Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-30
2002-04-30
Geist, Gary (Department: 1627)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S012000, C435S014000, C435S015000, C435S025000, C435S026000, C435S188000
Reexamination Certificate
active
06380380
ABSTRACT:
TECHNICAL FIELD
The invention is directed to the use of NAD analogs and NADP analogs as enzyme cofactors in the measurement of enzyme activities, metabolites and substrates using enzymatic procedures which require the use of NAD and/or NADP cofactors for their determination.
BACKGROUND OF THE INVENTION
NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) are enzyme cofactors which are widely used in the measurement of enzyme activities and metabolites. The popularity of these cofactors for analytical measurements arises from the fact that in their oxidized forms, i.e. NAD and NADP they have virtually no absorbance at wavelengths longer than about 320 nm. When they are reduced, for example during an enzyme or substrate assay, NADH and NADPH have absorbances in the ultraviolet region of the spectrum with a maximum at 340 nm. At this wavelength both reduced cofactors have a molar absorbtivity of 6.22×10
3
. This unique property of the oxidized forms having virtually no absorbance at 340 nm and the reduced forms having a well defined absorbance maxima at 340 nm is the basis for their analytical use, especially in diagnostic test procedures.
Despite their popularity, these two cofactors are not without their shortcomings. One limitation is their somewhat weak oxidation potential, E
o
=−0.32 for NAD and NADP (Lehniger, A. L.,
Biochemistry
, Worth Publishers, Inc., New York, 1970). For many analytes and substrates this is not a serious problem, but for some substrates the reaction has to be “engineered” in order for it to proceed in the forward direction. An example of an “engineered” reaction is the enzymatic measurement of ethanol using alcohol dehydrogenase according to the following reaction.
ethanol+NAD alcohol dehydrogenase→acetaldehyde+NADH+H
+
To quantitatively measure ethanol in a sample the reaction needs to be at an alkaline pH, an excess of NAD has to be present and a means of removing the acetaldehyde must be incorporated into the reaction system to drive the reaction to completion. Typically a reactive primary amine e.g. 1,3-diamino-2-hydroxypropane is present to form a Schiff base with the acetaldehyde thereby removing it from solution (Kaufman et al., U.S. Pat. No. 5,141,854). The alkaline pH removes protons generated during ethanol oxidation which also helps drive the reaction in the forward direction. The alkaline pH, however, also brings with it other undesirable effects. NAD and NADP start to become unstable above pH 7 and their instability increases with increasing pH. In order to have an all liquid, single component reagent ready to use, with reasonable shelf life e.g. 18 months at 2° to 8° C., for measuring ethanol in analytical samples, the NAD must be stored in a separate container at a lower pH to maintain its stability. Thus at a minimum, a two vial reagent configuration is needed for an ethanol reagent to have reasonable shelf life stability. The NAD instability has been somewhat overcome by the addition of aliphatic zwitterionic secondary and tertiary amines, and maintaining the pH below 8 (Dorn et al., U.S. Pat. No. 5,804,403). But this technique still employs a two vial system which is more costly than single vial systems from both a labor and materials perspective. A trapping agent such as TRIS is also required to drive the reaction which further adds to costs. Other examples of metabolites which exhibit an unfavorable equilibrium for oxidation by NAD or NADP are glycerol, acetaldehyde, lactic acid and 3-hydroxybutyric acid.
A second limitation of NAD and NADP is their somewhat limited sensitivity. At 340 nm the molar absorbtivity of the reduced cofactors is 6.22×10
3
. For most enzymes and metabolites measured by diagnostic procedures this is usually not a problem. With some diagnostic procedures, however, if a more sensitive cofactor were available better assay precision could be obtained or sample volumes could be reduced resulting in less interferences from endogenous serum sample components e.g. bilirubin, lipemia and hemoglobin. Examples of such clinical assays are the measurement of CK-MB activity which is an early and specific marker for patients having a myocardial infarction, some EST™ assays where the analyte is very low e.g. digoxin and tetrahydrocannabinol (THC) and the determination of serum steroids and bile acids which are present in serum at very low concentrations (EMIT is a registered trademark of Dade Behring Inc., Deerfield, Ill.).
A third limitation of NAD and NADP is the somewhat limited wavelengths at which the reduced cofactors absorb. In the near-UV region of the spectrum the reduced cofactors have their maximum absorbance at 340 nm. At longer wavelengths their absorbances fall off rapidly and at wavelengths longer than 400 nm they exhibit no absorbance at all. To develop assays based on the reduced cofactors where a colored product absorbs in the visible region of the spectrum is desired, formazon dyes and diaphorases have been used. Diaphorases catalyze the reduction of an oxidized formazon dye using a reduced cofactor according to the following reaction.
formazon dye+NAD(P)H diaphorase→reduced formazon dye+NAD(P) colorless dye highly colored dye
Assays for measuring serum triglycerides have been developed using formazon dyes to avoid the “clearing effect”, which is observed at 340 nm, and is due to the decrease in turbidity in the reaction mixture as the triglycerides in the sample are hydrolyzed by lipase(s) to fatty acids and glycerol.
Thus, given the inherent limitations for NAD and NADP mentioned above, there exists a need for enzyme cofactors with more favorable oxidation potentials, broader absorbance spectra in the near-UV and visible regions of the spectrum, and with higher sensitivities than NADH and NADPH.
NAD analogs and NADP analogs overcome many of the limitations mentioned above regarding NAD and NADP. For example, depending on the analog, many have more favorable Eo values which would facilitate oxidation reactions at lower pH (Anderson et al., J. Biol. Chem. 221, 1219, 1959). Kaplan et al. (J. Biol. Chem., 221, 823, 1956) found that the equilibrium constant for oxidation of ethanol to acetaldehyde using 3-acetylpyridine-NAD was 200 hundred times more favorable than with NAD. The absorbance maxima, depending on the reduced analog, absorbs not only in the ultraviolet region of the spectrum but well into the visible region as well (Siegel et al., Arch. Biochem. Biophys. 82, 288, 1959, and Stein et al., Biochemistry 2, 5, 1963).
In addition to the reduced analogs having ultraviolet and visible absorbance properties they also, depending on the analog, have significantly higher molar absorbtivities (see Siegel et al. and Stein et al. above). Siegel et al. observed that the molar absorbtivities of 3-acetylpyridine-NADH at 363 nm was 9.1×10
3
and 3-pyridinealdehyde-NADH at 358 nm was 9.3×10
3
. Stein et al. found the molar absorbtivity of reduced thionicotinamide adenine dinucleotide at 398 mn was 11.9×10
3
and the corresponding thio-NADPH analog at 399 nm was 11.7×10
3
. For a more complete listing of molar absorbtivities of NADH and NADPH analogs see
Pyridine Nucleotide Coenzymes
, edited by David Poulson and Olga Arramovic, Coenzymes and Cofactors, Vol III, Part A, John Wiley, New York, 1987.
In some developed procedures using NAD and NADP, Ueda et al. (U.S. Pat. No. 5,780,256) used a two coenzyme cycling technique, where the reagent contained two coenzymes e.g. NAD(P) and thio-NAD(P), to measure ammonia, bile acids (U.S. Pat. No. 5,286,627) and 3-hydroxybutyric in biological samples. In these methods one coenzyme is continuously recycled between two dehydrogenase enzymes while the other coenzyme is continually reduced to increase assay sensitivity. In another procedure, Makler (U.S. Pat. No. 5,124,141) found 3-acetylpyridine-NAD to be useful for diagnosing the presence lactic acid dehydrogenase originating from Plasmodium falcipanum (malaria) in human serum since human lac
Crane L. E.
Geist Gary
Roberts & Mercanti LLP.
Specialty Assays, Inc.
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