Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1995-11-29
1998-01-06
Leary, Louise
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 26, 435 14, 435 4, 435 791, 435832, 435853, 435963, 424 941, 424 943, 424 944, 436 63, 436 74, C12Q 126, C12Q 132, C12Q 154, C12N 100
Patent
active
057053561
DESCRIPTION:
BRIEF SUMMARY
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 enz
REFERENCES:
patent: 4394449 (1983-07-01), Modrovich
patent: 5116728 (1992-05-01), Crowther et al.
Leary Louise
Trace Scientific Limited
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