After cooling, 1 M Tris buffer (pH
After adding 1 ml of a mixed solution of 9.2) and 4N hydrochloric acid, 3
427 nm fluorescence was measured using 27 nm excitation light.
【００１９】ｃ）ＰＱ法 試料液１ｍｌ（１Ｎ水酸化ナトリウム溶液）に０．１ｍ
ＭＰＱ−ジメチルホルムアミド溶液１ｍｌを加え混和し
た後、室温で４０分間反応させた。濃塩酸１ｍｌ及び水
２．５ｍｌを加えて混ぜ合わせた後、３１０ｎｍの励起
光を用い、３９０ｎｍの蛍光を測定した。C) PQ method 0.1 m per 1 ml of sample solution (1N sodium hydroxide solution)
After adding and mixing 1 ml of an MPQ-dimethylformamide solution, the mixture was reacted at room temperature for 40 minutes. After 1 ml of concentrated hydrochloric acid and 2.5 ml of water were added and mixed, fluorescence at 390 nm was measured using excitation light at 310 nm.
【００２０】[0020]
【発明の効果】５−ヒドロキシクレアチニンのニンヒド
リン、ベンゾイン、ＰＱ試薬等の蛍光試薬に対する反応
性が極めて低いため、微量分析は困難であったが、本発
明方法に従ってメチルグアニジンに変換した後、これら
蛍光試薬と反応させることにより、高感度で定量分析で
きることが明らかになった。本発明のＨＰＬＣや陽イオ
ン交換樹脂を用いた５−ヒドロキシクレアチニン定量分
析系は再現性も優れ、実際の生体試料の分析に十分適用
可能であった。例えば、ＨＰＬＣを用いた本発明測定法
の定量性を検討した結果、１．５ｎｍｏｌまで原点を通
る直線が得られることを確認できた。The reactivity of 5-hydroxycreatinine with fluorescent reagents such as ninhydrin, benzoin, and PQ reagents was extremely low, so that microanalysis was difficult, but after conversion to methylguanidine according to the method of the present invention, these fluorescent It became clear that high-sensitivity quantitative analysis was possible by reacting with the reagent. The 5-hydroxycreatinine quantitative analysis system using the HPLC and the cation exchange resin of the present invention has excellent reproducibility and was sufficiently applicable to the analysis of actual biological samples. For example, as a result of examining the quantitativeness of the measurement method of the present invention using HPLC, it was confirmed that a straight line passing through the origin up to 1.5 nmol was obtained.
【００２１】陽イオン交換樹脂を用いた実施例２の方法
では、ニンヒドリン法、ベイゾイン法、ＰＱ法など種々
の蛍光試薬による測定法について比較したが、各検出方
法において操作性、蛍光の安定性、選択性等の点で少々
差があったがいずれも充分利用可能であり、血清中の５
−ヒドロキシクレアチニンを検出限界０．５〜１ｎｍｏ
ｌ／ｍｌの感度で測定できることが明らかになった。こ
の方法の再現性は高く、且つＨＰＬＣを用いた系の測定
値と良好な相関が認められた。In the method of Example 2 using a cation exchange resin, the methods of measurement using various fluorescent reagents such as the ninhydrin method, the benzoin method, and the PQ method were compared. Although there was a slight difference in selectivity, etc., all of them were sufficiently available and 5
-Hydroxycreatinine with a detection limit of 0.5 to 1 nmo
It was found that the measurement could be performed with a sensitivity of 1 / ml. The reproducibility of this method was high, and a good correlation was observed with the measured value of the system using HPLC.
【００２２】糖尿病性腎症及び慢性腎炎、ループス腎
炎、腎硬化症等の非糖尿病性腎疾患の患者の血中及び尿
中のクレアチニン、メチルグアニジン並びに５−ヒドロ
キシクレアチニンの各値を本発明方法により測定した。
その結果、これら３物質の血清中の値は各々正の相関関
係が見られた。一例として糖尿病性腎症における血清ク
レアチニン値と血清５−ヒドロキシクレアチニン値との
相関関係を第２図に示す。血清５−ヒドロキシクレアチ
ニン値は血清クレアチニン値と強い相関を示すことよ
り、本発明５−ヒドロキシクレアチニン定量法は腎機能
障害の診断に有用であることが示された。The values of creatinine, methylguanidine and 5-hydroxycreatinine in blood and urine of patients with non-diabetic renal diseases such as diabetic nephropathy and chronic nephritis, lupus nephritis, renal sclerosis and the like are determined by the method of the present invention. It was measured.
As a result, a positive correlation was found between the serum values of these three substances. As an example, FIG. 2 shows the correlation between the serum creatinine level and the serum 5-hydroxycreatinine level in diabetic nephropathy. The serum 5-hydroxycreatinine level shows a strong correlation with the serum creatinine level, indicating that the 5-hydroxycreatinine quantification method of the present invention is useful for diagnosis of renal dysfunction.
【００２３】メチルグアニジンは血清中のクレアチニン
値が約２ｍｇ／ｄｌ以上でなければ検出されないのに対
して、５−ヒドロキシクレアチニンはクレアチニン値が
２ｍｇ／ｄｌより低い場合でも検出されるため、症状が
軽い初期の腎疾患の早期発見、早期治療を可能とするも
のである。また腎不全患者において、５−ヒドロキシク
レアチニンはメチルグアニジンの１０倍程度含有されて
おり、例えばメチルグアニジン含量が検出限界の１０分
の１の血清でも５−ヒドロキシクレアチニンを検出する
ことができ、本発明測定法は初期の腎機能障害の診断に
特に有用なものである。Methylguanidine cannot be detected unless the serum creatinine value is at least about 2 mg / dl, whereas 5-hydroxycreatinine is detected even when the creatinine value is lower than 2 mg / dl, so the symptoms are mild. It enables early detection and early treatment of early stage kidney disease. In patients with renal insufficiency, 5-hydroxycreatinine is contained about 10 times that of methylguanidine. For example, 5-hydroxycreatinine can be detected even in serum whose methylguanidine content is one-tenth of the detection limit. The assay is particularly useful for diagnosing early renal dysfunction.
【００２４】腎機能が正常であれば５−ヒドロキシクレ
アチニンは尿に速やかに***されるため血中には蓄積せ
ず、健常人血中では検出されない。しかし、腎機能の低
下とともに５−ヒドロキシクレアチニンの血中での蓄積
が増加する。また、腎機能障害が重症になるにつれて尿
毒素でありメチルグアニジンの前駆体である５−ヒドロ
キシクレアチニンの総量は増加し、それに伴い体液中並
びに尿中の５−ヒドロキシクレアチニン量が増加するも
のと考えられる。従って、血液等の体液中や尿中の５−
ヒドロキシクレアチニン値の測定は、腎機能障害を診断
するうえで非常に重要である。If renal function is normal, 5-hydroxycreatinine is rapidly excreted in urine and does not accumulate in blood, and is not detected in blood of healthy humans. However, the accumulation of 5-hydroxycreatinine in the blood increases with a decrease in renal function. In addition, it is considered that as renal dysfunction becomes severe, the total amount of 5-hydroxycreatinine, which is a urinary toxin and a precursor of methylguanidine, increases, and the amount of 5-hydroxycreatinine in body fluids and urine increases accordingly. Can be Therefore, 5- in blood and other body fluids and urine
Measurement of hydroxycreatinine levels is very important in diagnosing renal dysfunction.
【００２５】以上の述べたように、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て測定する本発明
定量法は、蛍光試薬との反応性が極めて低い５−ヒドロ
キシクレアチニンの微量分析を可能としたものである。
特に血中の５−ヒドロキシクレアチニン値を測定するこ
とは腎機能障害の早期診断に有用であり、本発明測定法
は腎不全、***、慢性腎炎、尿路閉塞等の各種腎障害
を早期診断可能な検査法として有用性が高い。As described above, the quantitative method of the present invention, in which 5-hydroxycreatinine is converted to methylguanidine for measurement, enables microanalysis of 5-hydroxycreatinine having extremely low reactivity with a fluorescent reagent. It is.
In particular, measuring the 5-hydroxycreatinine level in the blood is useful for early diagnosis of renal dysfunction, and the measurement method of the present invention enables early diagnosis of various renal disorders such as renal failure, uremia, chronic nephritis and urinary tract obstruction. It is highly useful as a possible test method.
【図１】５−ヒドロキシクレアチニンのＨＰＬＣを用い
た定量分析システムの流路構成図である。FIG. 1 is a flow path configuration diagram of a quantitative analysis system using 5-hydroxycreatinine HPLC.
【図２】糖尿病性腎症における血清クレアチニン値と血
清５−ヒドロキシクレアチニン値を測定し、その相関関
係を調べた結果である。FIG. 2 shows the results of measuring serum creatinine and serum 5-hydroxycreatinine in diabetic nephropathy and examining the correlation.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 家永 和治 兵庫県加東郡社町木梨字川北山442番１ 日本臓器製薬株式会社 生物活性科学 研究所内 (56)参考文献 特許2948649（ＪＰ，Ｂ２) (58)調査した分野(Int.Cl.7，ＤＢ名) G01N 33/70 ＷＰＩ（ＤＩＡＬＯＧ)──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kazuharu Ienaga 442-1, Kawakitayama, Kinashi-sha, Kato-gun, Hyogo Japan Nippon Organ Pharmaceutical Co., Ltd. Bioactivity Science Research Institute (56) Reference Patent 2948649 (JP, B2) (58) Field surveyed (Int. Cl. 7 , DB name) G01N 33/70 WPI (DIALOG)
Claims (1)
よりメチルグアニジンに変換し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 converting 5-hydroxycreatinine into boric acid
A method for quantifying 5-hydroxycreatinine, which comprises measuring the amount of 5-hydroxycreatinine after conversion into methylguanidine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30520091A JP3147953B2 (en) | 1991-10-23 | 1991-10-23 | Biological substance measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP30520091A JP3147953B2 (en) | 1991-10-23 | 1991-10-23 | Biological substance measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05119038A JPH05119038A (en) | 1993-05-14 |
| JP3147953B2 true JP3147953B2 (en) | 2001-03-19 |
Family
ID=17942260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP30520091A Expired - Fee Related JP3147953B2 (en) | 1991-10-23 | 1991-10-23 | Biological substance measurement method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3147953B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002107350A (en) | 2000-10-03 | 2002-04-10 | Nippon Zoki Pharmaceut Co Ltd | Measuring method of 5-hydroxycreatinine |
| JP2009517677A (en) * | 2005-11-30 | 2009-04-30 | モザイク・ダイアグノステイツクス・アンド・テラピユーテイツクス・アー・ゲー | Polypeptide markers for diagnosis and evaluation of renal pelvic and ureteral junctions |
| RU2019121662A (en) * | 2016-12-16 | 2021-01-18 | Мерк Патент Гмбх | METHODS FOR USING GALECTIN-3-BINDING PROTEIN DETECTED IN URINE FOR MONITORING SEVERITY AND PROGRESSION OF LAVE JADE |
-
1991
- 1991-10-23 JP JP30520091A patent/JP3147953B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| JPH05119038A (en) | 1993-05-14 |
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