JPH01231896A - Measurement of calcium - Google Patents
Measurement of calciumInfo
- Publication number
- JPH01231896A JPH01231896A JP5894688A JP5894688A JPH01231896A JP H01231896 A JPH01231896 A JP H01231896A JP 5894688 A JP5894688 A JP 5894688A JP 5894688 A JP5894688 A JP 5894688A JP H01231896 A JPH01231896 A JP H01231896A
- Authority
- JP
- Japan
- Prior art keywords
- amount
- calcium
- specimen
- measuring
- thioester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000011575 calcium Substances 0.000 title claims description 24
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title claims description 16
- 229910052791 calcium Inorganic materials 0.000 title claims description 16
- 238000005259 measurement Methods 0.000 title description 5
- -1 phosphorylcholine thioester Chemical class 0.000 claims abstract description 8
- 229950004354 phosphorylcholine Drugs 0.000 claims abstract description 5
- 239000000758 substrate Substances 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 4
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 3
- 102000015439 Phospholipases Human genes 0.000 claims description 3
- 108010064785 Phospholipases Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- BDPQVGIMLZYZQA-UHFFFAOYSA-N 2-hexadecanoylthio-1-ethylphosphorylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)SCCOP([O-])(=O)OCC[N+](C)(C)C BDPQVGIMLZYZQA-UHFFFAOYSA-N 0.000 abstract description 5
- 102100037611 Lysophospholipase Human genes 0.000 abstract description 5
- 108010058864 Phospholipases A2 Proteins 0.000 abstract description 5
- 229910052751 metal Inorganic materials 0.000 abstract description 5
- 239000002184 metal Substances 0.000 abstract description 5
- 150000002739 metals Chemical class 0.000 abstract description 5
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 abstract description 4
- 238000004040 coloring Methods 0.000 abstract description 4
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 239000003086 colorant Substances 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 abstract 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 230000035945 sensitivity Effects 0.000 abstract 1
- 150000007970 thio esters Chemical class 0.000 abstract 1
- 150000003573 thiols Chemical class 0.000 abstract 1
- 210000002966 serum Anatomy 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000001052 yellow pigment Substances 0.000 description 2
- NJNWCIAPVGRBHO-UHFFFAOYSA-N 2-hydroxyethyl-dimethyl-[(oxo-$l^{5}-phosphanylidyne)methyl]azanium Chemical class OCC[N+](C)(C)C#P=O NJNWCIAPVGRBHO-UHFFFAOYSA-N 0.000 description 1
- CPBJMKMKNCRKQB-UHFFFAOYSA-N 3,3-bis(4-hydroxy-3-methylphenyl)-2-benzofuran-1-one Chemical compound C1=C(O)C(C)=CC(C2(C3=CC=CC=C3C(=O)O2)C=2C=C(C)C(O)=CC=2)=C1 CPBJMKMKNCRKQB-UHFFFAOYSA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 208000020084 Bone disease Diseases 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 102000018997 Growth Hormone Human genes 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000001057 purple pigment Substances 0.000 description 1
- 239000003998 snake venom Substances 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、他の金属の影響を受けることなく特異性が高
くかつ操作が簡便なカルシウムの測定法に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for measuring calcium that is highly specific and easy to operate without being influenced by other metals.
（従来の技術）
生体内のカルシウム（Ｃａ）のほとんど（９９％）は骨
中に存在するが、その他＠量ながら体中のあらゆる組織
に含まれている。(Prior Art) Most (99%) of calcium (Ca) in living bodies exists in bones, but other amounts are also contained in all tissues throughout the body.
血清中のカルシウムのうち５０〜６０％はイオン（Ｃａ
”）として存在し、他は血漿蛋白と結合している。臨床
的意義を持つのはイオン型の方で、血液凝固、筋収縮、
刺激の伝達および酵素活性等に必須の物質である。50-60% of calcium in serum is ion (Ca
”), and others are bound to plasma proteins.The ionic type is clinically significant, and is involved in blood coagulation, muscle contraction,
It is an essential substance for the transmission of stimuli and enzyme activity.
カルシウムの代謝は各種のホルモン（副甲状腺ホルモン
、カルシトニン、成長ホルモン等）と密接な関連を持ち
、血清カルシウム量はカルシウム吸収異常、骨疾患の池
内分泌疾患でも変動する。Calcium metabolism is closely related to various hormones (parathyroid hormone, calcitonin, growth hormone, etc.), and serum calcium levels fluctuate due to calcium absorption abnormalities and bone diseases.
このように血清カルシウム量は各種の病態を反映する一
つの指標となるため、無機リンの測定と共に主要な無機
質測定項目の一つとなっている。As described above, the serum calcium level is one of the indicators that reflects various pathological conditions, and therefore, along with the measurement of inorganic phosphorus, it is one of the main mineral measurement items.
臨床検査室で従来から用いられているカルシウムの測定
法にｏｃｐｃ法がある。この方法は、０−クレゾールフ
タレインコンプレクソンがアルカリ下で血清中のカルシ
ウムと反応して生成する赤紫色の色素を測定して血清中
のカルシウムを求めるものである。The OCPC method is a method for measuring calcium that has been conventionally used in clinical laboratories. In this method, calcium in serum is determined by measuring a reddish-purple pigment produced when O-cresolphthalein complexone reacts with calcium in serum under alkaline conditions.
（発明が解決しようとする課題）
上記した０ＣＰＣ法は、操作が簡単ではあるが、■低値
のカルシウムを測定できない、■温度の影響を受ける、
■着色性が強く機器・を汚す、■発色後の安定性が悪い
、■他の金属の影響を受ける、等の多くの欠点があった
。(Problems to be Solved by the Invention) Although the above-mentioned 0CPC method is easy to operate,
It had many drawbacks, such as: ■ Strong coloring property that stains equipment, ■ Poor stability after color development, and ■ Being affected by other metals.
本発明方法は、上記欠点を解決するためにホスホリパー
ゼＡ２のカルシウム依存性に着目して開発されたもので
、■Ｒａｔｅ Ａｓ５ａｙができる、■他の金属の影響
を受けることがなく特異性が高い、■着色性が少ない、
■操作が簡便である、ようにしたカルシウムの測定法を
提供することを目的とするものである。The method of the present invention was developed focusing on the calcium dependence of phospholipase A2 in order to solve the above-mentioned drawbacks, and it has the following properties: 1. Rate As5ay is possible; 2. It is not affected by other metals and has high specificity. ■Less colored,
(2) The purpose is to provide a method for measuring calcium that is easy to operate.
（課題を解決するための手段）
上記目的を達成するために、本発明方法においては、ホ
スホリルコリンチオエステルを基質に用い、検体中のカ
ルシウム量に応じて変化するホスホリパーゼＡ２の酵素
活性を測定することによって、検体中のカルシウム量を
測定するようにしたものである。(Means for Solving the Problems) In order to achieve the above object, the method of the present invention uses phosphorylcholine thioester as a substrate and measures the enzymatic activity of phospholipase A2, which changes depending on the amount of calcium in the sample. , which measures the amount of calcium in a sample.
本発明で使用できるホスホリルコリンチオエステルの代
表例としては、下記式に示される２−ヘキサデカノイル
チオ−１−エチルホスホリルコリンがあるが、これに限
定されるものではなく、下記式中のＲを脂肪属及び芳香
域の炭化水素とした化合物及びその誘導体が包含される
。さらに、チオグリコールのみならず、ホスホリルコリ
ン類のチオール化合物も広範囲に使用できる。A representative example of the phosphorylcholine thioester that can be used in the present invention is 2-hexadecanoylthio-1-ethylphosphorylcholine shown in the following formula, but is not limited thereto. and aromatic hydrocarbon compounds and derivatives thereof. Furthermore, not only thioglycol but also thiol compounds such as phosphorylcholines can be used in a wide range of ways.
ホスホリパーゼＡ２は、ブタ膵、ヘビ毒等の由来のもの
が使用できる。Phospholipase A2 derived from pig pancreas, snake venom, etc. can be used.
本発明方法の反応を弐で示せば下記の通りである。The reactions of the method of the present invention are shown below.
Ｏ−ＣＨ。O-CH.
（上式中Ｒを−（Ｃ１１□）＋４−ＣＨ５）とすれば、
２−ヘキサデカノイルチオ−１−エチルホスホリルコリ
ンである。）
ホスホリパーゼＡ２０ ＤＴＮＢ
一一一１・・→Ｒ・Ｃ・ＳＨ・→黄色色素Ｃａ ”
（ＤＴＮＢは５．５°−ジチオビス（２−ニトロ安息香
酸）である。）
（作用）
本発明方法の測定原理は、ホスホリルコリンチオエステ
ル、例えば２−ヘキサデカノイルチオ−１−エチルホス
ホリルコリン（チオグリコールレシチン）にＣａ”の存
在下で活性化するホスホリパーゼＡ２を作用させチオー
ル化合物を生成させ、−ＳＨ基を発色させる発色剤、例
えばＤＴＮＢ等を作用させて黄色色素を生成せしめ、こ
れを比色定量してＣａ”を測定するものである。(If R in the above formula is -(C11□)+4-CH5),
2-hexadecanoylthio-1-ethylphosphorylcholine. ) Phospholipase A20 DTNB 1111...→R・C・SH・→Yellow pigment Ca” (DTNB is 5.5°-dithiobis(2-nitrobenzoic acid).) (Effect) Measurement of the method of the present invention The principle is that phospholipase A2, which is activated in the presence of Ca, acts on phosphorylcholine thioester, such as 2-hexadecanoylthio-1-ethylphosphorylcholine (thioglycol lecithin), to generate a thiol compound, and the -SH group develops color. A yellow pigment is produced by the action of a coloring agent such as DTNB, which is then colorimetrically quantified to measure Ca''.
（発明の効果）
以上のように、本発明方法によれば、■Ｒａｔｅ Ａｓ
５ａｙができる、■他の金属の影響を受けることがなく
特異性が高い、■着色性が少ない、■操作が簡便である
という大きな効果を奏する。(Effect of the invention) As described above, according to the method of the present invention, ■Rate As
5ay, (1) high specificity without being affected by other metals, (2) less coloring, and (2) easy operation.
（実施例） 以下に本発明の実施例を挙げて説明する。(Example) Examples of the present invention will be described below.
実施例１
試薬ニ
ドリスマレイン酸緩衝液・・・・・２００ｍＭ、 ｐＨ
７，５２−ヘキサデカノイルチオ−１−エチルホスホリ
ルコリン・・・・・・・・・・・・・・・０，１ｍＭＤ
ＴＮＢ ・・・・・・・・・・・・・曲・１ ｍＭホス
ホリパーゼＡｚ ・’・・・・・０，８０／ ｍ測定
条件：
試薬２ｍｉ！を予め３７°Ｃで予備加温し、試料２０μ
ｐを加えたのち、４１０ｎｍで１分間当たりの吸光度変
化と比較して検体中のＣａ”量を測定した。Example 1 Reagent Nidris maleate buffer...200mM, pH
7,52-hexadecanoylthio-1-ethylphosphorylcholine 0.1mMD
TNB ・・・・・・・・・・・・Song・1 mM Phospholipase Az ・'・・・・・・0,80/m Measurement conditions: Reagent 2mi! Prewarm the sample at 37°C, and
After adding p, the amount of Ca'' in the sample was measured by comparing the absorbance change per minute at 410 nm.
■直線性
Ｃａ”°ｌ０ｍｇ／ｄ１以下５段階希釈して八Ｅ ／
ｍｉｎを測定した結果、第１図に示した如く、原点を通
る良好な直線性が得られ力。■Linearity Ca"°l0mg/d1 or less after dilution in 5 steps 8E/
As a result of measuring min, as shown in Fig. 1, good linearity passing through the origin was obtained.
■反応の経時変化
Ｃａ”°１１０１ｕ／ｄＩｌを加えて３７°Ｃにおける
反応の経時変化をとったところ、第２図に示したごとく
直線的に反応が進むことが確認された。(2) Time course of reaction When the time course of the reaction was measured at 37°C by adding Ca''°1101 u/dIl, it was confirmed that the reaction proceeded linearly as shown in FIG.
【図面の簡単な説明】
第１図は実施例１の直線性を示すグラフ及び第２図は実
施例１の経時変化を示すグラフである。
特許出願人 三光純薬株式会社BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing linearity of Example 1, and FIG. 2 is a graph showing changes over time in Example 1. Patent applicant: Sanko Pure Chemical Industries, Ltd.
Claims (1)
体中のカルシウム量に応じて変化するホスホリパーゼＡ
＿２の酵素活性を測定することによって、検体中のカル
シウム量を測定する方法。(1) Phospholipase A that uses phosphorylcholine thioester as a substrate and changes depending on the amount of calcium in the sample
A method for measuring the amount of calcium in a specimen by measuring the enzyme activity of _2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5894688A JP2659982B2 (en) | 1988-03-12 | 1988-03-12 | How to measure calcium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5894688A JP2659982B2 (en) | 1988-03-12 | 1988-03-12 | How to measure calcium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01231896A true JPH01231896A (en) | 1989-09-18 |
| JP2659982B2 JP2659982B2 (en) | 1997-09-30 |
Family
ID=13098997
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP5894688A Expired - Lifetime JP2659982B2 (en) | 1988-03-12 | 1988-03-12 | How to measure calcium |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2659982B2 (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5369219A (en) * | 1991-06-18 | 1994-11-29 | Multimedia Design, Inc. | Multi-layer printed circuit board apparatus and method for making same |
| WO1994028167A1 (en) * | 1993-05-31 | 1994-12-08 | Kyowa Medex Co., Ltd. | Method of assaying ionized calcium |
| WO1995005479A1 (en) * | 1993-08-17 | 1995-02-23 | The Regents Of The University Of California | Assay and substrate for arachidonoyl-specific phospholipase a¿2? |
| US5618684A (en) * | 1992-02-07 | 1997-04-08 | Oriental Yeast Co., Ltd. | Method of determination of calcium |
| EP0776979A1 (en) | 1995-11-28 | 1997-06-04 | Oriental Yeast Co., Ltd. | Method and reagent for measuring an ion by using maltose derivatives |
| CN100354630C (en) * | 2002-10-31 | 2007-12-12 | 世诺临床诊断制品株式会社 | Reagent for testing calcium in sample and test method thereof |
-
1988
- 1988-03-12 JP JP5894688A patent/JP2659982B2/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5369219A (en) * | 1991-06-18 | 1994-11-29 | Multimedia Design, Inc. | Multi-layer printed circuit board apparatus and method for making same |
| US5618684A (en) * | 1992-02-07 | 1997-04-08 | Oriental Yeast Co., Ltd. | Method of determination of calcium |
| WO1994028167A1 (en) * | 1993-05-31 | 1994-12-08 | Kyowa Medex Co., Ltd. | Method of assaying ionized calcium |
| WO1995005479A1 (en) * | 1993-08-17 | 1995-02-23 | The Regents Of The University Of California | Assay and substrate for arachidonoyl-specific phospholipase a¿2? |
| US5464754A (en) * | 1993-08-17 | 1995-11-07 | The Regents Of The University Of California | Assay and substrate for arachidonoyl-specific phospholipase A2 |
| EP0776979A1 (en) | 1995-11-28 | 1997-06-04 | Oriental Yeast Co., Ltd. | Method and reagent for measuring an ion by using maltose derivatives |
| US5948632A (en) * | 1995-11-28 | 1999-09-07 | Oriental Yeast Co., Ltd. | Method and reagent for measuring chlorine and calcium ions using a maltose derivative |
| CN100354630C (en) * | 2002-10-31 | 2007-12-12 | 世诺临床诊断制品株式会社 | Reagent for testing calcium in sample and test method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2659982B2 (en) | 1997-09-30 |
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