STABILIZATION OF NADPH OR NADH IN AMMONIA DETECTION ASSAYS

Abstract

The present disclosure deals with the biochemistry of reagents useful in the detection of ammonia in liquid samples. Specifically, the present disclosure is directed to a technical improvement of an enzyme-based test for ammonia that can be used for analysis of plasma samples taken from patients in clinical settings, among other uses. In this regard, stability of a reagent containing NAD(P)H is improved, enhancing shelf life and results in the detection of ammonia. In an exemplary reagent ammonia released as a result of NAD(P)H decay is scavenged using an enzymatic reaction to convert the ammonia using GLDH, NAD(P)H and 2-oxoglutarate, thereby forming L-glutamate, NAD(P).sup.+ and H.sub.2O in the NAD(P)H containing reagent.

Claims

1. An assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit containing 2-oxoglutarate, glutamate dehydrogenase capable of reacting NAD(P)H as a co-substrate (=GLDH), and NAD(P)H, wherein the kit comprises a first and a second container, wherein the first container contains a first aqueous reagent with a first amount of GLDH, the first reagent having a pH capable of maintaining enzymatic activity of GLDH, wherein the second container contains a second aqueous reagent with NAD(P)H, 2-oxoglutarate, and a second amount of GLDH, wherein the GLDH enzymatic activity per mL of the first reagent is higher than the GLDH enzymatic activity per mL reagent in the second reagent, and wherein the pH of the second reagent is capable of maintaining enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H and 2-oxoglutarate thereby being able of forming L-glutamate, NAD(P).sup.+ and H.sub.2O in the second reagent.

2. The assay kit of claim 1, wherein NAD(P)H is present only in the second reagent.

3. The assay kit of claim 1, wherein the pH of the first reagent is from pH 7 to pH 9.

4. The assay kit of claim 1, wherein the pH of the second reagent is from pH 8 to pH 11.

5. The assay kit of claim 1, wherein the ratio between the GLDH enzymatic activity per mL of the first reagent and the GLDH enzymatic activity per mL of the second reagent is from 15:1 to 1.5:1.

6. The assay kit of claim 1, wherein relative to the total GLDH enzymatic activity provided by the reagents comprised in the kit the second reagent contains an amount of GLDH enzymatic activity form 1% to 30%.

7. The assay kit of claim 1, wherein the first reagent contains a buffer selected from N,N-bis(2-hydroxyethyl)-glycine and triethanolamine.

8. The assay kit of claim 1, wherein the first reagent contains a detergent.

9. A method to provide an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample, the kit comprising two different aqueous reagents, the reagents containing in aqueous solution 2-oxoglutarate, glutamate dehydrogenase capable of reacting NAD(P)H as a co-substrate (=GLDH), and NAD(P)H, the method comprising the steps of preparing a first reagent by dissolving GLDH in an aqueous solution with a pH capable of maintaining enzymatic activity of GLDH, preparing a second reagent by dissolving in an aqueous solution 2-oxoglutarate, GLDH, and NAD(P)H, and adjusting the pH of the second reagent to be permissive for maintaining enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H, and 2-oxoglutarate, thereby allowing formation of L-glutamate, NAD(P).sup.+ and H.sub.2O in the second reagent, providing the first and the second reagent in separate containers, and combining the containers in a kit of parts, thereby providing an assay kit for quantitatively determining the concentration of ammonia in an aqueous liquid sample.

10. (canceled)

11. An assay kit of claim 1, or an assay kit obtained from practicing the method of claim 9, wherein the concentration of ammonia in the second reagent is about 1.5 μM or less.

12. The assay kit of claim 11, wherein the second reagent is obtained by the steps of (a) dissolving in an aqueous solution 2-oxoglutarate, GLDH, and NAD(P)H, and adjusting the pH of the second reagent to be permissive for maintaining enzymatic activity of GLDH in the second reagent to convert ammonia, NAD(P)H, and 2-oxoglutarate, followed by (b) incubating the aqueous obtained from step (a) solution at 35° C. for 14 d, wherein the concentration of ammonia in the second reagent is from about 0.02 μM to about 1.3 μM.

13. The assay kit of claim 11, wherein in the second reagent ammonia released by decomposition of NAD(P)H is removed from the aqueous solution by means of GLDH enzymatic activity reacting ammonium ions, NAD(P)H and 2-oxoglutarate to L-glutamate, NAD(P).sup.+ and H.sub.2O.

14. A mixture comprising (i) an aqueous liquid sample suspected of containing ammonia, and (ii) the second reagent of the assay kit of claim 13.

15. The mixture of claim 14, wherein the sample is a biological sample.

16. The mixture of claim 14, wherein the mixture additionally comprises the first reagent of the assay kit.

17. An automated device capable of forming a mixture of claim 14, wherein the device is combined with (i) an aqueous liquid sample in a sample container and (ii) a kit of claim 1, or an assay kit obtained from practicing the method of claim 9.

Description

DESCRIPTION OF THE FIGURES

[0054] FIG. 1 Chemical formulas of NADP.sup.+ and NADPH

[0055] FIG. 2 Experimental UV/vis spectra of NADP.sup.+ and NADPH as reported in De Ruyck J. et al. Chem Phys Lett 450 (2007) 119-122.

[0056] FIG. 3A-C Depiction of working instructions for an improved diagnostic assay for ammonia detection and quantification, according to the teachings of the present report.

[0057] FIG. 4A-H Depiction of the molecular structures of oxidized forms of co-substrates for GLDH: (A) NAD.sup.+, (B) NADP.sup.+, (C) 3-Acetylpyridine-NAD.sup.+, (D) 3-Acetylpyridine-NADP.sup.+, (E) 3-Pyridinealdehyde-NAD.sup.+, (F) 3-Pyridinealdehyde-NADP.sup.+, (G) Thionicotineamide-NAD.sup.+, (H) Thionicotineamide-NADP.sup.+.

[0058] FIG. 5 Graphic representation of the results of the experiments listed in Table 1 (see Example 4 and Table 1).

[0059] FIG. 6 Comparison of (i) the original second reagent (R3) [solid line] working solution as described in Example 2 (containing NADPH) and (ii) modified working solution R3′ (modified second reagent according to the present disclosure) in which NADPH was replaced by NADH [dotted line], for further details see Example 5. Extinction readings for NADPH [solid line] and NADH [dotted line].

EXAMPLE 1

Diagnostic Determination of Ammonia in a Plasma Sample Using an Assay Kit of the Prior Art (Assay Designation “NH.SUB.3.”)

[0060] A Roche Cobas® NH.sub.3 (Ammonia) assay kit for Roche/Hitachi analyzers (including the MODULAR P platforms; assay kit obtained from Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 11877984216 with Cat. Nos. 20751995190, 20752401190, 20753009190 as calibrators) was provided.

[0061] Prior to use, R2 was prepared in three steps, thereby bringing in solution a solid (i.e. water-free) preparation of NADPH:

(1) Providing each of one bottle 2a and one bottle 2b which are supplied as parts of the assay kit, followed by reconstituting the contents of bottle 2b by adding 0.5 mL of reagent from bottle 2a into bottle 2b and mixing, followed by incubating the mixture for 10 minutes at room temperature, with occasional gentle swirling, thereby obtaining a first solution in bottle 2b.
(2) Providing one bottle 2 supplied as part of the assay kit and the bottle 2a from step (1), followed by reconstituting the contents of bottle 2 by adding 4.5 mL of reagent from bottle 2a into bottle 2, and mixing by gentle inversion, thereby obtaining a second solution in bottle 2.
(3) Providing the bottle 2b with the first solution obtained as a result of step (1), and providing the bottle 2 with the second solution obtained as a result of step (2), adding 150 μl of the first solution from bottle 2b to the second solution in bottle 2, and mixing by gentle inversion, thereby obtaining ready-to-use R2 working solution.

[0062] The working solution R1 provided as part of the kit contained triethanolamine buffer: 151 mmol/L, pH 8.6; 2-oxoglutarate: 16.6 mmol/L; ADP: ≥1.2 mmol/L; and additionally preservatives.

[0063] The ready-to-use working solution R2 obtained as a result of step (3) detailed above contained NADPH: ≥458 μmol/L; GLDH (bovine liver; 25° C.): ≥24.3 U/mL; triethanolamine buffer: 151 mmol/L, pH 8.6; 2-oxoglutarate: 16.6 mmol/L; ADP: ≥1.2 mmol/L; and additionally preservatives.

[0064] The assay kit, specifically R1 and R2 was/were used according to the instructions of the manufacturer on a Roche/Hitachi MODULAR P platform.

EXAMPLE 2

[0065] Scavenging of Ammonia by Way of Adding Different Amounts of GLDH into R2

[0066] R2 working solution was prepared as described in Example 1. Aliquots were mixed with different amounts of GLDH

EXAMPLE 3

[0067] Diagnostic Determination of Ammonia in a Plasma Sample Using the Novel Assay Kit with Enhanced Stabilization of NADPH (Assay Designation “NH.sub.3L2”)

[0068] A newly developed Cobas® NH.sub.3L2 (Ammonia) assay kit for Roche/Hitachi analyzers (including the cobas c platforms c 311, c 501/502; assay kit composed by the inventors, using Cat. Nos. 20751995190, 20752401190, 20753009190 as calibrators) was provided. The kit comprises two different working solutions, R1 and R3 which are ready-to-use.

[0069] The working solution R1 provided as part of the kit contained BICINE (=N,N-bis(2-hydroxyethyl)-glycine) buffer: 300 mmol/L, pH 8.3; GLDH (microbial): ≥16.7 μkat/L; detergents; preservative.

[0070] The working solution R3 provided as part of the kit contained GLDH (microbial): ≥5.0 μkat/L; 2-oxoglutarate: 78 mmol/L; NADPH: ≥1.3 mmol/L; stabilizer; nonreactive buffer.

[0071] The assay kit, specifically R1 and R3 was/were used according to instructions as depicted in FIGS. 3A-C on a Roche/Hitachi cobas c platform.

EXAMPLE 4

[0072] Scavenging of Ammonia by Way of Adding Different Amounts of GLDH into R2

TABLE-US-00001 TABLE 1 Amount of GLDH added to solution, NH.sub.3- Incubation Incubation # in U/mL spiked time temperature 1 None (=0) No n.a. 2° C. to 8° C. 2 None (=0) Yes n.a. 2° C. to 8° C. 3 None (=0) No  7 d 35° C. 4 None (=0) No 10 d 35° C. 5 None (=0) No 14 d 35° C. 6 1 No n.a. 2° C. to 8° C. 7 1 Yes n.a. 2° C. to 8° C. 8 1 No  7 d 35° C. 9 1 No 10 d 35° C. 10 1 No 14 d 35° C. 11 2 No n.a. 2° C. to 8° C. 12 2 Yes n.a. 2° C. to 8° C. 13 2 No  7 d 35° C. 14 2 No 10 d 35° C. 15 2 No 14 d 35° C. 16 5 No n.a. 2° C. to 8° C. 17 5 Yes n.a. 2° C. to 8° C. 18 5 No  7 d 35° C. 19 5 No 10 d 35° C. 20 5 No 14 d 35° C. 21 10 No n.a. 2° C. to 8° C. 22 10 Yes n.a. 2° C. to 8° C. 23 10 No  7 d 35° C. 24 10 No 10 d 35° C. 25 10 No 14 d 35° C. 26 14 No n.a. 2° C. to 8° C. 27 14 Yes n.a. 2° C. to 8° C. 28 14 No  7 d 35° C. 29 14 No 10 d 35° C. 30 14 No 14 d 35° C.

[0073] An aqueous solution was freshly prepared, the solution contained 2-oxoglutarate: 78 mmol/L; NADPH: ≥1.3 mmol/L; and nonreactive buffer. Different amounts of GLDH were added to aliquots of the aqueous solution. One aliquot was additionally spiked with a known amount of ammonia. Different incubation conditions were applied to the individually prepared test solutions.

[0074] Table 1 summarizes compositions and conditions. FIG. 5 is a graphical representation of the results, i.e. the ammonia concentration (also referred to as “c[NH.sub.3]”) detected after each experiment is depicted by the height of each individual block in the 3D diagram. The more a depicted block is extended along the Y-axis given by the arrow in FIG. 5 (i.e. the taller the respective block in the diagram is), the higher the ammonia concentration that was detected in the respective experiment. Each block represents an experiment as given in the numbered list of Table 1, and the number designations given to the single blocks in FIG. 5 correspond to the numbering [#] in Table 1.

[0075] Remarkably by means of the presence of GLDH the concentration of ammonia could be kept reproducibly in a range of between 0.01 μM and 1.5 μM. The GLDH-related blocks in the diagram of FIG. 5 correspond to values from 0.02 μM to 1.3 μM.

[0076] At the end of the 35° C. incubation of experiments ##10, 15, 20, 25 and 30 (i.e. at the end of the 14 d interval) the surviving GLDH activity remaining in the aqueous solution was determined. Table 2 summarizes the results.

TABLE-US-00002 TABLE 2 Initial GLDH activity, surviving GLDH activity in U/mL # in in U/mL after 14 d at 35° C. 10 1 0.13 15 2 0.91 20 5 1.66 25 10 3.53 30 14 5.18

[0077] It should be noted that the incubation conditions applied were designed to mimic the strain provided by a longer time at ambient conditions (e.g. room temperature) on the reagent, and in particular on the enzymatic activity comprised therein. Thus, the implication on reagent shelf life caused by the presence of a GLDH-based scavenging mechanism for ammonia becomes apparent by the data reported here.

[0078] It is interesting to see that even the lowest amount of GLDH (experiment #10) was successful in removing any ammonia that was formed during the incubation period. When this is compared to the result of the same solution but in the absence of GLDH (experiment #5) it becomes apparent that even under constraint, i.e. under conditions which are non-optimal for GLDH activity and/or stability, it is well possible to find conditions under which the enzyme is active enough to ensure efficient removal of ammonia from decomposition of the co-substrate.

[0079] As the co-substrate is present at a saturating concentration, consumption in the scavenging process was found to be insignificant, i.e. irrelevant for the performance of the newly developed Cobas® NH.sub.3L2 (Ammonia) assay kit.

EXAMPLE 5

[0080] Stabilized Working Solution with NADH Instead of NADPH

[0081] A working solution R3 as given in Example 2 was prepared, and a working solution R3′ in which NADH replaced NADPH. Each solution was filled into a cuvette, and the extinction at or near the UV absorption maximum of NADH or NADPH was constantly measured. At one point in time (after 15 min) an ammonia solution was added into the cuvette, adjusting the ammonia concentration in the respective working solution to 100 μM. Extinction was further recorded. It was found that in both working solutions concentration of the co-substrate decreased, as became apparent by decreasing extinction readings. From these data it was concluded that the mechanism of scavenging ammonia from the working solution (by means of GLDH enzymatic activity) worked in the presence of both co-substrates. Measurements were taken at 10° C.

[0082] FIG. 6 depicts the extinction readings for NADPH (solid line) and NADH (dotted line). The time point of the addition of ammonia is indicated by the thin arrow which also marks the start of co-substrate consumption, indicated by decreasing extinction.

[0083] It should be noted here that the amount of ammonia added was very high and was chosen in this concentration to indicate that a low amount of GLDH, even under suboptimal conditions, is capable of reacting ammonium and co-substrate to yield L-glutamate and water, thereby scavenging ammonia from the solution.