JPS58148964A - Determination of serum iron - Google Patents
Determination of serum ironInfo
- Publication number
- JPS58148964A JPS58148964A JP3154982A JP3154982A JPS58148964A JP S58148964 A JPS58148964 A JP S58148964A JP 3154982 A JP3154982 A JP 3154982A JP 3154982 A JP3154982 A JP 3154982A JP S58148964 A JPS58148964 A JP S58148964A
- Authority
- JP
- Japan
- Prior art keywords
- serum
- iron
- soln
- added
- color
- 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
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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Inorganic Chemistry (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】
本発明は血清鉄の定量方法、更に詳しくは極少量の血清
から正確且つ簡易迅速に該血清中の鉄分を定量し得る方
法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying serum iron, and more particularly to a method for accurately, simply and rapidly quantifying iron in serum from a very small amount of serum.
妊婦その他の貧血乃至貧血気味患者の健康状態を管理す
る指標として、該患者の血清鉄含量を知ることは極めて
重要である。It is extremely important to know the serum iron content of pregnant women and other patients with anemia or anemia as an indicator for managing their health status.
血液中に含まれる総鉄分の11500に相当する血清中
の鉄分、すなわち血清鉄は、アルブミンやグロプミンそ
の他の蛋白質及び糖類等の各種の化合物群からなる血清
中において、蛋白質であるトランスフェリンに3価鉄の
状態で結合して存在し、標準的には、男性の場合で80
〜200μg7dl。Serum iron, which corresponds to 11,500% of the total iron contained in blood, is composed of various compound groups such as albumin, glopmin, other proteins, and sugars, and trivalent iron is added to the protein transferrin. It exists in combination in a state of 80% in a male.
~200μg7dl.
女性の場合で70〜180μg/dl含まれている。In women, it is 70 to 180 μg/dl.
従来、かかる血清鉄の定量は、一般に次のようないわゆ
る国際法によっている。これは、所定量(2ml程度)
の血清に酸性の蛋白変性剤であるトリクロル酢酸を加え
て、該血清中の蛋白質を変性する一方で鉄分を遊離し、
次いでこれを遠心分離して除蛋白した上澄液にpH調整
用のリン酸緩衝液及び遊離した3価鉄還元用のチオグリ
コール酸を加え、3価鉄を2価鉄となし、2価鉄発色剤
であるバソフェナンスロリン(以下これをBPTと略記
する)を加えて一定のpHで発色せしめ、静置して発色
の安定を図った後に535nmの波長で吸光分析する方
法である。そしてこの場合、トリクロル酢酸に代えて例
えば塩酸グアニジンが、チオグリコール酸に代えて例え
ばアスコルビン酸が、またBPTに代えて例えばフェロ
チンが用いられること等もある。Conventionally, the determination of serum iron has generally been carried out using the following so-called international method. This is the specified amount (about 2ml)
Add trichloroacetic acid, an acidic protein denaturing agent, to the serum of the patient to denature the proteins in the serum while liberating iron,
Next, this was centrifuged to remove protein, and phosphate buffer for pH adjustment and thioglycolic acid for reducing liberated trivalent iron were added to the supernatant, converting trivalent iron to divalent iron. In this method, bathophenanthroline (hereinafter abbreviated as BPT), which is a coloring agent, is added to develop color at a constant pH, and after being allowed to stand to stabilize the color development, absorption analysis is performed at a wavelength of 535 nm. In this case, for example, guanidine hydrochloride may be used instead of trichloroacetic acid, for example ascorbic acid may be used instead of thioglycolic acid, and ferrotin may be used instead of BPT.
しかし、この種の従来法には、定量方法の性質上、検体
である血清を21程度必喪とするためそれだけ患者に採
血の苦痛を強いることとなり、また第一段階で除蛋白し
たものを第二段階で発色させて定量するといういわば二
段階操作のため作業が煩わしく面倒で且つ時間を要する
欠点がある。However, due to the nature of the quantitative method, this type of conventional method requires about 210 ml of serum as a specimen, which forces the patient to suffer from blood collection. Since it is a so-called two-step operation in which color is developed and quantified in two steps, the work is troublesome, troublesome, and time-consuming.
本発明は、斜上の従来欠点を解消する改善された血清鉄
の定量方法を提供するもので、その目的は、極少量の血
清からいわば一段階操作で該血清中の鉄分を定量し得る
ようにすることにより、患者に与える採血の苦痛を著る
しく軽減し、併せて定量操作を簡易迅速にする点にある
。The present invention provides an improved method for quantifying serum iron that overcomes the conventional drawbacks of sloping.The purpose of the present invention is to enable the determination of iron in serum using a very small amount of serum in a so-called one-step operation. By doing so, it is possible to significantly reduce the pain of blood collection for the patient, and also to simplify and speed up the quantitative operation.
以下、本発明の構成を詳細に説明する。Hereinafter, the configuration of the present invention will be explained in detail.
本発明者等は、前述の目的に即して鋭意研究した結果、
血清中のトランスフェリンの如き蛋白質を部分変性して
結合している3価鉄を2価鉄イオンの状態に遊離させる
酸性還元剤を用い、遊離した2価鉄イオンを2価鉄発色
剤で発色せしめ、しかる後に部分変性した蛋白質を微ア
ルカリ性緩衝液を加えて溶解させることにより、そのま
ま吸光分析し得ることを見出し、本発明を完成するに至
つた0
すなわち本発明は、所定量の血清にアスコルビン酸やチ
オグリコール酸等の酸性還元剤を加え、該血清中のトラ
ンスフェリンの如き蛋白質を部分変性して、結合してい
る3価鉄を2価鉄イオンに還元しつつ遊離し、次いでこ
れをBPTやフェロチン等の2価鉄発色剤により発色さ
せ、更に部分変性した蛋白質が再溶解して全体が透明と
なるようにリン酸緩衝液やトリス緩衝液等を加えてpH
調整し、要すれば発色の安定を得るべく数分間静置した
後、吸光分析するものである。As a result of intensive research in accordance with the above-mentioned purpose, the present inventors found that
Using an acidic reducing agent that partially denatures proteins such as transferrin in serum and liberates bound trivalent iron into divalent iron ions, the liberated divalent iron ions are colored with a divalent iron coloring agent. They then discovered that by adding a slightly alkaline buffer to dissolve the partially denatured protein, it was possible to perform absorption analysis as it was, leading to the completion of the present invention. By adding an acidic reducing agent such as or thioglycolic acid, proteins such as transferrin in the serum are partially denatured, and the bound trivalent iron is reduced and released to divalent iron ions, which are then treated with BPT or thioglycolic acid. Color is developed using a divalent iron coloring agent such as ferrotin, and then the pH is adjusted by adding phosphate buffer, Tris buffer, etc. so that the partially denatured protein is redissolved and the whole becomes transparent.
After adjustment and, if necessary, allowing the mixture to stand for several minutes to stabilize the color development, absorption analysis is performed.
従来一般に行われている前述の国際法によれば、血清か
ら除蛋白した上澄液を発色させるため、遠心分離による
除蛋白段階で検体の移し換え作業が避けられず、いわば
二段階操作とならざるを得ないため、作業が煩わしく面
倒で且つ時間を要するのであるが、本発明によれば、血
清から除蛋白するわけではなく、したがって検体の前記
のような移し換え作業も必要でなく、そのままの状態で
発色させて吸光分析するいわば一段階操作でよいため、
定量操作が簡易迅速になし得る。そして、かかる定量操
作の性質上、前述の国際法によれば21程度必要である
血清が、本発明によれば、100μjあれば充分である
。According to the above-mentioned international method, which has been generally practiced in the past, in order to develop color in the supernatant after protein removal from serum, sample transfer work is unavoidable during the protein removal step by centrifugation, resulting in a so-called two-step operation. However, according to the present invention, the protein is not removed from the serum, and therefore there is no need for the above-mentioned transfer of the sample, and the work can be done as it is. It is a one-step operation in which the color is developed and the absorption is analyzed.
Quantitative operations can be performed easily and quickly. Further, due to the nature of such quantitative operation, according to the above-mentioned international law, approximately 21 μj of serum is required, but according to the present invention, 100 μj is sufficient.
第1図は、本発明による場合の血清鉄濃度(μg/di
’)と535nmにおける吸光度との関係を例示する
グラフである。この場合、血清鉄濃度が既知の血清に意
識的に鉄分を添加したものを対象とし、該血清100μ
lに、3俤アスコルビン酸200μ110.06チBP
T300μ11及び05モルリン酸緩衝液(pH7,8
) 900μlを各々前述の本発明にしたがって用い、
5分間静置して発色の安定を図ったものを、前記BPT
に代えて同量の蒸留水を用いたものをブランクとして測
定した。血清鉄濃度とその吸光度との関係は図面に例示
する通り直線である。Figure 1 shows the serum iron concentration (μg/di) according to the present invention.
It is a graph illustrating the relationship between the absorbance at 535 nm and the absorbance at 535 nm. In this case, the target serum is one in which iron has been intentionally added to serum with a known serum iron concentration, and 100μ of the serum is
l, 3 tbsp ascorbic acid 200 μl 110.06 tBP
T300μ11 and 05 molar phosphate buffer (pH 7,8
) 900 μl each according to the invention described above,
After leaving it for 5 minutes to stabilize the color development, the BPT
The blank was measured using the same amount of distilled water instead. The relationship between serum iron concentration and its absorbance is a straight line as illustrated in the drawing.
第2図は、前記第1図の関係を利用した本発明による場
合の血清鉄濃度(μg/dl、以下これをXで表示する
)と前述の国際法による場合の血清鉄濃度(μg/dl
、以下これをyで表示する)との関係を相対的に比較し
て例示するグラフである。この場合、血清に意識的に鉄
分を添加したもの等を含んで36個の検体を対象とし、
Xは前記第1図の場合と同様に測定した。Xとyとの相
関係数は0.999であり、また回帰式は)’ = 1
.007 x −0,18が得られ、第2図からも、本
発明によって正確に血清鉄を定量し得ることが明らかで
ある。Figure 2 shows the serum iron concentration (μg/dl, hereinafter expressed as
, hereinafter referred to as y). In this case, 36 samples were used, including serum with iron added intentionally.
X was measured in the same manner as in the case of FIG. 1 above. The correlation coefficient between X and y is 0.999, and the regression equation is )' = 1
.. 007 x -0.18 was obtained, and it is clear from FIG. 2 that serum iron can be accurately determined by the present invention.
以上説明した通り、本発明による血清鉄定量法を用いれ
ば、極少量の血清からいわば一段階操作で該血清中の鉄
分を定量することができ、患者に与える採血の苦痛を著
るしく軽減し、併せて定量操作を正確且つ簡易迅速にす
ることができる特長をもつ。As explained above, by using the method for quantifying serum iron according to the present invention, the iron content in serum can be determined in a so-called one-step operation from a very small amount of serum, and the pain of blood sampling for patients can be significantly reduced. In addition, it has the advantage of making quantitative operations accurate, simple, and quick.
・実施例1
血清lOOμtに3%アスコルビン酸200μjを加え
て振とうし、次いで0.06%BPT300μ/を加え
て再び振とうし、更に0.5モルリン酸緩衝液(pH7
,8)900μjを加えて均一化した後、25℃で5分
間静置し、前記BPTに代えて蒸留水300μlを加え
たものをブランクとして、波長535nmで吸光度を測
定した。予め求めておいた検量線から得られる血清鉄濃
度は、国際法と同一結果であり、所望通りであった。・Example 1 200 μj of 3% ascorbic acid was added to 10 μt of serum and shaken, then 300 μj of 0.06% BPT was added and shaken again, and further 0.5 molar phosphate buffer (pH 7) was added.
, 8) After adding 900 μj and homogenizing it, it was allowed to stand at 25° C. for 5 minutes, and the absorbance was measured at a wavelength of 535 nm using a blank prepared by adding 300 μl of distilled water instead of the BPT. The serum iron concentration obtained from the calibration curve determined in advance was the same result as the international method and was as desired.
・実施例2
実施例1と同じ使用割合となるように、予め用意シてお
いだアスコルビン酸とBPTとの混合溶液を用い、その
他は実施例1と同様にした。結果は実施例1と同じであ
った。- Example 2 A mixed solution of ascorbic acid and BPT prepared in advance was used in the same ratio as in Example 1, and the other conditions were the same as in Example 1. The results were the same as in Example 1.
・実施例3
実施例1と同じ使用割合となるように、予め用意してお
いたBPTとリン酸緩衝液との混合溶液を用い、その□
他は実施例1と同様にした。結果は実施例1と同じであ
った。・Example 3 Using a mixed solution of BPT and phosphate buffer prepared in advance so that the usage ratio is the same as in Example 1, the □
The rest was the same as in Example 1. The results were the same as in Example 1.
第1図は本発明による場合の血清鉄濃度(μg/dl
)と波長535nmにおける吸光度との関係を例示する
グラフ、第2図は本発明による場合の血清鉄濃度(μg
/di)と従来の国際法による場合の血清鉄濃度(μg
/di)との関係を相対的に比較して例示するグラフで
ある。
第1図
11番It C/’1/d )Figure 1 shows the serum iron concentration (μg/dl) according to the present invention.
) and the absorbance at a wavelength of 535 nm, and FIG. 2 is a graph illustrating the relationship between the serum iron concentration (μg
/di) and serum iron concentration (μg
/di) is a graph illustrating a relative comparison. Figure 1 No. 11 It C/'1/d)
Claims (1)
を2価鉄イオンとして遊離させ、次いでこれを2価鉄発
色剤により発色させ、更に全体が透明溶液となるように
緩衝液を加えてpH調整したものを吸光分析する血清鉄
の定量方法。1 Add an acidic reducing agent to a predetermined amount of serum to liberate the iron in the serum as divalent iron ions, then color it with a divalent iron coloring agent, and add a buffer solution so that the entire solution becomes transparent. In addition, a method for quantifying serum iron that involves absorbance analysis after adjusting the pH.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3154982A JPS58148964A (en) | 1982-02-27 | 1982-02-27 | Determination of serum iron |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3154982A JPS58148964A (en) | 1982-02-27 | 1982-02-27 | Determination of serum iron |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS58148964A true JPS58148964A (en) | 1983-09-05 |
Family
ID=12334264
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP3154982A Pending JPS58148964A (en) | 1982-02-27 | 1982-02-27 | Determination of serum iron |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS58148964A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0343592A2 (en) * | 1988-05-26 | 1989-11-29 | Roche Diagnostics GmbH | Method and reagent for the determination of iron |
EP0599474A2 (en) * | 1992-11-19 | 1994-06-01 | Halliburton Company | Controlling iron in aqueous well fracturing fluids |
JP2014514575A (en) * | 2011-04-27 | 2014-06-19 | ナルコ カンパニー | System parameter determination method and apparatus for reducing corrosion of crude equipment |
-
1982
- 1982-02-27 JP JP3154982A patent/JPS58148964A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0343592A2 (en) * | 1988-05-26 | 1989-11-29 | Roche Diagnostics GmbH | Method and reagent for the determination of iron |
EP0599474A2 (en) * | 1992-11-19 | 1994-06-01 | Halliburton Company | Controlling iron in aqueous well fracturing fluids |
EP0599474A3 (en) * | 1992-11-19 | 1994-11-02 | Halliburton Co | Controlling iron in aqueous well fracturing fluids. |
US9453798B2 (en) | 2010-12-01 | 2016-09-27 | Nalco Company | Method for determination of system parameters for reducing crude unit corrosion |
JP2014514575A (en) * | 2011-04-27 | 2014-06-19 | ナルコ カンパニー | System parameter determination method and apparatus for reducing corrosion of crude equipment |
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