JP2010148526A - Quantative method for cholesterol in low density lipoprotein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method avoiding the influence of high TG (triglyceride) in a sample in a method eliminating cholesterol in lipoprotein excluding LDL in a first process and quantifying the LDL cholesterol in a second process. <P>SOLUTION: The quantative method for cholesterol in low density lipoprotein in a test sample comprises a first process of eliminating the cholesterol in lipoprotein excluding low density lipoprotein in the test sample and a second process of determining residual cholesterol in the test sample, wherein the first process is carried out in the presence of albumin. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for quantifying cholesterol in low-density lipoprotein in a test sample.

  Cholesterol in low-density lipoprotein (hereinafter referred to as “LDL cholesterol”) plays a major role in cholesterol transport in the blood, and particularly cholesterol deposited on the blood vessel wall in atherosclerosis is mainly derived from LDL. Increased LDL is one of the main risk factors for arteriosclerotic diseases, and differential quantification is clinically useful.

  Conventional methods for quantifying LDL cholesterol include a method obtained from two operations of fractionation and cholesterol quantification, and a calculation method based on the Friedewald equation obtained from total cholesterol, HDL cholesterol, and triglyceride values.

  Fractionation methods include ultracentrifugation, precipitation, immunization, etc., but the sample must be centrifuged or filtered, making it difficult to spread in clinical laboratories in terms of convenience and economy. . Also, the calculation method based on the Friedewald equation is limited in its use, and there is a problem in accuracy because it does not take into account individual differences.

  However, an LDL cholesterol quantification method (Publication No. JP-A-11-318496) that does not require a fractionation operation has recently been reported, and is currently being applied as a clinical test reagent in the field of testing. In this method, cholesterol in lipoproteins other than LDL in the sample is selectively eliminated in the first step (deletion is the degradation of ester-type cholesterol and free cholesterol, so that the degradation product is not detected in the second step. In the second step, LDL cholesterol is quantified.

  However, special clinical specimens have a problem in that a negative measurement error occurs when the triglyceride (TG) value shows an abnormally high value. Therefore, avoiding this problem increases the usefulness as a measurement method, and the method has been desired.

JP 11-318496

  It is an object of the present invention to provide a method for avoiding the effects of high TG in a sample in a measurement method for eliminating cholesterol in lipoproteins other than LDL in the first step and quantifying LDL cholesterol in the second step. .

  As a result of investigating the cause of the negative measurement error, the present inventors have found out that cholesterol in LDL that should not be erased starts to be erased if a high concentration of TG is present in the sample at the time of elimination in the first step. It was. Furthermore, the presence of a high concentration of TG means that there are many lipoproteins such as chylomicron (CM) and very low density lipoprotein (VLDL) containing a lot of TG, and these induce the elimination of LDL cholesterol. (These lipoproteins including LDL form a particle system as an aggregate of cholesterol, TG, phospholipid, and protein, and each lipoprotein has a difference in protein type, cholesterol, and TG amount. ).

Specifically, if CM and VLDL are present in the sample, CM,
Cholesterol in VLDL is decomposed and eliminated, but debris such as other proteins and phospholipids remain. Therefore, as the concentration of CM and VLDL in the sample increases, the concentration of these debris also increases, and adsorption of some debris to LDL begins to occur. It has become clear that the adsorbed LDL is recognized as a lipoprotein other than LDL and starts to disappear.

  Therefore, as a result of examining a method for suppressing the adsorption of the debris to LDL, by adding albumin during the first step, the debris was adsorbed to albumin and the influence on LDL could be avoided.

  That is, the present invention comprises a first step of eliminating cholesterol in lipoproteins other than low density lipoprotein in a test sample, and then a second step of quantifying residual cholesterol in the test sample. In the method for quantifying cholesterol in a low-density lipoprotein in a test sample, the method comprises performing the first step in the presence of albumin.

  According to the method of the present invention, by adding albumin, the effect of other lipoproteins can be avoided, and the effect of being able to quantify cholesterol in LDL with higher accuracy than the conventional method is achieved.

  Cholesterol contained in lipoprotein includes ester-type cholesterol (cholesterol ester) and free-type cholesterol. In the present specification, the term “cholesterol” includes both of them.

  The test sample used in the method of the present invention may be any sample as long as it may contain lipoproteins such as HDL, LDL, VLDL, and CM, for example, body fluids such as blood, serum, and plasma. However, it is not limited to these.

  The method of the present invention comprises a first step and a second step. In the first step, cholesterol in HDL, VLDL and CM in the test sample is eliminated, and in the subsequent second step, residual cholesterol in the test sample is erased. Quantify. Since cholesterol in HDL, VLDL and CM is erased in the first step, cholesterol quantified in the second step is mainly cholesterol in LDL in the test sample.

  “Elimination” in the first step means degrading cholesterol and preventing the degradation product from being detected in the next second step. Examples of a method for selectively eliminating cholesterol contained in lipoproteins other than LDL, that is, HDL, VLDL, CM, and the like include the following methods.

  That is, cholesterol esterase and cholesterol oxidase are allowed to act in the presence of a surfactant that acts on lipoproteins other than low-density lipoprotein, thereby eliminating the generated hydrogen peroxide.

  As a method of eliminating hydrogen peroxide, catalase is used to decompose it into water and oxygen, and peroxidase is used to react phenolic or aniline hydrogen donor compound with hydrogen peroxide to convert it into colorless quinone. Although a method can be mentioned, it is not limited to these.

  The cholesterol esterase concentration in the reaction solution in the first step is preferably about 0.2 to 1.0 U / mL, and as the origin, those produced from Pseudomonas bacteria are effective. The concentration of cholesterol oxidase is preferably about 0.1 to 0.7 U / mL, and those derived from bacteria or yeast are preferably used. Furthermore, the concentration of catalase is preferably about 40 to 100 U / mL. In addition, the concentration of peroxidase when converting hydrogen peroxide to colorless quinone is preferably 0.4 to 1.0 U / mL, and the concentration of the phenolic or aniline hydrogen donor compound is 0.4 to 0.8 mmol / L is preferred.

  Preferable examples of surfactants that act on lipoproteins other than LDL used in the first step include polyalkylene oxide derivatives having an HLB value of 13 to 15, preferably 13 to 14. Examples of the derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercaptan condensates, and alkylphenol condensates. The method for calculating the HLB of the surfactant is well known, and is described in, for example, “New Surfactant”, Hiroshi Horiuchi, 1986, Sankyo Publishing.

  Preferable specific examples of polyalkylene oxide derivatives having an HLB value of 13 to 15 include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octyl phenyl ether. Examples thereof include compounds having an HLB value of 13 or more and 15 or less, such as polyoxyethylene nonylphenyl ether, but are not limited thereto.

  The concentration of the surfactant used in the first step is preferably about 0.1 to 10 g / l, more preferably about 0.5 to 5.0 g / l.

  The first step is preferably performed in a buffer solution having a pH of 5 to 9, and the buffer solution is preferably a buffer solution containing an amine such as Tris, triethanolamine or Gut's buffer solution. In particular, Bis-Tris, PIPES, MOPSO, BES, HEPES and POPSO which are good buffer solutions are preferable, and the concentration of the buffer solution is preferably about 10 to 500 mM.

  In the first step, a divalent metal ion may be included in the reaction solution in order to suppress the reaction with LDL and further enhance elimination of other lipoproteins. Copper ions, iron ions, and magnesium ions can be used as the divalent metal ions, but magnesium ions are particularly preferable. The concentration of the divalent metal ion is preferably about 5 to 200 mM.

  In addition, lipoproteinase can be optionally added to the reaction solution in the first step. It is preferable to add this enzyme because cholesterol in VLDL is particularly easily reacted. The concentration of the enzyme in the reaction solution is preferably about 5.0 to 10.0 U / mL.

  The reaction temperature in the first step is suitably about 30-40 ° C, most preferably 37 ° C. The reaction time may be about 2 to 10 minutes.

  In the method of the present invention, the first step is performed in the presence of albumin. The albumin is not limited as long as it is albumin, and commercially available albumin such as serum albumin can be suitably used. The origin of albumin is not limited in any way, and it may be any animal such as human, bovine, porcine or equine. In particular, widely used bovine serum albumin can be suitably used. The concentration of the albumin in the reaction solution in the first step is preferably 0.1 to 5.0 g / dL, and more preferably 0.3 to 3.0 g / dL.

  In the subsequent second step, the residual cholesterol in the test sample is quantified. This can be performed, for example, by adding at least a surfactant that acts on LDL and quantifying hydrogen peroxide generated by the action of cholesterol esterase and cholesterol oxidase added in the first step. Here, the surfactant that acts at least on LDL may be a surfactant that acts selectively only on LDL, or a surfactant that acts on all lipoproteins.

  Preferable examples of surfactants that act on all lipoproteins include polyalkylene oxide derivatives having an HLB value of 11 or more and less than 13, preferably 12 or more and less than 13. Examples of the derivatives include higher alcohol condensates, higher fatty acid condensates, higher fatty acid amide condensates, higher alkylamine condensates, higher alkyl mercaptan condensates, and alkylphenol condensates.

  Preferable specific examples of the polyalkylene oxide derivative having an HLB value of 11 or more and less than 13 include polyoxyethylene lauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene higher alcohol ether, polyoxyethylene octyl phenyl ether, Examples of the compound having an HLB value of 11 or more and less than 13 such as polyoxyethylene nonylphenyl ether are not limited thereto.

  Moreover, an anionic surfactant can be mentioned as surfactant which acts selectively only on LDL. Although it does not specifically limit as an anionic surfactant used here, The thing which has a C4-C18 linear or branched alkyl group couple | bonded with the aromatic ring is preferable. Here, the aromatic ring is preferably composed of only carbon and hydrogen, such as benzene, naphthalene, diphenyl and the like. Further, those in which a hydrophilic group such as a sulfonate is bonded to the aromatic ring are preferable. Examples of such preferable anionic surfactants are shown in the following formulas (I) to (V).

However, in Formula (I)-(V), R shows a C4-C18 linear or branched alkyl group. Moreover, higher alcohol sodium sulfate etc. can be mentioned as a preferable anionic surfactant used at a 2nd process.
The concentration of the surfactant used in the second step is preferably about 0.1 to 100 g / l, more preferably about 1 to 50 g / l.

  Other preferable reaction conditions in the second step are the same as the preferable reaction conditions in the first step.

  The present invention will be specifically described below based on examples of the present invention. However, the present invention is not limited to the following examples.

(reagent)
An LDL cholesterol quantification reagent having the following composition was prepared. Furthermore, a reagent prepared by adding 1.0 g / dL of bovine serum-derived albumin to the first reagent was prepared, and the effect was confirmed using two kinds of reagents (the second reagent was commonly used).

First reagent
PIPES buffer pH7.0 50 mmol / L
HDAOS 0.7 mmol / L
Cholesterol esterase 0.8 U / mL
Cholesterol oxidase 0.5 U / mL
Catalase 80 U / mL
Magnesium chloride 10 mmol / L
Surfactant Emulgen B66 (Kao Corporation) 0.2%

Second reagent
PIPES buffer pH7.0 50 mmol / L
4-Aminoantipyrine 4.0 mmol / L
Peroxidase 2.4 units / mL
Sodium azide 0.1%
TritonX100 3.0%

(sample)
Six types of CM samples were prepared, and each of the six types of samples had the same LDL cholesterol concentration, and only the CM concentration was changed (the CM concentration difference is shown as a TG value). Six types of VLDL samples were prepared in the same manner as the CM samples.

(Measuring method)
300 μL of the first reagent preheated to 37 ° C. was mixed with 4 μL of each sample and reacted at 37 ° C. for 5 minutes, and then 100 μL of the second reagent was added and reacted for 5 minutes, and the absorbance at 600 nm was measured. The LDL cholesterol concentration was calculated by comparing the measured absorbance with the absorbance of a known concentration sample measured in advance.

(Unit: mg / dL)

  As shown in Table 1, it can be seen that the reagent added with albumin does not decrease the measured value even when the CM concentration increases, and avoids the influence.

(Unit: mg / dL)

  For VLDL in Table 2, the effect of albumin can be sufficiently confirmed as in CM.

Claims (8)

  A low density in the test sample comprising a first step of eliminating cholesterol in lipoproteins other than the low density lipoprotein in the test sample, and then a second step of quantifying residual cholesterol in the test sample In the method for quantifying cholesterol in lipoprotein, the first step is carried out in the presence of albumin.   The method according to claim 1, wherein the albumin concentration in the reaction solution of the first step is 0.1 to 5.0 g / dL.   The method according to claim 1 or 2, wherein the reaction in the first step is performed at a pH of 5 to 9 and a temperature of 30 to 40 ° C.   The said 1st process consists of making cholesterol esterase and cholesterol oxidase act in the presence of surfactant which acts on lipoproteins other than low density lipoprotein, and erase | eliminates the generated hydrogen peroxide. the method of.   The second step comprises adding a surfactant that acts on at least a low-density lipoprotein to the product of the first step, and quantifying hydrogen peroxide generated by the action of the cholesterol esterase and cholesterol oxidase. Item 5. The method according to Item 4.   The method according to claim 5, wherein the surfactant that acts on at least the low-density lipoprotein acts on all lipoproteins.   The surfactant which acts on lipoproteins other than the low density lipoprotein used in the first step is a polyalkylene oxide derivative having an HLB value of 13 or more and 15 or less. The method described.   The method according to claim 5, wherein the surfactant that acts on all lipoproteins used in the second step is a polyalkylene oxide derivative having an HLB value of 11 or more and less than 13.