JP6548115B2 - Arteriosclerosis marker and use thereof - Google Patents

Description

  The present specification relates to a marker for arteriosclerosis and its use.

  It is known that excessive production of reactive oxygen in the living body leads to the onset and progress of diseases by damage to in vivo molecules such as lipids and proteins. Among such disorders, non-enzymatic modification of proteins with electrophilic small molecules generated in vivo reflects the in vivo state and condition and can be regarded as a biomarker for diseases.

  One example of non-enzymatic modification is HbA1c, which is known as a glycated modification of hemoglobin (Hb), which is the major protein in red blood cells occupying about 45% of the blood volume. Hemoglobin and glucose are bonded by non-enzymatically attacking a carbon of aldehyde group of glucose non-enzymatically by non-covalent electron pair possessed by nitrogen of amino group of hemoglobin by nucleophilic attack. Among these bound substances, hemoglobin A1c is a combination of valine at the N-terminal end of hemoglobin and glucose. A1c is stable and accounts for a large proportion of glycated hemoglobin.

Hemoglobin A _1c is thus produced nonenzymatically, and the ratio to hemoglobin depends on the blood glucose concentration (blood glucose state), and the life is relatively long, about 120 days. For this reason, it is used as a marker of diabetes and an indicator of glycemic control in the treatment of diabetes as a thing which well reflects the glycemic state.

  On the other hand, oxidized fatty acids produced under oxidative stress or inflammatory conditions are known to react with proteins to form a wide variety of adducts. A method for exhaustively analyzing such adducts has been reported (Non-patent Document 1).

Takahiro Shibata et al., Proceedings of the 2014 Annual Conference of the Japanese Society of Agricultural Chemistry 3B03a11

  So far, no comprehensive analysis has been conducted on modified forms of erythrocyte proteins such as hemoglobin. For this reason, little was known about modifiers other than glycation of hemoglobin.

  The present specification provides a novel diagnostic marker for arteriosclerosis and its use and a screening method of the diagnostic marker.

  The present inventors focused on red blood cells. Erythrocytes consist mainly of hemoglobin (Hb) to which most of the oxygen molecules present in the body are bound, and this binding is activated in the presence of iron ions that can also catalyze lipid peroxidation, etc. This is because the oxidative potential of erythrocytes was estimated to be higher than that of other biological components. In addition, from such a background, it was speculated that generation of a new diagnostic marker is expected by analyzing post-translational modification of erythrocyte proteins. From the above viewpoints, the present inventors searched for protein adducts in erythrocytes using LC-MS / MS, and as a result, found an increase in specific adducts in arteriosclerosis model mice. This specification provides the following means based on this finding.

(1) A marker for arteriosclerosis, which is a compound having one or more structural parts selected from the group consisting of structural parts represented by the following formula (1).
(In the above formula, R represents a peptide or a protein or a part thereof)
(2) A test method for arteriosclerosis comprising the step of detecting the marker according to (1).
(3) The test method according to (1), wherein the detection step is a step of detecting using an antibody that specifically binds the marker according to (1).
(4) A diagnostic agent for arteriosclerosis, which is a reagent for detecting the marker according to (1).
(5) The diagnostic agent according to (4), wherein the reagent is an antibody that specifically detects the marker according to (1).
(6) A diagnostic kit for arteriosclerosis comprising means for detecting the marker according to (1).
(7) A method of screening a marker for diagnosis, comprising
Screening an adduct of blood-derived protein specific to a group to be diagnosed with unsaturated fatty acid or derivative thereof,
A screening method comprising:
(8) A method of screening a marker for diagnosis, comprising
Information on separation by liquid chromatography on adducts of unsaturated fatty acids or derivatives thereof with proteins derived from blood of a group to be diagnosed by histidine or lysine, and mass by mass spectrometry for the adducts Acquiring first adduct information including information;
Liquid chromatography of the first protein adduct information and an adduct of unsaturated fatty acid or a derivative thereof to a blood-derived protein of a reference group to the group to be diagnosed, wherein the adduct is mediated by histidine or lysine And second adduct information including mass information on mass spectrometry of the adducts, to obtain third adduct information on adducts specific to the group to be diagnosed. Process,
Identifying a polyunsaturated fatty acid derivative to which a protein is added via histidine or lysine using the third adduct information;
A screening method comprising:
(9) The screening method according to claim 7 or 8, which is a screening method for markers of erythrocyte damage due to oxygen toxicity.

It is a figure which shows the adductome analysis result of histidine in ApoE deficient mouse and control mouse. It is a figure which shows the ApoE deficient mouse specific adduct obtained from the adductome analysis result of the histidine in ApoE deficient mouse and control mouse. It is a figure which shows the comparison result of an ApoE deficient mouse specific histidine adduct and a 2-octenoic acid-His acid standard preparation. It is a figure which shows the result of having confirmed the presence or absence of 2-octenal-His and 2-octenoic acid-His in albumin and hemoglobin. It is a figure which shows the production | generation state of 2-octenoic acid-His by various iron oxidation systems. It is a figure which shows the production | generation pathway of 2-octenoic acid-His. It is a figure which shows the confirmation result of the 2-octenoic acid addition site | part in histidine in hemoglobin.

  The disclosure herein relates to arteriosclerosis markers and uses thereof. The present inventors purified proteins from plasma, white blood cells and red blood cells as blood components, respectively, and performed LC / MS / MS analysis, and it was found that adducts in which lysine and histidine were added to the protein were in the other fractions. In comparison, it was found that the red blood cell fraction was produced more. In addition, the arteriosclerosis marker disclosed herein is a protein adduct in which 2-alkenoic acid is added to a protein through histidine, which is specifically found in a population of arteriosclerosis.

  According to the present inventors, one of these protein adducts, that is, a protein adduct in which 2-octenoic acid was added to a protein via histidine, was found. Further examination of this protein adduct has shown that it is ultimately produced nonenzymatically by the iron coordinated by the heme which the hemoglobin holds.

  Because such protein adducts are specifically found in atherosclerotic individuals, we have identified them as arteriosclerosis markers.

  The arteriosclerosis marker disclosed in the present specification can be a mammal as a detection target, preferably, primates including humans, land mammals such as dogs, cats, cattle, horses, pigs, etc., sea Raw mammals etc. are mentioned.

  Hereinafter, the disclosure of the present specification will be described in detail.

(Arteriosclerosis marker)
The arteriosclerosis marker (hereinafter referred to as the present marker) disclosed in the present specification is a compound having a structural portion represented by the following formula (1).

  In the above formula (1), R can represent a peptide or a protein or a part of these. R may be a peptide or a protein. Although a peptide and a protein are not clearly distinguished, generally, a peptide is a compound in which 2 to several tens of amino acids are polymerized by peptide bond, and a protein is a peptide bond of about 100 or more amino acids. Refers to the compound polymerized in As part of a peptide or protein, it contains not only one amino acid but also degradation products of known peptides and proteins.

  R in the above formula preferably represents a carbon atom of the main chain of the peptide or protein or part thereof. Furthermore, R in the above formula is preferably a protein derived from blood or a part thereof, more preferably a protein derived from erythrocytes or a part thereof. More preferably, it is hemoglobin or a part thereof. This marker is found from the hemoglobin fraction of erythrocytes.

  In addition, it is preferable that the marker has a structure in which a hydrogen atom of a nitrogen atom of an amino group of a histidine residue or histidine side chain of a peptide or protein is substituted by a carbon atom at position 2 of octanoic acid. Such compounds are considered to be obtained, for example, by adding the nitrogen atom of the side chain of the histidine residue of erythrocyte hemoglobin to the 2-position carbon atom of 2-octenoic acid.

  The easily detectable form of this marker is a compound represented by the following formula, in which R in the above formula (1) is a carbon atom of histidine. By constructing the present test method, the present diagnostic agent and the present diagnostic kit so as to detect this compound as the present marker, good detection and / or measurement of detection sensitivity and reproducibility becomes possible.

  The marker is found in blood (whole blood), but is preferably found in the blood cell fraction, more specifically in the red blood cell fraction. Therefore, this marker preferably targets blood cell fractions or red blood cell fractions from which plasma and serum have been removed. When the marker has a dissociative group such as a carboxyl group or an amino group as represented by the above formula, the dissociative group may be either dissociative or nondissociative, the marker It is included in

(Test method of arteriosclerosis)
The method of testing for arteriosclerosis disclosed herein can comprise the step of detecting the marker from a test sample containing red blood cells. The marker can be detected from a test sample containing red blood cells, and the conditions such as onset, progress, improvement and prognosis of arteriosclerosis can be detected based on the content etc., and diagnosis can be assisted or diagnosed. .

  The test sample in this test method contains at least red blood cells. Test samples containing such red blood cells include blood (whole blood) collected from an individual to be tested, blood samples such as serum or plasma, or tissues, organs, organs and the like containing such blood samples. The test sample is preferably a so-called blood sample obtained by blood collection. The test sample is more preferably a blood cell fraction obtained from such various test samples, and still more preferably a red blood cell fraction. For fractionation of blood cells and red blood cells from blood etc., methods known to those skilled in the art can be applied.

  Various methods can be employed to detect this marker. The marker can also be detected by physicochemical means. For example, separation means such as liquid chromatography, mass spectrometry, LC / MS (liquid chromatography / mass spectrometry), LC / MS / M liquid chromatography / tandem mass spectrometry S can be used. For example, by applying a test sample to LC / MS / MS, based on the retention time in liquid chromatography and the m / z value of the tandem mass spectrometry (the m / z value of this marker and its histidine fragment) , This marker can be detected and quantified.

  As a physicochemical detection means, preferably LC-MS / MS can be used. With this method, it is possible to separate each of a protein hydrolyzate histidine adduct and a lysine adduct by LC, and further, by subjecting them to MS / MS and performing mass analysis, an adduct of histidine or lysine is obtained. And remaining mass numbers and structures can be identified relatively easily.

  The markers can also be detected by immunochemical means. For this purpose, an antibody that specifically binds to the present marker can be used. As used herein, the term "antibody" refers to polyclonal antibodies, monoclonal antibodies, fragments of monoclonal antibodies, F (ab ') 2 -conjugated antibodies, F (ab')-conjugated antibodies, short chain antibodies (scFv), diabodies, and May include mini-bodies.

  The method for detecting the present marker using an antibody is also not particularly limited, and a known immunoassay can be used, and examples of the immunoassay include enzyme immunoassay, radioimmunoassay, latex agglutination method, western blotting method, immunochromatography method, etc. Be Immunoassays include direct methods, indirect methods, sandwich methods, competition methods and the like, and these various aspects can be used as appropriate. In the immunoassay, if necessary, the antibody may be labeled, and an antibody as a primary antibody that binds to the present marker and a secondary antibody that binds to the present marker (labeled for detection etc. Can be used as a detection reagent as appropriate. In addition, in the immunoassay of various embodiments using an antibody, the antibody can be used in a state of being immobilized on a suitable solid phase. The method for producing the antibody will be described in detail later.

The blood sample collected from the test subject is preferably applied to the present detection step, for example, by processing as follows. It is preferable that the blood sample is first treated with a reducing agent to introduce various reaction products such as active oxygen into the reductant to suppress further decomposition. For reduction, a reducing agent which does not reduce carbon-carbon double bond, carboxyl (COOH) group, ester group and the like is used, for example, sodium borohydride (NaBH ₄ ), sodium cyanoborohydride (NaCNBH ₃ ), triphenyl And phosphine (triphenylphosphine) and the like.

  The reduction reaction advantageously proceeds by using a reducing agent in an amount of 1 equivalent to excess and reacting in water, an alcohol such as methanol or ethanol, or a water-containing alcohol at a temperature of about 0 ° C. to 50 ° C. for about 1 minute to 16 hours. Do.

  After reduction treatment, the protein is neutralized by using a known protein insolubilizing agent such as trichloroacetic acid, neutralized, and optionally washed with acetone or diethyl ether, and an acid of an appropriate concentration for the recovered protein (for example, 2-Octenoic acid-His etc. can be generated by appropriately heating and hydrolyzing at about 110 ° C. for about 24 hours under a reduced pressure condition by a vacuum pump etc. with 5 to 10 N HCl and the like).

  The hydrolysis may be carried out in water, or solvents such as tetrahydrofuran, acetonitrile, ethanol, methanol, isopropanol, butanol, acetone, DMF, DMSO, N-methylpyrrolidone, etc. may be mixed with water.

  By such hydrolysis, a test sample suitable for the step of detecting the marker can be prepared from the blood sample of the test subject individual. In addition, after alkali treatment, it is desirable to filter with a filter. As a filter used for filtration, a cutoff value is about 2000-100000 Da, preferably about 3000-50000 Da, preferably about 5000-30 000 Da. As a filter, for example, a YM10 filter (manufactured by Nippon Millipore Co., Ltd.) can be used.

In addition, 2-octenoic acid-His which is one aspect of this marker and can be used as a standard product can be obtained by synthesis by a known method. For example, as one method, a protein such as human serum albumin is reacted with 2-octenal in PBS at 37 ° C. for about 6 hours, and after the reaction, reduction treatment is performed with NaBH ₄ at room temperature for several hours, and then Then, NaClO ₂ was added and the mixture was allowed to stand overnight at room temperature or the like to oxidize the aldehyde to a carboxylic acid. After this, after insolubilizing the protein with trichloroacetic acid or the like, 2-octenoic acid-His can be obtained by hydrolyzing the recovered protein with about 6N HCl at about 110 ° C. for about 24 hours.

  The step of detecting the present marker is preferably a step of not only detecting the present marker but also measuring the concentration or content of the present marker. By doing this, it is possible to more specifically diagnose the onset and progress of arteriosclerosis.

  In the detection step, in order to quantify the concentration or the like of the present marker, it can be quantified based on a known method using the present marker as a standard prepared separately. In addition, indicators and thresholds useful for diagnosis and the like can be obtained from individuals at various stages of arteriosclerosis, and the concentration of this marker can be measured using a test sample to measure the development of arteriosclerosis. The threshold of each stage can be determined.

  For example, the threshold value of the present marker that can be diagnosed as arteriosclerosis can be set by performing the present examination method for the group of healthy subjects and the arteriosclerosis diagnosis group diagnosed as arteriosclerosis. Furthermore, it is possible to set a threshold that can be said to be a boundary area between a healthy subject and arteriosclerosis. In addition, by appropriately setting the target group and performing the present examination method, one skilled in the art can set an appropriate threshold according to the development of arteriosclerosis.

(Diagnostic agent for arteriosclerosis)
The diagnostic agent for arteriosclerosis disclosed herein can include a reagent that detects this marker. According to the present diagnostic agent, it is possible to easily diagnose the onset, progress, improvement and prognosis of arteriosclerosis from blood samples. Such a reagent is not particularly limited, but as described above, it may be a reagent for detecting the present marker physicochemically, or may be a reagent such as an antibody for detecting immunochemically. Good.

(Diagnostic kit for arteriosclerosis)
The arteriosclerosis diagnostic kit disclosed herein can comprise means for detecting the marker. Such means is, for example, a reagent for detecting the present marker, and examples thereof include an antibody that specifically binds to the present marker. Furthermore, the kit can comprise reagents for preparing a test sample. The reagent for preparing the test sample may contain any or two or more of various reducing agents as described above, a protein insolubiliser such as trichloroacetic acid, and a protein hydrolyzing agent such as an acid. It may contain an alkaline solution such as NaOH to be used together with the reducing agent.

  When an antibody that specifically binds to the present marker is included as a reagent for detecting the present marker, it is appropriately labeled to bind to a labeling reagent for detection and a primary antibody that specifically binds the present marker. Secondary antibodies etc. may also be mentioned.

  The kit can further include a carrier such as immunochromatography on which the antibody is immobilized. Furthermore, the kit can appropriately contain a buffer solution, a solvent and the like. Buffers include PBS for dispersing red blood cells. Further, examples of the solvent include acetone, ether and the like for washing the insolubilized protein. Further, the above-mentioned solvents which can be used for the reduction reaction are mentioned.

  Furthermore, the kit can contain 2-octenoic acid-His as a standard.

  In addition, the manufacturing method of the antibody which is used for this test method, is this diagnostic agent, and is an element of this diagnostic kit which detects this marker specifically, is not specifically limited. According to a known method, an animal is immunized by a known method using 2-octenoic acid-His as an immunogen, a method known in the art or widely used, such as the method of Keller, Milstein et al. (Nature, 256: 495-97, 1975) or it can manufacture according to it. The preparation of monoclonal antibodies is described in detail below. In addition, although a monoclonal antibody is obtained using the cell fusion technique using a myeloma cell as shown below, it is not limited only when a myeloma cell is used.

  As an antigen, 2-octenoic acid-His can be used as it is by mixing it with an appropriate adjuvant to immunize the animal, but as an immunogenic conjugate, it can be mixed with an appropriate adjuvant if necessary. It is desirable to immunize. In addition, an immunogenic conjugate can be produced by linking an immunogen containing this antigen to various carriers via an appropriate condensing agent.

  When the carrier is a protein, carrier proteins can be activated. The activation can be carried out by forming an active ester (a nitrophenyl ester group, a pentafluorophenyl ester group, a 1-benzotriazole ester group, an N-succinimide ester group, etc.). As the carrier, keyhole limpet hemocyanin (KLH), serum albumin (BSA, HSA), ovalbumin, globulin, polypeptides such as polylysine and polyhistidine, bacterial cell components such as BCG and the like can be mentioned.

For immunizing an animal, for example, Muramatsu Shigeru, et al., Experimental biology course 14, Immunobiology, Maruzen Co., Ltd., Showa 60, Japan Biochemical Society, Ed. Conduct according to the method described in Tokyo Chemical Doujin, 1986, or the Japan Biochemical Society, Neochemistry Experiment Course 12, Molecular Immunology III, Antigens, Antibodies, Complements, Tokyo Chemical Dojin, 1992 etc. Can. Examples of the adjuvant used together with the antigen include Freund's complete adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, liposome, aluminum hydroxide, silica and the like. Immunization is performed using an animal such as a mouse such as BALB / c. The dose of the antigen is, for example, about 1 to 400 μg / animal for mice, generally injected intraperitoneally or subcutaneously to the host animal, and every one to four weeks thereafter, more preferably every one to two weeks. Repeat immunization 2 to 10 times in the interior, subcutaneously, intravenously or intramuscularly. As mice for immunization, in addition to BALB / c mice, F1 mice of BALB / c mice and other mice can also be used. If necessary, an antibody titer measurement system can be prepared, and the antibody titer can be measured to confirm the degree of animal immunity.

  An infinitely proliferating strain (tumor cell line) used for cell fusion can be selected from cell lines that do not produce immunoglobulin, for example, P3-NS-1-Ag4-1 (NS-1, Eur. J. Immunol) , 6: 511-519, 1976), SP 2/0-Ag 14 (SP 2, Nature, 276: 269-270, 1978), P3-X63-Ag 8-U1 (P3U1, Curr) derived from the mouse myeloma MOPC-21 cell line. Immunol., 81: 1-7, 1978), P3-X63-Ag8 (X63, Nature, 256: 495-497, 1975), P3-X63-Ag8-653 (653, J. Immunol., 123). : 1548-1550, 1979) can be used.

  The animals immunized according to the above steps, such as mice, for example, have their spleens removed 2 to 5 days after the final immunization to obtain a splenocyte suspension. In addition to splenocytes, lymph node cells in various parts of the body can be obtained and used for cell fusion. The splenocyte suspension thus obtained and the myeloma cell line are placed in a cell culture medium such as minimal essential medium (MEM medium), DMEM medium, RPMI-1640 medium, and a cell fusion agent such as polyethylene glycol is added. . As the cell fusion agent, those known in various other relevant fields can be used, and examples thereof include inactivated Sendai virus (HVJ: Hemagglutinating Virus of Japan) and the like. Preferably, for example, 0.5 to 2 ml of 30 to 60% polyethylene glycol can be added, and polyethylene glycol having a molecular weight of 1,000 to 8,000 can be used, and further, a molecular weight of 1,000 to 4,000. Of polyethylene glycol can be more preferably used. The concentration of polyethylene glycol in the fusion medium is preferably, for example, 30 to 60%. If necessary, for example, a small amount of dimethyl sulfoxide or the like can be added to promote fusion. The ratio of splenocytes (lymphocytes): myeloma cell line used for fusion can be, for example, 1: 1 to 20: 1, and more preferably 4: 1 to 10: 1. The fusion reaction is carried out for 1 to 10 minutes and then cell culture medium such as RPMI-1640 medium is added. The fusion reaction process can also be performed multiple times. After the fusion reaction treatment, the cells are separated by centrifugation or the like and then transferred to a selection medium.

  The culture medium for selection of hybridoma includes, for example, culture medium (eg, HAT medium) such as FCS-containing MEM medium containing DMEM, aminopterin and thymidine, DMEM medium, DMEM medium, IMDM medium, RPMI-1640 medium and the like. The selective medium replacement method can be generally performed by adding an equal volume to the volume dispensed to the culture plate on the next day, and then exchanging half volume each with HAT medium every 1 to 3 days, but this may be appropriately carried out. You can also make changes to In addition, on the 8th to 16th days after fusion, the medium can be changed every 1 to 4 days with so-called HT medium except aminopterin. As feeders, it is also possible, for example, to use mouse thymocytes, which may be preferred.

  The culture supernatant of a culture well where proliferation of hybridomas is active can be measured using, for example, a measurement system such as radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence immunoassay (FIA), or fluorescence-induced cell separator (FACS) Then, screening is performed by using 2-octenoic acid-His as an antigen or measuring a target antibody using a labeled anti-mouse antibody. Thereafter, a hybridoma producing a target antibody is cloned. Cloning is carried out by picking up colonies in agar medium or by limiting dilution. More preferably, it can be carried out by the limiting dilution method. Preferably, cloning is performed multiple times.

  The obtained hybridoma strain is cultured in a suitable growth medium such as MEM medium containing or without FCS, DMEM medium, DMEM medium, IMDM medium, RPMI-1640 medium and the like to obtain a desired monoclonal antibody from the supernatant of the medium. be able to. In order to obtain a large amount of antibodies, ascites the hybridomas. In this case, each hybridoma is transplanted into the peritoneal cavity of a histocompatible animal syngeneic with a myeloma cell-derived animal, and each hybridoma is transplanted or propagated into a nude mouse or the like, and then propagated into the ascites fluid of the animal. The produced monoclonal antibody can be recovered and obtained. Prior to transplantation of hybridomas, animals can be intraperitoneally administered mineral oil such as pristane (2,6,10,14-tetramethylpentadecane), and after the treatment, hybridomas are grown and ascites fluid is collected. It can also be done. The ascites fluid is used as it is or in a conventionally known method, for example, salting out such as ammonium sulfate precipitation, gel filtration with sephadex, ion exchange chromatography, electrophoresis, dialysis, ultrafiltration, affinity chromatography It can be purified by high performance liquid chromatography or the like and used as a monoclonal antibody. More preferably, the ascites fluid containing the monoclonal antibody is fractionated with ammonium sulfate and then purified and separated using an anion exchange gel such as DEAB-Sepharose and an affinity column such as a protein A column. More preferably, using affinity chromatography in which an antigen or an antigen fragment (eg, synthetic peptide, recombinant antigen protein or peptide, a site specifically recognized by an antibody, etc.) is immobilized, affinity chromatography in which protein A is immobilized, etc. It can be purified and separated.

  It is also possible to determine the nucleic acid sequence of the antibody thus obtained in large amounts, or to produce the antibody by genetic recombination technology, using the nucleic acid sequence encoding the antibody obtained from the hybridoma strain.

  Furthermore, these antibodies may be treated with an enzyme such as trypsin, papain or pepsin and optionally used as an antibody fragment such as Fab, Fab ′ or F (ab ′) 2 obtained by reduction. The antibody may be labeled with an enzyme (peroxidase, alkaline phosphatase or β-D-galactosidase etc.), a chemical substance, a fluorescent substance or a radioactive isotope.

(Method of screening markers for diagnosis)
The screening method disclosed herein is an addition in which unsaturated fatty acid derivatives such as unsaturated fatty acids or oxides thereof are added to proteins derived from blood specific to a group to be diagnosed via histidine or lysine. Screening the body. According to this screening method, it is possible to screen, as a diagnostic marker, an adduct for diagnosis specific to a disease or condition in which a group to be diagnosed is afflicted.

  In addition, as unsaturated fatty acid, a C6-C20 or less unsaturated fatty acid, a C7-C12 unsaturated fatty acid is mentioned preferably. Typically, various 2-alkenoic acids can be mentioned. Moreover, aldehyde etc. are mentioned as a derivative of unsaturated fatty acid. For example, typically, various 2-alkenals are mentioned.

  In addition, as the adduct, various unsaturated fatty acids and aldehydes as their oxides were added via the N atom of the side chain amino group of lysine residues and histidine residues of proteins such as albumin derived from blood The thing is mentioned.

  As disclosed herein, screening for an adduct in which unsaturated fatty acid or a derivative thereof is added to a protein derived from blood, in particular red blood cells, is associated with arteriosclerosis, hyperlipidemia, methemoglobin plasma, brimaquin hypersensitivity. Markers related to diseases or conditions related to lipid oxidation such as triglycerol in blood, acid or stress such as sickle cell disease, etc. (these markers are collectively referred to as a marker of erythrocyte damage due to oxygen toxicity). be able to. Therefore, the screening method is useful for screening markers such as arteriosclerosis and hyperlipidemia.

  In screening for such adducts, it is preferable to use a blood sample such as whole blood or a blood cell fraction of the subject, more specifically, a red blood cell fraction. Such a blood sample can be obtained, as described above, in the same manner as obtaining a test sample suitable for the detection step of the present detection method, to obtain a test sample of the present screening method. The test sample can contain a protein in which various unsaturated fatty acids or their derivatives are linked via His or Lys.

  Next, the adducts in the test sample are screened. For screening of adducts, various methods described above can be employed. In order to comprehensively screen adducts, for example, liquid chromatography, mass spectrometry, LC / MS (liquid chromatography / mass spectrometry), LC / MS / M liquid chromatography, tandem mass spectrometry S, etc. Means of separation can be used. For example, as described above, by applying the test sample to LC / MS / MS, the m / z value of the retention time such as retention time in liquid chromatography and tandem mass spectrometry (this marker and The adduct can be detected based on mass information such as m / z value, mass-to-charge ratio of the histidine fragment.

  As a physicochemical detection means, preferably LC-MS / MS can be used. With this method, it is possible to separate each of a protein hydrolyzate histidine adduct and a lysine adduct by LC, and further, by subjecting them to MS / MS and performing mass analysis, an adduct of histidine or lysine is obtained. And remaining mass numbers and structures can be identified relatively easily.

  For example, in LC / MS / MS, various adducts can be detected on a matrix of retention time and mass to charge ratio in LC. In the matrix, relative values of peak areas detected on the LC can be displayed as area values such as circles. A matrix of adducts obtained from test samples of a diagnosis target group in which red blood cell damage due to certain types of oxygen toxicity is estimated and a matrix of adducts obtained from test samples of a suitable reference group such as healthy persons By contrast in terms of time, mass information and relative values of peak areas, it is possible to find adducts characteristic of the group to be diagnosed.

  Characteristic adducts, as disclosed in the examples below, use LC / MS / MS analysis matrices to deduce and identify adduct structures from retention times and mass to charge ratios in the LC. be able to.

  The adducts found in such screening methods are related to the oxidation state of proteins derived from blood, particularly proteins derived from erythrocytes, and can be useful markers for diagnosis of erythrocyte damage due to oxygen toxicity and the like.

  Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples.

(Aductome analysis of ApoE deficient mouse erythrocyte proteins)
In this example, adductome analysis of erythrocyte proteins of ApoE deficient mice and their control mice, balb / c mice, was performed. Blood was collected from each mouse at 14 weeks of age to prepare erythrocytes. The red blood cell proteins were reduced, acid hydrolysis was performed, test samples were prepared, and adductome analysis by LC-MS / MS was performed. Preparation of erythrocytes, reduction treatment and acid hydrolysis, and adductome analysis were carried out by the following method. The results are shown in FIGS. 1 and 2.

(1) Isolation of Erythrocytes EDTA was added to blood samples, centrifuged at 3500 rpm and 4 ° C. for 10 minutes, and the supernatant was removed. Then, add an equal volume of isotonic solution (10 mM phosphate buffer-152 mM NaCl (pH 7.4)) to the precipitate, shake well, and then centrifuge at 3500 rpm and 4 ° C for 20 minutes. Removed. This operation was repeated three times to isolate erythrocytes.

(2) Reduction treatment Mouse erythrocytes were diluted with PBS so that the number of erythrocytes was 2.0 × 10 ⁷ cells / ml. NaBH ₄ was dissolved in 1N aqueous NaOH solution so as to reach 1,100 mM, 1/10 volume of the reaction solution was added, and reduction treatment was performed at room temperature for 3 hours. After reduction treatment, 2N HCl was added in an equal amount to the aqueous NaOH solution to neutralize.

(3) Acid hydrolysis An equivalent amount of 20% TCA was added to a microtube and a reduction treatment sample, and the mixture was allowed to stand on ice for 1 hour to insolubilize proteins. The protein was precipitated by centrifugation at 4,000 × g (8,000 rpm, 6 cm) at 4 ° C. for 30 minutes, and the supernatant was removed so as not to disturb the precipitate. Thereafter, 500 μl of ice cold acetone was added and the mixture was gently stirred, and then centrifuged similarly to remove the supernatant. Further, 750 μl of diethyl ether was added, and after gently stirring, the supernatant was removed by centrifugation in the same manner. A small amount of 6N HCl was poured into the hydrolysis tube, and the sample-containing microtube was placed, and then vacuum conditions were applied using a vacuum pump. It was placed in a 110 ° C. oven and hydrolyzed for 24 hours. After completion of the hydrolysis, HCl remaining in the microtube was distilled off by a desiccator.

(4) Analysis of adductome The test sample after hydrolysis was redissolved in 500 μl of EtOH per 1 mg of protein. In the case of low solubility, sonication was applied, and the sample was passed through a 0.45 μm filter to obtain a sample for LC-MS / MS. The measurement was performed under the following conditions using LC-MS / MS.
After measurement, the mass-to-charge ratio of each peak, the retention time in the LC part, the peak area, and the peak area of valine were recorded, and an adduct up and a heat map were made. The peak area between samples was corrected by the peak area of valine.

(Conditions for adductome analysis of modified proteins (LC-MS / MS))
Injection: 10μl
Cone potential: 25 eV
Collision energy: 25 eV
Ion mode: ESP +
Precursor ion: m / z 150 to 450
Production: m / z 84 (modified lysine) and m / z 110 (modified histidine).
The valine measured m / z 118> 72.
Column: Developil C30-UG-5 (2.0 / 100)
Flow rate: 0.3 ml / min
Solvent A: H2O / 0.1% HCOOH
Solvent B: MeOH
Gradient

  As shown in FIG. 1, numerous adducts were detected in both ApoE deficient mice and balb / c mice. On the other hand, more adducts were detected in ApoE deficient mice compared to balb / c mice. As a result of comparing the area for each peak, it was revealed that the mass-to-charge ratio 298 shown in No. 1 in FIG. 1 and the amount of adduct generated for a retention time of 4.9 minutes were significantly increased.

Histidine is an amino acid having a mass-to-charge ratio of 156 represented by a composition formula of C ₆ H ₉ N ₃ O ₂ . From this, it was suggested that, for the one having a mass-to-charge ratio of 298, the modifying moiety has a structure represented by C ₉ H ₁₇ O or a structure represented by C ₈ H ₁₅ O ₂ . For C ₈ H ₁₅ O ₂ , 2-octenoic acid having a carboxylic acid in the molecule and having 8 carbon atoms was expected.

  Therefore, 2-octenoic acid-His was prepared by the following method, and identity with an adduct generated in erythrocytes of ApoE deficient mice was confirmed by LC / MS / MS under the following conditions. The results are shown in FIG.

(Preparation of 2-octenoic acid-His)
HSA (1.0 mg / ml) and 2-octenal (1.0 mM) were reacted in PBS at 37 ° C. for 6 hours. After the reaction, NaBH ₄ was dissolved in 1N aqueous NaOH solution so as to reach 1,100 mM, 1/10 of the reaction solution was added, and reduction treatment was performed at room temperature for 3 hours. DMSO was added to 2 equivalents of the reaction solution, and 3 equivalents of NaClO ₂ (0.5 mM) was added with stirring at 200 μl / min. After standing overnight at room temperature, oxidation of aldehyde to carboxylic acid was performed. After the oxidation treatment, acid hydrolysis was performed as described above. In addition, 2-2 octenoic acid-His was fractionated using the mobile phases A and B under the following conditions so that the ratio of A would be as follows.

«Preparation conditions of 2-octenoic acid-His»
Column: Sunniest RP-AQUA (diameter 10 mm, 250 mm)
Flow rate: 2.0 ml / min
Detection: 200-650 nm
Eluent: AH ₂ O / 0.1% TFA, B: Acetonitrile / 0.1% TFA

<< LC / MS / MS analysis conditions for 2-octenoic acid-His >>
Injection: 10μl
Cone potential: 40 eV
Collision energy: 30 eV
Ion mode: ESP ⁺
Precursor ion: 298
product ion: 110
Column: Developil C30-UG-5 (2.0 / 100)
Flow rate: 0.3 ml / min
Solvent A: H ₂ O / 0.1% HCOOH
Solvent B: MeOH

  As shown in FIG. 3, the synthesized 2-octenoic acid-His matched the retention time with the adduct generated in ApoE deficient mouse erythrocytes. From this, it was suggested that 2-octenoic acid-His is generated significantly in erythrocytes of the arteriosclerosis model mouse. Incidentally, it was also confirmed that 2-octenoic acid-His was not detected from plasma components of blood samples of ApoE-deficient mice (see FIG. 3).

  From the above, it was found that the adduct specifically generated in erythrocytes of ApoE deficient mice has a structure of 2-octenoic acid-His.

(Study of the formation pathway of 2-octenoic acid-His: Examination of the oxidation of 2-octenal-His to 2-octenoic acid-His)
With regard to 2-octenal, it was considered from its structure that the reactivity with histidine in the protein was high, and the reaction between 2-octenal and the protein sufficiently produced 2-octenal-His. Thus, in this example, attention was focused on the formation of 2-octenoic acid-His from 2-octenal-His.

  In Example 1, it was shown that the formation of 2-octenoic acid-His was not confirmed from the plasma component but was specifically confirmed to the red blood cell component. Based on this fact, we first compared albumin, which is the major protein of plasma, with hemoglobin, which is the major protein of red blood cells. For comparison, 1 mg / ml albumin or hemoglobin and 1 mM 2-octenal are reacted for 24 hours, and after reduction / acid hydrolysis shown in Example 1, in the same manner as Example 1, LC-MS / MS 2- The presence or absence of octenal-His and 2-octenoic acid-His was confirmed. The results are shown in FIG.

  As shown in FIG. 4, the formation of 2-octenal-His was confirmed from the reaction of both albumin and hemoglobin, but the formation of 2-octenoic acid-His was confirmed only by the reaction with hemoglobin.

  From this, it was suggested that the characteristic properties of hemoglobin are involved in the production of 2-octenoic acid-His.

(Examination of the contribution of iron etc. to the formation of 2-octenoic acid-His)
In this example, it was predicted that iron coordinated to hemoglobin might be involved in the oxidation of 2-octenal-His to 2-octenoic acid-His.

  After reacting 1 mM histidine and 1 mM 2-octenal at 37 ° C. for 6 hours to form 2-octenoic acid-His, examination was made on oxidation systems using various irons shown in FIG. The results are shown in FIG.

  As shown in FIG. 5, the formation of 2-octenoic acid-His from a typical iron-based oxidation system of Fe / AsA (ascorbic acid) was confirmed, and the heme analogue in hemoglobin was also confirmed. The formation of iron from the system of hemin coordinating iron was confirmed, and the formation was not confirmed from the system of protporphyrin ix from which iron was eliminated from hemin.

  From the above results, it is strongly supported that the non-enzymatic oxidation shown in FIG. 6 is a production pathway of 2-octenoic acid-His.

(Identification of the modification site of 2-octenoic acid-His)
In this example, identification of modification sites by MALDI-TOF MS was carried out as to which histidine in hemoglobin has been modified.

1 mg / ml hemoglobin and 1 mM 2-octenal were reacted at 37 ° C. for 24 hours. After that, the reaction was stopped by NaBH ₄ reduction, and after performing reductive alkylation treatment with DTT and IAA, trypsin was added to perform enzyme digestion. The prepared peptide mixture was fractionated by nano-LC, and analyzed by MALDI-TOF MS according to a conventional method. Furthermore, a search was conducted using MASCOT, a protein identification search software. The results are shown in FIG.

  As shown in FIG. 7, for the hemoglobin α chain, although the sequence coverage was as low as 40%, 2-octenoic acid modification was confirmed in the 21st and 59th histidines. Moreover, when identification of the modification site by MALDI-TOF MS was performed, it was confirmed that the 21st and 59th histidines in the hemoglobin α chain had been modified. There is also a report that the 21st histidine is present on the molecular surface, and by reacting 4-oxo-2-nonenal with hemoglobin, the 21st histidine in the hemoglobin α chain is preferentially modified. , It is considered to be a part susceptible to modification. Moreover, since the 59th histidine is located in the vicinity of heme, oxidation of 2-octenal-His to 2-octenoic acid-His by heme iron was suggested. Furthermore, since the 59th histidine is an active center in the binding of hemoglobin and oxygen, it is suggested that 2-octenoic acid may influence the oxygen binding ability of hemoglobin.

Claims (6)

An arteriosclerosis marker, which is a compound having one or more structural parts selected from the group consisting of structural parts represented by the following formula (1).

(In the above formula, R represents a peptide or a protein or a part thereof)   A method of examining arteriosclerosis, comprising the step of detecting the marker according to claim 1.   The test method according to claim 2, wherein the detection step is a step of detecting using an antibody that specifically binds the marker according to claim 1.   A diagnostic agent for arteriosclerosis, which is a reagent for detecting the marker according to claim 1.   The diagnostic agent according to claim 4, wherein the reagent is an antibody that specifically detects the marker according to claim 1. A diagnostic kit for arteriosclerosis comprising means for detecting the marker according to claim 1.