Method for determining a transaminase in a biological fluid and reagent combination for use in the method

Claim 2

In a method for determining glutamate oxalacetate transaminase or glutamate pyruvate transaminase present in a biological fluid, wherein L-glutamate is produced from alphaketoglutarate and an amino acid by transamination in the presence of the transaminase in the fluid, said amino acid being L-aspartate in the determination of glutamate oxalacetate transaminase and L-alanine in the determination of glutamate pyruvate transaminase, and the L-glutamate is oxidatively deaminated in a oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of the transaminase in the fluid, said fluid also containing an endogenous substance which reacts in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to produce reduced beta-nicotinamide adenine dinucleotide, thereby interfering with the determination, the improvement for obviating the interference caused by the endogenous substance which comprises: reacting said endogenous substance present in said fluid in an oxidation-reduction reaction with beta-nicotinamide adenine dinucleotide in the presence of glutamate hydrogenase to consume the endogenous substance by conversion thereof to an oxidation product of the reaction with the production of reduced beta-nicotinamide adenine dinucleotide, reacting the resulting reduced betanicotinamide adenine dinucleotide in said fluid with pyruvate in the presence of lactate dehydrogenase to return the reduced beta-nicotinamide adenine dinucleotide to its oxidized form with the reduction of pyruvate to lactate, thereafter inhibiting the lactate dehydrogenase activity in said fluid, and thereafter producing in said fluid a quantity of L-glutamate from alpha-ketoglutarate and said amino acid by said transamination for said oxidative deamination thereof and production of reduced beta-nicotinamide adenine dinucleotide in an amount proportional to the content of said transaminase in the fluid.

Claim 3

A method as defined in claim 1 wherein said biological fluid is human blood serum.

Claim 4

A method as defined in claim 2 wherein said endogenous substance comprises an amino acid.

Claim 5

A method as defined in claim 1 wherein the lactate dehydrogenase activity is inhibited by the anions of oxamic acid and oxalic acid.

Claim 6

A method as defined in claim 1 including the additional step of reacting the reduced beta nicotinamide adenine dinucleotide product with 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride to produce the reduced form of the latter compound in an amount proportional to the content of said transaminase in the fluid.

Claim 7

A method as defined in claim 5 wherein the reaction of said additional step is conducted in the presence of diaphorase.

Claim 10

A method as defined in claim 7 wherein said biological fluid is human blood serum and said endogenous substances include an amino acid.

Claim 11

A method as defined in claim 8 wherein in step (3), oxamate and oxalate are admixed with the resulting mixture to inhibit the lactate dehydrogenase enzymatic activity.

Claim 12

A method as defined in claim 7 including the additional step of providing in the resulting mixture diaphorase and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride to produce the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 13

A method as defined in claim 10 wherein said biological fluid is human blood serum and said endogenous substances include an amino acid, and in step (3), oxamate and oxalate are admixed with the resulting mixture to inhibit the lactate dehydrogenase enzymatic activity.

Claim 16

A method as defined in claim 12 wherein said biological fluid is human blood serum and said endogenous substances include an amino acid.

Claim 17

A method as defined in claim 13 wherein in step (3), oxamate and oxalate are admixed with the resulting mixture to inhibit the lactate dehydrogenase enzymatic activity.

Claim 18

A method as defined in claim 12 including the additional step of providing in the resulting mixture diaphorase and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride to produce the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 19

A method as defined in claim 15 wherein said biological fluid is human blood serum and said endogenous substances include an amino acid, and in step (3), oxamate and oxalate are admixed with the resulting mixture to inhibit the lactate dehydrogenase enzymatic activity.

Claim 20

A two-reagent assay system for determining glutamate oxalactate transaminase present in a biological fluid, which system includes a first reagent comprising beta-nicotinamide adenine dinucleotide, glutamate dehydrogenase, pyruvate, and lactate dehydrogenase, said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising oxamate and oxalate, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase, and each of L-aspartate, alpha-ketoglutarate, diaphorase, and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride included in one of said reagents, said diaphorase and said 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride being included in different reagents, for then producing the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 21

A two-reagent assay system for determining glutamate oxalactate transaminase present in a biological fluid, which system includes a first reagent comprising beta-nicotinamide adenine dinucleotide, glutamate dehydrogenase, pyruvate, and lactate dehydrogenase, said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising oxamate and oxalate, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase, and each of L-aspartate, alpha-ketoglutarate, diaphorase, and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride included in one of said reagents, said diaphorase and said 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride being included in different reagents, for then producing the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 22

A two-reagent assay system for determining glutamate oxalacetate transaminase present in a biological fluid, which system includes a first reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal L-aspartate 100-200 millimoles Akali metal pyruvate 0.25-1 millimole Alkali metal adenosine-5'-diphosphate 1-5 millimoles Beta-nicotinamide adenine dinucleotide 2-5 millimoles Glutamate dehydrogenase 40-80 I.U. × 10.sup.3 Lactate dehydrogenase 0.2-0.5 I.U. × 10.sup.3 Diaphorase 1-3 I.U. × 10.sup.3 ______________________________________ said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing the beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal alpha-ketoglutarate 10-50 millimoles Alkali metal oxalate 25-75 millimoles Alkali metal oxamate 10-30 millimoles 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyl- tetrazolium chloride 0.2-1 millimole ______________________________________ each of said reagents also including 25-150 millimoles of a buffer selected from the group consisting of alkali metal phosphate, tris(hydroxymethyl)aminomethane, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase and then producing the reduced form of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride in an amount proportional to the content of said transaminase in said fluid.

Claim 23

A two-reagent assay system for determining glutamate oxalacetate transaminase present in a biological fluid, which system includes a first reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal L-aspartate 100-200 millimoles Akali metal pyruvate 0.25-1 millimole Alkali metal adenosine-5'-diphosphate 1-5 millimoles Beta-nicotinamide adenine dinucleotide 2-5 millimoles Glutamate dehydrogenase 40-80 I.U. × 10.sup.3 Lactate dehydrogenase 0.2-0.5 I.U. × 10.sup.3 Diaphorase 1-3 I.U. × 10.sup.3 ______________________________________ said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing the beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal alpha-ketoglutarate 10-50 millimoles Alkali metal oxalate 25-75 millimoles Alkali metal oxamate 10-30 millimoles 2-p-Iodophenyl-3-p-nitrophenyl-5-phenyl- tetrazolium chloride 0.2-1 millimole ______________________________________ each of said reagents also including 25-150 millimoles of a buffer selected from the group consisting of alkali metal phosphate, tris(hydroxymethyl)aminomethane, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase and then producing the reduced form of 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride in an amount proportional to the content of said transaminase in said fluid.

Claim 24

An assay system as defined in claim 18 wherein each of said reagents has a pH of 7.3-8.

Claim 25

A two-reagent assay system for determining glutamate pyruvate transaminase present in a biological fluid, which system includes a first reagent comprising beta-nicotinamide adenine dinucleotide, glutamate dehydrogenase, pyruvate, and lactate dehydrogenase, said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising oxamate and oxalate, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase, and each of L-alanine, alpha-ketoglutarate, diaphorase, and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride included in one of said reagents, said diaphorase and said 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chlorie being included in different reagents, for then producing the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 26

A two-reagent assay system for determining glutamate pyruvate transaminase present in a biological fluid, which system includes a first reagent comprising beta-nicotinamide adenine dinucleotide, glutamate dehydrogenase, pyruvate, and lactate dehydrogenase, said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising oxamate and oxalate, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase, and each of L-alanine, alpha-ketoglutarate, diaphorase, and 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride included in one of said reagents, said diaphorase and said 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chlorie being included in different reagents, for then producing the reduced form of the latter compound in an amount proportional to the content of said transaminase in said fluid.

Claim 27

A two-reagent assay system for determining glutamate pyruvate transaminase present in a biological fluid, which system includes a first reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ L-Alanine 150-300 millimoles Alkali metal pyruvate 0.25-1 millimole Alkali metal adenosine-5'-diphosphate 1-5 millimoles Beta-nicotinamide adenine dinucleotide 2-5 millimoles Glutamate dehydrogenase 40-80 I.U. × 10.sup.3 Lactate dehydrogenase 0.2-0.5 I.U. × 10.sup.3 Diaphorase 1-3 I.U. × 10.sup.3 ______________________________________ said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing the beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal alpha-ketoglutarate 10-50 millimoles Aklali metal oxalate 25-75 millimoles Alkali metal oxamate 10-30 millimoles 2-p-Iodophenyl-3-p-nitrophenyl- 5-phenyl tetrazolium chloride 0.2-1 millimole ______________________________________ each of said reagents also including 25-150 millimoles of a buffer selected from the group consisting of alkali metal phosphate, tris(hydroxymethyl)aminomethane, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase and then producing the reduced form of 2-p-iodophenyl-3-p -nitrophenyl-5-phenyl tetrazolium chloride in an amount proportional to the content of said transaminase in said fluid.

Claim 28

A two-reagent assay system for determining glutamate pyruvate transaminase present in a biological fluid, which system includes a first reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ L-Alanine 150-300 millimoles Alkali metal pyruvate 0.25-1 millimole Alkali metal adenosine-5'-diphosphate 1-5 millimoles Beta-nicotinamide adenine dinucleotide 2-5 millimoles Glutamate dehydrogenase 40-80 I.U. × 10.sup.3 Lactate dehydrogenase 0.2-0.5 I.U. × 10.sup.3 Diaphorase 1-3 I.U. × 10.sup.3 ______________________________________ said first reagent to be admixed with said fluid initially for oxidizing endogenous substances present in the fluid so as not to interfere with the determination and then providing a product containing the beta-nicotinamide adenine dinucleotide in its oxidized form, and a second reagent comprising an aqueous solution of the following materials in the proportions indicated per liter of solution: ______________________________________ Material Amount ______________________________________ Alkali metal alpha-ketoglutarate 10-50 millimoles Aklali metal oxalate 25-75 millimoles Alkali metal oxamate 10-30 millimoles 2-p-Iodophenyl-3-p-nitrophenyl- 5-phenyl tetrazolium chloride 0.2-1 millimole ______________________________________ each of said reagents also including 25-150 millimoles of a buffer selected from the group consisting of alkali metal phosphate, tris(hydroxymethyl)aminomethane, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, said second reagent to be admixed with said product for inhibiting the enzymatic activity of said lactate dehydrogenase and then producing the reduced form of 2-p-iodophenyl-3-p -nitrophenyl-5-phenyl tetrazolium chloride in an amount proportional to the content of said transaminase in said fluid.

Claim 29

An assay system as defined in claim 21 wherein each of said reagents has a pH of 7.3-8.