JPH0653078B2 - Method for measuring inorganic phosphorus - Google Patents
Method for measuring inorganic phosphorusInfo
- Publication number
- JPH0653078B2 JPH0653078B2 JP61292192A JP29219286A JPH0653078B2 JP H0653078 B2 JPH0653078 B2 JP H0653078B2 JP 61292192 A JP61292192 A JP 61292192A JP 29219286 A JP29219286 A JP 29219286A JP H0653078 B2 JPH0653078 B2 JP H0653078B2
- Authority
- JP
- Japan
- Prior art keywords
- inorganic phosphorus
- nad
- measuring
- fructose
- dehydrogenase
- 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
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、ビリルビン、アスコルビン酸、ヘモグロビン
等の影響を回避することができ、かつ自動分析器に容易
に適用可能な無機リンの測定法に関する。TECHNICAL FIELD The present invention relates to a method for measuring inorganic phosphorus which can avoid the effects of bilirubin, ascorbic acid, hemoglobin and the like and can be easily applied to an automatic analyzer. .
（従来の技術） 従来、無機リン（Ｐｉ）の測定によく用いられている方
法としては、無機リン（Ｐｉ）でリンモリブデン酸を還
元しモリブデン青として測定するリンモリブデン酸法が
知られている。この方法は還元性物質の影響を強く受
け、しかも硫酸等の強い酸を使うため機器を傷めてしま
い、よい方法とは言えない。(Prior Art) Conventionally, a phosphomolybdic acid method in which phosphomolybdic acid is reduced with inorganic phosphorus (Pi) and measured as molybdenum blue is known as a method often used for measuring inorganic phosphorus (Pi). . This method is not a good method because it is strongly affected by reducing substances, and since strong acids such as sulfuric acid are used, the equipment is damaged.
それに代わって、いくつかの酵素的測定法が開発されて
いる。例えば、ホスホリラーゼａを用い、具体的には次
に示すよう方法が知られている。Instead, several enzymatic assays have been developed. For example, a method using phosphorylase a is specifically known as shown below.
この方法は、グリコーゲンの分子量が一定でなく管理し
にくい欠点がある。 This method has a drawback that the molecular weight of glycogen is not constant and is difficult to control.
また、グリセリンアルデヒド−３−ホスフェートデヒド
ロゲナーゼの反応を用いるものも知られている。しか
し、この方法はあまり使われていない。Also known is one that uses the reaction of glycerinaldehyde-3-phosphate dehydrogenase. However, this method is rarely used.
最近よく使われる方法に、イノシンとプリンヌクレオシ
ドホスホリラーゼを加え、加リン酸分解によって生成す
るヒポキサンチンをキサンチンオキシダーゼにより酸化
し、生成するＨ２Ｏ２をペルオキシダーゼ発色系に共役
させて比色する方法がある。これは非常に簡便であるも
のの、生体中のビリルビン、アスコルビン酸、ヘモグロ
ビン等の還元性物質の影響を受け、また乳びの影響も受
けるという欠点がある。In addition to inosine and purine nucleoside phosphorylase, hypoxanthine produced by phosphorolysis is oxidized by xanthine oxidase, and the produced H 2 O 2 is coupled to a peroxidase coloring system to perform colorimetry. is there. Although this is very simple, it has a drawback that it is affected by reducing substances such as bilirubin, ascorbic acid, and hemoglobin in the living body, and is also affected by chyle.
又、ごく最近では、ヌクレオシドとヌクレオシド−ホス
ホリラーゼの存在下で無機リン（Ｐｉ）を反応させて生
じた塩素またはリボース−１−ホスフェートを定量する
ことによって無機リン（Ｐｉ）を測定する方法が、特開
昭６０−１６０８９８号公報に開示されている。In addition, most recently, a method of measuring inorganic phosphorus (Pi) by quantifying chlorine or ribose-1-phosphate generated by reacting nucleoside and inorganic phosphorus (Pi) in the presence of nucleoside-phosphorylase has been particularly characterized. It is disclosed in Japanese Unexamined Patent Publication No. 60-160898.
（発明が解決しようとする問題点） 本発明者らは、上記した従来方法を改良すべく研究を重
ねた結果、ビリルビン、アスコルビン酸、ヘモグロビン
等の影響を回避し、かつ自動分析器に容易に適用可能な
無機リンの測定法を発明したものである。(Problems to be Solved by the Invention) As a result of repeated studies to improve the above-mentioned conventional method, the present inventors have avoided the effects of bilirubin, ascorbic acid, hemoglobin, and the like, and have an automatic analyzer easily. It is an invention of an applicable method for measuring inorganic phosphorus.
（問題を解決するための手段） 本発明の要旨は、シュクロースホスホリラーゼを介在さ
せてシュクロースと無機リンとを反応させてα−グルコ
ース−１−リン酸とフラクトースを生成させ、この生成
したフラクトースを測定することによって無機リンを測
定することを特徴とする無機リンの測定法に存する。(Means for Solving the Problem) The gist of the present invention is to react sucrose with inorganic phosphorus via sucrose phosphorylase to produce α-glucose-1-phosphate and fructose, and the produced fructose. It exists in the measuring method of inorganic phosphorus characterized by measuring inorganic phosphorus by measuring.
さらに、具体的に言えば、該生成したフラクトースに脱
水素酵素を反応させ、ＮＡＤ（Ｐ）からＮＡＤ（Ｐ）Ｈ
を、又はＮＡＤ（Ｐ）ＨからＮＡＤ（Ｐ）を生成させる
ことによって無機リンを測定するものである。Furthermore, specifically speaking, the produced fructose is reacted with a dehydrogenase to convert NAD (P) to NAD (P) H.
Or by producing NAD (P) from NAD (P) H.
本発明で用いられる脱水素酵素としては、マンニットデ
ヒドロゲナーゼ、フラクトースデヒドロゲナーゼ、ソル
ビトールデヒドロゲナーゼ、D-Iditol dehydrogenase、
ガラシトールデヒドロゲナーゼ等が用いられる。The dehydrogenase used in the present invention, mannitol dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, D-Iditol dehydrogenase,
Galacitol dehydrogenase or the like is used.
シュクロースホスホリラーゼを用いてシュクロースと無
機リンとを反応させるとき、フラクトースとグルコース
−１−リン酸を生じる。この方法で無機リンを測ろうと
する際、グルコース−１−リン酸を測定する系とフラク
トースを測定する系とが考えられる。前者については、
本特許出願人がすでに特開昭６１−１９１２７７号とし
て提案してある。When sucrose is reacted with inorganic phosphorus using sucrose phosphorylase, fructose and glucose-1-phosphate are produced. When measuring inorganic phosphorus by this method, a system for measuring glucose-1-phosphate and a system for measuring fructose are considered. For the former,
The applicant of the present patent has already proposed it as JP-A-61-191277.
本発明は、さらに簡便に測定できるようにフラクトース
をもって無機リンを測定する系を提案せんとするもので
ある。The present invention proposes a system for measuring inorganic phosphorus with fructose so that it can be measured more easily.
本発明方法をもう少し具体的に示すと、 フラクトースに脱水素酵素の存在下で、ＮＡＤ（Ｐ）Ｈ
又はＮＡＤ（Ｐ）を反応させると、ＮＡＤ（Ｐ）Ｈから
ＮＡＤ（Ｐ）を、ＮＡＤ（Ｐ）からＮＡＤ（Ｐ）Ｈを生
成する。More specifically, the method of the present invention will be described. In the presence of dehydrogenase on fructose, NAD (P) H
Alternatively, when NAD (P) is reacted, NAD (P) H produces NAD (P) and NAD (P) produces NAD (P) H.
本発明方法を実施例するにあたっては、血清又は尿中の
ビリルビンやヘモグロビン等の影響を避けるために初速
度法が好ましいが、勿論無機リン（Ｐｉ）を消費させる
終末法でも可能である。ここで、ＮＡＤはニコチンアミ
ドアデニンジヌクレオチド、ＮＡＤＰはニコチンアミド
アデニンジヌクレオチドホスフェートである。In carrying out the method of the present invention, the initial rate method is preferable to avoid the influence of bilirubin or hemoglobin in serum or urine, but it is also possible to use the terminal method of consuming inorganic phosphorus (Pi). Here, NAD is nicotinamide adenine dinucleotide and NADP is nicotinamide adenine dinucleotide phosphate.
緩衝液としては、無機リンが混入しないものを用いるこ
とは当然で、トリス，トリス・マレイン酸、トリス・酢
酸や、ＰＩＰＥＳ 〔Ｐｉｐｅｒａｚｉｎｅ−Ｎ，Ｎ’
−ｂｉｓ （２−ｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃ
ｉｄ）〕，ＭＥＳ〔２−（Ｎ−Ｍｏｒｐｈｏｌｉｎｏ）
ｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃｉｄ，ｍｏｎｏｈ
ｙｄｒａｔｅ〕，ＢＥＳ〔Ｎ，Ｎ−Ｂｉｓ （２−ｈｙ
ｄｒｏｘｙｅｔｈｙｌ）−２−ａｍｉｎｏｅｔ ｈａｎ
ｅｓｕｌｆｏｎｉｃ ａｃｉｄ〕，ＨＥＰＥＳ（Ｎ−２
−Ｈｙｄｒｏｘｙｅｔｈｙｌ ｐｉｐｅｒａｚｉｎｅ−
Ｎ’−２−ｅｔｈａｎｅｓｕｌｆｏｎｉｃ ａｃｉ
ｄ），ＴＥＳ〔Ｎ−Ｔｒｉｓ（ｈｙｄｒｏｘｙｍｅｔｈ
ｙｌ）ｍｅｔｈｙｌ−２−ａｍｉｎｏｅｔｈａｎｅ ｓ
ｕｌｆｏｎｉｃ ａｃｉｄ〕等のグッドバッファーと称
されるものが用いられる。これに限らず、無機リンがふ
くまれておらずかつ緩衝能力を維持できるものであれ
ば、他の緩衝液を用いることができることはいうまでも
ない。これらの緩衝液の濃度は、２０ｍＭ〜２００ｍ
Ｍ、そしてpHは６．５〜８．０で用いることができる。As the buffer solution, it is natural to use one that is not mixed with inorganic phosphorus, such as tris, tris maleic acid, tris acetic acid, PIPES [Piperazine-N, N '
-Bis (2-ethane sulfonic ac
id)], MES [2- (N-Morpholino)
ethane sulphonic acid, monoh
hydrate], BES [N, N-Bis (2-hy
droxyethyl) -2-amino et han
esulfonic acid], HEPES (N-2
-Hydroxyethyl piperazine-
N'-2-ethanesulfonic aci
d), TES [N-Tris (hydroxymeth
yl) methyl-2-aminoethanes
ufonic acid] or the like called a good buffer is used. It goes without saying that other buffer solutions can be used as long as they do not contain inorganic phosphorus and can maintain the buffer capacity. The concentration of these buffers is 20 mM to 200 m
M, and pH can be used from 6.5 to 8.0.
ＮＡＤ（Ｐ）Ｈの濃度は、１．２〜２ｍＭ、シュクロー
スホスホリラーゼは０．１〜２Ｕ／ml、マンニットデヒ
ドロゲナーゼ又はフラクトースデヒドロゲナーゼは、１
０〜６０Ｕ／mlが適当である。The concentration of NAD (P) H is 1.2 to 2 mM, sucrose phosphorylase is 0.1 to 2 U / ml, and mannitol dehydrogenase or fructose dehydrogenase is 1
0-60 U / ml is suitable.
（実施例） 以下に、実施例をあげて本発明方法をさらに具体的に説
明する。(Examples) Hereinafter, the method of the present invention will be described more specifically with reference to Examples.
実施例１試薬組成 ピペス……………………５０ｍＭ，pH７．０ シュクロース………………………２００ｍＭ シュクロースホスホリラーゼ…０．４Ｕ／ml マンニットデヒドロゲナーゼ……３０Ｕ／ml ＮＡＤＨ………………………………１．８ｍＭ 上記組成の試薬２．５mlを予め加温し、検体量５０μｌ
を添加した後、３４０nmにおいて、ΔＥ／minを測定し
た。その結果、無機リン（Ｐｉ）５０mg／dl以下で原点
を通る直線性を示した。Example 1 Reagent composition Pipettes ...... 50 mM, pH 7.0 Sucrose …………………… 200 mM Sucrose phosphorylase… 0.4 U / ml Mannit dehydrogenase… 30 U / ml NADH… ……………………………… 1.8 mM Preheat 2.5 ml of the above-mentioned reagent to give a sample volume of 50 μl.
Was added, the ΔE / min was measured at 340 nm. As a result, linearity passing through the origin was shown when the inorganic phosphorus (Pi) was 50 mg / dl or less.
また、検体について、従来法（フィスクサバーロー法）
との相関を求めた結果、良好な相関をえた。ｒ＝０．９
８、ｙ＝１．０３ｘ−０．２（ｘ：フィスクサバロー
法、ｙ：本発明）と良好な相関が得られた。In addition, the conventional method (Fisks-Barlow method) for specimens
As a result of obtaining the correlation with, a good correlation was obtained. r = 0.9
8, good correlation was obtained with y = 1.03x−0.2 (x: Fisk-Sabarrow method, y: present invention).
Claims (2)
シュクロースと無機リンとを反応させてα−グルコース
−１−リン酸とフラクトースを生成させ、この生成した
フラクトースを測定することによって無機リンを測定す
ることを特徴とする無機リンの測定法。1. A sucrose phosphorylase-mediated reaction between sucrose and inorganic phosphorus to produce α-glucose-1-phosphate and fructose, and the resulting fructose is measured to measure inorganic phosphorus. A method for measuring inorganic phosphorus, which is characterized in that
応させ、ＮＡＤ（Ｐ）からＮＡＤ（Ｐ）Ｈを、又はＮＡ
Ｄ（Ｐ）ＨからＮＡＤ（Ｐ）を生成させることによって
無機リンを測定することを特徴とする特許請求の範囲第
１項記載の無機リンの測定方法。2. The produced fructose is reacted with a dehydrogenase to convert NAD (P) to NAD (P) H or NA.
The method for measuring inorganic phosphorus according to claim 1, wherein the inorganic phosphorus is measured by producing NAD (P) from D (P) H.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61292192A JPH0653078B2 (en) | 1986-12-08 | 1986-12-08 | Method for measuring inorganic phosphorus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61292192A JPH0653078B2 (en) | 1986-12-08 | 1986-12-08 | Method for measuring inorganic phosphorus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63146800A JPS63146800A (en) | 1988-06-18 |
| JPH0653078B2 true JPH0653078B2 (en) | 1994-07-20 |
Family
ID=17778736
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61292192A Expired - Lifetime JPH0653078B2 (en) | 1986-12-08 | 1986-12-08 | Method for measuring inorganic phosphorus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0653078B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9001486B2 (en) | 2005-03-01 | 2015-04-07 | X2Y Attenuators, Llc | Internally overlapped conditioners |
| US9019679B2 (en) | 1997-04-08 | 2015-04-28 | X2Y Attenuators, Llc | Arrangement for energy conditioning |
| US9036319B2 (en) | 1997-04-08 | 2015-05-19 | X2Y Attenuators, Llc | Arrangement for energy conditioning |
| US9054094B2 (en) | 1997-04-08 | 2015-06-09 | X2Y Attenuators, Llc | Energy conditioning circuit arrangement for integrated circuit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57163494A (en) * | 1981-04-01 | 1982-10-07 | Ajinomoto Co Inc | Determination of fructose |
-
1986
- 1986-12-08 JP JP61292192A patent/JPH0653078B2/en not_active Expired - Lifetime
Non-Patent Citations (2)
| Title |
|---|
| ＡｎａｌＢｉｏｃｈｅｍ，１４２，Ｐ．５５６−５６１，１９８４ |
| ＺＬｅｂｅｎｓｍＵｎｔｅｒｓＦｏｒｓｃｈ，１７１（６），Ｐ．４４３−４４５，１９８０ |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9019679B2 (en) | 1997-04-08 | 2015-04-28 | X2Y Attenuators, Llc | Arrangement for energy conditioning |
| US9036319B2 (en) | 1997-04-08 | 2015-05-19 | X2Y Attenuators, Llc | Arrangement for energy conditioning |
| US9054094B2 (en) | 1997-04-08 | 2015-06-09 | X2Y Attenuators, Llc | Energy conditioning circuit arrangement for integrated circuit |
| US9001486B2 (en) | 2005-03-01 | 2015-04-07 | X2Y Attenuators, Llc | Internally overlapped conditioners |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63146800A (en) | 1988-06-18 |
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