JPS58148964A - Determination of serum iron - Google Patents

Abstract

PURPOSE:To determine serum iron quickly and accurately by adding an acidic reducing agent to a prescribed amt. of serum to liberate the iron in the serum as Fe<++>, allowing the same to color with an Fe<++> color former, further adding a buffer soln. thereto to control pH so as to make the entire part into a transparent soln. then determining the serum iron by absorptiometry. CONSTITUTION:An aq. soln. of 3% ascorbic acid or thioglycolic acid is added to a prescribed amt. (100mul) of serum to liberate the iron bound with transferrin, etc. in the state of Fe<3+> in the serum by reducing the same to Fe<++>. A color former for Fe<++> such as basophenanthroline or the like is added thereto under shaking, and further a phosphoric acid buffer soln. is added thereto to dissolve protein and to make the entire part into a transparent soln. The absorbancy at 535nm wavelength of said soln. is determined. The concn. of the serum iron is known from a predetermined calibration curve. The iron in the serum is thus determined accurately, easily and quickly at the amt. of the serum to be used about 1/20 that in the prior art without the need for laborious work for centrifugal sepn., etc. for the purpose of protein removal as in the prior art.

Description

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.

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.

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.

In women, it is 70 to 180 μg/dl.

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.

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.

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.

Hereinafter, the configuration of the present invention will be explained in detail.

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.

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.

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.

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.

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.

・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.

- 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.

・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.

[Brief explanation of the drawing]

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)

[Claims] 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.