US3887332A

US3887332A - Method of determining unsaturated iron binding capacity in serum

Info

Publication number: US3887332A
Application number: US348097A
Authority: US
Inventors: Kazuei Takase; Junji Morikawa
Original assignee: Eiken Chemical Co Ltd
Current assignee: Eiken Chemical Co Ltd
Priority date: 1972-04-12
Filing date: 1973-04-05
Publication date: 1975-06-03
Anticipated expiration: 1992-06-03
Also published as: JPS48104597A; JPS529160B2

Abstract

Unsaturated iron binding capacity of transferrin, a serum protein, is determined by adding an iron solution containing a known quantity of excess iron to the serum to saturate the iron binding capacity of the serum transferrin, removing the remaining surplus iron with an absorbent and making a colorimetric determination by causing iron to be released from transferrin under an acidic condition. Since the iron solution is unstable in alkaline zone, it is stabilized by forming it into nitrilotriacetic acid iron chelates. The stability of iron chelates of sodium bathophenanthroline sulfonate is greater than that of nitrilotriacetic acid. This fact is utilized, and the surplus quantity of nitrilotriacetic acid chelates remaining after the saturation of the iron binding capacity of tranferrin is measured by using sodium bathphenanthroline sulfonate. The sodium bathophenanthroline sulfonate iron chelates have maximum absorbance at the wavelength of 535 nm, and Beer''s law holds for an iron concentration range up to 600 mg/100 ml.

Description

Unite States atent [1 1 Takase et al.

[ METHOD OF DETERMINING UNSATURATED IRON BINDING CAPACITY IN SERUM [75] Inventors: Kazuei Takase; Junji Morikawa,

both of Tokyo, Japan {73] ,Assignee: Eiken Chemical Co., Ltd., Tokyo,

Japan [22] Filed: Apr. 5, 1973 [21] ,Appl. No; 348,097

OTHER PUBLICATIONS R, J. Henry, Clinical Chemistry Principles and Technics, Harper & Row, 1964, pp. 391-395 relied on, RB40.H4.

G. W. Bates, Dissert. Abstr. 27B (ll), 3816 (1967). Bates et al., Chem. Abstr. 67, 7925x (1967).

R. L. Searcy, Diagnostic Chemistry, McGraw Hill, 1969, p. 329 relied on, RB40.54.

Frankel et al, ed, Gradwohls Clinical Laboratory [451 June 3,1975

Methods and Diagnosis, C. V. Mosby C0., 1970. Vol. I, pp. 205-368, 441 relied on, RB37.G7.

Primary ExaminerRobert M. Reese Attorney, Agent, or Firm-Stevens, Davis, Miller & Mosher [5 7] ABSTRACT Unsaturated iron binding capacity of transferrin, a serum protein, is determined by adding an iron solution containing a known quantity of excess iron to the serum to saturate the iron binding capacity of the serum transferrin, removing the remaining surplus iron with an absorbent and making a colorimetric determination by causing iron to be released from transferrin under an acidic condition. Since the iron solution is unstable in alkaline zone, it is stabilized by forming it into nitrilotriacetic acid iron chelates. The stability of iron chelates of sodium bathophenanthroline sulfonate is greater than that of nitrilotriacetic acid. This fact is utilized, and the surplus quantity of nitrilotriacetic acid chelates remaining after the saturation of the iron binding capacity of tranferrin is measured by using sodium bathphenanthroline sulfonate. The sodium bathophenanthroline sulfonate iron chelates have maximum absorbance at the wavelength of 535 nm, and Beers law holds for an iron concentration range up to 600 mg/lOO ml.

4 Claims, 4 Drawing Figures Absorption Spectra of Fe(II)-BPT Complex Sample serum,

tilisoronnce will! MOO 450 Standard iron so|ution,(2.5 .|g)

Serum blank.

Wpve Length,

Reference.- water MFHTE JU? 3 "975 SHEET Mbsorpflon Spectra of Fe(II)-BPT Complex \PH: 8.6

Reference: wcl'rer \Effect of pH on serum UIBC value ll (PH) \M-Tris.HCL Buffer 'Y Buffer rgwgr-nrmmws 191s u 15% 8 87 332 Relationship of Rate of Color Development to time and Temperature.

M15 20 Standard Iron Solution,

D4 Somp|e Serym Serum B a 5 Mb 1'5 2'0 2'5 3'0 35 (min) Reference Water Fe pg/IOOml METHOD OF DETERMINING UNSATURATED IRON BINDING CAPACITY IN SERUM The present invention relates to a method of determining the unsaturated iron binding capacity in serum and, more particularly, to a method of readily and exactly determining the unsaturated iron binding capacity of the serum transferrin.

The clinical significance of blood iron was made clear by Heilmeyer (in 1937). Of the total quantity of iron contained in the adult body, 65 percent is present in red corpuscles as hemoglobin component (red corpuscle iron I), and about 30 percent is stored in liver, pancreas, marrow of bones and kidney (stored iron). While iron contained in blood {blood iron) is very slight in quantity, no greater than mere 0.1 percent (3 to 5 milligrams), the quantity of iron getting into and out of blood through this blood iron is as great as 30 milligrams or more per day. This to milligrams of iron is utilized for the production of hemoglobin in the marrow of bones, so that it may be said that the blood iron maintains the blood producing function of the marrow.

In the serum the blood iron is bound to transferrin which belongs to ,B-globulin. its normal content is 90 to I50 mg/100 ml for men and 80 to 120 mg/lOO ml for women. The serum is capable of binding iron up to 300 to 360 mg/100 ml. This total capacity is termed total iron binding capacity, and the difference between the total iron binding capacity and the actual blood iron content is termed unsaturated iron binding capacity.

The determination of the total iron binding capacity and unsaturated iron binding capacity is utilized in the classification of various amenic diseases and in the determination and diagnoses of liver diseases and acute and chronical infectious diseases.

If the determination of the total iron binding capacity and unsaturated iron binding capacity is done along with the determination of the content of the blood iron, the diagnoses of various diseases stemming from abnormal iron metabolism can be done more precisely.

Major prior-art methods of determining the unsaturated iron binding capacity are as follows;

1. Magnesium carbonate method This method consists of adding an excess iron solution, for instance a ferric chloride solution, to the serum to saturate the iron binding capacity of transferrin in the serum, subsequently removing surplus iron with magnesium carbonate, removing protein, thereafter estimating total iron binding capacity through a colorimetric process, and then subtracting the separately determined iron content value from the total iron binding capacity value, thereby determining the unsaturated iron binding capacity.

2. Peters method This method consists of adding a ferric ammonium cyanate solution to the serum to saturate the iron binding capacity of the serum transferrin, then removing the surplus iron with a cation-exchange resin, and after the removal of protein determining the unsaturated iron binding capacity in the same way as mentioned above.

3. RI method This method consists of adding a predetermined quantity of excess radioactive isotope Fe59 and iron to the serum to saturate the iron binding capacity of the serum transferrin, causing the residual surplus iron to be absorbed in a resin sponge and measuring the radioactivity of the absorbed iron.

Of the above prior-art methods, the first two involve many operation steps and are very tedious. The last method requires special equipment and uses expensive reagents, so that it is difficult to be carried out in the usual examination room.

To solve the above problems encountered in the prior-art methods, it has been proposed to directly determine the unsaturated iron binding capacity by using a tris hydrochloric acid buffer (1.0 M, pH being 8.1), a standard iron solution (prepared by using ferrous ammonium sulfate and containing 500 mg/ ml of iron and also used by adding 0.5 mg/lOO ml of ascorbic acid) and 0.5 percent of a bathophenanthroline sulfonate solution. However, the above salt of iron is unstable and forms hydroxides of iron in alkaline liquid. Therefore, it is dissolved in an acidic solution, to which an alkaline buffer is subsequently added for color. development and colorimetric determination, so that operation is involved.

An object of the present invention is to provide a method of speedily determining the unsaturated iron binding capacity of transferrin with a reduced number of operational steps.

Another object of the invention is to provide a method of determining the unsaturated iron binding capacity of transferrin with high accuracy and which enables one to obtain satisfactory reproducibility.

A further object of the invention is to provide a reagent (II)-BPT in the determination of unsaturated iron binding capacity of transferrin and one capable of perservation for a long time.

The above and other objects, features and advantages of the invention will appear more fully from the following description.

FIG. 1 is a graph showing an absorptive spectral of Fe (II)- BPT complex,

FIG. 2 is a graph showing the effect of pH on unsaturated iron binding capacity value,

FIG. 3 is a graph showing a relationship of rate of color development to time and temperature, and

FIG. 4 is a graph showing the calibrating curves representing the effect of Fe buffer solutions.

According to this invention there is provided a method of determining unsaturated iron binding capacity of transferrin by adding an iron solution containing a known quantity of excess iron to the serum to saturate the iron binding capacity of the serum transferrin and measuring the remaining surplus iron.

To carry out the above method, the problem has been studied of whether or not the number of operation steps may be reduced by adding a standard iron solution to a tris hydrochloric acid buffer. It has been found that it is best to establish the standard iron solution in the form of iron chelate compound in order to preserve the solution in the tris hydrochloric acid buffer for a long time.

As the chelating agent there may be used ethylenediamine tetraacetic acid (EDTA), bathophenanthroline sulfonate (BPT), tripyridyl-s-triadine (TPTZ) and nitrilotriacetic aicd (NTA). These substances can bind iron to form the so-called iron chelate compound, but their force of binding iron is different in the following way;

BPT EDTA NTA TPTZ It constitutes the principle underlying the invention that the unsaturated iron binding capacity of serum is determined by preparing a standard solution by dissolving an iron chelate compound in water, adding a predetermined quantity of sample serum to the standard solution and measuring the difference of absorbance before and after the addition of the serum.

More particularly, according to the invention a tris hydrochloric acid buffer containing the iron chelate compound is added to the serum to saturate the iron binding capacity of the serum transferrin, the surplus iron not bound to transferrin is removed, ascorbic acid is added, followed by the addition of bathophenanthroline sulfonate for colorimetric determination.

At this time, if the iron chelate compound is based on BPT or EDTA, the transferrin in the serum cannot derive iron from the chelates since the bond in the chelates is too strong. On the other hand, NTA and TPTZ have less strong iron binding forces than those of BPT and EDTA, so that transferrin can take up a requisite quantity of iron from the iron chelate compound based on NTA or TPTZ. TPTZ-Fe compound, however, is unstable in the tris hydrochloric acid buffer, so that it not suited for practical use. From these grounds, it has been recognized that the NTA-Fe compound is suitable for use.

Considering another aspect, the unsaturated iron binding capacity of serum differs with pH. To examine the effect of pH the unsaturated iron binding capacity has been measured of transferrin in the same serum with the pH adjusted to various values by using M-tris hydrochloric acid buffer for a pH range between 7 and 9 and M-glycine sodium hydroxide buffer for a pH range between 9 and l l. The conclusion is reached that a highly unsaturated iron binding capacity can be maintained within a pH range between 8.4 and 8.7, as will be described hereinafter in connection with FIG. 2.

EXAMPLE Preparation of reagents Nitrilotriacetic acid iron buffer:

121.14 grams of tris (hydroxymethyl) aminomethane was dissolved in 800 millilitres of distilled water, and 0.08 gram of nitrilotriacetic acid and then 0.01 17 gram of ferrous ammonium sulfate were added to and dissolved in the resultant solution. Then, the pH of the obtained solution was adjusted with 6N-hydrochloric acid. Then, distilled water was added to the resultant solution to adjust the total Volume to 1,000 milliliters. The concentration of iron in the final solution is 166.7mg/100 ml.

Nitrilotriacetic acid buffer:

It was prepared in the same way as the aforementioned and has the same composition as the aforementioned nitriloacetic acid iron buffer except for the fact that ferrous ammonium sulfate was not added.

Bathophenanthroline sulfonate solution:

It was prepared by dissolving 0.5 gram of sodium bathophenanthroline sulfonate in distilled water such that the total volume was 100 millilitres.

Reducing agent:

A small quantity of sodium ascorbate was used as the reducing agent.

The apparatus used for the preparation of the reagents and for the operation were all deironated.

Operation Four test tubes A, B, C and D were prepared. 1.5 millilitres of nitrilotriacetic acid iron bufferwas poured into each of the test tubes A, B and C, while 1.5 millilitres of nitriloacetic acid buffer was poured into the test tube D. Then, 10 milligrams of the reducing agent was poured into each test tube to produce each blend solution. Then, 0.5 millilitre of sample serum was poured into each of sample serum test tube A and serum blank test tube B, while 05 millilitre of distilled water was poured into each of standard iron solution test tube C and reagent blank test tube D. After leaving these tubes for 10 minutes, 0.05 millilitre of sodium bathophenanthroline sulfonate solution was poured into each of the tubes A, C and D, while 0.05 millilitre of distilled water was poured into the remaining tube B. After sufficiently stirring the individual resultant liquids and then leaving then at room temperature for 20 minutes, the absorbance of the samples, in the individual test tubes at the wavelength of 535 nm was measured with a spectrophotometer by using water as a reference.

By so doing, the unsaturated iron binding capacity (UIBC) of the serum can be determined from an equation A 1553:" UIBC ug/ml) 500 500 where E E E and E are values of the absorbance of the samples in the respective test tubes. Absorbance curve Sodium bathophenanthroline sulfonate iron chelates develop red color in alkaline solution, and FIG. 1 shows the resultant absorbance spectra in a wavelength range between 400 and 600 nm. Maximum absorbance was recognized at the wavelength of 535 nm. In the subsequent experiments the determination of the absorbance was made only for this wavelength. Effect of pH on unsaturated iron binding capacity value of the serum FIG. 2 shows the results of measurements of the unsaturated iron binding capacity value using M-tris hydrocloric acid buffer for a pH range between 7 and 9 and M-glycine sodium hydroxide buffer for a pH range between 9 and l 1. As is shown, a substantially constant maximum value was obtained within a pH range between 8.4 and 8.7. Relationship of rate of color development to time and temperature As shown in FIG. 3, at temperatures about room temperature (20C) the absorbance of both sample serum and standard iron solution settled to respective steady values in about 5 minutes. At temperatures of 15C the time required until the settling was extended, particularly for the sample serum is about 15 minutes. Effect of time for the action of serum on nitrilotriacetic acid iron buffer and sodium ascorbate on the unsaturated iron binding capacity value Time until the serum transferrin was saturated with iron by deriving and binding with iron from the nitrilotriacetic acid iron buffer was determined. The unsaturated iron binding capacity was settled to a constant value after the sample had been left for 5 minutes.

Relationship between absorbance and Fe concentration in nitrilotriacetic acid iron buffer A calibration curve was produced with the final Fe content adjusted to 0, 100, .200, 300, 400 and 500mg/l00 ml for each nitrilotriacetic acid iron buffer and by using reagent blank as reference. The curve obtained was straight up to 500mg/ l00 ml and passed the origin, as shown at plot 1 in FIG. 4. Also, the absorbance was measured in the same way up to the Fe contact of .500mg/100 ml in the nitrilotriacetic acid iron buffer but using a nitrilotriacetic acid buffer containing serum as reference. In this case, a straight plot parallel to plot 1 but lower than it by the absorbance value corresponding to the unsaturated iron binding capacity of the serum (in this case 220mg/100 ml) was obtained. as shown at plot 2. It is to be noted that this plot shows absorbance of 0.005 to 0.01 for Fe contents of 100 and 200mg/100 ml. This is due to the pink coloring of the iron in the transferrin. This is re-confirmed from the fact that the absorbance of plot 3 obtained in case of adding only nitrilotriacetic acid iron buffer and reducing agent but not adding sodium bathophenanthroline sulfonate to the serum increases linearly from zero Fe content to the saturation point of 220g/100 m1, and then it remains unchanged for greater Fe contents. Results of determination of unsaturated iron binding capacity value in human serum Unsaturatd iron binding capacity value for 32 human serum samples was determined by the method according to the invention and also through calculation based on the Peter method and magnesium carbonate method. Table (1) lists the analytical results. Excellent results were found to be obtained by the method according to the invention, with the co-relation coefficient and regression line with respect to the value (y) according to the Peter method being respectively 0.98 and (y) 1.1.97 10.5 and those with respect to the value 1v) according to the magnesium carbonate method being respectively 0.90 and (y) 0.98 10.4

the recovery of iron by adding 10, 30 and mg/100 ml of Fe(ll) to each sample. Table (11) shows the results of the experiments. The recovery was 89.2 to 103.7 percent and 94.4 percent in the average.

Table (II) Recovery of iron added to serum Sample Fe added Fe found Recovery No. (pg/ml) (pg/100ml) (pg/100ml) 30 As has been described in the foregoing, the method according to the invention permits a simple and speedy operation. Also, according to the invention it is possible to obtain satisfactory precision of determination and recovery. Further, the correlation with the priorart methods using iron absorbants is substantially satisfactory. Thus, the method according to the invention is thought to be suitable as practical analytic method for the determination of the unsaturated iron binding capacity in serum.

Table 11) t tnalytical results of UIBC value in human serum lug/ 100ml) Sample Proposed Peters MgCQ, Sample Proposed Peters MgCO Nummethod imethod method Nummethod method method ber ber Reproducibility What we claim is:

The same human serum sample (with unsaturated iron binding capacity value of 1.89mg/l00 ml) was an- I. A method of determining the unsaturated iron binding capacity of transferrin in serum comprising the alyzed ten times, and the variation factor was found to 65 steps of:

be 1.3 percent. Recovery Two human serum samples were used experimenting preparing a standard solution of iron chelate in which a known amount of iron is bound with nitrilotriacetic acid by using a buffer solution of pH 8.4 to 8.6;

adding the said standard iron solution to a sample serum in order to saturate the iron binding capacity of transferrin in the said serum;

reducing the excessive iron remaining unbound with transferrin by adding a salt of ascorbic acid to the said saturated solution;

developing a color by adding sodium bathophenanthroline to the said reduced solution, and determining the absorbance of the said colored solution.

2. The method as defined in claim 1, wherein said standard solution contains a small quantity of sodium ascorbate to serve as a reducing agent.

3. A method of determining the unsaturated iron binding capacity of serum comprising the steps of: pre paring four test tubes; preparing a sample serum by pouring 1.5 millilitres of a nitrilotriacetic acid buffer, 10 milligrams of a reducing agent, 0.5 millilitre of the serum and 0.05 millilitre of sodium bathophenanthro line sulfonate into a first one of said test tubes; preparing a serum blank by pouring 1.5 millilitres of a nitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent, 0.5 millilitre of said serum and 0.05 millilitre of distilled water into a second one of said test tubes; preparing a standard iron solution by pouring 1.5 millilitres of said nitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent, 0.5 millilitre of distilled water and 0.05 millilitre of a sodium bathophenanthroline sulfonate solution into a third one of said test tubes; preparing a reagent blank by pouring 1.5 millilitres of said nitrilotriacetic acid buffer, 10 milligrams of said reducing agent, 0.5 millilitre of distilled water and 0.05 millilitre of said sodium bathophenanthroline sulfonate solution into the last one of said test tubes; determining the absorbance at the Wavelength of 535 nm of the respective samples in said test tubes by using water as reference; and calculating the value of the unsaturated iron binding capacity from the values of the absorbance of said respective sample.

4. A composition for determining the unsaturated iron binding capacity in serum comprising in proportion 1.5 millilitres of a nitrilotriacetic acid iron buffer, 10 milligrams of a salt of ascorbic acid, 0.5 millilitres of distilled Water and 0.05 millilitres of a sodium bathophenanthroline sulfonate solution.

Claims

1. A METHOD OF DETERMINING THE UNSATURATED IRON BINDING CAPACITY OF TRANSFERRIN IN SERUM COMPRISING THE STEP OF: PREPARING A STANDARD SOLUTON OF IRON CHELATE IN WHICH A KNOWNAMOUNT OF IRON IS BOUND WITH NITRILOTRIACETIC ACID BY USING A BUFFER SOLUTION OF PH 8.4 TO 8.6; ADDIBG THE SAID STANDARD IRON SOLUTION TO A SAMPLE SERUM IN ORDER TO SATURATE THE IRON BINDING CAPACITY OF TRANSFERRIN IN THE SAID SERUM;

1. A method of determining the unsaturated iron binding capacity of transferrin in serum comprising the steps of: preparing a standard solution of iron chelate in which a known amount of iron is bound with nitrilotriacetic acid by using a buffer solution of pH 8.4 to 8.6; adding the said standard iron solution to a sample serum in order to saturate the iron binding capacity of transferrin in the said serum; reducing the excessive iron remaining unbound with transferrin by adding a salt of ascorbic acid to the said saturated solution; developing a color by adding sodium bathophenanthroline to the said reduced solution, and determining the absorbance of the said colored solution.

3. A method of determining the unsaturated iron binding capacity of serum comprising the steps of: preparing four test tubes; preparing a sample serum by pouring 1.5 millilitres of a nitrilotriacetic acid buffer, 10 milligrams of a reducing agent, 0.5 millilitre of the serum and 0.05 millilitre of sodium bathophenanthroline sulfonate into a first one of said test tubes; preparing a serum blank by pouring 1.5 millilitres of a nitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent, 0.5 millilitre of said serum and 0.05 millilitre of distilled water into a second one of said test tubes; preparing a standard iron solution by pouring 1.5 millilitres of said nitrilotriacetic acid iron buffer, 10 milligrams of said reducing agent, 0.5 millilitre of distilled water and 0.05 millilitre of a sodium bathophenanthroline sulfonate solution into a third one of said test tubes; preparing a reagent blank by pouring 1.5 millilitres of said nitrilotriacetic acid buffer, 10 milligrams of said reducing agent, 0.5 millilitre of distilled water and 0.05 millilitre of said sodium bathophenanthroline sulfonate solution into the last one of said test tubes; determining the absorbance at the wavelength of 535 nm of the respective samples in said test tubes by using water as reference; and calculating the value of the unsaturated iron binding capacity from the values of the absorbance of said respective sample.