CA1144458A - Process for the carrying out of enzymatic determinations - Google Patents
Process for the carrying out of enzymatic determinationsInfo
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- CA1144458A CA1144458A CA000331836A CA331836A CA1144458A CA 1144458 A CA1144458 A CA 1144458A CA 000331836 A CA000331836 A CA 000331836A CA 331836 A CA331836 A CA 331836A CA 1144458 A CA1144458 A CA 1144458A
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- 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
- C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
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Abstract
ABSTRACT OF THE DISCLOSURE
A process and reagent for carrying out enzymatic determinations in which NAD(P)H is formed and intermediate products and non-specifically-reacting disturbing substances are removed in a preliminary reaction, during which, there is simultaneously carried out an NAD(P)H-consuming enzymatic reaction which completely oxidises the NAD(P)H formed to give NAD(P); the NAD(P)H-consuming reaction is stopped at the start of the measurement reaction.
A process and reagent for carrying out enzymatic determinations in which NAD(P)H is formed and intermediate products and non-specifically-reacting disturbing substances are removed in a preliminary reaction, during which, there is simultaneously carried out an NAD(P)H-consuming enzymatic reaction which completely oxidises the NAD(P)H formed to give NAD(P); the NAD(P)H-consuming reaction is stopped at the start of the measurement reaction.
Description
The present invention is concerned with a process for the determination of substrates or enz~mes in serum, bodv fluids or other sample material, in which the form-ation of reduced nicotinamide-adenine-dinucleotide (-l~ADH) or of reduced-nicotinamide-adenine-dinucleotide phosphate (~DPH) is the measurement signal and is also concerned with a reagent and a test kit for carrying out this process~
In clinical and pharmaceutical chemistry, in bio-chemistry and in foodstuff chemistry, enzymatic reactions which proceed with the formation of NADH or NADPH are of great interest because, as a rule, they are characterised by good speci~icity and the ability of being auickly carried out. If a sufficiently specific enzymatic reaction seauence is present, then disturbances are only to be expected from those substances which can ocrur as inter-mediate products of the main reaction. Non-specificities of the adjuvant and indicator enzymes can also bring about the reaction of disturbing materials in the sample n~aterial. It is, therefore, conventional to carry out blank reactions at the same time in order to take into account the influence of these substances on the measure-ment. However, it has to be accepted that a part of the measurement range is there~y already no longer`available.
~ithout adiustment of the photometer used, normally only extinctions of up to 2 can be measured. If, however, the concentration of distuxbing substances in a sample are very high, then, in the case of each measurement, an adiustment of the photometer can be necessary. The position is even more difficult when the ratio of the concentration of the disturbing material to the zctual measurement concentration is large. The small difference between two large measurement signals is then the actual measurement value. Such measurement processes involve unacceptably large errors since the smallest measurement errors also have a great effect in the large measurement signals and place unfulfillable reauirements on the exact-itude of pipetting of the sample and on the exactitude of measurement of the apparatus.
In the case of determinations of enzyme activities, the presence of disturbing materials of the ad~uvant and indicator reactions in the sample have the effect that, before the actual linearly proceding reaction, a pre-run is measured. Here, too, the rneasurement range can already be severely lirnited by the pre-run, as mentioned above.
However, a furthex distuxbance can here also arise: many enzyme activities display a so-called lag phase, i.e. it takes a certain time until the maximum reaction rate is achieved. The rneasurernent can then only be carrie~ out after cessation of this lag phase. A part of the measure-ment range available without alteration of the photomeler is herebv also utilised. It is also to be adcled that many automatic analysers are not able, after ~he--start, to wait with the measurement, without addition of the -~ -4-sample, until the lag phase has ceased. Enzymatic measurement processes which display a pre-run, a lag phase or both cannot be adapt~ed to such automatic devices.
In principle, the destruction of disturbing materials with a reaction other than the measurement ` reaction could be of help. Thus, disturbing glucose can, in the case of a saccharose determination, be removed by means of glucose oxidase (see H.U. Bergmeyer, Methoden der enzymatischen Analyse, 2nd edition, Verlag Chemie, Weinheim 1970, Vol. II, page 1145). In the amylase test, endogenous glucose and glucose formed during the lag phase can also be removed by means of glucose oxidase (see Clin.
Chem., 23, 1149/197?). In each case, the glucose liberated n the measurement reaction of actual interest is subse-auently measured vla NADH or NADPH formation in the hexolclnase/glucose-6-phosphate dehydrogenase system.
However, it is obvious that this method cannot be applied to reactions in which materials other than glucose disturb.
As has already been stated, however, in principle all _ enzymatic measurement sequences are disturbed by the appearance of intermediate products in the sample.
* Therefore, it is an ob~ect of the present invention to provide a process with the help of which reactions giving rise to NADH and NADPH can be carried o~t in such a manner that neither the appearance of intermediate products nor of substrates for ~oreign and side activities disturb the ad~uvant and indicator enzymes in the sample.
.
. ., At the same time, this process is to be suitable for the elimination of lag phases which possibly occur in the case of enzyme measurements.
Thus, according to the present invention, there is provided a process for carrying out enzyrnatic determin-ations which proceed with the formation of NAD(P)H and in ~hich intermediate products and non--specifically-reacting disturbing substances are removed in a prelimin-ary reaction, wherein, during the preliminary reaction, an ~AD(P)H-consuming enzymatic reaction is carried out simultaneously which completely oY~idises the MAD(P)H
formed to giv~ ~AD~P) and, at the start of the measurement reaction, the NAD(P)H-consuming reaction is stopped.
Thus, according to the present invention, during the preliminary reaction in which inter~.ediate products and non-speci.fically-reacting disturbing substances are removed and possibly in the case of enzyrne reactions the lag phase proceeds, an NADH- or ]\~.DP~-consuminc~ reaction is sirnultaneously carried out which oxidises all the N.~DH
or NADPH fo~ned.
It is surprisiny that it is possible to accomplish the present invention in the stated manner. It could not have been foreseen that the simultaneous carrving out of two counter-runninq reac~ions wowld be possible without diEficulties. ~urthermc)re, it was to have been assumed that the rec~uired second substrate, the inhibitor and the substance which is employed for the destruction of s~
the seeond substrate would influence the actual measurement reaction since enz~matie reactions react extremely sensi-tively to changes of the reaction conditions. Thus, it was not to ha-~e been ex~ected that the counter~reaction would take place at all under the conditions of the measurement reaction which certainly, as a rule, differ very mueh from the optimum conditions of the counter-reaction and ought not to be changed.
The reaction conditions with regard to pH value, bufEer, reagent eoneentrations and the like ,or these reaetions, as well as for the main reaction, are well known and, taking into account these eonditions, the particular, best-suited ~P~D~P)H-consuming reactions ean be selected.
The start of the actual measurement reaction takes plaee by stopping the NAD(P~H-consuming reactlon, which can be aecomplished by addina an inhibitor which completely inhibits the NADH- or ~ADP~-consuminq reaetion, or by adding a substance ~hich instantaneously eompletely reaets the second substrate of the ~DH- or NADPH-consuming reaetion. If it is a auestion of a substrate measurement, then the starting enzyme is simultaneously added thereto.
Enzyme reaetions, on the other hand, dG not require this addition and can be read off immeditely~ The starting time point can be freely chosen and is so selected that all the disturbing reac',_ions have already run to com~letion.
In principle, within the scope of the proeess according to the present inven~ion, all ~ADH- and ~ADPH-consuming reactions can be employed if there is available either a suitable inhibitor or a reaction which practic-ally instantaneously reacts the second substrate. A
number of examples Gf such counter-reactions which can he used within the scope of the present invention are set out in the following Table 1:
T A B I. F, nzyme (EC No.) Isubstrate of i sto~ping of the counter-the counter- I reaction I reaction I inhibitor ¦reacting awav of ¦ ~ the 2nd substrate I .
alcohol dehydro- INADH/various I N~DP, ~, ketone acceptors, aenase 1.1.1.1 ¦aldehydes I ADP such as hydra-I l zine, hydrazones, I ¦ ¦semicarbazides dltto 1.1.1.2 INADPTI/various ¦ ¦ketGne acceptors ¦aldehydes L-xylulose ¦NADPH~ I excess reductase xylulose I xylulose 1.1. 1.10 .
L-iditol- NADH/ 2,3-dithio-reductase fructose l-pro~anol (sorbitol dehydroge~ase) 1.1.1.14 mannitol-l- NAD~I/ phosphatase ~hosphate de- fructose--6-hvdroqenase phosphate 1;~.1.17 aldose reduct- ! NADtP)H/ ~-KG, ase 1.1.1.21 various aceto !aldoses and 1 acetate, !aldehydes I L-oxo-I valerate 5~
TABI.E 1 ~continued) enzyme (EC No.) I substrate of stopping of the counter-I the counter- reactlon ¦ reaction inhibitor ! reacting away of l i the 2nd substrate _~ ! __ glyoxalate NADH/ ~ ketone reductase glyoxalate acce~tors 1.1.1.26 (hydro~v-pyruvate) lactate de- NADH/ LDH- keione hydrogenase pyruvate inhibitor, acce~tors 1.1.1.27 fluo~o-pyruvate, oxalate glycerate de- NAD~P)H/ ketone hydrogenase hydroxy- acceptors 1.1.1.29 pyruvate 3-hydroxy- NADH/aceto- D lactate bu yrate DH acetate oxaloglycerate NAD(P)H/ S0 2-reductase dihydroxy- 3 1.1.1092 fumarate diiodophenyl- NADH/di- 2,5-di-pyruvate iodophenyl ¦ hydroxy-reductase pyruvate benzoate 1.1.1,95 mannitol NADP/ 2,3-dithiol-dehydroqenase fructose l-propanol 1.1.1.13~
qlyoxalate NADPH/ tris dehydrogenase oxaloyl-CoA
1.2.1.17 alanine NADH/ keto acids ketone dehydrogenase ~yruvate/ acceptors 1.4.1.1 NH3 GlDH 1.4.1.3 ~AD(P)H/ I GPT ketone ~-KH/~3 acceptors D-Droline NADH/D- 5-amino- ketone j reductase proline valerate aCceptorc ~ .1.6 _9_ TABLE 1 (continued~
n~yme (EC NoO ~ substr,te of ¦ stopping of the counter-the counter- ¦ reaction reaction linhibitor ! reacting away of j I the 2nd substrate pyroline-2- N~H/ ~ 2-amino-carboxylic pyroline--2- octanoate acid reductase carboxylic 1.5.1 1 acid glutathione- NP~(P)H/ thiocide, dithiothreitol reductase GSSG ¦5-nitro-1.6.4.2 furaldehyde ~-nitro-benzene-sulphonic hydroxylamine NP~(P)H~ hydrazine ketone or 1~7~99.1 amine aldehyde NADH-POD NPDH/ bromide catalase 2 1.11.1.1 2 2 ascorbate SO3 pyrocatechol/
peroxidase glutathione/
_ . lutathione The fol.lowing NP~H- and ~P~Ph-consuming reactions are especially preferred:
1. NP~D~I + pyruvate + I~ lactate + NAD
LD~I = lactate dehydrogenase The start takes place with LDH inhibitor.
~ SDH
In clinical and pharmaceutical chemistry, in bio-chemistry and in foodstuff chemistry, enzymatic reactions which proceed with the formation of NADH or NADPH are of great interest because, as a rule, they are characterised by good speci~icity and the ability of being auickly carried out. If a sufficiently specific enzymatic reaction seauence is present, then disturbances are only to be expected from those substances which can ocrur as inter-mediate products of the main reaction. Non-specificities of the adjuvant and indicator enzymes can also bring about the reaction of disturbing materials in the sample n~aterial. It is, therefore, conventional to carry out blank reactions at the same time in order to take into account the influence of these substances on the measure-ment. However, it has to be accepted that a part of the measurement range is there~y already no longer`available.
~ithout adiustment of the photometer used, normally only extinctions of up to 2 can be measured. If, however, the concentration of distuxbing substances in a sample are very high, then, in the case of each measurement, an adiustment of the photometer can be necessary. The position is even more difficult when the ratio of the concentration of the disturbing material to the zctual measurement concentration is large. The small difference between two large measurement signals is then the actual measurement value. Such measurement processes involve unacceptably large errors since the smallest measurement errors also have a great effect in the large measurement signals and place unfulfillable reauirements on the exact-itude of pipetting of the sample and on the exactitude of measurement of the apparatus.
In the case of determinations of enzyme activities, the presence of disturbing materials of the ad~uvant and indicator reactions in the sample have the effect that, before the actual linearly proceding reaction, a pre-run is measured. Here, too, the rneasurement range can already be severely lirnited by the pre-run, as mentioned above.
However, a furthex distuxbance can here also arise: many enzyme activities display a so-called lag phase, i.e. it takes a certain time until the maximum reaction rate is achieved. The rneasurernent can then only be carrie~ out after cessation of this lag phase. A part of the measure-ment range available without alteration of the photomeler is herebv also utilised. It is also to be adcled that many automatic analysers are not able, after ~he--start, to wait with the measurement, without addition of the -~ -4-sample, until the lag phase has ceased. Enzymatic measurement processes which display a pre-run, a lag phase or both cannot be adapt~ed to such automatic devices.
In principle, the destruction of disturbing materials with a reaction other than the measurement ` reaction could be of help. Thus, disturbing glucose can, in the case of a saccharose determination, be removed by means of glucose oxidase (see H.U. Bergmeyer, Methoden der enzymatischen Analyse, 2nd edition, Verlag Chemie, Weinheim 1970, Vol. II, page 1145). In the amylase test, endogenous glucose and glucose formed during the lag phase can also be removed by means of glucose oxidase (see Clin.
Chem., 23, 1149/197?). In each case, the glucose liberated n the measurement reaction of actual interest is subse-auently measured vla NADH or NADPH formation in the hexolclnase/glucose-6-phosphate dehydrogenase system.
However, it is obvious that this method cannot be applied to reactions in which materials other than glucose disturb.
As has already been stated, however, in principle all _ enzymatic measurement sequences are disturbed by the appearance of intermediate products in the sample.
* Therefore, it is an ob~ect of the present invention to provide a process with the help of which reactions giving rise to NADH and NADPH can be carried o~t in such a manner that neither the appearance of intermediate products nor of substrates for ~oreign and side activities disturb the ad~uvant and indicator enzymes in the sample.
.
. ., At the same time, this process is to be suitable for the elimination of lag phases which possibly occur in the case of enzyme measurements.
Thus, according to the present invention, there is provided a process for carrying out enzyrnatic determin-ations which proceed with the formation of NAD(P)H and in ~hich intermediate products and non--specifically-reacting disturbing substances are removed in a prelimin-ary reaction, wherein, during the preliminary reaction, an ~AD(P)H-consuming enzymatic reaction is carried out simultaneously which completely oY~idises the MAD(P)H
formed to giv~ ~AD~P) and, at the start of the measurement reaction, the NAD(P)H-consuming reaction is stopped.
Thus, according to the present invention, during the preliminary reaction in which inter~.ediate products and non-speci.fically-reacting disturbing substances are removed and possibly in the case of enzyrne reactions the lag phase proceeds, an NADH- or ]\~.DP~-consuminc~ reaction is sirnultaneously carried out which oxidises all the N.~DH
or NADPH fo~ned.
It is surprisiny that it is possible to accomplish the present invention in the stated manner. It could not have been foreseen that the simultaneous carrving out of two counter-runninq reac~ions wowld be possible without diEficulties. ~urthermc)re, it was to have been assumed that the rec~uired second substrate, the inhibitor and the substance which is employed for the destruction of s~
the seeond substrate would influence the actual measurement reaction since enz~matie reactions react extremely sensi-tively to changes of the reaction conditions. Thus, it was not to ha-~e been ex~ected that the counter~reaction would take place at all under the conditions of the measurement reaction which certainly, as a rule, differ very mueh from the optimum conditions of the counter-reaction and ought not to be changed.
The reaction conditions with regard to pH value, bufEer, reagent eoneentrations and the like ,or these reaetions, as well as for the main reaction, are well known and, taking into account these eonditions, the particular, best-suited ~P~D~P)H-consuming reactions ean be selected.
The start of the actual measurement reaction takes plaee by stopping the NAD(P~H-consuming reactlon, which can be aecomplished by addina an inhibitor which completely inhibits the NADH- or ~ADP~-consuminq reaetion, or by adding a substance ~hich instantaneously eompletely reaets the second substrate of the ~DH- or NADPH-consuming reaetion. If it is a auestion of a substrate measurement, then the starting enzyme is simultaneously added thereto.
Enzyme reaetions, on the other hand, dG not require this addition and can be read off immeditely~ The starting time point can be freely chosen and is so selected that all the disturbing reac',_ions have already run to com~letion.
In principle, within the scope of the proeess according to the present inven~ion, all ~ADH- and ~ADPH-consuming reactions can be employed if there is available either a suitable inhibitor or a reaction which practic-ally instantaneously reacts the second substrate. A
number of examples Gf such counter-reactions which can he used within the scope of the present invention are set out in the following Table 1:
T A B I. F, nzyme (EC No.) Isubstrate of i sto~ping of the counter-the counter- I reaction I reaction I inhibitor ¦reacting awav of ¦ ~ the 2nd substrate I .
alcohol dehydro- INADH/various I N~DP, ~, ketone acceptors, aenase 1.1.1.1 ¦aldehydes I ADP such as hydra-I l zine, hydrazones, I ¦ ¦semicarbazides dltto 1.1.1.2 INADPTI/various ¦ ¦ketGne acceptors ¦aldehydes L-xylulose ¦NADPH~ I excess reductase xylulose I xylulose 1.1. 1.10 .
L-iditol- NADH/ 2,3-dithio-reductase fructose l-pro~anol (sorbitol dehydroge~ase) 1.1.1.14 mannitol-l- NAD~I/ phosphatase ~hosphate de- fructose--6-hvdroqenase phosphate 1;~.1.17 aldose reduct- ! NADtP)H/ ~-KG, ase 1.1.1.21 various aceto !aldoses and 1 acetate, !aldehydes I L-oxo-I valerate 5~
TABI.E 1 ~continued) enzyme (EC No.) I substrate of stopping of the counter-I the counter- reactlon ¦ reaction inhibitor ! reacting away of l i the 2nd substrate _~ ! __ glyoxalate NADH/ ~ ketone reductase glyoxalate acce~tors 1.1.1.26 (hydro~v-pyruvate) lactate de- NADH/ LDH- keione hydrogenase pyruvate inhibitor, acce~tors 1.1.1.27 fluo~o-pyruvate, oxalate glycerate de- NAD~P)H/ ketone hydrogenase hydroxy- acceptors 1.1.1.29 pyruvate 3-hydroxy- NADH/aceto- D lactate bu yrate DH acetate oxaloglycerate NAD(P)H/ S0 2-reductase dihydroxy- 3 1.1.1092 fumarate diiodophenyl- NADH/di- 2,5-di-pyruvate iodophenyl ¦ hydroxy-reductase pyruvate benzoate 1.1.1,95 mannitol NADP/ 2,3-dithiol-dehydroqenase fructose l-propanol 1.1.1.13~
qlyoxalate NADPH/ tris dehydrogenase oxaloyl-CoA
1.2.1.17 alanine NADH/ keto acids ketone dehydrogenase ~yruvate/ acceptors 1.4.1.1 NH3 GlDH 1.4.1.3 ~AD(P)H/ I GPT ketone ~-KH/~3 acceptors D-Droline NADH/D- 5-amino- ketone j reductase proline valerate aCceptorc ~ .1.6 _9_ TABLE 1 (continued~
n~yme (EC NoO ~ substr,te of ¦ stopping of the counter-the counter- ¦ reaction reaction linhibitor ! reacting away of j I the 2nd substrate pyroline-2- N~H/ ~ 2-amino-carboxylic pyroline--2- octanoate acid reductase carboxylic 1.5.1 1 acid glutathione- NP~(P)H/ thiocide, dithiothreitol reductase GSSG ¦5-nitro-1.6.4.2 furaldehyde ~-nitro-benzene-sulphonic hydroxylamine NP~(P)H~ hydrazine ketone or 1~7~99.1 amine aldehyde NADH-POD NPDH/ bromide catalase 2 1.11.1.1 2 2 ascorbate SO3 pyrocatechol/
peroxidase glutathione/
_ . lutathione The fol.lowing NP~H- and ~P~Ph-consuming reactions are especially preferred:
1. NP~D~I + pyruvate + I~ lactate + NAD
LD~I = lactate dehydrogenase The start takes place with LDH inhibitor.
~ SDH
2. fructose + NP~H ~ H ` sorbitol + NAD
SDH = sorbitol dehyclroyenase The start takes place with 2,3-dithiol-1-propanol as SDH
inhibitor.
SDH = sorbitol dehyclroyenase The start takes place with 2,3-dithiol-1-propanol as SDH
inhibitor.
3. dialutathione disulphide + NAD(P)H + H
2 glutathione ~ NADtp)+
GR = glutathione reductase The start takes place with dithiothreitol which instant-aneousl~ reduces GSSG to GSH.
~ I~7ADH-POD +
2 glutathione ~ NADtp)+
GR = glutathione reductase The start takes place with dithiothreitol which instant-aneousl~ reduces GSSG to GSH.
~ I~7ADH-POD +
4. H202 ~ NADH ~ H -- --- 2 NADH-POD = NADH peroxidase The start can take place either with catalase or with HSO3 or with glutathione/glutathione peroxidase which instantaneously destroys the second substrate, hydrogen peroxide.
In principle, the process according tv the present invention can be used for all enzyme and suhstrate determinations proceeding with the Eormation o~ N~(P1H.
Typical examples are set out in the following Table 2, which also indicates the enzyme~substrate pair. However, enzyme activ:ities or substrates can also be used in coupled reactions when -thev proceed with the formati.on of NAD(P)H. This is explained hereinafter in more detail -- ~in the specific E~amples.
In principle, the process according tv the present invention can be used for all enzyme and suhstrate determinations proceeding with the Eormation o~ N~(P1H.
Typical examples are set out in the following Table 2, which also indicates the enzyme~substrate pair. However, enzyme activ:ities or substrates can also be used in coupled reactions when -thev proceed with the formati.on of NAD(P)H. This is explained hereinafter in more detail -- ~in the specific E~amples.
5~
j enæymej substrate I detectable in coupled reaction ¦
1.1.1~21UDP glucose/ ! UDP-glucose-4- IATP, ADP, UTP, UDP, I
UDP-glucose ~AD ¦ epimerase, CMP, U~, UDP- ¦
dehydrogenase saccharose glucose, saccharose synthetase 1.1.1.41 threo-isocitrate isocitrate dehydrogenase NAD
1.1.1.44 phospho- creatine, glucose, fructose, phospho- gluconate/ ¦ kinase, maltose, ATP, I
glyconate NAD(P) pyruvate creatine phosphate, dehydrogenase kinase, oligosaccharides, hexokinase, starch a-amylase, a-glucosidase, ~-glucosidase 1.1.1.40 galactose/ a~glactosidase, lactose, galactose-galactose N~D ~-glactosidase l-pnosphate, dehydrogenase ¦ yalactose-6-¦ phosphate 1.1.1.49 Iglucose-6- creatine glucose, fructose, qlucose-6- ¦phosphate/ I kinase~ maltose, oligo-~hosphate NAD(P) j pyruvate saccharides, dehydrogenase I kinase, starch hexokinase, a-amylase, I
a-glucosidase, ~-glucosidase 1~2.11 ~o~aldehyde/ ¦ methanol formaldehyde N~) dehydrogenase 1~2.1.2 ~ormate/NAD oxalate, methanol, formate I iformaldehyde, dehydrogenase l sarcosine 1.2.1.3- acetaldehyde/ ¦ alcohol oxid- various alcohols - 1.2.1.5 NAD(P) ¦ ases, alcohol and aldehydes, aldehyde I DH, diamino glycerol dehydrogenase 1 oxidase 1.2.1.14 1~ linosine-5- ¦ creatine kin- ~, A~P, GMP, dehydrogenase phosphate/NAD ¦ ase,myo~inase IC~, U~
1.2.1.16 and isuccinate I GOT, GPT, ¦~Pr creatinine, 1.2.1.24 isemialdehyde/ 'I pyruvate la-KG, pyruvate, succinate INA~(P) j ~inase, GlDH loxalacetate, ~- j semialdehvde I laminobutyrate, dehydrogenase alanine, glutamate -The present invention also provides a reagent for carrying out enzymatic determinations, comprising a system for determining a substrate or enzyme which leads to the formation of NAD(P)H, ~7herein it additionally contains an enzymatic systern which completely oxidises ~AD(P).~l to I~AD(P).
Furthermore, the present invention pro~ides a test kit for carrying out the process according to the present invention which comprises the above-described reagent according to the present invention and a stopping agent for the NAD(P)H~consuming reaction.
As already mentioned, by means of the pre~ent invention, it is possible to exclude foreign and side activities in the case of the detennination methods to be carried out and to eliminate lag phases. The result of this is that such reactions can also be carried out in automatic analysers which are themselves not sui~able for en2ymatic measurement processes which display a pre-run or a lag phase. Furthermore, the measurement signals become more precise and better determinable, especially in the case of high concentrations of disturbing materials.
- In the following Examples, the following abbreviations are em~ioyed:
GSSG glutathione, oxidised SDH sorbitol dehydrogenase GR glutathione reductase GSH glutathione 1 ~ ~ L~ 4 5 ~3 Glyc-DH glycerol dehydrogenase Al-DH aldehyde dehydrogenase Gluc-DH glucose dehydrogenase GPT glutamate-pyruvate transaminase a-KG a-ketoglutarate GAB-GT ~-aminobutyrate-~-ketoglutarate transaminase SS Al-DH succinate semialdehyde dehydrogenase ADP adenosine-5'-diphosphate AMP adenosine monophosphate ATP adenosine-5'-triphosphate PEP phosphoenol pyruvate PK pyru~ate kinase N~ nicotinamide-adenine-dinucleotide NADH nicotinamide adenine-dinucleotide, reduced NADP nicotinamide-adenine-dinucleotide phosDhate NAD~P) NADP or NAD
NADPH nicotina~ide-adenine-dinucleotide, reduced NAD(P)H NADPH or ~ADH
LDH lactate dehydrogenase CK creatine kinase NADH POD NADM peroxidase HK hexokinase ~6p-DH glucose-6-phosphate dehydrogenase Tris tris-~hydroxymethyl~-aminomethane The follo~ing Examples are given for the purpose of illustrating the present invention:-Example 1.
Removal of free glycerol and disturbing aldehvdes inthe triglyceride test.
In the case of a triglyceride test, depending upon the reaction sequence:
esterase f id glycerol + Glyc-DH
b) glycerol + NAD
glyceraldehyde + ~ADH + H
c) glyceraldehyde ~ NAD Al-DH
glyceric acid ~ NADH ~ H~
free glycerol and aldehydes, which react non-specifically with the A1-DH (aldehyde dehydrogenase), can give rise to an increased blank value. This disturbance can be over-come by the following reaction:-NADH ~ pyruvate + H~ lactate ~ NRD+
The following Table explains the carrying out ofthe reaction':
1144~58 ~ABLE 3 starting solution sample blank conc in the Tris.HCl, pH 8.31.5 ml.1.5 ml. 50 mMol/l.
lOO mMol/l.
~AD, 27 mMol/lØ1 mlØ1 ml. 0.9 mMOL/l.
glycerol-DH0.1 ml. 0.-1 ml. 3.3 U~ml.
100-U/ml.
Al-DH, 200 U/ml.O.05 ml.O.05 ml. 3.3 U/ml.
pyruvate, 0.05 ml. O.05 ml. 2 mMol/l.
120 mMol/l.
sample 0.05 ml. _ LDH, 100 U/ml. o.oS ml. 1.65 U/ml.
In~the sample and blank, glycerol and aldehydes now react with ~ADH formation which is again immediately oxidioed by pyruvate and LDH to ~AD. After 5 minutes, the start takeA place with:
esterase, 2000 U/ml.) 33 U/ml.
LDH inhibitor+), ) O,~ 1. 0 1 0.33 mMol/l. ¦
+ ) B.B.A. 54, 210 (1961).
_ The LDH inhibitor now completely inhibits the LDH.
The esterase splits glycerol from the triglycerides which ; reacts further with ~ADH formation. The ~ADH remains unchanged~-since the LDH is no~longer active. Therefore, after 5 minutes, the extinction difference between the sample and the blank can be measured and the triglyceride content calculated therefrom.
. .
~4~58 The reaction conditions for this test are determined by the optimum conditions for the triglyceride test.
According to the present invention, only the addition of pyruvate, LDH and LDH inhibitor are needed. The pyruvate concentration is determined by the amount of disturbing substances to be removed and can vary from 0.005 mMol/l.
to 100 mMoljl., 0.5 to 5 mMol/l. being preferred. The concentration of the LDH is determined by the incubation time. It can vary from 0.001 U/ml. to 100 U/l., 0.5 to 5 U/ml. being preferred. The minimum concentration of the LD'H inhibitor must suffice in order quantitatively to inhibit the LDH. This is~the case with 0.2 mMol/l.
but higher concentrations of up to 5 mMol/l. do not disturb. A concentration of from 0.2 to 2 mMol/l. is preferred.
Exam~le 2.
Removal of disturbina creatine ADP ~vruvate and a-KG
in the determination of creatinine.
}n the case of the determinatlon of creatinine in serum according to the following reaction seauence:
creatininase B a) creatinine + H20 ~ creatine b) creatine + ATP ~ creatine phosphate + ADP
c) ADP + PEP 3 pyruvate + ATP
d) pyruvate + glutamate ~ a-KG + alanlne e) a-KG + 4 aminobutyrate succinate semialdehyde + glutamate f) succinate semialdehyde + ~AD+ -~succinate + NADH + H
creatine, pyruvate and ~-KG originating from the serum disturb, as well as, in particular, the ADP present in the ATP, because tney give too high blank values. The disturbance can be removed by the followincs reaction:-g~ NADH ~ fructose + H S H_~ sorbitol ~ NAD
The following Table explains the carrying out of the reaction:
TAE~LE a starting solution ~ sample blank c-~- the I glycine buffer, 1~0 ml. 1 1.0 ml. 0.1 mol/l.
0.2 Mol/l., pH 8~0 ~ADP, 80 mMol/l. O.05 ml. I O.05 ml.¦ 2 mMol/l.
I ATP, 40 mMoL/l. O.05 ml. 0.05 ml.¦ 1 mMol/l, PEP, 16 mMol/l. 0.05 ml. 1 0.05 ml.l 0.4 mMoljl.
glutamate, 0.05 ml. 1 0.05 ml.~ 1.1 mMol/l.
44 mMol/l.
~'-aminobutyrat:e, 0.5 ml. 0.5 ml. ~5 mMol/l.
380 mMol/l.
magnesium chloride, 0.1 ml. 0.1 ml. 2 mMol/l~
40 mMol/l.
-fructose, 80 mMol/l. I O.G5 ml. 0.05 mlO 2 mMol/l.
CK, 400 U/ml. ) l I 10 U/ml.
~ 0.05 ml. 0.05 ml.l PK, 160 U/ml. ) l 4 U/ml.
SDH, 20 U/ml. ) ~ 0.5 U/ml.
¦ GPT, 160 U/ml. ) j I a u~ml-I GkB-CT, 160 U/ml. ) I 0.05 ml.I ~- U/ml.
¦ SS-Al-DEI, 160 U/ml.) ~
I' sam~le 1 0.5 ml.
` ~2 ¦ - ! ~ 5 ml. I I
5~
Incubate for 10 minutes. During this time, creatine, pyruvate, ~-~G and ADP are reacted. The NADH formed is removed by fructose/S~H. Then start with:
creat nase ¦ l l 10 U/ml.
2,3-dithiol-1- ~
propanol, l O.05 ml. 1 0.05 ml. 2 mMol/l.
~O m~lol/l ) I ~
By means of the start with creatininase, the creatinine determination comes into operation. The 2,3-dithiol-l-propanol inhibitor completely inhibits the SDH
so that the NADH formation originating from the creatinine reaction can now be measured.
The reaction conditions are determined by the optimised creatinine determination. Eor the addition, accordin~ to the present invention, of fructose, SDH and 2,3-dithiol-1-propanol, there applies that stated in Example 1. Correspondingly, the fructose concentration is from 0.002 to 200 mMol~l. and preferably rom 0.5 to 10 mMol/l. The SDH concentration can vary from 0.002 to 5~ U/ml. and is preferably 0.1 to 5 U/ml. In the case of 2,3-dithiol~ ropanol, the concentration is from 0~1 to 100 mMol~l., from 1 to 10 mMol/l. bein~ preferred.
Exam~le 3.
Removal of a laa_phase in the aetermination of creatine ~inase.
In the case of the determination of C~ in serum, depending upon the reaction se~uence:
9L5~
C a) creatin.ine phosphate ~ ADP ~ ATP + creatinine b) ATP + glucose ~ glucose-6-phosphate ~ ADP
c) glucose-6-phosphate ~ N~DP ~
gluconate-6-phosphate + NADPH + H
a lag phase sometimes occurs which, in the case of the measurement, utilises a part of the measurement range and, furthermore, makes difficult or impossible the adaptation to certain automatic analysers. The disturb-ance is overcome by the following reaction:
diglutathione disulphide + NAD~P3H + H ~
2 glutathione + NAD(P) The following Table explains the carrying out of the reaction:
5~
I
starting solution I sample I conc. in test ', imidazole buffer, I 1.0 ml. ~l 0.1 mol/l.
pH 6.7, 0.2 mol/l.
glucose, 0.8 mol/l. 0.05 ml. ¦ 20 mMol~
Mg acetate, 400 mMolJl~ 0.05 ml. 1 10 mMol/l.
ADP, ~0 mMol/l. O.05 ml. ~ 2 rn~lol/l.
hMP, 0.2 mol/l. O.0~ mlO I 5 m~1ol~
diadenosine penta- 0.05 ml~ 1 0.01 mMol/l.
phosphate, 0.4 mMol/l.
NADP, 80 m~ol/1. O.05 ml. , 2 mMol/l.
HX, 100 U/ml. 0.05 ml. Il 2.5 U/ml.
G6P-DH, 60 U/ml. 1 0.05 r~. 1 1.5 U/ml.
GR, 20 U/ml. 0.1 ml. 1 U/ml.
N-acetylcysteine, 0.1 ml. , 20 mMol/l.
400 mIMol/l.
GSSG, 40 mMol/1. O.1 ml. 2 mMol/l.
sample ¦ 0.1 ml.
Incubate for 5 minutes. Durin~ this time, the lag phase of the CK takes ~lace. The NADPH thereby formed is immediately further oY~idised by GSSG/GR so that no indication takes place. Then start with:
dithiothreitol, ~ 0.2 rnl. 5 mMol/l.
50 mMol/l. I
- Due to the complete utilisat,on of the GSSG wi~h 2,3-dithio-1-propanol, the measurement signal of the C~
reaction becomes visible since l~DPH is no longer oxidisedu The measurement conditions are optimised for the CK
measurement. For the addition, according to the present invention, of GSSG, GR and dithiothreitol there applies that stated in Example 1. The range of variation for GSSG is from 0.001 mMol/l. to 200 m~lol/l., 0.5 to ~ m~lol/l.
being preferred. GR can vary from 0.02 to 50 U~ml., 0.5 to 5 U/ml. being preferred. The dithiothreitol instant-aneously reduces GSSG to GSH. Thus, it must be at least e~uimolar to GSSG. Therefore, the range of variation is from 0.001 to 250 mMol/l., 0.5 to 10 m~Iol/l. being preferred.
xample 4.
~emoval of endoqenous_alucose and laq phase in _ a-amvlase test.
. .
In the case o~ the determination of r~-arnylase in serum according to the reaction se~uence:
a-amylase D a) maltoheptaose + I-I20 ~ ~ triose -~ tetraose a-glucosidase ~) triose ~ tetraose + 5I-I20 ~ 3 7 gluccse c) 7 ~lucose + 7 ATP --~ 7-glucose-6-phosphate + 7ADP
+ G~P-DH
d) 7 glucose-~-phosphate ~ 7 NAD
7 glucon2te-6-phosphate ~- 7 l~ADH ~ 7 H
a large part of the measurement range is utilised ~y the - glucose in the serum. The lag phase of the ~-am~lase in this determination utilises a ,urther part of the measure-ment range and prevents the ad2~ta-tion to automatic an21ysers. I~le ~isturbance is overcome bv the following reaction:
-21~
~ NADH-POD
H202 + NADH ~ H - ~ 2 H20 + NAD
The following Table explains the carrying out of the reaction:
- I ' starting solution ¦ sam~le I blank I conc. in the test phosphate buffer, ¦ ~ ¦ NaCl 50 m~lol/l.
pH 7, 0.1 mol/l., ¦ I acetate 5 ~Mol/l.
containing 0.1 1.0 ml. ¦ 1.0 ml. I phosphate Mol/l. NaCl and 1 1 50 mMol/l.
10 m~5Ol/l. Mg acetate maltoheptaose, 0.1 ml. 0.1 ml. 10 mol/l.
0.2 mol/l.
NAD, 40 mMol/l. 0.1 ml. 0.1 ml.2 r~ol/l.
ATP, 24 mMol/l. ~ 0.1 mlO 0.1 ml.1.2 mMol/l.
H202, 80 m~lol/l. I, 0.05 ml. 0.05 ml.¦ 2 mMol/l.
~, 80 U/ml. 1 0.05 ml. 0.05 ml.~ 2 U/ml.
G5P-DH, 80 U/ml. ~ 0.05 ml. 0.05 ml, 2 U/ml.
~-glucosidase, 0.02 ml. 0.02 ml.i 10 U/ml.
1000 U/ml.
NADH-POD, 100 U/ml. O.01 ml. O.01 ml. O.5 U/ml.
seru~ 0.02 ml.l, -H20 0.45 ml.¦ 0.45 ml.
- '_ The Mixture i5 incubated for 15 minutes, During this time, the endogenous glucose and lag phase take place. The NADH forme~ i5 again removed immediately by H202/NP~H-POD. The start then takes place with NaHS03,0.16 mol/l. 0.05 ml.l 0.05 ml. I 4 m~lol/l. or or catalase, 1,000,000 U/ml. ' 0.05 ml.l 0.05 ml. 25,000 U/ml.
. -22-The hydrogen peroxide is instantaneously destroyed by HSO3 or catalase. The NADH formed can thus no longer be decomposed and the a-amylase activity can be measured immediately and without disturbance. Instead of HSO3 or catalase, glutathione/glutathione peroxidase can al~o be used. The reaction conditions are determined by the optimum conditions of the a-amylase.-For the addition, according to the present invention,of hydrogen peroxide, NADH-POD and ~aHS03 or catalase, there applies that stated in Example 1. The hydrogen peroxide concentration can be from 0.001 to 50 mMol/l., 1 to 5 mMol/l. being preferred. The ~ADH-POD concentration can be from 0.001 to 100 U/ml., 0.1 to 5 U/ml. being preferred. The HSO3 must be added thereto in an amount at least equimolar to the hydrogen peroxide so that an amount of from 0.001 to 250 mMol/l. is possible, 1 to 10 mMol/l. being preferred. For catalase, the range which can be chosen is from 10 to 100,000 U/ml. and preferably from 500 to 50,000 U/ml.
Figure 1 of the accompanying drawing shows a . . .
typical curve pattern which is obtained with and without the use of the present invention. Curve 1, without the addition according to the present invention of NADrI-POD/
H202 and catalase, demonstrates the mar]~ed curve brought about by endogenous glucose which then passes over in the lag phase of the a-amylase. The linearity is first achieved after about 14 minutes ( ~ ). Curve 2 shows, in the first part, the decomposition of the NADH formed s~
~23-by endogenous glucose, after reaching the base extinction, the value no longer changes up to the start t~ ) with catalase. Then, however, the linear course of the ~-amylase reaction, which can be measured directly, begins immediately.
Example 5.
Removal of endoqenous qlucose and laq phase in the a-amvlase test.
In the case o~ the removal of the a-~mylase activity according to the following reaction sequence:
~-amylase E a) maltoheptaose ~ H20 ~ triose -~ tetraose ~glucosidase b) triose + tetraose + 5H20 ~ 7 glucose ) 7 1 d 7 AD~ Gluc~ 7 1 t +
7 NADH -~ 7 H
the same possibilities of disturbance occur as described above in Example 4. They are removed by the following reaction:
~. SDiI
fructose ~ NAD~I + H ` sorbltol + NAD
The ,ollowing Table explains the ca.rrying out of the rea.ction:
L9~
--~4~
TABLE
! starting solution I sanmple I blan~ conc. in the test j phosphate buffer, osphate 50 mMol/l. ,¦
j pH 7 . 0, O . l mol/l ., I ¦ maltohep-taose I containing mal to- I lO mMol/l., heptaose 20 m~lol/l ., 1 ~ O ml . 1 . O ml . NaC1 50 m~lol/l ., NaCl O . l mol/l ., MgC1 2 2 ~ ol/l .
Mc~C l 2 4 mMol /l .
7~AD 40 mMol/l . 0 . l ml . 0. l ml . 2 mMol/l .
glucose dehydro- () . l ml . 0 .1 ml . 18 U/ml .
genase 360 U/ml.
f ructose 0 . 6 mol/l . O .1 ml . O . l ml . 30 mMol/l .
SDH 3 U/rnl . 0. l m:L. 0. l ml . 0 . l5 U/I
se rum 0 . 0 2 ml . _ H20 ~ 0. 5 ml n 0 . 5 ml . ¦
.. . . , . , _ .. ~
The ~rixture is incubated for 15 minutes, during which time the endogenous qlucose and lag phase of the a-amylase determinacion take place. I'he NADH fonned is imrnecliately again removecl by fructose/SDI~ he start then takes place wi th:
2, 3-dithiol-] - ' O . l ml . I O .1 rnl . 2 m~5Ol ~l .
~ro~anol, 40 mMol/l. ~
The SDI:l is instantaneously cruantitatively inhibited by the addition of: 2, 3-dithi,ol-l-pro7~anol. I'he ~ADH
~ormed can thereby no longer be clecomt~osec1 and thus the u-amylase acti~,rity can be measured irnmediately and without disturbance. Ficsure 2 of the accom~anying c1rawing shows a typical curve pattern obtained with and without the use of the pre~ent invention. Curve ~, without the addition according to the present invention of f~-uctose~SDH and 2,3-dithiol-l-propanol~ clearly shows the marked curve caused by endogenous ~lucose which leads to extinctions via l and is concluded after about 15 minutes (~) and passes over into the linear range of the a-amylase reaction. Curve 2, with the additions of fructose/SDH
according to the present invention, still shows, in the first part, the decomposition of the ~rADH formed from endogenous glucose. P.fter achieving the base value, the eY~tinction does not change any more up to the start with 2,3-dithiol-l-propanol ('I 3. Thereafter, from the linear course of the curve, the a-amylase activity can be read off immediately and calculated.
The reaction conditions are determined by the optimum conditions of the a-amylase reaction~ For the addition, according to the present invention, of fructose/
SD~ and 2,3-d:ithiol-l-propanol, there apply the statements made in Examp:Le 1. The admissible concentrations corres-pond to those of Example 2.
j enæymej substrate I detectable in coupled reaction ¦
1.1.1~21UDP glucose/ ! UDP-glucose-4- IATP, ADP, UTP, UDP, I
UDP-glucose ~AD ¦ epimerase, CMP, U~, UDP- ¦
dehydrogenase saccharose glucose, saccharose synthetase 1.1.1.41 threo-isocitrate isocitrate dehydrogenase NAD
1.1.1.44 phospho- creatine, glucose, fructose, phospho- gluconate/ ¦ kinase, maltose, ATP, I
glyconate NAD(P) pyruvate creatine phosphate, dehydrogenase kinase, oligosaccharides, hexokinase, starch a-amylase, a-glucosidase, ~-glucosidase 1.1.1.40 galactose/ a~glactosidase, lactose, galactose-galactose N~D ~-glactosidase l-pnosphate, dehydrogenase ¦ yalactose-6-¦ phosphate 1.1.1.49 Iglucose-6- creatine glucose, fructose, qlucose-6- ¦phosphate/ I kinase~ maltose, oligo-~hosphate NAD(P) j pyruvate saccharides, dehydrogenase I kinase, starch hexokinase, a-amylase, I
a-glucosidase, ~-glucosidase 1~2.11 ~o~aldehyde/ ¦ methanol formaldehyde N~) dehydrogenase 1~2.1.2 ~ormate/NAD oxalate, methanol, formate I iformaldehyde, dehydrogenase l sarcosine 1.2.1.3- acetaldehyde/ ¦ alcohol oxid- various alcohols - 1.2.1.5 NAD(P) ¦ ases, alcohol and aldehydes, aldehyde I DH, diamino glycerol dehydrogenase 1 oxidase 1.2.1.14 1~ linosine-5- ¦ creatine kin- ~, A~P, GMP, dehydrogenase phosphate/NAD ¦ ase,myo~inase IC~, U~
1.2.1.16 and isuccinate I GOT, GPT, ¦~Pr creatinine, 1.2.1.24 isemialdehyde/ 'I pyruvate la-KG, pyruvate, succinate INA~(P) j ~inase, GlDH loxalacetate, ~- j semialdehvde I laminobutyrate, dehydrogenase alanine, glutamate -The present invention also provides a reagent for carrying out enzymatic determinations, comprising a system for determining a substrate or enzyme which leads to the formation of NAD(P)H, ~7herein it additionally contains an enzymatic systern which completely oxidises ~AD(P).~l to I~AD(P).
Furthermore, the present invention pro~ides a test kit for carrying out the process according to the present invention which comprises the above-described reagent according to the present invention and a stopping agent for the NAD(P)H~consuming reaction.
As already mentioned, by means of the pre~ent invention, it is possible to exclude foreign and side activities in the case of the detennination methods to be carried out and to eliminate lag phases. The result of this is that such reactions can also be carried out in automatic analysers which are themselves not sui~able for en2ymatic measurement processes which display a pre-run or a lag phase. Furthermore, the measurement signals become more precise and better determinable, especially in the case of high concentrations of disturbing materials.
- In the following Examples, the following abbreviations are em~ioyed:
GSSG glutathione, oxidised SDH sorbitol dehydrogenase GR glutathione reductase GSH glutathione 1 ~ ~ L~ 4 5 ~3 Glyc-DH glycerol dehydrogenase Al-DH aldehyde dehydrogenase Gluc-DH glucose dehydrogenase GPT glutamate-pyruvate transaminase a-KG a-ketoglutarate GAB-GT ~-aminobutyrate-~-ketoglutarate transaminase SS Al-DH succinate semialdehyde dehydrogenase ADP adenosine-5'-diphosphate AMP adenosine monophosphate ATP adenosine-5'-triphosphate PEP phosphoenol pyruvate PK pyru~ate kinase N~ nicotinamide-adenine-dinucleotide NADH nicotinamide adenine-dinucleotide, reduced NADP nicotinamide-adenine-dinucleotide phosDhate NAD~P) NADP or NAD
NADPH nicotina~ide-adenine-dinucleotide, reduced NAD(P)H NADPH or ~ADH
LDH lactate dehydrogenase CK creatine kinase NADH POD NADM peroxidase HK hexokinase ~6p-DH glucose-6-phosphate dehydrogenase Tris tris-~hydroxymethyl~-aminomethane The follo~ing Examples are given for the purpose of illustrating the present invention:-Example 1.
Removal of free glycerol and disturbing aldehvdes inthe triglyceride test.
In the case of a triglyceride test, depending upon the reaction sequence:
esterase f id glycerol + Glyc-DH
b) glycerol + NAD
glyceraldehyde + ~ADH + H
c) glyceraldehyde ~ NAD Al-DH
glyceric acid ~ NADH ~ H~
free glycerol and aldehydes, which react non-specifically with the A1-DH (aldehyde dehydrogenase), can give rise to an increased blank value. This disturbance can be over-come by the following reaction:-NADH ~ pyruvate + H~ lactate ~ NRD+
The following Table explains the carrying out ofthe reaction':
1144~58 ~ABLE 3 starting solution sample blank conc in the Tris.HCl, pH 8.31.5 ml.1.5 ml. 50 mMol/l.
lOO mMol/l.
~AD, 27 mMol/lØ1 mlØ1 ml. 0.9 mMOL/l.
glycerol-DH0.1 ml. 0.-1 ml. 3.3 U~ml.
100-U/ml.
Al-DH, 200 U/ml.O.05 ml.O.05 ml. 3.3 U/ml.
pyruvate, 0.05 ml. O.05 ml. 2 mMol/l.
120 mMol/l.
sample 0.05 ml. _ LDH, 100 U/ml. o.oS ml. 1.65 U/ml.
In~the sample and blank, glycerol and aldehydes now react with ~ADH formation which is again immediately oxidioed by pyruvate and LDH to ~AD. After 5 minutes, the start takeA place with:
esterase, 2000 U/ml.) 33 U/ml.
LDH inhibitor+), ) O,~ 1. 0 1 0.33 mMol/l. ¦
+ ) B.B.A. 54, 210 (1961).
_ The LDH inhibitor now completely inhibits the LDH.
The esterase splits glycerol from the triglycerides which ; reacts further with ~ADH formation. The ~ADH remains unchanged~-since the LDH is no~longer active. Therefore, after 5 minutes, the extinction difference between the sample and the blank can be measured and the triglyceride content calculated therefrom.
. .
~4~58 The reaction conditions for this test are determined by the optimum conditions for the triglyceride test.
According to the present invention, only the addition of pyruvate, LDH and LDH inhibitor are needed. The pyruvate concentration is determined by the amount of disturbing substances to be removed and can vary from 0.005 mMol/l.
to 100 mMoljl., 0.5 to 5 mMol/l. being preferred. The concentration of the LDH is determined by the incubation time. It can vary from 0.001 U/ml. to 100 U/l., 0.5 to 5 U/ml. being preferred. The minimum concentration of the LD'H inhibitor must suffice in order quantitatively to inhibit the LDH. This is~the case with 0.2 mMol/l.
but higher concentrations of up to 5 mMol/l. do not disturb. A concentration of from 0.2 to 2 mMol/l. is preferred.
Exam~le 2.
Removal of disturbina creatine ADP ~vruvate and a-KG
in the determination of creatinine.
}n the case of the determinatlon of creatinine in serum according to the following reaction seauence:
creatininase B a) creatinine + H20 ~ creatine b) creatine + ATP ~ creatine phosphate + ADP
c) ADP + PEP 3 pyruvate + ATP
d) pyruvate + glutamate ~ a-KG + alanlne e) a-KG + 4 aminobutyrate succinate semialdehyde + glutamate f) succinate semialdehyde + ~AD+ -~succinate + NADH + H
creatine, pyruvate and ~-KG originating from the serum disturb, as well as, in particular, the ADP present in the ATP, because tney give too high blank values. The disturbance can be removed by the followincs reaction:-g~ NADH ~ fructose + H S H_~ sorbitol ~ NAD
The following Table explains the carrying out of the reaction:
TAE~LE a starting solution ~ sample blank c-~- the I glycine buffer, 1~0 ml. 1 1.0 ml. 0.1 mol/l.
0.2 Mol/l., pH 8~0 ~ADP, 80 mMol/l. O.05 ml. I O.05 ml.¦ 2 mMol/l.
I ATP, 40 mMoL/l. O.05 ml. 0.05 ml.¦ 1 mMol/l, PEP, 16 mMol/l. 0.05 ml. 1 0.05 ml.l 0.4 mMoljl.
glutamate, 0.05 ml. 1 0.05 ml.~ 1.1 mMol/l.
44 mMol/l.
~'-aminobutyrat:e, 0.5 ml. 0.5 ml. ~5 mMol/l.
380 mMol/l.
magnesium chloride, 0.1 ml. 0.1 ml. 2 mMol/l~
40 mMol/l.
-fructose, 80 mMol/l. I O.G5 ml. 0.05 mlO 2 mMol/l.
CK, 400 U/ml. ) l I 10 U/ml.
~ 0.05 ml. 0.05 ml.l PK, 160 U/ml. ) l 4 U/ml.
SDH, 20 U/ml. ) ~ 0.5 U/ml.
¦ GPT, 160 U/ml. ) j I a u~ml-I GkB-CT, 160 U/ml. ) I 0.05 ml.I ~- U/ml.
¦ SS-Al-DEI, 160 U/ml.) ~
I' sam~le 1 0.5 ml.
` ~2 ¦ - ! ~ 5 ml. I I
5~
Incubate for 10 minutes. During this time, creatine, pyruvate, ~-~G and ADP are reacted. The NADH formed is removed by fructose/S~H. Then start with:
creat nase ¦ l l 10 U/ml.
2,3-dithiol-1- ~
propanol, l O.05 ml. 1 0.05 ml. 2 mMol/l.
~O m~lol/l ) I ~
By means of the start with creatininase, the creatinine determination comes into operation. The 2,3-dithiol-l-propanol inhibitor completely inhibits the SDH
so that the NADH formation originating from the creatinine reaction can now be measured.
The reaction conditions are determined by the optimised creatinine determination. Eor the addition, accordin~ to the present invention, of fructose, SDH and 2,3-dithiol-1-propanol, there applies that stated in Example 1. Correspondingly, the fructose concentration is from 0.002 to 200 mMol~l. and preferably rom 0.5 to 10 mMol/l. The SDH concentration can vary from 0.002 to 5~ U/ml. and is preferably 0.1 to 5 U/ml. In the case of 2,3-dithiol~ ropanol, the concentration is from 0~1 to 100 mMol~l., from 1 to 10 mMol/l. bein~ preferred.
Exam~le 3.
Removal of a laa_phase in the aetermination of creatine ~inase.
In the case of the determination of C~ in serum, depending upon the reaction se~uence:
9L5~
C a) creatin.ine phosphate ~ ADP ~ ATP + creatinine b) ATP + glucose ~ glucose-6-phosphate ~ ADP
c) glucose-6-phosphate ~ N~DP ~
gluconate-6-phosphate + NADPH + H
a lag phase sometimes occurs which, in the case of the measurement, utilises a part of the measurement range and, furthermore, makes difficult or impossible the adaptation to certain automatic analysers. The disturb-ance is overcome by the following reaction:
diglutathione disulphide + NAD~P3H + H ~
2 glutathione + NAD(P) The following Table explains the carrying out of the reaction:
5~
I
starting solution I sample I conc. in test ', imidazole buffer, I 1.0 ml. ~l 0.1 mol/l.
pH 6.7, 0.2 mol/l.
glucose, 0.8 mol/l. 0.05 ml. ¦ 20 mMol~
Mg acetate, 400 mMolJl~ 0.05 ml. 1 10 mMol/l.
ADP, ~0 mMol/l. O.05 ml. ~ 2 rn~lol/l.
hMP, 0.2 mol/l. O.0~ mlO I 5 m~1ol~
diadenosine penta- 0.05 ml~ 1 0.01 mMol/l.
phosphate, 0.4 mMol/l.
NADP, 80 m~ol/1. O.05 ml. , 2 mMol/l.
HX, 100 U/ml. 0.05 ml. Il 2.5 U/ml.
G6P-DH, 60 U/ml. 1 0.05 r~. 1 1.5 U/ml.
GR, 20 U/ml. 0.1 ml. 1 U/ml.
N-acetylcysteine, 0.1 ml. , 20 mMol/l.
400 mIMol/l.
GSSG, 40 mMol/1. O.1 ml. 2 mMol/l.
sample ¦ 0.1 ml.
Incubate for 5 minutes. Durin~ this time, the lag phase of the CK takes ~lace. The NADPH thereby formed is immediately further oY~idised by GSSG/GR so that no indication takes place. Then start with:
dithiothreitol, ~ 0.2 rnl. 5 mMol/l.
50 mMol/l. I
- Due to the complete utilisat,on of the GSSG wi~h 2,3-dithio-1-propanol, the measurement signal of the C~
reaction becomes visible since l~DPH is no longer oxidisedu The measurement conditions are optimised for the CK
measurement. For the addition, according to the present invention, of GSSG, GR and dithiothreitol there applies that stated in Example 1. The range of variation for GSSG is from 0.001 mMol/l. to 200 m~lol/l., 0.5 to ~ m~lol/l.
being preferred. GR can vary from 0.02 to 50 U~ml., 0.5 to 5 U/ml. being preferred. The dithiothreitol instant-aneously reduces GSSG to GSH. Thus, it must be at least e~uimolar to GSSG. Therefore, the range of variation is from 0.001 to 250 mMol/l., 0.5 to 10 m~Iol/l. being preferred.
xample 4.
~emoval of endoqenous_alucose and laq phase in _ a-amvlase test.
. .
In the case o~ the determination of r~-arnylase in serum according to the reaction se~uence:
a-amylase D a) maltoheptaose + I-I20 ~ ~ triose -~ tetraose a-glucosidase ~) triose ~ tetraose + 5I-I20 ~ 3 7 gluccse c) 7 ~lucose + 7 ATP --~ 7-glucose-6-phosphate + 7ADP
+ G~P-DH
d) 7 glucose-~-phosphate ~ 7 NAD
7 glucon2te-6-phosphate ~- 7 l~ADH ~ 7 H
a large part of the measurement range is utilised ~y the - glucose in the serum. The lag phase of the ~-am~lase in this determination utilises a ,urther part of the measure-ment range and prevents the ad2~ta-tion to automatic an21ysers. I~le ~isturbance is overcome bv the following reaction:
-21~
~ NADH-POD
H202 + NADH ~ H - ~ 2 H20 + NAD
The following Table explains the carrying out of the reaction:
- I ' starting solution ¦ sam~le I blank I conc. in the test phosphate buffer, ¦ ~ ¦ NaCl 50 m~lol/l.
pH 7, 0.1 mol/l., ¦ I acetate 5 ~Mol/l.
containing 0.1 1.0 ml. ¦ 1.0 ml. I phosphate Mol/l. NaCl and 1 1 50 mMol/l.
10 m~5Ol/l. Mg acetate maltoheptaose, 0.1 ml. 0.1 ml. 10 mol/l.
0.2 mol/l.
NAD, 40 mMol/l. 0.1 ml. 0.1 ml.2 r~ol/l.
ATP, 24 mMol/l. ~ 0.1 mlO 0.1 ml.1.2 mMol/l.
H202, 80 m~lol/l. I, 0.05 ml. 0.05 ml.¦ 2 mMol/l.
~, 80 U/ml. 1 0.05 ml. 0.05 ml.~ 2 U/ml.
G5P-DH, 80 U/ml. ~ 0.05 ml. 0.05 ml, 2 U/ml.
~-glucosidase, 0.02 ml. 0.02 ml.i 10 U/ml.
1000 U/ml.
NADH-POD, 100 U/ml. O.01 ml. O.01 ml. O.5 U/ml.
seru~ 0.02 ml.l, -H20 0.45 ml.¦ 0.45 ml.
- '_ The Mixture i5 incubated for 15 minutes, During this time, the endogenous glucose and lag phase take place. The NADH forme~ i5 again removed immediately by H202/NP~H-POD. The start then takes place with NaHS03,0.16 mol/l. 0.05 ml.l 0.05 ml. I 4 m~lol/l. or or catalase, 1,000,000 U/ml. ' 0.05 ml.l 0.05 ml. 25,000 U/ml.
. -22-The hydrogen peroxide is instantaneously destroyed by HSO3 or catalase. The NADH formed can thus no longer be decomposed and the a-amylase activity can be measured immediately and without disturbance. Instead of HSO3 or catalase, glutathione/glutathione peroxidase can al~o be used. The reaction conditions are determined by the optimum conditions of the a-amylase.-For the addition, according to the present invention,of hydrogen peroxide, NADH-POD and ~aHS03 or catalase, there applies that stated in Example 1. The hydrogen peroxide concentration can be from 0.001 to 50 mMol/l., 1 to 5 mMol/l. being preferred. The ~ADH-POD concentration can be from 0.001 to 100 U/ml., 0.1 to 5 U/ml. being preferred. The HSO3 must be added thereto in an amount at least equimolar to the hydrogen peroxide so that an amount of from 0.001 to 250 mMol/l. is possible, 1 to 10 mMol/l. being preferred. For catalase, the range which can be chosen is from 10 to 100,000 U/ml. and preferably from 500 to 50,000 U/ml.
Figure 1 of the accompanying drawing shows a . . .
typical curve pattern which is obtained with and without the use of the present invention. Curve 1, without the addition according to the present invention of NADrI-POD/
H202 and catalase, demonstrates the mar]~ed curve brought about by endogenous glucose which then passes over in the lag phase of the a-amylase. The linearity is first achieved after about 14 minutes ( ~ ). Curve 2 shows, in the first part, the decomposition of the NADH formed s~
~23-by endogenous glucose, after reaching the base extinction, the value no longer changes up to the start t~ ) with catalase. Then, however, the linear course of the ~-amylase reaction, which can be measured directly, begins immediately.
Example 5.
Removal of endoqenous qlucose and laq phase in the a-amvlase test.
In the case o~ the removal of the a-~mylase activity according to the following reaction sequence:
~-amylase E a) maltoheptaose ~ H20 ~ triose -~ tetraose ~glucosidase b) triose + tetraose + 5H20 ~ 7 glucose ) 7 1 d 7 AD~ Gluc~ 7 1 t +
7 NADH -~ 7 H
the same possibilities of disturbance occur as described above in Example 4. They are removed by the following reaction:
~. SDiI
fructose ~ NAD~I + H ` sorbltol + NAD
The ,ollowing Table explains the ca.rrying out of the rea.ction:
L9~
--~4~
TABLE
! starting solution I sanmple I blan~ conc. in the test j phosphate buffer, osphate 50 mMol/l. ,¦
j pH 7 . 0, O . l mol/l ., I ¦ maltohep-taose I containing mal to- I lO mMol/l., heptaose 20 m~lol/l ., 1 ~ O ml . 1 . O ml . NaC1 50 m~lol/l ., NaCl O . l mol/l ., MgC1 2 2 ~ ol/l .
Mc~C l 2 4 mMol /l .
7~AD 40 mMol/l . 0 . l ml . 0. l ml . 2 mMol/l .
glucose dehydro- () . l ml . 0 .1 ml . 18 U/ml .
genase 360 U/ml.
f ructose 0 . 6 mol/l . O .1 ml . O . l ml . 30 mMol/l .
SDH 3 U/rnl . 0. l m:L. 0. l ml . 0 . l5 U/I
se rum 0 . 0 2 ml . _ H20 ~ 0. 5 ml n 0 . 5 ml . ¦
.. . . , . , _ .. ~
The ~rixture is incubated for 15 minutes, during which time the endogenous qlucose and lag phase of the a-amylase determinacion take place. I'he NADH fonned is imrnecliately again removecl by fructose/SDI~ he start then takes place wi th:
2, 3-dithiol-] - ' O . l ml . I O .1 rnl . 2 m~5Ol ~l .
~ro~anol, 40 mMol/l. ~
The SDI:l is instantaneously cruantitatively inhibited by the addition of: 2, 3-dithi,ol-l-pro7~anol. I'he ~ADH
~ormed can thereby no longer be clecomt~osec1 and thus the u-amylase acti~,rity can be measured irnmediately and without disturbance. Ficsure 2 of the accom~anying c1rawing shows a typical curve pattern obtained with and without the use of the pre~ent invention. Curve ~, without the addition according to the present invention of f~-uctose~SDH and 2,3-dithiol-l-propanol~ clearly shows the marked curve caused by endogenous ~lucose which leads to extinctions via l and is concluded after about 15 minutes (~) and passes over into the linear range of the a-amylase reaction. Curve 2, with the additions of fructose/SDH
according to the present invention, still shows, in the first part, the decomposition of the ~rADH formed from endogenous glucose. P.fter achieving the base value, the eY~tinction does not change any more up to the start with 2,3-dithiol-l-propanol ('I 3. Thereafter, from the linear course of the curve, the a-amylase activity can be read off immediately and calculated.
The reaction conditions are determined by the optimum conditions of the a-amylase reaction~ For the addition, according to the present invention, of fructose/
SD~ and 2,3-d:ithiol-l-propanol, there apply the statements made in Examp:Le 1. The admissible concentrations corres-pond to those of Example 2.
Claims (23)
1. A process for carrying out enzymatic determinations which proceed with the formation of NAD(P)H and in which intermediate products and non-specifically-reacting disturbing substances are removed in a preliminary reaction, comprising:
removing intermediate products and non-specifically-reacting disturbing substances in a preliminary reaction, during said preliminary reaction, simltaneously carrying out an NAD(P)H-consuming enzymatic reaction which completely oxidises NAD(P)H formed to give NAD(P), carrying out a measurement reaction, and stopping the NAD(P)H-consuming reaction at the start of said measurement reaction.
removing intermediate products and non-specifically-reacting disturbing substances in a preliminary reaction, during said preliminary reaction, simltaneously carrying out an NAD(P)H-consuming enzymatic reaction which completely oxidises NAD(P)H formed to give NAD(P), carrying out a measurement reaction, and stopping the NAD(P)H-consuming reaction at the start of said measurement reaction.
2. A process according to claim 1, wherein the NAD(P)H-consuming reaction is stopped by the addition of an inhibitor for this reaction.
3. A process according to claim 1, wherein the NAD(P)H-consuming reaction is stopped by removal of the second substrate of this reaction.
4. A process according to claim 2, wherein as NAD(P)H-consuming reaction, there is used the reaction with pyruvate in the presence of lactate dehydrogenase (LDH), the reaction being stopped by the addition of an LDH inhibitor.
5. A process according to claim 2, wherein as NAD(P)H-consuming reaction, there is used the reaction with fructose in the presence of sorbitol dehydrogenase, the reaction being stopped by the addition of 2,3-dithiol-1-propanol.
6. A process according to claim 3, wherein as NAD(P)H-consuming reaction, there is used the reaction with glutathione disulphide in the presence of glutathione reductase, the reaction being stopped by the addition of dithiothreitol.
7. A process according to claim 3, wherein as NAD(P)H-consuming reaction, there is used the reaction with hydrogen peroxide in the presence of NADH-peroxidase, the reaction being stopped by the addition of catalase or sulphite or glutathione/glutathione peroxidase.
8. A reagent for carrying out enzymatic determinations, comprising a system for the determination of a substrate or enzyme which leads to the formation of NAD(P)H, and an enzymatic system which completely oxidises NAD(P)H to NAD(P).
9. A reagent according to claim 8, wherein the system which oxidises NAD(P)H comprises a dehydrogenase or reduc-tase and its substrate differing from NAD(P)H.
10. A reagent according to claim 9, wherein the said system comprises lactate dehydrogenase and pyruvate.
11. A reagent according to claim 9, wherein the said system comprises sorbital dehydrogenase and fructose.
12. A reagent according to claim 9, wherein the said system comprises glutathione reductase and diglutathione disulphide.
13. A reagent according to claim 9, wherein the said system comprises NADH peroxidase and hydrogen peroxide or a hydrogen peroxide-yielding compound.
14. A test kit for carrying out enzymatic determinations which proceed with the formation of NAD(P)H and in which intermediate products and non-specifically-reacting disturbing substances are removed in a preliminary reaction, comprising a reagent including a system for the determination of a substrate or enzyme which leads to the formation of NAD(P)H, an enzyme system which completely oxidises NAD(P)H
to NAD(P) and an inhibitor for the NAD(P)H-oxidising enzyme system.
to NAD(P) and an inhibitor for the NAD(P)H-oxidising enzyme system.
15. A test kit according to claim 14, wherein said enzyme system comprises lactate dehydrogenase and pyruvate, and further including an LDH inhibitor.
16. A test kit according to claim 14, wherein said enzyme system comprises sorbitate dehydrogenase and fructose, and further including 2,3-dithiol-1-propanol.
17. A test kit according to claim 14, wherein said enzyme system comprises glutathione reductase and digluta-thione disulphide, and further including dithiothreitol.
18. A test kit according to claim 14, wherein said enzyme system comprises NADH peroxidase and hydrogen peroxide, and further including sulphide or glutathione and glutathione peroxidase.
19. A test kit according to claim 15 for the determination of triglyceride, comprising NAD, glycerol dehydrogenase, aldehyde dehydrogenase, pyruvate, LDH and buffer and, separately therefrom, esterase and LDH inhibitor.
20. A test kit according to claim 16 for the determi-nation of creatinine in serum comprising NADP, ATP, phos-phoenol pyruvate, glutamate, ?-aminobutyrate, magnesium chloride, fructose, creatine kinase, pyruvate kinase, sorbitol dehydrogenase, glutamate-pyruvate transaminase, .gamma.-aminobutyrate transaminase, succinate-semialdehyde dehydrogenase and, separately therefrom, creatininase and 2,3-dithiol-1-propanol.
21. A test kit according to claim 17 for the determi-nation of creatine kinase, comprising buffer, glucose, magnesium acetate, ADP, AMP, diadenosine-pentaphosphate, NADP, hexokinase, glucose-6-phosphate dehydrogenase, gluta-thione reductase, N-acetyl-cysteine and diglutathione disulphide and, separately therefrom, dithiothreitol.
22. A test kit according to claim 18 for the determi-nation of ?-amylase, comprising buffer, sodium chloride, magnesium acetate, maltoheptaose, NAD, ATP, hydrogen peroxide, hexokinase, glucose-6-phosphate dehydrogenase, ?-glucosidase, NADH-peroxidase and, separately therefrom, sodium bisulphite or catalase.
23. A test kit according to claim 16 for the determi-nation of .alpha.-amylase, containing buffer, sodium chloride, magnesium chloride, maltoheptaose, NAD, glucose dehydroge-nase, fructose, sorbitol dehydrogenase and, separately therefrom, 2,3-dithiol-1-propanol.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2834705A DE2834705B1 (en) | 1978-08-08 | 1978-08-08 | Method and reagent for carrying out enzymatic determinations |
| DEP2834705.9 | 1978-08-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1144458A true CA1144458A (en) | 1983-04-12 |
Family
ID=6046494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000331836A Expired CA1144458A (en) | 1978-08-08 | 1979-07-13 | Process for the carrying out of enzymatic determinations |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0008341B1 (en) |
| JP (1) | JPS5526895A (en) |
| AT (1) | AT362081B (en) |
| AU (1) | AU514173B2 (en) |
| CA (1) | CA1144458A (en) |
| DD (1) | DD145429A5 (en) |
| DE (2) | DE2834705B1 (en) |
| DK (1) | DK320279A (en) |
| YU (1) | YU192879A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4777131A (en) * | 1986-01-13 | 1988-10-11 | Imperial Tobacco Limited | Method of determining sugar content of tobacco using a discrete analyzer |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4338395A (en) * | 1980-07-21 | 1982-07-06 | Technicon Instruments Corporation | Method for the analysis of triglycerides |
| EP0048347B1 (en) * | 1980-09-19 | 1983-07-27 | Roche Diagnostics GmbH | Process and reagent for the determination of glycerol |
| FR2547925B1 (en) * | 1983-06-23 | 1985-12-20 | Biotrol Sa Lab | PROCESS FOR THE SUCCESSIVE DETERMINATION OF VARIOUS COMPONENTS OF A BIOLOGICAL SAMPLING FROM A SAME TEST IN THE SAME REACTION ENCLOSURE AND REAGENTS USED |
| JPS6114364U (en) * | 1984-06-29 | 1986-01-28 | 新日本製鐵株式会社 | Molten metal leak detection device |
| JPS6120636U (en) * | 1984-07-04 | 1986-02-06 | 株式会社 ニチエイ | ceiling edge |
| US4983513A (en) * | 1986-03-28 | 1991-01-08 | Beckman Instruments, Inc. | Sulfhydryl compounds for suppressing the inhibitory effects of NAD degradation products on LD-L activity |
| AUPN200695A0 (en) * | 1995-03-28 | 1995-04-27 | Trace Scientific Pty Ltd | Transaminase reagent |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE1959410C3 (en) * | 1969-11-26 | 1974-02-28 | Boehringer Mannheim Gmbh, 6800 Mannheim | Indicator for determining the reduced pyridine coenzymes |
| US3956069A (en) * | 1974-04-29 | 1976-05-11 | Abbott Laboratories | Enzymatic assays for glucose, creatine phosphokinase or plasma ammonia |
| JPS5441520B2 (en) * | 1974-10-23 | 1979-12-08 | ||
| NL7500951A (en) * | 1975-01-28 | 1976-07-30 | Akzo Nv | FLUORIMETRIC DETERMINATION AND DETERMINATION OF A REDUCED CO-ENZYME OR DERIVATIVE IN AN Aqueous SYSTEM. |
| DE2625834B2 (en) * | 1976-06-09 | 1978-10-12 | Boehringer Mannheim Gmbh, 6800 Mannheim | Method for the determination of substrates or enzyme activities |
-
1978
- 1978-08-08 DE DE2834705A patent/DE2834705B1/en not_active Withdrawn
-
1979
- 1979-06-13 AT AT423079A patent/AT362081B/en not_active IP Right Cessation
- 1979-07-03 EP EP79102248A patent/EP0008341B1/en not_active Expired
- 1979-07-03 DE DE7979102248T patent/DE2961234D1/en not_active Expired
- 1979-07-13 CA CA000331836A patent/CA1144458A/en not_active Expired
- 1979-07-26 AU AU49276/79A patent/AU514173B2/en not_active Ceased
- 1979-07-30 DK DK320279A patent/DK320279A/en not_active Application Discontinuation
- 1979-08-02 DD DD79214767A patent/DD145429A5/en unknown
- 1979-08-07 YU YU01928/79A patent/YU192879A/en unknown
- 1979-08-08 JP JP10032179A patent/JPS5526895A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4777131A (en) * | 1986-01-13 | 1988-10-11 | Imperial Tobacco Limited | Method of determining sugar content of tobacco using a discrete analyzer |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0008341A1 (en) | 1980-03-05 |
| EP0008341B1 (en) | 1981-11-04 |
| DE2834705B1 (en) | 1980-01-24 |
| DD145429A5 (en) | 1980-12-10 |
| AT362081B (en) | 1981-04-27 |
| JPS5526895A (en) | 1980-02-26 |
| DE2961234D1 (en) | 1982-01-14 |
| DK320279A (en) | 1980-02-09 |
| ATA423079A (en) | 1980-09-15 |
| AU514173B2 (en) | 1981-01-29 |
| YU192879A (en) | 1983-01-21 |
| AU4927679A (en) | 1980-02-14 |
| JPS5731439B2 (en) | 1982-07-05 |
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