CN1211321A

CN1211321A - Methods for measurment and treatment predicated on presence of advanced glycosylation endproducts in tobacco and its combustion byproducts

Info

Publication number: CN1211321A
Application number: CN96180126A
Authority: CN
Inventors: A·瑟拉米; R·J·布卡拉; H·维拉萨拉; H·W·富恩德斯; C·J·瑟拉米
Original assignee: Alteon Inc; Picower Institute for Medical Research
Current assignee: Picower Institute for Medical Research; Synvista Therapeutics Inc
Priority date: 1995-12-26
Filing date: 1996-12-23
Publication date: 1999-03-17
Also published as: IN191280B; JP2000507346A; EP0877943A1; AU1345997A; WO1997023783A1; CA2241924A1; AU723649B2

Abstract

Methods are provided for measuring the accumulation of advanced glycosylation endproducts (AGEs), and for lowering the accumulation of advanced glycosylation endproducts, which are predicated on the discovery that such AGEs and their precedent glycotoxins are present in tobacco and its byproducts. More particularly, the methods focus on the observation that individuals who smoke or otherwise use tobacco have increased levels of AGEs relative to non-smoking individuals. The present methods relate to the measurement of AGE levels in both individuals and in tobacco and its byproduct, smoke, and to the treatment of such individuals with agents capable of reacting with glycosylation products to either avert or diminish the accretion of AGEs in the body. Methods are also provided for the evaluation of the tobacco products to determine their storage status and organoleptic capacity and potential, for the treatment of the ambient atmosphere to lower AGE levels, and for the treatment of the tobacco products and combustion byproducts to lower AGE levels therein. For example, air or other samples may be taken and evaluated by a dosimeter or like device, to determine whether AGE levels exceed normal, after which measures could be implemented to remediate the ambient condition. Likewise, filters and similar devices for removing AGEs from tobacco smoke are provided. All such methods and corresponding materials are contemplated and included.

Description

Mensuration and processing based on the advanced glycosylation end product that exists in tobacco and the combustion by-products thereof

Method

Technical field of the present invention

Put it briefly, the present invention relates to use tobacco product, such as the correlativity between the existence of cigarette and advanced glycosylation end product (AGEs) (advanced glycosylationendproduct), and relate to diagnosis, treatment and the industrial applications that can help to relate to this observations and relation.More particularly, the present invention itself relates to a kind of like this observations, it is the amount that the consumption of tobacco product can increase AGEs in the body, concomitant circumstances is the harm and the incidence of disease increase of accumulating relevant disease with smoking and AGE, and the present invention relates in the smog of tobacco, tobacco and the extract thereof and the detection of AGEs in the tobacco user body, comprise and detect unwanted excessive AGE level and estimate the tobacco of commercial scale and the material of being fuming.The invention still further relates to AGEs and form the application of inhibitor in various scopes so that treat or prevent unwanted excessive AGE level and the tobacco and the material of being fuming that improve commercial scale.

Background of the present invention

Smoking has obtained proof widely to the illeffects of health.In other situation, with regard to the smoker, this class disease, increase significantly on the incidence of disease and seriousness such as cancer and coronary artery disease, and this class patient is showing significant change aspect lipoprotein distribution and the oxidation LDLs rising equally.In other situation, the incidence of disease and the seriousness of also observing coronary artery disease and dyslipidemia in the patient that advanced glycosylation end product (AGEs) level raises increase, and the consistance of these observationss provides stage for this discovery that constitutes basis of the present invention.

Non-enzyme reaction between glucose and the protein obtained confirming many years, and the biology and the medical consequences of nonenzymatic glycosylation still manifest at present in the molecule detail drawing of these reactions and the body.The nonenzymatic glycosylation of Que Rening takes the form of in the cooked food process and brown pigment occurs the earliest, and this is definite in 1912 by Mei Lade (maillard), and term " brown stain " is used for this Food Chemistry.U.S. rad observe glucose or other reducing sugar spontaneously with contain amino compound, form original schiff bases adduct such as amino acid and reactive polypeptide.This then shrinkage product carries out a series of other spontaneous dehydration, rearrangement and other reactions and forms various types of brown pigment, is called advanced glycosylation end product (or AGEs) at present.

This initial discovery in later several years, studied Maillard reaction the Food Chemistry furniture body and determined and stored and can carry out non-enzymatic browning and the improvement of primitive reaction between as glucose and polypeptied chain through heat treated food, and determine that as a result of protein cross and protein correspondingly show the bioavilability of reduction.In this, determine that the pigment that causes protein glycosylation (or advanced glycation) toffee to manifest also has characteristic absorption spectrum and fluorescent characteristic.

The reducing sugar of the above-mentioned discussion of discovered in recent years and the reaction between the food component are parallel in vivo to be carried out.Therefore, the non-enzyme that adds the original schiff bases that glucose forms by the free amine group on protein is reset and can be formed stable amino, and 1-deoxidation ketose base (ketosyl) adduct is called the Amadori product with this adduct.(parallel reactor that relates to reduction ketose rather than aldose generates the early glycosylation product that is called the Heyns rearrangement product).Proved accumulating of this early stage saccharification adduct and haemoglobin taken place, wherein with the primitive reaction of glucose after, the aminoterminal rearrangement of haemoglobin beta chain forms the haemoglobin modified, is called hemoglobin A _Ic, it is a kind of important marker that is used for the contrast of diabetes glucose clinically.Also taken place with various other body protein, such as the saccharification react of lens crystalline, collagen neuroprotein and low-density lipoprotein and DNA and amino phosphatide.

U.S. rad brown stain process generates the advanced glycosylation product of a large amount of varying numbers, and each in them all presents very low yield.This species diversity makes determining of concrete AGEs and structure determination becomes knotty problem.At United States Patent (USP) 4,665, in 192, from the polypeptide of some browning, such as separating bovine serum albumin(BSA) and the poly-L-Lysine and having discerned fluorescent chromophore 2-(2-furoyl)-4 (5)-2 (furyl)-1H-imidazoles.This has successfully promoted the identification of other subsequently advanced glycosylation end product and has impelled the research of more attempting to understand the chemistry of protein ageing process and discerning related special reaction thing, intermediate product and product, thereby has improved method and the reagent that is used to suppress saccharification.

Recently, confirmed other advanced glycosylation product, such as AFGP (on May 21st, 696,1991 authorized for Farmar etc., United States Patent (USP) 5,017); Pyrraline (Hayase etc., " wearing out of protein: the pyrroles's who forms in the Maillard reaction process in the body of glucose induction immune detection ", " journal of biological chemistry " 263:3758-3764 (1989)), and pentosidine (Sell, D. with Monnier V. " from the structure explanation of the aging cross-linking agent of human body extracellular matrix ", " journal of biological chemistry " 264:21597-21602 (1989)).

A large amount of evidence proofs of having collected are done the basis that as a whole Mei Lade product constitutes various normal and pathogenic activity and the reaction that take place when AGEs accumulates in vivo.This class activity can be directly, for example the chemically reactive result of saccharification product and adduct; Or non-directly, it is by the cell recognition mediation by the saccharification adduct of AGE binding proteins specific or acceptor.

Although the research of the majority of the pathogenic effects that AGE accumulates in the description body concentrates on AGE-protein and the AGE-peptide, but (for example) atheroma that is reflected at of having confirmed to form between fat and particularly low-density lipoprotein (LDL) and the glucose fat-AGEs plays pathogenic effects in forming, and wherein the formation of foam cells shows accumulating of atherosclerotic plaque.The protein of low-density lipoprotein (LDL) and the oxidation of fat composition and saccharification cause the cell ldl receptor can't discern apo B composition, have prolonged the circulating half-life of this LDL by means of macrophage " scavenger " acceptor, AGE acceptor and other special cells mechanism simultaneously and have caused absorbing better the LDL (ox-LDL) of oxidation or the LDL that modifies in addition.The ox-LDL menses tube wall macrophage encytosis that increases is transformed into them and shows that the foam cells with fully loaded fat that early atherosclerosis damages is relevant.Above-mentioned research proves that also the AGE of LDLs modifies the potentiality that increased lipid oxidation.

AGEs " family " comprises can be separated and be characterised in that the metastable kind of chemical constitution, and other kind is unstable or reactive and their structure determination has become knotty problem thus.Can " catch " unstable or reactive AGEs by special chemical reagent, and for therapeutic purposes, this class reaction and capture-process are used to not only understand structure in depth but also be used to suppress saccharifying.AGE-fat can also be stable, unsettled or reactive.

Disturb the chemical process of (or inhibition) advanced glycation can have huge curative effect to the evaluation prompting of the pathogenic potentiality of AGEs.In this respect, having had been found that a series of reagent, is representative instance with aminoguanidine (also claiming Pimagedine), and they are useful saccharification inhibitor.Infer in theory this compound (with similar its other compound) can with the carbonyl moiety reaction of the early stage glycation product of target protein (or other biomolecule), described target protein with the original non-enzyme reaction of glucose or another kind of reducing sugar after form, and prevented the reaction of further formation advanced glycosylation end product thus.

Various other inhibitor of advanced glycation reaction also are known, think effective especially to the Maillard reaction stage of the inhibiting effect that is produced by aminoguanidine and analog thereof outside the most responsive stage.For example, has effective inhibitor that substituent some compound of active aldehydes (such as acetaldehyde) or its composition are the advanced glycation approach.Think that this activity is that the glycosyl-amino partial reaction of the saccharification product that forms in the starting stage by this class active aldehydes reagent and Maillard reaction produces, promptly these reagent and Amadori and Heyns rearrangement product (they are early stage saccharification products) react.Other reagent that can have similar capabilities is that those comprise conservative material in conjunction with primitive, the described hydrophilic peptide ring district of containing total 17-18 amino acid cysteine combination in conjunction with primitive of guarding, this is initial discovery and definite in antiseptic protein lysozyme and lactoferrin.More particularly, typical reagent comprises the molecule with hydrophilic loop district, and this hydrophilic loop district has the R of meeting ₁Z ₁Xaa _nZ ₂R ₂The structure of (SEQ ID NOS:1-6), wherein Z ₁And Z ₂It is the residue that can form cross-linking agent; R ₁And R ₂Be polypeptide independently, C ₁-C ₁₂Alkyl, aryl, assorted alkyl, or heteroaryl, or hydrogen; Xaa is L-or D-amino acid arbitrarily; And n=13-18.This class reagent and application thereof appear in the International Application No. WO 96/31537, and this application is open on October 10th, 1996, and the document is incorporated herein by reference.And by with saccharification in late period product reaction, other reagent, effective especially to preventing advanced glycation, and even can make the formation of advanced glycation end-product and relevant crosslink part reversible such as some thiazolium compounds.

Think the compound of using among the present invention (and composition) and early stage glycation product reaction, prevent from thus to form advanced glycosylation end product from the same substance in late period, these advanced glycosylation end products can cause crosslinked and cause molecule thus or protein is aging and other harmful molecules influence.

The saccharification approach restrainer different with all these classes early stage and reaction of saccharification in late period product and other compound (independent or with combination) can be applicable to improve the pathogenic potentiality of the AGEs that accumulates in vivo.

AGEs can accumulate in the following manner: the recombination (reattachment) of the AGEs that forms again in vivo by contact (for example by smoking) with external source AGEs, discharged by cytoactive in vivo or as by the disclosed in this article mode of applicant.Therefore, the meaning of AGEs reaction path and in tobacco and smog viewed their existence needs are monitored individual smoking habit and are all had further importance and promotion property for the foundation of the effective technology that reduces potent agent by using the AGEs that tobacco transforms and device.This class monitoring technology can be used for collecting this class smoker's smoking history, and also is used to monitor their internal disease and the particularly procatarxis of these diseases relevant with the AGEs level that raises.Measuring AGEs in tobacco can promote evaluation of tobacco crop and the index that is provided at the AGEs amount among the smoker of transferring in the process of consumption by the material of being fuming of above-mentioned crop preparation; In this respect, can also in smog, estimate AGEs.Effectively therapeutic strategy, method and reagent are mentioned the AGEs that suppresses (in the material of being fuming) in the tobacco and are comprised improving and be used for smoking or preventing or treating smoking or contact smoking or contact the filtration unit and the similar substance of the individuality of smog colony in this respect.All above-mentioned purposes involved in the present invention are tending towards realizing.

Summary of the present invention

The present invention derives from a kind of like this discovery, be tobacco and combustion by-products thereof, contain advanced glycosylation end product (AGEs) such as smog, these AGEs react with the biomolecule that contains amine (amine) (particularly protein) as one group, and therefore smoking or the increase of using the individuality of tobacco generally to show AGE level in whole blood and the tissue in addition.It is relevant with the increase of the pathologic incidence of disease that such level raises, and such as the harm that coronary artery disease, atheroma formation and other disease are increased, these diseases are relevant with accumulating of AGEs.

Therefore, the present invention confirm AGEs be present in tobacco, as in tobacco product that uses by smoking and result of combustion of tobacco accessory substance and the biological sample of taking from the smoker.In aspect first, the present invention relates to measure tobacco be fuming in the material, in the result of combustion of tobacco accessory substance and take from AGEs in smoker's the biological sample, thereby determine AGE level and convenient thus possibility or historical judgement to individual this class of contact AGEs in the smoking process, or to the situation of tobacco or tobacco product, comprise that time limit, smell and/or the storage case (freshness) of (for example) estimation tobacco crop provide information.

More particularly, mensuration and evaluation to the smoker can be used for the patient and the practitioner of any pathological conditions are estimated, described disease can have very high outbreak possibility, if or such disease occurs, it need experience careful periodic monitoring and/or show needs the treatment measure, to smoker's mensuration with estimate and can be used for estimating degree that the smoker tolerates according to smoking habit and harm subsequently again.The latter's opinion will add in the evaluation as the participant's of the purpose step that is used for giving up smoking patient, both estimated the length of the smoking history that detects the experimenter, evaluate safety or similarly research purpose again, thus confirm to stop using tobacco (or because of its shortage).

Can carry out the mensuration of AGEs by handling tobacco sample to form the mode of suspecting the extract that wherein has AGEs, subsequently to this extract carry out this classification analysis.In one embodiment, handle sample to form smog, then smog is carried out the existence of AGEs and the check of amount by burning.On the other hand, this extract can produce by tobacco is carried out chemical treatment separately.Can also check smoker's body fluid and tissue to measure level and the degree of the AGEs that occurs because of smoking.Yet compare with these results with from the standard of setting up with the patient's to be checked of the total same medical science distribution plan of non-smoking individuality.

The present invention includes preparation and be used for the reagent of the specific recognition AGEs relevant,, thereby help the detection of the AGEs relevant and the evaluation of smoker's medical conditions with tobacco such as the antibody of the AGEs that finds in anti-tobacco of specificity and the smog with tobacco.This class reagent introduced in test kit and the analog and be used to carry out such evaluation.

In related aspect, use the substrate or the carrier of reservation and carrying saccharification target molecule (such as the part that contains amine (amine)) to make dosimeter, the immobilization of described saccharification target molecule combines with substrate or carrier, also can react with the AGEs that is present in the sample, and described sample is the surrounding air sample or takes from patient's biological sample.Dosimeter can be used together with this paper listed diagnosis scheme and test kit.

The invention further relates to the method that is used to detect and measure the individual middle-and-high-ranking AGEP level of smoking, this method is undertaken by following steps: collect from the biological sample of this class individuality, after this these samples are carried out the existence of advanced glycosylation end product and the check of amount.Collected sample can relate to cell, tissue, and serum, saliva, urine or ight soil, and detection and determination techniques were improved and used according to AGEs in the past.

In one aspect of the method, the present invention relates to prepare antagonist, such as the antibody of the AGEs that finds in anti-especially tobacco and the smog, in conjunction with the chemical inhibitor of primitive and advanced glycation.

The invention still further relates to the method and the related reagent that are used for the treatment of the disease in tobacco user or the smoker's individuality, comprise these class methods and reagent are used for the aging of Profilin matter, and particularly this class reagent can react with glycation product, thereby stops their formation and/or harmful activity of this class AGEs.

Especially, be used to suppress to be selected from the group that following material is formed: aminoguanidine, antagonist, analog, congener, homologue (cognate) and analogies thereof and their potpourri because of advanced glycosylation end product forms the aging reagent of protein that causes.

This class reagent can also comprise and can be selected from can with the material of the glycosyl-amino partial reaction of early stage glycation product (being also referred to as Amadori and Heyns product), described early stage glycation product forms with the biomolecular reaction that the responsive part of saccharification (such as the amino on the protein) is provided by glucose (or other reaction sugar), make this early stage glycation product stable thus, and prevent its further reaction formation advanced glycosylation end product.Therefore, for example, have substituent compound of active aldehydes or composition and be stable more particularly such as the such compound of acetaldehyde.Think that this activity is produced by the glycosyl-amino partial reaction of the saccharification product that forms in this class active aldehydes reagent and the Maillard reaction initial period, be the reaction of these reagent and Amadori and Heyns rearrangement product, Amadori and Heyns rearrangement product are early glycosylation products.

More particularly, these reagent can contain the compound that participation has the product formation of general formula I or II

Wherein R contains the amino peptide or the residue of protein, they prepare with the reagent reacting that contains active aldehyde radical by the glycosyl-amino part of early stage glycation product (being also referred to as Amadori or Heyns product), and described early stage glycation product forms with proteins react by glucose (or other reaction sugar).The provisional application serial number is to have described this quasi-representative reagent in 60/006,752 (" aging the improving one's methods and reagent of Profilin matter ", the application on November 15 nineteen ninety-five), and the document is incorporated herein by reference.

By with the reaction of saccharification in late period product, other reagent, effective especially to preventing advanced glycation, and even can reverse the formation of advanced glycation end-product and relevant crosslink part such as some thiazolium compounds.Think that effective opposing is pre-existing in or the typical compound of saccharification in late period adduct and composition by be present in α-dicarbapentaborane fragment, this class α-dicarbapentaborane fragment reaction that particularly in the AGE structure that forms intermolecular or intramolecular crosslinking, occurs among the AGEs and cause cracking that they work.This compound based on thiazole preferably works in this respect with a kind of catalytic way original, active thiazole of regenerating, and described thiazole can the catalysis cracking of the AGE part of existence in addition.This quasi-representative reagent can contain the thiazolium compounds with following general structure: R wherein ¹And R ²Independently be selected from the group that following group is formed: hydrogen, hydroxyl (rudimentary) alkyl, acetate (rudimentary) alkyl, low alkyl group, low-grade alkenyl, or R ¹And R ²Together with its ring carbon can be a kind of aromatic condensed ring, and it can be arbitrarily by one or more amino, and halogen or alkylidene dioxygen (alkylenedioxy) replace;

Z is hydrogen or amino;

Y is amino, a kind of group of following general formula

Wherein R is a low alkyl group, alkoxy, and hydroxyl, amino or aryl, described aryl can be arbitrarily by one or more rudimentary a heatable brick bed bases, lower alkoxy, halogen, dialkylamino, hydroxyl, nitro or alkylidene dioxygen replace;

A kind of group of following general formula

-CH ₂R’

Wherein R ' is a hydrogen, or low alkyl group, low-grade alkynyl, or aryl;

Or a kind of group of following general formula

R wherein " be hydrogen and R " ' be low alkyl group, can be replaced by aryl arbitrarily, or aryl, described aryl can be arbitrarily by one or more low alkyl groups, halogen, or alkoxy carbonyl replaces; Or R " and R " ' both are low alkyl group;

X is a halogenide, toluene sulfonate, the ion of mesylate or sulfonate;

And composition thereof.

Think the compound of using among the present invention (and composition) and early stage glycation product reaction, stop thus to form advanced glycosylation end product thereafter, these advanced glycosylation end products can cause, and crosslinked (and thus) causes molecule or protein to wear out and other harmful molecules influence.In addition, think described compound and composition and the reaction of the advanced glycosylation end product that formed to reduce the amount of this class product.At application Ser. No is more specifically to have described these reagent in 08/375,155, and the document is incorporated herein by reference.

The invention further relates to especially the smoker or consume in addition in the individuality of tobacco product and reduce the method that advanced glycosylation end product is accumulated, this method is undertaken by following steps: uses the known reagent that is used for suppressing advanced glycosylation end product formation, treat this class individuality such as those above listed reagent.Described reagent is applied to the incidence of disease or seriousness several diseases and the pathologic condition relevant with the consumption of tobacco, particularly smoking especially, such as cardiovascular, the cerebrovascular and peripheral vascular disease, they comprise, for example heart and coronary artery disease, arrhythmia cordis, atherosclerotic, apoplexy, hypertension, pulmonary infection, thromboangiitis obliterans, the periphery occlusive vascular disease, Raynaud's disease is walked lamely and kidney failure; Pulmonary disease, such as chronic obstructive disease of lung, chronic bronchitis, pulmonary emphysema, asthma, and pulmonary eosinophilic granuloma; Various cancers are including, but not limited to lung cancer, the cancer of oral cavity, head and neck, celiothelioma, the cancer of oesophagus, bladder, neck, endometrium, pancreas and nephrocyte; Digestibility, stomach and esophageal ulcer; High fat of blood and dyslipidemia; Osteoporosis; (snuff) keratosis and leukokeratosis of oral mucosa that chorionitis, snuff cause; Immunosupress; And tooth and skin dyeing.

It is external to remove directly treatment, this method also relate to handle tobacco material itself with reduce this class AGEs to consumer or onlooker's generation and pass through.Therefore, in first kind of situation, this method comprises by chemical method or other method handles tobacco material itself, thereby reduces the formation or the existence of advanced glycosylation end product.When handling some tobacco with sugar, these class methods can relate to handles handled tobacco so that finish the step of saccharification toxin (glycotoxins) content that reduces AGEs defined herein or its precursor.

Another embodiment of handling the tobacco material comprises that preparation is used for the proper filter media of using with tobacco end-product or cigarette, uses this filter medium such as those reagent of this paper inhibitor listed, that be known as advanced glycosylation and handles.Then this class reagent is distributed in the filter medium, is convenient to by this way carry out with it during by filter that the surface contacts and liquid holdup, thereby catch and/or prevent the formation of advanced glycosylation end product at smog.In this back one side, the present invention relates to comprise the filter of porous medium, it is prepared by fiber or other similar suitable material, on its active surface, comprised a certain amount of, known be used for suppressing advanced glycosylation maybe can be by the reagent of catching with the AGEs reaction of the plume that is present in tobacco.Filter tip that this class is derived and filter medium not only can be applied to reduce or eliminate the AGEs in the initial plume that directly consumes the tobacco product generation by smoking, and when pointing out near the smog that the onlooker produces in other people's smoking activity sucking, in described filter tip and filter medium can reduce or eliminate with tobacco smoke contact the environment, such as the AGEs on every side in " secondary smoking ".The technology that is used to prepare this class filter medium also is known, and they itself do not constitute part of the present invention.Certainly, enter this particular procedure of activity site on this class filter medium, in fact this class technology part that is this paper with regard to the reagent that the present invention is used.

A special and important aspect of the present invention is a kind of like this discovery, be that tobacco and combustion by-products thereof contain and be very similar to those observed reactions sugar in other food and in body fluid and tissue, these reaction sugar to the smoker and indirectly absorption smog person's body system has toxic action.When carrying out the processing of tobacco under can causing reacting the condition that sugar forms, present inventor's check is also confirmed subsequently that tobacco and tobacco smoke contain and propagate these and knownly is used for increasing the reactive material that AGE forms in the body.This paper is called these reactive materials " saccharification toxin (glycotoxins) ".Used term " saccharification toxin (glycotoxins) " is defined as the reactive material that can participate in the Maillard reaction process or can form in the Maillard reaction process herein, they are made up of independent carbohydrates or carbohydrates and the adduct that contains amino material, protein for example, DNA, fat and all this class materials.

As what this paper proved, saccharification toxin (glycotoxins) and proteins react, show special fluorescence, cross-linked proteins and be mutagenic.The listed digital proof tobacco smoke in this paper back contains the saccharification toxin (glycotoxins) of significant quantity, and this saccharification toxin (glycotoxins) can enter the circulation system and react and formation AGEs with serum and histone.The increase that contacts with saccharification toxin (glycotoxins) can make the atherosis increase of smoker's medium sized artery and cancer very general, and listed as this paper, think effective therapy be keep using and give the inhibitor of advanced glycation, such as aminoguanidine.

Therefore, the present invention is the basis with the formation of advanced glycosylation end product and manufacturing and the pass between the consumption of tobacco.Follow the saccharification react thing of existence in tobacco and the tobacco smoke, enter such as wherein reaction sugar and they that this situation is that various application lay the foundation in smoker and the onlooker's body, described application include but not limited to diagnose with estimate application, with suppress advanced glycosylation end product accumulate and the tobacco material is used and handled to treatment that harmful sequelae is relevant strengthening its organ sensory characteristic and storage case, and the more important thing is the illeffects that reduces their application.Main result of study that obtains from the advanced glycosylation phenomenon that the present inventor studies in great detail and experience and special discovery disclosed herein and observations all help to realize mechanism of the present invention.

Therefore, first purpose of the present invention provides and a kind ofly is used for measuring tobacco and accessory substance thereof and consumes degree that the individual middle-and-high-ranking AGEP of this series products forms and the method for level.

Further purpose of the present invention provides a kind of above-mentioned method that can be used in diagnostic purpose, so that the medical history of monitoring smoker and other tobacco user and preventing thus follows advanced glycosylation end product to accumulate development with the serious pathologic complication of smoking.

Another object of the present invention provides a kind of above-mentioned relating to and estimates tobacco to measure the method for its storage case and organ sensation potential.

Another object of the present invention provides diagnostic test method and relevant material, they can discern effectively the advanced glycosylation end product relevant and they with tobacco in special tobacco product existence and can discern their similarly accumulating in consumer's body equally.

Another object of the present invention is the modification method that improves tobacco and tobacco product, and these modification methods are used to reduce formation and the propagation of advanced glycosylation end product to the tobacco product consumer.

Further purpose of the present invention provides inhibition, catch or in addition in and AGEs and particularly be present in the filter of the saccharification toxin (glycotoxins) (comprising reaction AGEs) in the tobacco smoke.

Another object of the present invention provides and is used for evaluation of hazard degree methods and relevant material, if possible, individuality can be used as the function of AGEs level in the environment, thus realize or the methods of treatment of adjusting this class individuality to prevent or to reduce the illeffects of the individual contact of this class AGE.

Another object of the present invention provides the above-mentioned method and relevant material that is used for regulating at described environment described AGEs level.

Another object of the present invention provides and is used for tobacco user, smoker or contacts individuality with cigarette suppressing AGEs and the aging reagent of protein, thereby prevents that in described individuality and colony the harmful of AGEs from accumulating.

Owing to consider the specific descriptions of proceeding the back with reference to following illustrative embodiments, so other purpose and advantage it will be apparent to those skilled in the art that.

Brief description of drawings

Accompanying drawing 1 is the synoptic diagram of expression cigarette tobacco water extract AGE content.According to the competitive AGE immunoassay of standardization the AGE level in the phosphate-buffered liquid extract of cigarette sample is carried out quantitatively and with it being calculated as total AGE unit/cigarette.The trade mark of cigarette is: Gau, Gauloises; Mar, Marlboro; ML, Marlboro Light; Par, Parliament; Sal, Salem Ultra Lights.

Accompanying drawing 2 be illustrated in experimentally with serum that tobacco smoke contacts in the synoptic diagram of AGE level.AGE level in the blood serum sample that the smoke from cigarette bubbling is passed through according to the competitive AGE immunoassay of standardization carries out quantitatively and with it being calculated as AGE unit/ml serum.The similar blood serum sample (smog that does not contain tobacco) of reference substance=only contact with air-flow.

Accompanying drawing 3 is that expression is present in saccharification toxin (glycotoxins) in the tobacco smoke condensation product aqueous solution and reaction sugar and AGEs at external and synoptic diagram proteins react.To contact with the smoke from cigarette condensation product of phosphate buffer solubilising with the assay plate of glue primordial covering, and being determined at the AGEs that forms on the assay plate hole that collagen handles by a kind of immune measure, the increase of final absorbance log (optical density or OD) reflects that AGE crosslinked and that accumulate increases with the collagen stroma of assay plate on the collagen stroma of assay plate thus.

Accompanying drawing 4 is synoptic diagram that expression Pimagedine (aminoguanidine) suppresses external smoke from cigarette condensation product extract and proteins react.Under the situation that is with or without variable concentrations Pimagedine, will contact with the water extract of smoke from cigarette condensation product by the assay plate of glue primordial covering.Be determined at the AGEs that forms on the assay plate hole of collagen processing by a kind of immune measure, the increase of final absorbance log (optical density or OD) reflects that AGE crosslinked and that accumulate increases with the collagen stroma of assay plate on the collagen stroma of assay plate thus.

Accompanying drawing 5 is the synoptic diagram that are illustrated in the saccharification toxin of finding in the water extract of tobacco and tobacco smoke (glycotoxins) (reaction AGEs or saccharification intermediate product).In the water extract of tobacco, measure saccharification toxin (glycotoxins) according to competitive AGE determination method (A) and directed binding immunoassay determination method (B).To in PBS, extract from the tobacco sample of U.S.'s cigarette of four different trades mark 24 hours, carry out aseptic filtration and according to the AGE content of above-mentioned competitive AGE ELISA test sample.Each bar shaped frame is represented the cigarette (A) of the different trades mark.Total AGE unit/cigarette is calculated as the index of extract A GE content.Tobacco sample extracted 1 hour, aseptic filtration and utilize the mouse tail tendon collagen that is fixed in the 96 hole microtitration flat boards to come reaction sugar (saccharification toxin (glycotoxins)) content (B) in the test sample in PBS.

Accompanying drawing 6 is coordinate diagram that another series of tables is shown in the saccharification toxin of finding in the tobacco smoke (glycotoxins).

(A) form determination method according to external AGE and in the smoke from cigarette condensation product, measure saccharification toxin (glycotoxins).The smoke from cigarette of serial dilution added to collagen wrap in the hole of quilt in advance and seal with the PBS/0.05% Tween-20.Use the anti-AGE polyclonal serum of a kind of rabbit to show the formation of AGE.

(B) can remove saccharification toxin (glycotoxins) by means of smog by aminoguanidine.The smoke from cigarette that makes new system is by standard filter (black bar), standard filter+500mg sodium sulphate (bar of band line bar) and standard filter+500mg aminoguanidine (white bars).

(C) can be from the saccharification toxin (glycotoxins) of mainstream smoke with the crosslinked RNA enzyme of the mode of covalent bond A albumen (and aminoguanidine can suppress this crosslinked).RNA enzyme A is contacted 0,5 with the main flow smoke from cigarette, 8,24 and 72 hours.In order to estimate the dipolymer of formation, sample is carried out SDS PAGE under reducing condition, use a kind of and the anti-RNA enzyme of the rabbit HRP conjugation A antibody with above-mentioned sample then carry out cellulose nitrate and immunoblotting subsequently.Circle=independent RNA enzyme A; RNA enzyme A after square=with smoke from cigarette is cultivated; RNA enzyme A after triangle=usefulness smoke from cigarette and 5mM aminoguanidine are cultivated; RNA enzyme A after rhombus=usefulness smoke from cigarette and 50mM aminoguanidine are cultivated.

(D) saccharification toxin (glycotoxins) has autofluorescence.RNA enzyme A is contacted 0,5 with the main flow smoke from cigarette, 8,24 and 72 hours, then by when 370nm excites, detecting the specificity fluorescent of saccharification toxin (glycotoxins) in 440 nm places mensuration emission situation.Circle=independent RNA enzyme A; Square=with smoke from cigarette cultivation after RNA enzyme A; RNA enzyme A after triangle=with smoke from cigarette and 5mM aminoguanidine is cultivated; RNA enzyme A after rhombus=with smoke from cigarette and 50mM aminoguanidine is cultivated.

Accompanying drawing 7 is synoptic diagram that proof mutagenesis saccharification toxin (glycotoxins) is.With the smoke from cigarette by aminoguanidine is contrast (white bars), with the smoke from cigarette condensation product of serial dilution Salmonella bacterial strain TA98 is cultivated 1 hour (black bar), carries out flat board then and fixes.After 48 hours the clump count on each flat board is counted.Each bar frame is represented the mean+/-standard deviation of three flat boards.

Accompanying drawing 8 is a series of synoptic diagram that expression smoking causes the level of interior apoB-AGE of body and serum AGE to increase.In the non-diabetic smoker of the health that works in Picower Institute and healthy, ND non-smoker, measure serum apoB-AGE (A) and total AGE level (B).ApoB-AGE level among the smoker (324+/-140U/ml, n=9) be significantly higher than (p＜0.01, non-matching StudentShi t check) non-smoker (177+/-33, n=10).(202+/-76 AGE U/ml n=23) is significantly higher than the serum AGE level (146+/-31 AGE U/ml) among (p＜0.02) non-smoker to serum AGE level among [apoB-AGE＞300U/ml in the diabetic] smoker.(serum AGE level among the diabetic=200-400).With the AGE-BSA standard is that benchmark is represented AGE unit.Experimenter's age, sex or smoking year number unrestricted.

Accompanying drawing 9 is expressions by the spray synoptic diagram of assay of the tobacco of handling of the aminoguanidine that uses certain limit concentration, and purpose is to determine the effect of this class disposal route that AGEs is formed.This check forms determination method with reference to accompanying drawing 6 described external AGE and carries out.

Specific descriptions of the present invention

The present invention derives from and consumes tobacco and improve the individual middle-and-high-ranking AGEP water of this class Fixed relation between flat the increasing. Initial observed result is in the special test colony ApoB-AGE level in the research of advanced glycosylation end product level and particularly this types of populations Increase and coronary heart disease takes place simultaneously and to the individuality one of the level that shows such increase As be the correlation of the such fact of tobacco product consumer or the discovery of relation. Initial Observed result has been impelled the research to tobacco product itself, and this has produced such discovery, Be this series products, comprise that combustion by-products (smog) contains the advanced glycosylation end of high concentration Product and the saccharification toxin (glycotoxins) that impels their to form. These are found at first The result promoted further research and caused relating to according to tobacco, cigarette The progress of the mensuration of mist, smoker and AGEs and the each side of the present invention of processing mode.

More particularly, the present invention relates in aspect first the experimenter of smoking history is arranged Test and better determine their smoking history, this process is advanced in the following manner OK: to derive from the non-smoking colony that has in other respects similar or identical medical condition Quantitatively measure the increase of advanced glycosylation end product for benchmark. By the check test experimenter Various bodily tissues and body fluid (such as blood, urine and ight soil) can implement these class methods. In addition, the existence of mensuration AGEs and saccharification toxin (glycotoxins) and level are to helping The prediction experiment experimenter at least part of with AGEs and saccharification toxin (glycotoxins) Known mutagenesis characteristic is useful for the sensitiveness of the disease on basis.

In similar mode, can check and estimate tobacco product itself with definite they It is latent to promote in the tobacco user that AGE and saccharification toxin (glycotoxins) level increase Energy. In this case, can check this class to tobacco product itself and combustion by-products thereof Advanced glycosylation end product and/or form their the depositing of saccharification toxin (glycotoxins) . In a related aspect, can check identical product and mark advanced glycosylation The level of end-product is in order to measure the storage stability of gained tobacco crop and potential smell. As possible situation, change according to some benchmark or the standard of taking from similar sample or colony Advance this class assay method.

In this respect, the filter of the various special devices of protein brown stain determination method and seizure AGE Device can be made according to the present invention, and they provide and exemplified main parts, require this A little parts are to be convenient to measure the individual cigarette that contacts with ETS (the particularly smog of tobacco) Mist contact agent measuring device, based on detect will with the amino of protein or other sensitiveness chemistry saccharification The AGEs that the target thing is crosslinked. The applicant think use occur in as utilization relevant with tobacco smoke Saccharification toxin (glycotoxins), AGEs and protein or other with free amine group or Other plays the substituent intermolecular covalent reaction of saccharification target molecule effect and the dosage made Device can be estimated the dilution factor of tobacco smoke in the air,, described saccharification target molecule thus To with the AGEs relevant with tobacco and the chemical reaction of saccharification toxin (glycotoxins) being Responsive, cause described AGEs and saccharification toxin (glycotoxins) and described sugar The change target molecule carries out crosslinked with covalent and therefore can examine by mode as herein described Go out.

Use dosimeter of the present invention can estimate the harm of contact environment tobacco smoke, described Dosimeter of the present invention can connect saccharification target molecule and the atmospheric environment that needs monitoring smog content Touch, wherein said dosimeter carries AGEs for the described reactive gas that derives from tobacco smoke Spontaneity and the AGE target branch that is presented on the matrix with saccharification toxin (glycotoxins) The combination of son has consisted of the collecting part of dosimeter thus. By well-known in the art The method of any a lot of identification AGE, include, but is not limited to use anti-AGE mentioned above The AGE ELISA method of antibody, according to the step that consists of the dosimeter display unit can be qualitatively, Semi-qualitative ground or observe quantitatively with dosimeter and show the AGEs that amino matrix is combined. With Not only comprise the well-known AGE sensitivity that is in the suitable target molecule of introducing the dosimeter display unit The range protein of property target molecule, such as BSA, RNA enzyme, collagen and polylysine, and Comprise with to any other biomolecule of the responsive amino of AGEs reaction or organise Compound.

Preferably this class saccharification target immobilization is being helped they submissions to monitor sky On the surface of the substrate in the gas; Such as a kind of paper surface for showing that amino is derived, a kind of The surface of similar nitrocellulose filter of deriving, a kind of being similarly provides amino as the saccharification target Thing and the surface of the polystyrene filter membrane of deriving, or the saccharification target molecule is provided and is positioned over and institute The matrix of interactional any other relative inertness of monitoring of environmental air. By impelling air Any mode (such as using a kind of fan) that flows through collecting part can be passively (for example By convection current) or affect on one's own initiative the air of smog to be measured and the saccharification target branch that matrix is fixed Contact process between the son and described air flow through the cyclic process of described collecting part. In addition On the one hand, can will provide amino target molecule to be dispersed in a kind of liquid, and can be in the future Took out described liquid, for example pass through to foam from the air of institute's monitoring of environmental. Two kinds of situations In, all by any suitable AGE detection method, all as mentioned open for detection of AGEs The special device of common AGE ELISA determination method check environment sky with the need monitoring The saccharification target molecule (no matter being placed on the matrix or in liquid phase) of gas contact, so that To with the AGEs of saccharification target molecule reaction or the detection table of saccharification toxin (glycotoxins) Understand the pollution level with the surrounding air of smog. The corresponding face of land of the application of this class dosimeter Bright individuality has contacted the indirect smog in the environment.

Similar assay method can be used for monitoring tobacco product saccharification toxin (glycotoxins) existence and amount, and estimate thus and judge that they are to same cigarette The tendentiousness of grass consumer and onlooker's illeffects. For example, for this classification, Can form determination method with the listed external AGE of this paper and measure sugar in the tobacco extract Change toxin (glycotoxins).

Above-mentioned dosimeter and corresponding assay method can be used for certain therapeutic domain, with Just monitor and estimate the therapeutic process of especial patient, these especial patient diseases are characterised in that AGEs level in body fluid and the tissue raises. Therefore, the sample of taking from patient can be carried out Check (is for example passed through AGE ELISA with the extent of reaction of determining to be present in the AGEs in the sample Or the similar method of inspection is carried out) so that the progress of the announcement patient doctor in charge in treatment. The doctor can select to revise modestly therapeutic modality then, but will be take assay as the basis. Equally relevant is that sample and the patient who takes from patient is in wherein the overwhelming majority times The measurement result of surrounding air prompting response environment transform to reduce patient and AGEs or Other source of saccharification toxin (glycotoxins), no matter be from the smog of tobacco also Be the contact in the source in other external world, therefore, this result further helps the doctor to nurse disease The people.

Follow foregoing, be oriented to by preparation and in tobacco and tobacco accessory substance, find The antibody of advanced glycosylation end product and saccharification toxin (glycotoxins) can be formulated Various diagnostic test methods, generate a reagent box and analog, they are to have especially in the present invention With. Can generate thus this antibody-like and with they as in the diagnostic test and with the tobacco phase The AGE that closes and the standard in saccharification toxin (glycotoxins) chemical structure characteristic.

With regard to the therapeutic strategy and reagent that the present invention relates to, the increasing of advanced glycosylation end product Add and the application of the material of being fuming between correlation impel application true according to institute's disease for the treatment of Fixed methods for the treatment of in described disease, thinks that the increase of advanced glycosylation end product rises Certain effect. So, if if special entity have tobacco product the consumption history and because of This methods for the treatment of comprises the inhibitor that this class individuality is given advanced glycosylation end product, so Above-mentioned these class methods are adaptable in this case.

Suitable advanced glycation inhibitor is former to be determined, and they comprise, for example ammonia Base guanidine (Pimagedine), its analog, antagonist, congener, homologue and simulation Thing and the mixture of suitable they wherein. Other suitable inhibitor can be selected from activity Aldehyde or have the substituent compound of corresponding active aldehydes (as mentioned determined, they with The early stage Amadori of Maillard reaction and the glycosyl of Heyns product-amino part is anti-Should) and wherein mixture and the combination of suitable they. Suitable inhibitor is passable in addition Be selected from above those thiazoles of determining and other compound and composition thereof (they with α among the AGEs that exists-dicarbapentaborane fragment reaction), particularly in automatic regeneration, catalysis Impel or cause the cracking in the AGEs of described existence of described α-dicarbapentaborane fragment in the mode This compounds. Administration really butt formula will be decided by experienced doctor.

Another strategy for the smoker is directly to process the material of being fuming that consumes. This class Material can be processed by similar inhibitor, so that accept the like this tobacco product of processing The quantity of the AGEs that has reduces (and saccharification toxin (glycotoxins) and instead particularly Answer the quantity of AGEs to reduce) or so that promote to form in consumer's body the energy of this class AGEs Power reduces.

On the other hand, the filtrate that the present invention relates to prepare tobacco product, comprises such as cigarette Matter so that this medium itself can suppress, catch or neutralize can impel senior Formation and the propagation of AGEP (for example saccharification toxin (glycotoxins)) increase The AGEs relevant with tobacco that adds or relevant chemical entity. Medium be those routinely The material that provides and be used for cigarette, and they normally are in the fibrous material of pencil And be cylindrical shape in the cigarette of finally making. This fiber material is (wherein representative Be cellulose fibre) can contain in its surface as catch AGEs in conjunction with or neutralization Other saccharification product, such as a kind of suitable inhibition of saccharification toxin (glycotoxins) Agent or reactant are in order to prevent from forming in use AGEs.

In another embodiment, can prepare and play inhibition, catch or in addition neutralization Can impel the formation of advanced glycation end-product and propagate increase relevant with tobacco or smog The filter of the effect of AGEs or relevant body and be used for purification general or surrounding air, example As reduce or eliminate AGEs and/or the saccharification toxin (glycotoxins) of surrounding air, They are not absorbed with non-direct, passive or " indirectly " smoking regime with needing. No matter be by the contact environment air or by directly to sample (biological or other shape Formula) use this class material or substrate, prepare this class filter or similar substance so that can be right Environment carries out sampling inspection, and wherein AGEs maybe can form their saccharification toxin (glycotoxins) concentration falls under suspicion; Purpose is to formulate to reduce this class AGE level Assay method. This class assay method can comprise environment is carried out suitable processing to transform The AGE level is not expected the zone that raises or is formulated or adjust the individuality that is in the sort of environment Methods for the treatment of to reduce level or the content of AGE in the individual body of this class. This special class is treated Method comprises and gives the related reagent such as this paper, and they play inhibitor that AGE forms Effect or play impels or realize the effect that AGEs removes from health.

In general fashion and various concrete enforcement, protein brown stain determination method of the present invention With suppress AGE, catch AGE or in and the filter of AGE also provide and exemplified main section Part, requiring these parts is to be convenient to the individual and ETS (the particularly smog of tobacco) of estimation The smog contact agent measuring device of contact is based on detection amino or other sensitiveness with protein Learn the crosslinked AGEs of saccharification target thing. The applicant thinks by occurring in and tobacco smoke for utilizing Relevant AGEs and/or saccharification toxin (glycotoxins) and protein or other Anti-with free amine group or other substituent intermolecular covalency that plays the effect of saccharification target molecule The doser of answering and making can be estimated the dilution factor of tobacco smoke in the atmosphere, and is thus described The chemical reaction of saccharification target molecule pair and the AGEs relevant with tobacco be responsive, cause institute The AGEs that states and described saccharification target molecule carry out crosslinked with covalent and therefore can lead to Crossing mode as herein described detects.

The following example that is provided is used for determining this relation and promoting the experiment of the embodiment realization of foregoing invention to make an explanation.

Embodiment 1

In order to determine whether AGEs is present in the tobacco product, preparation is from the tobacco sample of several commercially available brand of cigarette that get in 0.02M phosphate buffer (pH7.4), the concentration of tobacco is that 66.7mg/ml is at room temperature after the overnight incubation, make tobacco precipitation and the water extract of tobacco is carried out aseptic filtration by centrifugal, diluted by 1: 800, and be used to analyze the content of AGE.Use anti-AGE monoclonal antibody, the AGE content of the tabacco water extract of dilution detected (referring to following content) according to the competitive AGE immunoassay of standardization, this detection test can be measured the immunoreactive unit of AGE, and bovine serum albumin(BSA) (AGE-BSA) the reference standard competitor preparation of modifying with AGE is a benchmark.As can be seen, total AGE unit/cigarette is calculated as the index of tobacco AGE content from this testing result, and as shown in accompanying drawing 1, the AGE content of these tobacco samples that extracted is about 50,000-150, the scope of 000AGE unit/cigarette.From this evidence, the applicant reaches a conclusion: have high AGE para-immunity reactivity in the water extract of tobacco.This conclusion prompting applicant tobacco product, comprise (for example) snuff with chew cigarette or from the cigarette of flammable form, consume tobacco such as the consumer of cigar, cigarette or pipe smoking tobacco by them and can contact with a large amount of AGEs.In addition, because known AGEs has accumulated a period of time with temperature and sugared dependence mode, and because known AGEs has taste and smell, so this information also points out the applicant that tobacco is processed into the tobacco of special batch in various consumer products and the sales process subsequently or the AGE content of crop can be a kind of useful indicator of the potential smell of tobacco and storage and processing history commercial.

Carry out competitive ELISA according to the common immunoassay program of this area routine to AGEs.In brief, by pressing 50mg/ml in the 0.5M glucose under 37 ℃, in the sealed tube BSA is cultivated (through SDS-PAGE, Calbiochem, Cat.#12657, fraction V, purity＞99%) prepare the main material (Master Stock) of the BSA (AGE-BSA) that AGE modifies 8 weeks, wherein said 0.5M glucose is preparation and to wherein leading to nitrogen in 7.4 the phosphate buffered saline (PBS) (PBS) containing 1.0mM EDTA, pH.Detected dull and stereotyped (NUNC Maxisorp) with the standard antigen bag by the commercially available 96 hole microtitrations that get, this process is carried out in the following manner: under 4 ℃, the bag in each hole is cushioned liquid (0.1M NaHCO ₃/ 0.02% azide is cultivated 100 μ l AGE-BSA solution by the concentration of 30 μ g/ml in pH9.6) and is spent the night, and covers.Use the dull and stereotyped scrubber of automatic ELISA then, will detect hole flushing 6 times, reverse and blot with 200 μ l lavation buffer solutions (the Trisaminomethane buffer saline that contains 0.05% polysorbas20).Packing in each hole, 200 μ l sealing damping fluid (pH7.4) and in the time of 37 ℃ cultivated flat board, cover 1 hour by PBS/2% normal goats serum/0.2%BSA/0.02% azide.After washing as mentioned above, promptly be ready for the detection flat board of the standard A GE-BSA modification of competitive ELISA.

By in each hole of the detection flat board of AGE-BSA bag quilt, adding the sample of 50 μ l equal portions or the detection method that AGE-BSA standard competitor begins to carry out present embodiment, immediately add the anti-AGE antibody preparation of the suitable titration of 50 μ l.At room temperature flat board is cultivated, covered 2 hours then, the AGEs in the test specimen (or AGE-BSA reference material) combines with the elementary or anti-AGE antibody competition that opposing is fixed on the AGEs on the detection flat board that forms AGE-BSA during this period.Finally, in this emulative immunoassays form, the bag that the AGEs of higher amount reflects having the corresponding minimizing of final read output signal in the sample is had higher competitiveness by AGE-BSA, and its intensity reflects the combination degree in the hole of elementary anti-AGE antibody and AGE-BSA bag quilt.In the present embodiment, after 2 hours, will resist AGE monoclonal antibody (called after 4G9) under dilutability, to use with about 1.5 optical density (OD) unit in the final cultivation of the Kong Zhongyu chromogenic substrate that does not contain competitor.Have the series concentration of scope and prepare the AGE-BSA standard competitor of a series of dilutions by AGE-BSA main material (Master Stock) is diluted in about 0.1-2 μ g AGE-BSA/ hole, and from this standard competition curve can in push away the respective concentration of the AGE-BSA standard competitor of the final OD that meets each sample well.Be the concentration (in BSA mg/ml) that requires in this competitive AGE ELISA form, to have 50% inhibiting standard competitor AGE-BSA in the use with an AGE unit definition.

After this cultivation of primary antibody and competitor (sample or reference material), with the hole with lavation buffer solution washing 6 times, reverse and blot.Add according to the producer's suggestion then and cultivate that commercially available the secondary antibody that gets-the enzyme conjugate (for example, the anti-mouse IgG antibody and the alkaline phosphatase coupling that raise in the goat body) preparation generally is under 37 ℃ duplicate samples such as 100 μ l to be diluted 45 minutes by 1: 1200.Certainly, according to general immunoassays mechanism well-known in the art, can replace the method for various elementary (anti-AGE) antibody (polyclone or monoclonal or its potpourri) and these primary antibody combinations of various detection in this course.

In the present embodiment, washing plate and according to adding the substrate chromophore (being p-nitrophenyl phosphoric acid (PNPP) in this case) of 100 μ l equal portions by the concentration of producer suggestion and they are cultivated (generally being) as mentioned above at 37 ℃ of following 1-2 hours, thus the OD that has in the control wells that is detected under the situation without any competitor is a 1.5-1.7 unit.Commodity in use microtitration plate reader can be read the absorbance log (OD) in each hole easily, in this case, determines to read at the 410nm place, and be contrast with reference wavelength 570nm.Then can be with the standard competitor AGE-BSA (in BSA mg/ml) that pushes away in the AGE level in any sample is from the standard competition curve to respective concentration, and generally this value is changed into AGE unit, wherein 1 AGE unit is the concentration that produces 50% inhibiting standard competitor AGE-BSA in this competitive AGE ELISA method.

Embodiment 2

In order to determine to be present in saccharification toxin (glycotoxins) (the reaction sugar in the tobacco smoke, AGEs etc.) whether with the proteins react of health, use a kind of " water pipe " or water pipe smoking device that makes up by little glass side neck flask that smoke from cigarette was taken out 1ml normal human serum sample, described little glass side neck flask not only is used for or makes the plume foaming by contained blood serum sample but also can make smog be accumulated on the above-mentioned sample and contact with it, and this contact is kept between contact automatic " being fuming " mutually later culture period.Filter tip is removed from used cigarette, and cigarette being contained in passed the flask plug make on the terminal pasteur pipet that immerses in the blood serum sample of tube head.In case install, then be connected the absorption of the syringe on the side neck and this device is used for repeatedly and will takes out blood serum sample from the smog of five (5) cigarettes by use.For control reaction, except that smoke from cigarette by the side neck, use the air that same device will about equal volume to take out independent blood serum sample.Measure these AGE levels in the blood serum sample that experiment is handled by the competitive AGE ELISA of above-mentioned standardization (referring to embodiment 1) then.This experiment is carried out twice independently, and the comparative result in each situation is as shown in accompanying drawing 2.Therefore, when the mean value of control serum samples is during less than 25AGE unit/ml, the blood serum sample that contacts with smog shows as about 150AGE unit/ml.From this evidence, the applicant reaches a conclusion: the AGEs that (active or passive) is present in the tobacco smoke during smoking is reactive and may combines with human body protein, this has increased smoker and onlooker's AGE content, and serves as simple and easy label with reference to the intensity that recent smoking activity is provided or has contacted with tobacco smoke in environment in the recent period with the appropriate control sample from non-smoking colony.

Embodiment 3

In further testing, in order to determine whether reaction sugar, saccharification toxin (glycotoxins) etc. are present in the smog of tobacco and whether can to form AGEs with proteins react, the content of estimating the reaction AGE of tobacco smoke condensation product as described below for definite this class saccharification toxin (glycotoxins).Set up the plug that is connected with the pasteur pipet that is connected with a short section proofed sleeve for side neck flask.The syringe that is contained on the side neck is used for this flask being immersed in acetone/the dry ice bath during the condensation product of collecting smog propping up this flask of smog suction of cigarette from amounting to 3-5 repeatedly.The smog of cigarette condenses on the flask walls of cooling, and by being extracted in the warm neutral aqueous solution with this condensation product solubilising and with them with the phosphate buffer flushing flask interior of 5mls, 0.02M.This condensation extract is carried out aseptic filtration also to be diluted by 1: 5 with other phosphate buffer.

Form the existence that determination method is estimated saccharification toxin (glycotoxins) in these extracts according to the AGE that uses anti-AGE antibody test with the collagen layer covalent bond AGEs that fixes then.Briefly, the commercially available 96 hole microtitration flat boards (joint study) that get that at room temperature will contain the mouse tail tendon collagen that is fixed on the hole surface contact with the smog condensation extract of above-mentioned dilution and overnight incubation, covering.The immunity tolerance program that is used for competitive AGE detection according to above-mentioned summary detects by being fixed on lip-deep AGEs with the interaction of mouse tail tendon collagen: the flat board that washes the glue primordial covering of smog extract processing successively, they are contacted with 4G9 monoclonal anti AGE antibody, flushing, contact with the commercially available mountain sheep anti mouse IgG/ alkaline phosphatase conjugation thing that gets, flushing, and it is additional with suitable chromogenic substrate (for example PNPP), thereby produce the signal of being determined by the optical density at 410nm place, this signal reflects that quantitatively with the reference substance without smog condensation extract-treated be benchmark, by contact the amount of the AGE para-immunity reaction that is accumulated on the flat board with the condensation extract of tobacco smoke.

As shown in Figure 3, the amount of the more AGE para-immunity reactions of Kong Zhongyou contacts with the condensation extract of tobacco smoke, and the signal from these AGEss relevant with smog decays with diluted extract, and this process strictness is carried out as the reference material of this class comparison according to the AGEs of the glucose induction of external formation, dilution.This prompting applicant contacts tobacco smoke can correspondingly make the experimenter contact saccharification toxin (glycotoxins) with the native protein qualitative response.

In a series of additional experiments, comprise in the incubated overnight process of tobacco smoke condensation extract that in the flat board that mouse tail tendon collagen is handled plaing of variable concentrations suppresses the Pimagedine (aminoguanidine) (a kind of AGE inhibitor) that AGE is cross-linked to form effect in other situation.As shown in accompanying drawing 4, Pimagedine has reduced the amount that has the AGEs that detects by immunity tolerance in the 50% inhibiting dose dependent mode at about 50mM Pimagedine place.As the inhibition of this inhibition AGE pharmacologic agent result's who exists final AGE signal or weaken be further proof, promptly with the smog condensation product to the processing of experimentizing property of tail tendon collagen after tangible AGE para-immunity reactivity on the molecule detail drawing with external and body in contact reducing sugar and on protein the AGEs of spontaneous formation similar, promptly can shared immunochemistry determinant from the AGEs in all these sources and their formation and/or crosslinking active be subjected to the special inhibition of Pimagedine.Being present in reaction AGEs in the tobacco smoke can be subjected to Pimagedine and suppress this evidence and also point out the applicant to can be used for reducing the excessive AGE content of smoker's self contact by the consumption tobacco by the corresponding anti-AGE measure of handling tobacco, tobacco smoke or treatment smoker and producing.

Embodiment 4

Carry out a series of experiment with the relation between the incidence of the AGEs rising of further research and proof tobacco and combustion by-products (tobacco smoke) and formation.Particularly and listed as this paper, these experiment confirm tobaccos and accessory substance thereof contain the soluble reaction saccharification intermediate product (this paper is called saccharification toxin (glycotoxins)) of significant quantity, this intermediate product can promote the formation of AGE and produce with aging and diabetes in AGE accumulate relevant whole secondary molecules and physiological complication, and they are mutagenesis, and subsequently they can participate in causing disease, such as cancer.This scheme and experiment subsequently are as follows.360mg/ml is pressed in the preparation of material and method tabacco water extract in PBS concentration by jolting tobacco sample was extracted 1 hour and (micropore, Bedford MA) carry out aseptic filtration by 0.45 μ m Millex-HA filter device.The preparation of smoke from cigarette condensation product

" water pipe " smoking device is used for and operation so that plume contact with aqueous solution.Aqueous solution (3mls) is put into glass Alan Mei Shi (erhlemeyer) flask that 25ml has the side neck, cigarette being contained in passed on the 500 μ l suction pipe tube heads in the hole in the flask plug.500 μ l suction pipe heads are extended to opening by flask side neck downwards, but do not enter aqueous solution.In case device installed then vacuum (about 20mmHg) fed the side neck of flask and lights cigarette.Change the tube head of suction pipe in some experiments: the commodity cigaratte filter of a slice 2 * 2mm is used to clog the suction pipe head, then with the 500mg aminoguanidine hydrochloride or sodium sulphate is placed between small pieces filter tip and the cigarette or do not let alone what material between them.In any experiment, from cigarette, remove the filter tip of the commercial preparation that connects in advance.With 5 cigarette lighters and smog is sucked among the PBS of 3ml equal portions.Before using the water condensate of smoke from cigarette is filtered by 0.45 μ m Millex-HA filter device (micropore).The detection that external AGE forms

To contain the microtitration flat board (joint study of fixing mouse tail tendon collagen, Cambridge, HA) cultivated 1 hour with TBS (PBS/0.05% Tween-20), the PBS hydroponics of the tobacco extract of usefulness serial dilution or smog contact under 37 ℃ is 18 hours then, with TBS washing 5 times, with the anti-AGE serum of rabbit polyclonal (the pAb RU 9.9.93 that in the TBS/0.1% lowlenthal serum, dilutes) cultivate 1 hour, with TBS washing 5 times and use and cultivate with the anti-rabbit igg of alkaline phosphatase (Sigma) conjugation and with TBS washing 5 times.Subsequently, by being to cultivate the AGEs colour developing that made combination in 45 minutes with 250mg/ml p-nitrophenyl phosphoric acid in 9.5 the diethanolamine and go up flat board to be carried out reading at 100mM, pH at the 405nm place at ELISA plate reader (Dynatech, MR 5000).Biolynx2.0 is used to generate typical curve and data extrapolation method.Saccharification toxin (glycotoxins) crosslinking active

The PBS solution that will contain 2mM EDTA as mentioned above contacts with tobacco smoke.To be dissolved in and contain 0,5 or the 40mg RNA enzyme A of the PBS/EDTA of 50mM aminoguanidine and identical 8 cigarettes contacts and in the time of 37 ℃, in the dark cultivated 0,5,8,24,48 and 72 hour.(Amicon, Beverly MA) remove unconjugated and low-molecular-weight material to use Centricon 10 concentrators.In order to estimate the formation of RNA enzyme dipolymer, use the separation gel [" nature " 227:680-685,1970] of 5% spacer gel and 12%, under reducing condition, sample carried out discontinuous SDS-PAGE and they are transferred on the nitrocellulose paper.Under 20 ℃, with lock solution (the not fatty milk of PBS/5%/1%BSA/0.2% Tween-20) with trace sealing 1 hour, use the anti-ribonuclease A antibody of the rabbit (BiodesignInternational with the horseradish peroxidase conjugation of in lock solution, diluting, Kennebunkport, ME) cultivate 45 minutes, wash up hill and dale and it launched with the PBS/0.2% Tween-20 according to the producer's instructions.Use AdobePhotoshop and the fluorograph of gained is processed processing with the light densitometry of the image of NIH image program determination (level and smooth, sharp-pointed and contrast enhancing).Fluoroscopic examination

In order to estimate the AGE specificity fluorescent, in the RNA enzyme sample of contact smoke from cigarette condensation product (preparation as mentioned above), measure when exciting at the 370nm place emission situation at the 440nm place with LS 50B fluorescence spectrometer (Perkin-Elmer).Determination of protein concentration fluorescent value with 0.5mg/ml.Mutagenicity research

As described in [] such as Ames, carry out the mutagenicity detection test.Briefly, in Oxoid nutrient broth (Difco) under 37 ℃ with Salmonella bacterial strain TA98, TA100, TA1535 and TA1537 (present that provides by doctor B.N.Ames) overnight incubation, with they in 0.1M sodium phosphate buffer (pH7.4) with the smoke from cigarette condensation product of serial dilution cultivate 1 hour, parallelly do three parts, the concentration fixed of then they being pressed the 0.1ml/ flat board is on minimum glucose flat board.Down should the flat board overnight incubation and the revertant number on each flat board counted at 37 ℃.The mensuration of serum AGE-apoB

Use aforesaid monoclonal anti AGE capture antibodies and anti-apoB-HRP conjugate (JBC, 267:5133-5138,1992), pass through sandwich ELISA measures the apoB (AGE-apoB) of AGE modification in human serum amount.Give value of sample and be that unit represents from typical curve with apoB-AGE U/ml serum.The apoB-AGE of a unit is defined as the activity of 1 μ g apoB (LDL).The mensuration of AGE

Use as (PNAS USA 90:6434-6438 and above) described in the generation glucose AGE epi-position of deriving AGE monoclonal antibody specific (IgG1 subclass), measure AGEs in the human serum by competitive ELISA.Become to belong to the hole that the sample of the range of linearity is tested in this detection for serial dilution, measure the AGE immunoreactivity in the parallel mode of doing three parts.With synthetic AGE-BSA reference material is that benchmark calculates AGE unit.The susceptibility of this test is in 5 U AGE/ml and the test and the coefficient of variation of test bay is respectively 5% and 8%.The result contains the tobacco of the saccharification toxin (glycotoxins) that promotes that AGE forms in the body and the water extract of smoke from cigarette

Beginning is extracted water-soluble reaction saccharification toxin (glycotoxins) by the mode that immerses PBS from the cigarette tobacco sample.Whether can induce AGE to form in order to detect saccharification toxin (glycotoxins) in the tobacco, this water extract be added in the flat board of glue primordial covering, continue 18 hours.Use the anti-AGE polyclonal antibody of specificity to detect the existence of the AGEs of up-to-date formation on the tropocollagen molecule.Accompanying drawing 5A represents the comparable situation that formed by the AGE that U.S.'s cigarette of 8 kinds of trades mark is induced.Impelled AGE formation partly from all extracts of accepting the detection trade mark.It should be noted that trade mark E (the brand of cigarette D of " soft " amount) has the activity that this higher class forms AGE than trade mark D.This discovery (when with other " common " and " soft " trade mark carry out paired comparisons the time continue to reappear) reflect the difference in the tobacco processing course probably and show that the content of tar and other assay method of cigarette " intensity " do not rely on saccharification toxin (glycotoxins) content.

For saccharification toxin (glycotoxins) volatilization that determines whether tobacco to be derived, we form in the test at AGE and have detected the smoke from cigarette condensation product.In order to collect any volatility saccharification toxin (glycotoxins), we have installed a kind of smoking device, and wherein the smog suction of the cigarette of spontaneous combustion in the future is equipped with in the flask of PBS solution.In a kind of trial of closer simulating the mode that lung tissue is contacted smoke from cigarette, the smog of experiment is contacted with PBS at drag, but do not pass through solution foaming.Immediately the PBS (smoke from cigarette condensation product) of smog contact is added to after the preparation with in the microtiter well of glue primordial covering and their are reacted spend the night.As leaf tobacco extract, the smoke from cigarette condensation product can rapid induction forming by the AGEs of the anti-AGE polyclonal antiserum identification of rabbit.The AGEs that is produced by smoke from cigarette depends on concentration (accompanying drawing 5B) and time (unlisted data), and by solubility AGE inhibitor (aminoguanidine) directly being added to (unlisted data) in the hole or can in the dose dependent mode, suppressing AGEs by the smoke from cigarette generation by means of making smog pass through aminoguanidine crystallization post (accompanying drawing 5C).The saccharification toxin (glycotoxins) that the tobacco that smog by sodium sulphate contrast post contains smog derives forms AGEs on the target thing ability does not influence (accompanying drawing 5C).The smoke from cigarette condensation product is low 10 times at the specific activity tabacco water extract that impels AGE to form, and this prompting can make some saccharification toxin (glycotoxins) be damaged by combustion process or the system that is used to catch volatility saccharification toxin (glycotoxins) and AGEs is invalid.It is unsettled that the AGE that finds in leaf tobacco extract and smoke from cigarette condensation product forms activity: if in the time of-20 ℃ freezing this solution and before detection, melt or be retained in and keep on ice or at room temperature more than 5 hours, then AGE forms activity and can't detect.

Also can estimate the ability that smoke from cigarette saccharification toxin (glycotoxins) induces AGE to form by on the RNA enzyme A of contact smoke from cigarette condensation product, measuring AGEs characteristic fluorescence pattern.Shown in accompanying drawing 6A, for the time that increases, the RNA enzyme A of contact smoke from cigarette condensation product shows AGE type fluorescence and depends on the time increase, (promptly exciting the back) in the emission of 410nm place at the 370nm place, this inhibition that is increased in the aminoguanidine that reaches capacity after 24 hours and in incubation, existed.Saccharification toxin (glycotoxins) can cross-linked proteins

One of feature of reaction reducing sugar is that they can impel between the susceptibility functional group in the molecule and the formation of intermolecular cross-linking.In order to prove that the reactive saccharification toxin (glycotoxins) in tobacco and the smog also has this characteristic, we have detected saccharification toxin (glycotoxins) and the crosslinked ability of RNA enzyme.With the sample culturing different time of smoke from cigarette condensation product with RNA enzyme A, by the molecular weight on the SDS-PAGE gel they are separated, make their colour developings with anti-RNA enzyme A antiserum then.Use this technology, the applicant can determine that the formation of RNA enzyme A-RNA enzyme A dipolymer depends on the time and reached capacity in 24 hours.In a kind of dose dependent mode, the formation of dipolymer can be subjected to the inhibition of aminoguanidine, and this shows that cross-linking process depends on the saccharification toxin (glycotoxins) (accompanying drawing 6B) that contains carbonyl.Saccharification toxin (glycotoxins) is mutagenesis

Above-mentioned research has proved that reducing sugar can react and induced mutation in bacterium and mammalian cell with DNA external.The reducing sugar of high intracellular concentration (Robison ester) causes mutation rate to increase in the Escherichia coli.Equally, the mouse tire of the hyperglycaemia that causes because of the parent diabetes of contact shows the mutation rate that doubles.

In this experiment, a large amount of bacillus typhi murium bacterial strains (TA98, TA100 and TA1537) are contacted with the smoke from cigarette condensation product in typical A mes mutagenesis testing mode.In bacterial strain TA98, noticed active sudden change activity, but in other bacterial strain, do not found.As observable in the accompanying drawing 7, TA98 contacts the sudden change that causes dose dependent to be increased with the smoke from cigarette condensation product of the recruitment of up-to-date preparation.When TA98 contacted with the mutagen that can cause frameshift mutation also original sudden change to be replied in histidine operon, its can be grown on the nutrient culture media that lacks histidine.With the quantity that has reduced revertant before PBS contacts by the smog (saccharification toxin (glycotoxins) molecule of significantly removing or having neutralized and contained carbonyl) of the dry post of aminoguanidine.In the reaction of DNA and above-mentioned reducing sugar, observed similar mutagenic activity.This is that the applicant recognizes that the mutagenic activity in the smoke from cigarette does not need to carry out the report first that metabolic activates by the P450 system.May also not observe this activity in the past, for example Yi Qian worker mainly in organic solvent, prepared smog extract and at noticeable time bar post analysis mutagenic activity.The instability of saccharification toxin (glycotoxins) requires the express-analysis mutagenic activity in the water extract.The water smoke from cigarette condensation product that requires at room temperature or on ice to cultivate more than 5 hours no longer is a mutagenicity.Saccharification toxin (glycotoxins) impels AGEs to form on serum proteins in vivo

Research will react the possibility that saccharification toxin (glycotoxins) or AGEs absorb in vivo behind the smoke from cigarette in contact below, and this process is undertaken by the amount of the AGEs that check forms on the serum proteins as the total surrogate markers thing of body burden.Blood sample from other healthy smoking person and non-smoker

In this research, detect AGE serum proteins level and albumen apoB-AGE.AGE-apoB level among the smoker (324+/-140U/ml, n=9) be significantly higher than (p＜0.01, non-matching StudentShi t-check) non-smoker (177+/-33, n=10) and the serum AGE level among the smoker (202+/-76 AGE U/ml n=23) is significantly higher than the serum AGE level (146+/-31 AGE U/ml) among (p＜0.02) non-smoker.In the smoking group all are inhaled the above cigarette of 1 bag individual every day, but do not limit their smoking history.Similarly research relate to be present in blood in the amount and the relation that contacts the smoke from cigarette degree of AGE of apoB or other haemocyanin combination.The sum of this contact will can be used for directly and the analysis of indirect contact smog situation.In fact, this test is similar to the hemoglobin A of the blood-glucose total amount that the preceding 30 day time limit of contact obtain _ICTest.Level that it should be noted that AGE haemocyanin and AGE-apoB among the smoker almost with identical [the AGE haemocyanin 200-400U/ml of observed situation in the patient who suffers from diabetes; AGE-apoB 300U/ml].

Embodiment 5

For determine whether can measure the reactive raising of AGE para-immunity behind the clear and definite contact tobacco smoke, from the experimenter of custom smoking, get blood sample, at first get blood sample after 18 hours, get blood sample after the experimenter continuously inhales 5 cigarettes again when finishing at interval in 18 hours then in the interval cigarette smoking.Use standardization sandwich type ELISA program in from the LDL fraction of these blood samples, to compare the AGE degree of modification of apoB so that detect AGE-apoB.In carrying out this standardization detection test, anti-AGE antibody is fixed on the surface of detecting the hole catching the AGE ornamental equivalent of sample, and will commercially available anti-apoB monoclonal antibody/horseradish peroxidase (HRP) conjugate that gets be used to detect by the mutually combine apoB of any AGE modification of fixing of the specificity with the anti-AGE antibody of being fixed.

Briefly, the serum fraction for preparing low-density lipoprotein (LDL) enrichment by following steps: the polyglycol (molecular weight is 8000) with 900 μ l 6.66% is handled 100 μ l blood serum samples 15 minutes, centrifugal (14,000rpm, 10 minutes) and in μ l 0.5%SDS, gained is precipitated solubilising and spend the night.In order to make up test panel, to resist AGE antibody (being monoclonal antibody 4G9 in this case) to be fixed on the surface of microtitration flat board by under the dilutability of institute's titration, cultivating, after this block non-specific binding by BSA solution replacement capture antibodies solution with 1%.Be that 0.05% SDS cultivated 1 hour with three parts of parallel cultivations of the serum fraction of LDL enrichment (or suitably LDL reference material dilution of titration) and with their in test panel with the ultimate density in the damping fluid then, the conjugate (be in this case available from and anti-apoB/ horseradish peroxidase (HRP) conjugate of the BiodesignInternational that uses by dilution in 1: 500) that this moment flat board is washed and adds the commercially available anti-apoB monoclonal antibody/enzyme that gets is to detect the apoB of any combination.Washing is dull and stereotyped and add suitable colour developing HRP substrate (such as o-phenylenediamine (o-phenyline diamine) (OPD)) after 1 hour cultivation, and behind suitable culture period with the reference wavelength of 570nm for to read OD to impinging upon the 450nm place.Generally according to principle well-known in the art, the optium concentration of all ingredients (for example coated antibody and detect antibody) and best pH, detergent concentration, damping fluid composition, culture period and other special characteristic contrasted each other changing so that the sandwich type immunity tolerance determination method of modifying at the AGE of this apoB of being used for makes the sample signal maximization and the non-specific background " noise " that will disturb drops to bottom line.In this experiment, the AGE-apoB value before the smoking be 218 ± 1 and smoking after value be 241 ± 3 apoB-AGE units/mg apoB (wherein being the activity of the commercially available LDL reference material that gets of 1 μ g/ml (for example available from Cappel)) with the apoB-AGE unit definition.The applicant has studied this evidence, has been the AGE modification that smoking can increase special blood constituent (apoB of LDL) tempestuously.The mensuration of AGE level can provide relevant experimenter the information of recent smoking history on this prompting sample composition.In addition, because that the AGE of LDL modifies is relevant with the pathological mechanisms of deleterious effect health, so this further points out applicant smoker can have benefited from limiting measure as the AGE of the excessive contact of tobacco consumption result generation.

Embodiment 6

In order to find easily whether the tissue or the fluid zone of sampling reflect smoking history, the applicant has checked comfortable North Shore University Hospital, Manhasset, the order patient who has carried out operation in advance of NY screening.Among these patients some (chance) is smoker's (the 1st group), and other people is non-smoker's (the 2nd group).In this viewing test, patient's colony is divided into several levels or in addition some variable smoking intensity, sex, age, health condition or the further feature that can cause serum AGE level is controlled.Use monoclonal anti AGE antibody, measure serum AGE level by competitive AGE ELISA method, so as with AGE-BSA standardization preparation be benchmaring AGEs (as among the above-mentioned embodiment 1 specifically as described in).The average serum AGE level of the 1st group (N=16) is 200 ± 80, and Dui Bi the 2nd group (N=13) is 150 ± 30 therewith.The comparative result of these data is checked on the probability level that shows 0.05 through the t-of independent solution significant difference.

Embodiment 7

Tobacco and tobacco product, comprise (for example) tobacco smoke AGEs also as antigen or haptens, so that guiding antibody is oriented to the AGE structure relevant with tobacco especially.This antibody-like (identical with the present invention) is used to successively discern and suppresses and relevant with tobacco successively AGE structure.For example, by making up the immunoassay of using the relevant AGE antibody of the anti-tobacco of the present invention, can measure degree by the AGEs modifying protein relevant with tobacco.Discuss as above-mentioned, and depend on the half life period of the protein of modification like this, the immunochemistry of AGE epi-position on protein example (such as haemocyanin) measured the index that recent tobacco consumption (for example by smoking) is provided.Equally, the immunochemistry of AGE epi-position on circulation and/or histone can be detected the therapeutic process that is used to monitor with reagent of the present invention, by with tobacco or tobacco smoke in AGEs reaction, these reagent relate to inhibition and accumulate by the excessive of AGEs that uses tobacco to produce.

By from tobacco or from smog, separate the AGEs relevant with tobacco, for example by preparing the water extract that contains AGE (as mentioned above) of tobacco or tobacco smoke condensation product, can prepare the immunogene of the various haptens, antigen and the conjugation that meet the present invention AGEs relevant easily with tobacco.Can be with this extract as a kind of various immunogenes that increase the antibody of identification specificity epi-position or its characterization of molecules, this immunogene can be in a preferred embodiment, regard the AGEs of extract as haptens, according to the widely used scheme in this area, use a large amount of well-known divalence coupling reagents arbitrarily, all as if the such carbodiimide of EDC, with these haptens correspondingly with arbitrarily several preferred carrier protein couplings, described preferred carrier protein comprises, keyhole  hemocyanin (KLH) for example, thyroglobulin, and bovine serum albumin(BSA) most preferably.No matter the source how, can be with the AGEs relevant with tobacco, no matter be independent or with the carrier protein coupling and no matter be pure or partial-purified form is used to confirm good immunization protocol, thereby be created in the extensive application is useful antibody and relevant immunoreagent, and this is owing to the specificity that is used for the antibody of the characterization of molecules of tobacco associated products.

Be a preferred scheme below, any several animal species carried out immunity inoculation be oriented to the polyclonal antiserum of the protein conjugate of the AGE relevant that described animal comprises, for example mouse, rat, hamster, goat, rabbit, and chicken with tobacco with generation.First three kind of above-mentioned animal species is the selection that needs especially, because of they can be used for producing subsequently the hybridoma that can secrete the hapten specificity monoclonal antibody.Can finish generation easily by any several schemes of this area general implementation, and described scheme has been described and has been suitable for by merging the condition of the immortalization of the immunity inoculation splenocyte that produces with suitable clone (for example myeloma cell line) from the described hybridoma of immunity inoculation animal splenocyte.The described scheme that is used to produce hybridoma also provides certain methods, and these methods can be used for selecting and clone immune spleen cell/myeloma cell's hybridoma and are used to discern the hybridoma clone that secretion stably is oriented to the antibody of required epi-position.To be used to generate polyclonal antiserum more at large such as rabbit and the such animal species of goat, but no matter finally whether need polyclonal antiserum or monoclonal antibody, generally begin carrier protein and a kind of adjuvant of hapten transformation, together carry out administration such as complete Freund ' s adjuvant (Complete Freund ' s Adjuvant).Can give immunity inoculation by any approach, generally by in the peritonaeum, intramuscular or intradermal approach; Come preferred some approach according to the kind and the final type that produces antibody of institute's immunity inoculation in the art.Subsequently, general with a kind of adjuvant, give booster immunization such as alum or incomplete Freund ' s adjuvant (Incomplete Freund ' s Adjuvant).After original immunity inoculation, give booster immunization at set intervals; General 1 month is suitable interval, and the 1-2 behind each booster immunization gets blood sample between week.On the other hand, sometimes various so-called hyperimmune schemes (generally with the nearer booster immunization in space be feature) timely are used to produce a kind of trial that is better than the antihapten antibody in the anti-carrier protein antibody.

For in any several easy modes, comprise (for example) Ouchterlony diffusion gel and the direct hapten specificity immunity titre in the ELISA scheme, can compare the antibody titer after raising in the blood sample.In a kind of typical directly ELISA, a kind of definite antigen is fixed on the surface of detecting the hole (generally in 96 holes or microtitration flat board), detect the hole by flushing subsequently and separate a series of cultures to remove unconjugated binding partner.As non-restrictive example, detect dull and stereotyped hole and can hold a kind of dilution of haptens/carrier conjugate, the aqueous solution of buffering, preferred situation is that wherein carrier protein is different from the used albumen that is used for the generation antibody animal that immunity inoculation detects; For example can detect serum to the detection hole of modifying with the AGE/BSA conjugate relevant of immunity inoculation with tobacco from the AGE/KLH conjugate immunity inoculation animal of being correlated with tobacco.On the other hand, detect the surface by cultivating to modify with haptens separately.In general, the surface of detecting the hole is contacted with the solution of a kind of irrelevant protein (such as casein) with unappropriated position on the sealing frosting.After neutral buffered solution flushing, one of a series of antiserums of hole and the serial dilution that is prepared by significant blood sample are contacted (elementary antiserum) with salt that generally contains a kind of degree that non-specific interaction is minimized and detergent.Once more after the flushing, be fixed on the quantity that detects the detection antibody on the hole by cultivating with the commercially available enzyme that gets-antibody conjugates can estimate by interacting with required haptens or haptens/carrier conjugate, the antibody moiety of wherein this hyperconjugation thing directly is oriented to and is used to produce elementary sero-fast kind; For example, if elementary antiserum increases in the rabbit body, can will in the goat body, increase and use as secondary antibody so with the commodity preparation of the anti-rabbit antibody of several enzymes (such as horseradish peroxidase) conjugation.After producer's regulated procedure, in colorimetric detection method, can estimate the amount of this secondary antibody quantitatively by the activity of correlation conjugate enzyme.Can change many relevant ELISA or radio-immunity tolerance scheme (such as competitive ELISA s or sandwich ELISAs) arbitrarily, all these all are well-known in the art, thereby determine the antiserum of required high titre; Be that special antiserum provides correct positive findings (for example greater than 1/1000 and more preferably greater than 1/10,000) when high dilution.

Similar immune tolerance scheme can be used to estimate the culture supernatants of the hybridoma that comes free immunity inoculation animal splenocyte preparation.In the antiserum or hybridoma supernatant of so characterization, require to use the various control cultures (for example with different carrier protein together) of relevant and haptens that structure is different or antigen, and omit all ingredients in the immune metrics process and minimize and discern from false positive result and false negative result's the antibody specificity and the reliable measurement result of titre so that will detect non-specific signal in testing.The kind of the control cultures of Shi Yonging is well-known in this respect.In addition, identical common immune tolerance scheme is used subsequently together (being that the antibody of being found is the specificity structure determinative that has high titre and relate to the AGEs relevant with tobacco) with the antiserum of being discerned by said process.The relevant AGE antibody (no matter being polyclonal or monoclonal) of the required anti-tobacco that this class latter can be used and instructions are also packed in handled easily person's the kit formulation with other useful reagent (including, but not limited to the molecular criteria thing of one group of AGES relevant with tobacco) and thinning agent arbitrarily.

As mentioned above the relevant AGE antibody of the anti-tobacco of Sheng Chenging (and particularly this class monoclonal antibody) also can be used for discerning, separation, AGE epi-position that enrichment is relevant with tobacco with purifying.This process can be finished by technology well known in the art, for example passes through following steps: the known preparation that contains meaningful epi-position is contacted, rinses out the material of unconjugated material, wash-out specificity combination then and analyzes its chemical constitution so that measure the specific molecular form of the AGEs relevant with tobacco with the sessile antibody that is used in particular for described epi-position.

Embodiment 8

For further proof use AGE inhibitor of the present invention and AGE replying agent prevent protein on a kind of surface (on or skin or mucomembranous surface) such as the dental surface that occurs in the smoker go up this situation of decolouring, carry out following surperficial brown stain experiment.The substitute of the dental surface that covers as a kind of skin, mucous membrane or mycoderm is used to carry stick one piece of cloth or paper on top of another ankyrin (gelatin, i.e. collagen) surface on the material of a kind of simple and easy paper mounting with photographic paper unexposed and that launch.This class photographic paper is broken into the disk thing of 5mm, and with in the solution of several immersion tobacco extracts in these disk things or tobacco smoke condensation product (preparation as mentioned above), used or need not AGE inhibitor of the present invention or the AGE replying agent described tobacco extract or tobacco smoke condensation product have been carried out suitably dilution in a kind of suitable water damping fluid (as mentioned above).General by in the dark, the temperature of control keeps suitable time bar with this class experiment culture between 20 ℃-60 ℃, this time limit can be at several hours to the scope in several weeks.At different times place, cultivation beginning back, reclaim the disk thing, the color of observation brown stain is also taken pictures to be used for keeping records.In a kind of dyeing way of skin, mucomembranous surface or the tooth mycoderm that is similar to tobacco user, cause protein surface to become having the color of the AGE pigment of yellow and brown from the suction-operated of the reaction AGEs of tobacco or smog extract.Proved that as accumulating the method for preventing the method that prevents that AGEs accumulates determines that the AGE inhibitor can be used for scope of the present invention by the yellow/brown pigment that with the contrast disk thing of not cultivating is reference with detectable.

Be used for AGE at another and reply active detection test, carry out " pre-brown stain " by cultivating the disk thing that protein is covered, and the AGE replying agent that the present invention is selected adds another time bar in the culture solution to described dilution tobacco extract above or tobacco smoke condensation extract.With the contrast disk thing do not cultivated with detectable is reference, and inhibition that the yellow/brown pigment that is produced by detectable of the present invention is accumulated and return action have determined to be particularly suitable for the candidate as the AGE replying agent in the scope of the invention.So AGE inhibitor and the AGE replying agent of determining is specially adapted to be made into mouth rinse or tooth powder, unwanted tooth dyeing and pigment that they could prevent and reply other oral surfaces form, described unwanted tooth dyeing and pigment form with using tobacco to take place, no matter be the oral cavity use chew cigarette and snuff or by the cigarette of enfleuraging, cigar and tobacco pipe.Can also missible oil, emulsifying agent or the analog that active detectable is mixed with the hand that is used in smoker's dyeing and points will be had in this protein brown stain determination method.This method is also as the dosimetric example that is used to monitor contact environment smog.

Embodiment 9

Although generally accept smoking is this fact of a kind of hazard factor that causes heart, lung, blood vessel, tumour and other disease, and the molecule mechanism that constitutes this cause-effect relationship basis is not still understood fully.Pointed out chronic and acute process prior to using the pathological reaction of tobacco, can cause AGE level rising in blood and the tissue but the applicant confirms tobacco consumption (particularly by smoking) for the first time.Situation about noticing in the experiment of the immunochemistry dyeing of using anti-AGE antibody shows that AGEs has been covered with the smoker's who suffers from vascular diseases coronary artery thick and fast, and the applicant attempts also to confirm that chronic contact smog can cause controlling good animal model inner blood and organize AGEs to raise in the body.

Use whole body to suck the contact chamber, the male and female rats (for example laboratory strains F344) in 6 weeks of growth is contacted different time bar in several thoughtful 2 years scopes with the gas of the filtered air of the smoke from cigarette particulate that is loaded with the constant concentration through diluting the mainstream smoke generation.For example, level according to the total particle matter of 250mg (TPM)/cubic meter contacts the air that this class is carried tobacco smoke, contact 6 hours every day, contact 5 days weekly, the material that this process causes inhaling tobacco smoke sediment and every day in people's the air flue of 5 cigarette packages is identical.(J.Chen etc. for example as more complete description in the open program, 1989, " inhalation toxicology " (Inhalation Toxicol.) 1: 331-347), use the machine of being fuming continuously, study with cigarette (tobacco health research institute (Tobacco HealthReaserch Institute) as the no filter tip of IR3 from type; Lexington, KY) the middle mainstream smoke that produces.Same control-animal packed into and the air-flow of contact filtration (not containing smoke from cigarette) only.By measure the level of smog contact with reference to the gravimetric analysis of the filtered sample of taking from the contact chamber.Described particularly the chemistry of this contact smog air and physical features (referring to above with J.Chen etc., 1992, " pharmaceutical aerosol " (Aerosol Med.) 5: 19-30).

After the desired contact time limit, put to death experimental animal and collect various organs and tissue, comprise (for example) whole blood and with their sampling be used to dissect, micro-and such as the quantitative such biochemical analysis of AGE level.In a kind of preferable methods, serum diluted by 1: 5 in phosphate-buffered saline and Proteinase K is added into 1/100 of total serum protein content in the sample.In the time of 37 ℃, make haemocyanin digestion then by cultivation, and by give birth to temperature to 70 ℃ 1 hour and with the Proteinase K deactivation.Simple centrifugal make it clarification after, collect supernatant and before analyzing AGE content, it is moved to by AGE ELISA mentioned above 1mM phenylmethylsulfonyl fluoride (phenymethylsulfonyl fluoride) (PMSF) in.

In the Primary Study of carrying out according to this program, average (± standard deviation) AGE level that demonstrates collection from the haemocyanin digest that contacts 6-85 time limit in week rat with smoke from cigarette is 22.0 ± 4.9AGE unit/ml serum (N=13), and the average serum albumin A GE level that the control rats that does not contact with smoke from cigarette shows is 13.5 ± 2.9 AGE units/ml serum (N=7).Said method also provides a kind of easy determination method, use this determination method estimation reagent of the present invention and filtration unit suppressing, prevent or replying the activity that the AGEs that causes because of the consumption tobacco accumulates, for example according to the present invention, can handle the animal of the tobacco smoke of tobacco itself, original or main flow, the air that is loaded with smog or contact smog, and the tissue of check test animal with blood so that measure the degree of accumulating by the AGE that reduces more thus, prevents or reply with the control sample that is untreated or do not contact.

Embodiment 10

The filter tip that the following examples proofs is removed saccharification toxin (glycotoxins) has also increased the organ sensation potential of cigarette, and has particularly removed " stimulation " from the cigarette tobacco smog taste that produces " softer smog ".

For the cigarette that is used for this test is installed, following material is put into a kind of standard manual tobacco roller press: a kind of standard merchandise filter tip (cutting down), the commodity cigarette that the sodium chloride of 500mg aminoguanidine or equivalent and the 3cm on its original paper are long from Marlboro soft type cigarette.Use a roll extrusion paper that these materials are wrapped in together.By observing or touch the cigarette that is difficult to distinguish mutually the roll extrusion again that has other medium.

In order carry out this test, experimenter's mounted in pairing mode, comprise one of every class cigarette (being aminoguanidine or salt) and mark of this centering with yellow spotting.Then cigarette is put into the #10 packing and contain packing more for a short time of underlined (being that cigarette has yellow spotting).The cigarette that half that packing is interior has the built-in filter tip of aminoguanidine marks with yellow spotting with the yellow spotting mark and with second half cigarette that has the built-in filter tip of sodium chloride.

Each individuality to 10 experimenters (comprising a large amount of smokers and accidental smoker) gives the cigarette of one of above-mentioned packing and requires them to state the sensation of two kinds of cigarettes.By the reaction that the experimenter writes down the experimenter, the experimenter opens interior packing with show tags then.

All 10 experimenters all think two kinds of cigarettes " product get up to resemble a kind of cigarette ".Among 10 experimenters 8 can distinguish the smog that cigarette that subtle difference between two kinds of cigarettes and all feeling per capita contain the built-in filter tip of aminoguanidine produces and have " stimulation " or a kind of " soft smog " hardly.1 experimenter is described to: " soft must be also softer than new wine as old red wine ".All 8 experimenters that can distinguish two kinds of cigarette differences all prefer having the cigarette of the built-in filter tip of aminoguanidine.

Embodiment 11

Present embodiment proof saccharification toxin (glycotoxins) (AGE) is removed filter tip can prevent that smoke from cigarette dyeing albumen bag is by the surface.In the present embodiment, use microtitration flat board, and the result that will find is used for the surface of any albumen bag quilt, such as tooth, nail or skin by the glue primordial covering.

In embodiment 4, described 10 preparations of being with aminoguanidine or the filter-tip cigarette of sodium chloride, in equipment, be fuming.5 milliliters of PBS add in the glass beaker of 25 milliliters of band side arms, and 20 cigarettes that have one of two kinds of filter tips are put into the 500 microlitre suction nozzles in the hole that is inserted in bottle stopper in turn, and light.Side arm to flask applies 20 mm Hg, so that smog is sucked flask, contacts with PBS.After 20 cigarette combustion are intact, add 5 milliliters of fresh PBS, 2 ml mixtures are joined in per 3 holes of 6 orifice plates.Each 6 orifice plate has 3 holes to have to be exposed to the PBS of band aminoguanidine cigarette with filter tip smog, and there is the PBS that is exposed to band sodium chloride cigarette with filter tip smog in 3 holes.The plate lucifuge was incubated 48 hours in 37 degree temperature.With the PBS washing that contains 0.05% polysorbas20 6 times, the relatively color at the bottom of the hole.Remaining color is painted AGE in the hole, and it is saccharification toxin and or the result that reacts of the hole of reactive AGE and glue primordial covering.

Embodiment 12

Can eliminate saccharification toxin (glycotoxins) with aminoguanidine spraying tobacco

In the present embodiment, tobacco leaf is handled with aminoguanidine (a kind of AGE forms inhibitor), and observed the formation that saccharification toxin (glycotoxins) can not impel AGE molecule in the body.The loose tobacco leaf that equates with the amount of 5 cigarettes with the 5mls solution spray that contains following concentration aminoguanidine: 10mg/ml, 1mg/ml after the 0.1mg/ml spraying, ties up tobacco leaf and cultivated 7 days down at 37 ℃ with aluminium foil.Then tobacco leaf is rolled into cigarette and in the pipe device of embodiment 3 and 4, lights (mentioned above).Use external AGE mentioned above to form determination method and measure saccharification toxin (glycotoxins) content.As shown in accompanying drawing 9, the dose dependent of having observed saccharification toxin (glycotoxins) content reduces.

Discuss

Above-mentioned disclosure and embodiment have proved the biological and chemical relation and the effect of the protein glycation of before having illustrated, and are based on the pharmacokinetics that organ sensory characteristic and tobacco are handled and consume.Therefore, owing to also observed the many vascular complications relevant with smoking in the diabetic with high circulation and tissue bond AGE level, this paper has studied the possibility that smoking impels AGE to form.More particularly, the listed data of this paper comprise up-to-date content, and this content proof has also exemplified existence and the activity that this paper is defined as a group reagent of saccharification toxin (glycotoxins).The water extract of tobacco and smoke from cigarette contains saccharification toxin (glycotoxins), remarkable reactive reducing sugar or saccharification intermediate product, and they in vitro and in vivo can rapid induction AGE form on protein and at external generation dna mutation.Can from sample, remove these activity by making smog through the dry post of aminoguanidine (the effective and specific inhibitor that a kind of AGEs forms).

Have the characteristic of many uniquenesses from the saccharification toxin (glycotoxins) of smoke from cigarette, these characteristics can distinguish reducing sugar, glucose and the Robison ester of saccharification toxin (glycotoxins) with above-mentioned research.Most importantly their extreme reactivity: take glucose with reporting of being write down or Robison ester is opposite to induce the AGE that can measure to form a couple of days to several weeks, the saccharification toxin (glycotoxins) that tobacco derives can induce AGE to form in a few hours.When most reducing sugars (such as glucose) need transhipment to enter tenuigenin, can think saccharification toxin (glycotoxins) cell membrane by bacterial cell easily.In addition, in healthy people, can be absorbed and react with haemocyanin successively by lung from the saccharification toxin (glycotoxins) of smoke from cigarette.Although we do not measure the chemical constitution of saccharification toxin (glycotoxins), we can infer that they have carbonyl and may have dicarbapentaborane, and this is because can remove them or make their quenchings with aminoguanidine.Saccharification toxin (glycotoxins) may be a kind of product that the product that forms in the Maillard reaction that causes in the tobacco processing procedure is reset.The instability of saccharification toxin (glycotoxins) means when handling tobacco and when it is aging, can forming and consume they and other Mei Lade product (may be the reason that causes smog fragrance) consistently in the detachment process.

The AGEs that is formed by tobacco saccharification toxin (glycotoxins) in the body can partly cause the atherosis incidence of disease of smoker's medium sized artery to increase.Have a large amount of evidences to show that AGEs plays a role in the external and body on atherosclerotic pathology: (1) has proved AGEs and the linked groups's collagen cross-linking that is used to increase linked groups's rigidity.(2) AGEs of collagen cross-linking as catch the circulation haemocyanin with covalent, such as the reaction " focus " of lipoprotein.(3) the combination meeting of endothelial cell AGE acceptor increases permeability, synthesizing of the anti-set accelerator thrombomodulin of minimizing and the synthesizing of increase procoagulant tissue factor of blood vessel.(4) AGEs of tissue bond can make cell-derived nitrogen oxide quenching with chemical mode, suppresses the vasodilation of nitrogen oxide dependence thus.(5) the verified AGE that contains the phosphatide of amine modifies the oxidation that can cause fat.The cell ldl receptor can not discerned the LDL of oxidation, and macrophage scavenger receptor can be removed the LDL of oxidation.The vascular wall macrophage that fat carries is the characteristic foam cells of finding in fat line and the infringement of other class early atherosclerosis usually.(6) vascular tissue that significantly raises is relevant with the acceleration angiosis of diabetes kidney failure disease latter end with round-robin AGEs.(7) albumin that the external source AGE of normal immature rat of injection and rabbit modifies in the intravascular can produce and be similar to observed blood vessel change in the animal body of suffering from the vascular diseases relevant with diabetes with aging.With the animal of external source AGE albumin injection show that the vascular wall permeability increases, monocyte migratory activity and attacking the blood pressure response increase that detects the back with acetylcholine and nitroglycerine in interior subcutaneous and artery is all (periarteriolar) space.[PNAS 89: 12043-12047 such as Vlassara, 1992].It is desirable to expectation and have the activity identical with the AGEs that serum and histone reaction form with above-mentioned AGEs by saccharification toxin (glycotoxins).

Saccharification toxin (glycotoxins) can also make the incidence of disease of cancer among the smoker increase.Except that with protein and fat on amino reaction, reducing sugar can also with the amino reaction of nucleic acid.External use reducing sugar cultivation DNA or mononucleotide can produce to be similar to the AGE compound that combines with protein and fat viewed absorbance log and fluorescence are changed.Show noticeable mutation rate increase with the bacteria plasmid DNA of glucose or Robison ester cultivation or the cultivation of mammal shuttle vector DNA.(Bucala etc., 1984, PNAS, 81: 105-109; Bucala 1985, PNAS82: 8439-8442; Lee AT and Cerami, A.1987, PNAS84: 8311-8314).Because the reducing sugar of above-mentioned research can pass through bacterial cell membrane, so although they can be added in the bacterial growth media, they can not produce the sudden change in the bacterium.Yet, having proved that Robison ester can induce the sudden change in Escherichia coli (DF40 and DF2000) the glycolysis mutant, described mutant can be accumulated glucose-6 phosphoric acid in its tenuigenin.Universals in all these results of study are that the major part sudden change that is produced by reducing sugar is to insert body or deletant.

((1994) FASEB magazine 8: 545-550), promptly parent hyperglycaemia environment is to the influence of embryo growth in the mouse body in the work that the most compellent evidence of reducing sugar mutagenicity comes free Lee etc. to be done in the mammal.As eye's lens and red blood cell, embryonic cell does not need insulin that glucose is shifted by its film.To contain bacterial gene (lacI, a kind of evident characteristic mutagenesis label) integrates the trangenic mice embryo of duplicate and implants in the diabetes and contrast alternative precursor that chain urine rhzomorph induces.In the DNA from the embryo who implants the diabetes parent, the lacI mutation rate is significantly higher than from the situation of implanting control mice.

Although the mammal that suffers from diabetes has the serum level of glucose of rising, there is not verifiable evidence to prove that any class cancer increases in their bodies.Glucose can freely not enter mammalian cell; It needs insulin to exist to stimulate its migration.Therefore in ripe mammal body, concentration of glucose depends on serum level of glucose in the born of the same parents.Yet, to notice that importantly saccharification toxin (glycotoxins) is more effective more than glucose.As glucose and Robison ester, they can produce in a kind of aminoguanidine dependence mode inserts and/or deletion mutant, but does not resemble these other reducing sugar, and saccharification toxin (glycotoxins) can pass through cell membrane easily.

In a word, our discovery shows that saccharification toxin (glycotoxins) (finding) can partly cause the increment rate of observed atheroma vascular diseases and cancer among the smoker in smoke from cigarette.In smoking process, saccharification toxin (glycotoxins) the suction alveolar with high concentration in alveolar, can suck them blood flow and suck the pulmonary parenchyma cell.In case be in the blood flow, then saccharification toxin (glycotoxins) can be induced and be formed the part of AGE and quicken development of atherosclerosis thus on serum and vascular wall albumen.Saccharification toxin (glycotoxins) can also with the nucleic acid reaction in the pulmonary parenchyma nucleus, thereby induced mutation and possible cancer.

The present invention can be summarised in other form or otherwise carry out and can not break away from its essence or essential characteristic.Therefore regard the disclosure of the specification as illustrative and nonrestrictive in all respects, scope of the present invention represented by appended claim, and the institute in identical purpose and scope changes and includes therein.

Claims

1. one kind is used for the mensuration method that smoking history is arranged recently if possible in human experimenter's body, may further comprise the steps: in described subject, measure the serum levels of advanced glycosylation end product (AGEs), with this measurement result with take from database standard and compare with non-smoking individuality that similar medical history is arranged in other respects.

2. one kind is used for estimating the individual method to advanced glycosylation end product (AGEs) contact possibility of smoking process, may further comprise the steps: measure this class AGEs in the products of combustion (or smog) of tobacco and volatilization thereof.

3. a method that is used to measure the organ sensation potential of tobacco may further comprise the steps: existence and the amount thereof of measuring advanced glycosylation end product (AGEs) in described tobacco.

4. one kind is used to measure the time limit of tobacco and the method for storage case, may further comprise the steps: existence and the amount thereof of measuring advanced glycosylation end product (AGEs) in tobacco.

5. a method that is used to measure the organ sensation potential of tobacco may further comprise the steps: existence and the amount thereof of measuring advanced glycosylation end product (AGEs) in the smog that is produced by described result of combustion of tobacco.

6. one kind is used to measure the time limit of tobacco and the method for storage case, may further comprise the steps: existence and the amount thereof of measuring advanced glycosylation end product (AGEs) in the smog that is produced by described result of combustion of tobacco.

7. method that is used to measure the commercial value of special tobacco crop, may further comprise the steps: the organ sensation potential of measuring tobacco is also measured its time limit and storage case, this step by in its sample, measure advanced glycosylation end product (AGEs) and existence its measure and carry out.

8. the method for claim 2-7, wherein said AGEs measures by following steps:

A. the sample of handling described tobacco is to form the extract of suspecting the saccharification toxin (glycotoxins) that contains described AGEs or corresponding precursor; With

B. analyze described extract to detect any saccharification toxin (glycotoxins) wherein contain or existence and the amount of AGEs.

9. the method for claim 8 is wherein handled described sample by burning therefrom to form smog, and described smog is used to check the existence and the amount of saccharification toxin AGEs and relevant (glycotoxins) that wherein contains.

10. one kind is used to detect the existence of advanced glycosylation end product (AGEs) in the individual body of smoking and the method for amount thereof, may further comprise the steps:

A. collection of biological sample from described individuality, described biological sample is selected from by serum, saliva, the group of urine and ight soil composition; With

B. described sample is used to check existence and the amount thereof of described AGEs.

11. one kind is used for generating enrichment and is used for and is present in tobacco and combustion by-products thereof, comprises the method for antibody of advanced glycosylation end product (AGEs) the sample partial reaction of the smog of tobacco, may further comprise the steps: with immunogene animal is carried out immunity inoculation, described immunogene is selected from tobacco; The smog of tobacco; Its extract; The carrier protein of deriving with above-mentioned any material; And haptens, the crosslinked bridge joint reaction of a kind of difunctionality of non-imposed use.

12. relevant advanced glycosylation end product with tobacco with claim 11 antibodies.

13. one kind is used for reducing the method that advanced glycosylation end product (AGEs) is accumulated in tobacco or the individual body of smoking of using, may further comprise the steps: come individuality is treated by the reagent that gives amount of suppression, described reagent can with glycation product reaction with the formation that stops described AGEs and/or harmful active.

14. one kind is used to suppress to use the method that the advanced glycosylation end product (AGEs) relevant with using tobacco or smoking accumulated in the individual body of this class tobacco, may further comprise the steps: use a kind of reagent of amount of suppression to come tobacco is handled, described reagent can with the glycation product reaction with the formation that stops them and/or harmful activity of described AGEs.

15. the method for claim 13 or 14, wherein said reagent and early stage glycation product reaction, described reagent is selected from the group that following material is formed: aminoguanidine, antagonist, analog, congener, homologue and analogies thereof and their potpourri.

16. the method for claim 13 or 14, wherein said reagent contain the compound that a kind of product that participates in general formula I or II forms

Wherein R is the residue that contains amino peptide, protein or other biomolecule, this residue produces by the reaction of the glycosyl-amino part of early stage glycation product (being also referred to as Amadori or Heyns product), described early stage glycation product forms with described biomolecular reaction by glucose (or other reaction sugar), and wherein said reagent contains a kind of reaction aldehyde radical.

17. the method for claim 13 or 14, wherein said reagent contain a kind of thiazolium compounds with following general structure: R wherein ¹And R ²Independently be selected from the group that following group is formed: hydrogen, hydroxyl (rudimentary) alkyl, acetate (rudimentary) alkyl, low alkyl group, low-grade alkenyl, or R ¹And R ²Together with ring carbon can be a kind of aromatic condensed ring, can be arbitrarily by one or more amino, and halogen or alkylidene dioxygen replace;

Z is hydrogen or amino;

Y is amino, a kind of group of following general formula

Wherein R is a low alkyl group, alkoxy, and hydroxyl, amino or aryl, described aryl can be arbitrarily by one or more low alkyl groups, lower alkoxy, halogen, dialkylamino, hydroxyl, nitro or alkylidene dioxygen replace;

A kind of group of following general formula

-CH ₂R’

Wherein R ' is a hydrogen, or low alkyl group, low-grade alkynyl, or aryl;

Or a kind of group of following general formula

X is a halogenide, toluene sulfonate, the ion of mesylate or sulfonate;

And composition thereof.

18. the method for claim 14 is wherein handled the described tobacco or the material of being fuming by contacting with reagent.

19. the method for claim 18, wherein said contact process occur in before the described tobacco or the substance combustion of being fuming.

After 20. the method for claim 18, wherein said contact process occur in described tobacco or the substance combustion process of being fuming neutralizes.

21. the method for claim 19, wherein said contact process takes place by directly described reagent is applied to described tobacco before the described tobacco of collection is fuming material.

22. the method for claim 21, the described tobacco that wherein said contact process will produce through burning are fuming the passage of smoke of material contain described reagent filter medium passage and take place.

23. the method for claim 22, wherein said filter medium comprises porosint, contains a certain amount of described reagent in this porosint equably, and described reagent tends to and contacts from the smog that wherein passes through.

24. one kind is used to measure and monitor as dosimeter and is present in or derives from the AGEs of tobacco and/or its accessory substance or saccharification toxin (glycotoxins) or the existence of saccharification intermediate product and the device of level of its precursor, described device comprises a kind of substrate or carrier phase, a certain amount of saccharification target molecule is in this immobilization combination, and be suitable for any sample that is present in the AGEs of containing under a cloud, saccharification toxin (glycotoxins) or other reaction saccharification intermediate product in AGEs, saccharification toxin (glycotoxins) or other reaction saccharification intermediate product reaction.

Suspect and to contain the AGEs that derives from smog or the atmosphere of the saccharification toxin (glycotoxins) before other AGE 25. the device of claim 24, wherein said sample material comprise.

26. the device of claim 24, wherein said substrate are to have a kind of solid that is suitable for the surface that contacts with described sample material, and described AGE target molecule is fixed on the described surface.

27. the device of claim 24, wherein said carrier comprises a kind of liquid mutually and wherein accommodates described AGE target molecule, can make described sample material by described liquid thus, make the AGEs of any existence to react with described AGE target molecule.

28. filter that is used for catching and remove the saccharification toxin (glycotoxins) of the AGEs that is present in the tobacco smoke that therefrom passes through or other AGE precursor, comprise a kind of porosint that wherein contains a certain amount of reagent equably and be used for contacting with described smog, wherein said reagent can stop harmful activity of AGE formation and/or described AGEs with the glycation product reaction.

29. one kind is used for filtration of tobacco smoke suppressing advanced glycosylation end product and form and reduce its amount and active system, described system comprise be connected with the generator (source) of described tobacco smoke, according to a kind of filter of claim 28.

30. the system of claim 29, wherein said aerosol producer is a kind of cigarette, and described system comprises a kind of described generator and upright therein a kind of filter of arranging of being permanently connected.

31. the system of claim 29, wherein said tobacco smoke tobacco smoke generator is a kind of pipe, and described system comprises a kind of filter that is placed in liquid holdup place, and dried (stem) of described reservoir (bowl) is placed on the cigarette holder mouth and is equipped with between the reservoir of described tobacco.