METHOD FOR THE DETERMINATION OF CERULOPLASMIN ACTIVITY IN A SAMPLE

Abstract: The present invention privides a method for the determi­ nation of ceruloplasmin activity, wherein a member selected from the group consisting of (a) ferrocene or derivatives thereof (b) metallocene and (c) chelate metal containing iron or copper is reacted with a chromogen in the presence of ceruloplasmin to form colored material which is determined photometrically, as well as a reagent suitable therefor.

Description: The present invention relates to a method for the determination of ceruloplasmin activity in a sample, and a reagent suitable therefor. Ceruloplasmin is a copper-containing glycoprotein enzyme capable of oxidising iron from the divalent state to the trivalent state. It is a plasma protein and is usually present in α₂-globulin fractions at a concentration of about 0.5% based on total serum weight. The determination of ceruloplasmin activity is useful in the diagnosis of liver cirrhosis, chromic hepatitis, liver failure, Wilson's disease, etc. Known methods for the assay of ceruloplasmin activity include the single radial diffusion method for the immunologic quantitation of proteins (the SRID method), and colorimetric methods using diamine oxidase. The SRID method is time-consuming (usually more than 24 hours) and the results are not very accurate. The colorimetric method is simpler, but the reaction rate is slow and it still takes 15 to 30 minutes to complete. Not only that, but the colorimetric method cannot be used in automatic assay instruments due to the instability of the reagent. In accordance with the present invention, it has been found that ceruloplasmin activity in a sample can be determined by reacting the sample with a chromogen in the presence of (a) ferrocene or a ferrocene derivative, (b) a metallocene, or (c) a chelate metal complex containing iron or copper, and measuring the change in absorption due to the formation of pigment in the sample. Suitable derivatives of ferrocene used in the present invention are: α -hydroxyferrocene, benzoylferrocene, 1,1-ferrocene-dicarboxylic acid, acetylferrocene, and N,N-dimethylaminomethylferrocene. Suitable metallocenes are: cobaltocene, nickelocene, zirconocene, and titanocene, whilst suitable chelate metal complexes are: ferrous diethyl-dithiocarbamic acid (DDC-Fe), ferrous acetylacetonate, EDTA-Fe, and copper acetylacetonate. Each may be used in concentrations of from 10 µM to 1 mM. Suitable chromogens include: 4-aminoantipyrine (4AA)-phenol system, 4AA-N-ethyl-N-(3-methylphenyl)-N'-acetylethylene-diamine (EMAE) system, N,N-dimethyl-p-phenylenediamine (DPD), methylcarbamoyl-3,7-dimethylamine-10H-phenothiazin (MCDP), and bis[3-bis(4-chlorophenyl)-methyl-4-dimethylaminophenyl]amine (BCMA), and these may be used at concentrations of from 10 µM to 1mM. The reaction is carried out in a buffer solution such as acetate or citrate, at a pH of 4 to 6, preferably 5. If desired, a surfactant can be added to increase solubility or remove turbidity due, for example, to high concentrations of fat in the serum sample. Suitable surfactants are Triton X-100, polyoxyethylenelaurylether and sodium laurylsulfate. (Triton is a Registered Trade Mark.) The change in absorbance of the reaction solution due to the formed pigment is measured after a period of from 1 to 15 minutes, preferably at the wavelength of maximum absorption of the pigment, and usually in the range 600 to 800 nm. Ceruloplasmin activity can then be determined by reference to a standard curve. To illustrate the invention, 1 mg of each of the compounds indicated in Table 1 and 1.65 mg of MCDP were added to 100 ml quantities of acetate buffer to prepare a series of reagent solutions. 3 ml of each reagent solution were then incubated at 37°C. 30 µl of serum was added to each solution and the change in absorption (Δ E) at 663 nm was measured for 1 minute, 2 minutes after the reaction started. The results are shown in Table 1. Δ E x 10³/min control 1 ferrocene 63 α-hydroxyferrocene 61 benzoylferrocene 12 1,1-ferrocene-dicarboxylic acid 60 acetylferrocene 30 N,N-dimethylaminomethylferrocene 45 cobaltocene 3 titanocene 3 zirconocene 2 nickelocene 2 ferrous diethyl-dithio-carbamic acid 24 ferrous acetylacetonate 3 EDTA-Fe 2 copper acetylacetonate 3 The above procedure was repeated using 20 mg quantities of 4AA-EMAE, DPD and MCDP each with 1 mg of ferrocene to obtain the results shown in Table 2. Chromogen No addition of ferrocene Δ E x 10³/min Addition of ferrocene Δ E x 10³/min 4AA-EMAE 1 7 DPD 3 7 MCDP 1 63 The results obtained are not influenced by other substances present in serum because the reaction speed is very high, so that the ceruloplasmin activity can be measured very quickly and with high accuracy. The invention is further illustrated by the following example. EXAMPLE To 100 ml of a solution (pH 5.3) having the composition: 0.2M-acetate buffer 11.3 0.2M-sodium acetate solution 38.7 Triton X-100 0.1 Water 50 were added 1 mg of ferrocene and 1.65 mg of MCDP. 3 ml of the reagent solution was then poured into a cell and incubated at 37°C for 5 minutes. To the reagent solution were then added 30 µl of serum and the change in absorption (Δ E) of the reaction solution at 663 nm was measured for 1 minute, two minutes after the reaction started. The results are shown in Table 3. The same procedure was repeated, but using 10 mg of N,N-dimethylaminomethylferrocene (Test No. 2), 10 mg of titanocene (Test No. 3), 10 mg of nickelocene (Test No. 4), 7.5 mg of DDC-Fe (Test No. 5), or 10 mg of copper acetylacetonate (Test No. 6) instead of ferrocene. Finally in Test No. 7, 10 mg of N,N-dimethylaminomethylferrocene was used instead of ferrocene and 12.3 mg of oxalate of DPD instead of MDCP were used to obtain the results in Table 3. Test No. Sample 1 Sample 2 A U/ml S A U/ml S 1 63 79.0 30.2 110 138.0 52.8 2 45 56.0 30.2 78 98.0 52.3 3 3 3.7 30.0 5 6.3 50.0 4 2 2.5 28.0 4 5.0 56.0 5 24 30.0 31.2 41 51.0 53.3 6 3 3.8 30.0 5 6.3 50.0 7 15 19.0 30.0 26 33.0 52.0 A: Δ E/min x 10³ S: Content of ceruloplasmin (mg/dl) U: International unit Also in accordance with this invention there is provided a colorimetric reagent for the determination of ceruloplasmin activity in a sample comprising both a chromogen as defined and the said ferrocene or ferrocene derivative, metallocene or said iron- or copper-containing chelate metal complex.

Claims: A method for determining ceruloplasmin activity in a sample which comprises reacting the sample with a chromogen in the presence of (a) ferrocene or a ferrocene derivative, (b) a metallocene, or (c) a chelate metal complex containing iron or copper, thereby to form a pigment, and photometrically measuring the change in absorption of the sample due to the formation of said pigment. A method according to claim 1, wherein there is used a ferrocene derivative selected from α-hydroxyferrocene, benzoylferrocene, 1,1-ferrocene-dicarboxylic acid, acetylferrocene, and N,N-dimethylaminomethylferrocene. A method according to claim 1, wherein there is used a metallocene selected from cobaltocene, nickelocene, zirconocene, and titanocene, A method according to claim 1, wherein there is used a chelate metal selected from ferrous diethyl-dithiocarbamic acid, ferrous acetylacetonate, EDTA-Fe, and copper acetylacetonate. A method according to any one of claims 1 to 4, wherein there is used as said chromogen: 4-aminoantipyrine (4AA)-phenol system, 4AA-N-ethyl-N-(3-methylphenyl)-N'-acetylethylene-diamine (EMAE) system, N,N-dimethyl-p-phenylenediamine (DPD), methylcarbamoyl-3,7-dimethylamine-10H-phenothiazin (MCDP), or bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA). A method according to any one of claims 1 to 5, wherein the reaction is carried out in a buffer solution at a pH of 4 to 6. A method according to any one of claims 1 to 6, wherein said measurement is carried out at the wavelength of maximum absorption of the chromogen. A reagent for the determination of ceruloplasmin activity in a sample comprising (A) (a) ferrocene or a ferrocene derivative, (b) a metallocene, or (c) a chelate metal complex containing iron or copper, and (B) a chromogen selected from 4-aminoantipyrine (4AA)-phenol system, 4AA-N-ethyl-N-(3-methylphenyl)-N'-acetylethylene-diamine (EMAE) system, N,N-dimethyl-p-phenylenediamine (DPD), methylcarbamoyl-3,7-dimethylamine-10H-phenothiazin (MCDP), or bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA). A reagent according to claim 8, wherein component (A) is α-hydroxyferrocene benzoylferrocene 1,1-ferrocene-dicarboxylic acid acetylferrocene N,N-dimethylaminomethylferrocene cobaltocene titanocene zirconocene nickelocene ferrous diethyl-dithio-carbamic acid ferrous acetylacetonate EDTA-Fe or copper acetylacetonate.

Inventors: MATSUMORI, SHIGERU, SHIMIZU, YOSHIAKI, TATANO, TOSHIO
Applicants: KYOWA MEDEX CO. LTD.
Application year: 1985
Main classification: C12Q1
IPC codes: C12Q1, G01N33