CN1878787A - Detection of cholesterol ozonation products - Google Patents

Abstract

The invention relates to detection of cholesterol ozonation products that are generated by atherosclerotic plaque material, and to methods of detecting vascular conditions that relate to the accumulation and oxidation of cholesterol.

Description

The detection of cholesterol ozonation products

Related application

The application requires the right of priority of provisional application series number of submitting on September 5th, 2,003 60/500,593 and the provisional application series number of submitting on November 6th, 2,003 60/517,821, and the disclosure integral body of described provisional application is attached to herein.

Government rights statement

Invention described herein obtains United States Government that National Institutes of Health (National Institutes of Health) authorizes and subsidizes (subsidy PO1CA 27489) and finish.United States Government has some right of this patent.

Invention field

The present invention relates to cholesterol ozonation products and produce this discovery by atherosclerotic lesion (lesion).The invention provides diagnosis, detection and monitoring and suffer from the patient's of cholesterol related artery illness such as atherosclerosis and/or cardiovascular disorder method.

Background of invention

Masses are constantly suggested, answer careful monitoring serum ornitrol level, and masses are often cautioned that also eating and drinking without temperance system and shortage athletic meeting cause cholesterol to accumulate, and increase the danger of atherosclerosis and coronary heart disease in artery plaque.Yet though the index that high serum ornitrol level is this danger, it does not prove debatable atherosclerotic plaque accumulation physical presence.

Known serum ornitrol is main relevant with the Witepsol W-S 55 (VLDL-cholesterol) in low-density lipoprotein (LDL-cholesterol), high-density lipoprotein (HDL) (HDL-cholesterol) and the vldl.The statistic evidence of many long-term clinical trials shows that the HDL-cholesterol is relevant with lower relative risk with low ratio LDL-cholesterol at high proportion.The HDL-cholesterol is useful, and condition is that its level is only low, promptly is lower than 30mg/dL.The VLDL-cholesterol does not relate to any risk yet to be measured, but high Witepsol W-S 55 itself may be serious problems.On the other hand, LDL-cholesterol and low ratio HDL-cholesterol are the higher atherosclerosis and the index of coronary heart disease danger at high proportion.

Even have dependency closely between atherosclerosis and the high LDL-cholesterol level, some research shows that serum LDL-carry out very poorly with the measurement of HDL-cholesterol level often provides insecure result.Referring to Superko, H.R. etc., High-DensityLipoprotein Cholesterol Measurements--A Help or Hinderance inPractical Clinical Medicine, JAMA 256:2714-2717 (1986); Warnick, G.R. etc., HDL Cholesterol:Results of Interlaboratory Proficiency Test, Clin.Chem.26:169-170 (1980); And Grundy, S.M. etc., The Place of HDL inCholesterol Management.A Perspective from the National CholesterolEducation Program, Arch.Inter.Med.149:505-510 (1989).The article report of Grundy etc., the interlaboratory variation coefficient of HDL-cholesterol observed value is up to 38%.The report that ACP (College of American Pathologists) did to measure to identical HDL-cholesterol sample about more than 2,000 laboratories in 1987 shows that the observed value more than 33% and the difference of reference value surpass 5%.In using the group of same procedure, the interlaboratory variation coefficient is improved to 16.5% really, but that the variation of this degree still shows most of test results is too inaccurate, and being difficult in clinical setting has any predictive value.Therefore, in assessment of risks, do not re-use total cholesterol: the ratio of HDL-cholesterol.

In typical lipid profile type (profile) research, total cholesterol and Witepsol W-S 55 level are directly measured from serum sample.Use the agent treated sample then, to be settled out LDL-cholesterol and VLDL-cholesterol.The HDL-cholesterol is done this processing in the remaining supernatant liquor in back at sample and is measured.Think that the VLDL-cholesterol accounts for the fixed fraction of Witepsol W-S 55 (for example 0.2).By deduct HDL and the cholesteric value of VLDL from total cholesterol, indirect calculation goes out the LDL-cholesterol then.The propagation of error that occurs in these three independent measurements makes that the measurement of LDL-cholesterol is minimum one of resultant accuracy and precision, although it is most important for the assessment of cardiovascular danger.Because this inaccuracy is difficult to free burial ground for the destitute monitoring intentionally and determines to lower to pass in time in the therapy whether obtain bed progress finally at the LDL-cholesterol.

Therefore, serum LDL-cholesterol is measured inaccurate often.This inaccuracy adds that can not actual proof there be this fact of debatable atherosclerotic lesion in LDL-cholesterol level, and illustrating needs simple relatively, reliable and reproducible method to determine whether to exist among the patient debatable rich cholesterol atherosclerotic lesion.

Summary of the invention

According to the present invention, cholesterol ozonolysis product is present in the atherosclerotic plaque.In addition, to the detection of cholesterol ozonation products in the tissue that picks up from the patient and the body fluid and quantitatively, be the accurate index of physical presence atherosclerotic lesion whether among the patient.Therefore the present invention provides simply accurately, and method detects whether there is atherosclerotic lesion among the patient.Method of the present invention relates to the detection cholesterol ozonation products and whether is present in the sample that picks up from the patient.The present invention also imagines, and the amount of the cholesterol ozonation products that exists in the quantitative biological sample is as the means of diagnosis and monitoring Mammals medium sized artery atherosclerotic plaque formation degree.

One aspect of the present invention is the isolating cholesterol ozonation products that results from atherosclerotic plaque.This cholesterol ozonation products can for example have following arbitrary formula 4a-15a, 3c or 7c:

Another aspect of the present invention is the detected derivative of cholesterol ozonation products, described derivative comprises bisulfite adduct, imines, oxime, hydrazone, red sulphonyl hydrazone, semicarbazone or Tollins test product, and wherein said cholesterol ozonation products produces in atherosclerotic plaque.

Another aspect of the present invention relates to the hydrazone derivative of the cholesterol ozonation products with formula 4b or formula 4c:

Another aspect of the present invention relates to the hydrazone derivative of the cholesterol ozonation products with formula 5b:

Another aspect of the present invention relates to the hydrazone derivative of the cholesterol ozonation products with arbitrary formula 6b-15b or 10c:

Another aspect of the present invention relates to the red sulphonyl hydrazone derivative of the cholesterol ozonation products with formula 4d:

Another aspect of the present invention relates to the red sulphonyl hydrazone derivative of the cholesterol ozonation products with formula 5c:

Another aspect of the present invention is the haptens with formula 13a, 13b, 14a, 14b, 15a, 15c or 3c.

Another aspect of the present invention is can be in conjunction with the separation antibody of cholesterol ozonation products.Described antibody can be monoclonal antibody or polyclonal antibody.The combinative cholesterol ozonation products of antibody can be the compound with arbitrary formula 4a-15a, 3c, 4c, 7c.In certain embodiments, separation antibody can for example have the compound of arbitrary formula 4b-15b, 4c or 10c in conjunction with the hydrazone derivative of cholesterol ozonation products.Antibody of the present invention can for example produce at the haptens with formula 13a, 13b, 14a, 14b, 15a, 15c or 3c.

Another aspect of the present invention is a separation antibody, and wherein said separation antibody is derived from hybridoma KA1-11C5:6 or KA1-7A6:6 (ATCC searching number PTA-5427 or PTA-5428).

Another aspect of the present invention is a separation antibody, and wherein said separation antibody is derived from hybridoma KA2-8F6:4 or KA2-1E9:4 (ATCC searching number PTA-5429 and PTA-5430).

Whether another aspect of the present invention is by detecting available from existing cholesterol ozonation products to detect atherosclerotic method among the patient in patient's the sample.Ozonation products can be produced by atherosclerotic plaque.Sample can for example be serum, blood plasma, blood, atherosclerotic plaque material, urine or vascular tissue.Detect the amount that atherosclerotic method also can relate to the cholesterol ozonation products that exists in the quantitative sample.

In one embodiment, detecting atherosclerotic method can may further comprise the steps: make sample and hydrosulphite, ammonia, Schiff's base, aromatics or aliphatic hydrazine, red sulfonyl hydrazide, Gerard reagent, the reaction of Tollins test reagent, and detect the derivative of the cholesterol ozonation products that is formed by this reaction.

In another embodiment, detecting atherosclerotic method can comprise and make the reaction of sample and hydrazine compound to produce the hydrazone derivative of sterol ozonation products.For example, hydrazine compound can be a 2,4 dinitrophenyl hydrazine.

In another embodiment, detecting atherosclerotic method can comprise and make the reaction of sample and red sulfonyl hydrazide to produce the red sulphonyl hydrazone derivative of sterol ozonation products.For example, formed red sulphonyl hydrazone derivative can have formula 4d or 5c.

In another embodiment, detecting atherosclerotic method can comprise and make sample and can contact in conjunction with the antibody of cholesterol ozonation products.In this method, can use any antibody described herein.

Another aspect of the present invention relates to by detecting available from whether there being cholesterol ozonation products in patient's the sample, detect the method whether cholesterol ozonation products is discharged by the atherosclerotic plaque among the patient, wherein said ozonation products is the compound with formula 5a.Detect the amount that method that whether cholesterol ozonation products discharged by atherosclerotic plaque also can relate to the cholesterol ozonation products that exists in the quantitative sample.

Another aspect of the present invention relates to the atherosclerotic method that detects among the patient, and described method comprises: 2,4 dinitrophenyl hydrazine is joined in the sample available from the patient, whether have the hydrazone derivative of cholesterol ozonation products in the test samples.Detected hydrazone derivative can be for having the compound of arbitrary formula 4b, 4c, 5b, 6b, 7b, 8b, 9b, 10b, 10c, 11b, 12b, 13b, 14b or 15b.

Another aspect of the present invention relates to by scavenger cell being contacted with sample and determining that whether scavenger cell increases the picked-up of lipid, comes whether to exist in the test samples method of cholesterol ozonation products.

Another aspect of the present invention relates to the atherosclerotic method that detects among the patient, and described method comprises makes scavenger cell contact with sample available from the patient, and whether definite scavenger cell increases the picked-up of lipid.

Another aspect of the present invention relates to the method for the cholesterol ozonolysis product in the test samples, and described method comprises makes low-density lipoprotein contact with sample, and whether the secondary structure of observation low-density lipoprotein changes.

Another aspect of the present invention relates to the atherosclerotic method that detects among the patient, and described method comprises makes low-density lipoprotein contact with sample available from the patient, and whether the secondary structure of observation low-density lipoprotein changes.

Another aspect of the present invention relates to the method for the cholesterol ozonolysis product in the test samples, and described method comprises makes apoprotein B ₁₀₀Contact with sample, and observe apoprotein B ₁₀₀Secondary structure whether change.

Another aspect of the present invention relates to the atherosclerotic method that detects among the patient, and described method comprises makes apoprotein B ₁₀₀Contact with sample, and observe apoprotein B available from the patient ₁₀₀Secondary structure whether change.

Low-density lipoprotein or apoprotein B ₁₀₀Secondary structure can for example observe by circular dichroism.

The accompanying drawing summary

Figure 1A-1D shows that indigo carmine 1 can be formed isatin sulfonic acid 2 by human artery's atheromatous lesions oxidation that 4-β-phorbol 12-tetradecanoic acid 13-acetic ester (PMA) is handled.

Figure 1A illustrates indigo carmine 1 and is changed into the chemical transformation that takes place in the process of isatin sulfonic acid 2 by ozone.

Figure 1B illustrates the bleaching of PMA-activation atherosclerotic lesion to indigo carmine 1.Each vial contains the dispersion liquid of the atherosclerotic plaque (about 50mg weight in wet base) of equivalent, in the phosphoric acid buffers saline solution of indigo carmine 1 (200, μ M) and ox catalase (50 μ g) (150mM NaCl, pH 7.4 for PBS, 10mM sodium phosphate).The DMSO solution of PMA (10 μ L, 40 μ g/mL) is added photograph after 30 minutes in the bottle of the right.Left side bottle adds the DMSO of the no PMA of equal volume.The cumulative volume of reaction mixture is 1mL.

Fig. 1 C shows that the reversed-phase HPLC analysis draws, shown in Figure 1B+new HPLC peak appears in the supernatant liquor of PMA bottle.This new peak is corresponding to isatin sulfonic acid 2, retention time (R _T) about 9.71 minutes.

Fig. 1 D shows that the negative ion electrospray of supernatant liquor sprays mass spectrum, and this supernatant liquor activates human artery's atherosclerotic plaque material from the described PMA-with indigo carmine 1 reaction of centrifugal above Figure 1B.When using indigo carmine 1 indicator at H ₂ ¹⁸Suspend among the O PMA when activation of patch material, as in the mass spectrum of isolating split product isatin sulfonic acid 2 [M-H] ^-Shown in the outward appearance and relative intensity at 230 mass fragment peaks, the lactan ketonic oxygen of about 40% indigo carmine 1 mixes ¹⁸O.At normal water (H ₂ ¹⁶O) exist the isatin sulfonic acid 2 that generates by indigo carmine 1 down to have [M-H] ^-228 mass fragment peaks.

Fig. 2 A diagram relates to cholesterol 3 ozonolysiss and produces 5, and 6-sterol (secosterol) 4a that breaks changes into the reactions steps of 5a again by aldol condensation.Provide hydrazone derivative 4b and 5b respectively with 2,4 dinitrophenyl hydrazine (2mM is in 0.08%HCl) derivatize.The amount of the 5b that generates from 4a in the derivatize process is about 20%.The conformation of 5a and 5b is arranged by K.Wang, E.Bermudez, and W.A.Pryor, Steroids 58,225 (1993) determines.

Fig. 2 B shows [M-H] as about 18 minutes wash-outs among Fig. 3 ^-The structure of oxygen sterol 6a-9a that the standard substance at 579 peaks are studied and 2,4 dinitrophenyl hydrazine hydrochloride derivative 6b-7b.The conformation of 7a-7b is arranged based on using real synthetic 7b material ¹H- ¹H ROESY experiment.

Fig. 3 A-E diagram is used the analysis of liquid chromatography mass (LCMS) to the chemosynthesis authentic sample of patch material and hydrazone 4b, 5b and 6b.Condition: Adsorbosphere-HS RP-C18 post, 75% acetonitrile, 20% water, 5% methyl alcohol, 0.5mL/ minute flow velocity, 360nm detects, and the patch extract sprays mass spectrum (MS) (Hitachi M8000 chromatographic instrument) with negative ion electrospray in the laggard line of hydrochloric acid 2,4 dinitrophenyl hydrazine (DNPH HCl) derivatize.

Fig. 3 A diagram is without the PMA activation, but the lcms analysis with the patch material behind the 2,4 dinitrophenyl hydrazine derivatize as described herein.In with the atherosclerotic lesion before PMA (the 40 μ g/mL) activation, detect compound 4b (RT～14.1 minute), 5b (RT～20.5 minute) and 6b (RT～18 minute).

Fig. 3 B diagram is used PMA (40 μ g/mL) activation, extracting and as mentioned above with the lcms analysis of the patch material behind the 2,4 dinitrophenyl hydrazine derivatize.In with the atherosclerotic lesion after PMA (the 40 μ g/mL) activation, detect relatively large compound 4b (RT～14.1 minute) and more a spot of compound 6b (RT～18 minute).

The HPLC that Fig. 3 C illustrates true 4b analyzes; Illustration shows mass spectroscopy.

The HPLC that Fig. 3 D illustrates true 6b analyzes; Illustration shows mass spectroscopy.

The HPLC that Fig. 3 E illustrates true 5b analyzes; Illustration shows mass spectroscopy.

The HPLC-MS of the atherosclerotic material matter of Fig. 4 A-D diagram extracting and derivatize analyzes, and wherein uses 100 μ l volume injected, so that detect the trace hydrazone.Fig. 4 A show to use above describe in detail condition time-intensity LC trace.R _T26.7 be 7b (comparing) with true material.R _T～24.7 peak is [M-H] ^-461 unknown hydrazone.Fig. 4 B provides [M-H] ^-597 single ion monitoring.Fig. 4 C provides [M-H] ^-579 single ion monitoring.Fig. 4 D shows [M-H] ^-461 single ion monitoring.

Cholesterol ozonation products concentration in the atherosclerosis extract of Fig. 5 A-C diagram patient A-N.

Fig. 5 A shows to use by oneself the 4a of the patient's atherosclerotic lesion before and after the PMA activation behind extracting and derivatize, the bar graph of the measurement concentration of hydrazone 4b.This bar graph shows and carries out Student t test (bilateral) with the GraphPad Prism on the Macintosh (3.0 version) (numerical value of detection limit before and after the activation of determining n=14) is analyzed in p＜0.05.

Fig. 5 B shows to use by oneself the 5a of the patient's atherosclerotic lesion before and after the PMA activation behind extracting and derivatize, the bar graph (n=14) of the measurement concentration of 5b.

Fig. 5 C shows 5a from patient's plasma sample behind extracting and derivatize, the bar graph of the measurement concentration of 5b.Group A (n=8) patient carried out carotid endarterectomy program (behind the sample collection 3 days carry out plasma analysis) in 24 hours.Group B (n=15) patient is random choose (behind the sample collection 7 days carry out plasma analysis) in the middle of the patient of last general medical science clinic.Notice that in preliminary study, the blood plasma level of 5a descends about 5% every day.Under the condition of this test, the detection limit of 4b and 5b is 1-10nM.Therefore, under the situation that does not have tangible 4b or 5b, the level of 4b or 5b is lower than 10nM.

Fig. 6 A diagram 3,4a and 5a are to the cytotoxicity of B-cell (WI-L2) clone.Each data point is the mean value of at least twice replicate measurement.The IC of 4a (■) and 5a (▲) ₅₀± standard error is calculated by carrying out nonlinear regression analysis (Hill pattern analysis) with the GraphPad Prism on the macintosh computer (3.0 version).In this concentration range, do not observe the cytotoxicity of 3 ().

Fig. 6 B diagram 3,4a and 5a are to the cytotoxicity of T-cell (Jurkat) clone.Each data point is the mean value of at least twice replicate measurement.The IC of 4a (■) and 5a (▲) ₅₀± standard error is calculated by carrying out nonlinear regression analysis (Hill pattern analysis) with the GraphPad Prism on the macintosh computer (3.0 version).In this concentration range, do not observe the cytotoxicity of 3 ().

Fig. 7 A-B shows that cholesterol ozonolysis product 4a and 5a increase the lipid load (lipid-loading) of scavenger cell, produce foam cell.

Fig. 7 A shows few to the lipid effects of load of these scavenger cells with the LDL of J774.1 scavenger cell incubation.At first growth 24 hours in the RPMI-1640 that contains 10% foetal calf serum of scavenger cell, incubation 72 hours in the same medium that contains LDL (100 μ g/mL) then.Cell 4% formaldehyde fixed with phenodin and oil red O stain, makes lipid granule dye darker redness.Magnification * 100.

Fig. 7 B shows that the LDL with ozonolysis product 4a incubation induces the lipid load of scavenger cell, produces foam cell.The J774.1 scavenger cell was grown 24 hours in the RPMI-1640 that contains 10% foetal calf serum.Cell incubation 72 hours in the same medium that contains LDL (100 μ g/mL) and ozonolysis product 4a (20 μ m) then.Cell 4% formaldehyde fixed with phenodin and oil red O stain, makes lipid granule dye darker redness.Magnification * 100.Notice that ozonolysis product 4a can not distinguish with the effect of ozonolysis product 5a mutually to the effect of scavenger cell.

Fig. 8 A-C shows that circular dichroism detects finds that by being exposed to ozonolysis product 4a or 5a, the proteinic secondary structure among the LDL is changed.The result of report derives from least twice repeated experiments of each sample.

Fig. 8 A shows that the protein inclusion of normal LDL has αLuo Xuanjiegou (～40 ± 2%) and more a spot of beta structure (～13 ± 3%), βZhuan Jiao (～20 ± 3%) and the random coil (27 ± 2%) of larger proportion.Fig. 8 A shows the time-dependent manner circular dichroism spectrum of the LDL (100 μ g/ml) among 37 ℃ of following PBS (pH 7.4).

Fig. 8 B shows that 37 ℃ of following LDL cause the secondary structure of apoB-100 to be lost with ozonolysis product 4a incubation in PBS (pH 7.4).LDL (100 μ g/ml) among Fig. 8 A 37 ℃ of following PBS of demonstration (pH 7.4) and the time-dependent manner circular dichroism spectrum of 4a (10 μ m).

Fig. 8 C shows that 37 ℃ of following LDL cause the secondary structure of apoB-100 to be lost with ozonolysis product 5a incubation in PBS (pH 7.4).LDL (100 μ g/ml) among Fig. 8 A 37 ℃ of following PBS of demonstration (pH 7.4) and the time-dependent manner circular dichroism spectrum of 5a (10 μ m).

Fig. 9 illustrates the structure of red sulfonyl hydrazide (being respectively 4d and 5c) of cholesterol ozonation products 4a and 5a and the HPLC elution profile of these hydrazine derivatives.As shown in the figure, cholesterol ozonation products 4a produces the red sulphonyl hydrazone conjugate with different HPLC retention time with 5a.

Figure 10 diagram can be passed through gas phase-mass spectrum (GCMS) analysis, detects cholesterol ozonation products in people's carotid artery sample.Shown in color atlas be the characteristics of atherosclerotic plaque extract.It at the peak of 22.49 minutes wash-outs peak corresponding to cholesterol ozonation products 4a and 5a.Its m/z of material that the mass spectrum that inserts is shown in 22.49 minutes wash-outs is 354.

Figure 11 provides ID-GCMS quantitative analysis to two kinds of atherosclerotic plaques (P1 and P2).Cholesterol ozonation products 4a that detects and the amount of 5a are about 80-100pmol/mg tissue, analyze detect approximate with LC-MS.Each bar is represented the double extract, is expressed as mean value ± standard error of mean.

Detailed Description Of The Invention

The invention provides the method that detects cholesterol ozonation products. The present invention also provides kit and the reagent that detects cholesterol ozonation products. These methods, kit and reagent are for detection of the vascular disorder relevant with the cholesterine accumulation. For example, these methods, kit and reagent are used for diagnosis and monitoring inflammatory arterial disease such as atherosclerotic prognosis.

Cholesterol ozonation

According to the present invention, cholesterine atherosclerotic artery occurs in by reactive oxygen species such as ozone oxidation. Many cholesterol ozonation products are produced by this process, can detect in the tissue that picks up from the atherosclerotic or fluid sample. The detection of cholesterol ozonation products is diagnosis inflammatory arterial disease such as atherosclerotic foundation.

Cholesterine has following structure (3):

Although cholesteric high level is relevant with the possibility that forms atherosclerotic plaque in the blood, there is the pulse atherosclerosis patch in this cholesterine high level and the indefinite expression patient arterial system. Suffer from atherosclerotic lesion for determining that the patient is whether actual, use at present expensive method of testing, such as quick cat scan, with the dyestuff injection of image forming program, perhaps invasive endoscopy or cathterization method.

But, according to the present invention, can by detecting cholesterol ozonation products, detect the existence of actual atherosclerotic plaque. When cholesterine is deposited in the artery, can form atherosclerotic plaque. Although do not want to be confined to concrete mechanism, as if macrophage, neutrophil and other immunocytes drop in the atherosclerotic lesion, and discharge reactive oxygen species such as ozone. The reactive oxygen species that produces and the reaction of the cholesterine in the pathology become the multiple product that can detect with ornitrol oxidation in the patient. Therefore, two events occur successively, cholesterol ozonation products is appeared at pick up from patient's the sample. At first, cholesterine must in a large number accumulation in atherosclerotic plaque. Secondly, atherosclerotic must develop into the stage that wherein produces reactive oxygen species. The just formation that cholesterol ozonation products occurs side by side causing of these two events. Because cholesterine accumulation and ozone generating do not occur under other situations substantially, so the detection of cholesterol ozonation products is whether to have inflammatory disorder of artery such as atherosclerotic accurate index among the patient. In addition, according to the present invention, the amount of picking up from the cholesterol ozonation products that exists in atherosclerotic's the biological sample (for example serum) is that the patient suffers from atherosclerotic seriousness index.

According to the present invention, identified multiple cholesterol ozonation products. For example, when cholesterine 3 was oxidized, formed primary product was disconnected keto-aldehyde 4a and aldol adduct 5a thereof.

In addition, also observe the cholesterol ozonation products that structure is similar to compound 6a-15a and 7c.

According to the present invention, disconnected keto-aldehyde 4a, its aldol adduct 5a and related compound 6a-15a and 7c can be present in atherosclerotic's the atherosclerotic plaque and blood flow. In addition, disconnected keto-aldehyde 4a, its aldol adduct 5a and related compound 6a-15a are relevant with degree and the seriousness that patient's medium sized artery AP forms with 7c. For example, fully develop into eight Atheromatosis conditions and have reason to carry out have six to detect the aldol 5a (Fig. 5 C) of content in the 70-1690nM scope in the middle of the patient of endarterectomy. But, 15 parts from general medical science clinic patient in the middle of patient's the plasma sample of random choose, only have portion can detect 5a.

The present invention therefore imagination determine whether the patient suffers from atherosclerotic lesion, and definite circulation cholesterine is attached to the degree in the atherosclerotic plaque by detecting these cholesterol ozonation products.

The detection of ozone and cholesterine product

Can pass through the obtainable any method of those skilled in the art, detect or identify cholesterol ozonation products. For example, can detect the HPLC of (ELSD), ion detection (ID-GCMS), visible spectrum, ultraviolet spectra or infrared spectrum, thin-layer chromatography, electrophoresis, liquid chromatogram, nuclear magnetic resonance, wet chemistry mensuration, immunoassays (for example ELISA), immunohistochemistry, fluorescence spectrum, visible spectrum or the ultraviolet spectra with gaschromatographic mass spectrometry by high pressure liquid chromatography (HPLC), liquid chromatography mass (LCMS), gas-chromatography (GC), gaschromatographic mass spectrometry (GCMS), high pressure liquid chromatography mass spectrum (HPLC-MS), band evaporative light-scattering, perhaps by obtainable any other method of those skilled in the art, detect or identify these products.

In addition, also can be by observing cholesterol ozonation products to low-density lipoprotein (LDL), apoprotein B₁₀₀The effect of (apoB-100, the protein component of LDL) or macrophage detects the existence of these products. As described herein, cholesterine ozonolysis product 4a and 5a can promote to form foam cells from macrophage. In addition, the secondary structure of cholesterine ozonolysis product 4a and 5a modified LDL and apoB-100. Therefore, can whether can promote foam cells to form or change LDL or apoprotein B by determining sample₁₀₀Secondary structure, come the existence of cholesterine ozonolysis product in the test samples. These tests are for a more detailed description hereinafter.

In certain embodiments, make sample and the reagent reacting that helps to detect and identify cholesterol ozonation products. For example, can make sample with any fluorescence, phosphorescence or have color reagent to contact, described reagent can react with cholesterol ozonation products, and product can use fluorescence, visible light or UV-detector to detect. In other embodiments, do not adopt this reagent, cholesterol ozonation products is identified by its physical property or chemical property. This method is for a more detailed description hereinafter.

Ozone amount in the atherosclerotic plaque material also represents the amount of established atherosclerotic plaque. Therefore, the present invention's imagination is estimated the size of atherosclerotic plaque by the ozone in detection and/or the quantitative atherosclerotic plaque material. Can by using any reagent that can detect ozone, detect the ozone in the atherosclerotic plaque material. For example indigo carmine 1 is that color reagent is arranged, and its blueness disappears when itself and ozone reaction. As follows, in this process, form isatin sulfonic acid 2.

Therefore, can assess with the ozone detection method degree of atherosclerotic plaque accumulation.

But, though can in atherosclerotic material matter, detect ozone, in the blood flow of the patient with a large amount of atherosclerotic plaque materials, can detect cholesterol ozonation products. Therefore, for avoiding separating the atherosclerotic plaque material, those skilled in the art can select the separating blood sample, then detect whether there is cholesterol ozonation products. This is avoided expensive invasive method such as endarterectomy, provides reliable method for there being how many atherosclerotic plaque materials among the estimation patient.

Be diagnosing atherosclerotic, can detect any cholesterol ozonation products, for example disconnected keto-aldehyde 4a, its aldol adduct 5a and/or related compound 6a-15a and 7c. But that carries out up to now studies show that, aldol adduct 5a is one of primary product that can detect in serum.

In certain embodiments, but the cholesterol ozonation products that chemical modification obtains in biological sample, to promote detection. The reagent that can be used for this chemical modification comprises bisulfites, ammonia, schiff bases (using aliphatic series or aromatic amine such as aniline), aromatics or aliphatic hydrazine, red sulfohydrazide, Gerard reagent (semicarbazides), Tollins test reagent (formaldehyde and calcium hydroxide) etc. These reagent are when with cholesterol ozonation products reaction of the present invention, provide the distinctiveness product that can easily be detected by those skilled in the art, such as bisulfite adduct (easily with sodium salt crystal), imines, oxime, hydrazone, semicarbazones, Tollins test product etc.

For example, can easily form the hydazone derivative of disconnected keto-aldehyde 4a, its aldol adduct 5a or related compound 4c, 6a-15a and 7c, they are to determine whether the patient suffers from the useful sign of atherosclerotic lesion. These hydazone derivatives comprise the compound with the structure that is similar to compound 4b-15b and possibility 4c or 10c.

With low these hydazone derivatives that reach about 1nM to 10nM of HPLC mass spectrum concentrations. Use gaseous-mass spectrography, can detect the cholesterol ozonation products that is low to moderate 10fg/ μ L.

Cholesterol ozonation products can for example by reacting with hydrazine compound such as DNPH, change into hydazone derivative. In certain embodiments, reaction is carried out in organic solvent such as acetonitrile or alcohol (for example methyl alcohol or ethanol). Usually adopt sour environment and non-oxygen, the nonactive atmosphere of containing.

For example, blood plasma can obtain from the patient, and places EDTA. This sample can be with the washed with dichloromethane several, with the extracting cholesterol ozonation products. But vacuum evaporation carrene fraction, the residue that contains cholesterol ozonation products dissolves in the alcohol (for example methyl alcohol). The ethanolic solution that then can add 2,4-dinitrophenylhydrazine and 1N HCl. Described solution can be blown into the nitrogen short period (for example 5 minutes), to remove free oxygen. But agitating solution, mixing time are enough to make cholesterol ozonation products to change into its hydazone derivative (for example 2 hours). The primary product that detects in this method it is believed that it is the hydazone derivative of aldol adduct 5a. In addition, Primary Study discloses, can descend about 5% every day from the amount of the 5a of blood plasma extracting. Therefore, the fresh plasma sample will provide the measured value of aldol adduct 5a actual content in the sample more accurately.

Reagent of the present invention and method can be used to detect the atherosclerotic of any developing stage. According to being adopted by AHA and being used for new classification of the present invention, in atherosclerotic evolution, can distinguish eight kinds of pathology types.

I type pathology is formed by the little lipid deposits in the inner membrance (interior and huge the biting in the foam cells of born of the same parents), causes initial and minimum arterial blood tube wall to change. This variation can not thicken the arterial blood tube wall.

II type pathology is take fatty streak as feature, and described fatty streak is yellow cord or spot, and its thickness that increases inner membrance is less than 1 millimeter. They are comprised of the more greasiness matter accumulation of observing than I type pathology. Lipid content is about 20-25% of pathology dry weight. Most of lipids are mainly bitten in foam cells and the smooth muscle cell huge in born of the same parents. Born of the same parents' external space can contain fat drips, but they are less than intracellular, also contains little cryptomere particle. Chemically, lipid is by cholesterol ester (cholesteryl oleate and cholesterine linoleate), cholesterine and Lipid composition.

III type pathology is also referred to as prerolandic artery Rolando gruel type pathology. In III type pathology, endangium is only viewed slightly thick than II type pathology. III type pathology can the obstructing arterial blood flow. The outer lipid of born of the same parents and cryptomere particle with see the identical of II type pathology, but the amount that exists increases (approximately 25-35% dry weight), and begins to be accumulated in the corpusculum (small pool).

IV type pathology is relevant with atheroma. It is crescent, can increase the thickness of artery. Described pathology can not make arterial lumen become very narrow, but the very high people's exception (for many people, described pathology can not be seen by angiography) of blood plasma ornitrol level. IV type pathology is comprised of lipid outside the born of the same parents of theca interna excess accumulation (about 60% dry weight) (being sometimes referred to as fat nuclear (lipid core)). Fat is endorsed and is comprised small mineral matter heap (clamp). These pathologies are easy to break and are easy to form chamber wall thrombus.

The V-type pathology is relevant with the fiber atheroma.They have one or more layers fibrous tissue of mainly being made up of type i collagen.The V-type pathology has the wall that thickness increases, and along with development of atherosclerosis, tube chamber constantly dwindles.The feature that these pathologies have can further be segmented it.In Va type pathology, new organization is the part of the pathology of band fat nuclear.In Vb type pathology, other part calcifications (causing VII type pathology) of fat nuclear and pathology.In Vc type pathology, there is not lipid minimum usually (causing VIII type pathology) in fat nuclear.Usually, the disruptive pathology taking place is Va type pathology.They are softer relatively, have the cholesterol ester rather than the free cholesterol monohydrate crystal of high density.The V-type pathology can be broken, and forms chamber wall thrombus.

VI type pathology is the complex lesions with pathology surface ulceration such as crackle, erosion or ulcer (VIa type), hemotoncus or hemorrhage (VIb type) and thrombosis settling (VIc type), and it covers on IV type pathology and the V-type pathology.The pathology thickness of VI type pathology increases, and tube chamber often gets clogged fully.These pathologies can be converted into the V-type pathology, but they cause obstruction more greatly and more.

VII type pathology be with late period more pathology large stretch of mineral turn to the calcify lesion of feature.Mineralize and take the form of calcium phosphate and phosphatic rock, replace the accumulation resistates of the outer lipid of dead cell or born of the same parents.

VIII type pathology is the fibrosis pathology of mainly being made up of multilayer collagen, contains few lipid.VIII type pathology may be that the lipid of thrombus disappears or the lipid pathology changes into the result of collagen in a large number.These pathologies can be blocked the tube chamber of median size artery.

As described herein, cholesterol ozonolysis product 4a and 5a can promote to form foam cell from scavenger cell, but and modified low density lipoprotein (LDL) and apoprotein B ₁₀₀The secondary structure of (protein component of LDL).Do not activating in the presence of the mouse macrophage LDL with 4a or 5a incubation.The beginning lipid was loaded after these scavenger cells were exposed to 4a or 5a, and foam cell begins to occur (referring to Fig. 7) in reaction vessel.In addition, circular dichroism detects finds (Fig. 8 B, C), and people LDL (100 μ g/ml) causes the time-dependent manner of apoB-100 structure to change with 4a and 5a (10 μ M) incubation.Shown in Fig. 8 A, the albumen inclusion of normal LDL has αLuo Xuanjiegou (～40 ± 2%) and more a spot of beta structure (～13 ± 3%), βZhuan Jiao (～20 ± 3%) and the random coil (27 ± 2%) of larger proportion.But as LDL during with 4a and 5a incubation, secondary structure has considerable damage.The loss of secondary structure mainly is loss (4a～23 ± 5% of αLuo Xuanjiegou; 5a～20 ± 2%).Correspondingly, observe random coil (4a～39 ± 2% of higher per-cent; 5a～32 ± 4%).Therefore, 4a can directly cause some physiological change relevant with debatable atherosclerosis with 5a cholesterol ozonolysis product.

Therefore the present invention provides the debatable cholesterol ozonolysis product of diagnosis whether to be present in method in the sample.In certain embodiments, this method relates to the variation whether definite sample can cause scavenger cell picked-up lipid aspect.Picked-up increases if sample is observed lipid behind the scavenger cell incubation, and then sample contains cholesterol ozonolysis product, and the patient who obtains sample may suffer from debatable atherosclerosis.In another embodiment, but the invention provides by test samples whether modified LDL or apoprotein B ₁₀₀Secondary structure, the method for coming the cholesterol ozonolysis product in the test samples.Can use the obtainable method of those skilled in the art, for example LDL or apoprotein B are monitored or observed to circular dichroism or calorimetry ₁₀₀Secondary structure.

The quantitative measurment of cholesterol ozonation products can be used to diagnose the pathology that has which and/or what type in the animal that obtains biological sample in atherosclerosis stage in the biological sample.Test can be tabulating with the amount of cholesterol ozonation products in these samples from known biological sample with the definite pathology type or the patient colony in definite atherosclerosis stage.This form be statistical study with make atherosclerosis stage (or pathology type) and patient's sample in the amount of cholesterol ozonation products the related possibility that provides is provided.Can calculate the mean value and the scope of the cholesterol ozonation products amount of each patient colony, thus the atherosclerosis stage that exists among the measurable new patient of understanding to sterol ozonation products amount in new patient's sample.Similarly, also quantitatively biological sample can cause scavenger cell lipid load or cause low-density lipoprotein and/or apoprotein B ₁₀₀The degree of secondary structural change, and can with its with the atherosclerotic in the atherosclerosis stage and/or the pathology type that exist set up related.

Can carry out the quantitative measurment of the amount of cholesterol ozonation products in patient's sample by any obtainable method.For example, can be by determining to derive from high pressure liquid chromatography (HPLC), liquid chromatography mass (LCMS) but, visible spectrum, UV spectrum, infrared spectra, gas-chromatography, liquid chromatography or other those skilled in the art's preparation methods read area under the peak, carry out quantitative measurment.In other embodiments, the size of available thin-layer chromatography spot or electrophoretic band or optical density(OD) are come the amount of cholesterol ozonation products in the quantitative sample.The optical density(OD) of wet chemistry reaction assay mixture, color reaction or immunoassay (for example ELISA) also can be used to the amount of cholesterol ozonation products in the quantitative sample.When being exposed to sample, show the percentage of the scavenger cell that lipid is loaded or the quantitative measurment that quantity also can be used as the amount of cholesterol ozonation products in the sample.Similarly, LDL or apoprotein B when being exposed to sample ₁₀₀The intensity of variation of secondary structure or percentage can be used as the quantitative measurment of the amount of cholesterol ozonation products in the sample.

In another embodiment, this product can detect by immunoassay.The invention provides can be in conjunction with the antibody and the binding entity (binding entity) of any formula 3,4a-15a, 4b-15b, 3c, 4c, 7c or 10c compound.The invention further relates to haptens relevant on the structure with the hydrazone derivative of cholesterol ozonation products and this ozonation products.For example, the invention provides haptens with formula 3c, 13a, 13b, 14a, 14b, 15a or 15b, its can be used to produce can with the antibody of cholesterol ozonation products and the reaction of hydrazone product:

Antibody and binding entity

The invention provides antibody preparation and binding entity at cholesterol ozonation products, haptens and relevant cholesterol sample molecule, it can be used for detecting and identifying cholesterol ozonation products.For example, antibody of the present invention or binding entity can be in conjunction with arbitrary formula 3,4a-15a, 4b-15b, 3c, 4c, 7c or 10c compounds.Term binding entity used herein comprises that antibody and other can be in conjunction with the polypeptide of cholesterol ozonation products.

In one embodiment, antibody or binding entity are optionally in conjunction with arbitrary formula 3,4a-15a, 4b-15b, 3c, 4c, 7c or 10c compound.In another embodiment, antibody or binding entity can be in conjunction with not only a kind of formula 3,4a-15a, 4b-15b, 3c, 4c, 7c or 10c compounds.The specific examples of antibody preparation produces at formula 13a, 14a, 13b, 14b or 15a compound.Specifically, hybridoma KA1-11C5 and KA1-7A6 provide the antibody preparation that produces at formula 15a compound.Hybridoma KA2-8F6 and KA2-1E9 provide the antibody preparation that produces at formula 14a compound.

Clause according to budapest treaty, the hybridoma KA1-11C5 and the KA1-7A6 that will produce at formula 15a compound on August 29th, 2003 are preserved in American type culture collection (10801 University Blvd., Manassas, Va., 20110-2209 USA (ATCC)), the ATCC preserving number is PTA-5427 and PTA-5428.According to the clause of budapest treaty, the hybridoma KA2-8F6 and the KA2-1E9 that will produce at formula 14a compound on August 29th, 2003 is preserved in ATCC equally, and the ATCC preserving number is PTA-5429 and PTA-5430.

The present invention also provide by the preparation of existing method can be in conjunction with the antibody of cholesterol ozonation products.The binding domains of this antibody, for example the CDR district of these antibody can be transferred to or utilizes with any skeleton of binding entity easily.

Antibody molecule belongs to the plasma proteins family that is called immunoglobulin (Ig), and its basic building block---immunoglobulin folding or structural domain are used in many molecules of immunity system and other biological recognition system in a variety of forms.The antibody of standard is tetramer structure, is made up of two identical heavy chain immunoglobulins and two identical light chains, and molecular weight is about 150,000 dalton.

The heavy chain of antibody is made up of different structural domains with light chain.Every light chain has a variable region structural domain (VL) and a constant region structural domain (CL), and every heavy chain has a variable region structural domain (VH) and three or four constant region structural domains (CH).Referring to for example Alzari, P.N., Lascombe, M.-B.﹠amp; Poljak, R.J. (1988) Three-dimensional structure ofantibodies.Annu.Rev.Immunol.6,555-580.It is immunoglobulin folding that each structural domain of being made up of about 110 amino-acid residues is folded into characteristic β sandwich structure, and described β sandwich structure is formed by two βZhe Dies that are crowded together mutually.VH and VL structural domain respectively have three complementary determining regions (CDR1-3), and it is ring or corner, at the end connection β of structural domain chain.All make contributions to antigen-specific usually in the variable region of light chain and heavy chain, although always each independent chain is unimpartial to specific contribution.Antibody molecule is by using six randomization rings (CDR), and being evolved into can be in conjunction with a large amount of molecule.

According to the aminoacid sequence of its CH, immunoglobulin (Ig) can be classified as different classifications.Have at least five (5) to plant main immunoglobulin class: IgA, IgD, IgE, IgG and IgM.Several in these classifications can further be subdivided into subclass (isotype), for example IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.CH corresponding to IgA, IgD, IgE, IgG and the IgM classification of immunoglobulin (Ig) is called alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ).According to the aminoacid sequence of its constant region, the light chain of antibody can be classified as a kind of in two kinds of distinct types, and described two types are called kappa (κ) and lambda (λ).The subunit structure of different classes of immunoglobulin (Ig) and 3-d modelling are known.

With regard to the variable region of antibody, term " variable " refers to that some part of variable region structural domain between a kind of antibody and the another kind of antibody has this fact of very big difference on sequence.The variable region structural domain is to be used for bonded, and it determines each antibody specific to its concrete antigenic specificity.But mutability is uniform distribution in the middle of the antibody variable region structural domain not.On the contrary, mutability concentrates on three sections that are called complementary determining region (CDR is also referred to as the hypervariable region) in variable region of light chain structural domain and the variable region of heavy chain structural domain.

The conservative variable region structural domain of height partly is called framework (FR) district.Each self-contained four FR district that mainly take the βZhe Die configuration of the variable region structural domain of natural heavy chain and light chain, described FR district is connected by three CDR, forms the ring that connects the βZhe Die structure, constitutes the part of βZhe Die structure in some cases.CDR in each chain is close to together by the FR district, makes contributions with the formation of the antigen binding site of the CDR antagonist of other chains.The constant region structural domain does not directly relate to antibody and combines with antigenic, but shows various effector functions, participates in antibody dependent cellular cytotoxicity as antibody.

Therefore, it can be any various forms that imagination is used for antibody of the present invention, comprise complete immunoglobulin (Ig), antibody fragment such as Fv, Fab and similar fragment, the single-chain antibody that comprises variable region structural domain complementary determining region (CDR), and similar forms, all these forms all falls within the generality term used herein " antibody ".The present invention imagines any specificity of using polyclonal antibody or monoclonal antibody, is not limited to discern and with concrete cholesterol ozonation products or derivatives thereof immunoreactive antibody takes place.

In addition, the skeleton that the land or the CDR of antibody can be placed any appropriate combination entity polypeptide.In preferred embodiments, with regard to method as herein described, use the antibody, binding entity or its fragment that any formula 3-15 compound and haptens thereof and derivative are had (comprising hydrazone derivative) immunologic opsonin.

Term " antibody fragment " refers to the part of full length antibody, normally antigen binding domain or variable region.The example of antibody fragment comprises Fab fragment, Fab ' fragment, F (ab ') ₂Fragment and Fv fragment.Produce two segmental Fabs of the identical Fab of being called (each fragment has single antigen binding site) and remaining Fc fragment with papain digestion antibody.Therefore the Fab fragment has the part of a complete light chain and a heavy chain.With pepsin produce have two can with the F (ab ') of the Fab of antigen cross-linking ₂Fragment and be called the segmental remaining fragment of pFc '.After the antibody of gastric pepsin digestion is reduced, obtain Fab ' fragment, its part by complete light chain and heavy chain is formed.Each antibody molecule can obtain two Fab ' fragments.Fab ' fragment and Fab fragment difference are to add the minority residue at the C-terminal of heavy chain CH1 structural domain, comprise one or more halfcystines from antibody hinge region.

Fv is the minimum antibody fragment that comprises complete antigen identification and binding site.This district is by the dimer (V of a firm non-covalent associating variable region of heavy chain structural domain and a variable region of light chain structural domain _H-V _LDimer) forms.Three CDR of each variable region structural domain interact with this configuration just, establish V _H-V _LThe lip-deep antigen binding site of dimer.Six CDR give the antibody antigen binding specificity jointly.But, even single variable region structural domain also has (or only comprising three half Fv that resists original specific CDR) ability of identification and conjugated antigen, although lower than the avidity of complete binding site." function fragment " that relates to antibody used herein refers to Fv, F (ab) and F (ab ') ₂Fragment.

Other fragment can comprise double antibody, line style antibody, single-chain antibody molecule and the multi-specificity antibody that is formed by antibody fragment.Single-chain antibody is the genetically engineered molecule that comprises variable region of light chain and variable region of heavy chain, connects into genetically engineered by suitable polypeptide chain junctor and merges single chain molecule.This single-chain antibody is also referred to as " strand Fv " or " sFv " antibody fragment.Usually, the Fv polypeptide also comprise in addition between VH structural domain and the VL structural domain, make sFv can form the polypeptide chain junctor of the antigen integrated structure of needs.The summary of relevant sFv is referring to the ThePharmacology of Monoclonal Antibody of Pluckthun, and the 113rd rolls up, and Rosenburg and Moore edit, Springer-Verlag, N.Y., 269-315 page or leaf (1994).

Term " double antibody " refers to have the little antibody fragment of two antigen binding sites, and wherein said fragment comprises variable region of heavy chain structural domain (VH) that variable region of light chain structural domain (VL) connects (VH-VL) on same polypeptide chain.By using short two structural domain generation paired linkers that can not make on the same chain, described structural domain is forced to the complementary structure territory pairing with another chain, causes two antigen binding sites.Double antibody is at for example EP 404,097; WO 93/11161 and Hollinger etc. have among the Proc.Natl.Acad Sci.USA 90:6444-6448 (1993) more fully and describe.

Therefore, the antibody fragment of the present invention's imagination is not a full length antibody.But with respect to full length antibody, this antibody fragment can have similar or improved immunological properties.This antibody fragment can be as small as about 4 amino acid, 5 amino acid, 6 amino acid, 7 amino acid, 9 amino acid, about 12 amino acid, about 15 amino acid, about 17 amino acid, about 18 amino acid, about 20 amino acid, about 25 amino acid, about 30 amino acid or more.

In general, with respect to the antibody of specificity,, can have the highest any size restriction as long as antibody fragment of the present invention has similar or improved immunological properties in conjunction with cholesterol ozonation products.For example, less binding entity and light chain antibody fragment can have less than about 200 amino acid, less than about 175 amino acid, less than about 150 amino acid or less than about 120 amino acid, if described antibody fragment is relevant with light chain antibody subunit.In addition, bigger binding entity and heavy chain antibody fragment can have less than about 425 amino acid, less than about 400 amino acid, less than about 375 amino acid, less than about 350 amino acid, less than about 325 amino acid or less than about 300 amino acid, if described antibody fragment is relevant with heavy chain antibody subunit.

Antibody at cholesterol ozonation products of the present invention can prepare by any existing method.The method for preparing polyclonal antibody is that those skilled in the art are obtainable.Referring to for example Green etc., Production of Polyclonal Antisera, see: Immunochemical Protocols(Manson edits), 1-5 page or leaf (Humana Press); Coligan etc., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, see: Current Protocols in Immunology, 2.4.1 saves (1992), and described reference is attached to herein by reference.

Monoclonal antibody also can be applicable to the present invention.Term used herein " monoclonal antibody " refers to the antibody that obtains from basic homogeneous antibody colony.In other words, except having in more a spot of some antibody the accidental natural sudden change, the single antibody that constitutes colony all is identical.Monoclonal antibody is a high degree of specificity, can be at single antigen site.In addition, with the polyclonal antibody preparation difference that generally includes at the different antibodies of different determinants (epi-position), each monoclonal antibody is at the single determinant on the antigen.Except their specificity, the advantage of monoclonal antibody is that also they are next synthetic by the hybridoma cultivation, is not subjected to the pollution of other immunoglobulin (Ig)s.Modifier " mono-clonal " expression antibody can not be interpreted as requiring antibody to produce by any concrete grammar from the characteristic that basic homogeneous antibody colony obtains.

The monoclonal antibody of this paper clearly comprises " chimeric " antibody, in described " chimeric " antibody a part of heavy chain and/or light chain be derived from specific species or belong to the consistent or homology of corresponding sequence of the antibody of specific antibodies classification or subclass, the rest part of described chain be derived from another species or belong to the consistent or homology of corresponding sequence of the antibody of another antibody classification or subclass.Also can use the fragment of this antibody, as long as they show required biological activity.Referring to United States Patent (USP) the 4th, 816, No. 567; Morrison etc., Proc.Natl.Acad Sci.81,6851-55 (1984).

MONOCLONAL ANTIBODIES SPECIFIC FOR is conventional equally.Referring to for example Kohler ﹠amp; Milstein, Nature, 256:495 (1975); Coligan etc., the 2.5.1-2.6.7 joint; With Harlow etc., see: Antibodies:A Laboratory Manual, the 726th page (Cold Spring Harbor Pub. (1988)), these reference are attached to herein by reference.Can separate and monoclonal antibody purification from the hybridoma culture by various technology of having established.This isolation technique comprises albumin A sepharose affinity chromatography, size exclusion chromatography and ion exchange chromatography.Referring to for example Coligan etc., 2.7.1-2.7.12 joint and 2.9.1-2.9.3 save; Barnes etc., Purification ofImmunoglobulin G (IgG), see: Methods in Molecular Biology, the 10th volume, 79-104 page or leaf (Humana Press (1992).

External and method in-vivo procedures antibody is that those skilled in the art are obtainable.For example, the monoclonal antibody of using according to the present invention can prepare by above-mentioned hybridoma method, maybe can be by as United States Patent (USP) the 4th, 816, and the recombination method of description prepares in No. 567.Also can use at Clackson etc., the technology of describing among Nature 352:624-628 (1991) and the Marks etc., J.Mol Biol.222:581-597 (1991) is separated from phage antibody library and to be used for monoclonal antibody of the present invention.

The method for preparing antibody fragment also be well known in the art (referring to for example Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork, (1988), it is attached to herein by reference).Can prepare antibody fragment of the present invention by the proteolysis of antibody or by in appropriate host, expressing the segmental nucleic acid of encoding antibody.Can obtain antibody fragment by the ordinary method of stomach en-or papain digestion whole antibody.For example, can provide to be called F (ab ') by carry out the enzymolysis cutting with the stomach en-antagonist ₂The 5S fragment, produce antibody fragment.Can use thiol reductant and the optional blocking groups that uses the sulfydryl that obtains by cracked disulfide bond, further cut this fragment, produce 3.5S Fab ' unit price fragment.Perhaps, carry out the enzymolysis cutting with stomach en-and directly produce two unit price Fab ' fragments and a Fc fragment.These methods reach in the reference that wherein comprises for the 4th, 036, No. 945 and 4,331, No. 647 at for example United States Patent (USP) description.These patents integral body by reference are attached to herein.

Also can use other antibody cutting methods, as separate heavy chain with form unit price light-heavy chain fragment, segmental further cutting, perhaps other zymetologys, chemistry or genetics technology are as long as fragment is in conjunction with the antigen of being discerned by complete antibody.For example, the Fv fragment comprises V _HChain and V _LThe association of chain.This association can be non-covalent, and perhaps variable chains can be by the intermolecular disulfide bond connection or by chemical reagent such as glutaraldehyde cross-linking.Preferred Fv fragment comprises the V that connects by the peptide linker _HChain and V _LChain.By making up the V that encodes that comprises by the oligonucleotide connection _HStructural domain and V _LThe structure gene of the dna sequence dna of structural domain prepares these single chain antigen binding proteins (sFv).Structure gene is inserted in the expression vector, and the latter is incorporated into host cell such as intestinal bacteria subsequently.Recombinant host cell synthesizes the single polypeptide chain, two V structural domains of linker peptide bridge joint wherein.The method of producing sFv is by for example Whitlow etc., Methods:a Companion to Methods in Enzymology, the 2nd volume, the 97th page (1991); Bird etc., Science 242:423-426 (1988); Ladner etc., United States Patent (USP) the 4th, 946, No. 778; With Pack etc., Bio/Technology11:1271-77 (1993) describes.

The antibody fragment of another kind of form is the peptide of coding (coding for) single complementary determining region (CDR).CDR peptide (" atom ") often participates in antigen recognition and combination.Can obtain the CDR peptide by the gene of clone or structure coding purpose antibody CDR.This gene is for example by using the polymerase chain reaction to prepare from the RNA synthetic variable region of antibody producing cells.Referring to for example Larrick etc., Methods:a Companion to Methods in Enzvmology, the 2nd volume, the 106th page (1991).

The present invention imagines inhuman (for example mouse) antibody of people's form and humanization form.This humanized antibody is that gomphosis immunoglobulin, immunoglobulin chain or its fragment are (as Fv, Fab, Fab ', F (ab ') ₂Or other antigens of antibody are in conjunction with subsequence), it contains the minmal sequence that is derived from non-human immunoglobulin.To a great extent, humanized antibody is human normal immunoglobulin (receptor's antibody), is wherein replaced as the CDR residue of the required specificity of having of mouse, rat or rabbit, avidity and capacity from inhuman species (donor antibody) from receptor's complementary determining region (CDR) residue.

In some cases, the Fv framework residue of human normal immunoglobulin is by corresponding inhuman residue displacement.In addition, humanized antibody can comprise and neither sees receptor's antibody and also do not see the CDR of introducing or the residue of framework sequence.Causing these modifications is further to improve and optimization antibody performance.In general, humanized antibody comprises the whole of at least a, common two kinds of variable region structural domains substantially, wherein whole or whole substantially CDR districts is corresponding with non-human immunoglobulin, and whole or whole substantially FR districts is the human normal immunoglobulin consensus sequence.Humanized antibody preferably also comprises at least a portion constant region for immunoglobulin (Fc), normally some people constant region for immunoglobulin.More details is referring to Jones etc., and Nature 321,522-525 (1986); Reichmann etc., Nature 332,323-329 (1988); Presta, Curr.Op.Struct.Biol.2,593-596 (1992); Holmes etc., J.Immunol., 158:2192-2201 (1997) and Vaswani etc., Annals Allergy, Asthma ﹠amp; Immunol., 81:105-115 (1998).

Produce antibody though can obtain standardized means, the complicacy of six coupling collars that exist in the size of antibody, the multichained construction of antibody and the antibody has constituted the obstacle of improvement and production lot of antibodies.Therefore, the present invention further imagines the use binding entity, and it comprises can discern and in conjunction with the polypeptide of cholesterol ozonation products.

Numerous protein can serve as protein scaffolds, and the binding domains of cholesterol ozonation products can connect thereon, thereby forms suitable binding entity.Binding domains combines with cholesterol ozonation products of the present invention or interacts, and protein scaffolds is only supported and the stable bond structural domain, so that they can carry out combination.There is the range protein support to use.For example, can use the phage capsid protein.Referring to Clackson ﹠amp; Wells, the summary among the Trends Biotechnol.12:173-184 (1994).The phage capsid protein has been used as the support of display random peptide sequence, comprise ox pancreas trypsin inhibitor (Roberts etc., PNAS 89:2429-2433 (1992)), human growth hormone (Lowman etc., Biochemistry 30:10832-10838 (1991), Venturini etc., Protein Peptide Letters 1:70-75 (1994)) and streptococcic IgG binding domains (O ' Neil etc., Techniques in Protein Chemistry V (Crabb, L edits) the 517-524 page or leaf, Academic Press, San Diego (1994)).These supports have been showed single randomization ring or the district that can be modified with the binding domains that comprises cholesterol ozonation products.

The investigator has used also that little 74 amino acid whose αDian Fenmei inhibitor---tendamistat (Tendamistat) is as the support of presenting on the filobactivirus M13.McConnell，S.J.，&Hoess，R.H.，J.Mol.Biol.250：460-470(1995)。Tendamistat is the βZhe Die protein from streptoverticillium (Streptomyces tendae).The various features that it has makes it become attractive polypeptide in conjunction with support, comprises small size, stability and can obtain high-resolution NMR and the x-ray structure data.The overall topological structure of tendamistat is similar to the structural domain of immunoglobulin (Ig), and two βZhe Dies connect by a series of ring.Form with immunoglobulin domains and correlatedly to be, the βZhe Die of tendamistat is by two rather than a disulfide bonds, and this is the sizable reason of this protein stability.The ring of tendamistat can play the function that is similar to the CDR ring that sees immunoglobulin (Ig), and can be randomized by vitro mutagenesis easily.Tendamistat is derived from streptoverticillium, in the mankind antigenicity can be arranged.Therefore, adopt the binding entity of tendamistat preferably in external application.

Fibronectin III type structural domain also has been used as the protein scaffolds that can connect binding entity.Fibronectin III type is the part of the big subfamily (Fn3 family or s-type Ig family) of immunoglobulin superfamily.Sequence, carrier and the cloning process of this fibronectin III type structural domain as the protein scaffolds of binding entity (as the CDR peptide) for example provided in the U.S. Patent Application Publication 20020019517.In addition referring to Bork, P.﹠amp; Doolittle, R.F. (1992) Proposedacquisition of an animal protein domain by bacteria.Proc.Natl.Acad.Sci.USA 89,8990-8994; Jones, E.Y. (1993) The immunoglobulin superfamilyCurr.Opinion Struct.Biol.3,846-852; Bork, P., Hom, L.﹠amp; Sander, C. (1994) The immunoglobulin fold.Structural classification, sequencepatterns and common core.J.Mol.Biol.242,309-320; Campbell, I.D.﹠amp; Spitzfaden, C. (1994) Building proteins with fibronectin type III modulesStructure 2,233-337; Harpez, Y.﹠amp; Chothia, C. (1994).

In immunity system, select and specific amplification antibody (affinity maturation) from big library.Can pass through the mutagenesis and the generation of the combinatorial library of binding entity, simulate the combination technique that immunocyte uses.Therefore, the binding entity of variation, antibody fragment and antibody also can produce by the technology of showing type.The technology of this displaying type comprises for example phage display, retrovirus displaying, ribosomal display and other technologies.Can use the obtainable technology in this area to produce library and these libraries of screening of binding entity, the binding entity of selecting can carry out other ripening process, as affinity maturation.Wright and Harris, ibid, Hanes and PlucthauPNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene73:305-318 (1988) (phage display), Scott TIBS 17:241-245 (1992), Cwirla etc., PNAS USA 87:6378-6382 (1990), Russel etc., Nucl.Acids Research21:1081-1085 (1993), Hoganboom etc., Immunol.Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH 10:80-84 (1992) and United States Patent (USP) the 5th, 733, No. 743.

Therefore the present invention also provides and makes antibody, CDR or binding domains sudden change, needs the method for character with their avidity of optimization, selectivity, bonding strength and/or other.The sudden change binding domains refers to the variant amino acid sequence body of the binding domains (for example CDR) selected.In general, the one or more amino-acid residues in the sudden change binding domains are with different with reference to what exist in the binding domains.The sequence identity of this sudden change antibody and reference amino acid sequence or similarity must be less than 100%.In general, the sudden change binding domains with reference to the aminoacid sequence of binding domains at least 75% consensus amino acid sequence or similarity are arranged.Preferred sudden change binding domains has at least 80%, more preferably at least 85% even more preferably at least 90%, most preferably at least 95% consensus amino acid sequence or similarity with aminoacid sequence with reference to binding domains.

For example, use the affinity maturation of phage display to can be used as a kind of method that produces the sudden change binding domains.Use that the affinity maturation of phage display refers at Lowman etc., Biochemistry 30 (45): the process of describing among the 10832-10838 (1991), and in addition referring to Hawkins etc., J.Mol Biol.254:889-896 (1992).Though not strictly be confined to following description, this process can briefly be described as relating to the sudden change in a plurality of different loci of several binding domainss or antibody hypervariable region, and purpose is to produce all possible amino-acid substitution in each site.The binding domains mutant of Chan Shenging is shown as fusion rotein in the unit price mode from the filobactivirus particle like this.Usually the gene III product to M13 causes fusion.Express phage several wheel the capable of circulation of different mutants, to select purpose characteristic, for example binding affinity or selectivity.Separate purpose mutant and order-checking.This method is described in greater detail in United States Patent (USP) the 5th, 750, No. 373, No. the 6th, 290,957, United States Patent (USP) and Cunningham, and B.C. etc., EMBO are (11) J.13, among the 2508-2515 (1994).

Therefore, in one embodiment, the invention provides the nucleic acid of handling binding entity or antibody polypeptides or encoding them, have the method for the binding entity in conjunction with character, antibody and the antibody fragment of improved identification cholesterol ozonation products with generation.

The method of this part sudden change that makes existing binding entity or antibody relates to: the coding nucleic acid of the coded polypeptide of cholesterol ozonation products binding domains is fused to the coding nucleic acid of phage coat protein to produce the recombinant nucleic acid of encoding fusion protein, the recombinant nucleic acid sudden change that makes encoding fusion protein is to produce the mutant nucleic acid of encoding mutant fusion rotein, on the surface of phage, express the sudden change fusion rotein, and select phage in conjunction with cholesterol ozonation products.

Therefore, the invention provides and to discern and in conjunction with antibody, antibody fragment and the binding entity polypeptide of cholesterol ozonation products, haptens or cholesterin derivative.The present invention further provides and handle these antibody, antibody fragment and binding entity polypeptide, with optimization they need the method for character (for example stability, size, use easily) in conjunction with character or other.

Can modify this antibody, antibody fragment and binding entity polypeptide, can be used for detecting mark or the reporter molecules that antibody exists to comprise.Mark used herein or reporter molecules be can directly or indirectly link to each other with antibody and directly or indirectly cause measuring, any molecule of detectable signal.Many this marks that mix or be coupled to antibody or binding entity are that those skilled in the art are obtainable.The example that is fit to the mark that uses with antibody of the present invention or binding entity comprises radio isotope, fluorescence molecule, phosphorescent molecules, enzyme, second antibody and part.

Suitable fluorescently-labeled example comprises fluorescein (FITC), 5,6-carboxymethyl fluorescein, texas Red, oil of mirbane-2--1,3-diazole-4-base (NBD), tonka bean camphor, dansyl chloride, rhodamine, 4 '-6-diamidine base-2-phenylindone (DAPI) and cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.In certain embodiments, fluorescent mark is fluorescein (5-Fluoresceincarboxylic acid-N-hydroxy-succinamide ester) or rhodamine (5,6-tetramethyl-rhodamine).The fluorescent mark that is used for the combination polychrome (combinatorial multicolor) of some embodiment comprises FITC and cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7.The maximum absorption and the emission maximum of these fluorescent agents are respectively: FITC (490nm; 520nm), Cy3 (554nm; 568nm), Cy3.5 (581nm; 588nm), Cy5 (652nm; 672nm), Cy5.5 (682nm; 703nm) and Cy7 (755nm; 778nm), make them to detect simultaneously like this.This fluorescent mark can obtain from multiple commercial source, comprises Molecular Probes, Eugene, OR and Research Organics, Cleveland, Ohio.

The obtainable sensitive method of certification mark such as the available subsequently this area of vitamin H that is incorporated in antibody or the binding entity detects.For example, vitamin H can followingly detect: use the Streptavidin-alkaline phosphatase conjugate (Tropix. in conjunction with vitamin H, Inc.), can pass through suitable substrates (chemical luminous substrate CSPD:3-(4-methoxyl group spiral shell-[1 for example subsequently, 2,-dioxetane-3-2 '-(5 '-chlorine) three ring [3.3.1.1.sup.3,7] decane]-the 4-yl) disodium phenylphosphate; Tropix, chemoluminescence Inc.) detects.

Also can use in conjunction with two or more these reporter molecules or the molecule of certification mark in the present invention.Any known certification mark can use with disclosed antibody, antibody fragment, binding entity and method.The detection and the measuring method of the signal that is produced by certification mark also are that those skilled in the art are obtainable.For example, radio isotope can detect by scintillation counting or direct colour developing (visualization); Fluorescence molecule can detect with spectrophotofluorometer; Phosphorescent molecules can detect with scanner or spectrophotometer, or directly manifests with photographic camera; Enzyme can be by enzymic catalytic reaction the colour developing of product detect.This method can be directly used in the disclosed sterol ozonation products detection method.

The mensuration of cholesterol ozonation products

The obtainable any assay method of those skilled in the art can be used for detecting cholesterol ozonation products, comprises in order to the cholesterol haptens of detection indication cholesterol ozonation or the assay method of cholesterin derivative.For example, described method of testing can adopt mass spectrum, gas-chromatography or liquid chromatography, nucleus magnetic resonance, infrared spectra, UV spectrum, visible spectrum or high pressure liquid chromatography.In certain embodiments, can use immunoassay to detect any formula 3,4a-15a, 3c, 4c, 7c, 10c or 4b-15b compound.

Assay method can be in order to detect the cholesterol ozonation products from the sample that multiple source obtains, and described sample comprises for example from mammiferous serum, blood plasma, blood, lymph liquid, tissue (for example patch sample), saliva, urine, stool and other biological sample.In certain embodiments, sample is a tissue sample.But in other embodiments, sample is body fluid such as urine, blood or serum.Mensuration to this sample from mammalian object makes and can carry out non-invasive diagnostic to vascular disease.For example, can be from the mammalian object collection liquid, and cholesterol ozonation products measured as the constraint of the cytolemma on the factor that discharges in the sample liquids or the cell factor.

In certain embodiments, adopt immunoassay.This immunoassay can relate to the obtainable any measuring method of those skilled in the art.The example of immunoassay comprises radioimmunoassay, competitive binding assay, sandwich assay and immune precipitation determination.Can or be connected to detectable label as herein described with binding entity combination of the present invention.Depend on applicable cases, the selection meeting of used mark is different, can be selected by those skilled in the art.

In enforcement of the present invention, detectable label can be inner complex, vitamin H, fluorophore, chromophore, heavy metal, the inner complex of heavy metal, the impenetrable compound of X-ray or element, radio isotope or the radioisotopic inner complex of enzyme such as horseradish peroxidase or alkaline phosphatase, paramagnetic ion, paramagnetic ion.

The radio isotope that can be used as detectable label comprises following isotropic substance: iodo-123, iodine-125, iodo-128, iodine-131, the perhaps chelated metal ions of chromium-51, cobalt-57, gallium-67, indium-111, indium-113m, mercury-197, selenium-75, thallium-201, technetium-99m, lead-203, strontium-85, strontium-87, gallium-68, samarium-153, europium-157, ytterbium-169, zinc-62 or rhenium-188.

Paramagnetic ion as detectable label comprises following ion: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) or ytterbium (III).

Radioimmunoassay is used for radioactivity the measurement of the mixture between binding entity (for example antibody) and the cholesterol ozonation products usually.In this method, binding entity is radiolabeled.Make the reaction of binding entity and unlabelled cholesterol ozonation products.For example centrifugal by post precipitation then, never bond material separates radiolabeled mixture.In case the mixture between radiolabeled binding entity and the cholesterol ozonation products separates with bond material not, can be by direct measuring radiation or by observing the effect of radio-labeling, the amount of coming quantitative mixture to fluorescence molecule such as phenylbenzene  azoles (DPO).Back one method requires less radioactivity, and sensitive more.This be called scintigraphic method measure by radio-labeling as ³H or ³²The fluorescent emission of the dye solution that P excites.By measuring the intensity of fluorescence that discharges from fluorescent grain, determine the bonded degree.This advantage that is called the method for scintillation proximity assay (SPA) is to measure the binding entity mixture that original position forms, and need not the unconjugated radioactivity binding entity of flush away.

Competitive binding assay depends on the mark standard substance combine limited amount binding entity with the competition of sample analysis thing ability.The mark standard substance can be cholesterol ozonation products or immunoreactivity haptens or derivatives thereof.The amount of sample is inversely proportional to the amount that is attached to the standard substance of binding entity.For helping measuring the amount that the bonded standard substance take place, before or after competition, cause the binding entity that is adopted insoluble usually.Do like this is for the standard substance that are attached to binding entity can be separated with analyte with keeping unconjugated standard substance easily with analyte.

Sandwich assay relates to uses two kinds of binding entities, and every kind of binding entity separately can be in conjunction with the different immunogenicity parts or the epi-position of product to be detected.In sandwich assay, the sample analysis thing is immobilized in the first binding entity combination on the solid carrier, and second binding entity is in conjunction with described analyte then, thereby forms insoluble three part complex body (David ﹠amp; Greene, United States Patent (USP) the 4th, 376, No. 110).But the available test section of second binding entity itself mark (directly sandwich assay), but perhaps available detect in conjunction with second binding entity and by the 3rd binding entity of test section mark (sandwich assay indirectly).For example, one type sandwich assay is that ELISA measures, but the test section is an enzyme in this case.

Usually, sandwich assay comprises " forward " mensuration, the binding entity that wherein is attached to solid phase at first contacts with given the test agent, to pass through forming binary solid phase complex body between immobilization binding entity and cholesterol ozonation products, from sample extracting cholesterol ozonation products.At one section suitable incubation after the time, the washing solid carrier is removed unconjugated liquid sample, comprise unreacted cholesterol ozonation products (if any).Solid carrier is contacted with the solution of the mark binding entity that contains unknown quantity (playing the effect of mark or reporter molecules).Carry out the incubation second time,, remove unreacted mark binding entity so that after the reaction of the mixture between mark binding entity and immobilization binding entity and the cholesterol ozonation products, wash solid carrier once more.Such forward sandwich assay can be that simple " being/deny " measured, to determine whether there is cholesterol ozonation products in the sample.

Operable other types sandwich assay comprises that so-called " simultaneously " measured and " oppositely " measured.Mensuration relates to single incubation step simultaneously, and wherein mark is exposed to given the test agent simultaneously with unlabelled binding entity.Unlabelled binding entity is immobilized onto on the solid carrier, and the binding entity of mark is free in the solution that contains sample.After incubation finished, the washing solid carrier was to remove the unreacted sample and the mark binding entity of complexing not.As conventional " forward " sandwich assay, determine existence then with solid carrier bonded mark binding entity.

In " oppositely " mensuration, adopt progressively additive process, at first the solution with the mark binding entity adds in the sample, then carries out incubation, and then adds the unmarked binding entity that is attached on the solid carrier.Behind the incubation, wash solid phase in the usual way for the second time, remove the solution of given the test agent resistates and unreacted mark binding entity.As " simultaneously " mensuration and " forward " mensuration, measure and solid carrier bonded mark binding entity then.

Except their diagnosis effectiveness, binding entity of the present invention also can be used for coming the advancing of disease of monitoring target medium vessels by checking the level of cholesterol ozonation products different time in tissue, cell or the serum sample.The cholesterol ozonation products level can show further developing of object medium vessels disease or heart disease over time.

Vascular disease

Vascular disease by the present invention's diagnosis are Mammals blood vessel diseases.The word Mammals refers to any Mammals.Mammiferous some example comprises pet animals such as dog and cat; Farming animals such as pig, ox, sheep and goat; Laboratory animal such as mouse and rat; Primate such as monkey, ape and chimpanzee; And it is human.In certain embodiments, preferably come diagnosing human by method of the present invention.

The present invention relates to detect or diagnose and relate to cholesterol deposition and the vascular disorder of cholesterol ozonation or the method for circulatory disorders.This illness can be caused by loss, injury or the destruction of the vascular system in anatomy position or the system.Term " vascular disorder " or " vascular disease " refer to impaired or the vascular tissue's state that can be impaired of blood flow wherein.

Many pathological conditions can cause depositing the vascular disease that cause by cholesterol.The example of the vascular disorder of available the compositions and methods of the invention detection or diagnosis comprises atherosclerosis (or arteriosclerosis), preeclampsia, peripheral vascular disease, heart disease and stroke.Therefore, the present invention relates to treat for example method of following disease: sclerosis, four limbs disease (peripheral limb disease), intermittent claudication and diabetic complication (comprising ischemic heart disease, peripheral arterial disease, congestive heart failure, retinopathy, neuropathy and ephrosis) or thrombosis that primary and Secondary cases in apoplexy, atherosclerosis, acute coronary syndrome (comprising astable stenocardia, thrombosis and myocardial infarction), plaque rupture, coronary artery or the peripheral arterial (in the support) restenosis, transplanting cause.

Test kit

The present invention also comprises the test kit of the cholesterol ozonation products that is used for test samples.In one embodiment, described test kit comprises and comprises specificity in conjunction with the binding entity of cholesterol ozonation products or the container of antibody.Described binding entity or antibody can have direct connection or indirect bonded certification mark or reporter molecules.Binding entity or antibody also can provide by liquid form, and perhaps it can be connected to solid phase, for example are used for the needs of any method of immunity easily.

Test kit of the present invention also can comprise another container, and described container comprises cholesterol ozonation products, its can be for example in the mensuration of cholesterol ozonation products with comparing or standard substance.

Test kit of the present invention also can comprise another container, and described container comprises and can react the reagent that can easily detect by any binding entity of the present invention or antibody to produce with cholesterol.

Test kit of the present invention also can comprise the 3rd container, and described container comprises certification mark or reporter molecules, is used to detect the mixture between binding entity, antibody or binding entity/antibody and the cholesterol ozonation products.

These test kits also can comprise the container that the instrument that is used to collect sample (as blood, blood plasma, serum, urine, saliva and stool) is housed.This instrument comprises blood taking needle, test tube and thieving paper or the cloth in order to collection and stable blood; Swab in order to collection and stable saliva; Cup in order to collection and stable urine or stool sample.But optionally treating collection material such as test tube, paper, cloth, swab, cup etc. are with sex change or the irreversible adsorption of avoiding sample.The also available sanitas of these collection materials, stablizer or biocide are handled or are comprised them, to help to keep the integrity of sample.

The present invention further describes by following non-limiting example.

Embodiment 1: material and method

Present embodiment is provided for the material and the method for some experiment as herein described.

The operation of atherosclerotic artery sample separates and handles.Obtain tissue sample by carotid endarterectomy.Described sample contains atherosclerotic plaque and some adherent tunica intimas and middle film.The scheme that patch is analyzed will obtain patient's agreement by Scripps Clinic Human SubjectsCommittee approval before the surgical operation.Flesh tissue by carotid endarterectomy excision was analyzed in 30 minutes.Notice that the patch sample neither stores and also do not carry out rotproofing.All analysis operations are all finished in 2 hours behind excision.Do not add stationary liquid in the sample.

Atherosclerotic human artery's sample is to the oxidation of indigo carmine 1.Isolating as mentioned above endarterectomy sample (n=15) is divided into two portions of weight in wet base approximately equal (± 5%).With each sample place contain indigo carmine 1 (200 μ M, Aldrich) and the phosphate-buffered saline of ox catalase (100 μ g) (PBS, pH 7.4,1.8mL) in.Add indigo carmine 1 and serve as the chemical trap (chemical trap) of ozone.Takeuchi etc., Anal.Chim.Acta 230,183 (1990); Takeuchi etc., Anal.Chem.61,619 (1989).Add tetradecanoic acid phorbol ester (PMA, 40 μ g are in 0.2mL DMSO) or DMSO (0.2mL) activator as protein kinase C.With organizing each sample of clarifixator homogeneous 10 minutes, centrifugal then (10,000rpm 10 minutes).Supernatant decanted liquid by strainer (0.2 μ m), is analyzed the existence of isatin sulfonic acid 2 in the filtrate with quantitative HPLC.

Shown in Figure 1B, the visible absorbance of indigo carmine 1 is bleached, and reaction produces the novel chemical substance that available quantitative HPLC detects (table 1) and is accredited as isatin sulfonic acid 2 (in addition referring to Figure 1A).

Quantitatively the HPLC of isatin sulfonic acid 2 measures.Carrying out HPLC on the Hitachi D-7000 instrument of band L-7200 automatic sampler, L-7100 pump and L-7400 UV-detector (254nm) analyzes.Described L-7100 controls with the Hitachi-HSM software on the Dell GX150 Personal Computer.The LC condition is Spherisorb RP-C ₁₈Post and acetonitrile: water (0.1%TFA) (80: 20) moving phase 1.2mL/ minute.The retention time R of isatin sulfonic acid 2 _TBe about 9.4 minutes.By using the GraphPad v3.0 software on the Macintosh, peak area is compared to the typical curve of authentic sample concentration with peak area, carry out quantitatively (table 1).

Table 1

The isatin sulfonic acid 2 (ISA) that forms by activatory atherosclerosis arterial material.

Sample	ISA nmol/mg
		1	27.3
2	54.4
		3	27.6
4	1.0
		5	30.1
6	238.3
		7	39.4
8	152.9
		9	127
10	262.1
		11	27.9
12	64.6
		13	1.4
14	3.2
		15	32.1

Mean value ± standard error of mean=72.62 ± 21.69

At H ₂ ¹⁸Human artery's sample that the O medium sized artery is atherosis is to the oxidation of indigo carmine 1.This experiment is measured described carrying out by above indigo carmine, but following exception is arranged.The first, each patch sample (n=2) is joined in 95% above H ₂ ¹⁸Phosphate buffered saline buffer among the O (10mM, pH7.4) in.The second, filtrate desalination on the PD10 post is sprayed mass spectroscopy by negative ion electrospray on Finnegan electrospray mass spectrograph.Primary ion abundance data extract becomes Graphpad Prism (3.0 version) form, for demonstration.

These experiments show, at patch material and H ₂ ¹⁸O (＞95% ¹⁸O) exist down, ¹⁸The O isotropic substance is incorporated in the lactan carbonyl of isatin sulfonic acid 2.Because have only two keys that ozone can oxicracking indigo carmine 1, promote from H ₂ ¹⁸The isotropic substance of O is incorporated in the lactan carbonyl of isatin sulfonic acid 2, and ozone is likely the reactive oxygen species of oxidation indigo carmine 1.Therefore, in atherosclerotic lesion, produce ozone.In addition referring to P.Wentworth Jr. etc., Science298,2195 (2002); B.M.Babior, C.Takeuchi, J.Ruedi, A.Guitierrez, P.Wentworth Jr., Proc.Natl.Acad.Sci.U.S.A.100,3920 (2003); P.Wentworth Jr etc., Proc.Natl.Acad.Sci.U.S.A.100,1490 (2003).

Extracting and derivatization method from the aldehyde of atheroma artery sample.Isolating as mentioned above endarterectomy sample is divided into two portions of weight in wet base approximately equal (± 5%).With each sample place the phosphate-buffered saline that contains ox catalase (100 μ g) and tetradecanoic acid phorbol ester (40 μ g are in 0.2mLDMSO) or DMSO (0.2mL) (PBS, pH 7.4,1.8mL) in.With organizing each sample of clarifixator homogeneous 10 minutes.Behind the endarterectomy sample homogeneous, as above separate, use methylene dichloride (DCM, 3 * 5mL) washings then.The organic moiety that vacuum-evaporation merges.Resistates is dissolved in the ethanol (0.9mL), and adds the ethanolic soln of 2,4 dinitrophenyl hydrazine (100 μ L, 2mM and 1N HCl).Be blown into nitrogen 5 minutes in solution, stirred solution is 2 hours then.Gained suspension is filtered 0.22 μ m strainer, and filtrate is measured by HPLC and is analyzed (seeing below).(1-20 μ M) when handling under these conditions, do not form 4a or 5a when cholesterol 3.The amount of adding the 4b that detects before and after the PMA in atheroma artery extract is carried out Student sided t test Analysis, determining that PMA adds the significance (it is remarkable that p＜0.05 is thought) to 4a level in the artery extract, and measure described amount with the Graphpad v3.0 software on the Macintosh.

4a in the process of derivatize, in the concentration range (5-100 μ M) of 4a, has 20% 4a to change into 5b under these conditions approximately.These data show, the 5a of measured quantity (surpass the 4a that exists in the identical patch sample 20%) is produced by aldolize behind 3 ozonolysiss.Change in the concentration range (5-100 μ M) of the degree of 6b at 4a all the time＜2% at 4a under the derivatize condition that is adopted.These observationss show, the 6a amount that exists in the spot extract surpasses 2% of keto-aldehyde 4a amount, just exists before derivatize, are that β by water eliminates from ozonolysis product 4a and produces.

Except three kinds of main hydrazone product 4b-6b, in several patch extracts, also detect trace (＜5pmol/mg) 7a hydrazone derivative (being called 7b) (R _T～26 minutes, [M-H] ^-579, SOM Tu2 ﹠amp; 4).Compound 7a is the A ring dewatered product of 5a.Therefore the detection limit that the amount of 7b is measured near the HPLC that is adopted in the patch extract of derivatize does not carry out the analysis and research fully of this compound in all patch samples.The assignment of configuration of compound 7a and 7b is based on synthetic 7b's ¹H- ¹H ROESY experiment.

Utilize the synthetic products of compound 6b, 7a, 7b, 8a and 9a to identify to have R among Fig. 4 _T～26 minutes peak [M-H] ^-579 compound.

The HPLC-MS of hydrazone analyzes.Carrying out HPLC-MS on the HitachiD-7000 instrument of M-8000 ion trap mass spectrometer (in the negative ion mode) in band L-7200 automatic sampler (normal injection volume 10 μ l), L-7100 pump, L-7400 UV-detector (360nm) or L-7455 diode-array detector (200-400nm) and line analyzes.Described L-7100 and M-8000 control with the Hitachi-HSM software on the Dell GX150 Personal Computer.Use Vydec C ₁₈Reversed-phase column carries out HPLC.Adopt 0.5mL/ minute moving phase such as degree such as grade (75% acetonitrile, 20% methyl alcohol and 5% water).Measure peak height and peak area with Hitachi D7000 chromatographic working station software, relatively be converted into concentration by typical curve with true material.Under these conditions, the detection limit of hydrazone 4b-6b is between 1-10nM.Use this HPLC system, do not obtain the fractionation of cis and trans hydrazone isomer.

Extracting goes out also, and the representative HPLC-MS of the atherosclerotic material matter of derivatize shows in Fig. 4.The retention time and the mass ratio of the authentic sample of several crucial hydrazone compounds are displayed in Table 2.

The lcms analysis of the true hydrazone of table 2

Hydrazone	R T/ minute	[M-H] -
			4b	13.9	597
5b	20.3	597
			6b	18.0	579
7b	26.8	579
			a，d8b	26.6	579
b9b	16.5	579
			c10b	48.2	561

^aThe hydrazone of true aldehyde 8a is by above-mentioned derivatization method preparation, and aldehyde 8a does not synthesize separately and purifying. ^bThe hydrazone of commercially available ketone 9a does not synthesize separately and purifying by above-mentioned derivatization method preparation. ^cThe hydrazone of true aldehyde 10a does not synthesize separately and purifying by above-mentioned derivatization method preparation. ^d[measuring by Hitachi L-7455 diode-array detector (200-400nm)] made in distinguishing based on their UV spectrum between 8b and the 9b.α, the λ of β-unsaturated hydrazone 8b _MaxBe 435nm, and the λ of hydrazone 9b _MaxBe 416nm.

The analysis of aldehyde 4a and 5a in the plasma sample.Obtain plasma sample from the patient (n=8) who in 24 hours, carries out carotid endarterectomy according to plan.Analyzed the existence of 4a and 5a in all this plasma samples behind the sample collection in 3 days.Obtain the contrast plasma sample from the patient at random (n=15) of general medical science clinic, and analyzed in back 7 days in collection.In typical method, with methylene dichloride (DCM, the blood plasma among 3 * 1mL) the washing EDTA (1ml).The organic moiety that vacuum-evaporation merges.Resistates is dissolved in the methyl alcohol (0.9mL), and add 2,4 dinitrophenyl hydrazine (100 μ L, 0.01M, Lancaster) and the ethanolic soln of 1N HCl.Be blown into nitrogen 5 minutes in solution, stirred solution is 2 hours then.Gained suspension is filtered 0.22 μ m strainer, and filtrate is measured by HPLC and is analyzed (seeing above).Preliminary study discloses, and can descend about 5% every day from the amount of the extractive 5a of blood plasma.

The preparation of authentic sample 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a and 8b

General method.Unless otherwise specified, respond and all in the inert atmosphere that has siccative, solvent and flame-dried glass wares, carry out.All raw materials are all available from Aldrich, Sigma, Fisher or Lancaster, and former state is used when obtaining.Ketone 9a is available from Aldrich.All flash column chromatographies all use silica gel 60 (230-400 order) to carry out.Preparation type thin-layer chromatography (TLC) uses Merck (0.25,0.5 or 1mm) to be coated with shop silica gel Kieselgel 60 F ₂₅₄Plate carries out. ¹H NMR spectrum record on Bruker AMX-600 (600MHz), AMX-500 (500MHz), AMX-400 (400MHz) or AC-250 (250MHz) wave spectrum meter. ¹³C NMR spectrum record on Bruker AMX-500 (125.7MHz) or AMX-400 (100.6MHz) wave spectrum meter.Chemical shift is to report with the δ scale with respect to interior target 1,000,000/(ppm).High resolution mass spec is record on VG ZAB-VSE instrument.

3 beta-hydroxies-5-oxo-5,6-secocholestane-6-aldehyde (4a).This compound is pressed K.Wang, E.Bermudez, and W.A.Pryor, the general description among the Steroids 58,225 (1993) is synthesized.Under dry-ice temperature, make cholesterol 3 ((100ml) solution ozonize 10 minutes of 1g, chloroform-methanol 2.6mmol) (9: 1).Evaporation reaction mixture, (650mg, 10mmol stirred 3 hours in water-acetate (1: 9,50ml) in) at room temperature to use the Zn powder.Dilute the mixture that is reduced with methylene dichloride (100ml), and water (3 * 50ml) washings.The organic fraction dried over sodium sulfate that merges, vacuum-evaporation is to doing.Resistates obtains title compound 4a with silica gel chromatography [ethyl acetate-hexane (25: 75)] purifying, is white solid (820mg, 76%):

¹H NMR(CDCl ₃)δ9.533(s，1H，CHO)，4.388(m，1H，H-3)，3.000(dd，J＝14.0，4.0Hz，1H，H-4e)，0.927(s，3H，CH ₃-19)，0.827(d，J＝6.8Hz，3H，CH ₃-21)，0.782(d，J＝6.8Hz，3H，CH ₃)，0.778(d，J＝6.8Hz，3H，CH ₃)，0.603(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ217.90(C-5)，202.76(C-6)，70.81(C-3)，55.96(C-17)，54.26(C-14)，52.52(C-10)，46.70(C-4)，44.17(C-7)，42.43(C-13)，42.17(C-9)，39.75(C-12)，39.33(C-24)，35.85(C-22)，35.61(C-20)，34.58(C-8)，33.99(C-1)，27.87(C-25)，27.73(C-16)，27.52(C-2)，25.22(C-15)，23.62(C-23)，22.91(C-11)，22.70(C-27)，22.44(C-26)，18.44(C-21)，17.46(C-19)，11.42(C-18).

C ₂₇H ₄₆O ₃Na (M+Na) ⁺HRMALDITOFMS calculated value 441.3339, measured value 441.3355.

3 beta-hydroxies-5-oxo-5,2 of 6-secocholestane-6-aldehyde, 4-dinitrophenylhydrazone (4b).This compound is pressed K.Wang, E.Berm ú dez, and W.A.Pryor, the general description among the Steroids 58,225 (1993) is synthesized.With 2,4 dinitrophenyl hydrazine (52mg, 0.26mmol) and tosic acid (1mg 0.0052mmol) joins keto-aldehyde 4a (100mg, acetonitrile 0.24mmol) (10ml) solution.At room temperature stirred reaction mixture is 4 hours, and vacuum-evaporation is to doing.Resistates is dissolved in ethyl acetate (10ml) and water (3 * 20ml) washings.The organic moiety dried over sodium sulfate that merges, vacuum-evaporation is to doing.Resistates obtains title compound 4b by silica gel chromatography [ethyl acetate-hexane (1: 4)] purifying, for yellow solid (100mg, 70%), is the mixture of cis and trans-isomer(ide) (1: 4).Obtain trans 4b from hexane-methylene dichloride crystallization, be yellow spicule (30mg, 21%):

¹H NMR(CDCl ₃)：δ10.994(s，1H，NH)，9.107(d，J＝2.8Hz，1H，H-3′)，8.316(dd，J＝9.6，2.8Hz，1H，H-5′)，7.923(d，J＝9.6Hz，1H，H-6′)，7.419(dd，J＝6.0，3.6Hz，1H，H-6)，4.417(m，1H，H-3)，2.971(dd，J＝13.6，4.0Hz，1H，H-4e)，1.076(s，3H，CH ₃-19)，0.915(d，J＝6.4Hz，3H，CH ₃-21)，0.853(d，J＝6.4Hz，3H，CH ₃)，0.849(d，J＝6.4Hz，3H，CH ₃)，0.710(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ216.05(C-5)，150.84(C-6)，144.96(C-1′)，137.87(C-4′)，130.23(C-5′)，128.90(C-2′)，123.50(C-3′)，116.52(C-6′)，71.42(C-3)，56.07(C-17)，54.54(C-14)，52.69(C-10)，47.34(C-4)，42.61(C-13)，42.61(C-9)，39.82(C-12)，39.42(C-24)，36.99(C-8)，35.96(C-22)，35.67(C-20)，34.13(C-1)，32.65(C-7)，27.98(C-16)，27.93(C-25)，27.90(C-2)，25.31(C-15)，23.70(C-23)，23.12(C-11)，22.78(C-27)，22.52(C-26)，18.56(C-21)，17.77(C-19)，11.67(C-18)；

C ₃₃H ₅₀N ₄O ₆Na (M+Na) HRMALDITOFMS calculated value 621.3622, measured value 621.3622: λ _Max360nm, ε 2.57 ± 0.31 * 10 ⁴M ^-1Cm ^-1

Cholestane-6 β-formaldehyde (5a) falls in 3 beta-hydroxies-5 beta-hydroxies-B-.This compound is pressed T.Miyamoto, K.Kodama, and Y.Aramaki, R.Higuchi, R.W.M.Van Soest, the general description among the Tetrahedron Letter 42,6349 (2001) is synthesized.To keto-aldehyde 4a (800mg, acetonitrile-water 1.9mmol) (20: 1,100ml) solution add the L-proline(Pro) (220mg, 1.9mmol).At room temperature stirred reaction mixture is 2 hours, and vacuum-evaporation is to doing.Resistates is dissolved in ethyl acetate (50ml) and water (3 * 50ml) washings.The organic fraction dried over sodium sulfate that merges, vacuum-evaporation.Resistates obtains title compound 5a by silica gel chromatography [ethyl acetate-hexane (1: 4)] purifying, is white solid (580mg, 73%):

¹H NMR(CDCl ₃)δ9.689(d，J＝2.8Hz，1H，CHO)，4.115(m，1H，H-3)，3.565(s，1H，3β-OH)，2.495(broad s，1H，5β-OH)，2.234(dd，J＝9.2，3.2Hz，1H，H-6)，0.920(s，3H，CH ₃-19)，0.904(d，J＝6.4Hz，3H，CH ₃-21)，0.854(d，J＝6.8Hz，3H，CH ₃)，0.850(d，J＝6.8Hz，3H，CH ₃)，0.705(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ204.74(C-7)，84.26(C-5)，67.33(C-3)，63.89(C-9)，56.10(C-14)，55.67(C-17)，50.42(C-6)，45.47(C-10)，44.72(C-13)，44.22(C-4)，40.02(C-8)，39.67(C-12)，39.44(C-24)，36.15(C-22)，35.58(C-20)，28.30(C-16)，27.98(C-2)，27.91(C-25)，26.69(C-1)，24.55(C-15)，23.78(C-23)，22.78(C-27)，22.52(C-26)，21.54(C-11)，18.71(C-21)，18.43(C-19)，12.48(C-18).

C ₂₇H ₄₆O ₃Na (M+Na) ⁺HRMALDITOFMS calculated value 441.3339, measured value 441.3351.

2 of cholestane-6 β-formaldehyde falls in 3 beta-hydroxies-5 beta-hydroxies-B-, 4-dinitrophenylhydrazone (5b).This compound is pressed K.Wang, E.Berm ú dez, and W.A.Pryor, the general description among the Steroids 58,225 (1993) is synthesized.With 2,4 dinitrophenyl hydrazine (52mg, 0.26mmol) and hydrochloric acid (12M, 2) join aldehyde 5a (100mg, acetonitrile 0.24mmol) (10ml) solution.At room temperature stirred reaction mixture is 4 hours, and vacuum-evaporation is to doing.Resistates is dissolved in ethyl acetate (10ml) and water (3 * 20ml) washings.The organic moiety dried over sodium sulfate that merges, vacuum-evaporation is to doing.Resistates obtains title compound 5b yellow solid (90mg, 62%) by silica gel chromatography [ethyl acetate-hexane (1: 4)] purifying, is trans 5b phenylhydrazone:

¹H NMR(CDCl ₃)11.049(s，1H，NH)，9.108(d，J＝2.4Hz，1H，H-3′)，8.280(dd，J＝9.6，2.6Hz，1H，H-5′)，7.901(d，J＝9.6Hz，1H，H-6′)，7.561(d，J＝7.2Hz，1H，H-7)，4.214(m，1H，H-3)，3.349(s，1H，3β-OH)，2.337(dd，J＝9.2，6.8Hz，1H，H-6)，0.967(s，3H，CH ₃-19)，0.917(d，J＝6.8Hz，3H，CH ₃-21)，0.850(d，J＝6.4Hz，3H，CH ₃)，0.846(d，J＝6.4Hz，3H，CH ₃)，0.713(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ155.18(C-7)，145.12(C-1′)，137.51(C-4′)，129.91(C-5′)，128.64(C-2′)，123.57(C-3′)，116.36(C-6′)，83.35(C-5)，67.56(C-3)，56.34(C-17)，56.34(C-9)，55.56(C-14)，51.47(C-6)，45.50(C-10)，44.76(C-13)，43.62(C-4)，42.59(C-8)，39.66(C-12)，39.43(C-24)，36.16(C-22)，35.58(C-20)，28.50(C-16)，28.07(C-2)，27.98(C-25)，27.70(C-1)，24.67(C-15)，23.78(C-23)，22.78(C-27)，22.52(C-26)，21.63(C-11)，18.75(C-21)，18.67(C-19)，12.48(C-18)；

C ₃₃H ₅₀N ₄O ₆Na (M+Na) ⁺HRMALDITOFMS calculated value 621.3622, measured value 621.3625.HPLC-MS detects: R _T20.8 minute; [M-H] ^-597; λ _Max361nm, ε 2.47 ± 0.68 * 10 ⁴M ^-1Cm ^-1

5-oxo-5,6-secocholesta-3-alkene-6-aldehyde (6a).This compound is pressed P.Yates, S.Stiveer, and Can.J.Chem.66, the general description in 1209 (1988) is synthesized.With methylsulfonyl chloride (400 μ l, 2.87mmol) be added drop-wise to the keto-aldehyde 4a that stirs under the ice bath temperature (300mg, 0.72mmol) and triethylamine (65 μ l, CH 0.84mmol) ₂Cl ₂In the solution (15ml).Gained solution stirred 30 minutes under 0 ℃ of argon gas, and (400 μ l 2.87mmol), are warming up to room temperature with solution to add triethylamine then.After 2 hours, reaction mixture vacuum-evaporation is extremely done.Resistates is dissolved in methylene dichloride (15ml) and water (3 * 20ml) washings.The organic fraction anhydrous sodium sulfate drying that merges, vacuum-evaporation.Thick resistates is by silica gel chromatography [ethyl acetate-hexane (1: 9)] purifying.Evaporation gained fraction obtains aldehyde 6a (153mg, 53%), is water white oil.

¹H NMR (CDCl ₃) demonstration δ 9.574 (s, 1H, CHO), 6.769 (m, 1H, H-3), 5.822 (d, J=10Hz, 1H, H-4), 2.512 (dd, J=16.8,3.6Hz, 1H, H-7), 1.070 (s, 3H, CH ₃-19), 0.882 (d, J=6.8Hz, 3H, CH ₃-21), 0.845 (d, J=6.8Hz, 3H, CH ₃), 0.841 (d, J=6.8Hz, 3H, CH ₃), 0.674 (s, 3H, CH ₃-18); ¹³C NMR (CDCl ₃) δ 208.22 (C-5), 202.42 (C-6), 147.46 (C-3), 128.44 (C-4), 56.08 (C-17), 54.96 (C-14), 47.80 (C-10), 45.05 (C-7), 42.33 (C-13), 42.04 (C-9), 39.73 (C-12), 39.43 (C-24), 35.93 (C-22), 35.71 (C-20), 35.42 (C-1), 33.77 (C-8), 27.97 (C-25), 27.67 (C-16), 25.22 (C-15), 24.67 (C-2), 23.71 (C-23), 23.27 (C-11), 22.77 (C-27), 22.51 (C-26), 18.54 (C-21), 17.71 (C-19), 11.48 (C-18).

C ₂₇H ₄₅O ₂(M+H) ⁺HRMALDITOFMS calculated value 401.3414, measured value 401.3404.

5-oxo-5,2 of 6-secocholesta-3-alkene-6-aldehyde, 4-dinitrophenylhydrazone (6b).With 2,4 dinitrophenyl hydrazine (45mg, 0.23mmol) join keto-aldehyde 6a (80mg, 0.2mmol) and tosic acid (1mg, acetonitrile 0.0052mmol) (10ml) solution.At room temperature stirred reaction mixture is 2 hours, and vacuum-evaporation is to doing.Resistates is dissolved in methylene dichloride (10ml) and water (3 * 20ml) washings.The organic fraction dried over sodium sulfate that merges, vacuum-evaporation is to doing.Resistates obtains title compound 6b by silica gel chromatography [ethyl acetate-hexane (15: 85)] purifying, is yellow solid (70mg, 60%):

Trans-6b ¹H NMR (CDCl ₃) demonstration δ 10.958 (s, 1H, NH), 9.104 (d, J=2.4Hz, 1H, H-3 '), (8.288 dd, J=9.8,2.8Hz, 1H, H-5 '), 7.896 (d, J=9.6Hz, 1H, H-6 '), 7.337 (dd, J=5.6,5.6Hz, 1H, H-6), 6.771 (m, 1H, H-3), 5.822 (d, J=10Hz, 1-H, H-4), 2.600 (ddd, J=16.4,4.8,4.8Hz, 1H, H-7), 1.139 (s, 3H, CH ₃-19), 0.897 (d, J=6.4Hz, 3H, CH ₃-21), 0.840 (d, J=6.8Hz, 3H, CH ₃), 0.837 (d, J=6.8Hz, 3H, CH ₃), 0.703 (s, 3H, CH ₃-18); ¹³C NMR (CDCl ₃) δ 207.78 (C-5), 151.17 (C-6), 147.69 (C-3), (145.00 C-1 '), 137.61 (C-4 '), 129.97 (C-5 '), (128.52 C-2 '), 128.38 (C-4), 123.48 (C-3 '), (116.46 C-6 '), 56.05 (C-17), 54.68 (C-14), 47.87 (C-10), 42.30 (C-13), 41.69 (C-9), 39.72 (C-12), 39.37 (C-24), 36.35 (C-8), 35.91 (C-22), 35.66 (C-20), 35.34 (C-1), 32.84 (C-7), 27.93 (C-25), 27.73 (C-16), 24.93 (C-15), 24.68 (C-2), 23.69 (C-23), 23.24 (C-11), 22.74 (C-27), 22.48 (C-26), 18.52 (C-21), 17.81 (C-19), 11.58 (C-18);

C ₃₃H ₄₈N ₄O ₅Na (M+Na) ⁺HRMALDITOFMS calculated value 603.3517, measured value 603.3523.HPLC-MS detects: R _T18.3 minute; [M-H] ^-579; λ _Max360nm, ε 2.29 ± 0.23 * 10 ⁴M ^-1Cm ^-1

Courage steroid-3-alkene-6 β-formaldehyde (7a) falls in 5 beta-hydroxies-B-.This compound is pressed P.Yates, S.Stiveer, and Can.J.Chem.66, the general description in 1209 (1988) is synthesized.Under the room temperature argon atmospher, (0.5M, (50mg is in absolute methanol solution 0.125mmol) (10ml) 0.16mmol) to be added drop-wise to keto-aldehyde 4a with the sodium methylate in the methyl alcohol.After 30 minutes, vacuum is removed methyl alcohol, and resistates is dissolved in methylene dichloride (20ml) and water (3 * 20ml) washings.The organic fraction dried over sodium sulfate that merges, vacuum-evaporation.Resistates obtains title compound aldehyde 7a by silica gel chromatography [ethyl acetate-hexane (1: 9)] purifying, is water white oil (16mg, 32%):

¹H NMR(CDCl ₃)δ9.703(d，J＝3.2，1H，CHO)，5.716(m，2H，H-3 andH-4)，2.398(dd，J＝9.6，3.6Hz，1H，H-6)，0.953(s，3H，CH ₃-19)，0.904(d，J＝6.4Hz，3H，CH ₃-21)，0.854(d，J＝6.4Hz，3H，CH ₃)，0.849(d，J＝6.4Hz，3H，CH ₃)，0.706(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ204.41(C-7)，134.21(C-3)，126.66(C-4)，81.44(C-5)，64.49(C-9)，55.86(C-14)，55.55(C-17)，48.44(C-6)，45.12(C-10)，44.47(C-13)，39.92(C-8)，39.45(C-12)，39.40(C-24)，36.16(C-22)，35.57(C-20)，29.06(C-1)，28.31(C-16)，27.98(C-25)，24.73(C-15)，23.76(C-23)，22.78(C-27)，22.53(C-26)，21.69(C-2)，21.24(C-11)，18.74(C-21)，18.44(C-19)，12.37(C-18)；

C ₂₇H ₄₄O ₂Na (M+Na) ⁺HRMALDITOFMS calculated value 423.3233, measured value 423.3240.

2 of courage steroid-3-alkene-6 β-formaldehyde falls in 5 beta-hydroxies-B-, 4-dinitrophenylhydrazone (7b): with 2,4 dinitrophenyl hydrazine (8mg, 0.041mmol) and tosic acid (1mg, 5.2 (15mg is in acetonitrile solution 0.037mmol) (5ml) μ mol) to join aldehyde 7a.At room temperature stirred reaction mixture is 2 hours, vacuum-evaporation, and dilute with methylene dichloride (10ml).Organic layer water water (use dried over sodium sulfate, and be evaporated to dried by 3 * 20ml) washings.Resistates obtains hydrazone 7b by silica gel chromatography [ethyl acetate-hexane (1: 9)] purifying, is yellow solid (9mg, 41%):

¹H NMR (CDCl ₃) trans-7b 11.060 (s, 1H, NH), 9.119 (d, J=2.8Hz, 1H, H-3 '), (8.291 dd, J=9.2,2.0Hz, 1H, H-5 '), 7.930 (d, J=9.6Hz, 1H, H-6 '), 7.546 (d, J=7.2Hz, 1H, H-7), 5.761 (ddd, J=10.2,4.4,2.0Hz, 1H, H-3), 5.705 (d, J=9.6Hz, 1H, H-4), 2.485 (dd, J=10.4,7.6Hz, 1H, H-6), 0.977 (s, 3H, CH ₃-19), 0.917 (d, J=6.4Hz, 3H, CH ₃-21), 0.848 (d, J=6.8Hz, 3H, CH ₃), 0.844 (d, J=6.4Hz, 3H, CH ₃), 0.707 (s, 3H, CH ₃-18); ¹H- ¹H ROESY NMR significant correlation (H ₄-H ₆), (H ₆-H ₇), (H ₇-H ₈), (H ₇-H ₁₉), lack dependency (H ₃-H ₁₉), (H ₄-H ₇), (H ₄-H ₁₉), (H ₆-H ₁₉); ¹³C NMR (CDCl ₃) δ 154.62 (C-7), 145.09 (C-1 '), 137.59 (C-4 '), 133.89 (C-3), 129.94 (C-5 '), 128.68 (C-2 '), 127.12 (C-4), 123.57 (C-3 '), 116.42 (C-6 '), 80.91 (C-5), 56.83 (C-9), 56.07 (C-14), 55.39 (C-17), 49.58 (C-6), 45.00 (C-10), 44.58 (C-13), 42.50 (C-8), 39.44 (C-12), 39.44 (C-24), 36.17 (C-22), 35.54 (C-20), 30.46 (C-1), 28.53 (C-16), 27.98 (C-25), 24.91 (C-15), 23.74 (C-23), 22.77 (C-27), 22.52 (C-26), 21.79 (C-2), 21.31 (C-11), 18.76 (C-21), 18.76 (C-19), 12.34 (C-18).

HPLC-MS detects: RT 18.3 minutes; [M-H]-579; λ _Max364nm, ε 2.32 ± 0.17 * 10 ⁴M ^-1Cm ^-1

3 beta-hydroxies-B-fall courage steroid-5-alkene-6-formaldehyde (8a) with aldehyde 5a (50mg, 0.12mmol) and phosphoric acid (85%, (1: 1,4ml) reflux was 30 minutes for acetonitrile-dichloromethane solution 5ml).The vacuum-evaporation reaction mixture, with methylene dichloride (50ml) dilution, and water (3 * 20ml) washings.The organic layer dried over sodium sulfate, vacuum-evaporation.Resistates obtains title aldehyde by silica gel chromatography [ethyl acetate-hexane (1: 4)] purifying, is the α of 12mg (25%), β unsaturated aldehyde 8a:

¹H NMR (CDCl ₃) of 8a demonstration δ 9.958 (s, 1H, CHO), 3.711 (tt, J=10.8,4.5Hz, 1H, H-3), 3.475 (dd, J=14.1,4.8,1H, H-4), 2.563 (dd, J=11.0,11.0Hz, 1H, H-8), 0.953 (s, 3H, CH ₃-19), 0.941 (d, J=6.9Hz, 3H, CH ₃-21), 0.881 (d, J=6.6Hz, 3H, CH ₃), 0.876 (d, J=6.6Hz, 3H, CH ₃), 0.746 (s, 3H, CH ₃-18); ¹³C NMR (CDCl ₃) δ 189.44 (C-7), 168.74 (C-5), 139.21 (C-6), 70.88 (C-3), 60.16 (C-9), 55.40 (C-17), 54.48 (C-14), 46.35 (C-10), 46.19 (C-8), 45.27 (C-13), 39.86 (C-12), 39.55 (C-24), 36.26 (C-4), 36.22 (C-22), 35.64 (C-20), 33.93 (C-1), 31.32 (C-2), 28.62 (C-16), 28.09 (C-25), 26.65 (C-15), 24.00 (C-23), 22.90 (C-27), 22.64 (C-26), 20.80 (C-11), 19.02 (C-21), 15.73 (C-19), 12.59 (C-18);

C ₂₇H ₄₄O ₂Na (M+Na) ⁺HRMS calculated value 423.3233, measured value 423.3239.

From then on reaction obtains by product B-and falls courage steroid-3, and 5-diene-6-formaldehyde 12a is white solid (27mg, 60%):

¹H NMR(CDCl ₃)δ10.017(s，1H，CHO)，6.919(d，J＝10.2Hz，1H，H-4)，6.225(m，1H，H-3)，2.675(d d，J＝10.8，10.8Hz，1H，H-8)，0.950(d，J＝6.9Hz，3H，CH ₃-21)，0.914(s，3H，CH ₃-19)，0.882(d，J＝6.8Hz，3H，CH ₃)，0.877(d，J＝6.8Hz，3H，CH ₃)，0.769(s，3H，CH ₃-18)； ¹³C NMR(CDCl ₃)δ189.41(C-7)，163.33(C-5)，138.18(C-6)，135.75(C-3)，120.68(C-4)，59.54(C-9)，55.41(C-17)，54.30(C-14)，45.47(C-8)，45.08(C-10)，44.72(C-13)，39.79(C-12)，39.55(C-24)，36.27(C-22)，35.65(C-20)，34.18(C-1)，28.62(C-16)，28.09(C-25)，26.72(C-15)，24.00(C-23)，23.96(C-2)，22.90(C-27)，22.64(C-26)，20.72(C-11)，19.03(C-21)，14.87(C-19)，12.62(C-18)；

C ₂₇H ₄₃O (M+H) ⁺HRMALDITOFMS calculated value 383.3308, measured value 383.3309.

Keto-aldehyde 4a and amino acid whose aldol condensation.In typical method, keto-aldehyde 4a (2mg, 4.8 μ mol) is dissolved in the DMSO-d in the NMR pipe ₆(800 μ l) and D ₂O (80 μ l).Add 1 normal arbitrary to this solution: a) L-proline(Pro), b) glycine, c) L-Lysine mono Hydrochloride or d) two hydrochloric acid L-ethyl ester of lysine.Sample carries out at each time point ¹H NMR analyzes.By monitoring the variation of many resonance, customary observing response.

¹H NMR (DMSO-d ₆) ¹H NMR 5a demonstration δ 9.527 (d, J=3.2Hz, 1H, CHO), 3.876 (m, 1H, H-3), 0.860 (d, J=6.4Hz, 3H, CH ₃-21), 0.772 (d, J=6.8Hz, 3H, CH ₃), 0.767 (d, J=6.8Hz, 3H, CH ₃), 0.771 (s, 3H, CH ₃-19), 0.642 (s, 3H, CH ₃-18) .1H NMR 4a show δ 9.518 (s, 1H, CHO), 4.223 (m, 1H, H-3), 2.994 (dd, J=12.8,4.0Hz, 1H, H-4e), 0.858 (d, J=6.8Hz, 3H, CH ₃), 0.842 (s, 3H, CH ₃-19), 0.811 (d, J=6.8Hz, 3H, CH ₃), 0.807 (d, J=6.4Hz, 3H, CH ₃-21), 0.615 (s, 3H, CH ₃-18).

Under these conditions, at DMSO-d ₆(800 μ l) and D ₂The aldol condensation of 4a does not take place among the O (80 μ l).

The aldol condensation of disconnected keto-aldehyde 4a and atherosclerotic artery part and blood part.In typical method, (5mg 0.0012mmol) is dissolved in DMSO-d with keto-aldehyde 4a ₆(800 μ l) and D ₂Among the O (80 μ l).Add arbitrary a) atherosclerotic artery (2.1mg) to this solution, its homogeneous also freeze-drying in PBS (1ml) in organizing clarifixator, b) freeze dried human blood (1ml), c) freeze dried human plasma (1ml) or d) freeze dried PBS (1ml).Carry out at each time point draw samples ¹H NMR analyzes (seeing above).The aldol condensation of 4a when existing, freeze dried PBS does not take place under these conditions.

Carry out biological study with 4a and 5a

Some oxygen sterol that is produced by ornitrol oxidation in the body has been described.E.Lund，I.Bjrkhem，Acc.Chem.Res.28，241(1995)。In addition, isolate the 5a analogue that only has textural difference from sponge Stellettahiwasaensis, as the part of comprehensive screening of pair cell toxicity natural product at cholestane (cholestan) side chain.T.Miyamoto，K.Kodama，Y.Aramaki，R.Higuchi，R.W.M.Van Soest，Tetrahedron Lett.42，6349(2001)；B.Liu，Z.Weishan，Tetrahedron Lett.43，4187(2002)。But, in the mankind, examine the not report in the past of ruined derivative as steroid in sterol 4a and 5a.

Cytotoxic assay.WI-L2 people B-lymphocyte series, HAAE-1 people abdominal cavity aortic endothelial cell system, MH-S mouse pulmonary alveolar macrophage system and J774A.1 rat tissue macrophage system obtain from ATCC.Human aorta endotheliocyte (HAEC) and human smooth muscle cell (VSMS) obtain from Cambrex Bio Science.Jurkat E6-1T-lymphocyte is so kind as to give by doctor J.Kaye (TheScripps Research Institute).Cell is recommended to cultivate in the substratum at the ATCC that contains 10% foetal calf serum.Cell is at 37 ℃, 5 or 7%CO ₂Controlled atmosphere under incubation.Discharge mensuration for serum lactic dehydrogenase (LDH), by adding 0.05% trypsinase/EDTA or fetching the results attached cell by scraping.With the cell inoculation (25,000 cells/well) on 96 hole microtiter plates that obtains, allow it restore 24-48 hour.Washed cell gently, substratum is with the fresh culture displacement that contains 5% foetal calf serum.With 3,4a or 5a (0-100 μ M) handled two parts of multiple or more cell sample 18 hours.By measuring the serum lactic dehydrogenase (LDH) that discharges from cell in the culture, determine cytotoxicity then.Briefly, (Promega USA), measures cultured cells LDH activity in the cell conditioned medium liquid when keto-aldehyde 4a, aldol 5a or cholesterol 3 processing end in 96 orifice plates with CytoTox 96 on-radiation cytotoxic assay.100% cytotoxicity is defined as trypan blue repels the LDH maximum that dead cell discharges shown in the test, the LDH maximum amount that detects when perhaps cell is with 0.9%Triton X-100 cracking.By with the Graphpad on the Macintosh (version 3 .0) software, the original double repeating data of concentration pair cell toxicity (%) is carried out nonlinear regression analysis (Hill plot), determine IC ₅₀Value.

Lipid-loading measures (foam cell formation).At 37 ℃, 5 or 7%CO ₂Controlled atmosphere under, the J774.1 scavenger cell is recommended in the substratum in the ATCC that contains 10% foetal calf serum at incubation on the 8 vestibule formula slide glasss.Then with cell incubation 72 hours in containing the same medium of antioxidant 2,6 di tert butyl 4 methyl phenol toluene (100 μ M), diethylene triaminepentaacetic acid(DTPA) (100 μ M) and LDL (100 μ g/mL) or LDL (100 μ g/mL) and 4a (20 μ g/mL) or LDL (100 μ g/mL) and 5a (20 μ g/mL).During end, use twice of PBS (pH 7.4) washed cell.With 6% (v/v) paraformaldehyde fixed cell 30 minutes that is dissolved in PBS,, lipid was dyeed 8 minutes then with 5mg/ml oil red O with propylene glycol rinsing 2 minutes.With 45 seconds of Harris haematoxylin redyeing cell, remove background dyeing with 6% paraformaldehyde, then in PBS washing washing is once once and in tap water.With glycerine cover glass is placed on the slide glass, then slide sample is carried out the optical microphotograph inspection.Write down the number of rich lipocyte at least 100 cells altogether of being counted in the single visual field of every slide glass, it is expressed as the percentage ratio of total cell.Magnification with 100x is taken pictures.

Circular dichroism.Under 37 ℃, record is dissolved in circular dichroism (CD) spectrum of LDL (100 μ g/mL), LDL (100 μ g/mL) with 4a (10 μ M) and the LDL (100 μ g/mL) and the 5a (10 μ M) of PBS (pH 7.4, contain 1% Virahol) in the 0.1cm quartz sample pool of thermostatic control on the Aviv spectropolarimeter (± 0.1 ℃).(200-260nm) record circular dichroism spectrum in the scope of peptide.For improving signal to noise ratio, a plurality of spectrums (three s') mean value is all got in each measurement.With the CDPro software package on the Dell Personal Computer (by the NarasimhaSreerama exploitation of Colorado State University), going of the molar ellipticity spectrum of at every turn measuring is superimposed.

Embodiment 2: atherosclerotic plaque produces ozone and cholesterol ozonolysis product

Use aforesaid method, present embodiment shows, can produce ozone by carotid endarterectomy from the atherosclerosis tissue that 15 human patients (n=15) obtain, and ozone can be by detecting with indigo carmine 1 reaction.

The ozone that atherosclerotic plaque produces is to the bleaching of indigo carmine

The inventor before found, when in indigo carmine 1 (the chemical trap of ozone) solution, using the white corpuscle of protein kinase C activation thing 4-β-phorbol 12-tetradecanoic acid 13-acetic ester (PMA) processing antibody sandwich, the visible absorbance of indigo carmine 1 is bleached, and indigo carmine 1 is converted to isatin sulfonic acid 2.Referring to for example P.Wentworth Jr etc., Science 298,2195 (2002); B.M.Babior, C.Takeuchi, J.Ruedi, A.Guitierrez, P.Wentworth Jr., Proc.Natl.Acad.Sci.U.S.A.100,3920 (2003); P.Wentworth Jr etc., Proc.Natl.Acad.Sci.U.S.A.100,1490 (2003).The structure of isatin sulfonic acid 2 provides in Figure 1A.When these are tested at H ₂ ¹⁸O (＞95% ¹⁸When carrying out O), observe in the lactan carbonyl that oxygen isotope is incorporated into isatin sulfonic acid 2 (seeing above).This method with ozone and ¹O ₂ ^*Come with other oxygenants differences of also oxidable indigo carmine 1, because in the middle of the oxygenant that it is believed that with inflammation-related, have only two keys of ozone oxidation cracking indigo carmine 1, the while, isotropic substance was (from H ₂ ¹⁸Among the O) be incorporated in the lactan carbonyl of isatin sulfonic acid 2 (referring to above and Figure 1A).

As described in embodiment 1, it is believed that from 15 debatable atherosclerotic human patients obtains the patch material by carotid endarterectomy.Each spot is divided into two equates part (about 50mg weight in wet base is suspended among the 1mL PBS).Every part plaque substance is joined in phosphate-buffered saline (PBS, pH 7.4,10mM phosphate buffered saline buffer, the 150mM NaCl) solution (1mL) of indigo carmine 1 (200 μ M) and ox peroxidase (50 μ g/mL).Join in the aliquots containig or another aliquots containig of suspension patch material by DMSO solution, begin to analyze DMSO (10 μ L) or tetradecanoic acid phorbol ester (PMA, 10 μ L, 20 μ g/mL).

When adding PMA, in 15 spot samples, observe the bleaching (Figure 1B) of the visible absorbance of 14 appearance 1.Determine that through reversed-phase HPLC analysis (Figure 1A and C) formation of isatin sulfonic acid 2 is followed in this bleaching.Depend on the separation spot or the piece that are tried, the amount of formed isatin sulfonic acid 2 changes between 1.0-262.1nmol/mg.The mean vol of the isatin sulfonic acid 2 that is produced by different isolates is 72.62 ± 21.69nmol/mg.

When containing H ₂ ¹⁸The PBS (＞95% of O ¹⁸O) (n=2) in during with the PMA of indigo carmine 1 (200 μ M) the patch material that suspends activation, as in the mass spectrum (Fig. 1 D) of isolating split product isatin sulfonic acid 2 [M-H] ^-Shown in the relative intensity at 228 and 230 mass fragment peaks, nearly 40% indigo carmine 1 lactan ketonic oxygen mixes ¹⁸O.

These are produced by activatory atherosclerotic plaque material with studies show that indigo carmine 1 is carried out, ozone.

Cholesteric ozonolysis product

A kind of main lipid that exists in the atherosclerotic plaque is a cholesterol 3.D.M.Small，Arteriosclerosis 8，103(1988)。In chemical model research, the investigator shows, one group of oxygenant as ³O ₂, ¹O ₂ ^*, O ₂ ^-, O ₂ ^2-, hydroxyl radical free radical, O ₃And O ₂ ⁺And ozone O ₃In the middle of, have only ozone to make the Δ of cholesterol 3 ^5,6Two bond cleavages are separated, and produce 5, the 6-sterol 4a (Fig. 2 A) that breaks.This observations is consistent with other chemistry reports, and described chemistry report also points out, and 5, the disconnected sterol 4a of 6-is the primary product of cholesterol 3 ozonolysiss.Gumulka etc., J.Am.Chem.Soc.105,1972 (1983); Jaworski etc., J.Org.Chem 53,545 (1988); Paryzek etc., J.Chem.Soc.Perkin Trans.l, 1222 (1990); Comforth etc., Biochem.J.54,590 (1953).

Therefore, further experiment is concentrated in detection and is identified 5, and whether the disconnected sterol 4a of 6-or cholesteric other ozonolysis products are present in the atherosclerotic plaque.Therefore, investigated human artery's atherosclerotic plaque of 14 patients (n=14) in PMA activation front and back 5, the existence of the disconnected sterol 4a of 6-.

The improvement analytical procedure that Pryor is developed with the colleague is used for these research.Referring to K.Wang, E.Berm ú dez, W.A.Pryor, Steroids 58,225 (1993).This is improved one's methods and relates to organic solvent (methylene dichloride, PBS (the 1mL of 3 * 5mL) extraction homogeneous patch materials (about 50mg weight in wet base), pH 7) suspension, at room temperature use hydrochloric acid 2 then, the ethanolic soln of 4-dinitrophenylhydrazine (DNPH HCl) (2mM is in ethanol, and pH 6.5) was handled organic moiety 2 hours.Spray this reaction mixture of analytical reagent composition by negative ion electrospray on HPLC (direct injection, 360nm ultraviolet detection) and the line, seeking 4b is 2 of ozonolysis product 4a, the existing of 4-dinitrobenzene hydrazone derivative (Fig. 3).Do not activate in 14 that (1.4-200.6pmol/mg) detects hydrazone 4b in (6.8-61.3pmol/mg patch) among 11 of patch extract and all activation patch extract.In addition, judge by the mean vol of 4b, when activating with PMA in the patch material amount of 4a significantly increase.Concrete place says that when not using PMA, the mean vol of 4b is 18.7 ± 5.7pmol/mg.In contrast, when adding PMA, the mean vol of 4b is 42.5 ± 13.6pmol/mg (n=14, p＜0.05) (Fig. 3 A-B).

In the HPLC of patch extract analytic process, except that 4b, also observe two other main hydrazone peak.First peak has R _T～20.5 minutes, [M-H] ^-=597, second peak has R _T～18.0 minutes, [M-H] ^-=579 (Fig. 3 A, B).Hydrazone 4b distinguishes mutually with these peaks easily, because its retention time is about 13.8 minutes (R _T～13.8 minutes, [M-H] ^-597) (Fig. 3 A, B).Through comparing R with authentic sample _T～20.8 minutes peak is defined as the hydrazone derivative 5b (Fig. 2 and 3E) of aldol condensation product 5a.In chemistry model research, the main by product that Pryor had mentioned the hydrazine derivatize of 4a in the past is the hydrazone derivative 5b of aldol condensation product 5a, and its relative content is the function in acid concentration and reaction times.K.Wang，E.Bermúdez，W.A.Pryor，Steroids 58，225(1993)。

Under the derivatize condition that is adopted, in the 4a concentration range of being tried (5-100 μ M), the degree that 4a changes into 5b is about 20%.But, often observe the transformation efficiency more than 20%.20% the 5a measured quantity that surpasses the 4a that exists in the patch sample is probably by 3 ozonolysis and aldol condensation subsequently and produce.

But many biochemical component catalysis aldol condensations that contain amino or carboxyl.This component is present in spot and the blood, can promote the conversion of 4a to 5a.Further experiment shows, following amino acid and material promote the conversion of 4a to 5a: L-Pro (2 hours, conversion fully), Gly is (24 hours, transform fully), L-Lys HCl (24 hours, transform fully), L-Lys (OEt) 2HCl (100 hours, 62% transforms) and from the extract of atheroma artery (22 hours, conversion fully), whole blood is (15 hours, conversion fully), blood plasma (15 hours, transform fully) and serum (15 hours, transform fully).With respect to the speed of background response, all these materials all quicken the conversion of 4a to 5a.

As mentioned above, the amount of keto-aldehyde 4a increase in the patch when PMA activates.But PMA is undistincter to the effect that 5a forms.In some cases, the level that activates back 5a at PMA improves (Fig. 5 B, patient F and H), and in other cases, in the level decline (Fig. 5 B, patient C, G and N) of PMA activation back 5a.

Synthetic and analyzed many its 2,4-dinitrobenzene hydrazone derivative (Fig. 2 B) in mass spectrum has [M-H] ^-579 peaks contain carbonyl steroid derivative 6a-9a, to help to identify [M-H] that located in 18 minutes ^-579 peaks (Fig. 3 A, B).Through inject altogether with the HPLC of authentic sample, negative ion electrospray sprays mass spectrum and UV spectrum relatively, the peak of locating in～18 minutes is defined as 6b, i.e. the A of the hydrazone derivative of 6a and 4a ring dewatered product (Fig. 3 D).Studied under the selected derivatize standard conditions 4a to the transforming degree of 6b.Discovery in the 4a concentration range of being tried (5-100 μ M) this transforming degree all the time below 2%.These data show that the 6a amount of the keto-aldehyde 4a amount 2% in the extract that surpasses that exists in the patch extract promptly existed, and was produced by the β elimination of water by ozonolysis product 4a before derivatize.

Except these three kinds main hydrazone products of 4b-6b, also detect another kind of product 7b, and be defined as the hydrazone derivative of 7a and the A ring dewatered product of 5a.(＜5pmol/mg) existence, retention time is about 26 minutes ([M-H] to this product (7b) with trace in several spot extracts ^-579, Fig. 4).But, the detection limit that the amount of 7b is measured near the HPLC that adopted in the patch extract, as yet not to this compound in all spot samples existence or do not exist and carried out research comprehensively.

Two keys (being the chemical feature of ozone) of activation patch material oxicracking indigo carmine 1, and cholesteric Δ ^5,6Two keys by ozone exclusive approach (according to the known chemical principle) cracked experimental evidence provided convictive evidence, atherosclerotic plaque can produce ozone.In addition, because these unique cholesterol ozonation products also exist, in the evolution of atherosclerotic plaque, also produce ozone probably before the spot activation.

Confirm that now the ozone that external source gives has short scorching effect by the activation of interleukin (IL)-1 α, IL-8, Interferon, rabbit (IFN)-γ, platelet aggregation factor (PAF), grow relevant oncogene (Gro)-α, nf (NF)-κ B and tumour necrosis factor (TNF)-α in vivo.Except these generally well-known ozone effects in inflammation, also exist atherosclerotic plaque exclusive situation, the ozone that this situation can strengthen endogenous generation when producing at this position to the initiation of disease and the pathology effect of perpetuity.Cholesteric ozonolysis may be that patch is exclusive, because the high-concentrated ozone and the cholesterol that need only occur at this position, and other active substances that can catch the ozone of any generation do not exist.

As long as atherosclerotic artery contains antibody and activated macrophage and myeloperoxidase form ¹O ₂ ^*The generation system, atherosclerotic lesion can produce O by the water oxidative pathway of antibody catalysis probably ₃Really, 3 Δ ^5,6It is the further evidence that ozone produces by antibody catalysis in the inflammation that two bond cleavages are separated the observations that produces 4a.Known many oxygen sterols produce by cholesteric oxidation in vivo, and only exist the 5a analogue of textural difference to isolate from sponge Stelletta hiwasaensis on the cholestane side chain, as the part of comprehensive screening of pair cell toxicity natural product.T.Miyamoto，K.Kodama，Y.Aramaki，R.Higuchi，R.W.M.Van Soest，Tetrahedron Letter 42，6349(2001)；B.Liu，Z.Weishan，Tetrahedron Lett.43，4187(2002)。But do not report before as far as our knowledge goes, in the mankind and examine ruined derivative as steroid in sterol 4a-6a.Therefore, importantly initiate search, and study their biological function other this steroids and derivative thereof.

Embodiment 3: cholesterol ozonolysis product is present in atherosclerotic's the blood flow

The inventor confirms that in front ozone produces in the water oxidative pathway of antibody catalysis, and ozone can play a role in inflammation as effective oxygenant.P.Wentworth Jr etc., Science 298,2195 (2002); B.M.Babior, C.Takeuchi, J.Ruedi, A.Guitierrez, P.Wentworth Jr., Proc.Natl.Acad.Sci.U.S.A.100,3920 (2003); P.Wentworth Jr etc., Proc.Natl.Acad.Sci.U.S.A.100,1490 (2003).

Inflammation is considered to the factor in the incidence of atherosclerosis mechanism.R.Ross，NewEngl.J.Med.340，115(1999)；G.K.Hansson，P.Libby，U.Schnbeck，Z.-Q.Yan，Circ.Res.91，281(2002)。But, before the present invention, can not obtain the specificity noninvasive method that inflammatory artery disease and other inflammatory processes can be distinguished mutually.The product that the unique component of atherosclerotic plaque and atherosclerotic plaque are discharged in the blood flow can provide this method.Specifically, atherosclerotic lesion contains the cholesterol of high density.As shown here, ozone is produced by atherosclerotic lesion, and cholesterol ozonolysis product such as 4a and/or its aldol condensation product 5a are produced by atherosclerotic lesion.Therefore, carried out further experiment, whether can be used as inflammatory artery disease such as atherosclerotic mark to find out this cholesterol ozonolysis product.

Analyzed plasma sample, to seek existing of 4a or 5a from two groups of patients.Group A fully develops into the patient (n=8) who has reason to carry out endarterectomy by the Atheromatosis condition and forms.Group B patient is the patient of the general medical science clinic of random choose.Have 6 to detect aldol 5a in 8 patients of group A, (detection limit of this mensuration is～1-10nM) (Fig. 5 A-C) content in the scope of 70-1690nM.Have only one can detect 5a in 15 plasma samples of group B.In any patient's blood sample, all do not detect keto-aldehyde 4a (detection limit of this mensuration is～1-10nM).These data show, or are that 4a changes into 5a by contained catalyzer in the blood, or are that composition in the blood plasma has distinctiveness avidity to 4a and 5a.

In the past, because cholesterol autoxidation problem, the serum analysis of " oxygen sterol " is difficult.H.Hietter，P.Bischoff，J.P.Beck，G.Ourisson，B.Luu，Cancer Biochem.Biophys.9，75(1986)。But, as described herein, in the middle of all ornitrol oxidation products that produce by the biological dependency oxygenizement of cholesterol 3, steroid 4a and 5a be ozone exclusive product.These studies show that the existence of acetal product 5a in the blood plasma (detecting with its DNP hydrazone derivative 5b) can be used as the mark of atherosclerosis middle and advanced stage arterial inflammation.Therefore, the antibody catalysis of ozone produces and these irrelevant apparently in other respects factors of cholesterol accumulation, inflammation, oxidation and cell injury can be got in touch to causing atherosclerotic pathology cascade.

Some studies show that the ornitrol oxidation product has biological activity, as cytotoxicity, actuating arteries and veins gruel type and immunogenicity.H.Hietter，P.Bischoff，J.P.Beck，G.Ourisson，B.Luu，Cancer Biochem.Biophys.9，75(1986)；J.L.Lorenso，M.Allorio，F.Bernini，A.Corsini，R.Fumagalli，FEBS Lett.218，77(1987)；A.Sevanian，A.R.Peterson，Proc.Natl.Acad.Sci.U.S.A.81，4198(1984)。Suppose that ornitrol oxidation product 4a and 5a never are considered to occur in human body, these compounds of further research as described below are to the effect of the critical aspects of atheroma formation.

Embodiment 4: the cytotoxicity of cholesterol ozonolysis product

Some ornitrol oxidation product has biological activity, as cytotoxicity, actuating arteries and veins gruel type and immunogenicity.In the present embodiment, analyzed 4a and 5a cytotoxic effect to various kinds of cell system.

Following clone: Levy etc. in this research, have been adopted, the human B lymphocyte of describing among the Cancer 22,517 (1968) (WI-L2); Weiss etc., J.Immunol.133,123 (1984) the middle T lymphocyte clones of describing (Jurkat E6.1); Folkman etc., Proc.Natl.Acad.Sci.U.S.A.76,5217 (1979) middle vascular smooth muscle cell system (VSMC) and abdominal cavity aortic endothelial cell (HAEC) clones of describing; Ralph etc., J.Exp.Med.143,1528 (1976) the middle rat tissue scavenger cells of describing (J774A.1); With Mbawuike etc., J.Leukoc.Biol.46, the pulmonary alveolar macrophage clones of describing in 119 (1989) (MH-S).

The 4a of chemosynthesis and 5a are to the multiple known cell type that is present in atherosclerotic plaque---white corpuscle, vascular smooth muscle cell and endotheliocyte have cytotoxicity.The result shows in Fig. 6 and in the table 3.

Table 3

Clone	The IC of 4a 50	The IC of 5a 50
			WIL2	10.9±1.6μM	17.7±2.3μM
Jurkat E6.11	15.5±1.7μM	12.6±1.9μM
			HAEC	24.6±3.2μM	18.2±1.9μM
VSMC	21.9±2.2μM	29.8±2.8μM
			J774A.1	15.6±2.1μM	26.1±2.8μM
MH-S	11.2±1.2μM	13.6±1.1μM

4a and 5a are to the IC of all subject cell systems ₅₀Be worth closely similar.In addition, compound 4a and 5a are closely similar to the cytotoxicity curve of subject cell system.In view of significant textural difference between 4a and the 5a, these results are uncommon.But 4a and 5a be mutual really balance (on seeing) in by cellular constituent such as the promoted process of amino acid, and 4a and 5a can be in equilibrium state mutually in the time bar of cytotoxic assay.Therefore, compound 4a can have similar cytotoxicity in vivo with 5a.

The inventor uses similar method to confirm that compound 6a, 7a, 7c, 10a, 11a and 12a have cytotoxicity to leucocyte cell line, and disconnected keto-aldehyde 4a and aldol adducts 5a thereof have cytotoxicity to neuronal cell system.The 7c compound has following structure.

Ozone and cholesteric near causing producing cytotoxicity steroid 4a-12a and 7c, they are in position under the production, fully can be by promoting the damage of endotheliocyte or smooth muscle cell, perhaps, in the development of pathology, play effect (on seeing) by causing the inflammatory cell apoptosis in the atheroma.Cholesteric ozonolysis can be facilitated the patch instability in the aforementioned atherosclerotic plaque crystalline phase, and the patch instability is considered to the final step before the artery occlusion.

Embodiment 5: cholesterol ozonolysis product promotes foam cell to form

With change LDL and apoprotein B ₁₀₀Structure

Low-density lipoprotein (LDL) modification that improves low-density lipoprotein actuating arteries and veins gruel type is considered to the developing critical event of cardiovascular disorder.D.Steinberg，J.Biol.Chem.272，20963(1997)。For example, remove LDL or the apoprotein B that acceptor improves scavenger cell picked-up LDL by CD36 and other scavenger cells ₁₀₀(apoB-100, the protein component of LDL) oxidative modification is considered to the intraictal decisive pathology reason incident of atherosclerosis.The experiment that present embodiment is described shows that cholesterol ozonolysis product 4a and 5a can promote the formation of foam cell from scavenger cell, but and the structure of modified LDL and apoB-100.

As described in embodiment 1, in the presence of non-activated mouse macrophage (J774.1), with LDL (100 μ g/mL) with 4a or 5a incubation.These scavenger cells are exposed to after 4a or the 5a, and beginning lipid load, foam cell begin to occur (Fig. 7) in reaction vessel.

In addition, (Fig. 8 B C) detect to show circular dichroism, and people LDL (100 μ g/mL) causes the structure time of origin dependent change of apoB-100 with 4a and 5a (10 μ M) incubation.The circular dichroism analysis of total LDL discloses when no 4a and 5a, and in the time length of experiment (48 hours), the secondary structure of LDL keeps stable (Fig. 8 A) usually.Shown in Fig. 8 A, the albumen inclusion of normal LDL has αLuo Xuanjiegou (～40 ± 2%) and more a spot of beta structure (～13 ± 3%), βZhuan Jiao (～20 ± 3%) and the random coil (27 ± 2%) of larger proportion.But as LDL during with 4a and 5a incubation, though its spectrogram shape still keeps some similar (Fig. 8 B and C) to natural LDL, its secondary structure has considerable damage, mainly is loss (4a～23 ± 5% of αLuo Xuanjiegou; 5a～20 ± 2%), correspondingly the per-cent of random coil improves (4a～39 ± 2%; 5a 32 ± 4%).Therefore, as if 4a and 5a cholesterol ozonolysis product destroy the structural integrity of LDL.

Be the modified LDL structure, covalent reaction partly and between the epsilon-amino side group of apoB-100 lysine residue can take place in the aldehyde at 4a and 5a cholesterol ozonolysis product, form Schiff's base or enamine intermediate, its with the former viewed compounds of reaction between mda and 4-hydroxyl nonenal and apoB-100 seemingly.Steinbrecher etc., Proc.Natl.Acad.Sci.U.S.A.81,3883 (1984); Steinbrecher etc., Arteriosclerosis 1,135 (1987); Fong etc., J.Lipid.Res.28,1466 (1987).This Schiff's base or enamine intermediate can have obvious half life, can make the LDL of derivatize become and can be removed the form that acceptor is discerned by scavenger cell.Therefore, covalent reaction between 4a and 5a cholesterol ozonolysis product and the apoB-100-LDL can produce the apoB-100-LDL mixture of derivatize, it can be removed acceptor identification by scavenger cell and absorb with higher speed, thereby produces viewed foam cell among Fig. 7.

Unique known ornitrol oxidation form that contains the aldehyde composition is 4a and 5a ozonolysis product.Therefore, the reaction between this cholesterin derivative and the LDL/apoB-100 can provide heretofore the relation between the cholesterol of the unknown, foam cell formation and the artery plaque formation always.Therefore, the detection of high-caliber 4a and 5a ozonolysis product can provide these patients to suffer from directly measuring of atherosclerotic degree in patient's blood flow.

Embodiment 6: produce the antibody at cholesterol ozonation products

Present embodiment be described as producing at formula 13a, 14a or 15a haptens can with the antibody of cholesterol ozonation products and the reaction of hydrazone product.The haptenic structure of formula 13a, 14a and 15a is as follows:

Compound 13a is 4-[4-formyl-5-(4-hydroxyl-1-methyl-2-oxo-cyclohexyl)-7a-methyl-octahydro-1H-indenes-1-yl] valeric acid.

Method

The KLH conjugate of preparation compound 13a, 14a and 15a.With these KLH conjugates with the standard method immune mouse.Pipette spleen and dispersion from mouse, obtain splenocyte as antibody produced cell.

Splenocyte and the SP2/0-Ag14 cell (ATCC CRL-1581) that is derived from mouse myeloma are suspended in the serum-free RPMI-1640 substratum (pH 7.2) that is preheated to 37 ℃ altogether, and cell density is respectively 3 * 10 ⁴Individual cell/ml and 1 * 10 ⁴Individual cell/ml.Centrifugal suspension, collecting precipitation.In 1 minute, drip the serum-free RPMI-1640 substratum (pH 7.2) that 1ml contains the 50w/v% polyoxyethylene glycol, then in 37 ℃ of following incubation gained mixtures 1 minute to this precipitation.Further in mixture, drip serum-free RPMI-1640 substratum (pH 7.2), to final volume be 50ml, centrifugal collecting precipitation.Precipitation is suspended in the HAT substratum, is divided into the aliquots containig of 200 μ l, each aliquots containig joins a hole of 96 hole microtiter plates.37 ℃ of following one weeks of incubation, the result forms about 1,200 type hybridoma with microtiter plate.By combining of immunoassay and cholesterol ozonation products, analyze the supernatant liquor that derives from hybridoma.

Clause according to budapest treaty, the hybridoma KA1-11C5 and KA1-7A6 preservation U.S. typical case (the 10801 University Blvd. of DSMZ that will produce at formula 15a compound on August 29th, 2003, Manassas, Va., 20110-2209 USA (ATCC)), the ATCC preserving number is PTA-5427 and PTA-5428.According to the clause of budapest treaty, the hybridoma KA2-8F6 and the KA2-1E9 that will produce at formula 14a compound on August 29th, 2003 is preserved in ATCC equally, and the ATCC preserving number is PTA-5429 and PTA-5430.

Produce the storehouse (pool) of monoclonal antibody goods KA1-7A6:6 and KA1-11C5:6 (for the KLH conjugate at haptens 15a produces) and KA2-8F6 and KA2-1E9 (for the KLH conjugate at haptens 14a produces).By ELISA measure KA1-7A6:6 and KA1-11C5:6 monoclonal antibody (producing) by 15a to ozonation products 5a and cholesterol haptens 3c in conjunction with titre.Also carry out ELISA and measure, with determine KA2-8F6:4 and KA2-1E9:4 antibody (producing) by ozonation products 5a to 13b, 14b and cholesterol haptens 3c in conjunction with titre.

The structure of cholesterol haptens 3c below is provided.

The following ELISA that carries out measures.BSA conjugate with 13a, 14a, 3c, 13b, 14b or 15a joins high combination (hi-bind) 96 hole microtiter plates (FischerBiotech.) respectively, is allowed to condition at 4 ℃ of following standing over night.With PBS plate is thoroughly washed, add then newborn solution (1%w/v in PBS, 100 μ L.Allow plate at room temperature leave standstill 2 hours, wash with PBS then.Contain the culture of different antibodies goods with the PBS serial dilution, each diluent of 50 μ L is joined respectively in first hole of each row.After mixing and the dilution, allow plate 4 ℃ of following standing over night.With after the plate washing, add goat anti-mouse horseradish peroxidase conjugate (0.01 μ g, 5 μ L) with PBS.With plate 37 ℃ of following incubations 2 hours.With after the plate washing, add substrate solution (50 μ L) 3,3 ', 5,5 '-tetramethyl benzidine [0.1mg in the sodium acetate of 10mL (0.1M is pH6.0) and in the hydrogen peroxide (0.01%%w/v)].Plate was in the dark developed the color 30 minutes.Add sulfuric acid (1.0M, 50 μ L) quencher reaction, in 450nm place measuring light density.

The titre of report is 50% a serum dilution corresponding to maximum optical density.Data are analyzed with Graphpad Prism (3.0 version), and are reported as the mean value of at least twice replicate measurement.

The result

The result of ELISA test shows in table 4 and table 5.

Table 4: anti-15a antibody KA1-7A6:6 and KA1 11C5:6 are to 15a, ozonation products 5a

With cholesterol haptens 3c in conjunction with titre

Antibody	15a

	5a	3c
			KA1-7A6:6	32,000	32,000	16,000
			KA1 11C5:6	64,000	64,000	16,000

^*By the titre of ELISA measurement to the BSA conjugate of 15a, 5a and 3c.Absolute value be when in conjunction with the time corresponding to the extension rate of the antibody tissue culture supernatant solution of maximum absorbance 50%.

As shown in table 4, the apparent binding affinity of Ce Lianging is almost completely identical as mentioned above.

Table 5:KA2-8F6:4 and KA2-1E9:4 antibody (being produced by 5a) are to 15b, 14b and cholesterol

Haptens 3c in conjunction with titre

Antibody	15b	14b	3c
				KA2-8F6:4	32,000	32,000	16,000
				KA2-1E9:4	64,000	64,000	16,000

^*By the titre of ELISA measurement to the BSA conjugate of 15b, 14b and cholesterol haptens 3c.Absolute value is when in conjunction with the BSA conjugate of 13b, 15b and cholesterol haptens 3c, corresponding to the extension rate of the antibody tissue culture supernatant solution of maximum absorbance 50%.

These results show, can produce the high-affinity antibody goods at cholesterol ozonation products.

Embodiment 7: other cholesterol ozonation products detection method

Present embodiment explanation cholesterol ozonation products can detect by several different methods, comprises by the free aldehyde on these ozonation products being conjugated to the fluorescence part and passing through use and the antibody of these ozonation products reaction.

Material and method

General method

Unless otherwise specified, respond and all carry out with dried reagent, solvent and flame-dried glass wares.Unless otherwise specified, raw material is available from Aldrich Chemical Company, and former state is used when obtaining.Cholesterol-[26,26,26,27,27,27-D ₆] available from MEDICALISOTOPES INC.Flash column chromatography all uses silica gel 60 (230-400 order) to carry out.2 of cholesterol ozonation products 4a and 5a and ozonation products 4a and 5a, 4-dinitrophenylhydrazone (being respectively 4b and 5b) synthesizes by the description of previous embodiment.Thin-layer chromatography (TLC) uses Merck (0.25mm) to be coated with shop silica gel Kieselgel 60 F ₂₅₄Plate carries out, and dyes with p-anisaldehyde and develops the color. ¹H NMR spectrum record on Bruker AMX-600 (600MHz) wave spectrum meter. ¹³CNMR spectrum record on Bruker AMX-600 (150MHz) wave spectrum meter.Chemical shift is to report with the δ scale with respect to outer target 1,000,000/(ppm).

3 beta-hydroxies-5-oxo-5, the red sulphonyl hydrazone (4d) of 6-secocholestane-6-aldehyde synthetic

With red sulfonyl hydrazide (50mg, 0.17mmol) and tosic acid (1mg, (65mg is in acetonitrile solution 0.16mmol) (8ml) 0.0052mmol) to join cholesterol ozonation products 4a.Stirred reaction mixture is 2 hours under the room temperature argon atmospher, and vacuum-evaporation is to doing.Resistates is dissolved in methylene dichloride (10ml) and water (2 * 10ml) washings.The organic moiety dried over mgso, vacuum concentration.Thick yellow oil is by silica gel chromatography [ethyl acetate-hexane (1: 1; 7: 3)] purifying, obtain title compound 4d (70mg, 68%), be the mixture (cis: trans 8: 92) of geometrical isomer:

¹H NMR(CDCl ₃)δ9.341(s，1H)，8.567(d，J＝8.4Hz，1H)，8.358(dd，J＝7.2，1.2Hz，1H)，8.290(d，J＝8.4Hz，1H)，7.550(dd，J＝8.4，7.6Hz，1H)，7.539(dd，J＝8.4，7.6Hz，1H)，7.167(d，J＝7.6Hz，1H)，7.000(t，J＝4.0Hz，0.92H trans)，6.642(dd，J＝6.8，2.8Hz，0.08H cis)，4.273(bs，1H)，3.045(dd，J＝13.6，3.4Hz，1H)，2.869(s，6H)，2.233(d，J＝13.6Hz，1H)，2.097(dt，J＝18，4.4Hz，1H)，1.162(s，3H)，0.904(d，J＝6.4Hz，3H)，0.899(d，J＝6.8Hz，3H)，0.892(d，J＝6.4Hz，3H)，0.513(s，3H)； ¹³C NMR(CDCl ₃)δ209.66，151.77，149.49，133.52，131.20，130.99，129.64(2C) ^*，128.52，123.25，118.83，115.25，71.07，56.20，52.68，52.56，47.10，45.40，42.32，40.81，39.82，39.48，36.51，36.05，35.79，34.39，31.05，28.02，27.74，27.30，24.27，24.13，22.99，22.84，22.56，18.53，17.45，11.31；

C ₃₉H ₅₉N ₃O ₄SNa (M+Na) HRMALDIFTMS calculated value 688.4118, measured value 688.4152; R _f0.43[ethyl acetate-hexane (7: 3)]. ^*2C represents that this signal is considered to corresponding to two carbon signal (C from red sulphonyl part ₀Press gHSQC).

Red sulphonyl hydrazone (5c) synthetic of cholestane-6 β-formaldehyde falls in 3 beta-hydroxies-5 beta-hydroxies-B-

To cholesterol ozonation products 5a (30mg, tetrahydrofuran (THF) 0.072mmol) (5ml) solution add red sulfonyl hydrazide (25mg, 0.08mmol) and hydrochloric acid (dense, 0.05ml).By adding the white precipitate dissolving that water (0.2ml) will form immediately.Reaction mixture is evaporated to dried after stirring 3 hours under the room temperature argon atmospher uniformly.Red resistates is dissolved in ethyl acetate (10ml) and water (2 * 10ml) washings.The organic moiety dried over mgso, vacuum concentration.Thick yellow oil is at first by silica gel chromatography [ethyl acetate-methylene dichloride (1: 4-1: 1)] purifying, then by preparation HPLC (C18 Zorbax21.22mm and 25cm, 100% acetonitrile) purifying, obtain title compound compound 5c (14.5mg, 30%), be the mixture (cis: trans 17: 83) of geometrical isomer:

¹H NMR(CDCl ₃)δ8.557(d，J＝8.8Hz，1H)，8.372(dd，J＝7.2，1.2Hz，1H)，8.300(d，J＝8.8Hz，1H)，8.084(s，1H)，7.575(dd，J＝8.8，7.6Hz，1H)，7.554(dd，J＝8.8，7.6Hz，1H)，7.197(d，J＝7.6Hz，1H)，7.057(d，J＝7.2Hz，0.84H trans)，6.517(d，J＝5.2Hz，0.16H cis)，4.229(m，0.17H cis)，4.004(m，0.83H trans)，2.905(s，6H)，2.379(bm，4H)，1.913(dd，J＝9.6，7.2Hz，2H)，0.886(d，J＝6.8Hz，3H)，0.879(d，J＝6.4Hz，3H)，0.841(d，J＝6.8Hz，3H)，0.691(s，3H)，0.393(s，3H)； ¹³C NMR(CDCl ₃)δ154.081，133.425，131.367，130.912，129.695，128.611，123.350，115.121，83.268，70.469，67.079，55.773，55.677，55.280，51.652，45.429，45.038，44.372，43.129，42.443，39.488，36.143，35.585，28.580，28.458，27.984，27.766，23.850，22.825，22.549，21.389，18.659，18.063，12.192；

C ₃₉H ₅₉N ₃O ₄SNa (M+Na) HRMALDIFTMS calculated value 688.4118, measured value 688.4118; R _f0.41[ethyl acetate-methylene dichloride (1: 1)].

3 beta-hydroxies-5-oxo-5,6-disconnected [26,26,26,27,27,27-D ₆]-cholestane-6-aldehyde (D ₆-4a) synthetic

Under-78 ℃, the gas phase mixture of ozone in oxygen is blown into D ₆(this moment, solution became light blue-cholesterol for 50mg, 5mL chloroform-methanol (9: 1) solution 0.13mmol) 1 minute.Evaporation reaction mixture, at room temperature (40mg 0.61mmol) stirred 3 hours in 2.5mL acetate-water (9: 1) with the Zn powder.(10mL) dilutes this heterogeneous mixture with methylene dichloride, and water (3 * 5mL) and salt solution (5mL) washing.Organic moiety is also evaporated with dried over mgso.Resistates obtains title compound with silica gel chromatography (with hexane-ethyl acetate 5: 1,3: 1 and 2: 1 wash-outs) purifying, for white solid (44mg, 0.104mmol), yield: 81%.

¹H NMR 600MHz(δ，ppm，CDCl ₃)：9.61(s，1H)，4.47(s，1H)，3.09(dd，1H，J＝13.6Hz，4.0Hz)，2.25-2.40(m，3H)，2.15-2.19(m，1H)，1.01(s，3H)，0.88(d，3H，J＝6.1Hz)，0.67(s，3H). ¹³CNMR 150MHz(δ，ppm，CDCl ₃)：217.5，202.8，71.0，56.1，54.2，52.6，46.8，44.1，42.5，42.1，39.8，39.3，35.9，35.7，34.7，34.0，27.8，27.7，27.5，25.3，23.7，23.0，18.5，17.5，11.5.

3 beta-hydroxies-5 beta-hydroxies-B-norcholesterol-[26,26,26,27,27,27-D ₆]-6 β-formaldehyde (D ₆-5a) synthetic

To D ₆(26mg, (20: 1,5mL) solution added L-proline(Pro) (11mg) to acetonitrile-water 0.061mmol) to-4a.At room temperature stirred reaction mixture is 2.5 hours, vacuum-evaporation.Resistates is dissolved in ethyl acetate (10mL), water (2 * 5mL) and the salt water washing.The organic moiety dried over mgso, evaporation stays analytically pure white solid (26mg, 0.061mmol, yield: 100%), supply to carry out NMR.

¹HNMR 600MHz(δ，ppm，CDCl ₃)：9.69(s，1H)，4.11(s，1H)，2.23(dd，1H，J＝9.2Hz，3.0Hz)，0.91(s，3H)，0.90(d，3H，J＝6.6Hz)，0.70(s，3H)； ¹³C NMR150MHz(δ，ppm，CDCl ₃)：204.7，84.2，67.3，63.9，56.1，55.7，50.4，45.5，44.7，44.2，40.0，39.7，39.3，36.1，35.6，28.3，27.9，27.5，26.7，24.5，23.8，21.5，18.7，18.4，12.5.

4-(5-(4-hydroxyl-1-methyl-2-oxo cyclohexyl)-7 Alpha-Methyls-4-(2-oxoethyl)-octahydro-1H-indenes-1-yl) valeric acid 15a's is synthetic.Press D ₆The described ozonolysis that carries out 3 beta-hydroxy courage steroids-5-alkene-24-acid 3c of-5a.

¹HNMR 400MHz(δ，ppm，CDCl ₃)：9.60(s，1H)；4.47(s，1H)，3.40(dd，J＝13.6Hz，4Hz，1H)；1.00(s，1H)，0.91(d，J＝6.4Hz，3H)，0.67(s，3H). ¹³C NMR 100MHz(δ，ppm，CDCl ₃)：218.7，202.9，179.8，70.9，55.5，54.1，52.5，46.4，44.0，42.4，42.1，39.6，35.1，34.5，34.0，30.8，30.4，27.5，27.3，25.1，22.8，17.9，17.4，11.4.

The extracting of cholesterol ozonation products

With improved Bligh and Dyer method from blood sample and tissue sample extracting TL.Referring to Bligh EG, D.W.Can J Biochem Physiol 1959,37,911-17.Join the potassium primary phosphate (KH that adds a cover in the Glass tubing with being collected in Vacutainer pipe and being preserved in 4 ℃ contain citric acid or EDTA as the human plasma (200 μ L) of antithrombotics ₂PO ₄, 0.5M, 300 μ L) in.Add methyl alcohol (500 μ L), of short duration whirlpool stirred sample.Add chloroform (1mL), whirlpool stirred sample 2 minutes.With 3000rpm centrifugal 5 minutes, pipette organic layer.Repeat chloroform adding, whirlpool stirring and centrifugal this process.Merge organic layer, vacuum-evaporation.Obtain the endarterectomy sample from the patient who carries out the carotid artery endarterectomy according to conventional indication.TheScripps Green Hospital Institutional Review Board has ratified the human subjects scheme.Sample is freezing and-70 ℃ of following preservations before analysis.For analyzing, allow tissue sample be warming up to room temperature, then with organizing clarifixator (Tekmar) homogeneous in water-containing buffering liquid (KH ₂PO ₄, 0.5M, 1-2mL) in.Homogenizing fluid is joined methyl alcohol: chloroform (1: 3,6mL) in the solution, with 3000rpm centrifugal 5 minutes.Collect organic moiety.Chloroform (6mL) is joined remaining aqueous can mix part, centrifugal sample (3000rpm, 5 minutes).The organic moiety that merges of vacuum-evaporation then.

The red sulfonyl hydrazide derivatize of extractive cholesterol ozonation products and HPLC analyze

Blood extract after the evaporation or tissue extract's (on seeing) are suspended in again and contain red sulfonyl hydrazide (200 μ M) and H ₂SO ₄In the Virahol of (100 μ M) (200 μ L), in 37 ℃ of incubations 48 hours.Analytical procedure comprises, with isocratic elution moving phase acetonitrile: water (90: 10,0.5mL/ minute), use fluoroscopic examination (excitation wavelength 360nm, emission wavelength 450nm), carrying out HPLC in the Hitachi D-7000 HPLC system that links to each other with Vydec C-18RP post analyzes.Retention time (the R of the red sulfonyl derivative (5c) of ozonation products 5a _T) be about 8.1 minutes.The retention time of the hydrazine derivative of 5a (5b) is about 10.7 minutes.By using Macintosh Personal Computer and Prism (edition 4 .0) software,, determine concentration routinely by calculating peak area with reference to the true standard product.

Gas chromatography-mass spectrum

Sample after the evaporation is rebuild in methylene dichloride to the 1mL volume, and 100uL pyridine and 100uL N by containing 1% trimethylchlorosilane, two (the trimethyl silyl)-trifluoroacetamides of O-join in the spissated patch extract, carry out silylation.With 37 ℃ of following incubations in the sample 2 hours, rotary evaporation was to doing then.Each sample is suspended in the 100uL methylene dichloride before analysis again.By split stream sampling (Agilent 7673 automatic samplers) not the 2.5ul sample is expelled on the HP-5ms post, thickness 30m * 0.25mm ID * 0.25um, flow velocity 1.2ml/ minute, 290 ℃ of injector temperature, temperature program(me) kept 5 minutes since 50 ℃, then with 20 ℃ of/minute intensifications, up to 300 ℃, kept 12 minutes.Carry out mass analysis with Agilent model 5973 inert, sweep limit 50-700m/z is then by selecting ion detection (SIM) scanning m/z 354 and 360.MS four utmost point temperature are 150 ℃, and the MS source temperature is 280 ℃.

Haptens 5a is coupled to carrier proteins KLH and BSA

With hydrochloric acid 1-ethyl-3,3 '-(0.008mmol) (1.8mg 0.008mmol) is dissolved in 0.01mL H to dimethylaminopropyl-carbodiimide with the sulfo group N-hydroxy-succinamide for EDC, 1.5mg ₂Among the O, and (2.5mg is in 0.1mL DMF solution 0.006mmol) to join haptens.Whirlpool stirs the mixture, and at room temperature keeps 24 hours, under 4 ℃, join then BSA (5mg, in the PBS damping fluid (0.9ml, 0.05mM, pH=7.5) in) in.Descend maintenance after 24 hours, at 4 ℃ this final mixture-20 ℃ of following preservations.The following diagram of reaction that relates to KLH or the BSA conjugate of synthetic compound 5a.

Reaction a relates to as mentioned above and uses O ₃/ O ₂Ozonolysis compound 3c.Reaction b relates to EDC and HOBt (in DMF) processing compound 15a and spending the night, then with BSA or KLH at phosphate-buffered saline (PBS), incubation among the pH 7.4.

Monoclonal antibody is produced and is undertaken by standard method.Following carry out 8 age in week the 129GIX+ mouse immunity: every mouse is with 10ug KLH-15a conjugate (in 50uL PBS), and described PBS mixes with isopyknic RIBI adjuvant, every 3 days peritoneal injections once, totally 5 immunity.Measure serum titer by ELISA.After 30 days, injection (IV) last 50ug KLH-15a conjugate (in 100uL PBS) in tail lateral vein medium sized vein.After 3 days, put to death animal, take out spleen, for fusion.To in the RPMI substratum, mix with 5: 1 with X63-Ag8.653 myeloma cell from the splenocyte of immune animal, centrifugal, under 37 ℃, be suspended in again among the 1mLPEG 1500.Described PEG in 3 minutes with 9mL RPMI dilution, centrifugal then 37 ℃ of following incubations 10 minutes, be suspended in the substratum again after, plating is in 15 * 96 orifice plates.Carry out ELISA, screening is in conjunction with cholesterol ozonation products 4a or 5a but the cholesteric antibody of debond.In 2 generations of selecting of hybridoma subclone, are to guarantee monoclonicity.

Prepare tissue slice from the aorta ascent stage of ApoE knock-out mice

Make sample anxious freezing in liquid nitrogen.Obtain 10 microns sections, be placed on the slide glass.By sample is immersed in 1: 1 ethanol successively: diethyl ether 20 minutes, 100% ethanol 10 minutes and 95% ethanol were fixed in 10 minutes.In PBS, after the washing, grant 1: 200 diluent that cholesterol ozonation products is had specific antibody, and with organizing incubation 1 hour.40: 1 diluents with the anti-mouse IgG of FITC labelled goat (Calbiochem) carry out the secondary mark.Obtain image with optronics microfire digital camera, handle with Adobe Photoshop.

The result

The fluoroscopic examination of the red sulphonyl hydrazone of cholesterol ozonation products.

As previously described in the embodiment, available K.Wang, E.Berm ú dez, W.A.Pryor, improving one's methods of the analytical procedure of developing in the chemical research of Steroids 58,225 (1993) detects cholesterol ozonation products in the body.Organic solvent (methylene dichloride is arrived in these PBS (1mL) pH 7.4 suspension extractings that relate to homogeneous patch material (～50mg weight in wet base) of improving one's methods, in 3 * 5mL), at room temperature use hydrochloric acid 2, the ethanolic soln of 4-dinitrophenylhydrazine (DNPH HCl) (2mM, pH 6.5) was handled organic solvable fraction 2 hours.Spray mass spectroscopy gained reaction mixture by negative ion electrospray on reversed-phase HPLC (direct injection, 360nm place ultraviolet detection) and the line, analyzing 4b is 2 of 4a, and (2,4-DNP) derivative and 5b are 2 of 5a to the 4-dinitrophenylhydrazone, the existence of 4-DNP derivative.This technology not only fast but also highly sensitive.But this assay method has many restrictions when being applied to biological sample.These restrictions comprise: the interference of the other biological compound of uv-absorbing is arranged at the 360nm place, and 4b changes into 5b in the conjugation reaction process, and puts together reaction efficiency decline under the lower concentration cholesterol ozonation products.

Therefore, tested new method, whether can improve measurement sensitivity with conclusive evidence.This method relates to cholesterol ozonation products is conjugated to the hydrazine with fluorescence chromophore, carries out fluoroscopic examination and HPLC then and analyzes.Selected fluorescence chromophore is a dansyl group.This assay method relates under above-mentioned acidic conditions makes extractive cholesterol ozonation products derivatize with red sulfonyl hydrazide.The product of red sulfonyl hydrazide and cholesterol ozonation products 4a reaction is following illustrated 4d.

The product of red sulfonyl hydrazide and cholesterol ozonation products 5a reaction is following illustrated 5c.

In multiple solvent, assessed the reaction efficiency of red sulfonyl hydrazide derivatize, described solvent such as hexane, methyl alcohol, chloroform, tetrahydrofuran (THF), acetonitrile and Virahol (IPA).Analysis confirmation thus, with regard to reaction efficiency and the spontaneous generation aldol condensation of cholesterol ozonation products 4a generate the speed of 5a minimum with regard to, IPA is an optimum solvent.True red sulphonyl hydrazone standard substance 4d and 5c (Fig. 9) with chemosynthesis come quantitative reaction efficient by HPLC.Under 37 ℃, cholesterol ozonation products 4a and red sulfonyl hydrazide (200 μ M) and sulfuric acid (100 μ M) derivatize 48 hours in IPA forms retention time (R _T) be about 11.2 minutes 4a hydrazone derivative 4d, its efficient is 86.0 ± 8.0%.Importantly, in the derivatize process, have only 1.3% 5c to be formed by the aldol condensation of 4a or 4d.In the 5a concentration range of 0.01-100 μ M, 5a changes into its red sulphonyl hydrazone derivative 5c (R _T～19.4 minutes) efficient be 83 ± 11%.The level of sensitivity of red sulphonyl hydrazone 4d and 5c is～10nM.

For determining that 4a and 5a cholesterol ozonation products from the plasma sample extracting and by the efficient of derivatize, make human plasma sample's admixture 5a, extracting then, with 2,4-DNP or red sulfonyl hydrazide are puted together.The content of puting together hydrazone that two kinds of methods detect does not have significant difference; Have 37.5 ± 1.9% to derive to red sulphonyl hydrazone 5c, have 31 ± 8.9% with 2,4-DNP hydrazone 5b reclaims.

Mass spectrometry on isotopic dilution-gas-chromatography and the line (ID-GCMS)

At present, great majority are measured the analytical procedure of the oxygen sterol in rich cholesterol tissue such as blood (blood plasma) and the atherosclerosis artery, are based on the GC that the band flame ion detects (FID) or selects ion detection (SIM).The advantage that the SIM method is better than the FID method is the specificity that detects.In bio-matrix, analyze the oxygen sterol and require to have this specific specificity.Target was used in the key aspect of SIM strategy was.The most frequently used is 5 α-cholestane.Referring to Jialil, I.; Freeman, D.A.; Grundy, S.M.Aterioscler.Thromb.1991,11,482-488; Hodis, H.N.; Crawford, D.W.; Sevanian, A.Atherosclerosis 1991,89,117-126.But, preferable methods be to use deuterium-labeled in target GC-MS because its not only sensitivity but also specificity is arranged can be proofreaied and correct at the different rate of recovery of different analytes.Dzeletovic，S.；Brueuer，O.；Lund，E.；Diszfalusy，U.Analytical Biochem.1995，225，73-80。The target effect has double in the deuterate.At first, they are related by isotopic abundance and concentration are set up, and make and can carry out quantitatively.Secondly, the feasible extraction efficiency that can assess cholesterol ozonation products of deuterate molecule that before the extracting program, adds known quantity.Leoni，V.；Masterman，T.；Patel，P.；Meaney，S.；Diczfalusy，U.；Bjrkhelm，I.J.Lipid.Res.2003，44，793-799。

As follows from [26,26,26,27,27,27-D]-cholesterol (deuterium is for 3c) preparation six deuteriums for cholesterol ozonation products D ₆-4a and D ₆-5a.

In the synthetic the first step (a), under 78 ℃, ozone is blown into D ₆In the chloroform-methanol of-3c (9: the 1) solution, produce D ₆-4a.In second step (b), with D ₆-4a is dissolved among the DMSO, at room temperature with proline(Pro) reaction 2.5 hours, produces D ₆-5a.

D ₆-4a and D ₆-5a tests the sensitivity of GC/MS method as interior mark on inner Agilent GC/MS.In typical method,, make true cholesterol, 4a, 5a, D by under 37 ℃ of argon atmosphers, handling 2 hours with pyridine and BSTFA ₆-cholesterol, D ₆-4a and D ₆The sample of-5a changes into their trimethyl silyl ether.After vacuum is removed volatile matter, resistates is dissolved in the methylene dichloride, transfers to then in the automatic sampler bottle.

Carry out GC-MS being connected on the Agilant Technologies 6890GC of 5973 Inert MSD (band shunting/not split stream sampling system and 7683 automatic injector assemblies) then.Operating mass spectrograph under the ion scan pattern fully.Observed retention time (R _T) and M ⁺Ion is as follows: ozonation products 4a and 5a (R _T=29.6 minutes, M ⁺354); D ₆-4a and D ₆-5a (R _T=29.6 minutes, M ⁺360); Cholesterol (R _T=27.2 minutes, M ⁺329), D ₆-cholesterol (R _T=27.2 minutes, M ⁺335).The derivation fragment of cholesterol ozonation products 4a and 5a is as follows among the GC-MS.

As implied above, cholesterol ozonation products 4a and 5a produce M ⁺About 354 fragment.Deuterate (D ₆) 4a and 5a cholesterol ozonation products generation M ⁺About 360 fragment.

Therefore, in measuring, GC-MS do not observe the difference between cholesterol ozonation products 4a and the 5a, and may be because cholesterol ozonation products 4a changes into 5a in the silylation step.Therefore, M ⁺The amount of 354 (or 360) is the measuring of concentration of true 4a and 5a cholesterol ozonation products.The area and the concentration of 354 quasi-molecular ions are linear, and for cholesterol ozonation products, the sensitivity of the lower level of measuring up to now is 10fg/ μ L (detection limit that the LC/MS that is equivalent to describe among the embodiment of front measures improves about 2-logarithm).

By carotid plaques material extracting cholesterol ozonation products, verify that further GCMS measures from clinical excision.Obtain to be used for the carotid artery endarterectomy tissue (n=2) of routine analysis from the patient who carries out the carotid artery endarterectomy, with organizing the clarifixator homogeneous 10 minutes (under argon gas), CHCl is arrived in extracting then ₃Among/the MeOH.With the extract silylation, carry out GC-MS then and analyze (Figure 10 and 11) as mentioned above.Ion abundance shows the GC-MS trace of time, has many clear and definite oxygen sterols that still remain.But, can know and differentiate ozonolysis product 4a and the 5a (R that merges _T=22.49 minutes).

These data have been unequivocally established and have carried out whole extracting and GC-MS and measure feasibility with 4a in the analysis of biological samples and 5a cholesterol ozonation products, and have verified the result about the atherosclerotic plaque species analysis described in the embodiment of front.

The immunohistochemistry location of cholesterol ozonation products 4a and 5a

As mentioned above, use KLH conjugate (the being cholesterol ozonation products 4a analogue) immune mouse of compound 15a.Monoclonal antibody produces by hybridoma method.These two kinds of monoclonal antibodies of 11C5 and 7A7 have good binding avidity (＜1 μ M) to cholesterol ozonation products 5a, and cholesterol is had fabulous specificity (low 1000 times of avidity).

Not strange at the haptenic anti-5a production of antibodies of 4a analogue, because show that as above cholesterol ozonation products 4a joins and causes it to change into 5a immediately in the blood.

Aorta cryofixation section from the ApoE deficient mice is carried out immunohistochemical staining with the anti-IgG second antibody of antibody 11C5 and FITC mark, when its when comparing with the painted serial section of non-specific murine antibody, confirm that cholesterol ozonation products 5a is positioned the atherosclerosis zone in the tunica intima lower floor.The fluorescence of tunica intima lower floor is not eliminated in the cholesteric absorption of antibody and solubility.

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The state of the art that this paper reference or all patents of mentioning and publication are represented those skilled in the art in the invention, each of this referenced patent or publication is attached to herein by reference, and its degree is attached to herein by independent reference in its entirety as it or integral body proposes in this article.The present patent application people keeps and will be attached to right in this specification sheets fully from any and all material of any this referenced patents or publication and information.

Concrete grammar described herein and composition are represented embodiment preferred, and are exemplary, and are not intended to and limit the scope of the invention.When this specification sheets of research, other targets, aspect and embodiment it will be apparent to those skilled in the art that they are also included within the defined spirit of the present invention of scope of claims.It will be apparent for a person skilled in the art that and to make various replacements and modification to the present invention disclosed herein and do not deviate from scope and spirit of the present invention.The present invention that this paper illustrative is described can suitably implement not existing under any key element or restricted condition that clearly is not disclosed as necessity herein.The method that this paper illustrative is described can suitably be implemented the sequence of steps that described sequence of steps not necessarily is limited to herein or points out in claims by different sequence of steps with process.The singulative that uses in this paper and the appended claim book comprises plural implication, unless other situation clearly stipulated in context.Therefore, comprise a plurality of this host cells (for example culture or group) when for example mentioning " host cell ", the rest may be inferred.The present invention under any circumstance can not be construed as limited to the clear and definite disclosed specific embodiment of this paper or embodiment or method.The present invention under any circumstance can not be construed as limited to the statement that any auditor or any other official or employee did of patent and trademark office, unless this statement is with the conditioned disjunction preserve specially to adopt by the present patent application people in replying document clearly and not.

Term that has adopted and expression are as descriptive term rather than restricted term; and be not to be intended to use this term and expression to get rid of shown and the feature of description or any Equivalent of its part; but it should be understood that and in the claimed scope of the invention, have various modifications.Therefore, be to be understood that, although the present invention is open clearly by preferred embodiment and optional feature, those skilled in the art can adopt the modification or the variation of notion disclosed herein, and this modification or variation are considered to fall in the defined scope of the invention of appended claim book.

This paper has done to understand and general description to the present invention.Each the narrower kind and the subspecies class group that fall into the kind disclosure also constitute a part of the present invention.This comprises that having the present invention that any subject matter is excluded the precondition of kind or negativity restriction plants class description, no matter whether the material that is excluded is clearly enumerated in this article.

Other embodiments fall into following claims.In addition, feature of the present invention or aspect under the situation about describing with Ma Kushi prescription formula, those skilled in the art will recognize that therefore the present invention also describes with any single member of Ma Kushi group or the mode of member's subclass.

Claims (63)

1. isolating cholesterol ozonation products, described cholesterol ozonation products produces in atherosclerotic plaque.

2. the ozonation products of claim 1, described ozonation products has formula 4a:

3. the ozonation products of claim 1, described ozonation products has formula 5a:

4. the ozonation products of claim 1, described ozonation products has arbitrary formula 6a-15a or 7c:

5. detectable cholesterol ozonation products derivative, described derivative comprises bisulfite adduct, imines, oxime, hydrazone, red sulphonyl hydrazone, semicarbazone or Tollins test product, and wherein said cholesterol ozonation products produces in atherosclerotic plaque.

6. cholesterol ozonation products hydrazone derivative, wherein said cholesterol ozonation products produces in atherosclerotic plaque.

7. the hydrazone derivative of claim 6, described hydrazone derivative has formula 4b or formula 4c:

8. the hydrazone derivative of claim 6, described hydrazone derivative has formula 5b:

9. the hydrazone derivative of claim 6, described hydrazone derivative has arbitrary formula 6b-15b or 10c:

10. the red sulphonyl hydrazone derivative of claim 6, described red sulphonyl hydrazone derivative has formula 4d:

11. the red sulphonyl hydrazone derivative of claim 6, described red sulphonyl hydrazone derivative has formula 5c:

12. a haptens, described haptens has formula 13a or 13b:

Wherein said haptens can be used to produce can with the antibody of cholesteric ozonation products or the reaction of hydrazone product.

13. a haptens, described haptens has formula 14a or 14b:

Wherein said haptens can be used to produce can with the antibody of cholesteric ozonation products or the reaction of hydrazone product.

14. a haptens, described haptens has formula 3c:

Wherein said haptens can be used to produce can with the antibody of cholesteric ozonation products or the reaction of hydrazone product.

15. a haptens, described haptens has formula 15a:

Wherein said haptens can be used to produce can with the antibody of cholesteric ozonation products or the reaction of hydrazone product.

16. an isolated antibody, described antibody can be in conjunction with cholesterol ozonation products.

17. a monoclonal antibody, described monoclonal antibody can be in conjunction with cholesterol ozonation products.

18. the antibody of claim 16 or 17, wherein said cholesterol ozonation products has formula 4a:

19. the antibody of claim 16 or 17, wherein said cholesterol ozonation products has formula 5a:

20. the antibody of claim 16 or 17, wherein said cholesterol ozonation products has arbitrary formula 6a-14a or 7c:

21. the antibody of claim 16 or 17, wherein said antibody produces at the haptens with formula 15a:

22. an isolated antibody, described antibody can be in conjunction with the hydrazone derivative of cholesterol ozonation products.

23. the isolated antibody of claim 22, wherein said hydrazone derivative have formula 4b or formula 4c:

24. the isolated antibody of claim 22, wherein said hydrazone derivative has formula 5b:

25. the isolated antibody of claim 22, wherein said hydrazone derivative has arbitrary formula 6b-15b or 10c:

26. the isolated antibody of claim 22, wherein said isolated antibody produces at the haptens with formula 13a or 14a:

27. the isolated antibody of claim 22, wherein said isolated antibody produces at the haptens with formula 15a:

28. an isolated antibody, wherein said isolated antibody are derived from hybridoma KA1-11C5:6 or KA1-7A6:6 that the ATCC searching number is PTA-5427 or PTA-5428.

29. an isolated antibody, wherein said isolated antibody are derived from hybridoma KA2-8F6:4 or KA2-1E9:4 that the ATCC searching number is PTA-5429 and PTA-5430.

30. an atherosclerotic method that detects among the patient, described method comprises: detect available from whether there being cholesterol ozonation products in patient's the sample.

31. the method for claim 30, wherein said ozonation products is produced by atherosclerotic plaque.

32. the method for claim 30, wherein said sample are serum, blood plasma, blood, atherosclerotic plaque material, urine or vascular tissue.

33. the method for claim 30, wherein said ozonation products are the compounds with formula 4a:

34. the method for claim 30, wherein said ozonation products are the compounds with formula 5a:

35. the method for claim 30, wherein said ozonation products are the compounds with arbitrary formula 6a-15a or 7c:

36. the method for claim 30, wherein said method further comprises reacts sample and hydrosulphite, ammonia, Schiff's base, aromatics or aliphatic hydrazine, red sulfonyl hydrazide, Gerard reagent, Tollins test reagent, and detects the derivative of the cholesterol ozonation products that is formed by this reaction.

37. further comprising, the method for claim 30, wherein said method make sample and hydrazine compound reaction, to produce the hydrazone derivative of cholesterol ozonation products.

38. the method for claim 37, wherein said hydrazine compound is a 2,4 dinitrophenyl hydrazine.

39. the method for claim 37, wherein said hydrazone derivative have formula 4b or formula 4c:

40. the method for claim 37, wherein said hydrazone derivative has formula 5b:

41. the method for claim 37, wherein said hydrazone derivative has arbitrary formula 6b-15b or 10c:

42. further comprising, the method for claim 30, wherein said method make sample and the reaction of red sulfonyl hydrazide, to produce the red sulphonyl hydrazone derivative of cholesterol ozonation products.

43. the method for claim 42, wherein said red sulphonyl hydrazone derivative has formula 4d or 5c:

Make sample and can contact 44. the method for claim 30, wherein said method further comprise in conjunction with the antibody of cholesterol ozonation products.

45. the method for claim 44, wherein said antibody produces at the haptens with formula 13a or 14a:

46. the method for claim 44, wherein said antibody produces at the haptens with formula 15a:

47. the method for claim 44, wherein said antibody are derived from hybridoma KA1-11C5:6 or KA1-7A6:6 that the ATCC searching number is PTA-5427 or PTA-5428.

48. the method for claim 44, wherein said antibody are derived from hybridoma KA2-8F6:4 or KA2-1E9:4 that the ATCC searching number is PTA-5429 and PTA-5430.

49. the method for claim 44, wherein said antibody can be in conjunction with the compounds with formula 4a:

50. the method for claim 44, wherein said antibody can be in conjunction with the compounds with formula 5a:

51. the method for claim 44, wherein said antibody can be in conjunction with the compounds with arbitrary formula 6a-15a or 7c:

52. one kind is detected the method whether cholesterol ozonation products is discharged by the atherosclerotic plaque among the patient, described method comprises: detecting available from whether having cholesterol ozonation products, wherein said ozonation products in patient's the sample is the compound of formula 5a:

53. an atherosclerotic method that detects among the patient, described method comprises: 2,4 dinitrophenyl hydrazine is joined in the sample available from the patient, whether have the hydrazone derivative of cholesterol ozonation products in the test samples.

54. the method for claim 53, wherein said hydrazone derivative has formula 4b, 4c, 5b, 6b, 7b, 8b, 9b, 10b, 10c, 11b, 12b, 13b, 14b or 15b:

55. an atherosclerotic method that detects among the patient, described method comprises: red sulfonyl hydrazide is joined in the sample available from the patient, whether have the red sulphonyl hydrazone derivative of cholesterol ozonation products in the test samples.

56. the method for claim 55, wherein said red sulphonyl hydrazone derivative is the compound with formula 4d or 5c:

57. whether have the method for cholesterol ozonolysis product in the test samples, described method comprises makes scavenger cell contact with sample, and whether definite scavenger cell increases the picked-up of lipid.

58. an atherosclerotic method that detects among the patient, described method comprise scavenger cell is contacted with sample available from the patient, and whether definite scavenger cell increase to the picked-up of lipid.

59. the method for the cholesterol ozonolysis product in the test samples, described method comprises makes low-density lipoprotein contact with sample, and whether the secondary structure of observing low-density lipoprotein changes.

60. an atherosclerotic method that detects among the patient, described method comprise low-density lipoprotein is contacted with sample available from the patient, and whether the secondary structure of observing low-density lipoprotein change.

61. the method for the cholesterol ozonolysis product in the test samples, described method comprises makes apoprotein B ₁₀₀Contact with sample, and observe apoprotein B ₁₀₀Secondary structure whether change.

62. comprising, an atherosclerotic method that detects among the patient, described method make apoprotein B ₁₀₀Contact with sample, and observe apoprotein B available from the patient ₁₀₀Secondary structure whether change.

63. each method among the claim 57-62, wherein said low-density lipoprotein or apoprotein B ₁₀₀Secondary structure observe by circular dichroism.

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