JPH0578315B2 - - Google Patents
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- Publication number
- JPH0578315B2 JPH0578315B2 JP18752485A JP18752485A JPH0578315B2 JP H0578315 B2 JPH0578315 B2 JP H0578315B2 JP 18752485 A JP18752485 A JP 18752485A JP 18752485 A JP18752485 A JP 18752485A JP H0578315 B2 JPH0578315 B2 JP H0578315B2
- Authority
- JP
- Japan
- Prior art keywords
- placental
- alp
- isoenzyme
- activity
- type
- 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.)
- Expired - Lifetime
Links
- 108010044467 Isoenzymes Proteins 0.000 claims description 106
- 230000000694 effects Effects 0.000 claims description 76
- 230000003169 placental effect Effects 0.000 claims description 51
- 238000000034 method Methods 0.000 claims description 30
- 239000002253 acid Substances 0.000 claims description 11
- 210000000988 bone and bone Anatomy 0.000 claims description 9
- 210000004185 liver Anatomy 0.000 claims description 8
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 7
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 7
- 210000000813 small intestine Anatomy 0.000 claims description 7
- 108010031345 placental alkaline phosphatase Proteins 0.000 claims description 6
- 230000005764 inhibitory process Effects 0.000 claims description 5
- MKWKNSIESPFAQN-UHFFFAOYSA-N N-cyclohexyl-2-aminoethanesulfonic acid Chemical compound OS(=O)(=O)CCNC1CCCCC1 MKWKNSIESPFAQN-UHFFFAOYSA-N 0.000 claims description 4
- PJWWRFATQTVXHA-UHFFFAOYSA-N Cyclohexylaminopropanesulfonic acid Chemical compound OS(=O)(=O)CCCNC1CCCCC1 PJWWRFATQTVXHA-UHFFFAOYSA-N 0.000 claims description 2
- 101710170876 Antileukoproteinase Proteins 0.000 description 81
- 101710112538 C-C motif chemokine 27 Proteins 0.000 description 81
- 238000005259 measurement Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 11
- 239000008001 CAPS buffer Substances 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 6
- 239000008000 CHES buffer Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 210000002826 placenta Anatomy 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000000691 measurement method Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- 101000615334 Homo sapiens Antileukoproteinase Proteins 0.000 description 1
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229960005190 phenylalanine Drugs 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000009666 routine test Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】
「産業上の利用分野」
本発明は臨床検査において重要な、アルカリ性
ホスフアターゼ（EC3.1.3.1、以下ALPと略す）
のイソ酵素の一つである胎盤型ALPの測定法に
関する。[Detailed Description of the Invention] "Industrial Application Field" The present invention uses alkaline phosphatase (EC3.1.3.1, hereinafter abbreviated as ALP), which is important in clinical testing.
This paper relates to a method for measuring placental type ALP, which is one of the isoenzymes.
ALPは式(1)に示す反応を触媒する酵素である。 ALP is an enzyme that catalyzes the reaction shown in formula (1).
Ｒ−Ｏ−PO3Ｈ＋H2Ｏ→ROH＋H3PO4 式(1)
（Ｒはアルキル基またはアリル基を示す）
血清中のALPは日常臨床検査として広く行な
われており、肝障害、骨障害などの診断に重要な
測定項目である。血清中には、由来臓器の異なる
ALPイソ酵素が存在し、それらは肝、骨型群、
胎盤型、小腸型、の三種に分類される。そこで、
血清中のALPイソ酵素を分別定量することは、
疾患臓器の診断精度の向上に役立つ。この中で、
胎盤型ALPイソ酵素の測定は妊娠の推移を観察
するのに重要であるばかりでなく、ある種の悪性
腫よう（胎盤型ALPを産出する）の診断にも役
立つ。 R-O-PO 3 H + H 2 O → ROH + H 3 PO 4 Formula (1) (R represents an alkyl group or an allyl group) ALP in serum is widely performed as a routine clinical test, and is associated with liver damage, bone damage, etc. This is an important measurement item for diagnosis. In the serum, there are
There are ALP isoenzymes, which are found in the liver, bone type group,
It is classified into three types: placental type and small intestinal type. Therefore,
Differential determination of ALP isoenzyme in serum is
Helps improve diagnostic accuracy of diseased organs. In this,
Measuring placental-type ALP isoenzyme is not only important for monitoring the progress of pregnancy, but also useful for diagnosing certain malignant tumors (which produce placental-type ALP).
「従来の技術」
血清中のALPイソ酵素の測定は、それぞれの
イソ酵素の蛋白質としての性状の差、酵素化学的
な差などに基づいて分別定量する必要があり、既
に種々の方法が考案されている。その中には、電
気泳動あるいは、分子ふるいクロマトグラフイに
より分離分画し、分画された各イソ酵素を測定す
る方法、熱安定性の差に基づく方法、阻害剤に対
する挙動の差に基づく方法、各イソ酵素に対する
特異抗体を用いる方法などがある。これらの中
で、電気泳動や、分子ふるいクロマトグラフイに
よる方法は、各イソ酵素の一斉分析には適する
が、操作が複雑であるうえに、時間もかかるため
に日常検査としてはあまり適していない。胎盤型
イソ酵素は、他のイソ酵素に比べて熱安定性が高
いために、予め加熱処理して胎盤型イソ酵素を測
定する方法が考案されている。この方法は比較的
操作は簡単であり、しかも分別能も優れている
が、多くの種類の自動分析機では加熱処理ができ
ないために、この方法を自動分析機に適用するこ
とが難しい。またある種のアミノ酸で、ALPは
阻害を受け、その阻害はイソ酵素により異なる。
たとえば、Ｌ−フエニルアラニンは小腸、胎盤型
ALPイソ酵素を、Ｌ−ホモアルギニンは肝−骨
型ALPイソ酵素を阻害する。そこで、これらの
性質をALPイソ酵素の分別測定に応用されてい
るが、特異性に欠ける欠点がある。また、各イソ
酵素の特異抗体を用いる方法は、コストが高くな
るなどの欠点がある。``Prior art'' Measurement of ALP isoenzymes in serum requires separate quantification based on differences in the properties of each isoenzyme as a protein, differences in enzyme chemistry, etc., and various methods have already been devised. ing. Among them, methods include methods of separating and fractionating by electrophoresis or molecular sieve chromatography and measuring each fractionated isoenzyme, methods based on differences in thermostability, and methods based on differences in behavior toward inhibitors. , methods using specific antibodies for each isoenzyme, etc. Among these, methods using electrophoresis and molecular sieve chromatography are suitable for simultaneous analysis of each isoenzyme, but are not very suitable for routine testing because they are complicated and time-consuming. . Since placental isoenzyme has higher thermal stability than other isoenzymes, a method has been devised in which placental isoenzyme is measured by subjecting it to heat treatment in advance. Although this method is relatively easy to operate and has excellent separation performance, it is difficult to apply this method to automatic analyzers because many types of automatic analyzers cannot perform heat treatment. ALP is also inhibited by certain amino acids, and the inhibition varies depending on the isoenzyme.
For example, L-phenylalanine is present in the small intestine and placenta.
L-homoarginine inhibits the liver-bone type ALP isoenzyme. Therefore, these properties have been applied to the fractional measurement of ALP isoenzymes, but they have the drawback of lacking specificity. Furthermore, methods using specific antibodies for each isoenzyme have drawbacks such as high costs.
「発明が解決しようとする問題点」
上記のように従来のALPイソ酵素の測定法は、
操作法の複雑さ、特異性、コストなどの点で問題
がある。そこで、特異性が優れ、操作法も簡単で
しかも自動分析機への応用が容易であり、コスト
も低い日常検査として応用可能なALPイソ酵素
の分別測定法の開発が望まれる。そのような観点
から、本発明者らは胎盤型ALPイソ酵素の測定
法の開発を行なつた。“Problems to be solved by the invention” As mentioned above, the conventional method for measuring ALP isoenzyme is
There are problems with the complexity, specificity, and cost of the operating method. Therefore, it is desired to develop a method for differentially measuring ALP isoenzymes that has excellent specificity, is easy to operate, is easy to apply to automatic analyzers, and can be applied as a low-cost routine test. From this perspective, the present inventors developed a method for measuring placental ALP isozyme.
従来より活性測定に用いる、基質の違い、ある
いは、緩衝液の違いによりALPイソ酵素の間で、
反応性が異なることが知られている。そこで、本
発明者らは、種々の緩衝液によるALPイソ酵素
の活性におよぼす影響を詳細に検索した。その中
で、シクロヘキシルアミノアルキルスルホン酸誘
導体を用いた緩衝液において、胎盤型ALPイソ
酵素は強く阻害を受けるのに対して、その他の
肝、骨、小腸型ALPイソ酵素はあまり阻害を受
けないことを見いだし本発明を完成した。従来の
ALPイソ酵素の活性におよぼす緩衝液の影響の
検討の目的は各イソ酵素の活性におよぼす影響の
少ない緩衝液の検索に向けられていることが多
く、このように、シクロヘキシルアミノアルキル
スルホン酸誘導体が胎盤型ALPイソ酵素を特異
的に阻害することは予知できなかつたことであ
る。 Traditionally, there have been differences between ALP isoenzymes due to differences in substrates or buffers used for activity measurements.
It is known that they have different reactivities. Therefore, the present inventors conducted a detailed search for the effects of various buffer solutions on the activity of ALP isoenzyme. Among them, placenta-type ALP isoenzymes are strongly inhibited in buffer solutions using cyclohexylaminoalkylsulfonic acid derivatives, whereas other liver, bone, and small intestine-type ALP isoenzymes are not significantly inhibited. They discovered this and completed the present invention. Traditional
The purpose of studying the effect of buffers on the activity of ALP isoenzymes is often to find buffers that have less effect on the activity of each isoenzyme, and in this way, cyclohexylaminoalkylsulfonic acid derivatives It was not foreseeable that the placental type ALP isoenzyme would be specifically inhibited.
「問題点を解決するための手段」
すなわち、本発明は、以下の工程からなるシク
ロヘキシルアミノアルキルスルホン酸誘導体によ
る胎盤型アルカリ性ホスフアターゼイソ酵素の活
性阻害による活性測定値の変化に基づく胎盤型ア
ルカリホスフアターゼイソ酵素の分別定量法に関
する。"Means for Solving the Problems" That is, the present invention provides placental alkaline phosphatase isoenzyme activity inhibition based on the change in activity measurement value caused by the activity inhibition of placental alkaline phosphatase isoenzyme by a cyclohexylaminoalkylsulfonic acid derivative consisting of the following steps. This invention relates to a method for differentially quantifying phosphatase isoenzymes.
(A) 胎盤型アルカリ性ホスフアターゼ（胎盤型
ALP）イソ酵素を阻害しない条件下で検体中
のアルカリ性ホスフアターゼ総括性Ｈ及び胎盤
型ALPイソ酵素を阻害する条件下で検体中の
アルカリ性ホスフアターゼ総括性Ｌを求める工
程。(A) Placental-type alkaline phosphatase (placental-type
ALP) Step of determining the total alkaline phosphatase level H in the sample under conditions that do not inhibit the isoenzyme and the total level L of alkaline phosphatase in the sample under conditions that inhibit the placental type ALP isoenzyme.
(B) 胎盤型ALPイソ酵素を阻害しない条件下で
胎盤型ALPイソ酵素の活性ａ及び胎盤型ALP
イソ酵素を阻害する条件下で胎盤型ALPイソ
酵素の活性ｂを求める工程。(B) Activity a of placental-type ALP isoenzyme and placental-type ALP under conditions that do not inhibit placental-type ALP isoenzyme.
A step of determining the activity b of placental type ALP isoenzyme under conditions that inhibit the isoenzyme.
(C) 胎盤型ALPイソ酵素を阻害しない条件下で
胎盤型以外のALPイソ酵素（肝型、骨型、小
腸型）の活性ｃ及び胎盤型ALPイソ酵素を阻
害する条件下で胎盤以外のALPイソ酵素の活
性ｄを求める工程。(C) Activity of non-placental ALP isoenzymes (liver type, bone type, small intestine type) under conditions that do not inhibit placental-type ALP isoenzymes, and non-placental ALP under conditions that inhibit placental-type ALP isoenzymes. Step of determining the activity d of the isoenzyme.
(D) 上記工程(A)〜(C)によつて得られたそれぞれの
値から、下記式を用いて検体中の胎盤型ALP
イソ酵素活性を算出する工程。(D) From each value obtained in steps (A) to (C) above, placenta-type ALP in the sample is calculated using the following formula.
Step of calculating isoenzyme activity.
Ｈ＝Ｙ＋Ｚ
Ｌ＝Ｙ×K1＋Ｚ×K2
（ここでＹは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中の胎盤型ALPイソ
酵素活性、Ｚは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中に含まれる胎盤型以
外のALPイソ酵素の活性の和、K1はｂ／ａ、K2
はｄ／ｃ、を示す。）
以下、本発明を詳細に説明する。H=Y+Z L=Y×K1+Z×K2 (where Y is placental ALP isoenzyme activity in the specimen under conditions that do not inhibit placental ALP isoenzyme activity, and Z is under conditions that do not inhibit placental ALP isoenzyme activity. The sum of the activities of ALP isoenzymes other than the placental type contained in the sample, K1 is b/a, K2
indicates d/c. ) Hereinafter, the present invention will be explained in detail.
本発明によれば、胎盤型ALPイソ酵素に対す
るシクロヘキシルアミノアルキルスルホン酸誘導
体による特異的な阻害作用を利用することによ
り、各種ALPイソ酵素中の胎盤型ALPイソ酵素
を特異的に測定することができる。すなわち、胎
盤型ALPイソ酵素が阻害を受けないような測定
条件およびシクロヘキシルアミノアルキルスルホ
ン酸誘導体による胎盤型ALPイソ酵素を阻害す
る測定条件でALPの総括性を測定し、それらの
測定値と既知量の胎盤型ALPイソ酵素を含む検
体を用いて得られる測定値から胎盤型ALPイソ
酵素の活性が算出できる。 According to the present invention, by utilizing the specific inhibitory effect of cyclohexylaminoalkylsulfonic acid derivatives on placental-type ALP isozymes, placental-type ALP isoenzymes among various ALP isoenzymes can be specifically measured. . That is, the comprehensiveness of ALP is measured under measurement conditions in which placental ALP isoenzyme is not inhibited and in which placental ALP isoenzyme is inhibited by cyclohexylaminoalkylsulfonic acid derivatives, and these measured values and known amounts are compared. The activity of the placental ALP isoenzyme can be calculated from the measured value obtained using a sample containing the placental ALP isoenzyme.
すなわち、
Ｈ＝Ｙ＋Ｚ（式２）
Ｌ＝Ｙ×K1＋Ｚ×K2（式３）
（ここでＹは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中の胎盤型ALPイソ
酵素活性、Ｚは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中に含まれる胎盤型以
外のALPイソ酵素の活性の和、K1は胎盤型
ALPLPイソ酵素を阻害する条件下での胎盤型
ALPイソ酵素の活性／胎盤型ALPイソ酵素を阻
害しない条件下での胎盤型ALPイソ酵素の活性、
K2は胎盤型ALPイソ酵素を阻害する条件下での
胎盤型以外（肝型、骨型、小腸型）のALPイソ
酵素の活性／胎盤型ALPイソ酵素を阻害しない
条件下での胎盤型以外のALPイソ酵素の活性を
示す。）
上記式において、K1及びK2は、胎盤型ALPイ
ソ酵素を阻害しない条件下及び阻害する条件下に
おいて、精製した各ALPイソ酵素を用いて予め
それぞれの活性を測定して求めることができる定
数であり、測定条件に変更が無ければ、１度これ
らを求めておけば良い。そして、式２及び式３よ
り、求めようとする検体中の胎盤型ALPイソ酵
素活性Ｙは、
Ｙ＝（Ｈ×K2−Ｌ）／（K2−K1）（式４）
によつて求めることができる。 That is, H=Y+Z (formula 2) L=Y×K1+Z×K2 (formula 3) (where Y is placental-type ALP isoenzyme activity in the specimen under conditions that do not inhibit placental-type ALP isoenzyme activity, and Z is placenta-type ALP isoenzyme activity. K1 is the sum of the activities of ALP isoenzymes other than placental type contained in the sample under conditions that do not inhibit placental type ALP isoenzyme activity.
Placental type under conditions that inhibit the ALPLP isoenzyme
Activity of ALP isoenzyme/Activity of placenta-type ALP isoenzyme under conditions that do not inhibit placental-type ALP isoenzyme,
K2 is the activity of non-placental type ALP isoenzymes (liver type, bone type, small intestine type) under conditions that inhibit placental type ALP isoenzymes/activation of non-placental type ALP isoenzymes under conditions that do not inhibit placental type ALP isoenzymes. Shows the activity of ALP isoenzyme. ) In the above formula, K1 and K2 are constants that can be determined by measuring the activity of each purified ALP isoenzyme in advance under conditions that do not inhibit placental type ALP isoenzyme and under conditions that inhibit it. Yes, and if there are no changes to the measurement conditions, you only need to obtain these once. From Equations 2 and 3, the placenta-type ALP isoenzyme activity Y in the sample to be determined can be determined by Y = (H x K2-L)/(K2-K1) (Equation 4). can.
シクロヘキシルアミノアルキルスルホン酸誘導
体としては３−シクロヘキシルアミノプロパンス
ルホン酸（以下、CAPSと略す）や２−シクロヘ
キシルアミノエタンスルホン酸（以下、CHESと
略す）が好ましく用いられる。 As the cyclohexylaminoalkylsulfonic acid derivatives, 3-cyclohexylaminopropanesulfonic acid (hereinafter abbreviated as CAPS) and 2-cyclohexylaminoethanesulfonic acid (hereinafter abbreviated as CHES) are preferably used.
胎盤型ALPイソ酵素を阻害しない測定条件と
しては従来からある測定法を用いることができ
る、たとえば、カインド−キング法や、各国臨床
化学会や国際臨床化学会などにより勧告されてい
る測定法により測定できる、また、胎盤型ALP
イソ酵素が阻害を受けないような低濃度のCAPS
やCHES緩衝液で測定することもできる。 Conventional measurement methods can be used as measurement conditions that do not inhibit placental ALP isoenzymes, such as the Kind-King method or measurement methods recommended by national clinical chemistry societies and the International Society of Clinical Chemistry. Possible, also placental type ALP
CAPS at low concentrations so that isoenzymes are not inhibited
It can also be measured using CHES buffer or CHES buffer.
胎盤型ALPイソ酵素を阻害する測定条件とし
ては、胎盤型ALPイソ酵素が充分に阻害される
濃度のCAPSやCHES緩衝液の反応液で測定でき
る。 As measurement conditions for inhibiting placental ALP isoenzyme, measurement can be performed using a reaction solution containing CAPS or CHES buffer at a concentration that sufficiently inhibits placental ALP isoenzyme.
次にCAPSによる各ALPイソ酵素の阻害につい
て、参考例で説明する。 Next, the inhibition of each ALP isoenzyme by CAPS will be explained using reference examples.
参考例
15mM ｐ−ニトロフエニルリン酸、0.5mM塩
化マグネシユーム、1mM塩化亜鉛を含むPH
10.4CAPS−NaOH緩衝液（50mM，100mM，
200mM，300mM，400mM）をあらかじめ30℃
に予加温し、これに精製したヒトALPイソ酵素
を加え、30℃で、410nmの吸光度変化より活性を
求めた。第１図に各ALPイソ酵素（図中、各グ
ラフは１：肝、２：胎盤、３：骨、４：小腸の各
酵素）の各濃度のCAPS緩衝液における活性を
50mMCAPS緩衝液を用いたときの活性を100と
したときの相対活性をしめした。Reference example PH containing 15mM p-nitrophenyl phosphate, 0.5mM magnesium chloride, 1mM zinc chloride
10.4CAPS-NaOH buffer (50mM, 100mM,
200mM, 300mM, 400mM) at 30℃ in advance.
The purified human ALP isoenzyme was added to the mixture, and the activity was determined from the change in absorbance at 410 nm at 30°C. Figure 1 shows the activity of each ALP isoenzyme (1: liver, 2: placenta, 3: bone, 4: small intestine enzyme) in each concentration of CAPS buffer.
The relative activity is shown when the activity when using 50mMCAPS buffer is set as 100.
第１図から明らかなように、１：肝、３：骨、
４：小腸型イソ酵素はCAPSの濃度に影響を受け
ずに一定の活性を示すのに対し、２：胎盤型イソ
酵素はCAPSの濃度が高くなるに従つて活性が低
下し、300mM以上ではほぼ一定の活性をしめす。
また、CHESについてもCAPS同様の検討をおこ
なつたが、同様な結果を得た。 As is clear from Figure 1, 1: Liver, 3: Bone,
4: The small intestine isoenzyme shows a constant activity without being affected by the concentration of CAPS, whereas the activity of the placental isoenzyme decreases as the concentration of CAPS increases, and it almost disappears at 300mM or higher. Shows a certain level of activity.
We also conducted a study on CHES similar to CAPS, and obtained similar results.
「実施例」
以下、実施例により本発明を更に詳細に説明す
る。"Example" The present invention will be described in more detail below with reference to Examples.
操作 １
15mM ｐ−ニトロフエニルリン酸、0.5mM塩
化マグネシユーム、1mM塩化亜鉛を含むPH10.4
50mMCAPS−NaOH緩衝液１mlをあらかじめ30
℃に予加温し、血清検体20μを加え、30℃で、
410nmの吸光度変化より活性を求める。Procedure 1 PH10.4 containing 15mM p-nitrophenyl phosphate, 0.5mM magnesium chloride, 1mM zinc chloride
Add 1 ml of 50mMCAPS-NaOH buffer in advance to 30%
Prewarm to 30°C, add 20μ of serum sample, and incubate at 30°C.
Activity is determined from the change in absorbance at 410 nm.
操作 ２
15mM ｐ−ニトロフエニルリン酸、0.5mM塩
化マグネシユーム、1mM塩化亜鉛を含むPH10.4
400mMCAPS−NaOH緩衝液１mlをあらかじめ
30℃に予加温し、血清検体20μを加え、30℃
で、410nmの吸光度変化より活性を求める。Procedure 2 PH10.4 containing 15mM p-nitrophenyl phosphate, 0.5mM magnesium chloride, 1mM zinc chloride
Add 1 ml of 400mMCAPS-NaOH buffer in advance.
Prewarm to 30℃, add 20μ of serum sample, and incubate at 30℃.
Then, determine the activity from the change in absorbance at 410 nm.
以上の操作１および操作２によつて、検体の
ALPの総括性を測定し、その活性値より次のよ
うにして計算し、胎盤ALPイソ酵素の活性を求
める。 By the above operations 1 and 2, the sample
The overall activity of ALP is measured and the activity value is calculated as follows to determine the activity of placental ALP isoenzyme.
まず始めに、精製した各イソ酵素の既知量を含
む検体をもちいて、操作１および操作２により活
性を測定する。各イソ酵素の操作１による活性を
１としたときの操作２により得られる相対活性
は、胎盤型ALPイソ酵素で0.26（K1に対応）、胎
盤型以外のALPイソ酵素で0.97（K2に対応）とな
る。 First, using a purified sample containing a known amount of each isoenzyme, the activity is measured according to Steps 1 and 2. When the activity obtained in Step 1 for each isoenzyme is set as 1, the relative activity obtained by Step 2 is 0.26 for placental type ALP isoenzyme (corresponding to K1) and 0.97 for non-placental type ALP isoenzyme (corresponding to K2) becomes.
次に、各イソ酵素を含むような検体について行
なつた操作１および操作２よりえられたALPの
総括性をそれぞれＨ，Ｌとするとき、これらの値
を式４に代入することにより、次式から求めよう
とする検体中の胎盤型ALPイソ酵素活性Ｙを求
めることができる。 Next, let H and L be the comprehensiveness of the ALP obtained from Step 1 and Step 2, respectively, for samples containing each isoenzyme, and by substituting these values into Equation 4, the following can be obtained. The placental type ALP isoenzyme activity Y in the specimen to be determined can be determined from the formula.
Ｙ＝（0.97×Ｈ−Ｌ）／（0.97−0.26）
＝（0.97×Ｈ−Ｌ）／0.71
次に、本発明方法の実用性について試験例で説
明する。Y=(0.97×HL)/(0.97−0.26) =(0.97×HL)/0.71 Next, the practicality of the method of the present invention will be explained using test examples.
試験例 １
精製した各イソ酵素を実施例の操作１により測
定し、肝、骨、胎盤、小腸型のイソ酵素の活性が
それぞれ50単位／１になるように混合した検体に
ついて操作１および操作２により活性を測定した
ところ、操作１で200単位／１、操作２で160単
位／１を得た。これらの測定値を上式に代入して
胎盤イソ酵素の活性を求めたところ48単位／１で
あつた。この活性値は、試料調製時に加えた胎盤
イソ酵素の活性（50単位／１）によく近似してお
り、本発明方法の有効性を示している。Test Example 1 Each purified isoenzyme was measured according to Step 1 of Example, and Steps 1 and 2 were carried out on the mixed specimen so that the activity of liver, bone, placenta, and small intestine isoenzymes was 50 units/1, respectively. When the activity was measured, 200 units/1 in operation 1 and 160 units/1 in operation 2 were obtained. When these measured values were substituted into the above formula to determine the activity of placental isoenzyme, it was found to be 48 units/1. This activity value closely approximates the activity of placental isoenzyme (50 units/1) added during sample preparation, demonstrating the effectiveness of the method of the present invention.
試験例 ２
患者血清について、従来よりおこなわれている
熱処理法（血清を65℃、５分間加熱した後、活性
を測定し、加熱処理をしなかつたときの活性と比
較）および実施例による方法で胎盤型ALPを測
定し、全ALP活性に対する胎盤型ALPの活性の
比を比較した第２図のグラフにしめすように、両
方法による測定値は良い相関をしめており、本発
明方法の特異性をしめしている。なお、このグラ
フにおいてＮ＝14，ｒ＝．984746，Ｙ＝4.45951
＋．868449Xである。Test Example 2 Patient serum was tested using the conventional heat treatment method (heating the serum at 65°C for 5 minutes, measuring the activity, and comparing it with the activity without heat treatment) and the method described in the example. As shown in the graph of Figure 2, which compares the ratio of placental ALP activity to total ALP activity after measuring placental type ALP, the measured values obtained by both methods show a good correlation, demonstrating the specificity of the method of the present invention. It's showing. Note that in this graph, N=14, r=. 984746, Y=4.45951
+. It is 868449X.
「発明の効果」
以上から明らかな如く、本発明によれば、ヒト
血清中の胎盤ALPイソ酵素を胎盤イソ酵素を阻
害しない測定条件およびシクロヘキシルアミノア
ルキルスルホン酸誘導体による胎盤イソ酵素を阻
害した測定条件で測定することにより、該酵素の
活性を特異的に求めることができる。本発明方法
は、従来のALP測定における緩衝液をシクロヘ
キシルアミノアルキルスルホン酸誘導体に置き換
えるだけであり、極めて安価に測定が可能でしる
ばかりでなく、自動分析機に容易に適用できるな
ど、日常検査に適した極めて有用な方法である。"Effects of the Invention" As is clear from the above, according to the present invention, the placental ALP isoenzyme in human serum is measured under measurement conditions that do not inhibit the placental isoenzyme, and measurement conditions that inhibit the placental isoenzyme with a cyclohexylaminoalkylsulfonic acid derivative. By measuring this, the activity of the enzyme can be specifically determined. The method of the present invention simply replaces the buffer in conventional ALP measurement with a cyclohexylaminoalkylsulfonic acid derivative, and is not only capable of performing measurements at an extremely low cost, but also can be easily applied to automatic analyzers for routine testing. This is an extremely useful method suitable for
第１図は各ALPイソ酵素活性のCAPS濃度によ
る影響をCAPS濃度50mMのときの活性を100と
したときの相対活性でしめすグラフ図であり、第
２図は熱処理法および本発明の実施例による胎盤
型ALPの測定値の相関関係を全ALP活性に対す
る胎盤型ALPの活性の比（％）でしめすグラフ
図である。
Figure 1 is a graph showing the influence of CAPS concentration on each ALP isoenzyme activity as a relative activity when the activity at a CAPS concentration of 50mM is taken as 100, and Figure 2 is a graph showing the effect of the heat treatment method and the example of the present invention. FIG. 2 is a graph showing the correlation between measured values of placental ALP as a ratio (%) of placental ALP activity to total ALP activity.
Claims (1)
ルキルスルホン酸誘導体による胎盤型アルカリ性
ホスフアターゼイソ酵素の活性阻害による活性測
定値の変化に基づく胎盤型アルカリ性ホスフアタ
ーゼイソ酵素の分別定量法。 (A) 胎盤型アルカリ性ホスフアターゼ（胎盤型
ALP）イソ酵素を阻害しない条件下で検体中
のアルカリ性ホスフアターゼ総括性Ｈ及び胎盤
型ALPイソ酵素を阻害する条件下で検体中の
アルカリ性ホスフアターゼ総括性Ｌを求める工
程。 (B) 胎盤型ALPイソ酵素を阻害しない条件下で
胎盤型ALPイソ酵素の活性ａ及び胎盤型ALP
イソ酵素を阻害する条件下で胎盤型ALPイソ
酵素の活性ｂを求める工程。 (C) 胎盤型ALPイソ酵素を阻害しない条件下で
胎盤型以外のALPイソ酵素（肝型、骨型、小
腸型）の活性ｃ及び胎盤型ALPイソ酵素を阻
害する条件下で胎盤型以外のALPイソ酵素の
活性ｄを求める工程。 (D) 上記工程(A)〜(C)によつて得られたそれぞれの
値から、下記式を用いて検体中の胎盤型ALP
イソ酵素活性を算出する工程。 Ｈ＝Ｙ＋Ｚ Ｌ＝Ｙ×K1＋Ｚ×K2 （ここでＹは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中の胎盤型ALPイソ
酵素活性、Ｚは胎盤型ALPイソ酵素活性を阻害
しない条件下における検体中に含まれる胎盤型以
外のALPイソ酵素の活性の和、K1はｂ／ａ、K2
はｄ／ｃ、を示す。） ２ シクロヘキシルアミノアルキルスルホン酸誘
導体として２−シクロヘキシルアミノエタンスル
ホン酸を用いる特許請求の範囲第１項記載の胎盤
型アルカリ性ホスフアターゼイソ酵素の分別定量
法。 ３ シクロヘキシルアミノアルキルスルホン酸誘
導体として３−シクロヘキシルアミノプロパンス
ルホン酸を用いる特許請求の範囲第１項記載の胎
盤型アルカリ性ホスフアターゼイソ酵素の分別定
量法。[Claims] 1. A method for differentially quantifying placental alkaline phosphatase isoenzyme based on a change in the measured activity value due to activity inhibition of placental alkaline phosphatase isoenzyme by a cyclohexylaminoalkylsulfonic acid derivative, which comprises the following steps: . (A) Placental-type alkaline phosphatase (placental-type
ALP) Step of determining the total alkaline phosphatase level H in the sample under conditions that do not inhibit the isoenzyme and the total level L of alkaline phosphatase in the sample under conditions that inhibit the placental type ALP isoenzyme. (B) Activity a of placental-type ALP isoenzyme and placental-type ALP under conditions that do not inhibit placental-type ALP isoenzyme.
A step of determining the activity b of placental type ALP isoenzyme under conditions that inhibit the isoenzyme. (C) Activity of non-placental ALP isoenzymes (liver type, bone type, small intestine type) under conditions that do not inhibit placental-type ALP isoenzymes, and activity of non-placental-type ALP isoenzymes (c) under conditions that inhibit placental-type ALP isoenzymes. Step of determining the activity d of ALP isoenzyme. (D) From each value obtained in steps (A) to (C) above, placenta-type ALP in the sample is calculated using the following formula.
Step of calculating isoenzyme activity. H=Y+Z L=Y×K1+Z×K2 (where Y is placental ALP isoenzyme activity in the specimen under conditions that do not inhibit placental ALP isoenzyme activity, and Z is under conditions that do not inhibit placental ALP isoenzyme activity. The sum of the activities of ALP isoenzymes other than the placental type contained in the sample, K1 is b/a, K2
indicates d/c. 2. The method for fractional determination of placental alkaline phosphatase isoenzyme according to claim 1, wherein 2-cyclohexylaminoethanesulfonic acid is used as the cyclohexylaminoalkylsulfonic acid derivative. 3. The method for fractional determination of placental alkaline phosphatase isoenzyme according to claim 1, which uses 3-cyclohexylaminopropanesulfonic acid as the cyclohexylaminoalkylsulfonic acid derivative.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18752485A JPS6248400A (en) | 1985-08-28 | 1985-08-28 | Method of fractional determination of placental type alkalie phosphatase isoenzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18752485A JPS6248400A (en) | 1985-08-28 | 1985-08-28 | Method of fractional determination of placental type alkalie phosphatase isoenzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6248400A JPS6248400A (en) | 1987-03-03 |
| JPH0578315B2 true JPH0578315B2 (en) | 1993-10-28 |
Family
ID=16207589
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18752485A Granted JPS6248400A (en) | 1985-08-28 | 1985-08-28 | Method of fractional determination of placental type alkalie phosphatase isoenzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6248400A (en) |
-
1985
- 1985-08-28 JP JP18752485A patent/JPS6248400A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6248400A (en) | 1987-03-03 |
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