# US 4,394,449 A · Stabilization of coenzymes in aqueous solution > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,394,449 A - Document ID: US4394449A - Status: Granted - Kind: A - Application number: 06/121,225 - Filed: 1980-02-13 - Granted: 1983-07-19 - Earliest claimed: 1980-02-13 - Inventors: Ivan E. Modrovich - Web page: https://patentgrep.com/patent/US4394449A ## CPC - C12Q 1/58: involving urea or urease - C12P 1/00: Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes - C12Q 1/008: {for determining co-enzymes or co-factors, e.g. NAD, ATP} - C12Q 1/48: involving transferase - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} ## Abstract A stabilized aqueous coenzyme solution is disclosed for use in the clinical assay of a selected biological constituent through the enzymic reaction of an enzyme with a selective substrate for the enzyme and a coenzyme. The aqueous coenzyme solution comprises at least one first enzyme and a first substrate for selectively reacting with the first enzyme in the assay, a coenzyme for interacting with the first enzyme and first substrate in the assay which coenzyme degrades forming a coenzyme conversion product, and at least one second enzyme and second substrate selective for the second enzyme which react with the coenzyme conversion product to regenerate the coenzyme. ## Claims ### Claim 1 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 2 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); .alpha.-hydroxybuteric dehydrogenase (.alpha.-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 3 (depends on 1, 2) 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 4 (depends on 1, 2) 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 5 (depends on 1, 2) 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, .alpha.-HBD and BUN and wherein the coenzyme is NADH. ### Claim 6 (depends on 5) 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 7 (depends on 5) A stabilized aqueous coenzyme solution as recited in claim 5 wherein the enzyme and substrate system comprises glucose dehydrogenase and glucose. ### Claim 8 (depends on 1, 2) 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 9 (depends on 8) A stabilized aqueous coenzyme solution as recited in claim 8 wherein the enzyme and substrate system comprises ammonium sulfate, .alpha.-ketoglutarate and glutamate dehydrogenase. ### Claim 10 (depends on 1, 2, 6, 7, 9) 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 11 (depends on 1, 2) A stabilized coenzyme solution as claimed in claim 1 or 2 in which at least one polyhydroxy organic compound is present. ### Claim 12 (depends on 10) A stabilized coenzyme solution as claimed in claim 10 in which a polyhydroxy organic compound is present. ### Claim 13 A stabilized aqueous coenzyme solution for use in the biological assay of BUN, the aqueous coenzyme solution comprising urease, .alpha.-ketoglutarate, GLDH, ADP, glycerol, NADH, ATP, glucose, hexokinase, and G-6-PDH dissolved in an aqueous solution of triethanolamine. ### Claim 14 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 .alpha.-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 15 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; .alpha.-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 16 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; .alpha.-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 17 A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; .alpha.-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 18 A stabilized aqueous coenzyme solution for use in the biological assay of SGPT, the aqueous coenzyme solution comprising L-alanine; .alpha.-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 19 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 20 A method of stabilizing a labile coenzyme in an aqueous clinical assay solution comprising at least one first enzyme, selective first substrate for reacting with the first enzyme and a labile coenzyme for reacting with the first enzyme and first substrate in a diagnostic assay of a selected biological constituent, said coenzyme degrading in aqueous solution to produce a coenzyme conversion product, the method comprising adding to the clinical assay solution an enzyme and substrate system comprising at least one second enzyme and second substrate which selectively react with the coenzyme conversion product to produce the coenzyme. ### Claim 21 (depends on 20) A method as recited in claim 20 further comprising adding to the clinical assay solution coenzyme conversion product. ### Claim 22 A method of stabilizing a labile coenzyme for use in an aqueous clinical assay of a selected biological constituent, which coenzyme degrades to a coenzyme conversion product, the method comprising the steps of: forming an aqueous substrate solution comprising at least one first enzyme and first substrate for selectively reacting with said first enzyme in the assay of the selected biological constituent and an enzyme and substrate system comprising at least one second enzyme and second substrate capable of reacting with the coenzyme conversion product for regenerating the coenzyme; forming a coenzyme solution comprising a nonaqueous solution of the labile coenzyme useful in the clinical assay; and combining the substrate solution and coenzyme solution forming a stabilized coenzyme solution for use in an aqueous clinical assay of a selected biological constituent. ### Claim 23 A method of stabilizing the labile coenzyme NAD in an aqueous clinical assay solution comprising at least one first enzyme, selective first substrate for reacting with the first enzyme and a labile coenzyme for reacting with the first enzyme and first substrate in a diagnostic assay of a selected biological constituent, said coenzyme degrading in aqueous solution to produce a coenzyme conversion product, the method comprising providing to the clinical assay solution in which the labile coenzyme is NAD an enzyme and substrate system comprising at least one second enzyme and second substrate which selectively react with the coenzyme conversion product to form NAD, said provided enzyme and substrate system comprising ammonium sulfate, .alpha.-ketoglutarate and dehydrogenase. ### Claim 24 (depends on 23) A method as recited in claim 23 further comprising adding to the clinical assay solution coenzyme conversion product. ### Claim 25 A method of stabilizing the labile coenzyme NADH in an aqueous clinical assay solution comprising at least one first enzyme, selective first substrate for reacting with the first enzyme and a labile coenzyme for reacting with the first enzyme and first substrate in a diagnostic assay of a selected biological constituent, said coenzyme degrading in aqueous solution to produce a coenzyme conversion product, the method comprising providing in the clinical assay solution in which the labile coenzyme is NADH an enzyme and substrate system comprising at least one second enzyme and second substrate which selectively react with the coenzyme conversion product to form NADH, said provided enzyme and substrate system being selected from the system comprising ATP, glucose, hexokinase and G-6-PDH and the system comprising glucose dehydrogenase and glucose. ### Claim 26 (depends on 25) A method as recited in claim 25 further comprising adding to the clinical assay solution coenzyme conversion product. ### Claim 27 (depends on 23, 25) A method as claimed in claim 23 or 25 in which the formed aqueous clinical assay solution contains a polyhydroxy organic compound.