# US 5,700,653 A · Liquid stable thiol activator > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,700,653 A - Document ID: US5700653A - Status: Granted - Kind: A - Application number: 08/541,473 - Filed: 1995-10-09 - Granted: 1997-12-23 - Earliest claimed: 1994-05-06 - Assignees: Beckman Instruments, Inc. - Inventors: Carrie J. Lu; Fredrick S. Yein - Web page: https://patentgrep.com/patent/US5700653A ## CPC - C12Q 1/50: involving creatine phosphokinase - 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 ## Abstract An aqueous, liquid-stable thiol activator solution is disclosed. The solution can include a thiol activator, such as N-acetyl-L-cysteine and a stabilizing agent, such as mannitol. The thiol is stabilized in the aqueous solution for at least about one year under storage conditions and for at least about 28 days under open-bottle conditions. The solution can be used as a reactivator of creatine kinase catalytic activity. ## Claims ### Claim 1 A stabilized thiol activating composition, comprising: (a) a thiol which activates an enzyme; (b) a stabilizing agent which stabilizes the thiol, wherein the stabilizing agent is a free radical scavenger; (c) magnesium ion for facilitating activation of the enzyme; and (d) a liquid which solubilizes the thiol, the stabilizing agent, and the magnesium ion, wherein the thiol is stabilized in the liquid by the stabilizing agent, and wherein the composition is substantially free of any creatine kinase, said free radical scavenger is selected from the group consisting of inositol, tocopherol, mannitol, superoxide dismutase, catalase, glutathione peroxidase, N-2-mercaptopropionyl glycine, dimethyl thiourea, glutathione, 21-aminosteroids, deferoxamine, allopurinol, dimethyl sulfoxide and coenzyme Q. ### Claim 2 (depends on 1) The composition of claim 1, wherein the thiol is selected from the group consisting of glutathione, 2-aminoethylisothiouronium bromide, thioglycolic acid, cysteine, mercaptoethanol, dithiothreitol, monothioglycerol and N-acetyl-L-cysteine. ### Claim 3 (depends on 2) The composition of claim 2, wherein the thiol is N-acetyl-L-cysteine. ### Claim 4 (depends on 1) The composition of claim 1, further comprising an iron chelator for removing undesired iron ions from the liquid. ### Claim 5 (depends on 4) The composition of claim 4, wherein the iron chelator is present in the liquid in a concentration of from about 0.025 mmol/L to about 4 mmol/L. ### Claim 6 (depends on 4) The composition of claim 4, wherein the chelator is selected from the group consisting of ethylene diamine tetra acetic acid, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate and 2,2-bis(hydroxyethyl)-2,2',2"-nitrilotriethanol. ### Claim 7 (depends on 1) The composition of claim 1, wherein the thiol retains a substantially undiminished thiol activation ability for at least about one year under storage conditions. ### Claim 8 (depends on 1) The composition of claim 1, wherein the thiol retains a substantially undiminished thiol activation ability for at least about two years under storage conditions. ### Claim 9 (depends on 1) The composition of claim 1, wherein the thiol retains a substantially undiminished thiol activation ability for at least about 28 days under open bottle conditions. ### Claim 10 (depends on 1) The composition of claim 1, wherein the thiol retains a substantially undiminished thiol activation ability for at least about 60 days under open bottle conditions. ### Claim 11 (depends on 1) The composition of claim 1, wherein the stabilizing agent is present in the liquid at a concentration of from about composition 5 mmol/L to about 550 mmol/L. ### Claim 12 (depends on 1) The composition of claim 1, wherein the free radical scavenger is inositol. ### Claim 13 (depends on 1) The composition of claim 1, wherein the liquid comprises a buffered aqueous solution capable of maintaining the liquid at a pH between about pH 6 and pH 8. ### Claim 14 A method for making a liquid stabilized thiol activating solution, comprising the steps of: (a) selecting a thiol which activates an enzyme; (b) adding the thiol to a liquid which solubilizes the thiol; (c) adding magnesium ion to the liquid; and (d) adding a stabilized agent to the liquid, the stabilizing agent stabilizing the thiol solubilized in the liquid, wherein the stabilizing agent is a free radical scavenger selected from the group consisting of inositol, tocopherol, mannitol, superoxide dismutase, catalase, glutathione peroxidase, N-2-mercaptopropionyl glycine, dimethyl thiourea, glutathione, 21-aminosteroids, deferoxamine, allopurinol, dimethyl sulfoxide and coenzyme Q, thereby making a liquid stable thiol solution wherein the thiol is stable in the solution and retains its thiol activation ability substantially undiminished for at least about one year under storage conditions and for at least about 28 days under open bottle conditions; said solution being substantially free of treating kinase. ### Claim 15 A method for preparing a stabilized aqueous activating solution of N-acetyl-L-cysteine, comprising the steps of: (a) selecting an aqueous buffer; (b) adding ethylene diamine tetra acetate to an aqueous buffer; (c) adding N-acetyl-L-cysteine to the buffer; (d) adding magnesium ion to the buffer; and (e) adding mannitol to the buffer, wherein the buffer is capable of solubilizing the ethylene diamine tetra acetate and the mannitol, the method resulting in a stable aqueous solution of N-acetyl-L-cysteine, said solution being substantially free of creatine kinase. ### Claim 16 An assay for creatine kinase activity, comprising the steps of: (a) preparing a liquid stable thiol solution by adding ethylene diamine tetra acetate, N-acetyl-L-cysteine, magnesium ion, and mannitol to an aqueous buffer; (b) adding a test sample of a physiological fluid to the liquid stable thiol, wherein the physiological fluid is selected from the group consisting of blood, plasma, serum, urine, amniotic, pleural and cerebrospinal fluid; (c) spectrophotometrically detecting a change in light absorption of the solution; and (d) correlating the creatine kinase activity in the test sample with the change in light absorption. ### Claim 17 A liquid stabilized thiol activating composition comprising: (a) a thiol which activates an enzyme; (b) a free radical scavenger which stabilizes the thiol; (c) magnesium ion; (d) iron chelator; and (e) an aqueous buffer which solubilizes the thiol, the free radical scavenger, the magnesium ion and the iron chelator in the buffer, to thereby form a liquid stable thiol solution stable against significant deterioration of the thiol present in the solution for at least about one year under storage conditions and for at least about 28 days under open bottle conditions, said solution being substantially free of any creatine kinase, and wherein said free radical scavenger is selected from the group consisting of inositol, tocopherol, mannitol, superoxide dismutase, catalase, glutathione peroxidase, N-2-mercaptopropionyl glycine, dimethyl thiourea, glutathione, 21-aminosteroids, deferoxamine, allopurinol, dimethyl sulfoxide and coenzyme Q. ### Claim 18 A reagent for a creatine kinase assay, comprising: (a) an aqueous buffer; (b) an enzyme activating amount of N-acetyl-L-cysteine; (c) between about 0.025 mmol/L and about 4 mmol/L ethylene diamine tetra acetic add; (d) between about 5 mmol/L and about 550 mmol/L mannitol; and (e) about 12 mmol/L magnesium ion, said reagent being substantially free of creatine kinase.