Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1993-08-02
1995-05-30
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 18, 435 26, 435183, 435189, 435190, 435195, 435232, 436 74, 436 84, 436910, C12Q 1527, C12Q 100, C12Q 132, C12N 988
Patent
active
054200083
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to an assay method for serum iron, and more specifically, it relates to a highly-sensitive enzyme assay method in which iron bound to transferrin is liberated, apo-aconitase (EC 4.2.1.3) which exhibits no activity without iron is contacted therewith for activation, and isocitric acid produced from citric acid is subjected to a reaction with isocitrate dehydrogenase (EC 1.1.1.41, EC 1.1.1.42) to make an assay of the serum iron level, and relates to a reagent used for measuring.
The present invention further relates to an enzyme assay method for unsaturated iron binding capacity (hereunder abbreviated to UIBC), and more specifically, to a highly sensitive enzyme assay method for UIBC in which an excess amount of iron of known concentration is added to a subject test sample of body fluid, etc. and the iron is bound to transferrin to saturation, after which the unbound residual iron is contacted with apo-aconitase (EC 4.2.1.3) which exhibits no activity without iron, and isocitric acid produced from citric acid by a catalytic effect of aconitase is measured for its aconitase activity, using isocitrate dehydrogenase (EC 1.1.1.41, EC 1.1.1.42) as the detection enzyme, in order to determine UIBC, and relates to a reagent used for the assay.
PRIOR ART AND PROBLEMS THEREOF
The total amount of iron in the body is about 4 g, two-thirds of which exists in the erythrocyte hemoglobin, and the remaining one-third of which is kept in the tissues of the liver, spleen, bone marrow, etc. as stored iron. The total amount of serum iron is 3-4 mg, and all of it exists bound to transfertin belonging to a serum .beta.-globulin, (one molecule of transferrin combines with two molecules of iron). The serum iron concentration is regulated by the rate of production and destruction of erythrocytes, and if there is a decline in hematopoiesis in the bone marrow, the flow of serum iron stagnates, raising the serum iron concentration, while the serum iron concentration is lowered in the opposite case. This has led to the conclusion that the serum iron concentration is a reflection of the function of the hematogenous organs. In addition, the dynamics of stored iron also affects serum iron. For example, in diseases such as hepatitis, stored iron in the liver moves to other tissues, causing a rise in serum iron. As described above, serum iron plays a part not only in hemodyscrasia (iron deficiency anemia, aplastic anemia, pernicious anemia, hemolytic anemia, leukemia, polycythemia rubra, etc.), but also in a variety of other diseases (infectious diseases, acute hepatitis, liver cirrhosis, hemochromatosis, nephrosis, etc.), and therefore iron measurement is considered important in clinical examinations.
Assay methods for serum iron include the Matsubara method, the Standard method of International Committee for Standardization in Hematoloby (ICSH), the autoanalyzer method, the atomic absorption spectrophotometry, and so on. The principle of the Matsubara method and the Standard method of International Committee for Standardization in Hematology (ICSH) involve liberation of iron with an acid, deproteinization and reduction of the iron with a reducing reagent, followed by coloring with a color former. However, the molar absorption coefficient of BPT is small and the sensitivity is low, and therefore a large amount of serum is required. Further, as it is also susceptible to interference, it is difficult to deal with many samples simultaneously by to manual treatment. The autoanalyzer method is based on a simple automation of the principle used in the Matsubara method and the Standard method of Internation Committee for Standardization in Hematology (ICSH), without solving the problem of sensitivity, and thus interference by hemoglobin, billrubin, etc. is observed. The atomic absorption spectrophotometry is disadvantageous in that it necessitates pre-treatment and produces large variations in measurement values depending on differences in the procedure used, and the sensitivity is poor. In a
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Fujita Tsuyoshi
Nishida Hozumi
Nonobe Masatsugu
Gromet Nancy J.
Oriental Yeast Co. Ltd.
Wityshyn Michael G.
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