Radioenzymatic assay of catecholamines
AssigneeThe Upjohn Company
InventorsGarland A. Johnson, Jacob D. Peuler
Life of this patent
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Abstract
A new radioenzymatic assay for catecholamines utilizing the catechol-O-methyl transferase transfer of a methyl group from a labeled methyl donor to the catecholamines followed by isolation of the O-methylated (.sup.3 H)catecholamine and the subsequent measurement of radioactivity.
Claims81
A method for analyzing undifferentiated, total endogenous epinephrine and norepinephrine levels in a mammalian system selected from the group consisting of the supernatant of blood serum, plasma and urine which comprises
- a. contacting an aliquot of the mammalian system with the enzyme catechol-O-methyl transferase, the tritium labeled methyl donor S-adenosyl-L-methionine(.sup.3 H)methyl substantially free of unlabeled methyl donor, a cation of oxidation number +2 which allows the methyl transfer to proceed, a compound which stabilizes the enzyme-substrate system, and an amount of ethylene glycol bis-(amino ethyl ether)-N,N-tetracetic acid sufficient to remove transferase-inhibiting concentrations of calcium ions, to form a mixture,
- b. incubating the mixture for a time, temperature and pH sufficient to O-methylate (.sup.3 H) substantially all of the epinephrine and norepinephrine;
- c. extracting the O-methylated (.sup.3 H) epinephrine and norepinephrine with an organic solvent in which the O-methylated (.sup.3 H) epinephrine and norepinephrine are preferentially soluble;
- d. repartitioning the O-methylated (.sup.3 H) epinephrine and norepinephrine into an aqueous acid of sufficient strength to protonate the amine;
- e. oxidizing O-methylated (.sup.3 H) epinephrine and norepinephrine to vanillin-.sup.3 H,
- f. extracting vanillin-.sup.3 H from the aqueous solution with an organic solvent in which the vanillin-.sup.3 H is preferentially soluble, and
- g. counting the radiation emitted from the vanillin-.sup.3 H.
A method for differentially analyzing the levels of epinephrine and/or norepinephrine obtained from a mammalian system selected from the group consisting of blood serum, plasma and urine which comprises
- a. contacting an aliquot of said mammalian system with the enzyme catechol-O-methyl transferase, the tritium labeled methyl donor S-adenosyl-L-methionine(.sup.3 H)methyl substantially free of unlabeled methyl donor, a cation of oxidation number +2 which allows the methyl transfer to proceed, a compound which stabilizes the enzyme-substrate system and an amount of ethylene glycol bis-(aminoethylether) -N, N- tetracetic acid sufficient to remove transferase-- inhibiting concentrations of calcium ions, to form a mixture,
- b. incubating the mixture for a time and at a temperature and pH sufficient to O-methylate (.sup.3 H) substantially all of the epinephrine and norepinephrine,
- c. extracting the O-methylated(.sup.3 H)-epinephrine and O-methylated(.sup.3 H)-norepinephrine with an organic solvent in which the O-methylated(.sup.3 H)-epinephrine and O-methylated(.sup.3 H)-norepinephrine are preferentially soluble,
- d. repartitioning the O-methylated (.sup.3 H) epinephrine and O-methylated (.sup.3 H) norepinephrine into an aqueous acid of sufficient strength to protonate the O-methylated (.sup.3 H)-epinephrine and O-methylated (.sup.3 H) norepinephrine being essentially residue free upon evaporation,
- e. separating O-methylated (.sup.3 H) epinephrine from O-methylated (.sup.3 H) norepinephrine by chromatographic means,
- f. oxidizing O-methylated (.sup.3 H) epinephrine and/or O-methylated (.sup.3 H) norepinephrine to vanillin-.sup.3 H in separate containers,
- g. extracting the vanillin-.sup.3 H into an organic solvent,
- h. counting the radiation emited from the vanillin-.sup.3 H obtained from the O-methylated (.sup.3 H) epinephrine and/or O-methylated (.sup.3 H) norepinephrine.
In a composition which comprises in combination catechol-O-methyl transferase, a cation of oxidation number +2 selected from the group consisting of magnesium, cobalt, and manganese, a compund which stabilizes the catechol-O-methyl transferase-catecholamine enzyme-substrate system which is selected from the group consisting of glutathione, dithiothreitol, ascorbic acid, sodium metabisulfite, mercaptoethanol and cysteine, a mammalian system selected from the group consisting of blood serum, plasma, and urine, the methyl donor S-adenosyl-L-methionine-(.sup.3 H) methyl, the transferase, cation, stabilizing compound and methyl donor present in such quantities that substantially all the epinephrine, norepinephrine, and dopamine present in the mammalian system are O-methylated;
- the improvement consisting of the presence of an amount of ethylene glycol bis(aminoethylether)-N,N'tetraacetic acid sufficient to remove transferase-inhibiting concentrations of calcium ions.
A method for analyzing the level of dopamine obtained from a mammalian system selected from the group consisting of blood serum, plasma and urine which comprises
- a. contacting an aliquot of said mammalian system with the enzyme catechol-O-methyl transferase, the tritium labeled methyl donor S-adenosyl-L-methionine(.sup.3 H)methyl substantially free of unlabeled methyl donor, a cation of oxidation number +2 which allows the methyl transfer to proceed, a compound which stabilizes the enzyme-substrate system and an amount of ethylene glycol bis(aminoethylether)-N,N-tetra acetic acid sufficient to remove transferase-inhibitting concentrations of calcium ions, to form a mixture,
- b. incubating the mixture for a time, and at a temperature and pH sufficient to O-methylate (.sup.3 H) substantially all of the epinephrine, norepinephrine and dopamine,
- c. extracting the O-methylated (.sup.3 H)-epinephrine, O-methylated (.sup.3 H)-norepinephrine and O-methylated (.sup.3 H) dopamine with an organic solvent in which the said O-methylated compounds are preferentially soluble,
- d. repartitioning the said O-methylated (.sup.3 H) compounds into an aqueous acid of sufficient strength to protonate the said O-methylated (.sup.3 H) compounds said acid being essentially residue free upon evaporation,
- e. separating the O-methylated (.sup.3 H)-dopamine from the O-methylated (.sup.3 H)-epinephrine,
- f. oxidizing a solution containing O-methylated (.sup.3 H)-norepinephrine and O-methylated (.sup.3 H)-dopamine with periodate, to form vanillin-.sup.3 H,
- g. extracting the vanillin-.sup.3 H into an organic solvent,
- h. raising the pH of the aqueous periodate phase above 7,
- i. extracting O-methylated (.sup.3 H)-dopamine from the aqueous periodate phase with an organic solvent,
- j. counting the radiation emitted from the O-methylated (.sup.3 H)dopamine.
A method for analyzing the level of dopamine obtained from a mammalian system selected from the group consisting of blood serum, plasma and urine which comprises
- a. contacting an aliquot of said mammalian system with the enzyme catechol-O-methyl transferase, the tritium labeled methyl donor S-adenosyl-L-methionine(.sup.3 H)methyl substantially free of unlabeled methyl donor, a cation of oxidation number +2 which allows the methyl transfer to proceed, a compound which stabilizes the enzyme-substrate system,
- b. incubating together the components of "a" for a time, and at a temperature and pH sufficient to O-methylate (.sup.3 H) substantially all of the epinephrine, norepinephrine and dopamine,
- c. extracting the O-methylated (.sup.3 H)-epinephrine, O-methylated (.sup.3 H)-norepinephrine and O-methylated (.sup.3 H) dopamine with an organic solvent in which the said O-methylated compounds are preferentially soluble,
- d. repartitioning the said O-methylated (.sup.3 H) compounds into an aqueous acid of sufficient strength to protonate the said O-methylated (.sup.3 H) compounds said acid being essentially residue free upon evaporation,
- e. separating the O-methylated (.sup.3 H)-dopamine from the O-methylated (.sup.3 H)-epinephrine and the O-methylated (.sup.3 H)-norepinephrine by chromatographic means,
- f. eluting the separated O-methylated (.sup.3 H)-dopamine,
- g. extracting the separated O-methylated (.sup.3 H)-dopamine from the eluant at a basic pH with an organic solvent, and
- h. counting the radiation emitted from the O-methylated (.sup.3 H)-dopamine.
A container having within the container a multiplicity of smaller containers and contents, said contents comprising the items enzyme catechol-O-methyl transferase, the labeled methyl donor S-adenosyl-L-methionine(.sup.3 H)methyl essentially free of unlabeled methyl donor, a cation of oxidation number +2 which allows a catechol-O-methyl transferase catalyzed O-methylation of a catecholamine to proceed, a compound which stabilizes a catechol-O-methyl-transferase-catecholamine enzyme substrate system and an amount of ethylene glycol bis-(aminoethylether)-N,N-tetra acetic acid sufficient to remove transferase-inhibitting concentrations of calcium ions with the proviso that the S-adenosyl-L-methionine(.sup.3 H)methyl does not have any other item in its smaller container and with the overall proviso that each item is of such a quantity that the enzyme catalyzed transfer of methyl from the labeled donor to the catecholamine, initiated from the bringing together of all the items at a proper pH and temperature and in the presence of a catecholamine sample from a mammalian system selected from the group consisting of blood serum, plasma and urine O-methylate (.sup.3 H) substantially all of the catecholamines in the sample.
In a composition comprising in combination catechol-O-methyl transferase, magnesium, glutathione, S-adenosyl-L-methionine (.sup.3 H) methyl, a human blood serum or plasma sample, the transferase, magnesium, glutathione and methyl donor present in such quantities that substantially all the epinephrine, norepinephrine and dopamine present in the human blood serum or plasma are O-methylated;
- the improvement consisting of the presence of an amount of ethylene glycol bis(aminoethylether)-N,N'tetraacetic acid sufficient to remove transferase-inhibitting concentrations of calcium ions.
In a composition which comprises the enzyme catechol-O-methyl transferase, a tritium labeled methyl donor S-adenosyl-L-methionine (.sup.3 H) methyl essentially free of unlabeled methyl donor, a cation of oxidation number +2 which allows the methyl transfer to proceed, a compound which stabilizes the enzyme-substrate system and a mammalian system selected from the group consisting of blood serum, plasma and urine, the transferase, cation, stabilizing compound and methyl donor present in such quantities that substantially all the epinephrine, norepinephrine and dopamine present in the mammalian system are O-methylated-(.sup.3 H);
- the improvement consisting of the presence of an amount of ethylene glycol bis (aminoethylether)-N,N-tetra acetic acid sufficient to remove transferase-inhibitting concentrations of calcium ions.
Dependencies are inferred from the claim text.