US3887332A - Method of determining unsaturated iron binding capacity in serum - Google Patents

Method of determining unsaturated iron binding capacity in serum Download PDF

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US3887332A
US3887332A US348097A US34809773A US3887332A US 3887332 A US3887332 A US 3887332A US 348097 A US348097 A US 348097A US 34809773 A US34809773 A US 34809773A US 3887332 A US3887332 A US 3887332A
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iron
serum
binding capacity
solution
transferrin
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Kazuei Takase
Junji Morikawa
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Eiken Chemical Co Ltd
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Eiken Chemical Co Ltd
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    • 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
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/103332—Bilirubin or uric acid standard or control
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/108331—Preservative, buffer, anticoagulant or diluent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/109163—Inorganic standards or controls

Definitions

  • 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.
  • 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.
  • red corpuscle iron I red corpuscle iron I
  • liver, pancreas, marrow of bones and kidney stored iron
  • 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.
  • 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.
  • the diagnoses of various diseases stemming from abnormal iron metabolism can be done more precisely.
  • 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.
  • an excess iron solution for instance a ferric chloride solution
  • 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.
  • 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.
  • 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.
  • 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
  • FIG. 4 is a graph showing the calibrating curves representing the effect of Fe buffer solutions.
  • chelating agent there may be used ethylenediamine tetraacetic acid (EDTA), bathophenanthroline sulfonate (BPT), tripyridyl-s-triadine (TPTZ) and nitrilotriacetic aicd (NTA).
  • EDTA ethylenediamine tetraacetic acid
  • BPT bathophenanthroline sulfonate
  • TPTZ tripyridyl-s-triadine
  • NTA nitrilotriacetic aicd
  • 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.
  • 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.
  • the transferrin in the serum cannot derive iron from the chelates since the bond in the chelates is too strong.
  • 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 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.
  • the unsaturated iron binding capacity of serum differs with 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.
  • M-tris hydrochloric acid buffer for a pH range between 7 and 9
  • M-glycine sodium hydroxide buffer for a pH range between 9 and l l.
  • 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.
  • test tubes A, B, C and D were prepared. 1.5 millilitres of nitrilotriacetic acid iron buffer was 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.
  • 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.
  • 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:
  • 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
  • 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.

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US348097A 1972-04-12 1973-04-05 Method of determining unsaturated iron binding capacity in serum Expired - Lifetime US3887332A (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4224034A (en) * 1978-09-07 1980-09-23 American Monitor Corporation Assay of iron and iron binding protein reagents and methods
FR2496270A1 (fr) * 1980-12-11 1982-06-18 Sclavo Inst Sieroterapeut Procede pour la determination de la transferrine et composition adaptee a ce procede
US4459356A (en) * 1982-02-11 1984-07-10 Georgetown University Radioactive staining of gels to identify proteins
US4567150A (en) * 1981-11-20 1986-01-28 Sclavo, S.P.A. Method for determining transferrin and composition therefor
WO1986003011A1 (en) * 1984-11-07 1986-05-22 Ross Thomas Starr Measurement of total iron binding capacity
EP0137400A3 (en) * 1983-09-26 1986-05-28 Wako Pure Chemical Industries, Ltd. Determination of unsaturated iron-binding capacity
US4701420A (en) * 1985-04-01 1987-10-20 Eastman Kodak Company Analytical compositions, elements and methods utilizing reduction of ferric ion chelates to form detectable dyes
USRE32920E (en) * 1978-01-11 1989-05-09 Esa, Inc. Electrochemical testing system and method
WO1998038515A1 (en) * 1997-02-28 1998-09-03 London Health Sciences Centre Test for hemochromatosis
US6274382B1 (en) * 1998-04-22 2001-08-14 Roche Diagnostics Gmbh Method and reagent for the interference-free determination of iron
US6368866B1 (en) 1998-08-03 2002-04-09 Reference Diagnostics, Inc. Rapid separation assay for total iron binding capacity
WO2002021136A3 (de) * 2000-09-08 2003-06-12 Teraklin Ag Verfahren zur quantitativen bestimmung der vorhandenen bindungskapazität in einer wässrigen proteinlösung
US6627448B1 (en) * 1999-10-04 2003-09-30 Reference Diagnostics, Inc. Analyte-binding assay
CN111257549A (zh) * 2018-12-03 2020-06-09 深圳迈瑞生物医疗电子股份有限公司 检测血清中的不饱和铁结合力的试剂盒及方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995013544A1 (fr) * 1992-09-30 1995-05-18 Daiichi Pure Chemicals Co., Ltd. Reactif permettant de determiner l'insaturation de la capacite de fixation du fer insature

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Bates et al., Chem. Abstr. 67, 7925x (1967). *
Frankel et al., ed., Gradwohl's Clinical Laboratory Methods and Diagnosis, C. V. Mosby Co., 1970. Vol. I, pp. 205-368, 441 relied on, RB37.G7. *
G. W. Bates, Dissert. Abstr. 27B (11), 3816 (1967). *
R J. Henry, Clinical Chemistry Principles and Technics, Harper & Row, 1964, pp. 391-395 relied on, RB40.H4. *
R. L. Searcy, Diagnostic Chemistry, McGraw Hill, 1969, p. 329 relied on, RB40.54. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE32920E (en) * 1978-01-11 1989-05-09 Esa, Inc. Electrochemical testing system and method
US4224034A (en) * 1978-09-07 1980-09-23 American Monitor Corporation Assay of iron and iron binding protein reagents and methods
FR2496270A1 (fr) * 1980-12-11 1982-06-18 Sclavo Inst Sieroterapeut Procede pour la determination de la transferrine et composition adaptee a ce procede
US4567150A (en) * 1981-11-20 1986-01-28 Sclavo, S.P.A. Method for determining transferrin and composition therefor
US4459356A (en) * 1982-02-11 1984-07-10 Georgetown University Radioactive staining of gels to identify proteins
EP0137400A3 (en) * 1983-09-26 1986-05-28 Wako Pure Chemical Industries, Ltd. Determination of unsaturated iron-binding capacity
WO1986003011A1 (en) * 1984-11-07 1986-05-22 Ross Thomas Starr Measurement of total iron binding capacity
US4735904A (en) * 1984-11-07 1988-04-05 Starr Ross T Measurement of total iron binding capacity
US4886642A (en) * 1984-11-07 1989-12-12 Starr Ross T Measurement of total iron binding capacity
US4701420A (en) * 1985-04-01 1987-10-20 Eastman Kodak Company Analytical compositions, elements and methods utilizing reduction of ferric ion chelates to form detectable dyes
WO1998038515A1 (en) * 1997-02-28 1998-09-03 London Health Sciences Centre Test for hemochromatosis
US6274382B1 (en) * 1998-04-22 2001-08-14 Roche Diagnostics Gmbh Method and reagent for the interference-free determination of iron
US6368866B1 (en) 1998-08-03 2002-04-09 Reference Diagnostics, Inc. Rapid separation assay for total iron binding capacity
US6627448B1 (en) * 1999-10-04 2003-09-30 Reference Diagnostics, Inc. Analyte-binding assay
WO2002021136A3 (de) * 2000-09-08 2003-06-12 Teraklin Ag Verfahren zur quantitativen bestimmung der vorhandenen bindungskapazität in einer wässrigen proteinlösung
CN111257549A (zh) * 2018-12-03 2020-06-09 深圳迈瑞生物医疗电子股份有限公司 检测血清中的不饱和铁结合力的试剂盒及方法

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