Reagent for invitro diagnostic determination of bicarbonate

The invention provides a reagent for enzymatic determination of serum bicarbonate levels in a patient wherein the degree of oxidation of a coenzyme is measured and said reagent is stabilized against oxidation by a coenzyme reduction system comprising an enzyme and substrate pair selected to enable continuous regeneration of said coenzyme throughout storage of said reagent. The invention also provides an improvement in an enzymatic method of determination of the concentration of serum bicarbonate in a sample body fluid wherein the degree of oxidation of a coenzyme is measured, the improvement comprising stabilizing a reagent which comprises the coenzyme against oxidation by a coenzyme reduction system comprising an enzyme and substrate pair selected so as to enable continuous regeneration of the coenzyme throughout storage of the reagent.

This invention relates to reagents used in enzymatic methods of determining 
the concentration of serum bicarbonate in a sample body fluid. In 
particular, this invention relates to reagents used in methods wherein the 
quantity of an oxidized coenzyme in the reacted sample corresponds 
directly to the concentration of bicarbonate present in the sample. The 
invention also relates to improved methods for carrying out the 
determination of the bicarbonate concentration. 
The quantification of analytes, in particular, bicarbonate, in sample body 
fluids may involve contrasting a sample "blank" against a sample in which 
an enzymatic conversion of the analyte has taken place. 
To achieve enzymatic conversion of the bicarbonate, substrate specific 
enzymes are allowed to act upon enzyme substrates known for use in 
quantification of the serum bicarbonate. The change in the reaction 
composition with respect to the blank can be calculated by various methods 
measuring the change in absorbance of the composition. The change in 
absorbance correlates directly to the amount of bicarbonate present in the 
sample. 
Whilst traditional methods including colorimetric determination, partial 
pressure analysis and electrode testing have proved adequate, enzymatic 
analysis has been shown to be vastly more accurate, reliable and simpler 
than these other methods when it comes to the determination of serum 
bicarbonate levels. 
A commonly used method of quantification of total CO.sub.2 in a sample 
requires mixing the patient sample with the substrate phosphoenolpyruvate 
(PEP). A blank reading may be taken at this point. The substrate specific 
enzyme, phosphoenolpyruvate carboxylase (PEPC) is then added to the 
reaction mixture causing the conversion of PEP to oxaloacetate (OAA) and 
phosphate. 
##STR1## 
Although various methods may then be undertaken to correlate the 
oxaloacetate with the total CO.sub.2 in the sample the most common 
methodology requires the coupling of the oxaloacetate with reduced 
nicotinamide adenine dinucleotide (NADH) and malate dehydrogenase (MDH), 
the result of this reaction being the oxidation of nicotinamide adenine 
dinucleotide and the formation of malate. 
##STR2## 
The resulting concentration of NAD+ correlates to the concentration of 
total CO.sub.2 originally present in the sample. 
Historically, enzymatic Bicarbonate reagents have suffered from poor 
reconstituted stability. The cause of this instability could be attributed 
both to the deterioration of endogenous ingredients in solution coupled 
with the reagents uptake of atmospheric CO.sub.2. PEPC (phosphoenol 
pyruvate carboxylase), traditionally sourced from maize leaves, is 
particularly susceptible to oxidative degradation for example, by 
endogenous contaminants such as NADH oxidase and proteases which slowly 
degrade the potency of PEPC. NADH (Nicotinamide-adenine dinucleotide, 
reduced) will rapidly decompose in solution especially in an acidic 
medium, although storage of the NADH at an alkaline pH dramatically 
improves its reconstituted stability. 
Most reagents for the determination of serum bicarbonate are formulated at 
an alkaline pH of approximately 8.0. At this pH, atmospheric CO.sub.2 
becomes problematic. This exogenous CO.sub.2 absorption trigger a sequence 
of enzymatic reactions resulting in a subsequent loss of NADH and the 
consequent reduction in absorbance and reagent stability. Reagent 
reconstitution with poor quality laboratory water has the same detrimental 
effects as just described. 
Reagents thus have a very short effective life even when pampered to reduce 
exposure to atmospheric CO.sub.2. 
One means of overcoming this difficulty has been to generate reduced 
coenzyme in the reagent just prior to its use. 
One such method is described in Australian patent application AU-A-61906/90 
(02128191) Hoffmann La Roche AG. In this disclosure the reduced coenzyme 
is generated in situ either simultaneously with or prior to reoxidation of 
the coenzyme by the analyte, substrate and specific enzymes. This is 
achieved by including in the reaction mixture an enzyme and enzyme 
substrate enabling the reduction of the atmospherically oxidized coenzyme. 
The specific reaction disclosed and favoured by F. Hoffmann La Roche AG 
is: 
##STR3## 
This makes available reduced nicotinamide adenosine dinucleotide. 
The problem associated with this mode of generation of NADH is that a 
stable single vial reagent configuration is not possible. 
F. Hoffmann La Roche AG have to a certain extent overcome this problem by 
dividing the reagent system into 2 vials. The first reagent comprises the 
substrate specific enzymes; in the case of CO.sub.2 quantification, PEPC, 
MDH and G-6-P-DH, and the second reagent the enzyme substrates and the 
coenzyme, in the case of CO.sub.2 determination, PEP, G-6-P and NAD+ 
(oxidized state). The determination reaction thus proceeds as follows: 
##STR4## 
where (A) and (B) represent alternative, equivalent routes. 
Difficulties remain, however, with this reagent system. Apart from the fact 
that two reagent vials are required thus increasing cost, inventory and 
waste, very accurate levels of glucose-6-phosphate are required and 
moreover, the system is limited to use in specific chemical analyzers. As 
soon as the reagents are combined, generation of NADH from NAD+ occurs by 
exhaustion of glucose-6-phosphate. Because glucose-6-phosphate is thus 
exhausted, stability of the combined reagent could be severely affected if 
the two reagents were to be combined and not immediately used. If 
inaccurate or excess levels of glucose-6-phosphate are present, the timing 
associated with incubation of the reagent is critical. Results may be 
falsely low absorbance changes and grossly inaccurate results. 
One earlier solution described in U.S. Pat. No. 4,394,449(July 1983) to 
Modrovich uses substrate/enzyme pairs to generate the reduced coenzyme as 
does the Roche solution, however, in this case Glucose-6-phosphate is 
generated from Glucose in accordance with the following: 
##STR5## 
NAD+ then reacts with the formed Glucose-6-phosphate in the presence of 
the enzyme Glucose-6-phosphate Dehydrogenase to form NADH. Modrovich also 
includes both NADH and NAD+ in the formulation such that when NADH is 
oxidised or destroyed, the NAD+ present in the reagent will aid the 
regeneration of NADH. This is also a two vial reagent. 
A third alternative is provided by Crowther et al in U.S. Pat. No. 
5,116,728 (May 1992) In this invention the concept of enzyme/substrate 
pairs is utilized to regenerate NADH. This invention differs from those of 
Roche and Modrovich in that the regeneration reaction is slowed down to 
approximately 2% of the maximum rate by the use of very low levels of 
enzyme. In addition there is the inclusion of a stabilizer, which in 
effect is the addition of NAD+, at a theoretically pre-determined level 
which will set up an equilibrium between NADH and NAD+ and control the 
rate of generation. Both generation of NADH and regeneration of NADH occur 
within the system. It is not clear upon which the system is more reliant. 
The disadvantage of this system is that very low levels of enzyme in 
solution are very unstable and will affect the long term stability of the 
reagent. This is evident in the stability claimed for the ALT reagent 
which is indicated as being 22 days in a refrigerated environment. In 
addition it is a requirement that the reagent be configured in two parts. 
The general problem associated with the NADH generation mechanism adopted 
by each of the inventions described hereinabove, that is 
##STR6## 
is that a single step reaction using a single vial is not possible because 
as soon as the patient serum is added to the reagent, two simultaneous 
reactions occur: 
(a) a decrease in absorbance due to NADH being converted to NAD+, 
(b) generation of NADH from NAD+ resulting in an increase in absorbance. 
These two reactions occur at similar velocities with the net result being a 
falsely low absorbance change and grossly inaccurate results. 
Accordingly, it is an object of this invention to provide a reagent system 
for use in determination of serum bicarbonate levels which substantially 
ameliorates the problems of prior art reagent systems used in enzymatic 
analysis of serum bicarbonate levels relying on the oxidation of a 
coenzyme, particularly those problems which relate to endogenous or 
exogenous contamination of the reagent. It is a further object of this 
invention to provide an improved method of determination of the 
concentration of bicarbonate levels in a patient sample, the method 
overcoming the problems associated with prior art methods including 
premature oxidation of the coenzyme determinant and the necessity for a 
multi-vial system to minimize contamination of the reagent. 
To this end there is provided a reagent for enzymatic determination of the 
concentration of serum bicarbonate levels in a patient wherein the degree 
of oxidation of a coenzyme is measured, characterized in that said reagent 
is stabilized against oxidation by a coenzyme reduction system comprising 
an enzyme and substrate pair selected so as to enable continuous 
regeneration of said coenzyme throughout storage of said reagent. 
Preferably, the coenzyme reduction system comprises an enzyme and a 
substrate, said enzyme having incomplete specificity for said substrate 
thereby resulting in a reduced rate of cross reactivity. 
The reagent is preferably in a single vial configuration. 
Throughout this specification the term "incomplete specificity" is used 
with respect to enzyme and substrate pairs wherein the substrate selected 
is not the natural substrate of the enzyme selected and thus has less than 
100% cross specificity for the enzyme concerned. 
This invention is predicated on the discovery that by coupling an enzyme 
and substrate having incomplete specificity for each other, the rate of 
coenzyme reduction is considerably slowed. By slowing down the reduction 
reaction, the essential components of the reagent can be contained within 
one storage vial, the contents being stabilized against contamination by 
the low level continuous regeneration of the coenzyme. By slowing down the 
process, the regeneration of NADH can occur without affecting the 
measurement of serum bicarbonate. The regeneration can occur in the 
reagent when not in use and the velocity at which regeneration occurs can 
be fine tuned by adjusting the nature of the enzyme/substrate pair 
selected and the levels thereof. 
In an alternate embodiment of the invention, there is provided a reagent 
for use in an enzymatic method of determination of the concentration of 
serum bicarbonate levels in a patient wherein the degree of oxidation of a 
coenzyme is measured, characterized in that said reagent is stabilized 
against oxidation by a coenzyme reduction system comprising an enzyme and 
substrate pair selected so as to enable regeneration of said coenzyme at a 
rate of 0.10-0.90 mAbs/min at 20.degree.-25.degree. C. 
Preferably the rate of regeneration in a reagent according to this aspect 
of the invention is 0.20-0.80 mAbs/min at 20.degree.-25.degree. C. 
In a preferred embodiment of the invention, the degree of specificity 
between the substrate and enzyme of the coenzyme reduction system is 
preferably less than 100%, more preferably less than 50% and most 
conveniently less than 10% on an equimolar basis. Optimally, an 
enzyme/substrate pair having a cross-reactivity of less than 5% on an 
equimolar basis may be used. 
The coenzymes preferably used in the reagent according to the invention are 
reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide 
adenine dinucleotide phosphate (NADPH) although coenzyme analogs such as 
nicotinamide hypoxanthine dinucleotide phosphate or thio-NADH may also be 
suitable. 
Enzymes preferably utilised in the coenzyme reduction system for 
determination of the CO.sub.2 content of a serum sample may be 
glucose-6-phosphate dehydrogenase (G-6-P-DH) or glucose dehydrogenase. 
Enzymes such as formate dehydrogenase, glycerol dehydrogenase, leucine 
dehydrogenase, L-Alanine dehydrogenase, 3.alpha.-Hydroxy-steroid 
Dehydrogenase, L-lactate Dehydrogenase (from Lactobacillus sp.) or 
Glycerol-3-phosphate dehydrogenase may also be suitable. The preferred 
enzyme used for total CO.sub.2 determination reagents is 
glucose-6-phosphate dehydrogenase. This may be obtained from any suitable 
source such as Leuconostoc mesenteroides, Bacillus stearothermophilus, 
Zymomonas mobilus or yeast. 
Such enzymes are preferably derived from microbial sources. The 
incorporation into the reagent of enzymes from microbial sources has been 
found to eliminate the presence of endogenous contaminants such as NADH 
oxidase and proteases which previously severely affected the stability of 
the reagents. The microbial enzymes also have the added advantage of being 
more thermostable thereby improving their long term stability in solution. 
The more preferred source of glucose-6-phosphate dehydrogenase is from 
Leuconostoc mesenteroides. If glucose-6-phosphate from Bacillus 
stearothermophilus or Zymomonas mobilus is used, the rate of reaction is 
reduced. Similarly, if Yeast is used as the source of glucose-6-phosphate 
dehydrogenase, the coenzyme NADPH must be used as an alternative to NADH 
since yeast glucose-6-phosphate dehydrogenase only acts on NADP+. 
Bearing in mind that the selection of substrate and enzyme must be such 
that in the coenzyme reduction system they have incomplete specificity for 
each other, suitable substrates for use in the reagent according to the 
invention include Ribose-5-phosphate, Glucose-1-phosphate, 
6-phosphogluconic acid, 2-deoxyglucose-6-phosphate, 
2-deoxy-2-fluoroglucose-6-phosphate, 2-deoxy-2-chloroglucose-6-phosphate, 
2-deoxy-2, 2-difluoroglucose-6-phosphate, 2-O-methylglucose-6-phosphate, 
mannose-6-phosphate, glucosamine-6-phosphate, 3-deoxyglucose-6-phosphate, 
3-deoxy-3-fluoro-glucose-6-phosphate, 3-O-methylglucose-6-phosphate, 
allose-6-phosphate, ahrose-6-phosphate, 
4-deoxy-4-fluoroglucose-6-phosphate, galactose-6-phosphate, 
5-thio-glucose-6-phosphate, phosphonate analogs, glucose-6-stallate, 
.beta.-B-D-glucose, D-galactose, 2-deoxyglucose, arabinose, xylose, 
1-sorbose, D-mannose, D-fructose, D-lactose, D-sorbital, D-mannitol, 
saccarose, inositol, maltose. 
Using NAD+ as the preferred coenzyme in the reagent, the preferred 
enzyme/substrate combination is Glucose-6-phosphate dehydrogenase 
(G-6-P-DH)D-glucose. Preferred alternative substrates for D-glucose are 
those for which, relative to the specificity between Glucose-6-phosphate 
(G-6-P) and G-6-P-DH, the rate of reaction between the enzyme G-6-P-DH and 
the selected substrate is less than 50%, more preferably less than 10% and 
most preferably less than 5%. 
One preferred alternative to the use of D-Glucose/G-6-P-DH is the use of 
Glucose Dehydrogenase (GLD) according to the following reaction wherein 
D-Glucose is the 100% reactive substrate: 
##STR7## 
If glucose dehydrogenase is used as the enzyme, preferred substrates for 
reduction of the NAD coenzyme and their relative degree of cross 
reactivity when compared to D-Glucose are: 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
xylose 8.9% 
L-sorbose 0.3% 
D-mannose 2.4% 
D-fructose 0.8% 
D-galactose 0.1% 
D-lactose 1.2% 
D-sorbitol 0.1% 
inositol 0.2% 
maltose 3.9% 
______________________________________ 
wherein the figures in brackets represent the rate of reaction relative to 
that of Glucose-dehydrogenase 1.beta.-D-glucose with 1.beta.-D-glucose. 
Alternatively, using Glycerol Dehydrogenase (GLY.DH) as the enzyme, 
suitable substrates in the reaction 
##STR8## 
and their activity relative to glycerol (100%) are 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
Glycerol-.alpha.-monochlorohydrin 
48.5% 
Ethylene glycol 7.8% 
2,3-Butanediol 52.6% 
______________________________________ 
Wherein Leucine Dehydrogenase (L.D) is used as the enzyme according to the 
reaction 
##STR9## 
suitable substrates and their activity relative to L-leucine (100%) are 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
L-Valine 74% 
L-Isoleucine 58% 
L-Norvaline 41% 
L-Norleucine 10% 
L-Methionine 0.6% 
L-Cysteine 0.3% 
______________________________________ 
If L-Alanine Dehydrogenase (A.D) is used as the enzyme in a reaction system 
similar to that used for Leucine Dehydrogenase, a suitable substrate and 
its activity relative to L-alanine (100%) is 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
L-serine 5% 
______________________________________ 
3.alpha.-Hydroxysteroid Dehydrogenase (H.DH) may also be used as an enzyme 
in combination with the substrates listed below. Their activities relative 
to Cholic Acid are also listed. 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
Lithocholic Acid 
96% 
Etiocholic Acid 
60% 
______________________________________ 
Wherein, L-Lactate Dehydrogenase (LDH) from Lactobacillus sp. is used as 
the enzyme in the following reaction, 
##STR10## 
suitable substrates and their activity relative to L-Lactate are: 
______________________________________ 
Substrate Relative Activity 
______________________________________ 
2-oxoglutarate 
0.09% 
Oxoloacetate 36% 
______________________________________ 
Wherein NADP is the coenzyme, for example from Yeast, preferred 
substrate/enzyme combinations are: 
______________________________________ 
G-6-P--DH/galactose-6-P 
25% 
G-6-P--DH/2-deoxyglucose-6-P 
18% 
G-6-P--DH/glucosamine-6-P 
2% 
______________________________________ 
The figures in on the right hand side represent the relative reactivity to 
that of a G-6-P-DH/G-6-P pair. 
It is also possible using NADP as coenzyme to combine as enzyme/substrate 
glycerol-3-phosphate dehydrogenase with dihydroxy acetone phosphate. 
As described in the preamble to this specification, the other requirements 
of a reagent according to the invention for use in the determination of 
serum bicarbonate levels are phosphoenol pyruvate (PEP), phosphoenol 
pyruvate carboxylase (PEPC), nicotinamide-adenine dinucleotide, reduced 
(NADH) and malate dehydrogenase(MDH). 
Levels of PEP must be selected such that optimum performance of the reagent 
is achieved. This may vary according to the other components of the 
reagent selected, however, it has been found that a concentration in the 
range 1.5 mmol/L to at least 15 mmol/L is suitable in the context of this 
invention. Below 1.5 mmol/L substrate depletion occurs thereby affecting 
the stability of the reagent. Different salts of PEP may be used without 
compromising the stability of the reagent. It should be noted that the 
higher the level of PEP, the lower the pH of the reagent becomes. Thus 
buffer levels may need to be adjusted to achieve the optimum pH level. 
The level of coenzyme in the reagent will vary according to the following 
factors: 
linearity required in measurement 
wavelength chosen 
sample to reagent volume ratio 
photometric system of the analyzer selected. 
In general, increasing the sample volume improves the sensitvity but 
decreases the linearity of the reading obtained, whereas decreasing the 
sample volume improves linearity at the expense of losing sensitivity. 
The preferred wavelength of measurement is 320-400 nm, however, the level 
of coenzyme used should be adjusted so that the absorbance preferably does 
not exceed 2.0 A. The preferred wavelength of absorbance according to the 
invention is 380 nm. 
The level of PEPC is preferably selected so that the end point reaction is 
achieved within the desired time frame. For example, if a desired 
completion time is 5 minutes at 37.degree. with a 50 mmol sample, a level 
of at least 380 U/L may be selected. Preferably the PEPC is obtained from 
a microbial source so as to reduce the risk of endogenous contamination of 
the reagent. 
MDH is also preferably obtained from microbial sources so as to limit the 
risk of endogenous contamination. Appropriate levels are in the range 
150-1500 U/L, more preferably 200-400 U/L. 
The reagent according to the invention may include in addition to the 
coenzyme reduction system and other essential substrates and enzymes 
necessary to determine the analyte concentration, preservatives, chelating 
agents, surface active agents, protease inhibitors, buffers, cofactors, 
LDH inhibitors, antibacterials and other constituents which perform 
stability enhancing functions but do not materially affect the 
characteristics of the invention. 
The primary criteria for selecting a buffer is such that it will have good 
buffering capacity at the selected pH with minimal binding of divalent 
cation. A general rule of thumb is that a buffer may be considered 
effective if its pKa is .+-.1.0 pH units from the chosen pH. A preferred 
pH of the reagent according to the invention is 7-9, more preferably 8.0 
at 20.degree. C. At this preferred pH a compromise is reached between 
optimum enzyme activity and the stability of the enzymes and coenzyme in 
solution. A lower pH may result in increased degradation of the coenzyme. 
Suitable buffers are HEPES, 4-morpholine propanesulfonic acid (MOPS) or 
2-tris(hydroxymethyl)methylamino!-1-ethane-sulfonic acid (TES) or the 
other GOOD buffers, TRICINE, BICINE, TEA and TAPS. Preferred buffers 
according to the invention are TRIS and/or HEPES having a total ionic 
strength preferably of 30-100 mmol/L, and more preferably approximately 58 
mmol/L The sample to be tested may be diluted with any suitable diluent if 
desired, such as deionized water or saline. 
Magnesium ions are required as cofactor in the PEPC reaction described 
elsewhere in this specification. A concentration of 4-20 mmol/L is 
suitable. Appropriate sources of magnesium ions include Magnesium Sulphate 
(anhydrous), Magnesium Chloride and Magnesium acetate as well as other 
suitable magnesium salts. 
Preservatives such as sodium azide (NaN.sub.3), hydroxybenzoic acid, 
gentamicin, Thymol and mercury-free preservatives available from 
Boehringer Mannheim are suitable. The appropriate level is such that the 
preservative retains its preservative properties for at least 6-8 months 
when stored at 2.degree.-8.degree. without inhibiting the enzymes present 
in the reagent. A suitable range fulfilling these criteria is 0.1-1.0 g/L. 
Non-ionic surface active agents such as octyl phenoxypolyethoxy ethanol or 
a polyoxyethylene fatty alcohol ether are suitable. PMSF or Aprotinin are 
known protease inhibitors and, sodium oxamate, oxalic acid or gossypol 
will effectively inhibit interference due to Lactic Dehydrogenase. This 
last component may be required for patients having high levels of pyruvate 
since these cause interference in enzymatic bicarbonate reactions. A 
suitable level of for example, sodium oxomate is 1.0 g/L. Other suitable 
enzyme stabilizers include bovine serum albumin, bovine gamma globulin, 
N-acetyl cysteine and glycerol. 
A variety of chelating agents such as EDTA, EGTA, 
N-(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), etc. are also 
suitable. Suitable defoaming agents may also be added if desired. 
In one preferred embodiment of the invention, the reagent essentially 
comprises 
______________________________________ 
G-6-P--DH coenzyme reduction 
D-glucose system 
PEP substrate 
PEPC substrate specific 
MDH enzymes 
NADH coenzyme 
______________________________________ 
In addition, there is preferably included TRIS buffer, HEPES free acid, 
Sodium Oxamate, Sodium Azide, Bovine Serum Albumin and Magnesium Sulphate 
(anhydrous). 
One reagent formulated in accordance with the invention is as follows: 
______________________________________ 
MOLECULAR CONCEN- QUANTITY/ 
RAW MATERIAL 
WEIGHT TRATION LITER 
______________________________________ 
TRIS Buffer 21.14 40 mM 3.5-5.5 g 
HEPES free acid 
238.3 18 mM 3.5-5.3 g 
PEP-MCHA Salt 
267.1 6.0 mM 1.0-2.2 g 
Sodium Oxamate 
111.03 9 mM 0.5-1.5 g 
Sodium Azide 
65.01 7.7 mM 0.25-0.75 
g 
Bovine Serum 
0.1% 0.5-1.5 g 
Albumin 
NADH.Na.sub.2 3H.sub.2 O 
763.5 1.6 mM 0.6-1.80 
g 
Magnesium Sulphate 
120.4 8 mM 0.5-1.3 g 
(anhydrous) 
D-Glucose 180.16 0.25 mM 40.0-50.0 
g 
G-6-PDH 3000-4000 
U 
(Leuconostoc 
Mesenteroides) 
PEPC (microbial) 380-440 U 
MDH (microbial) 220-290 U 
______________________________________ 
In another aspect of the invention there is provided an improvement in an 
enzymatic method of determination of the concentration of serum 
bicarbonate in a sample body fluid wherein the degree of oxidation of a 
coenzyme is measured, the improvement comprising stabilizing a reagent 
comprising said coenzyme against oxidation by a coenzyme reduction system 
comprising an enzyme and substrate pair selected so as to enable 
continuous regeneration of said coenzyme throughout storage of said 
reagent. 
In a preferred method according to this aspect of the invention, the enzyme 
of the enzyme and substrate pair has incomplete specificity for said 
substrate thereby reducing the rate of cross reactivity between enzyme and 
substrate. 
In a preferred embodiment of this aspect of the invention, the coenzyme 
reduction system comprises an enzyme and substrate having a specificity 
for each other, relative to the specificity of the enzyme for its natural 
substrate, of less than 100%, preferably less than 50% and most 
conveniently less than 10%. Most conveniently, the specificity of the 
enzyme/substrate pair for each other, relative to the specifity of the 
enzyme for its natural substrate, is less than 5%, desirably approximately 
2%. 
The selection of coenzyme, substrate and enzyme may be made from those 
mentioned hereinabove in relation to the reagents of the invention, 
depending on the analyte to be assessed. 
In one embodiment of this aspect of the invention, the preferred components 
of the coenzyme reduction system used for determination of total CO.sub.2 
concentration are NADH, G-6-P-DH and D-glucose such that the stabilization 
reaction taking place is 
##STR11## 
Due to the low specificity of G-6-P-DH for D-glucose this stabilization 
reaction is slow and thus not competitive with the main reactions 
##STR12## 
which take place upon addition of the sample body fluid.

EXAMPLE 
The stability of one particular CO.sub.2 reagent formulated in accordance 
with the invention was tested as follows: 
______________________________________ 
FORMULATION: 
______________________________________ 
TRIS Buffer 4.86 g/L 
HEPES free acid 4.31 g/L 
PEP - MCHA Salt 1.60 g/L 
Sodium Oxamate 1.00 g/L 
Sodium Azide 0.50 g/L 
Bovine Serum Albumin 1.00 g/L 
NADH.Na.sub.2 3H.sub.2 O 
1.20 g/L 
Magnesium Sulphate (anhydrous) 
0.96 g/L 
D-Glucose 45.04 g/L 
G-6-PDH (L. Mesenteroides) 
3500 U/L 
PEPC (microbial) 410 U/L 
MDH (microbial) 250 U/L 
______________________________________ 
Optimum levels of D-glucose and G-6-PDH were determined by testing the rate 
of regeneration of NADH with varying levels of D-glucose and G-6-PDH. The 
rate of regeneration of NADH from NAD+ is proportional to the amount of 
enzyme G-6-PDH and to a lesser extent the level of D-glucose. 2 ml 
aliquots of bicarbonate formulation containing 250 U/L MDH, 400 U/L PEPC, 
6 mmol/L PEP and 50 mmol/L D-glucose at pH 8.0 were added to cuvettes and 
spiked with 20 .mu.l of a 50 mmol/L bicarbonate sample. The cuvettes were 
then sealed with parafilm to prevent further CO.sub.2 contamination, and 
the time course for NADH regeneration monitored at 380 nm and at 
25.degree. C. in a Shimadzu spectrophotometer over a 48 hour period. It 
was determined from these results that it was preferable to use 250 mmol/L 
of D-glucose as less G-6-PDH was used to obtain a similar rate of 
regeneration. The optimum level of G-6-PDH was determined to be in the 
range 2.5-5.0 KU/L since it did not effect the main reaction giving a 
stable end-point. A level of 3.5 KU/L of G-6-PDH was selected since this 
gave an acceptable regeneration rate of NADH over 6 months when the 
reagent was stored at 4.degree. C. capped. An acceptable regeneration rate 
is considered to be in the range 0.10-0.90mAbs/min at 
20.degree.-25.degree. C. 
STORAGE CONDITIONS: 
capped and refrigerated (2.degree.-8.degree. C.) 
______________________________________ 
SPECTROPHOTOMETRIC AMETERS 
(Shimadzu PC2101): 
______________________________________ 
reaction temperature 37.degree. C. 
reagent to sample volume 
100:1 
wavelength 380 nm 
cuvette path length 1 cm 
______________________________________ 
These spectrophotometric parameters were used to determine the following 
______________________________________ 
Initial absorbance of reagent 
at 380 nm 
Reagent Blank rate at 380 nm 
reaction .DELTA.Absorbance 
50 mmol/L standard used to check 
completion time at 37.degree. C. 
regeneration rate at 20.degree. C. 
(expressed in mAbs/min) 
______________________________________ 
The following results were obtained: 
__________________________________________________________________________ 
storage at 2-8.degree. C. 
(weeks) 6 9 12 16 21 24 28 fresh reagent 
__________________________________________________________________________ 
Initial absorbance 
1.81 
1.83 
1.76 
1.69 
1.65 
1.61 
1.56 
1.68 
Blank rate 0.02 
0.01 
0.01 
0.01 
0.01 
0.01 
0.01 
0.01 
(.DELTA.Abs/10 min) 
completion time (min) 
4' 4' 5' 5' 5' 5' 5' 4' 
.DELTA.Absorbance 
0.67 
0.7 
0.69 
0.68 
-- 0.68 
0.7 
0.68 
regeneration rate 
0.41 
0.38 
0.35 
0.31 
0.28 
0.25 
0.2 
0.47 
__________________________________________________________________________ 
CORRELATION STUDIES: 
Bicarbonate levels were determined on patient sera using fresh reagent, 
reagent stored for 6 months at 2.degree.-8.degree. C., and fresh regular 
reagent)not including the regeneration system). Results (mmol/L) were as 
follows: 
______________________________________ 
regeneration 
regeneration 
technology technology 
Regular fresh reagent 
(6 months old) 
(fresh) 
______________________________________ 
0 0 0 
15 17 15 
20 22 20 
21 22 21 
22 23 22 
23 25 23 
23 24 22 
23 24 23 
24 26 24 
24 24 23 
28 29 27 
28 29 28 
28 29 28 
29 32 30 
29 29 28 
30 31 30 
30 32 30 
30 31 30 
31 32 30 
33 34 33 
33 33 33 
33 33 33 
35 36 35 
35 36 36 
36 37 36 
37 38 37 
39 40 39 
40 41 40 
44 44 44 
44 45 44 
29 30 29 
______________________________________ 
From the results presented it is evident that the regeneration CO.sub.2 
reagent is exhibiting at least 6-7 months stability when stored capped at 
2.degree.-8.degree. C. The reagent must have an initial absorbance of 1.0 
A to be functional. After 7 months the reagent still has an absorbance of 
at least 1.5 A. The completion time has hardly shifted at 4-5 minutes as 
has the sensitivity which has remained at 0.67-0.70 A over the testing 
period, indicating good calibration stability. Studies of Linearity 
indicate that linearity is retained at at least 40 mmol/L in both the 
fresh reagent and in a 28 week old sample. 
The patient correlation data provides further evidence that there is no 
significant difference in performance when comparing fresh reagent versus 
reagent stored at 2.degree.-8.degree. C. for 6 months (r.sup.2 =0.997). 
The incorporation of the regeneration system according to the invention has 
resulted in an increase in reconstituted stability of a serum bicarbonate 
measurement capped reagent from 1 month at 2.degree.-8.degree. C. to at 
least 6-8 months at 2-8.degree. C. The stability of uncapped reagents has 
increased from one day at room temperature (18.degree.-25.degree. C.) to 
approximately 7 days at room temperature. Other major advantages of the 
reagent and method according to the invention are that the reagent is a 
single vial reagent thereby reducing space and inventory problems 
associated with prior art reagents, and that it is adaptable to varying 
instrumentation systems. 
It should be appreciated that there are numerous substrate/enzyme 
"unnatural" pairs which may be used to slow the regeneration of the 
coenzyme used in the reagent and method of the invention. In addition to 
those mentioned herein, there are others which are not commercially 
available or which are prohibitively expensive.