JPS6139040B2 - - Google Patents
Info
- Publication number
- JPS6139040B2 JPS6139040B2 JP2980378A JP2980378A JPS6139040B2 JP S6139040 B2 JPS6139040 B2 JP S6139040B2 JP 2980378 A JP2980378 A JP 2980378A JP 2980378 A JP2980378 A JP 2980378A JP S6139040 B2 JPS6139040 B2 JP S6139040B2
- Authority
- JP
- Japan
- Prior art keywords
- cholesterol
- serum
- color development
- oxidizing
- free cholesterol
- 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
Links
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 21
- 235000012000 cholesterol Nutrition 0.000 claims description 10
- 210000002966 serum Anatomy 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 238000011161 development Methods 0.000 claims description 5
- 150000001447 alkali salts Chemical class 0.000 claims description 4
- 108090001060 Lipase Proteins 0.000 claims description 3
- 102000004882 Lipase Human genes 0.000 claims description 3
- 239000004367 Lipase Substances 0.000 claims description 3
- 108010055297 Sterol Esterase Proteins 0.000 claims description 3
- 102000000019 Sterol Esterase Human genes 0.000 claims description 3
- 235000019421 lipase Nutrition 0.000 claims description 3
- 108010089254 Cholesterol oxidase Proteins 0.000 claims description 2
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 claims 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims 2
- 230000001590 oxidative effect Effects 0.000 claims 2
- 102000003992 Peroxidases Human genes 0.000 claims 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims 1
- GGCLNOIGPMGLDB-GYKMGIIDSA-N cholest-5-en-3-one Chemical compound C1C=C2CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 GGCLNOIGPMGLDB-GYKMGIIDSA-N 0.000 claims 1
- NYOXRYYXRWJDKP-UHFFFAOYSA-N cholestenone Natural products C1CC2=CC(=O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 NYOXRYYXRWJDKP-UHFFFAOYSA-N 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000011002 quantification Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 239000003518 caustics Substances 0.000 description 1
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 description 1
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
従来、コレステロール定量法には腐蝕性劇物で
ある濃硫酸や無水酢酸等が用いられていた。〔B.
Zak、R.C.Dickenman：Am.J.Clin.Path、24
1307（1954）〕
最近、コレステロールオキシダーゼ及びコレス
テロールエステラーゼが容易に大量に単離製造す
ることが可能になり、“酵素を用いるコレステロ
ール定量法”がその安全性、簡易性等の利点を有
するために用いられるようになつた。
然しこの方法に伴う大きな欠点は恐らく血清に
起因すると思われる混濁の出現による吸光度値の
上昇である。本発明者はコレステロールエステラ
ーゼを含まないリパーゼを作用せしめることによ
りこの混濁の出現を完全に除くことができること
を発見した。（図１参照）
さらにアルカリ塩を反応液中に添加することに
より発色速度を顕著に増大する事実を知つた。
（図２参照）
従つて本法を用いることにより、血清ブランク
値の測定を必要とせず、然も正確且精密な遊離コ
レステロール定量を行いうるに至つた。（図３参
照）。
アルカリ塩の濃度は図４にみられるが如く、大
なる程、発色速度を大とするが実用上、0.45M程
度の濃度とすれば充分である。
本発明を以下の実施例によつて説明するが、し
かしこれによつて本発明が限定されるものではな
い。
実施例 １
遊離コレステロールの定量
(1)試薬
Conventionally, corrosive substances such as concentrated sulfuric acid and acetic anhydride have been used in cholesterol quantitative methods. [B.
Zak, RCDickenman: Am.J.Clin.Path, 24
1307 (1954)] Recently, it has become possible to easily isolate and produce cholesterol oxidase and cholesterol esterase in large quantities, and the "enzyme-based cholesterol determination method" has been used because of its advantages such as safety and simplicity. I started to be able to do it. However, a major drawback with this method is the increase in absorbance values due to the appearance of turbidity, probably due to serum. The present inventor has discovered that the appearance of turbidity can be completely eliminated by using a lipase that does not contain cholesterol esterase. (See Figure 1) Furthermore, we have found that the color development rate can be significantly increased by adding an alkali salt to the reaction solution.
(See Figure 2) Therefore, by using this method, it has become possible to perform accurate and precise quantification of free cholesterol without requiring the measurement of serum blank values. (See Figure 3). As shown in FIG. 4, the higher the concentration of the alkali salt, the faster the color development rate will be, but for practical purposes, a concentration of about 0.45M is sufficient. The present invention will be illustrated by the following examples, but the invention is not limited thereto. Example 1 Quantification of free cholesterol (1) Reagent
【表】
上記試薬と精製水100mlに溶解し、測定試
液とする。
コレステロール標準血清
(2) 操作法
試験管に血清0.05ml又はコレステロール標準
血清0.05mlをとり、次いで測定試液3.0mlを加
えて37℃10分間放置後、試薬ブランクを対照と
して、500nｍにて各々の吸光度を測定する。
(3) 遊離コレステロール含量の求め方
遊離コレステロール含量（mg/dl）
＝検体の吸光度／標準血清の吸光度×標準血清の表
示値[Table] Dissolve the above reagent in 100 ml of purified water and use it as a measurement reagent. Cholesterol standard serum (2) Procedure: Take 0.05 ml of serum or 0.05 ml of cholesterol standard serum in a test tube, then add 3.0 ml of measurement reagent and leave it at 37℃ for 10 minutes. Using the reagent blank as a control, measure the absorbance of each at 500 nm. Measure. (3) How to determine free cholesterol content Free cholesterol content (mg/dl) = Absorbance of sample / Absorbance of standard serum x Displayed value of standard serum
図１はリパーゼ添加によるにごりの消失効果
図２はアルカリ塩添加による発色速度の増大 図
３は従来法であるジギトニン法と本発明法との相
関 図４は塩化ナトリウム（Nacl）添加による
発色速度の増大、を示す図である。
Figure 1 shows the effect of eliminating cloudiness by adding lipase.
Figure 2 shows the increase in color development rate due to the addition of an alkali salt. Figure 3 shows the correlation between the conventional digitonin method and the method of the present invention. Figure 4 shows the increase in color development rate due to the addition of sodium chloride (NaCl).
Claims (1)
ルオキシダーゼの作用によりコレステノンに酸化
し、同時に生ずる過酸化水素をパーオキシダーゼ
の存在下で、４−アミノアンチピリンとフエノー
ルとで酸化縮合せしめて生ずる赤色キノンを測定
する方法において、血清の混濁を消失するために
コレステロールエステラーゼを含まないリパーゼ
を用いると共に、アルカリ塩の添加により発色速
度を増大ならしめることを特徴とする遊離コレス
テロール定量法。1 A method of measuring red quinone produced by oxidizing free cholesterol in serum to cholestenone by the action of cholesterol oxidase, and oxidizing and condensing the hydrogen peroxide produced simultaneously with 4-aminoantipyrine and phenol in the presence of peroxidase. A method for quantifying free cholesterol, characterized in that a lipase not containing cholesterol esterase is used to eliminate turbidity in serum, and the rate of color development is increased by adding an alkali salt.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2980378A JPS54123095A (en) | 1978-03-17 | 1978-03-17 | Method and reagent for measuring quantity of cholesterol |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2980378A JPS54123095A (en) | 1978-03-17 | 1978-03-17 | Method and reagent for measuring quantity of cholesterol |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS54123095A JPS54123095A (en) | 1979-09-25 |
| JPS6139040B2 true JPS6139040B2 (en) | 1986-09-02 |
Family
ID=12286165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2980378A Granted JPS54123095A (en) | 1978-03-17 | 1978-03-17 | Method and reagent for measuring quantity of cholesterol |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS54123095A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0464327U (en) * | 1990-10-16 | 1992-06-02 |
-
1978
- 1978-03-17 JP JP2980378A patent/JPS54123095A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0464327U (en) * | 1990-10-16 | 1992-06-02 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS54123095A (en) | 1979-09-25 |
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