JP2019174432A - Simple measuring method and measuring device for oxidizable vldl (vldl susceptibility to oxidation) and oxidizable ldl (ldl susceptibility to oxidation) that are judged to have high risk of myocardial infarction and cerebral infarction due to atherosclerosis - Google Patents

Abstract

To provide a method for screening a person who is prone to myocardial infarction or cerebral infarction or a person who is difficult to cause by a simple blood test to determine that the risk of myocardial infarction or cerebral infarction is high because there is a person who cannot prevent the onset of myocardial infarction even if LDL cholesterol level is lowered based on the guideline for the prevention of arteriosclerotic myocardial infarction (2017 edition) of the Japanese Atherosclerosis Society.SOLUTION: In the present invention, the collected serum or plasma is heated as it is, or a sample with or without the addition of a thiol inhibitor is heated to a certain temperature (incubation). Dense LDL cholesterol (sLDLc) is measured, and the results are compared with each other. The increment in the increase is dominant, for example, 1.5 times, or the increase rate is superior, for example, 50% or more. Provided are an indirect oxidizable VLDL or oxidizable LDL measuring method, a measuring reagent, and an apparatus thereof that the risk of myocardial infarction or cerebral infarction due to atherosclerosis is judged to be higher.SELECTED DRAWING: Figure 2

Description

Fields related to simple lipoprotein testing methods and lipoprotein function testing methods that detect risk factors for arteriosclerosis and myocardial infarction, and can be used to prevent and treat myocardial and cerebral infarction.

Lipoproteins present in blood include chilomicron (CM), very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), small particle low density lipoprotein LDL (small dense). LDL, small size LDL, small LDL) and high-density lipoprotein (HDL). Particularly, cholesterol in LDL is well known as bad cholesterol (hereinafter referred to as LDLc). Among them, cholesterol in IDL and small particle LDL or small dense LDL cholesterol (hereinafter referred to as sLDLc) is also called super-bad cholesterol and has been said to be a causative substance of arteriosclerosis and myocardial infarction.

Total cholesterol (TC) and phospholipids are constituents of cell membranes and are raw materials for hormones and in vivo enzymes. Neutral fat (TG) is used as direct energy and is stored in fat cells as fat. It is an essential substance for living organisms.
Since TC, TG, and phospholipid cannot exist in the bloodstream as lipids, they are stored in lipoproteins such as CM, VLDL, and LDL, and are transported to every cell through the bloodstream. Therefore, when individual cells require TC, TG, or phospholipid, or to promote metabolism, the cells secrete lipoprotein lipase (LPL) and hepatic lipase (HTGL) that are anchored on the cell membrane surface. Then, TG in CM, VLDL, IDL, and LDL is hydrolyzed, and apoB100 (apoB100) and apoE that appear are recognized and cells (Cells) immediately via the B / E receptor (apoB / apoE receptor). ) Is taken in. In cells, VLDL or IDL, LDL, and sLDL are decomposed into fatty acids, glycerol (glycerol), cholesterol (cholesterol), etc., and consumed as energy, or accumulated as fat, or enzymes or cell membrane materials or hormones Or reused as a raw material for newly generated VLDL or LDL.

LPL and HTGL are enzymes that are secreted to deliver lipoproteins to cells. CM is mainly responsible for supplying lipids to the liver, and VLDL and LDL are responsible for supplying lipids to various cell groups including mast cells and liver. ing.

Conventionally, it is said that IDL and small particle LDL are causative substances causing myocardial infarction and cerebral infarction due to arteriosclerosis, and it is called super bad LDL or super bad cholesterol, and attention is paid to the measurement of cholesterol and sLDLc in small particle LDL. That is, various diagnostic drug manufacturers have already competed to develop a method for measuring sLDLc (Patent Literature 1, Patent Literature 2, Patent Literature 3, Patent Literature 4, Patent Literature 5, Patent Literature 6). In these measurement methods, small dense LDL and other LDL are fractionated with a special reagent or a surfactant, and then cholesterol in small dense LDL is measured (hereinafter, this method is collectively referred to as sLDLc direct quantification). The law).
In particular, Patent Document 3 is a method for diagnosing familial combined hyperlipidemia (FCHL) if the appearance of sLDLc is 40 mg / dL or more, but in the clinic, sLDLc is often increased temporarily. For example, when heparin is administered at the time of renal dialysis or cardiac catheterization, a large amount of small particles LDL and sLDLc appear temporarily in the blood, so that only the presence or increase of small particles LDL or sLDLc It cannot be diagnosed, and the appearance of small particles LDL and sLDLc itself cannot be determined as a causative substance of myocardial infarction or cerebral infarction.

In liquid chromatography (HPLC), LDL, LDLc, or sLDLc is measured by selecting an lipoprotein as an ion exchange resin or an eluent (Patent Document 7, Patent Document 8, Patent Document 9, and Patent Document 10). ). In particular, Patent Document 9 measures a subclass of LDL (including sLDLc measurement). Patent Document 10 attempts to measure sLDLc, which is a subclass of LDL, by a Gaussian waveform approximation method.

On the other hand, 3% polyacrylamide gel disc electrophoresis (PAGE method) (Non-patent Document 8) was originally said to analyze lipoproteins in the order of particle size (size). There was no report that the particle size was specified. Recently, with the advent of the LDL particle marker (Patent Document 11), the particle diameter can be measured from the LDL relative mobility (RM) and the calibration curve of the particle diameter. In literature 12), IDL, LDL, small particle LDL, small dense LDL or small size LDL and their particle sizes and their respective cholesterols can be measured, though indirectly.

In addition, it is said that the particle size of LDL, the amount of small LDL, and sLDLc can also be measured by an ion mobility spectrometry (IMS method) (non-patent document 2) using gas chromatography.

Other electrophoretic methods that have been used for a long time include, for example, concentration gradient type gradient gel electrophoresis image method (GGE method) (non-patent document 3) and agarose gel electrophoresis method (AGE method) (patent document 13). is there. The GGE method reports that pattern B is a small particle LDL. Moreover, although it describes that the modified | denatured lipoprotein is measured by AGE method, the measurement of small particle | grain LDL or small particle-like LDL may be intended.

Furthermore, the ultracentrifugation method (Non-patent Document 4) and the method of measuring by the dynamic light scattering method (DLS) (Patent Document 14) (Non-patent Document 5) that analyze the difference in the density of LDL particles include small particle LDL, It is supposed that small dense LDL or sLDLc can be measured. Recently, a method (patent document 20) for predicting the risk of myocardial infarction by calculation using a method of measuring the particle size of LDL or small particle LDL with a nuclear magnetic resonance apparatus (non-patent document 10) has appeared.

Recently, screening examinations for personal health management, such as postal examinations, have become widespread. Among them, since the appearance of a simple blood collection device (Patent Document 18) that allows individuals to collect blood at home and separate the serum, the number of measurement items has increased and cholesterol and sLDLc can be measured.

JP 2007-304012 A International Publication Number WO2004 / 1053500 International Publication Number WO2007 / 126099 International Publication Number WO2008 / 050636 JP 2010-148526 A JP 2012-165761 A JP 2001-324488 A JP 2004-2322 A JP 2002-139501 A JP H9-15225 A JP 2013-205411 A JP 2011-123039 A JP 2000-356541 A. JP 2010-48703 A JP 2005-121619 A JP 2004-258014 A JP 2001-192400 A JP 2003-270239 A Special table 2014-516953 gazette International Publication Number WO2005 / 043171

Masatune Ogura, Mika Hori, Mariko Harada-Shiba: Arterioscler Thromb Vas Biol, 181-188, January 2016 Clinical Chemistry 54-8, 1307-1316 (2008) Circulation vol. 82, no. 2, August 495-506, 1990 Clin. Chem. , 23, 2089 (1977). Anals of Clinical Biochemistry, vol 47, September, 476-481, 2010 Journal of Health Sciences, Journal of Health Science, No. 10: 19-24, 2013 Arteriosclerosis, Vol. 23 (7-8), 397-402, 1996 Clin. Chem. , 23, 2089 (1977). Lipid nutrition 8 (1), 11-24, 1999 J Clin Lipidol. Author manusscript; available in PMC 2012 March Japan Medical Newsletter, No. 4527 issued on January 29, 2011, p. 51-5

A simple blood test that can be predicted before the onset of myocardial or cerebral infarction is the most valuable discovery for humanity, but unfortunately there is no such test.
However, the phenomenon that occurs in the arteriosclerotic lesion is largely elucidated, and the oxidation of lipoprotein is the most suspicious. Among them, there are many prior arts that describe that the small particle LDL is unstable and easily oxidized. However, there is no prior art that clearly explains the relationship between the easy oxidation and the appearance of the small particle LDL. In other words, the claim that small particle LDL is a causative agent of myocardial infarction is inferior to FH patients who have few small particle LDL, type II a dyslipidemia, or healthy lipids with a small LDLc value and no small particle LDL. Myocardial infarction due to arteriosclerosis has appeared, and the presence of LDLc and small particle LDL itself was not an explanation for the grounds that can be declared as a direct causative substance of myocardial infarction.
In addition, many patients with cerebral infarction have little or no small particle LDL, and the concentration diagram of PAGE is very similar to the group of healthy lipids with type IIa dyslipidemia or LDLc in the normal range and no small particle LDL. It was.
Therefore, the inventors have continued research on the idea that it is necessary to reinforce the explanation that IDL and small particle LDL are easily oxidized.

About the mechanism by which small particle LDL appears in vivo.
When human cells take in CM or VLDL after meal, the particles immediately after becoming coarse by secreting LPL and HTGL using apo C as an activator of LPL and hydrolyzing TG in CM, VLDL, and LDL particles Recognize Apo B100 and Apo E in the cell, and by using Apo B receptor and Apo E receptor on the cell surface (B / E receptor, sometimes referred to generically as LDL receptor), CM, VLDL, IDL, and LDL are cells. Into.

At this point, almost all of CM (excluding specific diseases), most of VLDL (excluding diseases such as WHO classification V type) and part of LDL disappear. Immediately after hydrolysis, CM is taken up by the liver and other cell B / E receptors and disappears (except for some diseases), and most of VLDL is also extinguished (except for some diseases). Remnants and IDL and, to a lesser extent, small particle LDL or small particle-like LDL appear. (Hereafter, the small particle LDL is described as including a small particle-like LDL.)
However, the TG content ratio in LDL is as low as 10% (Table 1). Most of LDL that has not been taken up by cells remains, and this is the case where the fatty acid of phospholipid (lecithin) is hydrolyzed by phospholipase etc. in the blood In addition, the inventors thought that small particles LDL appeared. There is no interpretation that small-sized LDL appears only when free cholesterol (FC) in LDL is extracted by lecithin cholesterol acyltransferase (LCAT, Non-Patent Document 7) that is attached to HDL.

LDL and small particle LDL are thought to have retained in the blood because they have already lost apo C and cannot easily bind to the LDL receptor.
Note that the appearance of small particle LDL is not only the hydrolysis of TG by LPL or HTGL, but is proved by the dramatic decrease in LDL and small particle LDL when a recently released PCSK9 inhibitor (Patent Document 19) is administered. Is done.

Non-Patent Document 1 explains that the cause of atherosclerosis is the result of oxidization of oxidized LDL by macrophages and collection in the atherosclerotic lesion.
So it is not clear where LDL was oxidized.
Therefore, the inventors believe that VLDL and LDL are oxidized immediately after TG is hydrolyzed by LPL and HTGL, and VLDL and LDL that have not been incorporated into the B / E receptor are oxidized. We decided to call them VLDL (Easily Oxidizable VLDL, VLDL Susceptibility to Oxidation) and LDL (Easily Oxidized LDL, LDL Susceptibility to Oxidation).

In the living body (in vivo), there are many active oxygens that oxidize apo B100 (mainly C-terminal) and apo E in oxidizable VLDL and oxidizable LDL, and circulate in the blood. If it is unfortunately oxidized or acetylated during that time, it is not recognized by the B / E receptor or LCAT (Non-patent Document 7) and is taken up by macrophages, which are a type of white blood cell.
Oxidized or acetylated LDL taken up by macrophages is absorbed into the blood vessel wall, particularly atherosclerotic lesion, and promotes atherosclerosis (Non-patent Document 1). Cerebral infarction occurs when atherosclerosis occurs in the cerebral blood vessel or when a thrombosis or part of cells peeled off from other arteriosclerotic lesions occludes the cerebral artery.
That is, IDL and small particle LDL that appeared in blood were evidence that oxidizable VLDL or oxidizable LDL was present immediately after hydrolysis. Oxidized or acetylated VLDL or LDL may be simply referred to as modified VLDL or modified LDL.

Mechanism of appearance of small particle LDL in vitro.
After the collected blood serum or plasma is heated at 37 ° C. for 2 hours (hereinafter referred to as incubation), the appeared small particle LDL does not directly touch the cells that require lipids, so they are not absorbed by the cells. . In addition, since the content ratio of TG in LDL is about 10% (Table 1), there are too many small particle LDLs to conclude that small particle LDL has appeared just by hydrolysis of TG. .
Therefore, as a result of the activation of phospholipase (including LCAT, which is one type of phospholipase) by VLDL or LDL immediately after being hydrolyzed to hydrolyze phospholipid fatty acid, I discovered that there was nothing but presumed to have appeared.
In other words, small particle LDL that appeared in vitro at 37 ° C. for 2 hours in vitro includes VLDL or FCs in LDL and fatty acids of phospholipids transferred to HDL particles, but the amount of HDL cholesterol increased by incubation Is small (Table 3), and it cannot be said that all small particles LDL have been transferred to HDL by LCAT.

A small particle LDL newly or increased by measuring the sample before and after incubation at 37 ° C. for 2 hours by the measurement method of claim 4 of the present invention results in the presence of oxidizable VLDL and oxidizable LDL. It can be said that it is a proof of having been.

Familial hypercholesterolemia (FH) patients are a group of diseases that are said to be genetically at high risk of developing myocardial infarction. The PAGE disc electrophoretic image of this patient is a type IIa WHO phenotype (IIa type dyslipidemia or hyperlipidemia) that has only a slightly higher TC value than the group of healthy lipids and a low IDL and small particle LDL. It is known to exhibit. Therefore, there has always been a doubt that the appearance of small particle LDL is not the causative agent of myocardial infarction.

The inventors of the present invention used a serum of healthy lipid subjects to incubate the sample after blood collection at 37 ° C. for 2 hours and then migrated by the PAGE method. In most of the samples, small particle LDL did not appear, but FH patients In patients with old myocardial infarction, small LDL appeared or increased.
In vitro, small particle LDL that appeared in specimens of FH patients and old myocardial infarction patients after incubation at 37 ° C. for 2 hours has a considerable amount of residual LPL, HTGL, phospholipase, etc. present in serum or plasma. It is thought that TG and phospholipid in it appeared after hydrolysis. However, after incubation at 37 ° C. for 2 hours, HDL does not increase excessively (Table 3), so it cannot be said that small-particle LDL appeared only by the action of LCAT and HDL. Therefore, the inventors added a thiol group (R-SH) inhibitor (Non-patent Document 6) to specimens of FH patients and old myocardial infarction patients, incubated at 37 ° C. for 2 hours, and then lipoproteins by the PAGE method. As a result of electrophoresis, no new small particle LDL appeared, so it was estimated that phospholipase (including LCAT) was involved in the appearance of small particle LDL.

Based on the above experimental results, after incubating serum samples of healthy lipid subjects with little small LDL and patients with previous myocardial infarction (including patients with FH and old myocardial infarction) at 37 ° C. for 2 hours The study continued with the expectation that by measuring the small particle LDL by the PAGE method, it would be possible to distinguish between those who are prone to atherosclerotic myocardial infarction and cerebral infarction and those who are not.

The inventors have found that small-sized LDL, small dense LDL, or sLDLc, which appear or increase after a short incubation, is very small in quantity, and it has been necessary to develop a method for accurately measuring these.

Serum and plasma (in vitro) obtained by centrifugation after blood collection do not directly contact cell groups and macrophages including blood vessel walls and adipose tissue, so that oxidizable VLDL and oxidizable LDL are taken into each cell. Oxidized or acetylated LDL is not taken up by macrophages.
Therefore, IDL and small particle LDL that appeared or increased after incubating at 37 ° C. for 2 hours in vitro are not only TGs in LDL hydrolyzed by LPL or HTGL but also phospholipid fatty acids hydrolyzed by phospholipase. It can be said that it is missing.
That is, even if the content ratio of TG in LDL is about 8 to 11% (Table 1) and TG in LDL is hydrolyzed by the remaining LPL or HTGL by incubation at 37 ° C. for 2 hours, the amount is as follows: limited. Therefore, it cannot be completely said that all IDL and small particle LDL were produced by LPL and HTGL, and that all small particle LDL appeared because phospholipid fatty acid and FC were extracted by HDL by LCAT (after incubation). This is due to the fact that HDLc has not increased so much (Table 3).
Therefore, it is reasonable to think that LDL has been made into small particles because phospholipase in LDL is activated for some reason, phospholipids are hydrolyzed and small particles LDL appear.

CM of healthy lipids and part of VLDL and LDL are apo B100 and apo E are present in apo C on VLDL and LDL particles that are hydrolyzed by LPL and HTGL secreted by cells in vivo. It is considered that the cells were taken up by the B / E receptor of the cells and immediately disappeared.
Therefore, the healthy lipids originally had no oxidizable VLDL or oxidizable LDL even before LCAT and HDL worked, and even if they remained, the amount was small, which is the effect of LCAT and HDL. It seems that the small particles LDL were oxidized before oxidation, but the small particles LDL could not be detected in an extremely small amount.

In the FH patient group as well, CM and TG in VLDL and LDL are hydrolyzed by LPL, HTGL, etc., as in the healthy lipid group, and oxidizable VLDL and oxidizable LDL immediately become B / E receptors in cells. It is the same that it is captured and disappears immediately. However, in patients with myocardial infarction and FH patients, the B / E receptor function is decreased and LPL and HTGL are also secreted (Table 2). Regardless, it can be considered that oxidizable VLDL and oxidizable LDL remain without being taken up by cells. This can be confirmed by the fact that patients with emergency hospitalization due to myocardial infarction (not limited to FH patients) have more LPL and HTGL before heparin administration than normal (Table 2).

LPL and HTGL are enzymes that are anchored to the cell membrane, and are a group of enzymes that cannot be measured unless heparin is administered. Therefore, as a healthy value of LPL, a measured value after injecting test heparin is generally used. The LPL value at the time of fasting of a healthy lipid person who is not administered heparin is generally 0 to 20 ng / mL, although the healthy value itself is not established. However, as shown in Table 2, LPL measurement values before administration of heparin in patients (not limited to FH and IIa type) who developed myocardial infarction due to atherosclerosis are 2 to 5 normal values, although there are individual differences. It was characteristic that it exceeded twice. This can also be confirmed by the low VLDL value.

Patients with FH or II type a dyslipidemia or patients with myocardial infarction due to atherosclerosis despite TC or TG being in the normal range (part of Table 2) are originally low in CM and VLDL and It is a patient group with few IDL and small particle LDL, LPL and HT
In the LDL particles of patients with myocardial infarction due to (not limited to patients with FH or type IIa dyslipidemia), TG is low. And even if a large amount of heparin is administered, LDL itself does not disappear, so it cannot be explained that small particle LDL has appeared only by the action of LPL or HTGL.

From the above, the small particle LDL of patients with myocardial infarction due to atherosclerosis, such as FH patients and type IIa patients, is in an LPL-excess state although there are individual differences (Table 2). Even if oxidizable LDL is produced on a daily basis, it is immediately caught by the B / E receptor of the cell and disappears. Since it is transferred to HDL according to Patent Document 7), it is considered that small particles LDL that can be detected do not appear.
However, when incubating a test sample (serum or plasma) at 37 ° C. for 2 hours in vitro where there is no cell (receptor) to take up oxidizable VLDL or oxidizable LDL, small particle LDL that does not appear in healthy lipids is obtained. Appearance in FH patients and patients with myocardial infarction due to atherosclerosis (not limited to type IIa) (Table 3), appears to have appeared because phospholipase in LDL hydrolyzed phospholipid fatty acid be able to.

From another perspective, FH patients and patients with myocardial infarction due to atherosclerosis have high LPL and HTGL even on an empty stomach, and the probability that oxidizable VLDL and oxidizable LDL circulate in the blood. Apo B100 (C-terminal) and Apo E are oxidized or acetylated by active oxygen, etc., but are not taken up by the B / E receptor of the cells, but are taken up by macrophages and gradually become arterial walls. It can be considered that the atherosclerosis is accumulated and the myocardial infarction is developed as a result. Oxidized or acetylated VLDL or LDL may be referred to as modified VLDL or modified LDL.

Conversely, type IIb and type IV dyslipidemia groups that are not FH or IIa dyslipidemia are originally high VLDL patient groups, and there are many IDL and small particle LDL due to insulin resistance, etc. It is suspected that there are many oxidizable VLDL and oxidizable LDL.
IDL and small-particle LDL lowering therapy (Non-patent Document 11) adopted as the treatment method are treatment methods that try to eliminate oxidizable VLDL or oxidizable LDL before being oxidized and are logically very It has been proved effective.
That is, the treatment method according to Non-Patent Document 11 prevents in advance that the oxidizable VLDL or the oxidizable LDL undergoes oxidative modification while circulating in the blood and tries not to be taken up by macrophages in advance. It turns out that it was the treatment method to do.
However, this treatment method is also useful for preventing myocardial infarction and cerebral infarction by confirming the appearance or increase of small particle LDL before and after incubation at 37 ° C. for 2 hours according to the present invention.

In vitro, serum of 35 patients with hetero FH group was incubated at 37 ° C. for 2 hours, and each lipid was measured and small particle LDL was analyzed by polyacrylamide gel disc electrophoresis (Patent Document 16) and Gaussian waveform approximation method (Patent Document) When analyzed in 12), there was almost no difference except for the small particle LDL, but only the small particle LDLc showed an almost double increase rate (Table 3). This difference seems to be unique to patients who develop myocardial infarction because there is little appearance or increase in small particle LDL after 37 ° C. incubation in the group of healthy lipids.

From the above results, it can be seen that the appearance of small-particle LDL is clearly different after in vitro test specimens of healthy lipid subjects and patients with atherosclerotic myocardial infarction before incubation at 37 ° C. for 2 hours. It was. In other words, the inventors have incubated serum / plasma in vitro at 37 ° C. for 2 hours, and after incubation, small particle LDL appears, or the dominant increase is, for example, 1.5 times or the dominant increase is, for example, 50% or more. It was discovered that it can be judged that people who are prone to atherosclerotic myocardial infarction.
Know the existence of risk of myocardial infarction and cerebral infarction more accurately by examining not only the presence or appearance of small particle LDL or small particle LDL cholesterol, sLDLc, but also the amount or rate of increase in small particle LDL after incubation be able to.
The risk of myocardial infarction and cerebral infarction according to the test method of the present invention is not limited to an increase in small particle LDL or sLDLc after incubation being 1.5 times or more or an increase rate of 50% or more. And increase rate can be selected in a timely manner. Moreover, when the increase exceeds twice or the increase rate exceeds 100%, it can be determined that the risk of myocardial infarction or cerebral infarction is further increased. In addition, it is necessary to set the cut-off value, which is a judgment criterion for diagnosis, as a risk of myocardial infarction or cerebral infarction for each measurement method according to claim 4 such as the small particle LDL or sLDLc measurement method.

There are roughly two types of patient groups in which small particle LDL appears. One is a small particle LDL (typically TC or TG is a normal value) of a patient group represented by type IIa newly appearing by incubation at 37 ° C. for 2 hours. Small particle LDL, which already has small particle LDL, such as those with myocardial infarction) and IIb and type IV dyslipidemia groups (including diabetic patients), and increases further by incubation at 37 ° C. for 2 hours. It can be roughly classified into a certain group, and this can clearly explain why myocardial infarction has occurred in the group without FH, IIa and small particle LDL.

By the way, the difference in the particle size between healthy LDL and small particle LDL (small dense LDL) is only 1 to 2 nm, and the amount of small particles LDL and sLDLc that appear or increase after incubation at 37 ° C. for 2 hours is about 5 It was about ˜100 mg / dL, and how to specify the small particle LDL and how to measure the amount was a major issue. Currently, a direct quantification method for sLDLc is already sold as a research reagent (Patent Documents 1 to 6), but the sLDLc direct quantification method is a lipoprotein that reacts with a separation agent or a separating agent using a surfactant or the like. As can be seen, the basis of small particle LDL or sLDLc was abstract, and no explanation was given for the group without small particle LDL such as FH. However, even with this sLDLc direct quantification method, it is possible to examine the difference in the amount of increase or increase in the measured value before and after incubation at 37 ° C. for 2 hours (regardless of whether it is small particle LDL).

The PAGE method is said to be able to measure small particle LDL, but the particle size of the small particle LDL that appeared or the amount of sLDLc was unknown in the PAGE method currently used for clinical examination. Therefore, the inventors of the present invention use a PAGE method that is currently used for clinical examinations to detect a lipoprotein specimen (Patent Document 15, Non-Patent Document 8) and obtain a density map from an image. The Gaussian waveform approximation (Patent Document 12) is applied to the concentration map created using the "Method" (Patent Document 16) to create a Gaussian waveform group that divides lipoproteins into 8 types that are approximately 95% identical to the original concentration map. Relative mobility of each waveform center position (peak) (VLDL1 waveform peak is “0”, HDL waveform peak is “1”, and other waveform peaks are expressed as ratios: relative movement Degree: RM) from the cathode, VLDL1, VLDL2, IDL, lage-LDL (L / LDL), medium LDL (M / LDL), small LDL (sLDL), very mall was named LDL (vs · LDL).
However, the types and names of the fractions are not limited to the above, and for example, sLDL and vs · LDL may be integrated and referred to as small particle LDL.

Furthermore, in order to prove the diameter of each LDL particle analyzed by the PAGE method by a physical method, a colloidal gold particle labeled with bovine serum albumin (BSA) and purified by a gel filtration method is used as an analyte. Electrophoresis was performed, gels were extracted from IDL, lage-LDL (L / LDL), medium LDL (M / LDL), and small LDL (sLDL) with relative mobility (RM), and extracted with an electron microscope and light scattering particle size distribution ( The particle size of lipoprotein can be measured by the LDL particle marker (Patent Document 11) that determines the particle size by the RM value from the calibration curve of the particle size and the RM value. LDLc direct quantification of the total area ratio of IDL, L·LDL, M · LDL, sLDL, vs · LDL from a concentration diagram that approximates a Gaussian waveform (Patent Document 12) Cholesterol values of IDL, L·LDL, M · LDL, sLDL, vs · LDL by allocating to LDLc values obtained by LDLc calculation formula (F-LDLc = TC-HDLc-TG / 5) Practical use of the method of calculating The result coincided with the conventional small particle LDL having a particle size of 25.5 nm or less. Here, the small particles LDL = s · LDL + vs · LDL.

The small particle LDL, small particle LDL cholesterol, or small dense LDL cholesterol that have been described so far become a small particle-like state due to an increase in negative charge of the LDL particle, for example, oxidation or acetylation of LDL, due to future technological innovation. The present application contains them, although they may be regarded as modified LDL.

In addition, since fasting LPL, HTGL, or phospholipase was originally low in healthy lipid subjects, there was no appearance of small LDL particles that could be detected when the test sample was incubated at 37 ° C. for about 2 hours. However, patients with myocardial infarction (regardless of FH or IIa type) had new or increased appearance of small particle LDL.
As described above, it was found that a patient with myocardial infarction or a patient with a risk of causing myocardial infarction can be distinguished from a healthy lipid person.
The optimum incubation temperature and time is 37 ° C. for 2 hours, but it can be measured from 30 ° C. to 40 ° C. for 30 minutes to 4 hours due to the measurement sensitivity of the measurement method of claim 4. Alternatively, instead of incubation, it is possible to examine the amount of increase or the rate of increase in small particles of samples stored at room temperature for several days and samples stored refrigerated.

FIG. 1 is a PAGE concentration diagram and biochemical (lipid) data before incubation of an old myocardial infarction patient, and FIG. 2 is a PAGE concentration diagram after incubation at 37 ° C. for 2 hours in the same old myocardial infarction patient. And biochemical (lipid) data. Comparing the biochemical (lipid) data of both, it can be seen that a significant difference appeared only in small particle LDL (small dense LDL, s · LDL + vs · LDL).
When FIG. 1 and FIG. 2 are compared, there was a clear increase in small particle LDL after incubation at 37 ° C. for 2 hours, and the increase rate was more than twice.

Rather than the method of directly incubating the test sample, a certain amount of the collected serum / plasma is divided, and a thiol group inhibitor is added to one of them, and both are incubated at 37 ° C. for 2 hours, and then each of small particle LDL or small particle LDL cholesterol Alternatively, when sLDLc is measured and small particle LDL appears on the side where no thiol group inhibitor is added, it can be determined that the risk of causing myocardial infarction or cerebral infarction is high. It should be noted that the incubation temperature and time are not fixed. For example, even if the specimen is left at room temperature for 2 to 3 days and small particles LDL and sLDLc are measured by the measurement method of claim 4, myocardial infarction or cerebral infarction Judgment of risk is possible.
It should be noted that the task of dividing serum / plasma can be simplified by putting a thiol group inhibitor in a vacuum blood collection tube in advance.

It is known that there are patients with myocardial infarction in the absence of cholesterol ester transfer protein (CETP) despite high HDL cholesterol levels. In this patient, HDL is full of cholesterol esters, but due to lack of CETP, HDL cannot be transferred to VLDL, LDL, or liver, and ester-type cholesterol is saturated in HDL, making it impossible to recover more FC. When the oxidative VLDL or oxidizable LDL remaining in the blood undergoes oxidative degeneration and is taken into macrophages, atherosclerosis develops and myocardial infarction occurs.

WHO type III dyslipidemia is a group of diseases that develop myocardial infarction despite low LDL. This patient is a disease group having apo E2 / 2 genetically and is a disease group in which IDL is not taken into the liver apo E receptor and cannot be transferred to LDL.
That is, a group of diseases in which oxidative VLDL and IDL are oxidized by abnormal accumulation of VLDL and IDL and captured by macrophages, resulting in progression of atherosclerosis and development of myocardial infarction. However, in the type III dyslipidemia group, LDL is originally abnormally low, and small particle LDL may be hardly observed. Therefore, there may be a case where the small particle LDL appearing by incubation at 37 ° C. is small, but if the increase rate before and after the incubation exceeds, for example, twice, the risk determination by the measurement method of the present application is performed in consideration of type III dyslipidemia. It can be carried out. Therefore, there is a need to accurately determine WHO type III dyslipidemia that is easily overlooked.

V-type dyslipidemia is a disease group in which VLDL is abnormally high and there is almost no LDL.
However, when TG in VLDL is hydrolyzed by LPL or HTGL and oxidizable VLDL undergoes degeneration such as oxidation, it is captured by macrophages and accumulates in the arterial wall to cause atherosclerosis. However, in rare cases, type IV patients with poor metabolism may temporarily exhibit type V pathology, and WHO type determination is indispensable.
However, even if LDL and small particle LDL are small as in this case, as in the case of type III dyslipidemia group, if the increase rate before and after incubation exceeds 1.5 times, for example, V type dyslipidemia Considering this, it is possible to perform risk determination by the measurement method of the present application.
However, it can be determined that there is a risk when the increase rate before and after incubation exceeds 2.0 times in the case of V-type dyslipidemia, for example.
Therefore, there is a need to accurately determine WHO type V dyslipidemia that is easily overlooked.

In rare cases, the PAGE method has a fraction with a large LDL particle size (L / LDL fraction, fraction with a small relative mobility of LDL) and few small-sized LDLs appear after incubation at 37 ° C. for 2 hours. I have a patient with myocardial infarction.
It is currently unknown why there is a patient with an LDL fraction that has a slower mobility than the LDL fraction of healthy lipids. However, the risk determination method by the present measurement method is not limited to LPL or the like in serum or plasma after blood collection. The premise is that HTGL and phospholipase remain, and if there is little LPL or the like for some reason, it is an exception to the risk determination method based on the present measurement method.
Therefore, for this type of specimen, testing with a post-meal specimen is also an option.
Even if the amount of small-particle LDL is small, in the case of a sample with low LDL mobility, if the increase rate after incubation exceeds 1.5 times, it is judged that the risk of myocardial infarction is high. can do.
However, since this disease group includes diseases in which atherosclerosis is not directly caused, such as hypertension, coronary spasm, heart failure patients, etc., differentiation from these is necessary.

It is well known that the cause of myocardial infarction cannot be explained simply because of the high LDLc and sLDLc values. That is, it is said that the presence of LDLc and sLDLc is bad just because there are many cholesterol esters in the atherosclerotic lesion, but the basis for this is not clear. The present invention is the first in the world to clarify the mechanism of the appearance of small-sized LDL, predict the presence of indirect but easy-to-modify LDL particles such as oxidation, and treat it as oxidizable VLDL or oxidizable LDL. Named.
The present invention suggested that there are two mechanisms by which small particle LDL appears.
One of them is the group that is essentially free of small particle LDL in serum and plasma, typically represented by type IIa dyslipidemia, and the other in serum and plasma, typically represented by type IIb and type IV dyslipidemia. By distinguishing the group in which small particles LDL are seen and implementing both of the present invention, it is possible to know the possibility of myocardial infarction or cerebral infarction due to atherosclerosis before its onset, for prevention and treatment This is a simple method that can be used.

The Japanese Arteriosclerosis Society sets the LDLc value of field ward as a diagnostic criterion and a lipid management target value in the “Arteriosclerotic Disease Prevention Guidelines 2017 Edition”.
An LDLc of 140 mg / dL or more meets this criterion. However, treatment based on this criterion does not prevent myocardial infarction. In other words, myocardial infarction cannot be prevented if these diagnostic criteria are observed.
The present invention clarifies a part of the mechanism of the onset of myocardial infarction and cerebral infarction, and tries to distinguish between those who are at risk for myocardial infarction and cerebral infarction and those who are not at risk by TC and LDLc. Alternatively, this is an inspection method different from the conventional method in which determination is made based on the amount of sLDLc.
Therefore, the effect of the present invention has the effect of further promoting the prevention of myocardial infarction and cerebral infarction, which have been incomplete until now. For example, in the present invention, for people with a high risk of myocardial infarction or cerebral infarction, administration of oxidizable VLDL and antioxidant of oxidizable LDL, selection of therapeutic agents or development of new therapeutic agents, The development of foods for the purpose of preventing oxidation and the like, the development of immunological therapy for macrophages, etc. has been promoted, and the development of new therapeutic drugs, prevention methods, and treatment methods has progressed, and myocardial infarction and cerebral infarction can be prevented It can be expected to have great effects both socially and economically.

According to Patent Document 18, a system for diluting blood after blood collection with a lancet and separating serum for self-examination and post-examination is put into practical use. Using this system and claim 2 of the present invention makes it possible to easily check the risk of myocardial infarction and cerebral infarction while staying at home.

PAGE concentration measurement and biochemical (lipid) measurement results of small particle LDL before incubation in patients with old myocardial infarction PAGE concentration diagram and biochemical (lipid) measurement result of small particle LDL measurement after incubation of patients with the same myocardial infarction as in FIG.

Examples of measuring methods and measuring devices for oxidizable VLDL and oxidizable LDL that are judged to have a high risk of myocardial infarction and atherosclerosis by simple blood tests are shown below.

As the best example, it is sufficient that oxidizable VLDL and oxidizable LDL can be directly measured, but there is no such measurement method. Therefore, a method capable of measuring small particle LDL, small particle LDL cholesterol, or sLDLc that can be measured without adding anything to serum / plasma collected from a patient and without applying a great deal of physical gravity or pressure is ideal. At present, there is no such ideal measurement method, but the PAGE method is closest to the conditions.

The PAGE method is an orthodox test method in which VLDL, LDL, HDL, etc. are analyzed from a concentration map obtained by adding sudan black, which is a lipid staining solution, to serum / plasma collected from a patient and performing electrophoresis. 8). Although this test method does not directly measure small particle LDL, small particle LDL cholesterol or sLDLc, which are subclasses of LDL, it is a Gaussian waveform approximation method (Patent Document 12) of a concentration diagram made by the inventors. After making it possible to measure the LDL particle diameter and cholesterol amount from the calibration curve of the center position (RM value) of each LDL particle and the particle marker (Patent Document 11), As a result of measuring small particle LDL before and after incubation, there was a clear difference in sLDLc between healthy lipids and patients with old myocardial infarction (FH). FIG. 1 and FIG. 2 are comparisons of concentration diagrams and measured values before and after 37 ° C. incubation in patients with old myocardial infarction (FH). The small particle LDL cholesterol before the incubation was 11.1 mg / dL (FIG. 1 (7)), whereas the small particle LDL cholesterol after the incubation was 25.3 mg / dL (FIG. 2 (11)). As shown above, an increase exceeding 2 times was confirmed. A cutoff value such as sLDLc that increases is suitably 10 mg / dL or more, but is not limited. The dominant increase in small particle LDL after incubation can be determined in a timely manner, but it can also be determined that the risk of myocardial infarction or cerebral infarction is higher when the amount exceeds 2 times that before incubation. In this example (FIG. 2), the increase rate was 2.2 times.

The optimum incubation time was 37 ° C. for 2 hours, but can be determined in a timely range from 30 ° C. to 40 ° C. In addition, the incubation time may be shorter if the measurement sensitivity of small particle LDL cholesterol is obtained.
However, care should be taken because sLDLc also appears in healthy lipids when incubated for a long time.

In Example 1, the concentration value of sLDLc was obtained by the Gaussian waveform approximation method (Patent Document 12). However, as in Patent Document 15, small particles LDL were fractionated with a constant relative mobility (RM value), and small values were obtained. A method of analyzing the appearance of the particle LDL as a difference in fraction% value or an increase rate thereof can also be used qualitatively. The cut-off value can be appropriately selected depending on the sensitivity of measurement.

In agarose gel electrophoresis images (Patent Document 13) and GGE electrophoresis (Non-Patent Document 3), if the difference in measured values before and after incubation is assumed to be the difference in appearance of small particles LDL, fractionation The difference between the% value and the concentration value can confirm that the appearance or increase rate of the small particle LDL is increased, and it can be determined that the risk of myocardial infarction or cerebral infarction is high. Also in this embodiment, the cutoff value can be selected in a timely manner depending on the sensitivity of measurement.

In the HPLC method (Patent Documents 7 to 10), the small particle LDL or sLDLc is measured for the specimens before and after the incubation, respectively. As the small particle LDL or sLDLc appears, it increases predominately, for example, 1.5 times or the rate of increase. For example, when it exceeds 50%, it can be determined that the risk of developing myocardial infarction or cerebral infarction has increased.

The ultracentrifugation method (Non-Patent Document 4), in which small density LDL and particle LDL are individually separated according to the difference in specific gravity of lipoprotein and each cholesterol is measured (Non-Patent Document 4), is also based on a comparison of measured values before and after incubation. Can be regarded as having increased or increased.

Other analysis methods for measuring the particle size of lipoproteins include dynamic light scattering analysis (DLS, Patent Document 14, Non-Patent Document 5), ion mobility spectrometry (Non-Patent Document 2), and a nuclear magnetic resonance apparatus. Also in the method of measuring the particle LDL particle size (Non-patent Document 10) and the method of estimating in vivo small particle LDL (Patent Document 20), small particle LDL, small particle LDL cholesterol or As the appearance, increase, and increase rate of sLDLc increase, it can be captured.

Measuring reagents for sLDLc direct quantification using biochemical automatic analyzers have been released (Patent Documents 1 to 6) (sLDLc direct quantification). This is a method for measuring sLDLc by adding a surfactant, a lipid separation / separation agent, an enzyme reagent, or the like to a test sample.
As a measurement method, by adding several kinds of reagents to the test sample, apart from whether or not the small particle LDL is actually measured, the difference in the measured value before and after the incubation appeared or increased. Alternatively, it can be determined that the risk of myocardial infarction and cerebral infarction has increased because the increase rate was dominant.
However, since this direct quantification method uses an enzyme reagent such as cholesterol oxidase in the final stage of the measurement, it includes a step of incubating the test sample and the reagent at 37 ° C. at the time of measurement. In this measurement process, there is a possibility that LDL has become small particles, but as a test to see the difference in measured values before and after incubation (including the rate of increase), examine the risk of developing myocardial infarction or cerebral infarction. I can. Also in this example, the cutoff value is appropriate.

Although slightly different from the examples of Examples 1 to 7, the test sample was divided into two, and an appropriate amount of a thiol group inhibitor was added to one side, for example, NEM (N-ethylmaleimide) 2 mmol / L (Non-patent Document 6) and both After incubation at 37 ° C. for 2 hours, small particle LDL or small particle LDL cholesterol or sLDLc is measured by the measurement method of claim 4, respectively, and the appearance of more small particle LDL or the sLDLc value when NEM is not added It can also be determined that the risk of developing myocardial infarction or cerebral infarction is high due to the increase in the rate or the rate of increase.
In addition, the following reagent can also be used as a thiol group inhibitor (nonpatent literature 6).
HMCS (N- [6-Maleimidiocaproyloxy] sulfosuccinimide, sodium salt) or GMBS (N-4 [Maleimidobutyryloxy] sulfosuccinimide, sodium salt), EMCS (N- [6-Maleimidocaproyloxy] sulfosuccinimide, sodium salt), SIAB (N-SuccinimidylSulfosuccinimidyl { 4-iodoacetyl succinimidyl aminobenzoate), LC-SPDP (Succinimidyl 6- [3 ′ {2-pyridylylthio}}-propionamide Succinimidyl} -propiona medium] hexanoate).

As described in Patent Document 18, serum or plasma was collected with a blood sampling device for examination containing a diluted solution for obtaining serum or plasma using a simple blood collecting device such as a lancet, and a thiol group inhibitor was previously added to the diluted solution in the simplified blood collecting device. And serum and plasma obtained using a certain amount of preservatives are allowed to stand for 3 days to 1 week at room temperature, and then small particle LDL or sLDLc is measured using the method of claim 4 respectively. Then, the measurement results are compared, and it is confirmed that the small particle LDL is more frequently appearing or increasing or the increasing rate is increased in the measurement result of the sample not containing the thiol inhibitor, or the small particle is increased. It can be determined that there is a risk of myocardial infarction or cerebral infarction by confirming that the measured value of LDL cholesterol or sLDLc is increased or the rate of increase is increased.

The concentration diagram of automated PAGE electrophoresis or HPLC method is equipped with the program of Gaussian waveform approximation method pointed out in claim 3 of claim 4 and the test sample IDL and small particle LDL before and after incubation at 37 ° C for 2 hours And measuring the increase or rate of increase in small particle LDL by an apparatus that automatically measures the amount and rate of increase thereof, or increases the increase rate of small particle LDL cholesterol or It can be determined that there is a risk of myocardial infarction or cerebral infarction by confirming that the measured value of sLDLc is increasing or the rate of increase is increased.
In addition, it does not necessarily specify that the Gaussian waveform approximation method should be used as an apparatus for detecting small particles LDL, and it is detected that small particles LDL have appeared by fractionation with a constant relative mobility (RM value). It is also possible to do.

If the invention of this application is put into practical use, it will be possible to know the presence or absence of the risk with a simple blood test before the onset of myocardial infarction, which has not been known so far. Can be used for drug development.

In 2016, an inhibitor (PCSK9) such as a proprotein convertase subtilisin / kexin type 9 was released as a therapeutic agent that dramatically reduces LDLc (including small particle LDL) (Patent Document 19). PCSK9 inhibitors are therapeutic agents that inhibit enzymes that inhibit the activity of cellular LDL receptors, and are known to have no therapeutic effect in homozygous FH patients who are genetically lacking LDL receptors. Yes. However, in other disease groups, when a PCSK9 inhibitor was administered, LDLc and small particle LDL decreased dramatically, indicating that LDL was taken into cells.
PCSK9 inhibitors can deplete LDL, because it detoxifies enzymes that block receptors that transfer LDL to cells.
However, the use of PCSK9 inhibitors is approved only for patients with high LDLc and a high risk of developing myocardial infarction (except for homo FH patients). Currently, what patients have a high risk of developing myocardial infarction It is not clearly specified. This test method can be used as one of the conditions for administering a PCSK9 inhibitor.

(1) Concentration diagram of test sample (2) Graph of Gaussian waveform approximation (3) Concentration diagram of standard sample (solid line)
(4) Horizontal axis of graph (nm)
(5) Biochemical data of patient before incubation (6) LDL particle size of patient before incubation (7) Small particle LDL cholesterol value before incubation (8) Vertical axis of absorbance (absorbance)
(9) Patient biochemical data after incubation (10) LDL particle size after incubation (11) Small particle LDL cholesterol level after incubation

Claims (5)

Before or after a meal, blood collected from a patient is separated into serum or plasma, then small particle LDL, small particle-like LDL, small particle, small particle-like LDL cholesterol, or small dense LDL cholesterol is measured, and the serum of the same specimen Or plasma for a certain period of time (incubation), confirm the appearance of small particle LDL or small particle-like LDL again, or measure small particle or small particle-like LDL cholesterol or small dense LDL cholesterol. Oxidative VLDL that determines that there is a high risk of myocardial infarction or cerebral infarction due to atherosclerosis when a large number appears and a significant increase amount, for example, 1.5 times or a significant increase rate, for example, 50% or more Simple measurement method for easily oxidizable LDL and measurement test With the measuring device A sample collected from a patient before or after a meal is separated into serum and plasma, and then divided into two, and a predetermined amount of a thiol group inhibitor that inhibits enzyme activity such as lecithin cholesterol acyltransferase is added to one of them in advance or divided into two Without adding a certain amount of thiol group inhibitor in advance, each sample collected using a vacuum blood collection tube and serum or plasma was heated (incubated) to a certain temperature for a certain period of time, or from 3 days After storage at room temperature for about one week, small particle LDL, small particle-like LDL, small particle LDL cholesterol or small dense LDL cholesterol are measured, and the measurement results are compared with each other to measure the sample without thiol group inhibitor. As a result, the appearance of small particle LDL and an example of a significant increase For example, easy measurement of oxidizable VLDL and oxidizable LDL for determining that the risk of myocardial infarction or cerebral infarction due to atherosclerosis is high when the increase rate is 1.5 times or more dominant, for example, 50% or more. Method, measurement reagent and measurement apparatus The temperature at which the test sample is incubated at a constant temperature is 30 ° C to 40 ° C, and the incubation time is about 30 minutes to 4 hours. The myocardial infarction due to atherosclerosis or Easily oxidizable VLDL to determine that the risk of cerebral infarction is high, simple measurement method of oxidizable LDL, measurement reagent and measurement apparatus The method of confirming the presence or absence of the appearance of small particle LDL or small particle-like LDL, the method of measuring small particle LDL cholesterol or small dense LDL cholesterol, or the negative charge increased due to oxidative modification of LDL, etc. The oxidative VLDL and the oxidative oxidation that are judged to have a high risk of myocardial infarction or cerebral infarction due to atherosclerosis according to claims 1 to 3, including one or more of the following including a method for measuring degenerated LDL that has appeared. Convenient measurement method of LDL, measurement reagent and measurement device 1 Polyacrylamide gel disc electrophoresis (PAGE) method 2 High-performance liquid chromatography (HPLC) method 3 Concentration diagrams obtained by PAGE method and HPLC method, etc. Appearance of small particle LDL or small particle LDL with Gaussian waveform approximation Method of measuring lesterol or small dense LDL cholesterol 4 Ion mobility spectroscopy (IMS) method 5 Dynamic light scattering particle size distribution (DLS) method 6 Reagent or cholesterol that precipitates or separates part of lipoprotein Cholesterol Direct Measurement Method that Measures Small Particle LDL or Small Dense LDL Cholesterol Using Reagents for Measured Agarose Gel Electrophoresis (AGE) Method 8 Concentration Gradient Gel Electrophoresis (GGE) Method 9 Ultracentrifugation Method for measuring cholesterol after separating and separating small particle LDL with an apparatus 10 Method for measuring small particle LDL particle size with a nuclear magnetic resonance apparatus Before and after incubation of 30 minutes to 4 hours from 30 ° C. to 40 ° C. with or without serum or plasma as a test sample with or without a fixed amount of a thiol inhibitor added to serum or plasma, small particles LDL or small particles Like LDL small particles or LDL cholesterol or small dense LDL cholesterol, either individually or automatically, before and after incubation or without thiol group inhibitors. Comparison of measured values, appearance of small particle LDL or small particle-like LDL, or appearance of small particle or small particle-like LDL cholesterol or small dense LDL cholesterol after incubation or without thiol group inhibitor Increase It is determined that the risk of myocardial infarction or cerebral infarction due to atherosclerosis according to any one of claims 1 to 4 is detected individually or automatically when the amount is 1.5 times or a significant increase rate, for example, 50% or more. Easy oxidizable VLDL, easy measuring method of easily oxidizable LDL, measuring reagent and measuring device