JPH03504092A - Methods for predicting and treating arteriosclerosis - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Knowledge and treatment of kinematic sclerosis The present invention focuses on very low density lipoprotein (VLDL) targeting cells of blood vessel walls.

d<1.006g/ml) to treat arteriosclerosis, especially This invention relates to a method for predicting a subject's susceptibility to the development of atherosclerosis. base Essentially, this method uses only established chemical analysis techniques and Difficult biological methods are being avoided. Moreover, the present invention reduces such susceptibility. It also relates to treatment methods.

Cited documents 1 to 41 are for reference in the present invention.

established blood lipoprotein concentrations, atherosclerosis, and According to the correlation with sequelae of coronary heart disease (1,2,3), such It is clear that lipoproteins are closely related to the pathogenesis of the pathological process.

However, despite these correlations, riboprotein quantification has shown that subjects The degree of likelihood of developing such a disease in a patient is not known exactly.

Protective riboprotein [high-density lipoprotein (HDL) (3)] Ragged riboprotein [Very low density lipoprotein (VLDL) (2,6°7.8.9) , and low-density lipoprotein (LDL) (1)] is unfavorable ( 4,5) However, many people do not develop atherosclerosis, and Although the riboprotein ratio is favorable, atherosclerosis develops. There are many people who put it away.

21 known species in 431 patients undergoing cardiac catheterization who developed the disease. Examining risk factors, Holmes et al. (10) found that current risk factors Despite the highly significant correlation between children and the presence of documented coronary artery disease , these clues may not be diagnostically helpful when applied to individual patients. I have come to the conclusion that often.

The anti-injury response hypothesis regarding atherosclerosis dates back 100 years. This is also in line with Virchow's previous research (11).

However, the specific causes and mechanisms of endothelial damage have eluded researchers. It was.

In 1982, an increase in VLDL concentration caused damage to the endothelial cells of the aorta. was shown to be effective in vitro (12.13). Then, and as part of the invention Human serum contains an identifiable substance called toxicity inhibitory active substance (TxPA). and prevent this observed damage caused by elevated VLDL concentrations. It was discovered that (14)

In two types of clinical studies (14, 15), the main so-called protective factors (PFs) An atherogenic index incorporating TxPA as a protective component Patients with clinically proven coronary artery disease cannot be detected using lipoprotein levels alone. It was found that classification can be performed with much higher accuracy (~93%) than previously.

This index, in its most commonly applicable form, is specified in detail below. If TxPA is the protective component of PF and RF is the risk factor, then the quantified RF equals the numerical ratio of the quantified PF to the PF.

Using this atherogenic index called PF/RF, the range from normal to abnormal values can be calculated. Very accurate personal classification is possible using cholesterol concentrations within the range. VL Terms such as DL and VLDLC are used interchangeably here.

The present invention individualizes the likelihood of developing provable coronary artery disease associated with VLDLC toxicity. A method for evaluating individual individuals, in which the PF and PF contained in serum samples of said individuals are evaluated. PF/ calculating a value of RF; and comparing this value with the possibility evaluation criterion. It is defined as a method consisting of:

The main protective factor, TxPA, is an alkaline protein that forms part of the plasma protein fraction of serum. It was identified as a bumin substance, and according to measurements by isoelectric focusing, the isocenter is approximately It is 56. The amount of albumin at the peak with the second isoelectric point of 5.6 is It was determined using the Solgreen binding assay [Doumas m-as, BJ), Watson (W), and Pigges (H, G., Biggs): “Albumin standards and standards using bromcresol green. and the quantitative determination of albumin (Albumin 5 standards and the Measurement of Albumen with Bromcr esol Green) J, Clinica Chemiriki Acta (C1in, Che Acta), Volume 31 (1971), page 87].

This analytical method works well in serum with a fairly high concentration of TxPA at a pH of approximately 5.6. It is noteworthy that the marks are shown very precisely and clearly.

By the way, the therapeutically ineffective part of the albumin substance is mainly at a pH of about 4.8. migrate. Although this analytical method is very suitable for quantifying the concentration of TxPA, Other methods, and combinations thereof, such as adsorption chromatography, polarization chromatography, Matography, electrophoresis, reversed phase chromatography, affinity chromatography (especially by absorption of cibacron blue), with other support media, etc. Electrofocusing, indicator dye binding, ion exchange chromatography, high performance liquids Known methods for blood fractionation, such as chromatography, ligand binding, etc., can also be used with the present invention. are effective to varying degrees in carrying out the method.

RF, or risk factors, are substances known in the industry to contribute to cytotoxicity. This refers to the quality of very low density lipoprotein cholesterol (LDLC). consists of lipoprotein cholesterol, but generally not that of high-density (HDLC) It includes all terol substances and also total glycerides, especially in non-fasting subjects. This includes all persons.

Also, the protective factor, i.e. the molecule of this ratio, can be compared to other It is also possible to augment this with known protective factors, such as HDLC.

Therefore, the atherogenic index expressed as PF/RF is actually a comprehensive It is an index that includes various other ratios within its meaning.

These ratios are, for example, and TxPA/total trigditriglycerides.

Quantitation of HDLC and non-HDLC in serum samples is performed using well-established procedures, e.g. , Alain (C, A, A11ain), Boon ((, S, Poon), and Richmond (W.)'s article [“Total serum cholesterol by enzymes”] Enzymatic Determination of Tot alSerum Cholesterol) J, Clinica Chemiriki Acta , Vol. 31 (1971), p. 87] and Wanick, G.R. rnick) and Albers (J, J. Detailed evaluation of manganese precipitates: method for estimating the amount of high-density riboprotein cholesterol (A Comprehensive Evaluation of Hepar in Manga-nese Precipitation: A pro cedure for estimating high den-city 1ipoprotein cholesterol) J, Journal O Lipid Research (J, Lipid Res, ), Volume 19 (1978) ) using the method described in No. 65 Begeco.

To assess the likelihood of vascular disease in an individual, it is compared to the atherogenic index of that individual. The criteria used range from very high probability to very low probability. Consists of a sufficient range of numerical measures. Numerical data for this standard is very sera from a large number of subjects, ranging from a high probability to a very low probability. Therefore, a new model was created by correlating their vascular status with TxPA. It is.

The steps to create such data are shown below.

Collagenase in pig thoracic aorta [CLSII: Friedrich, New Chassis, USA] - Hold, Worthington Co., Ltd.] for 40 minutes. to obtain endothelial cells (12).

Earl's salts, NaHCOs 2.2g/l, amphotericin B 2.5μg/ ml.

Media containing gentamicin sulfate 50 μg/ml and 17% calf serum. 96 with a flat bottom, filled with 200 μl of the culture solution 199 (16) Human tissue culture cluster plate [Cambridge, Massachusetts, USA; Cells are seeded in a microtube (manufactured by Co5tar).

All tissue culture media and supplements are available in Grand Island, New York, USA. Location: Grand Island Biological Co., Ltd. (Grand 15land B) Obtained from iological Co., Ltd.

The medium is Forma Scientific (Forma 5 scientific) Model 3028 carbon dioxide incubator (Marietta, Ohio, USA) 3 in a high humidity atmosphere (95% relative humidity) containing 5% C in the air. Maintain at 7°C. After 18 hours, the culture medium was washed three times with Medium 199 and sample 2 0 μl, 30 μm of medium 199, and diabetic rat VLDL (17 , 18) ()-liglyceride is 0.90+ag/ml) and [methyl-sH]thi Midin [1,0 Physician lily/ml: Irvine, California, U.S.A. Currently, ICN Radiochemicals )] was dissolved in medium 199. turn into earth

After 3 days, the MASHU TI cell harvester [MASHU Ce1l Harve] Microbiologica, Carrsville, Maryland, USA Microbiological As5ociates .

Cells are collected onto glass fiber filter paper using a filter paper manufactured by INC, Inc.). V The procedure for preparing LDL is as follows.

Practical Rising Star Blood obtained from streptocytosine-induced diabetic rats was incubated at 37°C. Clot for 1 hour, then centrifuge at 300 x g for 10 minutes at 4°C. Prepare serum (supernatant). Next, Beckman L5-75B type and above was added to the serum. Centrifuge [SmithKline-Based, Philadelphia, Pennsylvania, USA] 5W-41 type rotor manufactured by Kline-Beckman (with Kline-Beckman) Centrifuge at 175,000 x g for 18 hours at 4°C using a rotor. trial Using a test tube cutter, separate the VLDL fraction (d<1.006g/ml) and d> Separate the lower supernatant of 1.006. The VLDL fraction was dissolved in 0.15 M cold NaC. 1 and subjected to wash swirl (175,000 x g) at 4°C.

Serum and serum fractions were electrophoresed in 0.8% agarose gels and oil-resolved. The electrophoretic mobility of lipoproteins is measured by staining with Naple (R,P). , Noble): Plasma riboproteins by electrophoresis in agarose gel Electrophoretic separation of pla sma 1ipoproteins in agarose gel) J, Ji Journal of Lipid Research, Volume 9 (1968) 693 pages].

If quantitative analysis of TxPA is required, the sample can be serially analyzed using Medium 199. 1.2.3.4.6.8.12, and obtain a concentration of 16% (50μ l). Next, diabetic rat VLDL and [methyl -IH] Add 50 ml of the mixture with thymidine to each well containing the cells, Culture the cells for an additional 3 days. Cell collection is performed as described above (14).

Toad regression (y=axhSy=cpffl, X=% of sample) best fits the data. It was used to calculate the values of a and b for each sample. Previous training In the study (15), one standard in which the concentration of TxPA was approximately 100 units/ml. Serum is used as a reference and is given a value of 100 units/ml. Then standard times The horizontal deviation of the regression line for each sample from the return line is determined by the serum concentration of 12% of each sample. is calculated using the values of a and b obtained from their individual regression analyses. Ru. This analysis provided a quantitative measure of TxPA in individual samples. .

By isofocus electrophoresis, standard serum with a TxPA concentration of 100 units/ml showed p It was found that the albumin peak of H5 and 6 contained 14 mg/ml of protein. Ta. The following evaluation criteria are derived from this serum analysis.

Cell-based atherogenic index x 14 = direct atherogenesis by albumin raw index 80 1.120 Regarding this criterion, in a clinical population of subjects who underwent experimental angiography, , a value up to approximately 280 indicates that the diameter of the coronary artery cross section has decreased by approximately 15% or more of the normal value. A value above 280 indicates a high probability that the artery is narrowed. , was found to be 15% or less. This data shows that almost 90% of the subjects examined It is noteworthy that it is effective for more than %.

This type of clinical population study is conducted, for example, in the following manner.

A population of subjects was tested using the Judkins method to determine coronary artery blood vessels. The study consisted of 29 boys (age 43±8 years) who underwent contrast imaging. 30″ from the right front Obtain an oblique left ventriculogram and perform multiple selective coronary angiograms.

Subjects undergoing catheterization may have abnormal motor tests, significant arrhythmias, or valvular disease. , or symptoms of left atrioventricular east block.

Angiography data were independently reviewed by two cardiologists blinded to the results of the clinical trial. Inspect and decipher it. Those with stenosis of 15% or less are considered normal; Those with stenosis greater than 5% are classified as having coronary artery disease.

None of the subjects had electrocardiographic evidence of a previous myocardial infarction, and no subjects had angina. Less than 10% were clearly suspected. Any medicine other than hydrochlorothiazide None of the subjects required medication, and none had diabetes.

For fasting serum samples, BMC-Autoflor Coles Terol reagent [Biodynamic, Indianapolis, Indiana, USA] Manufactured by Biodynamics/BMC, catalog number no. ASA-100 dichroic analyzer [No. 148393, Illinois, USA] Sujikago is located at Abot Laboratories. Total serum cholesterol and HDLC were quantified using es). bird Glycerides are Stat-Pak Enzymatic Triglycerides [St at-PckEnzymat, ic Triglyceride: U.S. Caliphate Calbiochem, located in Ugiola, Canton of California. - Behring), catalog number 869262], This was quantified enzymologically.

Hereinafter, the identification and characteristics of TxPA will be described in detail according to the following procedure.

4-place A 1 ml aliquot of serum was added to 4,005 molar HEPES, pH 7. 10%, 20%, or 30% ethanol, 5% or 10% in buffer % dimethyl sulfoxide, 10% glycerin, 10% dextrose, 8% sucrose , 1% N,N-dimethylformamide, or 01% 2-mercaptoethanol Dialyze for 48 hours at room temperature (RT). Next, only medium 199 dialysis is continued for 4 hours. Next, try TxPA as described above. Examine the fee immediately.

In a second experiment, serum was treated with an antioxidant dissolved in 0.005 molar HEPES; - Dialyze against mercaptoethanol and EDTA for 48 hours at room temperature.

Two samples each were then transferred to medium 199 and dialysis continued for an additional 4 hours. . The samples are then immediately assayed for TxPA.

Ahead-1 To characterize the activity of TxPA, (19 ), and (NH,), were tested for the precipitation potential of (19) in SO□. Ta.

Dilute 2 ml of serum with 18 ml of 0.15 molar NaC and add solid TCA to 0.15 molar NaC. Obtain a solution of Instead of this, solid (NH4), SO4 with 0.15 mol Na In addition to 1 ml of serum dissolved in 14 ml of C, add 10 to 40 moles of (NH,)ISO4. Also make a solution. Both precipitation by TCA and precipitation by (NH4), SO, Stir overnight at 4°C and centrifuge at 5,000 x g for 10 minutes at 4°C. . Pere-Soto obtained from precipitation with (NH4), So, 015 mol NaCl 5 ml and resuspended in (NH,), SO, at the same molar concentration as during the initial precipitation. Let it settle.

After centrifugation, precipitation with TCA and a second (NH4)ssOa The pellets obtained from each were redissolved in 0.15 molar NaCl (2 ml and 1ml).

The resulting solution and precipitation with TCA and a second (NH4)2SO4 The supernatant obtained from the precipitation by Roxyethylpiperazine-N-2-ethanesulfonate sodium [HEPES : Sigma V” Chemical Co., St. Louis, Missouri, USA (Mical Co., Ltd.) solution overnight, changing the solution three times.

TCA, (NH,)ffso, and NaC1 were Fisher 5ci-entific Co.: USA New (Fair Lawn, Chassis).

King Nierai Alliance 1 Cohn's fractions of human serum (1), (2), (1), (4), Vl and (19) were Purchased from Guma Chemical Company and dissolved in Medium 199 (30mg/ml) , the activity of TxPA was quantitatively tested.

column chromatography The sample was gelated using a 1.5 x 100 cm column of Sephadex G-100. Apply filtration. Columns, gels, and UV monitors are all manufactured in Sweden. It was obtained from Pharmacia, Inc., Sara. p H7,4 005 molar HEPES buffer, 041 molar NaCl 1,0,4 mmol ethylenediaminetetraacetic acid disodium salt (EDTA: from Fisher), and timed collection by elution with 50 μg/ml gentamicin sulfate. The fraction (approximately 8 m1) was purchased from Buchler (New Chassis, USA). The fraction was collected in a fraction collector located in Fort Lee, State.

In addition, diethylaminoethyl (DEAE) Sephacel column (1.5x 30c Separation of the samples by ion exchange chromatography using m) was also carried out. pH 7,4 0.05 molar HEPES buffer, 0.4 mmol EDTA, and 50 100 ml of a mixture of μg/ml gentamicin sulfate. and dissolved in the same buffer Using a linear concentration gradient of NaCl created with 200 ml of 0.4 M NaCl Elution from the column was performed. DEAE Sephacel column and Sephadec Elution from both sides of the G-100 column was carried out at room temperature by gravity gravity flow. Isoelectric focusing was carried out using a column called LKB8100-2 with a volume of 440+nl. This was carried out at 4°C. 5% to 50% of sweet potato using LKB's gradient forming device. Prepare a linear concentration gradient of sugar (from Sigma). sample and 0.04 molar EDTA Pass through this slope. Pharmacia, LKB (located in Broma, Sweden), or Bio-Rad (located in Suchmond, California, USA) Similar results were obtained using 1% Ampholine obtained from is obtained. The catholyte solution at the tip of the gradient was 0.25M NaOH, and the anolyte solution was 6 0.15 molar HIPO4 dissolved in 0% sucrose (20). Pharmacia Electrophoresis was performed for 18 hours at 30W using a 2000/300 type constant output power supply manufactured by Apply to the sample. Elution was performed by pumping H2O to the top of the column, and absorption at a wavelength of 280 nm was performed. I checked the luminosity. The pH of each fraction was determined by radial immunodiffusion (RID) at room temperature. LC-Partigen RID plate for Bumin and a M-Partigen RID plate for polypoprotein A (both manufactured in California, USA) (manufactured by Calbiochem, Ujora, State), using appropriately selected isoelectric focusing components. I did this for the image.

Yoshi-presentation Stored at -20°C or -70°C for 3 years or at 4°C for 6 months. The concentration of TxPA in the serum was approximately the same as that in the serum recently collected from the same individual. there were. Temperature of serum at room temperature for 2 days or at 60°C for 1 hour does not result in TxP. The concentration of A did not change significantly. At temperatures above 80°C, TxPA was completely destroyed in 3 minutes (data not shown).

Dialysis of serum against deionized water for 48 hours resulted in complete loss of TxPA. . Ethanol, dimethyl sulfoxide, glycerin, HEPES, dextrose sucrose, N,N-dimethylformamide, and 2-mercaptoethanol. Various substances were tested for their ability to protect against such deactivation.

It has been shown that only serum dialyzed against 2-mercaptoethanol maintains its activity. (data not shown). Next, 2-mercaptoethanol, which is a reducing agent, and di- Thiothreitol and the chelating agent EDTA were tested at various concentrations. Ta. By dialysis against either 0.04 molar or 0.04 molar EDTA, A high activity recovery resulted (Table 1). In subsequent experiments, the deactivation For prevention, EDTA at a concentration of 0.4 molar was used.

by established techniques to characterize the activity and as an initial step in purification. Precipitation of TxPA was utilized. TxPA is precipitated in 0.15 molar TCA and sufficient It was reconstituted with an activity recovery rate (85%) (Table 2). More than 30 moles of ammonium sulfate It was also found that Nium has precipitation activity and the activity recovery rate is 46-49%. (Table 2).

A commercially available source of starting material with some enhancement of TxPA activity was found. The Cohn fraction of human serum was tested with the intention of releasing it (Table 3).

Cohn fraction ■ was found to be toxic to endothelial cells and could not be tested.

Cohn fractions n, rv, and ■ did not contain TxPA. cone portion Activity was observed only in images 4 and V. Corn fraction treated for fatty acid removal V contained less TxPA activity than Cohn fraction ■.

To further purify and characterize TxPA, it was added to human serum and Cohn fraction V. , gel filtration and ion exchange chromatography. Toxicity inhibitory activity in serum The characteristics were observed in the 22nd to 32nd fractions eluted from the Sephadex G-100 column. (Figure 1).

TxPA from Cohn fraction V also eluted from a similar location (data not shown).

TxPA activity from Cohn's fraction V is the main component of albumin from DEAE Sephacel. It was observed in the portion eluted at the peak position (Figure 2). Therefore, gel filtration TXPA coelutes with albumin in both ion exchange and ion exchange.

However, in ion exchange, the TxPA peak is similar to the total albumin peak. TxPA is a lower component of albumin or a chroma of these two types. The number of independent components that are purified together with albumin in the column of It is suggested that there is a difference.

To solve this question, we used isoelectric focusing.

It was found that TxPA in Cohn fraction V migrates with pH 5 and 6 as its isoelectric point. (Figure 3). Column fractions were subdivided before and after adjusting their pH to 7.4. Cells were tested for TxPA and similar results were obtained. TXPA activity at pH 5 , 2 or less was never associated with albumin.

The presence of albumin was determined by radial immunodiffusion and the peak containing TXPA. This was measured using the binding of bromocresol green. Both of these methods However, it was found that total albumin and TxPA coincided (Fig. 3).

Three serum samples were collected from individuals with high and low TxPA concentrations. Electrofocusing was performed to determine the relationship between the absorbance in the pH 5 and 6 region and the amount of TxPA. I checked to see if there was.

Two representative examples are shown in FIGS. 4A and 4B. The serum run in Figure 4A is Contains 260 units/nl of TxPA, and has a main protein peak near the isoelectric point 56. This corresponds to the peak of aH-thymidine incorporation. Blood in Figure 4B The liquid has a TxPA of 10 units/m1 or less, and also has a small peak at the isoelectric point of 5.6. There is no detectable aH-thymidine incorporation above baseline.

The volume of serum passed through each column was the same (2 ml) and the column fractions tested were identical. was performed on cultured endothelial cells from a cell pool of Individuals with high concentrations of TxPA The remaining two sera from each of the individuals with low and low levels were also tested, and similar results were obtained.

11 TxPA was found to be extremely stable while present in serum, but dialysis, Alternatively, activity was gradually lost during multiple purification steps. In the refining process, examples are shown in Table 1. chelating agents, such as EDTA, or antioxidants and reducing agents, as indicated; For example, H2 or N, -appropriate charging, dithiothreitol, 2-mercaptoethyl Outperform the Cohn fraction throughout the purification step by including tanol and more The results show that recovery of TxPA activity is possible.

Complete inactivation usually occurs upon excessive purification. Human albumin contains, for example, S It is known that groups such as R are included, and to maximize TxPA activity, , which must be kept in reduced form.

In a number of isolation procedures, such as those described below, TxPA is co-purified with albumin. However, isoelectric focusing can easily separate TxPA. Ru.

TxPA contains 0.15 mol TCA and more than 3.0 mol (NH4) is precipitated in O4, which may also be the conditions that precipitate albumin. It has become clear (19). The majority of TxPA activity is associated with Cohn fraction V However, this fraction usually contains 96-99% albumin. significant amount Cohn fraction 4, which contains TxPA activity, also contains albumin (19 ), and there is a significant absorbance peak in the region of isoelectric point 5°6 ( (data not shown).

Serum or cone using gel filtration or ion exchange chromatography Fractionation of fraction V resulted in a single peak co-eluting with peak albumin. TxPA is obtained. In ion exchange columns, TXPA is more active than total albumin. TxPA also eluted as a much narrower peak (Figure 2), and TxPA A small molecule attached to a subcomponent of albumin, or albumin This suggests that it is either another protein that co-elutes with the protein.

TxPA was separated from bulk albumin by isoelectric focusing (third figure). The absorbance peak at 280 nm at pH 5 and 6 contains all TxPA. It was Album detectable with radial immunodiffusion and bromcresol green amine, and 8H-thymidine incorporation also at pH 5, 6 at a wavelength of 280 nm. It coincided with the absorbance peak. The peak fractions with equal weight of 5.6 were pooled and DEAE When chromatography is performed using a cell, only one protein peak is found. , even the purity of this fraction was demonstrated.

In plasma, albumin has two main isoelectric points, 48 and 56. (25.26) Degreasing operation before electrophoresis or electrophoresis process automation Removal of fatty acids by either gradual delipidation operations in the body lowers the isoelectric point 4 , 8 decreases, and the peak at isoelectric point 5.6 increases (26). Isoelectric point 4. The peak of 8 contains 2.5 to 10 moles of fatty acids per mole of albumin, The peak with an isoelectric point of 5.6 contains O~1.3 moles of fatty acids per mole of albumin. included (26).

The binding of one charge to albumin moves the isoelectric point by OO7 units. (27), the transition from 5.6 to 48 is due to several ionizable atoms. This appears to be due to changes in the tertiary structure that change the pKa of the progeny. did Therefore, toxicity inhibitory activity was identified as a component of albumin with an isoelectric point of 5.6. (Figure 3).

There is a particularly noticeable absorbance peak at 280 nm in serum with a high concentration of TxPA. (Figure 4A). 8H-thymidine uptake in the presence of harmful concentrations of VLDL The amount of TxPAO when measured by inclusion also corresponds to this peak. Ding x Serum from individuals with low concentrations of PA has absorption at a wavelength of 280 nm around pH 5 and 6. Although there is a small peak in luminosity, there is an increase in sH-thymidine above the background level in this region. There were no cases (Figure 4B).

The decisive factor, as determined in clinical studies (14, 15), is that the isoelectric point 6 albumin concentration and VLDLC + LDLCO ratio to HDLC appears to be the maximum concentration of VLDL in the non-fasting state when expressed using child These same components accurately represent the extent of damage in vitro. endothelial cells in vivo Damage is thought to occur during periods of increased concentration of VLDL.

The concentration of VLDL reaches its maximum value several hours after a meal, and this value is higher than the fasting concentration. It is variable (28). The ratio of VLDLC + LDLC/HDLC is VLD There is a correlation with the peak concentration of L (29), and in vivo data can be compared to in vitro data. This may help in interpreting the results. Actually involved in endothelial tissue damage The factors include: VLDL (measured by in vitro cell assay); Albumin has an isoelectric point of 5°6, but empirically, the formula for the atherogenic index is The ratio of VLDLC + LDLC/HDLC is the maximum concentration of LDC. Can be replaced with a value.

LDLC contributes to the main component of VLDLC+ LDLC in individuals with normal fat level and because LDLC and HDLC are relatively constant throughout the circadian cycle. , these can be easily measured to determine an individual's susceptibility to atherosclerosis. It can be used for.

Table 1 Effect of antioxidants on TxPA dithiothreitol 2-Mercaptoethanol EDTA (mmol) O, Q4 60.4 99 ．． 30.4 74.3 100.04.0 0 0 31.2 Table 2 Precipitation of TxPA from serum TxPA unit Control group (no precipitation) 100 (NH4)2SO4 [mol] Trichloroacetic acid [0.15 mol] Sedimentation 85 Supernatant 　　　　　　　0Table 3 TxPA content of corn fraction TxPA unit Total serum (control) 100 Corn V (no fatty acids) 9 corn ■ 0 figure! ”1 plate Figure 1: Use of TxPA from Sephadex G-100 ■ indicates the total content (using blue dextran). 2ml of human serum It was passed through a column and the absorbance at a wavelength of 280 nm was measured (-). Then, the elution fraction ( 20 μm) was incubated with primary cultured porcine aortic endothelial cells for 3 days, during which time the s Incorporation of H-thymidine (see Methods) was carried out in each fraction at pH 7.4. , 0,05 mol HEPES2 ml: dissolved Cohn fraction V (Ig) and 0 4 mmol EDTA was poured into the column and the absorbance at a wavelength of 280 nm was measured (-) . A 20 μm fraction of each fraction was incubated with primary cultured porcine aortic endothelial cells for 3 days. - Cohn fraction (VCIg) in which thymidine incorporation was measured, on a pH 4-6 gradient. Electrophoresis was performed for 18 hours, and the absorbance at a wavelength of 280 nm during elution was measured ( -).

The pH of the fractions was measured at room temperature (-). Using radial immunodiffusion method, each fraction 5μ The albumin of m was quantified (XX). Aliquot each fraction with endothelial cells. The device was used to quantify TxPA, and the incorporation of fin-thymidine was also measured (・ ...).

Figure 4A = 260 ml of TxPA is mistaken for isoelectricity of human blood. Serum (2 ml) was mixed into a pH 4-6 gradient and subjected to 18 hours of electrophoresis. . As shown in Figure 3, the absorbance (-) at a wavelength of 280 nm, pH (-), and 3H- The amount of TxPA (...) was determined as thymidine incorporation.

Figure 4B: Volume 1 of human blood with 10dIIll full TxPA The operating conditions are the same as in FIG. 4A.

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Although the present invention has been described in detail with particular reference to preferred embodiments thereof, the spirit of the invention and It is understood that variations and changes may be made without departing from the scope. To be.

FIGURE 1 FIGURE 2 FIGLIRE 3 + Yamabanmae CPM CPM X IQ-3 (・・・・・・-) International search report

Claims (9)

[Claims] 1. Individualized likelihood of developing provable coronary artery disease associated with VLDLC toxicity A method of evaluating, Determine the respective concentrations of PF and RF components contained in an individual's serum sample. A stage of quantitative measurement and a stage of calculating the value of PF/RF from the concentration of these components and comparing this value with the possibility evaluation criterion. evaluation method. 2. The PF component contains TxPA, and its concentration can be quantified using isoelectric focusing. The evaluation method according to claim 1, wherein the evaluation method is performed by: 3. The TxPA component of PF was kept in a non-oxidizing environment immediately before and during its quantification. The evaluation method according to claim 1, comprising: 4. The environment contains antioxidants, reducing agents, chelating agents, or mixtures of any of these. The evaluation method according to claim 3, wherein the evaluation method includes a compound. 5. The evaluation method according to claim 1, wherein the PF component consists of the total amount of TxPA×HDLC. 6. If the RF component is VLDLC, LDLC, or non-HDLC (LDL C+VLDLC), or total cholesterol, or VLDLC, LDL C, with or without addition of any or all of total cholesterol The evaluation method according to claim 5, comprising the total TG. 7. Protect against the toxic effects of VLDL on human vascular wall cells or A treatment method for alleviating the isoelectric point of approximately 5.6 and a considerable degree providing an albumin substance having TxPA activity of; providing a transport system containing the substance and mixing it into the bloodstream of the individual; floor and The transport system is used to increase the concentration of TxPA in the bloodstream, thereby reducing the atherogenic index. A treatment method characterized by comprising the steps of: causing the number to reach at least about 280. Law. 8. Determine the concentration of each of the PF and RF components contained in an individual's serum sample. Measure quantitatively, calculate the PF/RF ratio from the concentration of these components, and use this value as A claim that monitors and adjusts the concentration of TxPA by comparing it to an evaluation standard. 7. The treatment method described in 7. 9. The delivery system comprises (a) an injectable aqueous solution of an albumin substance; (b) an albumin substance; an orally administrable capsule in the form of an aqueous solution or a solid; (c) an albumin substance; and (d) an aqueous solution or solid of an albumin substance. The treatment method according to claim 7, wherein the treatment method is selected from among the suppositories containing the following.