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
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

• Glucose is determined enzymatically with a stable diagnostic agent mixture comprising hexokinase, glucose-6- phosphate dehydrogenase, ATP, magnesium sulfate, NADP, and a buffer containing an azide.
• the present invention is concerned with a diagnostic agent and with a process for the enzymatic determination of glucose by means of hexokinase and glucose-6-phosphate dehydrogenase.
• glucose-o-phosphate It is known to determine glucose enzymatically by converting it into glucose-fi-phosphate with adenosine triphosphate and hexokinase.
• the resultant glucose-o-phosphate is converted into gluconate-G-phosphate by means of nicotinamideadenine-dinucleotide-phosphate (NADP) in the presence of glucose-6-phosphate dehydrogenase (G-6- PDI-I).
• NADP nicotinamideadenine-dinucleotide-phosphate
• G-6- PDI-I glucose-6-phosphate dehydrogenase
• NADPH reduced nicotinamide adenine dinucleotide phosphate
• This known process is very dependable, specific and essentially not subject to disturbance and is especially useful for the determination of glucose in biological fluids, as well as in foodstuffs.
• a disadvantage of this known process is that the solutions of the individual reagents, namely, buffer, ATP, NADP, as well as magnesium sulfate, must be separately pipetted because solutions of ATP in the previously used mixtures of triethanolamine hydrochloride, sodium hydroxide or sodium carbonate and magnesium sulfate, are not stable and, after some time, show considerable turbidity.
• the instant invention provides a novel diagnostic composition, not subject to the inherent disadvantages of conventional diagnostic agents, as well as a novel method for determining glucose.
• Solutions which contain the ATP, magnesium sulfate, sodium hydroxide or sodium carbonate, triethanolamine hydrochloride and sodium azide show no turbidity and no decrease in the ATP content even after storage for 3 months at 33 C., whereas the previously used solutions, which did not contain sodium azide, could, at most, be kept in a refrigerator for up to 14 days. In the case of longer storage of these known solutions, the concentration of the ATP dropped 01f so considerably over time that, when using such a preparation, erroneous results are obtained.
• the new diagnostic reaction mixture provided by the present invention can also be stored in solid form, as a homogeneous powder mixture, in airand moisture-tight containers at ambient temperature so that the mixture can be supplied in this form. Surprisingly, the azide does not disturb the enzymatic reaction.
• a surfaceactive substance preferably digitonin or Sterox (an ethylene oxide adduct produced by the Monsanto Chemical Co.)
• the surface-active agent preferably being used in an amount of about 10 mg./ ml. of reagent solution.
• the present invention also provides a diagnostic agent for the enzymatic determination of glucose, which comprises hexokinase and glucose-6-phosphate dehydrogenase, as well as adenosine-triphosphate (ATP), magnesium sulfate, nicotinamide adenine dinucleotide phosphate (NADP) and a buffer which contains an azide.
• a diagnostic agent for the enzymatic determination of glucose which comprises hexokinase and glucose-6-phosphate dehydrogenase, as well as adenosine-triphosphate (ATP), magnesium sulfate, nicotinamide adenine dinucleotide phosphate (NADP) and a buffer which contains an azide.
• ATP adenosine-triphosphate
• NADP nicotinamide adenine dinucleotide phosphate
• the adenosine-triphosphate, magnesium sulfate, nicotinamide-adenine-dinucleotide phosphate, buffer substance and azide and optionally also a surface-active agent, such as dignitonin, are made up in the form of a homogeneous powder mixture, while the enzymes hexokinase and glucose-'6-phosphate-dehydrogenase are made up separately in the form of a suspension in an ammonium sulfate solution.
• the buffer substances preferably acomprises triethanolamine hydrochloride and sodium hydroxide or sodium carbonate, together with an azide, such as sodium azide.
• Other buffers which can be used include, for example, tris-hydroxy-methylaminomethane/succinic acid and imidazole/succinic acid, together with an azide, such as sodium azide, the pH value of the buffer being between 6.8 and 7.5.
• alkali metal or alkaline earth metal azides for example, sodium, potassium, lithium, magnesium or calcium azide, or also ammonium azide.
• the adenosine triphosphate, magnesium sulfate, buffer mixture and azide and optionally also a surface-active agent, such as digitonin are present in the form of an aqueous solution
• the nicotin-amide-adenine-dinucleotide phosphate is in solid form (for example, pressed to form a tablet) and the enzymes hexokinase and glucose 6-phosphate dehydrogenase in the form of a separate suspension in a solution of ammonium sulfate.
• the diagnostic agent according to the present invention preferably comprises 1-15% by weight magnesium sulfate heptahydrate, 0.5-6% by weight nicotinamideadenine-dinucleotide phosphate, 0.56% by weight adenosine triphosphate, 2.0-18% by weight sodium azide, 0.10-1.5% by Weight of a surface-active agent, such as digitonin, and 50-95% by weight of a buffer mixture, as well as the enzyme suspension.
• a surface-active agent such as digitonin
• the buffer mixture preferably comprises 1.56-1 1.0 parts by weight triethanolamine hydrochloride and 0.6 -3.5 parts by weight sodium carbonate; or 1.2-7.5 parts by weight tris-hydroxymethylaminomethane and 0.55-3.45 parts by weight succinic acid; or 0.745 parts by weight imidazole and 0.70-1.75 parts by weight succinic acid.
• the diagnostic agent if present in the form of a powder mixture, is simply dissolved before use in an appropriate amount of distilled water.
• the amount of unused solution can, if desired, be kept for several weeks in a refrigerator without loss of quality.
• the glucose-containing sample for example whole blood
• the enzyme mixture of hexokinase and glucose-6-phosphate dehydrogenase is added and the extinction again measured.
• the enzyme suspension can be kept for years without special precautions, especially when stored in a refrigerator.
• the process and diagnostic agent according to the present invention thus permit an extremely simple carrying out of the process for the determination of glucose and avoid the necessity for having to pipette out separately numerous individual reagents for each determination and also of having to prepare fresh solution repeatedly.
• adenosine triphosphate in aqueous solution achieved by means of the present invention can be appreciated from the comparative experiments described hereinafter.
• an aqueous solution of the diagnostic agent according to the present invention which contained adenosine triphosphate in the form of its disodium salt, magnesium sulfate, triethanolamine hydrochloride, sodium carbonate, digitonin and sodium azide, was compared with a solution made up in the same manner but with the omission of the sodium azide. The following results were obtained:
• the unused solution can be kept for months and can be used again without alteration of the procedure.
• a first reagent consisting essentially of adenosine triphosphate, magnesium sulfate, nicotinamide-adenine-dinucleotide phosphate, a buffer substance comprising triethanolamine hydrochloride, sodium hydroxide or sodium carbonate, and azide made up as a homogenous powder mixture free of hexokinase and GPDH, and
• a second reagent consisting essentially of the enzymes hexokinase and glucose-6-phosphate dehydrogenase made up separately in the form of a suspension in a solution of ammonium sulfate, the second reagent being free of azide.
• Diagnostic agent as claimed in claim 2 consisting essentially of 115% by weight magnesium sulfate, 0.5- 6% by weight nicotinamide-adenine-dinucleotide phosphate, 0.56% by weight adenosine triphosphate, 2.0-18% by weight of an azide, 0.10-1.5% by weight of a surfaceactive agent and 50-95% by weight of a buffer.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
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• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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Cited By (13)

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• 1969
  • 1969-08-11 DE DE1940816A patent/DE1940816C3/de not_active Expired
• 1970
  • 1970-07-11 IT IT27336/70A patent/IT961363B/it active
  • 1970-07-31 SE SE10543/70A patent/SE361362B/xx unknown
  • 1970-08-04 FR FR7028723A patent/FR2056563A5/fr not_active Expired
  • 1970-08-05 US US00061503A patent/US3778350A/en not_active Expired - Lifetime
  • 1970-08-06 IL IL35072A patent/IL35072A/xx unknown
  • 1970-08-07 ZA ZA705481A patent/ZA705481B/xx unknown
  • 1970-08-07 GB GB38118/70A patent/GB1276230A/en not_active Expired
  • 1970-08-10 CH CH1199870A patent/CH534357A/de not_active IP Right Cessation
  • 1970-08-10 NL NL7011769A patent/NL168049C/xx not_active IP Right Cessation
  • 1970-08-11 JP JP7034170A patent/JPS544280B1/ja active Pending
  • 1970-08-11 HU HUBO1243A patent/HU162212B/hu unknown

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