Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
  • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase

Definitions

• Stable, lyophilized diagnostic compositions are disclosed effective indetermining glutamic oxalic transaminase (GOT) and glutamic pyruvic transaminase (GPT) comprising malate dehydrogenase (for GOT) or lactate dehydrogenase (for GPT), a pyridine nucleotide and a buffer having a sodium content of less than about 600 meq./ 1. capable of regulating the pH of the composition in reconstituted form. Also disclosed are the aqueous reconstituted products, the method of determining the activity of GOT and GPT in a sample using the subject compositions and the process for making the disclosed diagnostic compositions.
• This invention relates generally to stable, lyophilized diagnostic compositions. More particularly, it relates to unusually stable novel diagnostic compositions useful in determining the activity of glutamic oxalic transaminase (GOT) and glutamic pyruvic transaminase (GP'T) in samples containing said enzymes.
• GOT levels Generally, if any heart muscle damage has taken place or is occurring, this will manifest itself as an increase in GOT levels. In addition to signaling heart disease, elevated GOT levels also indicate the possibility of severe liver disfunctions such as cirrhosis, cancer of the liver or hepatitis.
• diagnostic reagents are commonly prepared and used in the reconstituted state i.e., as an aqueous solution. It is unusual to observe any substantial degree of stability in diagnostic reagents in this form.
• compositions overcome these severe handicaps, the most crucial being the stability aspect.
• the reagent be used within a relatively short period of time. If not, decomposition which takes place renders the material totally useless and it must be discarded.
• the present invention does disclose such compositions, their effective use and the means for preparing same.
• this invention relates to two stable, lyophilized diagnostic compositions.
• the first is highly effective in determining glutamic oxalic transaminase (GOT) and comprises malate dehydrogenase (MDH); a pyridine nucleotide such as reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH); and a buffer having a sodium content of less than about 600 meq./l. capable of regulating the pH of the reconstituted product.
• GAT glutamic oxalic transaminase
• MDH malate dehydrogenase
• NADH reduced nicotinamide adenine dinucleotide
• NADPH reduced nicotinamide adenine dinucleotide phosphate
• the second is highly effective in determining glutamic pyruvic transaminase (GPT) and comprises lactate dehydrogenase (LDH); a pyridine nucleotide such as reduced nicotinamide adenine dinucleotide (NADH) orreduced nicotinamide adenine dinucleotide phosphate (NADPH); and a buffer having a sodium content of less than about 600 meq./ l. capable of regulating the pH of the reconstituted product.
• LDH lactate dehydrogenase
• NADH reduced nicotinamide adenine dinucleotide
• NADPH nicotinamide adenine dinucleotide phosphate
• each of these compositions is preferred and is prepared simply by adding a predetermined amount of water resulting in a solution having a pH preferably in the range from 6.0 to 8.0. It is possible and within the purview of this invention toprepare an intermediate reconstituted product outside the pH range of 6 to 8 which can then be adjusted subse quently by adding more buffer thereto.
• the dry reagent can be used in the analytical determination,
• compositions concerns the use of NADH as the pyridine nucleotide component.
• the reconstituted forms of these compositions are characterized by critical properties and limitations which must be present in order to have a diagnostic reagent having the highly desirable advantages described earlier.
• the MDH must be present in an amount to provide an activity of from about 0.5 units/ml. to about 10 units/mL, with a 3 units/ml. activity being the most preferred.
• the corresponding LDH activity upon reconstitution must be present in an amount to provide activity of from 8 units/ml. to about units/ml, with a 40 units/ml. activity being most pre ferred.
• the amount of pyridine nucleotide, preferable NADH, is present in both reconstituted compositions in an amount to obtain an optical density in the range of from 2.1 to 1.0 spectrophotometrically determined at340 nm using a 10 mm. light path. Most preferred is an optical density of 1.8.
• the buffer in both reconstituted compositions has a sodium content less than 600 meq./ l. and is capable of regulating the pH of the reconstituted product.
• sodium phosphate alone is used as the buffer, the amount used is dictated by the sodium content in the reconstituted product. It should not surpass a concentration of 600 meq./l. and is preferably about 400 meq./ I.
• potassium phosphate When potassium phosphate is the buffer, it is added in an amount to provide a concentration of from 0.05 M to 0.4 M, with a 0.1 M concentration most preferred.
• buffer which is of choice for purposes of this invention are salts derived from strong bases and weak acids.
• Sodium and potassium phosphate are typical examples.
• Others of this type include ammonium acetate, lithium phosphate and sodium borate.
• compositions concern the inclusion of albumin.
• concentration upon reconstitution is about 0. l 5 g./ 100 ml.
• compositions in the quantitative procedures for determining the particular enzyme.
• the usual procedure consists of mixing the sample with a substrate and subsequently analyzing for enzyme. This is usually effected in an instrument devised for that purpose.
• the inventive embodiment comprises combining the herein disclosed diagnostic composition with the reaction product and photometrically measuring the decrease in NADH concentration.
• the substrate is preferably an aspartic acid-a-ketoglutaric acid mixture whereas in the GPT determination, the substrate is preferably an alanine-a-ketoglutaric acid mixture.
• Another important embodiment of this invention deals with the preparation of the stable, lyophilized diagnostic compositions.
• the process is the same for both and comprises the commingling of MDH or LDH with pyridine nucleotide and buffer.
• the resulting aqueous solution is lyophilized.
• the reconstituted products having a pH in the range from 6.0 to 8.0 are characterized by the limiting properties described above.
• the amount of LDH which one adds to the GPT diagnostic reagent is calculated by determining the activity of a reconstituted product. For purposes of this invention, it is found that the reconstituted product should possess an LDH activity of 8 units/ml. to about 80 units/ml. An activity of 40 units/ml is preferred.
• NADH and NADPH are well known co-factors and are particularly useful in enzymatic reactions requiring oxidation.
• the amount of pyridine nucleotide in the diagnostic reagent is determined by measuring the optical density of the reconstituted product. It is found that an optical density in the range from 2.1 to 1.0 spectrophotometrically determined at 340 nm using a 10 mm. light path is necessary to provide the effective diagnostic reagents of this invention.
• the third component of the subject compositions namely, the buffer, is the most important. It appears to play a critical role in the stabilization of the lyophilized material as well as the reconstituted product. The manner which brings this about is not fully understood at this time.
• the type of buffer required for a satisfactory product should have a sodium content of less than 600 meq./l. capable of regulating the pH of the composition, preferably in the range from 6.0 to 8.0.
• the preferred buffer is potassium phosphate added in an amount to provide from In both the GOT and GPT diagnostic compositions,
• buffers of preference include sodium phosphate-potassium phosphate mixtures and sodium phosphate by itself.
• the process for preparing the subject diagnostic compositions comprises admixing MDH or LDH, pyridine nucleotide and buffer material. The resulting aqueous solution is then lyophilized to provide a dry, stable diagnostic reagent.
• the reconstituted product is relatively stable. It is stable for up to 12 hours at 4C without change in optical density and at 25C, the optical density changes less than .2 percent in 3 hours.
• the present invention relates in part to the improved modification of combining the dialysis product with the GOT or GPT diagnostic composition, usually in reconstituted form, and photometrically measuring the decrease in NADH concentration.
• the concentration of enzyme in question is directly proportional to the decrease in NADH concentration.
• the sample used in the GOT determination can be a serum sample (SGOT), a plasma sample, or it can be an aqueous solution, suspension or dispersion.
• SGOT serum sample
• plasma sample a plasma sample
• aqueous solution aqueous solution, suspension or dispersion
• albumin sample An example of the latter type is an albumin sample.
• the preferred substrate is an aspaitic -a-ketoglutaric acid mixture.
• an alanine -a-ketoglutaric acid mixture is the substrate of choice.
• the vials are then subjected to lyophilizing conditions and the resulting dry, stable compositions stored at 4C.
• the dry, lyophilized product can be stored indefinitely at a temperature of 4C and is stable for approximately 2 days at 45C.
• the reconstituted product is stable for up to 12 hours at 4C without change in optical density, and at 25C the optical density changes less than 0.2 percent in 3 hours.
• bovine albumin is included in the 0.5 M potassium.
• the properties of the reconstituted product are unchanged and the lyophilized materialhas-comparable stability.
• the .dry, lyophilized product can be stored indefinitely ata temperature of 4C and is stable for at least ,2 days at-45C.
• The: reconstituted product is stable for up to 12 hours at-4Cwithoutchange in optical density,.and at25C the optical density changes less than 0.2 percent in 3 hours.
• bovine albumin isincluded in the-800 ml.,portion of 0. 5 M potassium .phosphate buffer solution.
• the sample (about 0.3 ml.) is mixed with a substrate comprising an aspartic acid aketoglutaric acid mixture (75:1 by weight; about 0.4 ml.) dissolved in a phosphate buffer.
• Theproducts of the reaction dialyze through a dialyzer and are reacted with the reconstituted reagent as prepared according to Example II (about 0.3 ml.).
• the change in optical density is determined in a photometer at 340 nm.
• the activity of GOT is directly proportional to the decrease in the optical density of the NADH.
• Example VI The procedure of Example V is repeated for the determination of GPT activity in a sample except the reconstituted diagnostic reagent of Example IV is used in place of the reagent of Example 11 and the substrate is an alanine-a-ketoglutaric acid mixture.
• Example I The procedure of Example I is repeated except that instead of potassium phosphate, a buffer consisting of a mixture of potassium phosphate and sodium phosphate is used.
• the reconstituted product has a sodium content of 400 meq./ l. and exhibits comparable stability.
• Example X The procedure of Example I is repeated except that sodium phosphate buffer is used in place of potassium phosphate.
• the reconstituted product has a sodium content of 400 meq./1. and exhibits comparable activity.
• Example V The procedure of Example V is repeated for the determination of GPT activity of an unknown sample wherein instead of the diagnostic reagent prepared according to Example II, the one described in Example IV I is used instead.
• a stable, lyophilized diagnostic composition effective in determining glutamic oxalic transaminase (GOT) in a sample containing said enzyme which consists of malate dehydrogenase (MDH) present in an amount to provide an activity upon reconstitution of from about 0.5 units/ml to about 10 units/ml; a pyridine nucleotide selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), said pyridine nucleotide present in an amount to obtain an optical density upon reconstitution in the range of from 2.1 to 1.0 spectrophotometrically determined at 340 nm using a 10 mm light path; and a buffer selected from the group consisting of potassium phosphate and a mixture of potassium phosphate and sodium phosphate, said mixture having a sodium content less than 600 meq./1 upon reconstitution, which regulates the
• composition as claimed in claim 1 wherein said buffer is potassium phosphate present in an amount to provide a concentration of from about 0.05 M to 0.4 M upon reconstitution.
• composition as claimed in claim 1 wherein said pyridine nucleotide is NADH.
• composition as claimed in claim 1 wherein said activity of MDH is about 3 units/ml.
• composition as claimed in claim 2 wherein said. concentration is 0.1 M.
• a stable aqueous solution having a pH in the range from 6.0 to 8.0 which comprises the composition of claim 1 dissolved in water.
• composition as claimed in claim 9 wherein said buffer is potassium phosphate present in an amount to provide a concentration of from about 0.05 M to 0.4 M upon reconstitution.
• composition as claimed in claim 9 wherein said pyridine nucleotide is NADH.
• composition as claimed in claim 9 wherein said activity of LDH is about 40 units/ml.
• composition as claimed in claim 10 wherein said concentration is 0.1 M.
• the method of claim 17 including the further step of passing the reaction product through a dialysis membrane prior to combining with said composition.
• composition buffer regulates the reconstituted product in the range from 6 to 8.
• composition comprises malate dehydrogenase, NADH and potassium phosphate.
• composition is in the form of an aqueous solution having a pH in the range from 6.0 to 8.0.
• composition buffer regulates the reconstituted product in the range from 6 to 8.
• composition comprises lactate dehydrogenase, NADH and potassium phosphate.
• composition in the form of an aqueous solution having a pH in the range from 6.0 to 8.0.
• a process for the preparation of a stable, lyophilized diagnostic composition as claimed in claim I which comprises admixing malate dehydrogenase.
• a pyridine nucleotide selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH); and a buffer selected from the group consisting of potassium phosphate and a mixture of potassiumphosphate and sodium phosphate which regulates the pH of the reconstituted product in the range from 6 to 8; and lyophilizing the resulting aqueous product which upon reconstitution is characterized by an optical density within the range from 2.1 to 1.0 and an MDH activity of from 0:5 units/ml. to about 10 units/ml.
• a process for the preparation of a stable. lyophilized diagnostic composition as claimed in claim 9 which comprises admixing lactate dehydrogenase; a pyridine nucleotide selected from the group consisting of reduced nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH); and a buffer selected from the group consisting of potassium phosphate and a mixture of potassium phosphate and sodium phosphate which regulates the pH of the reconstituted product in the range from 6 to 8; and lyophilizing the resulting aqueous product which upon reconstitutionis characterized by an optical density within the range from 2.1 to 1.0 acid and an LDH activity of from 8 units/ml to about units/ml.

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Citations (2)

• 0
  • BE BE786291D patent/BE786291A/xx unknown
• 1971
  • 1971-07-20 US US00164415A patent/US3819488A/en not_active Expired - Lifetime
• 1972
  • 1972-07-05 IT IT26664/72A patent/IT962467B/it active
  • 1972-07-07 NL NL7209460A patent/NL7209460A/xx unknown
  • 1972-07-12 AU AU44464/72A patent/AU473850B2/en not_active Expired
  • 1972-07-17 CA CA147,278A patent/CA998925A/en not_active Expired
  • 1972-07-18 DE DE2235174A patent/DE2235174A1/de active Pending
  • 1972-07-19 GB GB3382672A patent/GB1399275A/en not_active Expired
  • 1972-07-19 FR FR7225965A patent/FR2146816A5/fr not_active Expired

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