US 3778350 A
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United States Patent O 3,778,350 DIAGNOSTIC AGENT AND METHOD FOR DETERMINING GLUCOSE Hans Ulrich Bergmeyer, Tutzing, Upper Bavaria, Erich Bernt, Munich, Wolfgang Gruber, Garatshausen, Felix Helmut Schmidt, Mannheim-Seckenheim, and Harald Stork, Lampertheim, Hesse, Germany, assignors to Boehringer Mannheim GmbH, Mannheim, Germany No Drawing. Filed Aug. 5, 1970, Ser. No. 61,503
Claims priority, application Germany, Aug. 11, 1969, P 19 40 816.3 Int. Cl. G01n 31/14 US. Cl. 195-103.5 C 8 Claims ABSTRACT OF THE DISCLOSURE 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.
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). In this way, there is formed an amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH) which is equivalent to the amount of glucose- 6-phosphate. The latter can very easily be determined photometrically.
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. However, 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.
We have now found that ATP, triethanolamine hydrochloride, sodium hydroxide or sodium carbonate and magnesium sulfate can all be kept together in solution when an azide is added to the solution. No turbidity then occurs.
Thus, 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.
The method according to the present invention for the enzymatic determination of glucose by conversion into glucose-6-phosphate by means of adenosine triphosphate (ATP) and hexokinase, oxidation thereof into glucose-6- phosphate by means of nicotinamide-adenine-dinucleotide phosphate (NADP) in the presence of glucose-6 phosphate dehydrogenase (C-6-PDH), with the formation of reduced nicotinamide adenine dinucleotide phosphate (NADP) and spectrophotometric determination of the latter, is characterized by using a buffer which contains an azide. Preferably, there is used a buffer which comprises a mixture of triethanolamine hydrochloride, sodium hydroxide or sodium carbonate and sodium azide.
By means of the method according to the present invention, it is possible to bring together the reagents needed for the reaction, apart from the enzymes and NADP, and
to use them in this form. 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.
Frequently, it is desirable, in the case of the method according to the present invention, also to add a surfaceactive substance, preferably digitonin or Sterox (an ethylene oxide adduct produced by the Monsanto Chemical Co.), to the solution of the reagents, 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.
In the diagnostic agent according to the present invention, 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.
As azide, there can be used the alkali metal or alkaline earth metal azides, for example, sodium, potassium, lithium, magnesium or calcium azide, or also ammonium azide.
According to another embodiment of the diagnostic agent according to the present invention, 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.
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.
For carrying out the determination of glucose with the diagnostic agent according to the present invention, 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. Subsequently, in the usual manner,. the glucose-containing sample, for example whole blood, is added and the extinction determined, whereupon 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.
The superior stability of adenosine triphosphate in aqueous solution achieved by means of the present invention can be appreciated from the comparative experiments described hereinafter. In these experiments, 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:
Cone. of ATP, Decrease of ATP,
mg./ml. cone. in percent With Without With Without Time azide azide azide azide Commencement 0. 425 0. 450 After-- 10 days.- 0.425 0. 450 0 20 days.-- 0. 420 0. 170 -1. 2 --62 50 days- 0. 418 0. 040 --1. 6 91 90 days 0.390 0. 000 --8 100 1 ATP=Adenosine triphosphate.
EXAMPLE 1 The following components were mixed together to form a homogeneous powder:
Grams Triethanolamine hydrochloride 11.0 Sodium carbonate 3.5 Magnesium sulfate heptahydrate 0.2 Nicotinamide-adenine-dinucleotide phosphate 0.1
Adenosine--triphosphate disodium salt trihydrate 0.1 Sodium azide 0.3 Digitonin 0.02
150 mg. amounts of this mixture were weighed out into bottles which were then closed in an airtight manner. The contents of a bottle were di solved before use by the addition of 2.0 ml. water and brief shaking up and then mixed with 0.0050.2 ml. of the sample to be investigated, such as urine, blood, serum or the like. The extinction E was measured at 366 nm. or at 340 nm. with a suitable photometer. Thereafter, there was added one drop of an enzyme suspension which contained about units of hexokinase and 75 units of glucose-6-phosphate-dehydrogenase per ml. 3 M ammonium sulfate solution.
After about 5-10 minutes, a constant extinction value E was obtained. The extinction difference AE=E E was multiplied by a calculation factor and gave directly the concentration of glucose in the sample.
The unused solution can be kept for months and can be used again without alteration of the procedure.
By use of the digitonin-containing solution, it was possible to add blood directly to the test mixture without carrying out a previous deproteinization or removal of the red blood corpuscles which interfere with an optical measurement. In the case of the present invention, only 0.005-0.01 ml. of blood was needed.
EXAMPLE 2 2.5 ml. of a solution containing:
Grams Triethanolamine hydrochloride 0.140 Adenosine5'-triphosphate 0.020 Magnesium sulfate heptahydrate 0.003 Sodium carbonate 0.016 Sodium azide 0.0025 Digitonin 0.00025 were pipetted into a measurement cuvette, a tablet of NADP was dissolved therein and 0.01 ml. whole blood was added. The haemolysis brought about by the digitonin present was finished practically immediately. Then, as described in Example 1, the extinction E was measured and the reaction commenced with the enzyme mixture, as in Example 1. The measurement and evaluation took place as in Example 1.
What is claimed is:
1. Diagnostic agent for the enzymatic determination of glucose which agent comprises two reagents:
(1) 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
(2) 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.
2. Diagnostic agent as claimed in claim 1 wherein the first agent additionally contains a surface active agent.
3. 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.
4. Diagnostic agent as claimed in claim 1, wherein the azide used is ammonium azide or an alkali metal or alkaline earth metal azide.
5. Diagnostic agent as claimed in claim 4, wherein the azige is sodium, potassium, lithium, magnesium or calcium az1 e.
6. Diagnostic agent as claimed in claim 2, wherein the surface active agent is digitonin.
5 6 7. Process for the enzymatic determination of glucose Pearse: Histochemistry-Theoretical and Applied, which comprises contacting the glucose-containing matepp. 560-561 (1960). rial with a diagnostic agent as claimed in claim 1. Bergmeyer: Methods of Enzymatic Analysis, pp. 117- 8. Process for the enzymatic determination of glucose 119 (1965). in serum which comprises contacting the glucose-contain- 5 Chemical Abstracts, II, 39:5275 (1945); ibid., III, ing serum with a diagnostic agent as claimed in claim 1. 43:269i (1949).
References Cited ALVIN E. TANENHOLT Z, Primary Examiner Bergmeyer: Methods of Enzymatic Analysis," pp. 970, M, D. HENSLEY, Assistant Examiner 975, 983-4 (1965). 10
Dixon et al.: Enzymes, p. 337 (1964). US. Cl. X.R.
Chemical Abstracts, 50:10838c (1956). 19599