Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1996-04-03
1998-11-24
Leary, Louise
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 4, 435967, 436 79, 436 74, 424 941, 424 946, 544358, 252374, C12Q 134, C12Q 100, H01L 21302
Patent
active
058405127
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method for measuring ionized calcium by using an enzyme.
BACKGROUND ART
Calcium contained in blood can be classified into the three kinds of protein-binding calcium, complexing calcium and ionized calcium. Among these, only ionized calcium is important in clinical tests and the amount of ionized calcium in blood is measured in a test for parathyroid functions, a cardiac operation and the like. As a method for selectively determining the amount of ionized calcium, the ion electrode method is known. However, this method requires much time for operation and cannot treat a large number of samples at one time. Therefore, it is not suitable for clinical tests.
As a method for treating a large number of samples at one time, there have been disclosed a method for quantitatively determining calcium by using a phospholipid, phospholipase D and (Japanese Unexamined Patent Publication No. 62-195297 and EP486997A); a method for quantitatively determining calcium by using phosphorylcholine thioester, phospholipase A.sub.2 and Tris-maleic acid buffer (Japanese Unexamined Patent Publication No. 1-231896); a quantitative determination method by using calmodulin (Japanese Unexamined Patent Publication No. 62-36199); and a quantitative determination method by using pyruvate kinase (Japanese Unexamined Patent Publication No. 2-142498). However, selective determination of ionized calcium is impossible by these methods.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a method by which a large number of samples can be treated at one time and, at the same time, the amount of ionized calcium in a sample can be determined selectively.
The method of the present invention for measuring ionized calcium belongs to a method for quantitatively determining calcium in a sample by using a phospholipase. The present method is characterized in that an enzyme reaction with the phospholipase is carried out in a buffer comprising a nitrogen heterocycle-binding sulfonic acid having a pK ranging from 6.6 to 7.6 or a salt thereof.
As a sample containing calcium, any sample may be used as long as it is miscible with the buffer. A preferable sample is a biosample such as plasma, cell extract and the like.
In the present invention, "phospholipase" means a general term for those enzymes which hydrolyze a phospholipid and a partial hydrolysate thereof.
As a method for quantitatively determining calcium by using a phopholipase, the following methods are illustrated. There are a method wherein phospholipase A.sub.1 or A.sub.2 and a substrate thereof are used. In these methods, the amount of calcium in a sample is determined by measuring activity of phospholipase A.sub.1 or A.sub.2 (Japanese Unexamined Patent Publication No. 1-231896). Furthermore, there is a method wherein phospholipase D and a substrate thereof are used. In this method, the amount of calcium in a sample is determined by measuring phospholipase D activity (Japanese Unexamined Patent Publication No. 62-195297 and EP486997A); and the like.
As a substrate for phospholipase A.sub.1 or A.sub.2, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and the like are enumerated. As a substrate for phospholipase D, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and the like are enumerated.
As a reaction product of phospholipase A.sub.1 or A.sub.2, acetic acid, fatty acid (monocarboxylic acid) and the like are enumerated. As a reaction product of phospholipase D, choline, glycerol, ethanolamine, inositol, serine and the like are enumerated.
As a method for determining the amount of a reaction product of phospholipase A.sub.1 or A.sub.2, there are a method which comprises reacting acetic acid with a dehydrogenase such as alcohol dehydrogenase in the presence of reduced nicotinamide adenine dinucleotide (NADH) or the following steps, adding phosphoenolpyruvic acid to adenosine diphosphate (ADP) produced by an acetate kinase reaction between ac
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Stryer, "Biochemistry", p. 39, 1975. Month not available.
Kayahara Norihiko
Miike Akira
Tadano Toshio
Umemoto Jun
Kyowa Medex Co., Ltd.
Leary Louise
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