GB2089505A

GB2089505A - Determination of Transferrine

Info

Publication number: GB2089505A
Application number: GB8136865A
Authority: GB
Original assignee: SCLAVO INST SIEROTERAPEUT
Current assignee: SCLAVO INST SIEROTERAPEUT
Priority date: 1980-12-11
Filing date: 1981-12-07
Publication date: 1982-06-23
Also published as: CA1165215A; ES8303698A1; GB2089505B; JPS57122361A; JPH027429B2; IT1148745B; ES508226A0; FR2496270B1; IT8026564A0; DE3147538A1; DE3147538C2; FR2496270A1

Abstract

The transferrine content of a sample is determined by adding to the sample a reagent consisting of a ferric salt adsorbed onto the particles of a suspension of a solid substrate, e.g. magnesium carbonate. The mixture obtained is centrifuged and the iron in the supernatant is determined e.g. colorimetrically. A reagent comprising a suspension of a solid substrate on which ferric salt is adsorbed is also claimed. <IMAGE>

Description

SPECIFICATION Determination of Transferrine This invention relates to a method for the determination of the iron-binding ability of transferrine, and to a reagent for use in the method.

Transferrine is a glycoprotein contained in blood. It serves to transfer iron, and each molecule thereof can bind two ferric ions.

The transferrine content of blood can be measured either directly by immunological methods, or indirectly by the determination of its iron-binding ability by chemical methods. With respect to the latter methods, there is known, from Clinica Chimica Acta, 2 221 (1957), a method which consists of the steps of (a) saturating the transferrine which is present in the sample, usually a serum sample, with an acidic solution which contains an excess of ferric ions, (b) removing, after incubation, the excess ferric ions by adding solid magnesium carbonate, and (c) determining, after centrifuging the slurry thus obtained, the amount of iron bonded to the transferrine contained in the supernatant, by means of a reagent suitable for the determination of iron.In other procedures, conversely, the excess amount of ferric ions is determined under appropiate conditions and the amount of iron removed by transferrine is then calculated, the amount of transferrine then being determined arithmetically.

We have now found that it is possible to determine the amount of tranferrine contained in a sample without previously saturating the sample with an excess of ferric ions in an acidic environment.

According to the present invention, there is provided a method of determining the transferrine content of a sample suspected of containing transferrine, which method comprises mixing the sample with a reagent comprising a suspension of a solid substrate on which substrate a ferric salt is absorbed, centrifuging the mixture, and determining the iron content of the resulting supernatant liquor.

The present invention also provides a reagent for use in determining the transferrine content of a sample suspected of containing transferrine, the reagent comprising a suspension of a solid substrate on which substrate a ferric salt Is adsorbed.

Thus, the present invention provides a method for the determination of the iron-binding ability of transferrine, in which method there is used a single reagent which comprises a solution of a ferric salt and a slurry of an appropriate solid adsorbant, the reagent being added to the sample whereafter the mixture is centrifuged and the iron bound to the transferrine contained in the supernatant is determined.

A number of advantages are achieved by the method of the invention, at least in preferred embodiments thereof, as compared to the prior art method outlined above. The most significant of them are (a) the procedure is simplified and the number of steps and incubation is lower; (b) fewer manipulative steps are required since it is not necessary to introduce magnesium carbonate in the solid state into the reaction mixture; (c) measurement of the amounts of reagent used is better reproducible; and (d) the tranferrine in the sample is saturated with ferric ions at a basic pH, the latter being suggested by the literature as the most suitable for the binding of transferrine to iron. We consider that these advantages result in a simplicity and an accuracy of determination which cannot be found in the prior art methods.

Preferably, the reagent has a pH of from 8 to 9, and accordingly it preferably contains a buffer capable of maintaining the pH at a value of from 8 to 9. The reagent preferably contains a colorimetric reagent for the determination of iron.

Examples of suitable solid substrates are basic magnesium carbonate and calcium carbonate, the former being preferred.

Examples of suitable buffers are H3PO4, H2PO-4, barbituric acid, and barbiturates. The preferred buffer is NaH2PO4.

If a colorimetric reagent is used, it can be a reagent known from the prior art. Also, it can be a reagent as disclosed and claimed in Italian Patent Application No. 201 77A/80, which was laid open to public inspection on the 26th August 1981.

The invention will now be illustrated by the following Examples.

Example 1 (Preparation of Reagent) In 100 ml of water there was dissolved 828 mg of NaH2PO4.H2O. Thereafter, 2.5 g of basic MgCO3 were added to form a suspension, and, to the suspension, there was added, with stirring, 1 ml of 0.01 normal HCI containing 500 mg/dl of Fe3+. The reagent was allowed to stand until its pH has stabilized.

Example 2 The reagent described in Example 1 was used for determing the iron-binding power of 48 samples of human blood serum. The average value was 293.71 micrograms/dl of iron, with a C.V. of 3.48%. In each of determinations, 1 ml of the reagent of Example 1 and 0.2 ml of serum were used, and incubation was carried out for 5 minutes at room temperature and centrifuging for 10 minutes at 4,000 RPM. The iron in the supernatant was drawn and determined with a conventional reagent Example 3 To fractional samples of a base serum whose iron-binding power had been determined, progressively increased amounts of human transferrine were added.The iron-binding power of the serum samples so enriched was determined according to the procedure set forth in Example 2 above and according to the Ramsey procedure disclosed in Clinica Chimica Acta, 2 221(1957). The values of the iron-binding power obtained by these two procedures were correlated with the amounts by weight of transferrine added, whereby graphs represented by the following equations were obtained: (A) y=0.90x+277.2 (correlation coefficient=0.998) (B) y=0.9 1 x+277.5 (correlation coefficient=0.997).

In the above equations, x is the amount of added tranferrine in milligrams and y is the ironbinding power in micrograms/dl of determined iron, equation (A) being that obtained by the procedure of Example 2 and equation (B) being that obtained by the Ramsey procedure.

The value of the iron-binding power of the base serum evaluated by the method of this invention was 281 micrograms/dl, and, by the Ramsay method, was 272 micrograms/dl, both these values being in satisfactory agreement with the values of the respective interceptions of the straight lines represented by the above equations.

The reagent used contained from 0.5 to 10 gidI of basic magnesium carbonate, from 1 to 100 mg/dl of Fe3+ and from 0 to 200 mg/litre of H2PO4.

Claims

1. A method of determining the transferrine content of a sample suspected of containing transferrine, which method comprises mixing the sample with a reagent comprising a suspension of a solid substrate on which substrate a ferric salt is adsorbed, centrifuging the mixture, and determining the iron content of the resulting supernatant liquor.

2. A method according to claim 1, wherein the solid substrate is basic magnesium carbonate or calcium carbonate.

3. A method according to claim 1 or 2, wherein the reagent contains a buffer capable of maintaining the pH of the reagent at a value of from 8 to 9.

4. A method according to claim 3, wherein the buffer is phosphoric acid, a phosphate, barbituric acid or barbiturate.

5. A method according to any of claims 1 to 4, herein the reagent contains a colorimetric reagent for the determination of iron.

6. A method according to any of claims 1 to 5, wherein the sample comprises blood or blood serum.

7. A method according to claim 1, substantially as described in either of the foregoing Examples 1 and 2.

8. A reagent for use in determining the tranferrine content of a sample suspected of containing transferrine, the reagent comprising a suspension of a solid substrate on which substrate a ferric salt is adsorbed.

9. A reagent as claimed in claim 8, wherein the solid substrate is basic magnesium carbonate or calcium carbonate.

10. A reagent as claimed in claim 8 or 9, containing a buffer capable of maintaining the pH of the reagent at a value of from 8 to 9.

11. A reagent as claimed in claim 10, wherein the buffer is phophoric acid, a phosphate, barbituric acid or a barbiturate.

12. A reagent as claimed in any of claims 8 to 11, wherein the reagent contains a colorimetric reagent of the determination of iron.

1 3. A reagent as claimed in claim 8, substantially as described in either of the foregoing Examples 1 and 3.