Stabilization of coenzymes in aqueous solution

Claim 2

A stabilized aqueous coenzyme solution for use in the clinical assay of a selected biological constituent through the enzymic reaction of an enzyme or enzymes with selective substrates for the enzymes and a labile coenzyme, said aqueous coenzyme solution comprising: (a) at least one first enzyme and a first substrate for selectively reacting with the first enzyme in the assay of the selected biological constituent; (b) a labile coenzyme for interacting with the first enzyme and first substrate, which labile coenzyme degrades in said aqueous solution to a coenzyme conversion product; and (c) an enzyme and substrate system comprising at least one second enzyme and a selective second substrate for the second enzyme, which second enzyme and second substrate react in said aqueous enzyme solution with the coenzyme conversion product to form the coenzyme.

Claim 3

A stabilized aqueous coenzyme solution for use in a clinical assay of a biological constituent selected from the group consisting of: glutamic-oxalacetic transaminase (SGOT); glutamic-pyruvic transaminase (SGPT); lactic dehydrogenase (LDH); creatine phosphokinase (CPK); α-hydroxybuteric dehydrogenase (α-HBD); glucose (via hexokinase-G-6-PDH); glucose (via glucose dehydrogenase); blood urea nitrogen (BUN); and triglycerides through the reaction of an enzyme or enzymes with a selective substrate for the enzymes, and a coenzyme, said aqueous coenzyme solution comprising: (a) at least one first enzyme and a first substrate for selectively interacting with the first enzyme in the assay for the selected biological constituent; (b) a labile coenzyme for interacting with the first enzyme and substrate in the assay to form a coenzyme conversion product and which labile coenzyme degrades in said aqueous solution to said coenzyme reaction product; and (c) an enzyme and substrate system comprising at least one second enzyme and a selective second substrate for the second enzyme which react in said aqueous coenzyme solution with the coenzyme conversion product of degradation to regenerate the coenzyme.

Claim 4

A stabilized aqueous coenzyme solution for use in a clinical assay of a biological constituent selected from the group consisting of: glutamic-oxalacetic transaminase (SGOT); glutamic-pyruvic transaminase (SGPT); lactic dehydrogenase (LDH); creatine phosphokinase (CPK); α-hydroxybuteric dehydrogenase (α-HBD); glucose (via hexokinase-G-6-PDH); glucose (via glucose dehydrogenase); blood urea nitrogen (BUN); and triglycerides through the reaction of an enzyme or enzymes with a selective substrate for the enzymes, and a coenzyme, said aqueous coenzyme solution comprising: (a) at least one first enzyme and a first substrate for selectively interacting with the first enzyme in the assay for the selected biological constituent; (b) a labile coenzyme for interacting with the first enzyme and substrate in the assay to form a coenzyme conversion product and which labile coenzyme degrades in said aqueous solution to said coenzyme reaction product; and (c) an enzyme and substrate system comprising at least one second enzyme and a selective second substrate for the second enzyme which react in said aqueous coenzyme solution with the coenzyme conversion product of degradation to regenerate the coenzyme.

Claim 5

A stabilized aqueous coenzyme solution as recited in claim 1 or 2 wherein the enzyme and substrate system comprises at least one second enzyme and selective second substrate different from the first enzyme and first substrate present in the solution for the assay of the selected biological constituent.

Claim 6

A stabilized aqueous coenzyme solution as recited in claim 1 or 2 wherein the coenzyme is selected from the group consisting of NADH and NAD.

Claim 7

A stabilized aqueous coenzyme solution as recited in claim 1 or 2 wherein the selected biological constituent assayed is selected from the group consisting of SGOT, SGPT, LDH, α-HBD and BUN and wherein the coenzyme is NADH.

Claim 8

A stabilized aqueous coenzyme solution as recited in claim 5 wherein the enzyme and substrate system comprises ATP, glucose, hexokinase and G-6-PDH.

Claim 9

A stabilized aqueous coenzyme solution as recited in claim 5 wherein the enzyme and substrate system comprises glucose dehydrogenase and glucose.

Claim 10

A stabilized aqueous coenzyme solution as recited in claim 1 or 2 wherein the selected biological constituent assayed is selected from the group consisting of CPK, glucose (via hexokinase-G-6-PDH) and glucose (via glucose dehydrogenase).

Claim 11

A stabilized aqueous coenzyme solution as recited in claim 8 wherein the enzyme and substrate system comprises ammonium sulfate, α-ketoglutarate and glutamate dehydrogenase.

Claim 12

A stabilized aqueous coenzyme solution as recited in claim 1, 2, 6, 7 or 9 wherein the enzyme and substrate system further comprises coenzyme conversion product.

Claim 13

A stabilized coenzyme solution as claimed in claim 1 or 2 in which at least one polyhydroxy organic compound is present.

Claim 14

A stabilized coenzyme solution as claimed in claim 10 in which a polyhydroxy organic compound is present.

Claim 15

A stabilized aqueous coenzyme solution for use in the biological assay of BUN, the aqueous coenzyme solution comprising urease, α-ketoglutarate, GLDH, ADP, glycerol, NADH, ATP, glucose, hexokinase, and G-6-PDH dissolved in an aqueous solution of triethanolamine.

Claim 16

A stabilized aqueous coenzyme solution for use in the biological assay of BUN, the aqueous coenzyme solution comprising urease, α-ketoglutarate, GLDH, ADP, glycerol, NADH, ATP, glucose, hexokinase, and G-6-PDH dissolved in an aqueous solution of triethanolamine.

Claim 17

A stabilized aqueous coenzyme solution for use in the biological assay of BUN, the aqueous coenzyme solution comprising from about 100 to about 10,000 IU/l urease; from about 0.1 to about 5 g/l α-ketoglutarate; from about twice the urease activity to about 60,000 IU/l GLDH; about 0.1 to about 20 g/l ADP; about 5 to about 40 percent by volume glycerol; at least about 0.25 mM of NADH; about 10 mg/l to about 10 g/l ATP; about 10 mg/l to about 10 g/l glucose; about 1 IU/l to about 50 IU/l hexokinase; and G-6-PDH in an amount providing an activity about one to about ten times the activity of the hexokinase dissolved in about a 0.025 to about 1 molar aqueous solution of triethanolamine.

Claim 18

A stabilized aqueous coenzyme solution for use in the biological assay of BUN, the aqueous coenzyme solution comprising from about 100 to about 10,000 IU/l urease; from about 0.1 to about 5 g/l α-ketoglutarate; from about twice the urease activity to about 60,000 IU/l GLDH; about 0.1 to about 20 g/l ADP; about 5 to about 40 percent by volume glycerol; at least about 0.25 mM of NADH; about 10 mg/l to about 10 g/l ATP; about 10 mg/l to about 10 g/l glucose; about 1 IU/l to about 50 IU/l hexokinase; and G-6-PDH in an amount providing an activity about one to about ten times the activity of the hexokinase dissolved in about a 0.025 to about 1 molar aqueous solution of triethanolamine.

Claim 19

A stabilized aqueous coenzyme solution for use in the biological assay of SGOT, the aqueous coenzyme solution comprising L-aspartic acid; tris(hydroxymethyl)aminomethane; gelatin; sodium azide; glycerol; MDH; LDH; α-ketoglutarate, glucose; glucose dehydrogenase; NADH; NAD; and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 20

A stabilized aqueous coenzyme solution for use in the biological assay of SGOT, the aqueous coenzyme solution comprising L-aspartic acid; tris(hydroxymethyl)aminomethane; gelatin; sodium azide; glycerol; MDH; LDH; α-ketoglutarate, glucose; glucose dehydrogenase; NADH; NAD; and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 21

A stabilized aqueous coenzyme solution for use in the biological assay of SGOT, the aqueous coenzyme solution comprising L-aspartic acid; tris(hydroxymethyl)aminomethane; gelatin, sodium azide; glycerol; MDH; LDH; α-ketoglutarate; glucose; hexokinase; G-6-PDH; NADH; ATP and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, and NaOH and mixture thereof.

Claim 22

A stabilized aqueous coenzyme solution for use in the biological assay of SGOT, the aqueous coenzyme solution comprising L-aspartic acid; tris(hydroxymethyl)aminomethane; gelatin, sodium azide; glycerol; MDH; LDH; α-ketoglutarate; glucose; hexokinase; G-6-PDH; NADH; ATP and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, and NaOH and mixture thereof.

Claim 23

A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; α-ketoglutarate; LDH; tris(hydroxymethyl) aminomethane; gelatin; sodium azide; glycerol; glucose; glucose dehydrogenase; NADH; NAD and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 24

A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; α-ketoglutarate; LDH; tris(hydroxymethyl) aminomethane; gelatin; sodium azide; glycerol; glucose; glucose dehydrogenase; NADH; NAD and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 25

A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; α-ketoglutarate; LDH; tris(hydroxymethyl)aminomethane; gelatin, sodium azide; glycerol; glucose; hexokinase; G-6-PDH; NADH; ATP and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 26

A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; α-ketoglutarate; LDH; tris(hydroxymethyl)aminomethane; gelatin, sodium azide; glycerol; glucose; hexokinase; G-6-PDH; NADH; ATP and 1,2-propanediol in an aqueous solution having a pH of about 7.8±0.2 provided by a buffering agent selected from HCl, NaOH and mixture thereof.

Claim 27

A stabilized aqueous coenzyme solution for use in the biological assay of CPK, the aqueous coenzyme solution comprising creatine phosphate; ADP; AMP; G-6-PDH; glycerol; sodium azide; phosphate tris buffer; glutamate dehydrogenase; NAD; AMP; hexokinase; magnesium acetate; tris(hydroxymethyl)aminomethane; glucose; N-acetyl cysteine; dithiothreitol; imidazole; and ammonium sulfate.

Claim 28

A stabilized aqueous coenzyme solution for use in the biological assay of CPK, the aqueous coenzyme solution comprising creatine phosphate; ADP; AMP; G-6-PDH; glycerol; sodium azide; phosphate tris buffer; glutamate dehydrogenase; NAD; AMP; hexokinase; magnesium acetate; tris(hydroxymethyl)aminomethane; glucose; N-acetyl cysteine; dithiothreitol; imidazole; and ammonium sulfate.

Claim 31

A method as recited in claim 20 further comprising adding to the clinical assay solution coenzyme conversion product.

Claim 36

A method as recited in claim 23 further comprising adding to the clinical assay solution coenzyme conversion product.

Claim 39

A method as recited in claim 25 further comprising adding to the clinical assay solution coenzyme conversion product.

Claim 40

A method as claimed in claim 23 or 25 in which the formed aqueous clinical assay solution contains a polyhydroxy organic compound.