JPS5950364A - Reagent for determination of iron - Google Patents

Abstract

PURPOSE:To obtain a reducing agent which is stable in a liquid state and adaptable to precision analysis, by using a specific compd. as a reducing agent for trivalent iron in colorimetric analysis of iron using a coloring reagent of bivalent iron. CONSTITUTION:Metabisulfic acid or salts thereof which can dissolve required amts. of alkali metal salt, ammonium salt and alkaline earth metal salt are used as a reducing agent. After 2.0ml buffer soln. is added to 0.5ml sample under mixing, the blind trial of the specimen is measure at the max. absorption wavelength of the coloring material used. A drop of a coloring reagent is added thereto with mixing, then the absorbancy at the max. absorption is again measured and the blind trial of the specimen is subtracted from the measured absorbancy and the iron value of the serum is calculated. The buffer soln. is prepd. by dissolving 5g sodium lauryl sulfate, 1g sodium metabisulfite, 4.15g sodium acetate in 90ml water, adjusting pH to 6.0 with hydrochloric acid and adding water to make 100ml in total. The reducing agent is stable for an extremely long period of time in a soln. state and has the substantial reducing power with which the measured values of L-ascorbic acid and thioglycolic acid coincide, while it has no malodor.

Description

[Detailed description of the invention] The present invention relates to an iron measuring device.

There are various methods for measuring iron, but they are minute: European analysis uses atomic absorption method,
Colorimetric analysis is the mainstream, and is especially advantageous in clinical chemical analysis where a large number of samples are processed. Approximately 273 of iron in living bodies exists as hemoglobin in red blood cells, and the remaining 1/3 exists as so-called stored iron in the liver, kidneys, bone marrow, etc., and the total electrical charge is approximately 4 g. Almost no iron is excreted in the urine, and only about once a day is caused by detachment of the intestinal mucosa.

Although it is lost due to the shedding of the skin, it maintains its balance by absorbing about 1cm of energy from things.Physiologically speaking, all of the wheat in the serum is a type of clopurin. Transferrin is a protein with a molecular weight of approximately 90,000 and has the ability to bind two atoms of iron. Normally, one-third of transferrin in serum exists in the form bound to iron. The remaining two-thirds of the iron is called serum iron, and the remaining two-thirds exists as transferrin that does not bind iron, and the amount is called the unsaturated iron-binding capacity.

When measuring serum iron, it is necessary to remove the chelate of transferrin heptaferron and obtain free iron. This method includes (11 adding hydrochloric acid and heating to remove protein, (2+
Color comparison is performed using a coloring agent that removes the bond with globulin at pH 3.0 or below and forms a chelate at low pH. (3j) Protein-pooring agents such as sodium lauryl deoxylate and basic guanidine are used to denature transferrin and release iron.On the other hand, as a method for measuring unsaturated iron binding capacity, excess iron is added to serum. Add the iron to bind all the transferrin and iron, precipitate the excess iron with an adsorbent such as magnesium carbonate, measure the iron concentration in the supernatant, and subtract the serum iron concentration, or use a known source of iron. The mainstream method is to measure the remaining iron combined with I-transferrin in addition to the serum, and determine the unsaturated iron binding capacity from the amount of decreased iron. Measured values can be used to differentiate various anemias such as iron deficiency anemia, aplastic anemia, pernicious anemia, chronic hemorrhagic anemia, polycythemia vera, and infectious anemia, as well as liver diseases such as acute hepatitis, diffuse hepatitis, and cirrhosis. It has important meaning.

Currently, the commonly used iron coloring agents are thiocyanate, α, α'' dipyridyl, 0-phenanthroline, bathophenanthroline (hereinafter abbreviated as B'PT), 2,4 .6 tripyridyl-S-triazine (hereinafter abbreviated as %TPTZ), 3-(2-pyridyl)-5゜6bis(4-sulfophenyl)-1,2,4
-triazine (hereinafter abbreviated as P'DT8) and recently developed 2-nitroso-5-(N-).

These include nitrosophenol derivatives represented by (ropyru-N-sulfopropylamino)phenol.

Among these coloring agents, the trivalent coloring agent for the legs is only thiocyanate, and the others are all divalent iron coloring agents.

If the vessel is used, it is necessary to use a reducing agent in combination. Known reducing agents for trivalent iron are the following 7 types (Table 1).

Table ■ Reducing agents for iron measurement Among these, L-ascorbic acid and thioglycolic acid are said to have sufficient reducing power for 4=Wm analysis, but these two reducing agents also For English use ± extremely serious flaws. In other words, L-ascorbic acid has relatively good stability in powder form, but extremely poor stability in solution form, and there is a fee to add powder to each sample when using it. In addition, thioglycolic acid is relatively stable even in a solution state in acidic conditions and has sufficient reducing power, but in neutral conditions or higher, its stability and reducing power are poor, and it takes a considerable amount of time to stabilize the color, which is unique to mercapto groups. The bad odor is also undesirable in use.

The present inventors despised these drawbacks, and as a result of intensive research into a reducing agent that is stable in solution state and has sufficient reducing power suitable for precise analysis, it was found that metasulfite or its salts satisfies these conditions. This led to the completion of the present invention.

The metasulfurous acid or its salt that can be used in the present invention is usually one that can dissolve the necessary amount among alkali metal salts, ammonium salts, and alkaline earth metal salts, but it is common sense to use sodium salts and potassium salts. It is true. Table 1 shows the stability of typical reducing agents that have been used in Takumai and the reducing agent of the present invention in a solution state. Conventionally used reducing agents lack stability, especially on the alkaline side, and the maximum period of use that can be used is a few minutes for L-ascorbic acid, 3 days for thioglycolic acid, and 3 months for hydroxylamine hydroxylamine. ,
Even after one year, the content of mekubisulfite decreases by only 3%, and its practical shelf life is well over two years when stored at room temperature. Furthermore, the stability is further improved at acidity ψ11.

Table Ill shows the results of color development using four representative coloring agents currently in use, varying the pH and H, and using one amount of each reducing agent. The three coloring agents that commonly develop color in a wide pH range are 7-ascorbic acid, thioglycolic acid, and the reducing agent of the present invention. The optimum concentration of mekumarnaceous acid or its salt varies depending on the used pI-7 and the coloring agent used, and the required amount increases as the pH increases, but it is usually in the range of 0.01% to 5.0%. It is preferable to use one serving, and an example will be shown next.

Example 1. (Measurement of serum iron) Buffer Sodium lauryl sulfate 5g Sodium metabisulfite 1g Sodium acetate 4.15g Dissolve the above in 90mt of water, adjust the pH to 6.0 with hydrochloric acid, and make a total fL of 1001M' with water.

Color development test solution A (BP'T) Dissolve 0.1 g of sodium bathophenanthrolin sulfonate in 1:1 (JTIIl) of water.

Coloring test solution B (PDTS) Dissolve 1 g of PDTSO in 20 #IA' of water.

Color reagent solution 0 (NO-PSAP) Dissolve 1 g of NO-P'S A PO in 2 parts of water (I+/).

Usage Sample Q, 5 ml of the above buffer W 2. After adding Oml and mixing, measure the sample blind at the thickest absorption wavelength of the coloring agent used, add 1 drop of coloring test solution A-0, and then mix.
After standing for 10 minutes, the absorbance at the maximum absorption wavelength is measured again, and the serum iron concentration is calculated by subtracting the sample blindness.

Table Iv compares the measured values of Kenpo with the values obtained when using conventionally used reducing agents such as ascorbic acid and thioglycolic acid. There is no significant difference between this and conventional reducing agents.

Example 2. (Measurement of unsaturated iron binding ability) Iron-containing buffer trishydroxylamine 1.21g citric acid
0.221g potassium metabisulfite
Dissolve 1.0 g of ferrous ammonium sulfate (100 μg/dz as F''e) in 90 cups of water, adjust the pH to 8.5 to 8.7 with hydrochloric acid, and drain to make a total volume of 100 m/dz.

Add 200 μt of serum to the above iron-containing buffer 2. '0, r
After adding nl and leaving it at room temperature for 15 minutes, the absorbance at 750° was measured. Next, coloring test solution 0 (described in Example 1) 1
After mixing, leave it at room temperature for 15 minutes and then test again at 750 nm.
Measure the absorbance of the sample and subtract the absorbance of the sample to determine the amount of iron reduction, and determine the unsaturated iron binding capacity.

Measured values in Kempo, conventional magnesium carbonate precipitation method, and using L-ascorbic acid as the reducing agent only according to Example 2 1
．． Table 3 shows the measured values when
．． 15g...Mixing...Distilled water Q, 5 ml
...Leave for 30 minutes...After centrifugation, supernatant Q, 5 ml + buffer solution (5% sodium lauryl sulfate 0.IM acetate as
Liquid p I-16,0)2. Qm/-1-L-ascorbic acid 15 ■...Mixture...No-PSAPo, 5
After leaving one drop of the % bath solution at room temperature for 5 minutes, the total iron binding capacity is determined by measuring the absorbance at 750 cm, and the serum iron value is separately measured and subtracted to determine the unsaturated iron binding capacity.

N op S A p direct method (L-ascorbic acid)
Example 2 is followed except that in place of the reducing agent potassium metabisulfite, about 10 L-ascorbic acid is added to the test tube as an IM powder.

No significant difference was observed in the measured values between the magnesium carbonate dilation method, the L-ascorbic acid reduction method, and the illegal method.

As described above, the reducing agent of the present invention is stable for an extremely long period of time in a solution state regardless of liquid properties, and has sufficient reducing power with measured values matching those of conventionally used L-ascorbic acid and thiochlorocholic acid, while also having no odor. Moreover, it is an extremely excellent product that does not cause any environmental health problems, and it makes a huge contribution to the business.

Patent applicant: Wako Pure Chemical Industries, Ltd.

Claims (1)

[Claims] A sample for measuring iron, characterized in that meta-type dish sulfuric acid or its salts are used as a reducing agent for trivalent iron in colorimetric analysis of iron using a coloring reagent for divalent iron.