U.S. patent application number 09/760205 was filed with the patent office on 2001-11-22 for stabilized coenzyme solutions and their use for determining dehydrogenases or substrates thereof.
Invention is credited to Zielenski, Ralf.
Application Number | 20010044128 09/760205 |
Document ID | / |
Family ID | 7627647 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010044128 |
Kind Code |
A1 |
Zielenski, Ralf |
November 22, 2001 |
Stabilized coenzyme solutions and their use for determining
dehydrogenases or substrates thereof
Abstract
Stabilized aqueous solution of a coenzyme for
hydrogen-transferring enzymes characterized in that the solution
contains NAD, NADP or a derivative thereof in an oxidized or
reduced form and one or several organic compounds or salts thereof
having a pKa value between 1.5 and 6.0 and/or a nitrogen compound
of the general formula (I) 1 in which the residues R.sup.1, R.sup.2
and R.sup.3 are the same or different and denote hydrogen, or a
saturated or unsaturated alkyl or aryl group as well as the use of
the solution to determine dehydrogenases in particular lactate
dehydrogenase or substrates thereof.
Inventors: |
Zielenski, Ralf;
(Benediktbeuern, DE) |
Correspondence
Address: |
Marilyn L. Amick
Roche Diagnostics Corporation
9115 Hague Road, Bld. D
P.O. Box 50457
Indianapolis
IN
46250-0457
US
|
Family ID: |
7627647 |
Appl. No.: |
09/760205 |
Filed: |
January 12, 2001 |
Current U.S.
Class: |
435/26 |
Current CPC
Class: |
C12Q 1/32 20130101 |
Class at
Publication: |
435/26 |
International
Class: |
C12Q 001/32 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 15, 2000 |
DE |
100 01 529.8 |
Claims
1. Stabilized aqueous solution of a coenzyme for
hydrogen-transferring enzymes characterized in that the solution
contains NAD, NADP or a derivative thereof in an oxidized or
reduced form and one or several organic compounds or salts thereof
having a pKa value between 1.5 and 6.0 and/or a nitrogen compound
of the general formula (I) 3in which the residues R.sup.1, R.sup.2
and R.sup.3 are the same or different and denote hydrogen, or a
saturated or unsaturated alkyl or aryl group.
2. Stabilized solution as claimed in claim 1, characterized in that
it contains an acid or a salt thereof with a buffering action in
the pH range of 1.0 to 7.0 as the organic compound.
3. Stabilized solution as claimed in claim 1 or 2, characterized in
that it contains citric acid or a citrate salt.
4. Stabilized solution as claimed in claim 3, characterized in that
it contains ca. 5 to 500 mM citric acid or a citrate salt.
5. Stabilized solution as claimed in one of the claims 1 to 4,
characterized in that the pH value of the solution is between 1.0
and 7.0.
6. Stabilized solution as claimed in one of the claims 1 to 5
containing a hydroxyl, O- or N-alkyl-hydroxyl,
O-benzylhydroxylamine and/or boric acid derivative.
7. Stabilized solution as claimed in claim 6, characterized in that
it contains a hydroxylamine derivative at a concentration between 2
and 300 mM.
8. Method for determining a hydrogen-transferring analyte or a
corresponding dehydrogenase in the presence of a hydrogen-accepting
coenzyme characterized in that the coenzyme is contained in a
stabilized aqueous solution as claimed in claims 1 to 7.
9. Method for the determination as claimed in claim 8,
characterized in that the analyte lactate, glutamate, ammonia,
alcohol, glyceraldehyde-3-phosphate or glucose is determined in the
presence of a lactate, glutamate, alcohol, glycerol-3-phosphate or
glucose dehydrogenase.
10. Method as claimed in one of the claims 8 or 9, wherein the pH
value is in a range of 8.5 to 10.0 and the final concentration of
citrate salt, boric acid and/or hydroxylamine derivative is in each
case between 2 and 50 mM.
11. Kit for determining a hydrogen-transferring analyte in a sample
comprising the following components: a first reagent which contains
a dehydrogenase in a suitable system buffering between pH 8.5 and
10.0 and a second reagent which contains a coenzyme for
hydrogen-transferring enzymes and an organic compound having a pKa
value between 1.5 and 6.0 and/or a nitrogen compound of the general
formula (I) 4in which the residues R.sup.1, R.sup.2 and R.sup.3 are
the same or different and denote hydrogen, or a saturated or
unsaturated alkyl or aryl group.
12. Kit for determining the enzyme activity of a dehydrogenase in a
sample comprising the following components: a first reagent which
contains a hydrogen-transferring analyte in a suitable system
buffering between pH 8.5 and 10.0 and a second reagent which
contains a coenzyme for hydrogen-transferring enzymes and an
organic compound having a pKa value between 2.0 and 4.0 and/or a
hydroxylamine derivative of the general formula (I) as claimed in
claim 11.
13. Kit as claimed in claim 11 or 12, characterized in that the
second reagent contains citric acid, a citrate salt, a boric acid
and/or hydroxylamine derivative.
14. Kit as claimed in claim 11 or 12, characterized in that the
first reagent contains a boric acid derivative and the second
reagent contains citric acid, a citrate salt and/or a hydroxylamine
derivative.
15. Kit as claimed in claim 11 to 14, characterized in that the
second reagent has a pH value between 1.0 and 7.0.
16. Kit as claimed in one of the claims 11 to 15, characterized in
that the second reagent has a pH value of about 3.0.
17. Kit as claimed in one of the claims 11 to 16, characterized in
that the first or second reagent contains approximately 5 to 200 mM
of a citrate salt, about 2 to 300 mM of a boric acid derivative
and/or 2 to 300 mM of a hydroxylamine derivative.
Description
[0001] The invention concerns stabilized aqueous solutions of a
coenzyme for hydrogen-transferring enzymes and their use for
determining a corresponding analyte (substrate) in a reduced form
or for determining the enzyme activity of a corresponding
dehydrogenase. The stabilized solution contains an organic compound
or appropriate salts having a pKa value between 1.5 and 6.0 and/or
a hydroxylamine derivative.
[0002] The determination of enzyme activities (or substrate
concentrations), especially in blood serum or plasma, plays an
important role in clinical chemical diagnostics. Test procedures
are often used for this which are based on the reduction of
nicotinamide adenine dinucleotide ("NAD") or nicotinamide adenine
dinucleotide phosphate ("NADP") and photometric detection of the
resulting change of the absorption behaviour in the ultraviolet
wavelength range (.lambda.=334, 340 or 365 nm). When suitable test
conditions have been selected, this change is linearly proportional
to the enzyme activity (or substrate concentration) to be
determined.
[0003] Nowadays the methods described in Eur. J. Clin. Chem. Clin.
Biochem. 31, 897 (1994) and Eur. J. Clin. Chem. Clin. Biochem. 32,
639 (1994) are generally recommended for determining the enzyme
activity of for example lactate dehydrogenase (LDH, E.C.1.1.1.27).
The test principle involves the oxidation of lactate to pyruvate
while a coenzyme such as NAD or NADP is simultaneously reduced to
NADH or NADPH. Such a conversion, in this case is for example
catalysed by LDH, takes place in an alkaline medium (pH 9.4). As a
result of this instability there is a relatively rapid increase in
absorbance (the so-called reagent blank) in the wavelength range
for the measurement and hence the reagent combination becomes
unusable already after a short time (3 months) even when stored in
a refrigerator (2.degree.to 8.degree. C.). This is a particular
problem for the production of ready-to-use liquid reagents with a
long shelf-life which are intended to enable the user to carry out
analyses in the daily routine in a simple and reliable manner.
[0004] A method for stabilizing aqueous coenzymes using chelating
agents and azides is known from JP 84/82398. However, a
disadvantage of this method is that it is necessary to add azide
which is nowadays classified as cancerogenic and which, moreover,
has an inhibitory effect on many enzymes.
[0005] It is also known that coenzyme solutions can be stabilized
by adding heavy metal salts, for example in the form of copper (II)
ions, and thus prevent an increase of the reagent blank (DE 195 43
493 or EP 0 804 610). However, degradation products may form during
long storage periods or at high storage temperatures (already above
10.degree. C.) which inhibit the dehydrogenase enzyme to be
determined and thus result in measured values that are too low. A
reagent that can be stabilized over a long period (3 months and
more) of constant quality which thus, among other things, does not
require repeated calibrations is not available at present.
[0006] Hence the object of the present invention is to provide an
improved stable liquid reagent containing a coenzyme for
hydrogen-transferring enzymes which is suitable for determining
dehydrogenase activity or corresponding substrates.
[0007] The object is achieved by an aqueous solution which contains
a coenzyme for hydrogen-transferring enzymes such as NAD, NADP or
an appropriate derivative in an oxidized or reduced form (so-called
regenerating systems) and one or several organic compounds or salts
derived therefrom with a pKa value between 1.5 and 6.0 and/or a
nitrogen compound of the general formula (I) 2
[0008] in which the residues R.sup.1, R.sup.2 and R.sup.3 are the
same or different and denote hydrogen, a saturated or unsaturated
alkyl or aryl group. Suitable alkyl groups are in particular those
which have one to ten carbon atoms. Furthermore the alkyl groups
can be straight-chained or branched. Suitable aryl groups according
to the invention are substituted or unsubstituted phenyl groups
which are optionally bound via an alkenyl group which can have 1 to
8 carbon atoms. Nitrogen compounds of the said general formula (I)
i.e. in particular hydroxylamine derivatives such as hydroxylamine,
O- or N-alkylhydroxylamine having one to six carbon atoms or
O-benzylhydroxylamine or salts derived therefrom such as sulfates,
phosphates or ammonium salts have proven to be particularly
suitable according to the invention. In addition suitable
hydroxylamine derivatives are characterized by a complexing effect
towards the degradation products of the coenzyme.
[0009] In addition the stability of the solutions can be further
improved when the solution additionally contains a complexing agent
i.e. a ligand which has two or more coordination positions.
Bidentate ligands such as ethylene diamine and tetradentate or
multidentate ligands such as ethylenediamine-N,N,N,N-tetraacetic
acid (EDTA) or appropriate salts thereof, especially the disodium
salt, crown ethers or cryptands have proven to be advantageous.
This corresponds to a concentration of the complexing agent of
about 0.5 to 30 mM, preferably of 1.0 to 5.0 mm.
[0010] Organic compounds or salts derived therefrom that are added
according to the invention with a pKa value between ca. 1.5 and 6.0
are especially organic acids which have a completing action and a
buffering action in the pH range of 1.0 to 7.0 such as citric acid
and water-soluble salts derived therefrom.
[0011] The concentrations of the organic compounds, salts or
hydroxylamine derivatives that are to be added according to the
invention can vary within wide limits i.e. between ca. 0.001 and
1.0 M. A concentration of ca. 5 to 200 mM has proven to be
particularly suitable for citric acid or citrate. In numerous cases
ca. 50 mM citric acid or citrate already resulted in the desired
effect. The preferred concentration range for the hydroxylamine
derivative according to the invention that is to be added in
addition to or in the absence of an organic compound with a
suitable pKa value is between about 2 and 300 mM. The pH value of
the stabilized aqueous solution can be between 1.0 and 7.0, a pH
value between ca. 2.0 and 4.0 or of ca. 3.0 having proven to be
particularly advantageous.
[0012] Moreover, it has proven to be particularly advantageous when
the reagent containing NAD or NADP contains a hydroxylamine
derivative and optionally additionally a citrate salt and when
boric acid or a borate salt is additionally present in an optional
further reagent that may be necessary to determine corresponding
hydrogen-transferring analytes that contains in particular buffers
necessary for the determination such as N-methylglucamine (MEG),
substrates and optionally other auxiliary substances. The
concentrations set forth above also apply to this special
embodiment. Furthermore, ca. 20 to 200 mM have proven to be
particularly advantageous for citrate or citric acid and ca. 10 to
150 mM for the respective hydroxylamine derivative. A concentration
range of ca. 50 to 200 mM has proven to be particularly suitable
for the boric acid derivative which is preferably added to the
substrate solution (so-called reagent 1) which does not contain NAD
or NADP.
[0013] Substances which have a good buffer capacity between ca. pH
8.5 and 10.0 such as the so-called Good buffers (tricine, bicine,
TAPS, AMPSO, CHES, CAPSO, AMP, CAPS), carbonates of alkali metal
ions, MEG, TRIS and phosphate buffer are basically suitable as
buffers for the reagent containing substrate. Mixtures of the said
buffer substances have also proven to be suitable for the solution
according to the invention. In addition it has proven to be
advantageous when the buffer concentration is between ca. 10 and
1000 mM, preferably between 200 and 600 mM. Furthermore, the
addition of boric acid or soluble salts and derivatives thereof to
the alkaline buffer solution (reagent 1) which primarily determines
the working pH value has proven to be advantageous. The
concentration of suitable boric acid components is preferably
between about ca. 50 and 200 mM, particularly preferably about 100
mM. Suitable coenzymes in the sense of the present invention are in
particular NAD and NADP, and also modified coenzymes such as
thioNAD(P) or NHXDP (=nicotinamide hypoxanthine dinucleotide
phosphate). The coenzymes can be present at a concentration of
approximately 1.0 to 100 mM in the reaction cuvette; a range of 5.0
to 15.0 mM is preferred.
[0014] The stabilized coenzyme solutions according to the invention
are preferably used in the form of aqueous solutions. Furthermore
the ready-to-use reagent is also stable over a wide time period as
a granulate, powder mixture and as a lyophilisate. Thus no signs of
reagent decomposition whatsoever are found at temperatures of
2.degree. to 8.degree. C. within 15 months. Under stress i.e. at a
temperature of ca. 35.degree. C. for 2 weeks or treatment at ca.
42.degree. C. for five days, it was shown that the solution
containing one or several additives according to the invention
remained qualitatively unchanged i.e. stable.
[0015] A further subject matter of the invention is a method for
determining a hydrogen-transferring analyte or a corresponding
dehydrogenase in the presence of a hydrogen accepting coenzyme
wherein the coenzyme is present in a stabilized aqueous solution as
described above.
[0016] The determination is carried out in particular in samples of
biological origin such as whole blood, serum or plasma, or other
human or animal sources or in plant extracts. The sample can be
prepared using physiological saline. In such a case a 0.9% NaCl
solution is advantageously used as a control value.
[0017] If it is intended to determine the enzyme activity of a
dehydrogenase such as a lactate dehydrogenase, a substrate solution
e.g. a lactate solution in a substance (mixture) buffering at ca.
pH 9.4 (37.degree. C.) is used. In this case the substrate can be
used in the usual concentrations known to a person skilled in the
art, preferably in a range of 40 to 80 mM.
[0018] In order to determine a hydrogen-transferring analyte such
as lactate, the respective dehydrogenase, e.g. LDH, is added first
in a substance buffering between pH 8.5 and 10.0. Usually a
dehydrogenase quantity of approximately 70 to 500 U/l, preferably
of 110 to 220 U/l is sufficient. The determination is usually
carried out at ca. 37.degree. C.
[0019] In addition to lactate which was described as an example, it
is also possible to similarly determine glutamate or ammonia,
alcohol, glyceraldehyde-3-phosphate, glucose or other parameters
that can be converted by a suitable coenzyme-dependent
dehydrogenase. This applies in a corresponding manner to the
determination of the enzyme activity of such dehydrogenases.
[0020] A further subject matter of the invention is a so-called
test kit for carrying out the enzyme or analyte determination. The
kit is essentially composed of two partial reagents. If it is used
to determine the activity of a dehydrogenase, the first reagent
contains a hydrogen-transferring analyte (substrate) in a suitable
system buffering between pH 8.5 and 10.0. The second reagent has a
coenzyme for hydrogen-transferring enzymes such as NAD or NADP and
an organic compound having a pKa value between 1.5 and 6.0 and/or a
hydroxylamine derivative according to the invention. The second
reagent can additionally contain other auxiliary substances such as
heavy metal salts or a complexing agent. This applies
correspondingly to the determination of an analyte or substrate
such as lactate.
[0021] Abbreviations
1 AMP = 2-amino-2-methyl-1-propanol AMPSO =
3-[(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxy- propanesulfonic
acid bicine = N,N-bis[2-hydroxyethyl]glycine CAPS =
3-[cyclohexylamino]-1-propanesulfonic acid CAPSO =
3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid CHES =
2-[N-cyclohexylamino]ethanesulfonic acid MEG = N-methylglucamine
TAPS = N-Tris[hydroxymethyl]methyl-3-aminopropane- sulfonic acid
Tricine = N-Tris[hydroxymethyl]methylglycine TRIS =
2-amino-2-(hydroxymethyl)-1,3-propanol
[0022] The invention is further elucidated by the following
examples:
EXAMPLE 1
[0023] Reagent 1: 390 mmol/l N-methylglucamine pH 9.4 (37.degree.
C.); 60 mmol/l lithium L-lactate.
[0024] Reagent 2: 60 mmol/l NAD(P) as a lyophilisate, powder
mixture, granulate or aqueous solution.
[0025] Incubation temperature: 37.+-.0.1.degree. C.; measurement
wavelength 340.+-.2 nm; path length 7 mm;
[0026] Preincubation: 5 minutes; lag phase: 2 minutes; measurement
time: 2 minutes.
[0027] Reagent 1=250 .mu.l; reagent 2=50 .mu.l; sample =7 .mu.l
NaCl solution (0.9% w/v).
[0028] The following determinations were carried out (IFCC:
recognised reference for the determination of LDH containing
lactate, NAD/NADP and N-methylglucamine, pH 9.4; Eur. J. Clin.
Chem. Biochem. vol. 32, p. 639-655 (1994)), Table 1:
2TABLE 1 blank calibrator calibrator blank value (BV) signal signal
value (BV) 5 days, -BV -BV unstressed 42.degree. C. unstressed 5
days 42.degree. C. reagent 1 reagent 2 [mA/min] [mA/min] [mA/min]
[mA/min] IFCC + IFCC 1.3 6.3 32.4 31.0 = 100 mmol/l 95.7% borate
IFCC IFCC + 2.1 6.2 35.2 33.4 = 100 mmol/l 95.4% citrate pH 3.0
IFCC + IFCC + 1.0 3.0 30.9 29.4 = 100 mmol/l 100 mmol/l 95.1%
borate citrate pH 3.0 IFCC IFCC + 0.9 3.2 34.4 33.8 = 50 mmol/l
98.3% hydroxylamine IFCC + IFCC + 0.7 1.8 31.1 30.5 = 100 mmol/l 50
mmol/l 98.0% borate hydroxylamine IFCC + IFCC + 0.7 1.2 29.8 30.3 =
100 mmol/l 50 mmol/l 101.7% borate hydroxylamine + 100 mmol/l
citrate pH 3.0 IFCC IFCC 1.5 11.5 35.7 33.7 = (prior 94.5% art)
[0029] Result: The inventive formulation containing appropriate
additives in the partial reagent 1 and/or partial reagent 2 shows a
considerably improved blank value with an almost unchanged
calibrator blank value compared to the IFCC reference method
especially under stress (5 days, 42.degree. C.).
EXAMPLE 2
[0030] The initial solutions described in example 1 and the
corresponding procedures were used. Citrate and/or various
hydroxylamine derivatives in different concentrations and
combinations were added to reagent 2 (table 2).
3TABLE 2 blank calibrator calibrator blank value (BV) signal signal
-BV value (BV) 5 days, -BV 5 days unstressed 42.degree. C.
unstressed 42.degree. C. reagent 1 reagent 2 [mA/min[ [mA/min]
[mA/min] [mA/min] IFCC = IFCC = reference 0.1 10.7 35.7 32.9 =
reference 92.1% IFCC IFCC + 20 mmol/l 0.8 2.5 31.6 31.2 = citrate +
50 mmol/l 98.7% hydroxylamine sulfate IFCC IFCC + 20 mmol/l 0.6 1.7
30.9 30.2 = citrate + 98.1% 50 mmol/l hydroxylamine phosphate IFCC
IFCC + 20 mmol/l -0.9 -0.6 34.8 33.3 = citrate + 50 mmol/l 95.7%
O-benzyl- hydroxylamine IFCC IFCC + 20 mmol/l -0.7 0.0 35.2 33.6 =
citrate + 50 mmol/l 95.5% O-methyl- hydroxylamine IFCC IFCC + 20
mmol/l 4.9 11.1 34.6 34.6 = citrate + 50 mmol/l 100.0% N-methyl-
hydroxylamine
[0031] Result: All compounds and salts added to the inventive
reagent 2 resulted in an improved recovery after stress (5 days,
42.degree. C.) compared to the IFCC reagent.
EXAMPLE 3
[0032] The recovery of the various isoenzymes was also demonstrated
using the formulation according to the invention. This must
correspond to the recovery of the recognized IFCC recommendation
(table 3).
[0033] The determinations were carried out using the IFCC reagent
described in example 1 (prior art) compared to a reagent according
to the invention.
4TABLE 3 activity activity activity activity activity iso- iso-
iso- iso- iso- enzyme 1 enzyme 2 enzyme 3 enzyme 4 enzyme 5 reagent
1 reagent 2 [U/l] [U/l] [U/l] [U/l] [U/l] IFCC IFCC 536 528 735 325
382 IFCC + IFCC + 536 523 736 301 368 100 mmol/l 50 mmol/l borate
hydroxylamine + 100 mmol/l citrate pH 3.0
[0034] Result: The recovery of the five LDH isoenzymes was
demonstrated with the reagent according to the invention. If the
said modifications were carried out on the formulation, it is
possible to provide a liquid LDH reagent which remains stable
during storage (>12 months) and transport (even at temperatures
>8.degree. C.). The resulting advantages for the user are
obvious and are shown in the description of the invention.
* * * * *