JPH0394698A - Integrated multi-layer analytic element containing ammonia determination reagent composition - Google Patents

Abstract

PURPOSE:To obtain the subject element capable of determining intermediate of protein metabolism, urea nitrogen, creatinine, ammonia, etc., with simple procedure in high accuracy by forming a specific ammonia reagent layer on a light-transmitting water-impermeable supporting member and laminating a developing layer on the reagent layer. CONSTITUTION:The objective integrated multi-layer analytic element is produced by forming (A) a reagent layer containing a part or total of the components of an ammonia determination reagent composition containing a glutamine synthetase, a pyruvic acid kinase, a pyruvic acid oxidase and a hydrogen peroxide-detecting reagent composition, wherein the content of the glutamine synthetase or the pyruvic acid kinase is the rate limiting amount on (B) a light- transmitting and water-impermeable supporting member and laminating (C) a developing layer on the reagent layer.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is applicable to analytes that can produce ammonium ions such as urea nitrogen, ammonia, and taleatinin.
This article relates to an integrated multilayer analytical element containing a reagent system for measuring and a reagent analysis method using the element. [Prior art] Urea nitrogen (B) is an intermediate and final product of protein metabolism.
Measurement of UN), ammonia, talleatinine, etc. is necessary for diagnosing renal disease, determining the course of treatment, etc. Conventionally,
These are analyzed by a wet method. For example, urea nitrogen is analyzed by directly reacting terephthalaldehyde and a phenylenediamine derivative under strongly acidic conditions, and colorimetrically quantifying the color development (D.J.
．． Jung+ CIin. Chem. ．． 21. 113
6 (1975)), ammonium ions produced by the action of urease on urea were quantified using Nessler's reagent or by colorimetric fixation using the indophenol method. The indophenol method has since been improved by many researchers (for example, H. Okud.
et al, Tokushima J. Exptl
．． Med. ．． 12. (1) 11-23 (1965)
). This method has problems in that the equipment is easily corroded because the reaction is carried out under strongly acidic conditions, and it also has problems with low sensitivity and the time required for measurements. Regarding the enzymatic method, a method has been developed that uses glutamate dehydrogenase to determine the amount of decrease in NADPH by measuring UV absorption (A. Mondzac et al., J.
．． Lab. CIin. Med. ．． 66, 526. (1
965)). This method also had problems with sensitivity and accuracy. In the presence of glutamic acid and ATP, glutamate synthesis enzyme is activated, and pyruvate kinase and pyruvate oxidase are activated to produce hydrogen peroxide, which is then colorimetrically determined using peroxidase and 4-aminoantipyrine. A method has also been developed to
Publication No. 00).

On the other hand, a dry method is also known as a method that allows rapid quantitative determination with simple operations. Examples of analytical instruments used in the dry method include:
Analysis Library 3 edited by the Japanese Society of Analytical Chemistry "Clinical Chemistry Analysis ■Nitrogen Containing Weight" 2nd edition (Tokyo Kagaku Doujin, published in 1979) 1
Pages 3-14, "Clinical Examination" Volume 5 (No. 6) 387-3
91 pages (published in 1961), US Pat. BU with an indicator layer (containing pH indicator bromcresol green) provided in this layer
A reagent strip for N quantitative analysis is disclosed. Japanese Patent Application Publication 1973-
Publication No. 93494 discloses a BUN assay method in which a filter paper piece is impregnated with a pH supporting agent promthymol blue, then further impregnated with a mixture of urease and a barrier-type substance made of a hydrophilic polymer, and finally an ethyl cellulose semipermeable membrane is provided. An analytical test strip is disclosed. Also, JP-A-52-34
Publication No. 88 (Japanese Patent Publication No. 58-19062) discloses a support drug layer on a water-impermeable transparent support, and a cellulose acetate layer that can be uniformly penetrated by ammonia vapor and impermeable to liquid water and alkaline obstructions. A BUN quantitative analysis unit consisting of a barrier layer made of a homogeneous (non-porous) thin layer of a hydrophobic polymer such as petyrate, a reagent layer containing urease and an alkaline agent, and a porous development layer, all laminated in this order. Body shape multi-layer analysis elements have been proposed.

[Problem to be solved by the invention]

In all conventional dry methods, ammonia gas is diffused within the test piece or analysis element, and there is a problem in that this diffusion lacks quantitative properties. The object of the present invention is to utilize urea nitrogen (BUN), an intermediate and final product of protein metabolism.
The object of the present invention is to provide an analytical element that can quantify , taleatinin, ammonia, etc. with high precision using a simple method.

[Means to solve the problem]

The inventors of the present invention have conducted extensive studies to achieve the above objectives, and have found that this is an ammonium ion detection method that can basically be used commonly for the measurement of protein metabolic intermediates and final biological products, and that is suitable for color reactions. As a related reaction principle, we have focused on a method in which the above-mentioned glutagon synthesis enzyme is combined with pyruvate kinase and pyruvate oxidase, and the precipitated hydrogen peroxide is colorimetrically fixed. However, when this reaction system was incorporated into a dry analytical element, a new problem arose in that pyruvic acid contained in the sample was detected at the same time, resulting in an error. Therefore, the present inventors further investigated the content of at least one of the above-mentioned glutamine binding enzyme and pyruvate kinase, and performed a rate assay, thereby subtracting the color produced by pyruvate in the sample. He was able to devise a means to quantify things into cash, and was able to achieve the above objective.

The present invention provides an integrated multilayer analytical element in which at least a light-transparent water-impermeable support, a reagent layer, and a developing layer are laminated in this order. The present invention relates to an integrated multilayer analytical element containing a detection reagent composition and an ammonia measurement reagent composition characterized in that the content of the glutamine synthase or pyruvate kinase is a rate-limiting amount.

As the light-transmitting and water-impermeable support, any known support used in general multilayer analytical elements may be used.

Specific examples thereof include polymers such as polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, and cellulose esters (cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) with a thickness of about 50 μm to about 1 μm.
Transparent supports in the range of mm, preferably about 80 pIQ to about 300 nm can be used. If necessary, a known undercoat layer or adhesive layer may be provided on the surface of the support as described in the above-mentioned patent specifications to strengthen the adhesion with a hydrophilic layer or the like provided on the support.

The reagent layer is a layer containing all or part of the enzyme and reagent composition described above, and is a non-porous, water-absorbing layer or a microporous, water-permeable layer.

The hydrophilic polymeric binder used in the substantially non-porous, water-absorbing reagent layer has a swelling ratio of approximately 1 at 30"C upon water absorption.
The natural or synthetic hydrophilic polymer ranges from 50% to about 2000%, preferably from about 250% to about 1500%. Examples include gelatin (acid-treated gelatin, deionized gelatin, etc.), gelatin derivatives (phthalated gelatin,
(hydroxyacrylate grafted gelatin, etc.), agarose, pullulan, pullulan derivatives, polyacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, and the like. The dry thickness of the substantially non-porous reagent layer ranges from about 3 Irm to about 50 mm, preferably from about 5 fm to about 30 mm, with a coverage of about 3 g/bo to about 50 g/bo.
/rd, preferably about 5g/rrf to about 30g/rd
rd range.

Preferably, the reagent layer is substantially transparent.

The microporous and water-permeable reagent layer is made of a fabric similar to the fabric interlayer described below, a paper made from organic polymer fiber valves described in JP-A-57-148250, and JP-A-57-125847.
A fibrous porous layer such as a porous layer formed by coating a dispersion of fibers and a hydrophilic polymer described in et al.; Japanese Patent Publication No. 53-21
677, U.S. Pat. No. 3,992,158, etc., the membrane filter layer (brushed polymer layer) is an isotropic porous structure containing continuous microvoids, in which fine particles such as polymer microbeads are bonded in point contact with a hydrophilic polymer binder. Polymer microbeads described in JP-A-55-90859 are contacted in a point-contact manner with a polymeric adhesive that does not swell with water. ), etc., or a microporous structure composed of solid particles and a hydrophilic polymer binder containing a reagent compound. This is a layer containing The dry layer thickness of the microporous reagent layer is about 7 μm to about 250 μm, preferably about 1 μm.
It ranges from 0 μm to about 250 μm.

When a developing layer is provided on the microporous reagent layer, the gap between the two layers is as described in JP-A-61-4959 and JP-A-62-13875.
It is preferable to use porous adhesion as described in No. 6 and the like.

One embodiment of the multilayer analytical element of the present invention is a structure in which the spreading layer contains the above-mentioned reagent composition, and a water absorption layer or a detection layer is provided below the spreading layer (via an adhesive layer if necessary). There are powerful multi-layered analysis elements. In another embodiment, the polymeric binder of the reagent layer is a polymer that is dissolved or solubilized in the solvent of the aqueous liquid sample, such as polyvinyl alcohol, polyvinylpyrrolidone, carboxymityl cellulose, sodium alginate, unhardened gelatin, agar. There is a multilayer analysis element having a structure in which a reagent layer is formed using a reagent layer, and a microporous thin sheet-like member used as the microporous reagent layer is laminated and bonded thereon as a porous detection layer.

The reagent layer can contain known pH buffer compositions, polymeric pH buffering agents, basic polymers, acidic polymers, polymeric mordants, and the like.

As a developing layer, a membrane filter (plush polymer N) disclosed in Japanese Patent Publication No. 53-21677, etc., polymer microbeads, glass microbeads, and continuous microvoid containing diatoms held in a hydrophilic polymer binder can be used. Porous layer, a continuous microvoid-containing porous layer (three-dimensional Non-fibrous isotropic porous spread layer such as lattice-like granular structure layer), JP-A-55-1643
56, JP-A-57-66359, etc., the woven fabric spreading layer disclosed in Japanese Patent Application No. 59-79158, the organic polymer fiber disclosed in Japanese Patent Application No. 57-148250, etc. Examples include a fibrous porous spread layer such as a spread layer made of made paper containing valves.

The spreading layer is laminated directly or via an adhesive layer if necessary.

In addition to the above-mentioned layers, the multilayer analysis element may include a detection layer or mordant layer disclosed in JP-A-51-40191, JP-A-53-131089, etc., and ξ disclosed in JP-A-54-29700, etc., as necessary. Gration prevention layer, JP-A-51-40191
It is possible to incorporate an intermediate layer disclosed in et al., a reagent-containing fine particle dispersed layer disclosed in JP-A-56-8549, and the like.

The reaction system for detecting ammonium ions in the analytical element of the present invention proceeds as follows.

C (GS: Glutamine Synthesis Enzyme PK: Pyruvate Kinase POP: Pyruvate Oxidase POD: Peroxidase Glutamine Synthesis Enzyme (EC6.3.1.2)
Escherichia col ATCC9637 etc.)
, Sac i order usstearothrmophilu
It can be isolated from sheep brain, bovine brain, etc. Bac
The enzyme produced by Illus stearothrmophilus is a thermostable enzyme that is commercially available and is particularly preferred for the analytical element of the present invention. Pyruvate kinase (E
C2 ・7-1 ・40) is Bacillus s
tearothrmophilusSBacillus
licheniformis etc. can be produced, as well as
It can be obtained from muscles of animals such as rats, rabbits, dogs, and chickens, pig hearts, etc. Pyruvate oxidase [
EC 1, 2, 3, 6] is Lactobactillus
It can be produced by various bacteria including S. delbrueckii. All of these enzymes are commercially available and can be purchased and used. On the other hand, these enzymes do not need to be pure products for use in the analytical elements of the present invention.
For example, it is also possible to use a fermentation liquid of a microorganism or a crude enzyme obtained from the fermentation liquid by means such as precipitation.

As the hydrogen peroxide detection reagent set, a reagent set containing peroxidase is preferable. Peroxidase is derived from plants such as wasabi, potatoes, radish, and figs, from animals such as leukocytes and milk, and from cochliobolus (Co
Chliobolus genus, Tarpuraria (Curvu)
Those derived from microorganisms such as the genus Laria are known, and any of these can be used, but it is convenient to use commercially available products.

In addition, this reagent composition further includes a coloring reagent composition.
The coloring reagent composition contains a chromogen (also called a hydrogen donor) and a coupler, and the chromogen and coupler are oxidized and combined with hydrogen peroxide in the presence of peroxidase, forming a quinone imine dye and forming a color. It is a compound that is oxidized by hydrogen peroxide in the presence of peroxidase to become a pigment and develops color.

As a chromogen, Ann. CIin. Biochem.
, 6. 24-27 (1969) (also known as 4-aminophenazone, i.e., 1-phenyl-2,3-dimethyl-4-ano-3-birazolin-5-one), JP-A-1986-59- 1-(2.4.6-}lichlorophenyl)-2 described in 54962 etc.
．． Tri-substituted-4-amino-3 such as 3-dimethyl-4-amino-3-birazolin-5-one, 1-(3.5-dichlorophenyl)-2,3-dimethyl-4-agono-3-birazolin-5-one -Virazolin-5-one, 1-phenyl-2.3 described in Japanese Patent Publication No. 55-25840, etc.
4- such as -dimethylano-3-virazoline-5-one, etc.
Anoantipyrine analogs can be used. Among these compounds, 4-aminoantivirine, 1-
(2.4.6-}licrophenyl)-2.3-dimethyl-4-amino-3-birazolin-5-one, 1-(3
．． Preferred are 5-dichlorophenyl)-2,3-dimethyl-4-amino-3-birazolin-5-one, and the like.

As a coupler, Ann. CIin. Biochen
．． , 61 24-27 (1969), Special Publication 1972
-25840, JP-A-5B-45599, JP-A-5B-18628, JP-A-55-164356, JP-A-56-124398, JP-A-56-155852, etc. Phenol; 2-hydroxy-1-benzenesulfonic acid , 4-hydroxy-1-benzenesulfonic acid, 3,
Phenolsulfonic acids (alkali metal salts, (including alkaline earth metal salts), 1-naphthol, 2-
Naphthol, dihydroxynaphthalenes such as 1,7-dihydroxynaphthalene; naphtholsulfonic acids such as l-hydroxy-2-naphthalenesulfonic acid and 1-hydroxy-4-naphthalenesulfonic acid (including alkali metal salts and alkaline earth metal salts) . There are other phenol or naphthol derivatives. Among these compounds, 1.
7-dihydroxynaphthalene, 1-hydroxy-2-naphthalene sulfonic acid (including Na salt, K salt, Li salt),
3.5-dichloro-2-hydroxy-1-benzenesulfonic acid (including Na salt, K salt, and Li salt) is preferred.

As a leuco dye, 2-
(4-hydroxy-3,5-dimethoxyphenyl)4,
Triarylimidazole leuco dyes such as 5-bis(4-(dimethylaξno)phenyl)iwarodazole; 2-(3.5-dimethoxy4-hydroxyphenyl)-4-[described in JP-A-59-193352; Diarylimidazole leuco dyes such as 4-(dimethylamino)phenyl]-5-phenethylidazole; JP-A-61-496
2-(2-phenyl-3-indolyl)-4 described in 0
．． Diarylindolylimidazole leuco dyes such as 5-di[4-(dimethylamino)phenyl]imidazole, 2-(4-hydroxy-3.5-dimethoxyphenyl)-3-acetyl-4 described in JP-A-61-229868; .5-bis[4-(jethylagono)phenyl]imidazole and other triarylmonoacylimidazole leuco dyes; 2-(4-hydroxy-3.5-'; methoxyphenyl)-3-methyl-4,5- Examples include triarylmonoalkylimidazole leuco dyes such as bis[4-(diethylaξno)phenyl]imidazole.

Glutamine synthetase, pyruvate kinase, pyruvate oxidase, and the reagent composition for detecting hydrogen peroxide may all be contained in the reagent layer, or some of them may be contained in other layers,
For example, it may be contained in a developing layer or the like.

In the analytical element of the present invention, the content of at least one of glutakamen synthase or pyruvate kinase is set to a rate limiting amount.
shall be. This means that the M (activity value) of glutamine synthetase or pyruvate kinase is such that it controls the rate at which the reaction proceeds with respect to the amount (activity value) of pyruvate oxidase and peroxidase. .

The rate-limiting amount is generally determined when the sample is plasma or serum.
Glutamine synthesis enzyme is about 200 to 20,000 IU/rrf,
And pyruvate kinase is about 200 to 20,010/bo. Pyruvate oxidase only needs to be present in sufficient amounts;
Usually about sooo to 40,000 IU/rrf, preferably 1
It is about 10,000 to 20,000/.

The spreading layer, reagent layer, water absorption layer, detection layer, adhesive layer, etc. can contain a surfactant, preferably a nonionic surfactant. Examples of nonionic surfactants include p-octylphenoxybolyethoxyethanol, P-nonylphenoxypolyglycidol, polyoxyethylene oleyl ether, polyoxyethylene sorbitan monolaurate, oxytyl glucoside, and the like. By including a nonionic surfactant in the reagent layer, water absorption layer, or detection layer, water in an aqueous liquid sample is easily absorbed substantially uniformly into the reagent layer, water absorption layer, or detection layer during analysis operations, and Liquid contact with the spreading layer is rapid and substantially uniform. By containing a nonionic surfactant in the spreading layer, the spreading action (metering action) of the aqueous liquid sample becomes better. The content of the nonionic surfactant in the spreading layer is in the range of about 10 g to about 3.0 g, preferably about 20 g to about 2.0 g per rrf of the spreading layer.

When the analytical element of the present invention is used for urea nitrogen analysis, for example, urease is used, and when it is used for taleatinine analysis, creatinine didinase, creatinine amidohydrolase, taleatinamidinohydrolase, and urease are used in the reagent layer, developing layer, or other layers. It may be contained in an appropriate layer.

The multilayer analytical element of the present invention can be prepared by known methods as described in the aforementioned patent specifications.

The multilayer analytical element of the present invention has a side of about 15 mm to about 30 mm.
Cut into small pieces such as squares or circles of approximately the same size,
Special Publication No. 57-28331, Real Publication No. 56-142454,
Japanese Patent Application Publication No. 57-63452, Utility Application Publication No. 58-323
50, Japanese Patent Publication No. 58-501144, etc., and used as a chemical analysis slide, it is preferable from various viewpoints such as manufacturing, packaging, transportation, storage, and measurement operations. Depending on the purpose of use, it can be used in the form of a long tape and stored in a cassette or magazine, or small pieces can be attached or stored in a card with an opening.

The multilayer analytical element of the present invention can analyze analytes in liquid samples by the operations described in the aforementioned patent specifications. For example about 5μe to about 30all, preferably 8μi
A drop of an aqueous liquid sample of whole blood, plasma, serum, urine, etc. ranging from
Incubation is carried out at a substantially constant temperature in the range of about 20° C. to about 40° C., preferably around 37° C., and color development or discoloration within the element is caused by exposure to visible or ultraviolet light. Rate assay is performed by measuring reflection photometry from the light-transmitting support side using light at or near the maximum absorption wavelength. This may be determined by continuously measuring the reflected optical density, or by measuring it twice or more over time. Color development in the analytical element of the present invention first occurs due to pyruvic acid previously contained in the sample, and then color development begins based on analytes such as urea nitrogen, ammonia, and creatinine. Color development due to pyruvic acid in the sample usually occurs about 30 seconds after the start of the reaction, and the color density state remains constant for about 1.5 to 2 minutes. This can be measured and determined as color development due to pyruvic acid previously contained in the sample. Color development based on the analyte begins one and a half to two minutes after the start of the reaction, and the rate of color development is correlated with the concentration of the analyte, so this color development rate is determined. This coloring speed may be determined by two measurements, or three or more measurements. In the case of two measurements, the timing is usually 3 minutes or later after the start of the reaction, and for example, the measurement can be carried out 3 minutes and 5 minutes later. The analyte concentration is determined in advance and calculated from the relationship between the analyte concentration and the rate of color development.

The measurement operation was performed using JP-A-60-125543 and JP-A-60-125543.
220862, JP 61-294367, JP 5
8-161867, JP-A-63-313063, etc., highly accurate quantitative analysis can be carried out with extremely easy operation.

[Effect]

In the reaction system for ammonia measurement adopted in the analytical element of the present invention, glutamine synthetase reacts ammonium ions with glutamic acid to convert them into glutamine, and pyruvate kinase converts the ADP produced at this time into phosphoenolpyruvate. React to produce pyruvate. Pyruvate oxidase converts this pyruvate into acetyl phosphate, and hydrogen peroxide is used as a substitute. This hydrogen peroxide is quantified colorimetrically using a hydrogen peroxide detection reagent composition. In such a reaction system, pyruvic acid previously contained in the sample is also detected, causing errors. In the analytical element of the present invention, the color development rate of the analyte is regulated by regulating the content of glutamine synthetase or pyruvate kinase, and the color development rate due to pyruvate contained in the sample is determined by determining this color development rate. This eliminates the influence of

〔Example〕

Example 1 185n thick colorless transparent PET with gelatin subbing layer
A second reagent layer according to the following composition was coated onto the film support so that the layer thickness after drying was approximately 1,000 ml, and dried.

Pyruvate oxidase 13000 [1/%
Peroxidase 22000 U/bothiaminpicophosphate 0.85 mmol/rr
rFAD 4.0 μmo/n
? Potassium dihydrogen phosphate 10 mmo/r
dM g” (magnesium sulfate) 3.0 mmo
/rd phenol 5.0 mmo
/rrf4-agonoantipyrine 2. O mm
ol/n {gelatin 14 g/bo The following first reagent layer was coated on the second reagent layer so that the layer thickness after drying was about 20 nm and dried.

Glutamine synthesis enzyme 500 U/Bopyruvate kinase 9500 U/BoL-glutamic acid 6.0 mmol/% Phosphoenolpyruvate4. O mmol/rh
ATP 5.4 IIIra
al/rrf gelatin 19
g/rrf Next, water is almost uniformly supplied to the surface of the first reagent layer to wet it, and cellulose acetate porous 1l9 (nominal pore diameter 3.0 μm, porosity approximately 85%, thickness 140 μm) is approximately applied thereon. Laminate the membrane by applying light pressure uniformly, and apply HEPES buffer (pH 7.0) to this porous membrane.
30 B mol and nonylphenoxypolyethoxyethanol (containing an average of 10 oxyethylene units) 0.2■
The analytical element (2) of the present invention was prepared by impregnating it by coating and drying it.

Example 2 In Example 1, the second composite bottom for detecting hydrogen peroxide was used.
Phenol and 4-aminoantipyrine in the reagent layer,
2-(3,5-dimethoxy4-hydroxyphenyl)
-4.5-bis(4-dimethylagonophenyl)imidazole 4 mmol/rd was used as analytical element (2) of the present invention.

Comparative Example 1 An analytical element (Comparative Example ■) identical to Example 1 was prepared except that the amount of glutamine-binding enzyme in the second reagent layer was increased to 150,000/nf in Example 2.

Examples 3 to 6 A reagent layer containing a certain amount of each of the above was applied in the same manner as in Example 1, and then a tricot knitted fabric made of polyester fibers was laminated in the same manner as in Example 1.

Example 7 The analytical elements of the above examples and comparative examples were 15 mm x 15 mm.
The chips were cut into square chips and placed in a plastic mount described in JP-A-57-63452 for measurement.

The following five types of human serum whose pyruvic acid concentration and ammonia concentration had been measured in advance were used as specimens.

? :1n? Amount of coverage per hit ←Same amount as left cover ■Unsuitable*
2-<2-phenyl-3-indolyl)-4,5-di[
4-(dimethylamino)phenyl]imidazole ('ltRFl{61-4960, IJS 4 7
21 67fC Memorandum) 1 1.0
1502 2.0
1503 0.5
1504 0.5
3005 0.5
600 Place 10 ue of the above serum on each analysis element, incubate at 37°C, and after 3 minutes (OD.) and 5 minutes.
After a minute (○D,), the reflected optical density was measured. The measurement wavelength is 510 nm for analytical elements (1), (2), and (2), and 570 nm for analytical elements V and (2). In addition, the measurement wavelength of the analytical element (■) and comparative product (■) is 625 nm.
It is.

The color development rate obtained for each analytical element is shown in the table below.

Color development rate = ODs-003 Color development rate (00/min) 0.112 0.222 [Effects of the invention] By using the analytical element of the present invention, the influence of pyruvic acid contained in the sample can be eliminated and ammonia and Using this measurement system, urea nitrogen, creatinine, etc. can be accurately quantified.

Claims (1)

[Claims] (1) An integrated multilayer analytical element in which a reagent layer and a developing layer are laminated in this order on a light-transparent water-impermeable support, the reagent layer containing glutamine synthetase, pyruvate kinase, pyruvate All or some of the components of an ammonia measurement reagent composition including an oxidase and hydrogen peroxide detection reagent composition are contained, and the content of the glutamine synthetase or pyruvate kinase is a rate-limiting amount. An integrated multilayer analytical element comprising an ammonia measurement reagent composition.