JP2948649B2 - Biological substance measurement method - Google Patents
Biological substance measurement methodInfo
- Publication number
- JP2948649B2 JP2948649B2 JP28965890A JP28965890A JP2948649B2 JP 2948649 B2 JP2948649 B2 JP 2948649B2 JP 28965890 A JP28965890 A JP 28965890A JP 28965890 A JP28965890 A JP 28965890A JP 2948649 B2 JP2948649 B2 JP 2948649B2
- Authority
- JP
- Japan
- Prior art keywords
- hydroxycreatinine
- methylguanidine
- renal
- blood
- 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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Description
【発明の詳細な説明】 （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。Description: TECHNICAL FIELD The present invention relates to a method for quantifying 5-hydroxycreatinine, which comprises measuring 5-hydroxycreatinine by converting it to methylguanidine.
（従来の技術） メチルグアニジ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。(Prior Art) Methylguanididine is known as one of the major urinary toxins that accumulate in the blood of patients with renal failure, and is considered to be produced from creatinine by active oxygen.
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昴進が認められる
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、尿毒症等各種腎臓障害の早期診断を可能と
したものである。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 present in the blood of healthy subjects, but is specifically found in the blood of patients with renal impairment, and the production of creatinine is confirmed to progress with the progression of the disease state. Was found to be a precursor of methylguanidine when methylguanidine is produced. The present invention, based on these facts, enables early diagnosis of various kidney disorders such as renal failure and uremic disease by measuring 5-hydroxycreatinine in blood or urine.
（発明が解決しようとする問題点） 本発明の目的は、腎臓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。(Problems to be Solved by the Invention) An object of the present invention is to provide a useful method for testing kidney disorders.
An object of the present invention is to provide a method for determining hydroxycreatinine.
（問題点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。(Means for Solving the Problems) The present invention is a method for quantifying 5-hydroxycreatinine, which comprises measuring 5-hydroxycreatinine by converting it to methylguanidine.
グアニジノ化合物の測定法としては、9,10−フェナン
スレンキノン（PQ試薬）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（低感度であるクレアチニンのさらに５
分の１に過ぎない）、微量分析が容易でない。As a method for measuring a guanidino compound, fluorescence analysis using 9,10-phenanthrenequinone (PQ reagent) is widely used. Although 5-hydroxycreatinine has a guanidino skeleton in the molecule, its reactivity with these reagents for fluorescence analysis is extremely low (5% less than creatinine, which has low sensitivity).
Trace analysis).
そこで、５−ヒドロキシクレアチニンは加水分解によ
ってメチルグアニジンとなることから、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。Therefore, since 5-hydroxycreatinine is converted into methylguanidine by hydrolysis, the present inventors have found a quantitative method of the present invention in which 5-hydroxycreatinine is converted to methylguanidine for measurement.
以下に本発明測定法について詳細に説明する。 Hereinafter, the measurement method of the present invention will be described in detail.
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。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. In addition, 5
A method of converting hydroxycreatinine into methylguanidine can also be used.
上記メチルグアニジン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、腎臓障害の病態の進行に伴い産生
昴進が認められる５−ヒドロキシクレアチニン及びメチ
ルグアニジンを両方を合わせて測定することも、腎臓障
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。Examples of the conversion treatment of methylguanidine include a method of fractionation using a cation exchange resin or the like, a method of separating 5-hydroxycreatinine by high performance liquid chromatography (HPLC) using the resin, and the like, followed by a hydrolysis treatment. 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 may be separately determined and corrected by subtracting this amount from the total methylguanidine amount. Measuring both 5-hydroxycreatinine and methylguanidine, where progress is observed, is also useful as a diagnosis of kidney damage. 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.
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、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
ルグアニジンを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 commonly used methylguanidine such as a fluorescence analysis method of reacting with a fluorescent reagent such as a PQ reagent and an enzymatic method using methylguanidine amidinohydrolase. The measurement method is available. For example, in the method using a PQ reagent, the PQ reagent is alkaline and reacts with a guanidino compound, so that 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.
（実施例） （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、
上清を凍結し測定時まで保存した。又、尿は24時間尿を
採取し、尿量を記録後、凍結し測定時まで保存した。上
記検体をトリクロロ酢酸により除蛋白処理した後以下の
測定法を行った。(Examples) (1) Method for preparing serum and urine Serum was collected on an empty stomach in the early morning and centrifuged at 4 ° C.
The supernatant was frozen and stored until measurement. Urine was collected for 24 hours, and the amount of urine was recorded, frozen, and stored until measurement. After the above sample was deproteinized with trichloroacetic acid, the following measurement method was performed.
（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。〔Journal of
Chromatography,162（1979）327−340〕 カラムは陽イオン交換樹脂（TOSO SCX、SHIMAZUISC−
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〕 （３）蛍光反応試薬 ポストカラムにおける蛍光反応試薬としては、2M水酸
化ナトリウム水溶液及びPQ試薬溶液（１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。(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. [Journal of
Chromatography, 162 (1979) 327-340] The column is a cation exchange resin (TOSO SCX, SHIMAZUISC-
07 / S1504) and the composition of the eluent was in accordance with the method of Higashidate et al. [Analytical Chemistry, 33 (1984) 366-370] (3) Fluorescent reaction reagent As a fluorescent reaction reagent in the post column, a 2M sodium hydroxide aqueous solution and a PQ reagent solution (prepared by dissolving 500 mg of PQ in 1 dimethylformamide) ) Was used.
（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0.5mm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495nmに設定し
た。(4) Measurement system for 5-hydroxycreatinine Fig. 1 shows a flow path configuration diagram of a quantitative analysis system for measuring 5-hydroxycreatinine by converting it to methylguanidine. By using a stainless steel tube having a length of 5 m and an inner diameter of 0.5 mm for the first reaction coil and setting the reactor at 125 ° C., the color development in the quantitative analysis system was maximized. The excitation wavelength of the fluorescence detector was set to 340 nm, and the fluorescence wavelength was set to 495 nm.
（作用） ５−ヒドロキシクレアチニンのPQ試薬に対する反応性
は極めて低いため、高感度で測定するのは困難であった
が、上記実施例の方法に従ってメチルグアニジンに変換
した後にPQ試薬と反応することにより、メチルグアニジ
ンの感度の約２分の１を確保できることが明らかになっ
た。(Action) Since the reactivity of 5-hydroxycreatinine with respect to the PQ reagent was extremely low, it was difficult to measure it with high sensitivity. It was found that about half of the sensitivity of methylguanidine could be secured.
この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。本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とを確認できた。This 5-hydroxycreatinine quantitative analysis system had excellent reproducibility and was sufficiently applicable to analysis of actual biological samples. As a result of examining the quantitative property of this method, it was confirmed that a straight line passing through the origin up to 1.5 nmol was obtained.
（効果） 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キレアチ
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。(Effects) The values of creatinine, methylguanidine, and 5-hydroxycreatinine in blood and urine of patients with non-diabetic renal diseases such as diabetic nephropathy, chronic nephritis, lupus nephritis, and renal sclerosis were measured. As a result, a positive correlation was found between the serum values of these three substances.
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。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 value shows a strong correlation with the serum creatinine value.
Hydroxycreatinine assay was shown to be useful for the diagnosis of renal dysfunction.
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、早期治療への道を可能とするものである。Methylguanidine has a serum creatinine level of about 2 mg /
5-hydroxycreatinine is detected even when the creatinine level is lower than 2 mg / dl, whereas 5-hydroxycreatinine is detected if it is not more than dl, enabling early detection and early treatment of early-stage renal disease with mild symptoms It is assumed that.
腎機能が正常であれば５−ヒドロキシクレアチニンは
尿に速やかに排泄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。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 the 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. Further, as the renal dysfunction becomes severe, the total amount of 5-hydroxycreatinine, which is a urinary toxin and a precursor of methylguanidine, increases, and accordingly, the amount of 5-hydroxycreatinine in body fluids and urine increases. Therefore,
Measurement of the 5-hydroxycreatinine level in body fluids such as blood and urine is very important in diagnosing renal dysfunction.
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、
蛍光反応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、尿毒症、慢性腎炎、尿路閉塞等の各種腎臓障害の早
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 and measured,
This enables trace analysis of 5-hydroxycreatinine, which has extremely low reactivity with a fluorescent reagent. In particular, measuring the 5-hydroxycreatinine level in blood is useful for early diagnosis of renal dysfunction, and the measurement method of the present invention is used for early diagnosis of various renal disorders such as renal failure, uremic disease, chronic nephritis, and urinary tract obstruction It is highly useful as a possible test method.
（１）第１図は５−ヒドロキシクレアチニンの定量分析
システムの流路構成図である。 （２）第２図は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。(1) FIG. 1 is a flow diagram of a 5-hydroxycreatinine quantitative analysis system. (2) FIG. 2 shows the results of measuring the serum creatinine value and serum 5-hydroxycreatinine value in diabetic nephropathy and examining the correlation.
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 ＥＸＰＥＲＩＥＮＴＩＡ，ＶＯＬ. 46，ＮＯ．５，（15 ＭＡＹ 1990）, Ｐ．470〜472 (58)調査した分野(Int.Cl.6，ＤＢ名) G01N 33/70 ──────────────────────────────────────────────────続 き Continuation of front page (56) References EXPERIENTIA, VOL. 46, NO. 5, (15 May 1990), p. 470-472 (58) Field surveyed (Int. Cl. 6 , DB name) G01N 33/70
Claims (1)
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 quantifying 5-hydroxycreatinine, which comprises measuring 5-hydroxycreatinine by converting it to methylguanidine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28965890A JP2948649B2 (en) | 1990-10-26 | 1990-10-26 | Biological substance measurement method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28965890A JP2948649B2 (en) | 1990-10-26 | 1990-10-26 | Biological substance measurement method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04161854A JPH04161854A (en) | 1992-06-05 |
| JP2948649B2 true JP2948649B2 (en) | 1999-09-13 |
Family
ID=17746080
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28965890A Expired - Fee Related JP2948649B2 (en) | 1990-10-26 | 1990-10-26 | Biological substance measurement method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2002107350A (en) | 2000-10-03 | 2002-04-10 | Nippon Zoki Pharmaceut Co Ltd | Measuring method of 5-hydroxycreatinine |
| WO2007063090A1 (en) * | 2005-11-30 | 2007-06-07 | Mosaiques Diagnostics And Therapeutics Ag | Polypeptide marker for the diagnosis and evaluation of ureteropelvic junction |
-
1990
- 1990-10-26 JP JP28965890A patent/JP2948649B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| ＥＸＰＥＲＩＥＮＴＩＡ，ＶＯＬ．４６，ＮＯ．５，（１５ ＭＡＹ １９９０），Ｐ．４７０〜４７２ |
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