CA1165215A - Method for determining transferrine and composition therefor - Google Patents
Method for determining transferrine and composition thereforInfo
- Publication number
- CA1165215A CA1165215A CA000391654A CA391654A CA1165215A CA 1165215 A CA1165215 A CA 1165215A CA 000391654 A CA000391654 A CA 000391654A CA 391654 A CA391654 A CA 391654A CA 1165215 A CA1165215 A CA 1165215A
- Authority
- CA
- Canada
- Prior art keywords
- transferrine
- determining
- iron
- reagent
- solution
- 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.)
- Expired
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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
- G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
ABSTRACT
A method for determining the iron-bonding capability of transferrine, consisting in adding to the sample being tested a reagent composed of a solution of a ferric salt absorbed onto a suspension of magnesium carbonate, centri-fuging the mixture so obtained and measuring the iron blocked by transferrine in the supernatant. The method is preferably carried out by utilizing a particular compo-sition consisting of a single reagent composed of a su-spension of a solution of a ferric salt absorbed on ma-gnesium carbonate, possibly a medium capable of maintain-ing the pull between 8 and 9, if desired, so colorimetric reagent for the determination of iron.
A method for determining the iron-bonding capability of transferrine, consisting in adding to the sample being tested a reagent composed of a solution of a ferric salt absorbed onto a suspension of magnesium carbonate, centri-fuging the mixture so obtained and measuring the iron blocked by transferrine in the supernatant. The method is preferably carried out by utilizing a particular compo-sition consisting of a single reagent composed of a su-spension of a solution of a ferric salt absorbed on ma-gnesium carbonate, possibly a medium capable of maintain-ing the pull between 8 and 9, if desired, so colorimetric reagent for the determination of iron.
Description
116~215 METHOD FOR DETERMINING TRANSFERRINE AND
COMPOSITION THEREFOR . -This invention relates to a novel method for the determination of the iron-binding ability of transfer-rine: it also relates to a particular reactive composi-tion adapted to the purpose .
Transferrine is a glycoprotein contained in the blood and serves to transfer the iron. Each proteinic molecule can bind two ferric ions.
The transferrine blood levels can be measured either directly with immunological methods, or via the determirlation of its iron-binding ability, with chemical methods.
With respect to the latter methods, it is known, from Chim. Clin. Acta, 2 , 221 (1957), a method which provides the steps of :
- saturating the transferrine which is present in the sample, usually a serum sample, by an acidic solution which contains an excess of ferric ions.
- removing, after an incubation time, the ferric ions in excess, by adding solid magnesium carbonate, and - the dosage, upon centrifuging the slurry thus ob-tained, of the iron bonded to the transferrine which is contained in the supernatant, by means of reagent ~p,
COMPOSITION THEREFOR . -This invention relates to a novel method for the determination of the iron-binding ability of transfer-rine: it also relates to a particular reactive composi-tion adapted to the purpose .
Transferrine is a glycoprotein contained in the blood and serves to transfer the iron. Each proteinic molecule can bind two ferric ions.
The transferrine blood levels can be measured either directly with immunological methods, or via the determirlation of its iron-binding ability, with chemical methods.
With respect to the latter methods, it is known, from Chim. Clin. Acta, 2 , 221 (1957), a method which provides the steps of :
- saturating the transferrine which is present in the sample, usually a serum sample, by an acidic solution which contains an excess of ferric ions.
- removing, after an incubation time, the ferric ions in excess, by adding solid magnesium carbonate, and - the dosage, upon centrifuging the slurry thus ob-tained, of the iron bonded to the transferrine which is contained in the supernatant, by means of reagent ~p,
2.
adapted to the determination of the iron contained in the serum.
Other procedures, conversely, dose, under appro-- priate conditions, the excess ferric ions and calculate, by difference, -the iron removed by transferrine, the amount of transferrine being then determined arithmeti-cally.
The present Applicants have now found that it is quite possible to dose the transferrine which is contain-ed in a sample without previously saturating the samplewith an excess of ferric ions in an acidic environment.
A primary obJective of the present invention is, in fact, to provide a method which permits the determina-tion of the iron-binding ability of transferrine, said method providing for the use of a single mixture which is composed of a solution of a ferric salt and a slurry of an appropriate solid ads~bant, the latter being added to the sample, whereafter the entire mixture is centri-fuged and the iron bonded to the transferrine which is contained in the supernatant is determined.
A number of advantages are achieved over theprior art method outlined above and the most significant of them can be summarized as follows :
- The procedure is simplified and the number of steps ancl incubation is lower, - Fewer manipulative steps are rcquired since it is no more necessary to introduce the magnesium carbonate in the solid state into the reaction mixture , 116S21~ -
adapted to the determination of the iron contained in the serum.
Other procedures, conversely, dose, under appro-- priate conditions, the excess ferric ions and calculate, by difference, -the iron removed by transferrine, the amount of transferrine being then determined arithmeti-cally.
The present Applicants have now found that it is quite possible to dose the transferrine which is contain-ed in a sample without previously saturating the samplewith an excess of ferric ions in an acidic environment.
A primary obJective of the present invention is, in fact, to provide a method which permits the determina-tion of the iron-binding ability of transferrine, said method providing for the use of a single mixture which is composed of a solution of a ferric salt and a slurry of an appropriate solid ads~bant, the latter being added to the sample, whereafter the entire mixture is centri-fuged and the iron bonded to the transferrine which is contained in the supernatant is determined.
A number of advantages are achieved over theprior art method outlined above and the most significant of them can be summarized as follows :
- The procedure is simplified and the number of steps ancl incubation is lower, - Fewer manipulative steps are rcquired since it is no more necessary to introduce the magnesium carbonate in the solid state into the reaction mixture , 116S21~ -
3.
- The dosage of the reagents is better reproducib]e, and - Transferrine is saturated in the sample with ferric ions and a basic pH, the latter being suggested by the literature as the most suitable for the ability of trans-ferrine to bind iron.
It is a considered opinion of the present Appli-cants that these facts entail a simplification and an accuracy of determination which cannot be found in the prior art methods.
More detailedly, the method according to the p~re-sent invention is carried out with the following steps:
- Adding to the sample being tested a single mi~ture consisting of a solution of a ferric salt and an adsorb-ing solid phase, at a pH comprised between 8 and 9,- Centrifuging the final slurry so obtained and - Measuring the iron bonded to the transferrine which is contained in the supernatant.
A second important objective of the present inven-20. tion is to provide, for carrying out the method outlinedabove, a particular composition, also an integral part of this invention, which consists of ra single reagent composed of a solution of a ferric salt adsorbed onto a solid substrate, possibly a medium capable of maintain-ing the pH between 8 and 9 and, possibly also, a colori-metric reagent for the determination of iron.
Solid substrates adapted to this purpose are mem-bers selected from the group consisting of magnesium 1 165~ 5
- The dosage of the reagents is better reproducib]e, and - Transferrine is saturated in the sample with ferric ions and a basic pH, the latter being suggested by the literature as the most suitable for the ability of trans-ferrine to bind iron.
It is a considered opinion of the present Appli-cants that these facts entail a simplification and an accuracy of determination which cannot be found in the prior art methods.
More detailedly, the method according to the p~re-sent invention is carried out with the following steps:
- Adding to the sample being tested a single mi~ture consisting of a solution of a ferric salt and an adsorb-ing solid phase, at a pH comprised between 8 and 9,- Centrifuging the final slurry so obtained and - Measuring the iron bonded to the transferrine which is contained in the supernatant.
A second important objective of the present inven-20. tion is to provide, for carrying out the method outlinedabove, a particular composition, also an integral part of this invention, which consists of ra single reagent composed of a solution of a ferric salt adsorbed onto a solid substrate, possibly a medium capable of maintain-ing the pH between 8 and 9 and, possibly also, a colori-metric reagent for the determination of iron.
Solid substrates adapted to this purpose are mem-bers selected from the group consisting of magnesium 1 165~ 5
4.
basic carbonate and CaCO3, magnesium basic carbonate is preferred.
The possible buffer adapted to maintain the pH at a constant value is a member selected ~rom the group consisting of H3PO~, H2PO4 , barbituric acid, barbiturates: NaH2PO4 is preferred.
If a colorimetric reagent is used, this shall pre-ferably be selected from among those suggested by the conven-tional art, or, also, it can be the reagent disclosed and claimed in the Canadian Patent Appln. N 371.506-0 filed on February 23, 1981 in the name of the Applicants hereof.
Additional details and the operative procedures for carrying out the metering of transferrine will become apparent from the scrutiny of the illustrative non-limiting examples reported hereinafter.
Preparation of a Rea~ent Suspension.-In 100 mls of water there are dissolved 828 mg ofNaH2PO4.H2O and, thereafter, 2.5 g of basic MgCO3 are slurried:
to the suspension so prepared there is added with stirring, 1 ml of 0.01-normal HCl containing S00 mg/dl of Fe (milli-grams per 0.1 liter). The reagent is allowed to stand until pH is stabilized.
The reagent described in EXAMPLE l is used for determining the iron-binding power of a pool of human sera for 48 times and an average value of 293.71 micro-~, .,~
grams/dl of iron is obtained, with a C.V. of 3.48%.
The procedure which has been adopted was as fol-1 ows - Reagent Suspension 1 ml
basic carbonate and CaCO3, magnesium basic carbonate is preferred.
The possible buffer adapted to maintain the pH at a constant value is a member selected ~rom the group consisting of H3PO~, H2PO4 , barbituric acid, barbiturates: NaH2PO4 is preferred.
If a colorimetric reagent is used, this shall pre-ferably be selected from among those suggested by the conven-tional art, or, also, it can be the reagent disclosed and claimed in the Canadian Patent Appln. N 371.506-0 filed on February 23, 1981 in the name of the Applicants hereof.
Additional details and the operative procedures for carrying out the metering of transferrine will become apparent from the scrutiny of the illustrative non-limiting examples reported hereinafter.
Preparation of a Rea~ent Suspension.-In 100 mls of water there are dissolved 828 mg ofNaH2PO4.H2O and, thereafter, 2.5 g of basic MgCO3 are slurried:
to the suspension so prepared there is added with stirring, 1 ml of 0.01-normal HCl containing S00 mg/dl of Fe (milli-grams per 0.1 liter). The reagent is allowed to stand until pH is stabilized.
The reagent described in EXAMPLE l is used for determining the iron-binding power of a pool of human sera for 48 times and an average value of 293.71 micro-~, .,~
grams/dl of iron is obtained, with a C.V. of 3.48%.
The procedure which has been adopted was as fol-1 ows - Reagent Suspension 1 ml
- 5 - lluman blood serum 0.2 ml - Incubation : 5 minutes at room temperature - Centrifuging : 10 minutes at 4,000 RPM
Iron in the supernatant was drawn and dosed with a conventional reagent.
To fractional samples of a base serum in which the iron-binding power has been determined, progressive-ly increased amounts of human transferrine have been added, The serum samples so enriched are sampled according to the procedure set forth in EXAI~lPLE 2 above and accor-ding to the Ramsay procedure disclosed in the above cited Chimica Clinica Acta, 2, 221 (1957). The values of the iron-binding power as obtained with said two procedures are correlated with the amount by weight of the added transferrine and the following regression lines are obtained :
x = milligrams (mg) of added transferrine, y = iron-binding power, in terms of micrograms/dl of found iron, Procedure according to this invention . -y = 0.90 x + 277.2 micrograms/dl with a correlation coefficient = 0.998 11~521~
Ramsay's Procedure .
y = 0.91 x + 277.5 micrograms/dl with a correla-tion coefficient = 0,997 The value of the iron-binding power of the base serum evaluated with the method of this invention was 281 micrograms/dl, and, with the Ramsay's method it was 272 micrograms/dl, both these values being in satisfact-ory agreement with the values of the respective intercep-tions on the equations of the straight lines reported 0 above.
Concentration ranges of the components of -the reagent :
Magnesium basic carbonate : 0.5 - 10 g/dl Fe+++ from 1 to 100 mg/dl H P0 ~ f 0 to 200 mg/liter
Iron in the supernatant was drawn and dosed with a conventional reagent.
To fractional samples of a base serum in which the iron-binding power has been determined, progressive-ly increased amounts of human transferrine have been added, The serum samples so enriched are sampled according to the procedure set forth in EXAI~lPLE 2 above and accor-ding to the Ramsay procedure disclosed in the above cited Chimica Clinica Acta, 2, 221 (1957). The values of the iron-binding power as obtained with said two procedures are correlated with the amount by weight of the added transferrine and the following regression lines are obtained :
x = milligrams (mg) of added transferrine, y = iron-binding power, in terms of micrograms/dl of found iron, Procedure according to this invention . -y = 0.90 x + 277.2 micrograms/dl with a correlation coefficient = 0.998 11~521~
Ramsay's Procedure .
y = 0.91 x + 277.5 micrograms/dl with a correla-tion coefficient = 0,997 The value of the iron-binding power of the base serum evaluated with the method of this invention was 281 micrograms/dl, and, with the Ramsay's method it was 272 micrograms/dl, both these values being in satisfact-ory agreement with the values of the respective intercep-tions on the equations of the straight lines reported 0 above.
Concentration ranges of the components of -the reagent :
Magnesium basic carbonate : 0.5 - 10 g/dl Fe+++ from 1 to 100 mg/dl H P0 ~ f 0 to 200 mg/liter
Claims (9)
1. A method for determining transferrine comprising the steps of adding to the sample being tested a reagent consisting of a solution of a ferric salt adsorbed on a suspension of a solid substrate , centrifuging the mix-ture thus obtained , and measuring the iron bonded by transferrine in the supernatant liquor .
2. Method for determining transferrine according to claim 1 , characterized in that the solid subs-trate is a member selected from the group consisting of magnesium basic carbonate and calcium carbonate .
3. Method for determining transferrine according to claim 1 , characterized in that the reagent composed of the solution of the ferric salt and -the suspension of the solid substrate contains a buffer capable of maintaining the pH between 8 and 9 .
4. Method for determining transferrine according to claim 2 , characterized in that the reagent composed of the solution of the ferric salt and the suspen-sion of the solid substrate contains a buffer capable of maintaining the pH between 8 and 9 .
5. Method for determining transferrine according to claim 3 or claim 4 characterized in that the buffer means is a member selected from the group consisting of H3P04 , H2P04 , barbituric acid and barbiturates .
6. A composition adapted to determining transferrine consisting of a solution of a ferric salt combined with a solid absorbant phase and a buffer means capable of maintaining the pH between 8 and 9 .
7. Composition adapted to determine transferrine according to claim 6 , characterized in that the adsorbant solid phase is a member selected from the group consisting of magnesium basic carbonate and calcium carbonate .
8. Composition adapted to determining trans-ferrine according to claim 6 or claim 7 , characterized in that the buffer means is a member selected from the group consisting of phosphoric acid , phosphates , barbituric acid and barbiturates.
9. Composition adapted to determine transferrine according to claim 6 additionally comprising a colorimetric reagent for the determination of iron .
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT26564A/80 | 1980-12-11 | ||
| IT26564/80A IT1148745B (en) | 1980-12-11 | 1980-12-11 | METHOD FOR DETERMINING TRANSFERRINE AND COMPOSITION SUITABLE FOR THE PURPOSE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1165215A true CA1165215A (en) | 1984-04-10 |
Family
ID=11219789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000391654A Expired CA1165215A (en) | 1980-12-11 | 1981-12-07 | Method for determining transferrine and composition therefor |
Country Status (7)
| Country | Link |
|---|---|
| JP (1) | JPS57122361A (en) |
| CA (1) | CA1165215A (en) |
| DE (1) | DE3147538C2 (en) |
| ES (1) | ES508226A0 (en) |
| FR (1) | FR2496270A1 (en) |
| GB (1) | GB2089505B (en) |
| IT (1) | IT1148745B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6069557A (en) * | 1983-09-26 | 1985-04-20 | Wako Pure Chem Ind Ltd | Method for measuring unsaturated iron bonding power |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1239121B (en) * | 1964-01-29 | 1967-04-20 | Haury Chem Fab Dr Heinz | Method for determining the total iron-binding capacity of body fluids, especially serum and urine |
| US3709985A (en) * | 1969-07-17 | 1973-01-09 | Abbott Lab | Method for determining total blood serum iron-binding capacity |
| US3996162A (en) * | 1970-02-27 | 1976-12-07 | Nuclear Medical Laboratories, Inc. | Analytical sorbent and method of use |
| JPS529160B2 (en) * | 1972-04-12 | 1977-03-14 | ||
| JPS5913521B2 (en) * | 1975-06-19 | 1984-03-30 | メイトウサンギヨウ カブシキガイシヤ | Method for producing magnetic iron oxide/dextran complex |
| US4154929A (en) * | 1976-08-16 | 1979-05-15 | American Monitor Corporation | 9-(2-Pyridyl)-acenaphtho[1,2-e]-as-triazines |
-
1980
- 1980-12-11 IT IT26564/80A patent/IT1148745B/en active
-
1981
- 1981-12-01 DE DE3147538A patent/DE3147538C2/en not_active Expired
- 1981-12-07 CA CA000391654A patent/CA1165215A/en not_active Expired
- 1981-12-07 GB GB8136865A patent/GB2089505B/en not_active Expired
- 1981-12-10 JP JP56197787A patent/JPS57122361A/en active Granted
- 1981-12-10 FR FR8123116A patent/FR2496270A1/en active Granted
- 1981-12-10 ES ES508226A patent/ES508226A0/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| GB2089505B (en) | 1984-03-21 |
| ES8303698A1 (en) | 1983-02-01 |
| GB2089505A (en) | 1982-06-23 |
| IT1148745B (en) | 1986-12-03 |
| DE3147538C2 (en) | 1983-09-08 |
| IT8026564A0 (en) | 1980-12-11 |
| JPH027429B2 (en) | 1990-02-19 |
| FR2496270B1 (en) | 1984-03-02 |
| FR2496270A1 (en) | 1982-06-18 |
| DE3147538A1 (en) | 1982-06-24 |
| ES508226A0 (en) | 1983-02-01 |
| JPS57122361A (en) | 1982-07-30 |
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| MKEX | Expiry |