Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1995-03-13
1998-07-14
Gitomer, Ralph
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435 15, 435 25, C12Q 132
Patent
active
057802564
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to a method for the quantitative determination of ammonia, an .alpha.-amino acid or an a-keto acid corresponding to the .alpha.-amino acid, or of a chemical substance producing any one of these compounds. The present invention also relates to an analytical composition for use in the above-mentioned quantitative determination.
BACKGROUND ART
Generally, in clinical diagnosis it is important to determine .alpha.-keto acids in biological samples, such as blood and urine, while in food chemistry it is important to determine .alpha.-amino acids, such as glutamic acid, contained in food. For example, organic monocarboxylic acids, such as pyruvic acid, .alpha.-ketoglutaric acid and the like, are Hayami Hyo, P.289, issued by Social Insurance Research Institute, Japan, 1990!. Especially, pyruvic acid, positioned at the intersections in various metabolic pathways, has been known to reflect the conditions of various diseases, and has generally been determined using lactate vol.47, P.496, 1989! or pyruvate oxidase (U.S. Pat. No.4,246,342 and Examined Japanese Patent Publication Specification No. 61-14794). The significance of L-alanine in blood as a criterion for determining the condition of a controlled diabetic has been acknowledged, and the determination of the L-alanine by the chemiluminescence method has been reported (Proceedings of Annual Meeting of Japan Society of Clinical Chemistry, vol.27, P.122, 1987).
It has also been known that the quantity of ammonia in blood is increased by a liver dysfunction or an enzyme deficiency in the urea cycle and, therefore, the determination of ammonia is useful in diagnosis of liver cirrhosis, uremia and the like. In addition, in view of the toxicity of ammonia, it is important to determine ammonia contained in food, drinking water, etc.
Conventionally, various methods are known for the determination of ammonia, .alpha.-keto acids and .alpha.-amino acids. They can be roughly classified into the microdiffusion method, ion exchange method, direct colorimetric method, enzymatic method and so on. In the microdiffusion method, an alkali is added to blood in a sealed container, and ammonia diffused from the blood is collected with an acid. In the ion exchange method, ammonium ions are adsorbed onto a cation exchange resin and then, eluted with an alkali, followed by colorimetry. In the direct colorimetric method, blood is deproteinized and then, ammonia in the blood is colorimetrically determined by indophenol reaction. These methods, however, have a drawback in that each of the methods necessarily involves two steps, i.e., a step of separating ammonia from a sample and a step of determining the separated ammonia, so that the whole procedure of each method inevitably becomes troublesome. In the meaning of the simplification of a determination process as well as the accuracy of determination, the enzymatic method is superior to the above-mentioned methods since this method utilizes a substrate specificity of an enzyme and an enzymatic reaction involved therein is conducted under ordinary physiological pH and temperature conditions, so that not only is it expected that determination results to be obtained should be closer to the real ammonia content value than those obtained by the above-mentioned other methods but the operation is also much simpler than that of the above-mentioned methods.
In the enzymatic method, ammonia, an .alpha.-keto acid or an .alpha.-amino acid is determined by measuring a change in amount of a reduced nicotinamide adenine dinucleotide (phosphate) (in terms of a change in absorbance at 340 nm with respect to the reaction mixture) in the following reversible reaction which is catalyzed by glutamate dehydrogenase (EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4): acid+NH.sub.3 +NAD(P)H+H.sup.+ 1978, and Examined Japanese Patent Publication Specification No. 57-21995). This method, however, has some problems. For example, this method is difficult to use in performing accurate determinations of low levels of substances in sa
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Kensa Tensu Hayami Hyo, p. 289, issued by Social Insurance Research Institute, Japan, 1990.
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Ikuta Shigeru
Misaki Hideo
Takahashi Mamoru
Ueda Shigeru
Asahi Kasei Kogyo Kabushiki Kaisha
Gitomer Ralph
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