DE1598326A1 - Laboratory reagent for the determination of urea - Google Patents

Laboratory reagent for the determination of urea

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Publication number
DE1598326A1
DE1598326A1 DE19671598326 DE1598326A DE1598326A1 DE 1598326 A1 DE1598326 A1 DE 1598326A1 DE 19671598326 DE19671598326 DE 19671598326 DE 1598326 A DE1598326 A DE 1598326A DE 1598326 A1 DE1598326 A1 DE 1598326A1
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Prior art keywords
sodium
reagent
urea
determination
laboratory
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DE19671598326
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German (de)
Inventor
Alfred Deutsch
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EMD Chemicals Inc
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Calbiochem Corp
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    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
    • 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
    • C12Q1/008—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/52—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving transaminase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/58—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving urea or urease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Description

■ MÖNCHEN J»■ MÖNCHEN J »

DR. ELISABETH JUNG UND DR. VOLKER VOSSIUSDR. ELISABETH JUNG AND DR. VOLKER VOSSIUS

PATENTANWÄLTE 1598326 PATENT LAWYERS 1598326

. ϋΕ0Ε··Τ«ΑββΕ2· · TtLEFON 3410« . TELEOBAMM-ADRE·«: INVBNT/MONCHEN. ϋΕ0Ε ·· Τ «ΑββΕ2 · · TtLEFON 3410«. TELEOBAM ADRE · «: INVBNT / MONCHEN

u.Z.: C 760 OTo/He)
ÖalMochem 1-2-BuZ: C 760 OTo / He)
ÖalMochem 1-2-B

OAIiBIOCHEM jOAIiBIOCHEM j

Lots Angeles, CaliforniaLots Angeles, California

Yq-V. AYq-V. A.

w Labor Et oriixnsreagens i3ur Bestimmung von Harnstoff w Laboratory et al. reagent for the determination of urea

Priorität: 30. Juni 1966, 7.St.A., Nr. 561757Priority: June 30, 1966, 7th St.A., No. 561757

Die Erfindung betrifft ein Lataoratoriumsreagens sum Kaohveie und eur Bestimmung von Harnstoff in biologischen flüssigkeiten, wie in Seru», und Methoden sum Test solcher Flüssigkeiten unter Verwendung des Laboratoriumsreagens.The invention relates to a laboratory reagent sum Kaohveie and eur determination of urea in biological fluids, as in Seru », and methods sum test such fluids below Use of the laboratory reagent.

Bas Reagens der Erfindung 1st in trockener Fora stabil, einfach und bequea verpackbar, es gibt reproduzierbare Ergebaiss* und ist einfach in der Anwendung. Dieses Beagens dient lur Arbel erleichterung für Biochemiker und Forscher auf ähnlichen OeThe reagent of the invention is stable, simple in dry form and conveniently packable, there are reproducible results * and is easy to use. This Beagens serves lur Arbel relief for biochemists and researchers on similar oe bleten«bleed "

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Das Iiaboratoriiiinsreagezjfl eier Erf inciting wird in Binheitsmezigen bereitet. Jede dieser Einheiten enthält gerade soviel Reagens» um einen einzigen Tost einer Probe durchstuf uhren. Um einen lest zu maehec, wird eine Packung ausgewählt, die des Reagens für diese besondere Bestimmung enthält. Das gesamte Reagens in der Packung ist vorher goxae3sen und von festgelegter Aktivität. Demzufolge kann 03 unmittelbar in einer Standardmenge Wasser zu einem flüssigen Reagens gelöst werden» DiesesThe laboratory experimentation is given in binary terms prepares. Each of these units contains just that much reagent » to step through a single toast of a sample. To one read to maehec, a pack is selected that contains the reagent for this particular purpose. All of the reagent The pack is previously goxae and has a fixed activity. As a result, 03 can be dissolved immediately in a standard amount of water to form a liquid reagent »This

* flüssige Reagens wird dann mit der biologischen Probe gemisoht und führt zu einer eneymatischen Reaktion. Die GruSe der Reaktionsgeschwindigkeit ist eine funktion der Menge oder der Aktivität der zu bestimmenden Substanz. In jedem !fest wird, unabhängig von der besonderen Art der Bestimmung, eis Coensym aus einer Fora in die andere überführt, wobei eine der beiden Formen bei einer bestimmten Wellenlänge Licht absorbiert. Die optische Dichte der Probe bei dieser Wellenlänge wird demzufolge als Funktion der eu bestimmenden Substans variieren.* Liquid reagent is then mixed with the biological sample and leads to an eneymatic response. Greetings from The rate of reaction is a function of the amount or the activity of the substance to be determined. In each! Becomes firm, regardless of the particular type of determination, eis coensym transferred from one fora to the other, one of the two Shapes at a certain wavelength absorbs light. The optical density of the sample at this wavelength will therefore vary as a function of the substances determining the eu.

) Durch Messung der optischen Dichte des Ansateee su verschiedenen Zeiten kamt man daher die Menge oder die Aktivität der su bestimmenden Sttbstans in der ursprünglichen Probe errechnen.) By measuring the optical density of the Ansateee su different Times one could therefore calculate the quantity or the activity of the substances to be determined in the original sample.

Das Lahoratoriuasreagens der Erfindung «ur Bestimmung von Harnstoff setst sich lusammen aus: .The lahoratorium reagent of the invention for the determination of urea is made up of:.

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Einem Substrat vrie eO ketoglutarsäure, genügend um mit dem Sticketoff des Harnstoffe in der Testprobe fm reagieren;One substrate contained eO ketoglutaric acid, sufficient to react with the substance of the urea in the test sample fm; den Enzymen Urease und GKLut^insäuredehydrogeiiaBejthe enzymes urease and GKLut ^ insäuredehydrogeiiaBej

einem Coensym aus dsr Gruppe der Pyridinnueleotideja coensym from the group of the Pyridinnueleotidej

einem Aktivator;an activator;

einem Stabilisator vie einer Sulifhydryl-Verbindung, s.B. Dithioerythrit, Cystein oder Glutathion, oder einem SaIs, wie Hatriura-Xalium-Tartrat. Natriumsulfat, Natriumtartrat»a stabilizer such as a sulfhydryl compound, see B. Dithioerythritol, cysteine or glutathione, or a SaIs such as Hatriura xalium tartrate. Sodium sulfate, sodium tartrate »

Kaliumsulfat oder If&triutaglutamat;Potassium sulfate or If? Triutaglutamate;

einem oder mehreren Puffern, wie Alkalimetallphosphate, Tris~(hydroxymethyl)-araino3iethant AlJcalimetallblcarbonate oder -carbonate und Glycin/ sowie einer Polyhydroxjrerbindung oder deren Polymer mit 1-5 Hydroxylgruppen je Monomereiniieit als stabilisierend wirkendem füllstoff. Ale Polyhydroxyrerbindung wird Mannit boToreugt. Andere Verbindungen sind Sorbit, Lftctoee und Polyvinylalkohol.one or more buffers, such as alkali metal phosphates, Tris ~ (hydroxymethyl) -araino3iethan t AlJcalimetallblcarbonate or carbonates and glycine / and a Polyhydroxjrerbindung or its polymer having 1-5 hydroxyl groups per Monomereiniieit a stabilizing acting filler. Mannitol is produced in the polyhydroxy compound. Other compounds are sorbitol, Lftctoee and polyvinyl alcohol.

Das Beispiel erläutert die Erfindung. (The example illustrates the invention. (

Belapiel . Belapiel .

Das Reagens but Bestimmung des Harnstoffgeh&ltes enthält in trockener Mischung folgende Substanzen:The reagent for determining the urea content contains in dry mixture of the following substances:

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EnzymeEnzymes

StabilisatorenStabilizers

Pufferbuffer

SubstrateSubstrates

CoenzymCoenzyme

AktivatorActivator

Füllstofffiller

Urease und GlutemlnBäure-Dehydrogenase Dithioerythrit und ffatrium-Xälium-Tartrat Tr is- (hydr os^naethyl) -aininoffie than ©6 -Ketoglutarsäure oder deren Salse Reduziertes Diphosphopyridinnucleotid (DPHH) Adenosindiphosphat, Natriumsals Mannit.Urease and gluten acid dehydrogenase Dithioerythritol and ffodium xaelium tartrate Tr is- (hydr os ^ naethyl) -aininoffie than © 6 -Ketoglutaric acid or its salts Reduced diphosphopyridine nucleotide (DPHH) Adenosine diphosphate, sodium as Mannitol.

Zur Herstellung einer großen Zahl von Einheitsmengen dieses Reagens wird das folgende Verfahren angewendet, welches eine Charge an trockenem Reagens liefert, die für 10 000 Kapseln ausreicht, von denen jede eine Testmischung von 3 al ergibt *To produce a large number of unit quantities of this Reagent, the following procedure is used which provides a batch of dry reagent sufficient for 10,000 capsules sufficient, each of which gives a test mixture of 3 al *

Die trockene Puffer-Subatrat-Misehung von Tris~(hrdroxynetliyl)-aminomethan und oi -Ketoglutarsäure wird durch Mischen und Vermählen der entsprsehenden Mengen der Komponenten erhalten. Wird die Mischung in Wasser gelöst, so bildet sich das 3?rie-(hydro^ymethyl)~aminomethanBalz der oC-Ketoglutarsäure, ein Puffer des geeigneten pg-Wertes. Trie-(hydro2ymethyl)-aminomethaa wird als Puffer vorgesogen; jedoch sind Phosphat-, (Jlyoin- oder Biearbonat-Puffer ebenso brauchbar.The dry buffer Subatrat-Misehung of Tris ~ (hrdroxynetliyl) aminomethane and oi ketoglutaric acid is obtained by mixing and grinding the entsprsehenden amounts of the components. If the mixture is dissolved in water, the 3? Rie- (hydro ^ ymethyl) ~ aminomethane salt of oC-ketoglutaric acid is formed, a buffer of the appropriate pg value. Trie- (hydro2ymethyl) -aminomethaa is pre-sucked as a buffer; however, phosphate, (jlyoin, or beer carbonate buffers are also useful.

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Urease wird aus fein gemahlenen Saubohnen mit verdünntem wässrigem Fatriumphosphat-Puffer (pg 6,3 bis 6,7) extrahiert· Der Extrakt wird zur Entfernung von Ammoniak dialysiert, durch Filtration geklärt, Dithioerythrit wird zugesetzt und die erhaltene lösung lyophilisiert. So entsteht ein trockenes, stabiles, Urease enthaltendes Pulver.Urease is extracted from finely ground broad beans with dilute aqueous sodium phosphate buffer (p g 6.3 to 6.7). The extract is dialyzed to remove ammonia, clarified by filtration, dithioerythritol is added and the resulting solution is lyophilized. The result is a dry, stable powder containing urease.

Im Anschluß daran wird durch Gtefriertrooknung ei» Pulver gewonnen, das G-lutamlnsäure-Dehydrogenaae enthält. Dies wird durch verwendung der folgenden Chemikalien in den angegebenen Mengen erreicht:The powder is then freeze-dried obtained which contains G-lutamic acid dehydrogenaae. this will achieved by using the following chemicals in the specified amounts:

&lutaminsäure~Dehydrogenaser 1 000 000 Internationale& lutamic acid ~ dehydrogenase r 1 000 000 international

Einheitenunits

Dithioerythrlt, 300 - 600 mgDithioerythrlt, 300-600 mg

Natrium-Kalium-tartrat, 25 al gesättigte Lösung.Sodium potassium tartrate, 25 al saturated solution.

Der Enzymlösung werden Dithioerythrit und Kalium-Natrium-tartrat Eugesetzt, und man erhält eine stabilisierte Lösung. Sobald dl· stabilisierte Lösung gemischt iet, wird sie eine ausreichend lange Zeit unter vermindertem Druck gehalten, um jeglioh· eingeschlossene Luft zu entfernen. Nun wird die Lösung lyophilisiert, und man erhält ein Trockenpulver aus Gluteroinsäu?e~Dehjdrogenms· und Stabilisatoren, die bewirken, daß die Aktivität dee J&usya* auf der gewünschten Höhe bleibt.Dithioerythritol and potassium sodium tartrate are added to the enzyme solution, and a stabilized solution is obtained. As soon as the stabilized solution is mixed, it is kept under reduced pressure for a sufficient time to remove any trapped air. The solution is now lyophilized, and a dry powder of gluterine acid (dehydrogenative) and stabilizers is obtained, which ensures that the activity of the drug remains at the desired level.

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Bind die lyophilisierten Pulver bereitet» die Urease u*d Glutaminsäure-Dehydrogenase enthalten, so werden aie getestet, um die Aktivität dee Enayms Urease im Pulver su bestimmen. Hierau wird ein Reagens verwendet, das die folgenden Chemikalien in den angegebenen Mengen enthält:Bind the lyophilized powder prepares »the urease and * d Contain glutamic acid dehydrogenase, they are all tested to determine the activity of Enaym's urease in the powder. A reagent is used that contains the following chemicals in the specified amounts:

Substrat Harnstoff (0,01 M)Urea substrate (0.01 M) Coensya DPNH (4 mg/ml)Coensya DPNH (4 mg / ml) Puffer 0,1 m, Pn 7,5, «4-Ketoglutarat enthaltendBuffer 0.1 m, P n 7.5, containing 4-ketoglutarate

I ■ ■■I ■ ■■

Aktivator Adenosindiphosphat 10 mg/ml.Activator adenosine diphosphate 10 mg / ml.

Wenn diese Lösungen in den angegebenen Konzentrationen hergestellt sind, so werden sie vermischt und ergeben ein flüssiges Reagens, mit dessen Hilfe der vorliegende Test ausgeführt werden kann. Eine passende Menge jedes der lyophilisierten Pulver, die die Enzyme Glutaminsäure-Dehydrogenase und Urease enthalten, wird in Wasser gelöst. Hierzu werden ungefähr 100 mg jedes Pulvere in ungefähr 10 ml Wasser gelöst su «wei getrenntem Ensymlösungen. Etwa 0,1 ml jeder Lösung wird mit den Reagens vereinigt. Sofort setsst die Umwandlung von DPIfH in VBB (Diphosphopyridinnucleotidy oxydierte Form) ein. Während diese Reaktion abläuft, wird die Lösung in ein geeignetes Spelrtralphotometer gestellt und die optische Dichte des Reagens su geeigneten Zelten, s.B. jede Minute, gemessen. Fach einigenWhen these solutions are made up at the indicated concentrations, they are mixed to produce a liquid reagent which can be used to carry out the present assay. An appropriate amount of each of the lyophilized powders containing the enzymes glutamic acid dehydrogenase and urease is dissolved in water. For this purpose, approximately 100 mg of each powder are dissolved in approximately 10 ml of water, as shown in separate ensym solutions. About 0.1 ml of each solution is combined with the reagent. The conversion of DPIfH into VBB (diphosphopyridine nucleotides oxidized form) starts immediately. While this reaction is proceeding, the solution is placed in a suitable spectral photometer and the optical density of the reagent is measured in suitable cells, for example every minute. Some subject

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Ablesungen wird, ein Mittelwert gebildet, der die Aktivität der Urease in dem Trookenpulver anzeigt. X)Ie Änderung der optischen Dichte von 0,001 pro Minute bedeutet 1 Aktivitäteeinheit. Hieran iat es möglich, die Kenge der Iyophilleierten, Urease und Glutaisineäure-Dehydrogenase enthaltenden Pulver su berechnen, die nötig ist, eine Aktivität zu entfalten, die die Reaktion (für 0,05 Mlkromol Harnstoff) in 5 bis 10 Mittaten -vollendet. . 'Readings are taken, averaging the activity which indicates urease in the trooken powder. X) Ie change of optical density of 0.001 per minute means 1 unit of activity. In this way it is possible to recognize the kenge of the lyophilized, Powders containing urease and glutaisinic acid dehydrogenase calculate su that is necessary to develop an activity, which the reaction (for 0.05 mlcromoles of urea) in 5 to 10 Accomplishments - accomplished. . '

Hierauf werden Puffor-Subeträte, Aktivatoren und Cofaktoren zu den Urease und Glutaminsäure-Dehydrogenaee enthaltenden trockenen Ijophilisierten Pulvern gegeben, gemahlen und in trockener Atmosphäre gemischt. Die resultierende Misohung kann in Kapseln verpackt werden. Zur Herstellung eines Reagens für 10 000 Test» werden die folgenden Chemikalien in den angegebenen Mengen eingeaetut:This is followed by puffing substances, activators and cofactors to those containing urease and glutamic acid dehydrogenaee given dry Ijophilized powders, ground and in mixed dry atmosphere. The resulting misogyny can packed in capsules. To make a reagent for 10,000 test »the following chemicals are inhaled in the specified quantities:

frie-(bydrox7methyl)-amlnoBi6than 300 - $00 £frie- (bydrox7methyl) -amlnoBi6than 300- $ 00 £

oC -.Ketoglutarsäure 50 - 69 goC - ketoglutaric acid 50 - 69 g

Adenoeindiphoaphat, Ratriumeals 5 - 15 gAdenoeindiphoaphate, sodium as 5-15 g

Mannit 500 -1000 gMannitol 500-1000 g

BPBH 5 - 6 gBPBH 5 - 6 g

Lyophilieierte Glutamiasäure-Dehydrogenaae 7 - 15 Internationale EinheitenLyophilized glutamic acid dehydrogenaae 7 - 15 international units

Igrophilisierte Urease 15 - 40 InterIgrophilized urease 15 - 40 inter

nationale Einheiten.national units.

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οι -Ketoglutarsäure, TriMhy^ODsymöthyl)-«einem* than, Adenosindiphosphat und Hannit wurden vermischt und mittels geeigneter Torrichtungen, wie einer Kugelmühle, 6 bis 10 Stunden vermählen« Xm Anschluß· an dieses üiieehen wird eine Probe von etwa 100 mg der Mischung in einer geeigneten Meaf* an Wasser, etwa $ al» gelöst. Der pg-Wert dieser Lösung soll «wischen 8,0 und 8,5 liegen.οι -Ketoglutaric acid, TriMhy ^ ODsymöthyl) - «a * than, Adenosine diphosphate and hannitol were mixed and by means of suitable gate directions, such as a ball mill, 6 to 10 Hours of marriage "Following this, there will be a marriage Sample of approximately 100 mg of the mixture in a suitable Meaf * in water, about $ al »dissolved. The pg value of this solution should be «Between 8.0 and 8.5 lie.

ψ Man hat nun ein Pulver erhalten, das die awel Eney»e susaxaen mit Mannit, Trie- (hydroxymethyl) -sninomethaa, oo-Zetoglutexeöure und Adenosindiphosphat enthält. Das Pulver wird im Vakuusi 2 Sage oder länger getrocknet, un sicher su gehen, daS das Pulver sehr stabil wird und eine lange lebensdauer hat, während Her die Enzymaktivität nicht serstQrt wird. ψ A powder has now been obtained which contains the awel Eney »e susaxaen with mannitol, tri- (hydroxymethyl) -sninomethaa, oo-zetoglutexic acid and adenosine diphosphate. The powder is 2 Speak in Vakuusi or more dried un, tHe the powder is very stable and has a long service life, while Her enzyme activity is not serstQrt sure su.

Vaeh dem Trocknen wird das BPBH unter trockenen Bedingungen sugesetct; die sugesetste Menge ist in der Regel ausreichend, ^ eine Absorption von 1 bei 340 Millimicron su erslelen. 8usatsllch enthält das Pulver einen Puffer, fiacyae, Idenosln* diphosphat-Vatriumsals und Φ -Ketoglutarsäure, die die er- «Unechte Reaktion bewirken» wenn ein Test durchgeführt wird.After drying, the BPBH is sugesetct under dry conditions; the sweetest amount is usually sufficient to achieve an absorption of 1 at 340 millimicrons. Usually, the powder contains a buffer, fiacyae, Idenosln * diphosphate sodium as and Φ- ketoglutaric acid, which cause the «false reaction» when a test is carried out.

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1598Ϊ281598-28

Dementsprechend wird dieses Pulver in eine Vielzahl kleiner feile geteilt, die gerade groß genug sind, ein flüssiges Reagens *u ergeben, das ausreicht, einen Test mit einer Probe zu machen. Sie gesamte Menge des Pulvers wird dann in Einheit einengen in Kapseln gefüllt, die die gewünschte Aktivität enthalten- .Accordingly, this powder becomes smaller in a multitude files that are just big enough to make a liquid one Reagent * u result in a test with a To make a test. You entire amount of powder is then put in The unit is filled into capsules containing the desired activity.

Ua eine der Kapseln ssu einer Earnatoffbestimmung la Serum zu verwenden, wird eine Probe eines Serums oder einer anderen biologischen Flüssigkeit von geeigneter Menge bereitet« Bine Reagenskapsel wird in einer passenden Menge Wasser gelöst. Die entstellende Lasung bildet ein flüssigee Reagens für einen einsigen lest des Serums. Sobald Reagens und Probe vermischt sind, wird folgende Reaktion ablaufen:Among other things, one of the capsules ssu an Earnatoff determination la serum To use it, a sample of a serum or other biological fluid is prepared in a suitable quantity «Bine The reagent capsule is dissolved in an appropriate amount of water. The disfiguring solution is a liquid reagent for you read some of the serum. Once reagent and sample are mixed the following reaction will take place:

Harnstoff + Wasser -SSSÜS* 2 KH* + HCOjUrea + water - SWEET * 2 KH * + HCOj

IHj * *>~Ketoglutarat + DPSH Olutaaat + BFNIHj * *> ~ ketoglutarate + DPSH Olutaaat + BFN

Da c£-Ket©glutarsäure, DPITH, Urease und Glutaminsäure-Dehydrogenaee in der Kapsel in genügender Menge vorliegen, wird das Ausmaß der beschriebenen Reaktionen nur durch die Renge asSince c £ -Ket © glutaric acid, DPITH, urease and glutamic acid dehydrogenaee are present in the capsule in sufficient quantities, this will be the case Extent of the reactions described only by Renge as

209810/0369 BAD ORlQlNAt209810/0369 BAD ORlQlNAt

15383261538326

Harnstoff in der zu untersuchenden Probe begrsnst werde». Durch- Einbringen der Probe in ein geeignetes Spektrslp&otoBwt und MeBSUHg der optischen Sicht® bei 340 Milliaioroii kann die Menge an DPHH bestimmt werden, die umgewandelt wird. Biss« wiederum erlaubt ee, die Menge an Harnstoff in der ursprünglichen Probe su bereöiinen.Urea is welcomed in the sample to be examined ». By introducing the sample into a suitable spectral lp & otoBwt and MeBSUHg of Optical Sight® at 340 Milliaioroii can die Determine the amount of DPHH that is converted. Bite" in turn, allows the amount of urea in the original sample to be checked.

20981Q/0369 - t. BAD ofuginal 20981Q / 0369 - t. BAD ofugin al

Claims (3)

!Patent an Sprüche! Patent to sayings 1) Laboratoriumsreögens zur Bestimmung Von Harnstoff in biologischen Plttssigksiten, enthaltend in trockener Fora *1) Laboratory test results for the determination of urea in biological fluids, contained in dry fora * a) Urease und GlutaMnsäure-Dehydrogenase,a) urease and glutamic acid dehydrogenase, b) <i* -Ketoglutarsäure,b) <i * -ketoglutaric acid, o) D&pnosphorpyridinnucleotid in reduzierter Form» d). eines Aktivator,o) D & pnosphorpyridinucleotide in reduced form » d). an activator, e) Dithioerythrit und Ratrium-Salium-tartrat,e) Dithioerythritol and Sodium Salium Tartrate, f) einen oder mehrere Puffer aus der Gruppe der natrium- oder Saliumphosphate, Tris-(hydroxymethyl)-euainometb.an», Hatriua- oder Kaliumbicarbonat, Natrium- oder Kaliumcarbonat und Glycin. ,.f) one or more buffers from the group of sodium or Saliumphosphate, Tris- (hydroxymethyl) -euainometb.an », Hatriua- or potassium bicarbonate, sodium or potassium carbonate and Glycine. ,. g) eine PolyhydroxyverMnclung oder deren Polymer mit 1 bis Hydroxylgruppen je Mpnamsreinheit als «tabilieierend wirkenden Sülls tof f. ·g) a polyhydroxy compound or its polymer with 1 to Hydroxyl groups per name purity as a stabilizing filler. 2) Laboratoriumsreagene nach Anspruch 1, dadurch g ekennseichnet» daß der Aktivator Adenosindi- , pho8phat~Natriumoalz ist.2) laboratory reagents according to claim 1, characterized g ekennseichnet »that the activator adenosine, pho8phat ~ is sodium salt. 3) Laboratoriumsreagens nach Anspruch 1, dadurch g ekennseichnet, dad der Füllstoff Mannit ist.3) laboratory reagent according to claim 1, characterized in that the filler is mannitol. 209810/0369209810/0369 BADORSQiNALBADORSQiNAL
DE19671598326 1966-06-30 1967-06-30 Laboratory reagent for the determination of urea Pending DE1598326A1 (en)

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US561757A US3413198A (en) 1966-06-30 1966-06-30 Reagents and method for assaying biological samples

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DE1598325A Granted DE1598325B1 (en) 1966-06-30 1967-06-30 Laboratory reagent for the determination of glutamate oxaloacetate transaminase
DE19671598321 Pending DE1598321A1 (en) 1966-06-30 1967-06-30 Laboratory reagent for the determination of creatine kinase
DE19671598326 Pending DE1598326A1 (en) 1966-06-30 1967-06-30 Laboratory reagent for the determination of urea

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DE1598325A Granted DE1598325B1 (en) 1966-06-30 1967-06-30 Laboratory reagent for the determination of glutamate oxaloacetate transaminase
DE19671598321 Pending DE1598321A1 (en) 1966-06-30 1967-06-30 Laboratory reagent for the determination of creatine kinase

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US (1) US3413198A (en)
BE (1) BE700778A (en)
CH (1) CH514841A (en)
DE (3) DE1598325B1 (en)
GB (3) GB1192046A (en)
IL (1) IL28098A (en)
NL (1) NL6708570A (en)

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US7744925B2 (en) 1994-12-02 2010-06-29 Quadrant Drug Delivery Limited Solid dose delivery vehicle and methods of making same
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BE700778A (en) 1968-01-02
IL28098A (en) 1976-02-29
CH514841A (en) 1971-10-31
GB1192046A (en) 1970-05-13
US3413198A (en) 1968-11-26
DE1598321A1 (en) 1972-02-24
DE1598325B1 (en) 1973-01-11
GB1163409A (en) 1969-09-04
NL6708570A (en) 1968-01-02
GB1191697A (en) 1970-05-13

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