CA2198790A1 - Test for hemochromatosis - Google Patents
Test for hemochromatosisInfo
- Publication number
- CA2198790A1 CA2198790A1 CA 2198790 CA2198790A CA2198790A1 CA 2198790 A1 CA2198790 A1 CA 2198790A1 CA 2198790 CA2198790 CA 2198790 CA 2198790 A CA2198790 A CA 2198790A CA 2198790 A1 CA2198790 A1 CA 2198790A1
- Authority
- CA
- Canada
- Prior art keywords
- hemochromatosis
- iron
- test
- uibc
- serum
- 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.)
- Abandoned
Links
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
Landscapes
- 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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Abstract Not Yet Available
Description
219879~
, TEST FOR HEMOC~RQ~ATOSIS
The invention relates to methods for diagnosing hemochromatosis.
Background of the Invention He~reditary hemochromatosis is a common autosomal recessive disease in Caucasians, with a prevalence of approximately 1 in 300(1 to 4). Excessive intestinal absorption of dietary iron in homozygotes leads to the progressive accumulation of iron in tissues. Life-threatening clinical manifestations, such as liver failure, diabetes, and heart failure, usually occur after a latent period of 40-60 years. The diagnosis of hemochromatosis is often missed, and the disease is commonly discovered during the management of incidental illnesses or periodic health examinations(5). Factors contributing to underdiagnosis include (1) asymptomatic status of most patients until irreversible tissue injury has occurred, (2) lack of specificity of symptoms when present, (3) confusion with aLcoholic liver disease, and (4) lack of awareness of hemochromatosis, including appropriate investigations. Early detection and treatment of the disease can prevent the development of impotence, heart failure, cirrhosis, and hepatocellular carcinoma and result in long-term survival similar to the general population(5-7).
The high prevalence, morbidity and mortality, and benefit of early diagnosis and treatment make hemochromatosis a prime target for population screening(4, 8).
The conventional screening test currently in use to diagnose hemochromatosis is the transferrin saturation test, which is a two stage test involving determination of both serum iron and total serum iron-binding capacity.
2198~9~
-Summary of Invention The present invention provides, in accordance with one embodiment, a less expensive and more convenient, one-step test for screening for hemochromatosis in a subject by determining the unsaturated iron-binding capacity (UIBC) of the serum of the subject. The method is an indirect colorimetric method for measuring the iron binding capacity in serum.
In accordance with a preferred embodiment, an assay method is employed wherein a known amount of iron is added to the serum sample in excess of that required to saturate any available iron-binding transferrin sites and the excess unbound iron is measured by interacting it with a chelator with which it forms a detectable chelate-iron complex. Preferably, the complex is a colouredcomplex which can be determined spectrophotometrically.
Ferrozine is a preferred chelator.
The screening test may be applied to a general population or to a specific population with an expected above-normal prevalence of hemochromatosis, such as siblings of patients.
EXAMPLES
The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Example 1 UIBC and percentage transferrin saturation (TS%) were measured in samples from about 130 subjects diagnosed with hemochromatosis.
TS% was measured using the Unimate 5 reagent system for total iron and the Unimate 7 system for UIBC, in accordance with the manufacturer's instructions (Hoffman-Ferrozine is a trade-mark 21987~
LaRoche, Mississauga, Ontario). TS% = total iron x 100 (total iron + UIBC).
UIBC was determined by a modification of the method described in the manufacturer's instructions accompanying the Unimate 7 reagent system for UIBC (Hoffman-La Roche, Mississauga, Ontario).
The modified method is scaled down so that reactions are carried out in microwell plates. This reduces the amount of sample required and facilitates automatic reading of the test results by a plate reader.
Materials Reagent R1: Buffer containing 250 mM Tris, pH 8.4, 50 mM
NaHCO3 and 41 ~M sodium azide.
Reagent R2: Chromogen solution containing 160 mM
hydroxylamine and 20 mM Ferrozine (disodium salt of 3-(2-pyridyl)-5,6-bis(4-sulphophenyl)-S-triazine).
Hemolysis-free blood samples were collected in iron-free tubes and serum was obtained from the samples.
Briefly, 40 ~l hemolysis-free serum was mixed with 200 ~l R1 at room temperature in Immulor I microtitre strips on a microtitre plate mixer for 10 minutes at setting 5.
The absorbance (A1) of the solution was read at 550 nm using a Titertek Plus microplate reader.
40 ~l R2 was then added and mixed for 20 minutes at setting 5 on microtitre plate reader. Absorbance at 550 nm was read again (A2).
Samples of the calibration standard provided by the manufacturer were similarly treated, and controls without serum were carried out to give reagent blank values.
UIBC value was calculated using the formula AA (Reagent blank) - ~A (test) UIBC = x concentration AA (Reagent blank) - ~A (calibration of calibration standard) standard - ~198790 UIBC values and TS% values were compared as shown in Figure 1. There was a close negative correlation between the UIBC screening test and TS% (r = -0.78). A TS% value of greater than 55% indicates a need to carry out further testing to confirm a diagnosis of hemochromatosis. Using the UIBC screening test of the present invention, a UIBC
value lower than 23% indicates that the subject requires further investigation.
References 1. Borwein, S.T. et al., (1983), Clin. Invest. Med., v.
6, pp. 171-179.
, TEST FOR HEMOC~RQ~ATOSIS
The invention relates to methods for diagnosing hemochromatosis.
Background of the Invention He~reditary hemochromatosis is a common autosomal recessive disease in Caucasians, with a prevalence of approximately 1 in 300(1 to 4). Excessive intestinal absorption of dietary iron in homozygotes leads to the progressive accumulation of iron in tissues. Life-threatening clinical manifestations, such as liver failure, diabetes, and heart failure, usually occur after a latent period of 40-60 years. The diagnosis of hemochromatosis is often missed, and the disease is commonly discovered during the management of incidental illnesses or periodic health examinations(5). Factors contributing to underdiagnosis include (1) asymptomatic status of most patients until irreversible tissue injury has occurred, (2) lack of specificity of symptoms when present, (3) confusion with aLcoholic liver disease, and (4) lack of awareness of hemochromatosis, including appropriate investigations. Early detection and treatment of the disease can prevent the development of impotence, heart failure, cirrhosis, and hepatocellular carcinoma and result in long-term survival similar to the general population(5-7).
The high prevalence, morbidity and mortality, and benefit of early diagnosis and treatment make hemochromatosis a prime target for population screening(4, 8).
The conventional screening test currently in use to diagnose hemochromatosis is the transferrin saturation test, which is a two stage test involving determination of both serum iron and total serum iron-binding capacity.
2198~9~
-Summary of Invention The present invention provides, in accordance with one embodiment, a less expensive and more convenient, one-step test for screening for hemochromatosis in a subject by determining the unsaturated iron-binding capacity (UIBC) of the serum of the subject. The method is an indirect colorimetric method for measuring the iron binding capacity in serum.
In accordance with a preferred embodiment, an assay method is employed wherein a known amount of iron is added to the serum sample in excess of that required to saturate any available iron-binding transferrin sites and the excess unbound iron is measured by interacting it with a chelator with which it forms a detectable chelate-iron complex. Preferably, the complex is a colouredcomplex which can be determined spectrophotometrically.
Ferrozine is a preferred chelator.
The screening test may be applied to a general population or to a specific population with an expected above-normal prevalence of hemochromatosis, such as siblings of patients.
EXAMPLES
The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Example 1 UIBC and percentage transferrin saturation (TS%) were measured in samples from about 130 subjects diagnosed with hemochromatosis.
TS% was measured using the Unimate 5 reagent system for total iron and the Unimate 7 system for UIBC, in accordance with the manufacturer's instructions (Hoffman-Ferrozine is a trade-mark 21987~
LaRoche, Mississauga, Ontario). TS% = total iron x 100 (total iron + UIBC).
UIBC was determined by a modification of the method described in the manufacturer's instructions accompanying the Unimate 7 reagent system for UIBC (Hoffman-La Roche, Mississauga, Ontario).
The modified method is scaled down so that reactions are carried out in microwell plates. This reduces the amount of sample required and facilitates automatic reading of the test results by a plate reader.
Materials Reagent R1: Buffer containing 250 mM Tris, pH 8.4, 50 mM
NaHCO3 and 41 ~M sodium azide.
Reagent R2: Chromogen solution containing 160 mM
hydroxylamine and 20 mM Ferrozine (disodium salt of 3-(2-pyridyl)-5,6-bis(4-sulphophenyl)-S-triazine).
Hemolysis-free blood samples were collected in iron-free tubes and serum was obtained from the samples.
Briefly, 40 ~l hemolysis-free serum was mixed with 200 ~l R1 at room temperature in Immulor I microtitre strips on a microtitre plate mixer for 10 minutes at setting 5.
The absorbance (A1) of the solution was read at 550 nm using a Titertek Plus microplate reader.
40 ~l R2 was then added and mixed for 20 minutes at setting 5 on microtitre plate reader. Absorbance at 550 nm was read again (A2).
Samples of the calibration standard provided by the manufacturer were similarly treated, and controls without serum were carried out to give reagent blank values.
UIBC value was calculated using the formula AA (Reagent blank) - ~A (test) UIBC = x concentration AA (Reagent blank) - ~A (calibration of calibration standard) standard - ~198790 UIBC values and TS% values were compared as shown in Figure 1. There was a close negative correlation between the UIBC screening test and TS% (r = -0.78). A TS% value of greater than 55% indicates a need to carry out further testing to confirm a diagnosis of hemochromatosis. Using the UIBC screening test of the present invention, a UIBC
value lower than 23% indicates that the subject requires further investigation.
References 1. Borwein, S.T. et al., (1983), Clin. Invest. Med., v.
6, pp. 171-179.
2. Edwards, C.Q., et al., (1988), N. Eng. J. Med., v.
318, pp. 1355-1362.
318, pp. 1355-1362.
3. Hallberg, L. et al., (1989), J. Int. Med., v. 225, pp. 249-255.
4. Edwards C.W., (1993), N. Engl. J. Med., v. 328, pp.
1616-1620.
1616-1620.
5. Niederau, C. et al., (1985), N. Engl. J. Med., v.
313, pp. 1256-1262.
313, pp. 1256-1262.
6. Adams, P.C. et al., (1991), Am. J. Med., v. 90, pp.
445-449.
445-449.
7. Adams, P.C. et al., (1991), Gastroenterology, v.
101, pp. 368-372.
101, pp. 368-372.
Claims
Claims Not Yet Available
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2198790 CA2198790A1 (en) | 1997-02-28 | 1997-02-28 | Test for hemochromatosis |
PCT/CA1998/000174 WO1998038515A1 (en) | 1997-02-28 | 1998-03-02 | Test for hemochromatosis |
AU62865/98A AU6286598A (en) | 1997-02-28 | 1998-03-02 | Test for hemochromatosis |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA 2198790 CA2198790A1 (en) | 1997-02-28 | 1997-02-28 | Test for hemochromatosis |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2198790A1 true CA2198790A1 (en) | 1998-08-28 |
Family
ID=4160069
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2198790 Abandoned CA2198790A1 (en) | 1997-02-28 | 1997-02-28 | Test for hemochromatosis |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU6286598A (en) |
CA (1) | CA2198790A1 (en) |
WO (1) | WO1998038515A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU3269101A (en) * | 1999-11-12 | 2001-06-06 | James C. Barton | Diagnostic test for hemochromatosis |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS529160B2 (en) * | 1972-04-12 | 1977-03-14 | ||
IT1100475B (en) * | 1978-11-08 | 1985-09-28 | R C O Ricerche Di Chimica Clin | METHOD AND COMPOSITIONS FOR THE DIRECT DETERMINATION OF IRON MEL STERO EMATICO |
JPS6069557A (en) * | 1983-09-26 | 1985-04-20 | Wako Pure Chem Ind Ltd | Method for measuring unsaturated iron bonding power |
DE3729502A1 (en) * | 1987-09-03 | 1989-03-23 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINING IRON |
EP0570588B1 (en) * | 1991-12-02 | 1998-09-09 | Oriental Yeast Co., Ltd. | Method and reagent for determination of serum iron or unsaturated iron binding capacity using aconitase |
-
1997
- 1997-02-28 CA CA 2198790 patent/CA2198790A1/en not_active Abandoned
-
1998
- 1998-03-02 WO PCT/CA1998/000174 patent/WO1998038515A1/en active Application Filing
- 1998-03-02 AU AU62865/98A patent/AU6286598A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
WO1998038515A1 (en) | 1998-09-03 |
AU6286598A (en) | 1998-09-18 |
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FZDE | Dead |