006-1Kinetic study of scrap tyre pyrolysis and combustion

Applied Catalysis A:General197(2000)165–173Kinetic of liquid-phase reactions catalyzed by acidic resins:the formation of peracetic acid for vegetable oil epoxidationR.L.Musante1,R.J.Grau2,M.A.Ba1tanás∗Instituto de Desarrollo Tecnológico para la Industria Qu´ımica(INTEC)Güemes3450,3000Santa Fe,ArgentinaAbstractA heterogeneous kinetic model which takes into account the complete physicochemical interaction of reactive species in a polar liquid phase with an ion-exchange resin acting both as selective sorbent and heterogeneous catalyst has been employed to analyze the peracetic acid synthesis from acetic acid and hydrogen peroxide in an aqueous solution.Model parameters were estimated using uncoupled data from phase equilibria,polymer sorption,chemical equilibrium and reaction kinetics.Activities rather than molar concentrations in the polymer phase and specific(dry weight of catalyst basis)rather than volume-based expressions were found to give the best constitutive equations for the heterogeneous reaction rate.©2000Elsevier Science B.V.All rights reserved.Keywords:Vegetable oil epoxidation;Heterogeneous models;Acidic resins1.IntroductionThe epoxidation of unsaturated triglycerides with percarboxylic acids is a common practice for obtain-ing low cost plasticisers of good performance from natural and renewable sources such as vegetable oils. Cost constrictions dictate the use of inexpensive per-acetic acid(PAA)as the active reagent,while safety concerns demand an in situ preparation of the reagent, to avoid the handling of a pre-formed concentrated peracid.The in situ process involves a heterogeneous system,as epoxidation reaction occurs in the organic phase whereas the formation of PAA takes place ∗Corresponding author.Tel.:+54-342-4559175;fax:+54-342-4550944.E-mail address:tderliq@.ar(M.A.Ba1tan´a s)1Research Assistant of U.N.L.2Professor at U.N.L.and member of CONICET research staff.in an aqueous medium.The latter step is slow and controls the overall reaction rate;then,high conver-sions are generally achieved after several hours of reaction.Traditionally,homogeneous acidic catalysts(e.g. sulfuric acid)have been used to facilitate the forma-tion of peracetic acid by reacting acetic acid(AA) and hydrogen peroxide(HP):CH3COOH(AA)+H2O2(HP)H+←−−→CH3COOOH(PAA)+H2O(W) Hydrogen peroxide(an oxygen source)reacts with acetic acid(an oxygen carrier)in the aqueous phase in the presence of the acidic catalyst(solvated pro-tons)to give peracetic acid(PAA).The latter,in turn, transfers to the organic phase and quickly attacks the double bonds of the unsaturated vegetable oil(VO)to form the oxirane ring in the homogeneous epoxidation0926-860X/00/$–see front matter©2000Elsevier Science B.V.All rights reserved. PII:S0926-860X(99)00547-5166R.L.Musante et al./Applied Catalysis A:General197(2000)165–173 reaction:R1CH=CHR2 (VO)+CH3COOOH (PAA)→R1CHOCHR2(EVO)+CH3COOH (AA)The co-produced AA returns to the aqueous phase to close the sequence and re-start the production cycle. Mass and heat transfer limitations may impose severe process constraints(epoxidations are highly exothermic)or may lead to undesirable side reac-tions.So,numerous workers have studied the epoxi-dation reaction pathway and a qualitative picture of it was agreed upon in the past decades[1–3].The rate-limiting step of the in situ process is the forma-tion of PAA in the aqueous phase.However,quan-titative determination of the kinetic parameters was lacking,and only for two-phase epoxidations using homogeneous mineral acids more rigorous kinetic models had begun to emerge in the recent past[4,5]. As mentioned,secondary(acid-catalyzed)side re-actions do appear.Invariably,they involve an opening of the oxirane ring and,consequently,lower yields of epoxidized vegetable oil[5]:R1CHOCHR2+CH3COOHH+→R1CH(OH)CH(OOCH3)R2R1CHOCHR2+CH3COOOHH+→R1CH(OH)CH(OOOCCH3)R2R1CHOCHR2H +→R1COCH2R2R1CHOCHR2+H2O H+→R1CH(OH)CH(OH)R2R1CHOCHR2+H2O2H+→R1CH(OH)CH(OOH)R2 Heterogeneous catalysts such as functionalized micro-reticular ion-exchange resins(IER)can be advanta-geously used instead,since only the small carboxylic acid molecules can enter into their gel-like structure, while the bulky epoxidized triglyceride molecules cannot.The oxirane ring can thence be protected from the attack of the protons which are confined inside the gel matrix and,as a result,further ring decomposition is prevented.Also,catalyst recovery and/or regeneration is much easier then.Several such strongly acidic sulfonic IER(e.g.Dowex50;Am-berlite IR120,Chempro C20)have been reported to contribute to minimizing oxirane ring opening[6–9]. Three phases are present in the in situ processes cat-alyzed by IER:(1)a polymer phase,whose behavior (notably its swelling properties)is highly dependent on the physicochemical properties of the system,to-gether with(2)an aqueous phase,immiscible with(3) the organic phase.A heterogeneous kinetic model of this three-phase system using IER has not been pre-sented so far.We are presently developing a complete study of the epoxidation process,uncoupling the reac-tion and transport system into sub-systems of increas-ing complexity to allow a better quantification of the relevant process parameters.This piece of work presents the modeling of one of these sub-systems whose understanding is crucial: the kinetics of formation of PAA in the heteroge-neous aqueous phase-polymer(acidic catalyst)phase system.For this,the partition of each component between the two phases,as well as the swelling ra-tio(relative increase of the volume of the resin)are first quantified.Selective sorption/swelling leads to values of the relative compositions in the reaction locus(i.e.the polymer resin)and of the reactants’polymer-phase concentration which are different from those in the liquid phase.Both aspects are taken into account in the modeling.Then,the chemical equilibrium constant and kinetic parameters are esti-mated from the equilibrium and kinetics experimental data,respectively,using operating conditions typical of the industrial stly,the activation en-ergy of the acid-catalyzed reaction is estimated from non-isothermal experimental data.The appropriate-ness of managing the kinetic equations in terms of activities,rather than concentrations,and mass of dry resin,rather than its volume,are discussed.2.Experimental2.1.Activation and conditioning of the ion-exchange resinRohm&Haas Amberlite IR-120microreticular gel-type resin,DVB-styrene matrix,8%cross-linking, d p(dry)=215–775␮m(54.5%<530␮m;75.3%<R.L.Musante et al./Applied Catalysis A:General197(2000)165–173167600␮m;94.8%<670␮m),functionalized withsulfonic groups,was used throughout this work.AsIER is commercially available in sodium form,hy-drochloric acid(10%w/w)was used to fully activateit in successive ion-exchange steps(7in total),withfurther washing(1:10w/w)using distilled demineral-ized water until complete elimination of the residualstly,glacial acetic acid(purity:>99.7%w/w)was used to replace water inside the ion ex-change resin.The exchange capacity of the resin,as determinedby titration using conventional volumetric tech-niques,was:[H+]o=4.507meq/g(dry basis).The dry polymer density,as measured by pycnometry usingn-heptane[10],was1.437kg/m3.A portion of the dryresin was crushed and sieved;successive washing anddecantation in distilled demineralized water allowedthe removal of undesiredfines adhered to the crushedparticles.Two fractions,<53and>595␮m,wereused to evaluate the possible impact of mass-transferresistances on the process rate.2.2.Determination of phase equilibrium partitioningof reactants between the aqueous and polymericphasesDifferent dilutions of either acetic acid or hydro-gen peroxide in water(40g)were added to the dryresin(15g).The system was kept at333K with occa-sional stirring until physicochemical equilibrium wasachieved in about4h.The equilibrium compositionof the liquid phase was measured by volumetric tech-niques(see below).A portion of the swollen resin wascentrifuged at2000g for5min to eliminate any resid-ual interstitial liquid[10].The amount of sorbed liq-uid retained by the resin was found by weighting thecentrifuged resin before and after drying in a stove at378K for12h[11].2.3.Reactor and experimental proceduresThe rate of formation of PAA was studied in exper-imental runs conducted in a1000cm3closed cylin-drical Pyrex reactor,furnished with a variable speed(0–1000rpm)mechanical stirrer.The device had fourteflon baffles to eliminate vorticity,an overhead re-flux condenser and a fast-sampling device for collect-ing representative liquid-resin samples of the reacting system.An internal cooling coil(OD:1/4 ;316SS), combined with an external cylindrical heating mantle (400W)linked to a PID temperature controller,en-sured operating within±0.1K.In a typical experiment,glacial AA and pre-activated IER were added to the empty reactor and slowly heated to the reaction temperature under stirring.Si-multaneously,the desired volume of a solution of HP (30%w/w)was also heated to that temperature,under reflux,and then added,at once,to the reactor(zero time of the reaction).Samples were periodically taken and immediatelyfiltered to separate the resin and stop the reaction.Next,aliquots of thefiltrates(1cm3) were diluted in ethanol(100cm3)and refrigerated for further analysis by GLC.2.4.Analytical techniquesThe concentration of AA and HP were measured by volumetric titration,using NaOH(0.l N)and KMnO4 (0.1N),respectively.The contents of PAA acid in the reaction samples was determined by GLC after a previous derivatization with methyl p-tolyl sulphide; n-octadecane was used as internal standard[12].A glass column(500×2mm)packed with3%FFAP on Chromosorb W AW DMCS(80–100mesh)was em-ployed.3.Results and discussion3.1.Intra-and extra-particle mass-transfer resistancesThe absence of intra-particle mass-transfer resis-tances to any significant extent was experimentally corroborated using two widely differing particle sizes of the resin:>595and<53␮m,under typical reaction conditions of the well-stirred mixture(375rpm).Both runs gave identical results as is shown in Fig.1.Also, the ratio of initial rates was close to unity even though particle diameters differed by more than10-fold. Hence,extra-particle mass-transfer resistances are of no concern either and,thus,the commercial un-crushed lER beads were used to perform the kinetic study.168R.L.Musante et al./Applied Catalysis A:General 197(2000)165–173Fig.1.Influence of particle size of the ion-exchange resin (Amberlite IR-120)on the reaction rate and final concentration of PAA at 333K (reactants molar ratio:H 2O 2/HOAc =1.1/0.5).The full line corresponds to model predictions.3.2.Kinetic modelAs we have pointed out in Section 1,the selec-tive sorption and swelling of the resin can have a strong influence on the observable reaction rate of this two-phase system,even though no change in the se-quence of involved reaction steps (as compared to the homogeneous reaction)is to be expected for a strongly acidic material being the active catalyst.Then,a pseu-dohomogeneous kinetic model such as the following:dd t C PAA =k 1C IER C HP C AA −1K C PAA C W(1)which considers IER merely as a source of protons in the multicomponent liquid mixture would be certainly insufficient.In fact,for the set of experimental runs summarized in Table 2,every attempt we made to get satisfactory data fittings by means of a non-linear re-gression algorithm was unacceptable.So,a new model was developed to include explicitly the presence of the polymer phase.Two approaches can be followed to solve this hur-dle.The first one focuses exclusively on the catalytic reaction pathway at the reaction locus,where two ex-treme situations are recognizable according to whether slightly dissociated sulphonic groups or free solvated protons are the catalytic agents [13–15].The first case applies whenever the resin is imbedded into a slightly polar medium;the classic LHHW formalism can then be applied to model the reaction kinetics [16,17].Here,the inclusion of adsorption parameters is both justifi-able and flexible enough so as to fit up any observ-able changes in catalytic activity due to the presenceof polar components in minor amounts.If the resin operates into a strongly polar solvent such as water,the sulphonic groups are fully dissociated and the free solvated protons catalyze the reaction through ionic mechanisms of protonation.In many such situations,specially if just small amounts of resin are used,a pseudohomogenous model describing the reaction rate in terms of power-law kinetics suffices,as it does in homogeneous catalysis.However,none of these models takes into account the selective sorption and swelling of the resin,which leads to values of the reactants’polymer-phase con-centration and of the relative composition of both reactants and products in the polymer phase (i.e.the reaction locus)that are different from those in the liquid phase.A second approach is then needed,to ex-plicitly differentiate the compositions of the aqueous and polymer phases whenever a significant amount of resin is used as a catalyst.For such purposes,the overall system can be considered as a two-phase sys-tem composed of a highly viscous multicomponent fluid phase containing N +l species (with the swollen polymer as (N +1)th species,)in physicochemical equilibrium with the N -component liquid phase,since the characteristic time for reaching phase-equilibrium conditions between them is usually of the order of a few minutes [18].3.3.Activities of the species in the liquid phase A predictive model is needed to compute the ac-tivity of the four species in the aqueous phase,rather than a correlative one,due to the scant informationR.L.Musante et al./Applied Catalysis A:General197(2000)165–173169 available in the open literature about this reactingsystem.For these components,just a pair of binarydata is available:water–hydrogen peroxide[19]andwater–acetic acid[20].Then,the UNIFAC group con-tribution method[21]is a suitable tool,as it does notinvolve any adjustable parameter.The central con-cept of the method rests on considering any mixtureas a solution of functional groups interacting amongthemselves.Most of the extensions and refinementof the original concept have rested onfinding newprocedures for calculating the molecular interactionparameters between components,which initially camefrom vapor–liquid equilibrium data(UNIFAC VLE).Later,a new set of interaction parameters has beenderived from the liquid–liquid equilibrium data(UNI-FAC LLE)for making predictions related to thesesystems[22].More recently,the dependence of theinteraction parameters with temperature has also beenincluded,in the Modified UNIFAC method[23].We used each of these methods to predict the activ-ities in the aqueous phase.The UNIFAC LLE methodwas found to give the closestfit when a comparison ofmodel predictions with experimental data taken fromthe available vapor–liquid equilibria was made.It wasthen adopted for further use in our calculations.3.4.Activities of the species in the polymer phaseThe activity a P i of the i th species of a multicompo-nent polymeric solution can be evaluated in the frame-work of the extended Flory–Huggins model[25]:ln a P i=1+ln v i−N+1j=1m ij v j+N+1j=1χij v j−N+1j=1j−1k=1m ik v j v kχkj+ηV i53v1/3P−76v P(2)where N is the number of components excluding thepolymer,which is the(N+l)th species;νandνP are thevolume fractions of the i th species and of the polymerin the polymer phase,respectively;m ij is the ratio ofmolar volumes of the i th and j th species(m iP=0); V i is the molar volume of i th species;ηrepresentsthe number of moles of active elastic chains per unitvolume andχij represents the molecular interaction between components i and j.The latter parameters are known to be temperature-dependent[24]. According to Eq.(2),11adjustable parameters would have to be determined for calculating the set of a P i of the four species in the polymer phase:η,and 10molecular interaction parameters,four of them corresponding to interactions between the polymer and the other species(χiP)and six binary interaction parameters among the liquid phase components(χij), sinceχij=χji andχii=0(see Ref.[18]for a full discussion on the subject).The elasticity parameter andfive of the binary interaction parameters were found independently (i.e.uncoupled),from the sorption equilibria of the water–acetic acid and water–hydrogen peroxide bi-nary mixtures in contact with the resin.For this purpose,the activities of each pair of components in the aqueous phase were calculated using the UNIFAC LLE routine,which does not involve any adjustable parameter.Next,as in thermodynamic equilibriuma L i=a P i(3) Eq.(2)was used for each binary mixture,using the Levemberg–Marquardt algorithm,to estimate the cor-responding interaction parameters.Fig.2shows representative sets of tie lines,in adsorbent-free mass coordinates(N=g dry resin/g ad-sorbate),obtained experimentally at333K.These data clearly indicate that water is more strongly sorbed than either acetic acid or hydrogen peroxide,and that the resin swelling is much higher in water than in acetic acid.It is also apparent that the polymer beads can expand even more(albeit slightly)with higher concentration of hydrogen peroxide.Fig.3re-plots the experimental data(tie lines) shown in Fig.2,as molar fractions in the polymer and liquid phases,for the water–acetic acid and water–hydrogen peroxide binary pairs,together with model predictions using the calculated parameters (the latter are included in Table1).The agreement is satisfactory.The experimental determination of the partition of PAA was not made owing to the obvious experi-mental difficulties and hazards involved in handling a highly concentrated peracid.Instead,the heuristic assumption was made that in the polymer phase the analogs—carboxylic acids(acetic and peracetic)—can be assumed to behave as identical molecules,as170R.L.Musante et al./Applied Catalysis A:General 197(2000)165–173Fig.2.Binary sorption and phase partition equilibria on Amberlite IR-120of (a)water–acetic acid and (b)water–hydrogen peroxide binary mixtures,obtained experimentally at 333K,in adsorbent-free mass coordinates (N =g dry resin/gadsorbate).parison of experimental data and model predictions for the water–acetic acid (a)and water–hydrogen (b)binary pairs shown in Fig.2(333K).far as their molecular interactions are concerned,and so χAA −PAA =0.Likewise,an uncoupled estimation of the interaction parameters of the reactive binary pairs,PAA–water and acetic acid–hydrogen peroxide cannot be made,for obvious reasons.Nevertheless,under the previous hypothesis χW −PAA is identical to χW −AA .Lastly,the remaining interaction parameter can be estimated from chemical equilibrium data us-Table 1Estimated values of the interaction parameters of the extended Flory–Huggins model [Eq.(2)],at 333K.χij j i AA HP Water PAA Resin AA 0−0.00880.339000.1592HP −0.00360−1.9670−0.00360.8809Water 0.1039−1.488600.1039−0.6673PAA−0.00880.33900.1592ing asymptotic compositions (i.e.after long enough contact times)of the experimental runs.The proce-dure is described below,in Section 3.5.As is shown in Table 1,the complete set of χij inter-action parameters indicates relatively low interaction between acetic acid,PAA and hydrogen peroxide,and moderate interactions of these with water and of the four liquid components with resin.The value obtained for the elasticity parameter was:η=0.022mol/cm 3,which is about one order of magnitude higher than the theoretical one.However,by imposing progres-sively lower values to this parameter there is worse agreement between model predictions and experimen-tal data results,owing to the tight correlation among the parameters in Eq.(2).This problem has been dis-cussed in a recent work on the kinetics of liquid-phase esterification of acetic acid with ethanol using Am-berlyst 15,where about two orders of magnitude dif-ferences were encountered [18].Despite these shortcomings of presently available phase partition equilibrium models,they allow one to reproduce rather satisfactorily the experimental data and seem sufficient to help in describing the kinetic behavior of these systems under reaction conditions.3.5.Chemical equilibriumBy solving the set of multicomponent sorption equi-librium equations,Eq.(3),together with the mass bal-ances of each of the i th species:n L i +n P i =n 0i +νi ξ(4)R.L.Musante et al./Applied Catalysis A:General 197(2000)165–173171Table 2Initial loading and operating conditions of the batch experimental runs Run Temp (K)AA (moles)HP (moles)Water (moles)Resin mass (g)13331.329 2.73411.4707.44723331.345 3.48015.2058.66733332.783 2.73411.945.22643335.497 2.73411.9474.820533310.945 2.73411.9475.61663330.768 2.73411.94711.99373330.574 2.73411.94721.37583231.329 2.73411.9477.44793431.3292.73411.9477.447and the condition of chemical equilibrium:K =(a P PAAa P W /a P AA a PHP )eq (5)it is possible to jointly estimate the interaction param-eters for the reactive couple acetic acid–hydrogen per-oxide and the equilibrium constant,K .Indeed,owing to a lack of reliable data,it was impossible to estimate K from G 0data,as the standard free energy of the formation of PAA is known within broad uncertainty limits.The calculated value of χHP −AA is given in Table 1.At 333K the equilibrium constant was found to be K =2.18.Values ranging from 0.7to 5,which were found to be dependent both on the initial molar ratio of reac-tants and on the catalyst concentration,calculated from the equilibrium concentrations,have been reported by Rangarajan et al.[5].With our approach,for the broad set of experimental conditions given in Table 2,a good fit of the data could be achieved using a single value of K ,as is shown in Fig.4.The values of K at 323and 343K,obtained exper-imentally,were 1.911and 2.778,respectively.Fig.4.Experimental and calculated concentrations of PAA in liquid phase at equilibrium conditions (333K).3.6.Kinetic equationFrom Eq.(4)it is straightforward to recognize thatin the well-mixed isothermal batch reactor the time rate of change of the observable degree of advance-ment of the reaction (ξ)is sufficient to fully describe the reaction process whenever equilibrium conditions between the bulk liquid and the polymer phase hold.Also,because the catalyzed reaction proceeds only in-side the polymer phase:dd tξ=W P R P (6)where W P indicates mass of dry resin placed in the system (an invariable property)and R P is the specific reaction rate (dry weight basis).Another choice,which is to write Eq.(6)in terms of a volumetric reaction rate,leads to awkward rate ‘constants’,as the volume of the polymer phase continuously changes during the reaction owing to the swelling properties of the resin.In addition,as the system is highly non-ideal,the kinetic equation describing the reaction rate has to be written in terms of activities [26],accounting for the172R.L.Musante et al./Applied Catalysis A:General 197(2000)165–173Fig.5.Concentration of PAA in the liquid phase as a function of time for various values of the initial composition of the reacting mixture:(a)Run 7;(b)Run 6;(c)Run 3.Full lines represent model predictions.chemical equilibrium as well (Eq.(5)).The simplest expression which satisfies these requirements is the following:R P =ka P AA a P HP [1−K −1a P PAA a P W /a P AA a PHP ](7)where k [mol s −1(g dry resin)−1]=k 0[H +]0.Its value was estimated by means of the Marquardt–Levemberg algorithm,solving for Eqs.(2),(3),(5)-(7)and using the experimental data from the runs indicated in Table 2,all of them obtained in the absence of mass-transfer limitations.The previous calculation of the interaction parameters and of the chemical equilibrium constants at each temperature allowed the uncoupled estimation of k .The values of the interaction parameters at 323and 343K were obtained from those at 333K using the well-tested derivation of Flory [24]:χ(T )T =χ(T )T .The estimated values of the pre-exponential factor and activation energy of k are (8.48±l.l6)×10mol s −1(g dry resin)−1and 48.4±0.47kJ mol −1,respectively,for a 95%confidence level.Given that the absence of mass transfer constraints was corroborated,this some-what low E act value,as compared with the onereportedFig.6.Concentration of PAA in the liquid phase as a function of time for various values of the reaction temperature:(a)Run 8;(b)Run 1;(c)Run 9.Full lines represent model predictions.in homogeneous systems,might be due to the steric constraints imposed on the acid-catalyzed bimolecu-lar rds of the reaction [3,4]by the microreticular resin rather than an incomplete degree of solvation [27].Figs.5and 6compare experimental results and model predictions which are fair.Additional fittings were made using empirical kinetic expressions for:(a)the specific reaction rate (dry weight basis)in terms of molar concentrations instead of activities of the re-actants,and (b)the volumetric reaction rate in terms of activities of the components,factorized by the resin volume.In both such cases the new predictions were poorer than the one using the more sound Eq.(7),as the residual sum of squares were 58and 238%higher,respectively.4.ConclusionsA two-phase model has been proposed to describe the catalyzed reaction of the formation of PAA from acetic acid and hydrogen peroxide under a broadR.L.Musante et al./Applied Catalysis A:General197(2000)165–173173range of conditions,using a sulphonated ion-exchange resin acting as both a sorbent and a heterogeneous catalyst.The developed model incorporates relevant aspects with regard to the different affinities of the reactive species toward the liquid and resin phases. The selective partitioning of each component be-tween the two phases and the relative increase of the volume of the resin(i.e.its swelling ratio)with vary-ing composition were corroborated,quantified and taken into account in kinetic modelling.These fea-tures,which have adequate literature support,had not been previously considered for this particular reactive system.A progressive,uncoupled estimation of the model parameters was made using:(a)phase equilib-rium/sorption data of unreactive pairs of components, to obtain the binary interaction parameters;(b)chem-ical equilibrium data,to estimate the thermodynamic equilibrium constant and binary interaction parame-ters of the reactive pairs,and(c)reaction rate data in absence of mass-transfer resistances to estimate the specific kinetic rate constant.Activities rather than molar concentrations and specific(dry weight of cat-alyst based)rather than volume-based reaction rates were used throughout the work.For processing purposes the preferential partition of water inside the catalytic polymer phase(the reaction locus)is inconvenient because,being both a diluent and a reaction product,water lowers the rate of for-mation of PAA from the reversible reaction which is involved.Yet,the use of microreticular ion-exchange resins as heterogeneous catalysts for epoxidizing un-saturated triglycerides is desirable.Protons are then confined inside the polymer phase,which prevents their further attack on the oxirane ring and,so,higher oxirane indexes than those achievable in homogeneous catalytic processes can be realised. 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Detoxification ofandA,two abundantmycotoxins,by lactic acid bacteriaS.Fuchsa ,G.Sontag a ,R.Stidl a ,V.Ehrlichb,*,M.Kundi c ,S.Knasmu ¨llerbaDepartment of Analytical and Food Chemistry,Faculty of Chemistry,University of Vienna,Vienna,AustriabInstitute of Cancer Research,Department of Medicine I,Medical University of Vienna,Vienna,Austria cInstitute of Environmental Center for Public Health,Medical University of Vienna,Vienna,AustriaReceived 9July 2007;accepted 10October 2007AbstractAim of the present study was to investigate the detoxification of two abundant mycotoxins,namely ochratoxin A (OTA)and patulin (PAT)which are frequently found in human foods,by lactic acid bacteria.The removal of the two mycotoxins from liquid medium by thirty different LAB strains was analyzed in a screening trial by the use of HPLC coupled with UV-or fluorescence detection.Two highly effective strains were identified;Lactobacillus acidophilus VM 20caused a decrease of OTA by P 95%and Bifidobacterium animalis VM 12reduced PAT levels by 80%.Subsequently experiments showed that the binding of these compounds depends on different parameters,i.e.the concentration of toxins,the cell density,the pH-value and on the viability of the bacteria.To proof that the decrease of the toxins LAB from liquid medium results in a reduction of their toxic properties,micronucleus (MCN)assays were conducted with a human hepatoma cell line (HepG2).Indeed,a substantial decrease (39–59%)of OTA and PAT induced MCN formation was observed the most effective strains detected in the chemical analyses.Furthermore,also the inhibition of the cell division rates by the toxins was significantly reduced.These findings indicate that certain LAB strains are able to detoxify the two toxins and may be useful to pro-tect humans and/or animals against the adverse health effects of these compounds.Ó2007Elsevier Ltd.All rights reserved.Keywords:Patulin;Ochratoxin A;Detoxification;Lactic acid bacteria;Micronuclei;HepG21.IntroductionLactic acid bacteria (LAB)are widely used for the pro-duction of fermented foods and are also part of the intesti-nal microflora.Numerous investigations indicate that LAB have beneficial health effects in humans (Ouwehand et al.,2002;Saxelin et al.,2005).One of the effects identified is the protection against toxins contained in foods such as heterocyclic aromatic amines,polycyclic aromatic hydrocarbons,mycotoxins and reactive oxygen species (Knasmu ¨ller et al.,2001;Stidl et al.,2007a ).The Food and Agriculture Organization esti-mated that about 25%of human foods and animal feed are contaminated with mycotoxins and strong efforts have been made to decontaminate them by the use of physical and chemical adsorbents but the success made so far is limited (Huwig et al.,2001;Shetty and Jespersen,2006).It has been shown earlier by El-Nezami et al.(2000)that LAB detoxify aflatoxin B 1(AFB 1),which is the most potent known human carcinogen and contributes to the high prevalence of liver cancer in regions such as Central Africa and China (IARC,2002).Also with other fungal toxins such as zearalenone,trichothecenes and fumonisins,binding effects have been observed in chemical analytical0278-6915/$-see front matter Ó2007Elsevier Ltd.All rights reserved.doi:10.1016/j.fct.2007.10.008Abbreviations:AFB 1,aflatoxin B 1;BNC,binucleated cells;cfu,colony forming units;DMEM,Dulbecco’s minimal essential medium;DMSO,dimethylsulfoxide;FCS,foetal calf serum;FLD,fluorescence detection;HPLC,high performance liquid chromatography;LAB,lactic acid bact-eria;MCN,micronuclei;MRS,de Man-Rogosa-Sharpe;OTA,ochratoxin A;PAT,patulin;PBS,phosphate buffered saline;UV–VIS,ultraviolet–visible.*Corresponding author.Tel.:+431427765147;fax:+43142779651.E-mail address:veronika.ehrlich@meduniwien.ac.at (V.Ehrlich)/locate/foodchemtoxAvailable online at Food and Chemical Toxicology 46(2008)1398–1407investigations(Kabak et al.,2006;El-Nezami et al.,2002b, 2002a;Shetty and Jespersen,2006;Niderkorn et al.,2006).In the present study,the removal of patulin(PAT)from liquid medium by a variety of LAB strains was investigated for thefirst time.PAT is found sometimes in higher concen-trations as other mycotoxins in foods as it is produced by a large number of fungi within several genera such as Bys-sochlamys,Eupenicillium,Penicillium,Aspergillus and Paeci-lomyces(Speijers,2004)and is predominantly found in vegetables and fruits(in particular in apples and apple derived products)(Thurm et al.,1979;Pohland and Allen, 1970;Moureau,2002;Paster et al.,1995).Its acute toxic effects in mammals are primarily due to impairment of kid-ney functions(Speijers et al.,1988).It is also well docu-mented that PAT causes oxidative damage(Liu et al., 2007),has a negative impact on reproduction in males via interaction with hormone production(Selmanoglu and Koc-kaya,2004)and affects the immune system(Llewellyn et al., 1998;Sharma,1992).PAT caused potent genotoxic effects in experiments with mammalian cells(Alves et al.,2000; Schumacher et al.,2005;Schumacher et al.,2006)but is devoid of activity in bacterial mutagenicity assays(Wu¨rgler et al.,1991;Wouters,1996;Ueno et al.,1978).Since DNA-damage is causally related to cancer induction and a number of other diseases(Fenech,2002b;2002a),it was suspected that PAT may possess carcinogenic properties,but in older studies(which do not meet the current standards)negative results were obtained and newer investigations are not avail-able at present(Speijers,2004).In addition,also experiments with ochratoxin A(OTA), which is produced by several species of Aspergillus and Pen-icillium(Aish et al.,2004),were included in the present study. This compound is present in human foods like beer,cereals, wine,cocoa,dried vine fruits,coffee and spices(JECFA, 2001;Birzele et al.,2000;Fazekas et al.,2002).OTA causes severe nephrotoxic effects in animals and humans and its genotoxic and carcinogenic effects are well documented (Aish et al.,2004).It has been shown in a few earlier investi-gations that LAB cause removal of OTA from liquid med-ium(Piotrowska and Zakowska,2000;Piotrowska and Zakowska,2005;Turbic et al.,2002;Skrinjar et al.,1996).In the present investigation,the protective properties of LAB towards PAT and OTA were studied in co-incubation experiments with chemical analytical methods.In total,30 different LAB strains were included in a screening trial. Subsequently,the dose dependency,as well as the impact of the viability of the bacteria on the binding of the toxins was tested with selected strains.Since chemical analytical studies do not provide afirm proof for the detoxification of mycotoxins,as active metab-olites may be formed,additional experiments were carried out,in which the influence of the incubation of bacteria with toxins was studied in human derived liver cells(HepG2).We investigated if the treatment of the toxins with the bacteria has an effect on the inhibition of cell division and on the formation of micronuclei(MCN)which are formed as a consequence of chromosome breakage(clastogenicity)and aneuploidy(Fenech,2000).The cell line we used has retained the activities of a broad variety of drug metabolising enzymes which are normally lost during cultivation and it was shown earlier that it detects the effects of genotoxic car-cinogens,which give negative results in conventional in vitro test systems(Knasmu¨ller et al.,1998,2004b).Also mycotox-ins such as citrinin,fumonisin B1and OTA,which can not be detected in conventional in vitro assays,caused positive results in this experimental model(Ehrlich et al.,2002a, 2002b;Knasmu¨ller et al.,2004a).2.Materials and methods2.1.Chemicals and mediaDe Man-Rogosa-Sharpe(MRS)broth,was obtained from Oxoid (Bassingstoke,England),Elliker Bouillon was purchased from Difco (Sparks,USA).Patulin(4-hydroxyl-4H-furo(3,2c)-pyran-2(6H)-on,CAS No.149-29-1,>98%)and ochratoxin A((-)-N-[(5-chloro-8-hydroxy-3-methyl-1-oxo-7-isochromanyl)carbonyl]-3-phenyl-alanine,CAS No.303-47-9,>98%)as well as cytochalasin B and L-cysteine,which were used for the cultivation of Bifidobacteria,were purchased from Sigma–Aldrich (Vienna,Austria).Dulbecco’s minimal essential medium(DMEM),anti-biotics and trypsin were obtained from PAA Laboratoires GmbH(Linz, Austria).Foetal calf serum(FCS)was obtained from Gibco(Paisley, Scotland).Inorganic salts(P99%)and DMSO(P99.9%)came from Merck(Darmstadt,Germany).Acetonitrile and methanol which were used for the HPLC analyses were purchased from Fluka(Hannover, Germany).Ammonia solution(33%)was obtained from Riedel-de Haen (Seelze,Germany).Stock solutions of PAT and OTA for HPLC analyses were prepared in MeOH(0.1mg/ml),for MCN-assays the two toxins were dissolved in sterilefiltered DMSO(1.0mg/ml).Sodium phosphate buffer (50mM,pH5.0)was prepared by mixing a50mM Na2HPO4-solution with a50mM NaH2PO4-solution.2.2.Storage and cultivation of LAB strainsLAB strains were either obtained from Lactosan(Kapfenberg,Aus-tria)or provided by the Institute of Milk Hygiene,Milk Technology and Food Science of the University of Veterinary Medicine(Vienna,Austria). The strains were coded(LA–Lactosan;VM–Veterinary Medicine)and stored deep frozen atÀ80°C.For preparation of overnight cultures, Lactobacilli were grown in MRS(pH6.2,37°C),Bifidobacteria were cultivated in MRS(pH6.2,37°C)with addition of0.03%of L-cysteine and Streptococci were grown in Elliker Bouillon(pH6.8,37°C).The Bifidobacteria were cultivated under anaerobic conditions,i.e.in anaerobic jars obtained from Oxoid(Basingstoke,England)with Anaerocult A (Merck,Darmstadt,Germany).The colony forming units(cfu)in the overnight cultures were determined by turbidimetry at600nm(Niderkorn et al.,2006)with a spectrophotometer(DU640,Beckman Industries Inc, Fullerton,CA,USA).2.3.Storage and cultivation of HepG2cellsThe cells were kindly provided by F.Darroudi(University of Leiden, The Netherlands),stored in deep frozen portions atÀ20°C and cultivated in DMEM,supplemented with15%FCS and addition of1.0%penicillin/ streptomycin in250cm2cultureflasks(Kremsmu¨nster,Austria).2.4.Preparation of LAB-strains for HPLC-analysis andMCN-assaysBacteria cultivated in overnight cultures were harvested by centrifu-gation(4000rpm,4°C,10min).The supernatants were discarded and the pellets resuspended in10mlThe washing step wasS.Fuchs et al./Food and Chemical Toxicology46(2008)1398–14071399repeated three times.Subsequently,the pellets were resuspended in physiological saline to obtain thefinal concentrations.The adsorption assays were performed in Eppendorf(1.5ml,safe lock)vials.For HPLC-analyses,50l l of stock solutions of the mycotoxins,250l l of the bacterial suspensions(final concentration109cfu/ml)and700l l sodium phosphate buffer(50mM;pH5.0)were used.To obtain heat inactivated cells,bac-teria were autoclaved for20min at121°C.The vials were vortexed (Vortex Genie2,Scientific Industries,Bohemia,NY,USA)shortly(3s) and incubated(37°C,4h)on a horizontal shaker table(IKA,Staufen, Germany)with soft agitation(500rpm).Subsequently,the incubation was terminated by centrifugation(10000rpm,4°C,10min)and700l l of the supernatants were transferred to Eppendorf vials and stored at4°C.In a set of preliminary experiments it was excluded that the preincubation of bacteria with the liquid medium had any effect on the outcome of the chemical analyses.For MCN-assays,the initial incubation mixtures consisted of50l l of the mycotoxin solution,500l l bacterial suspension(final concentration of 5·109cfu/ml)and450l l sodium phosphate buffer(50mM;pH5.0).For PAT,the concentrations were25,50and100l g/ml.In the case of OTA, concentrations in the range between100and300l g/ml were used.After the incubation with the bacteria,the mixtures were centrifuged,and ali-quots of the supernatants(50l l in the case of PAT-mixtures and250l l of OTA-mixtures)added to HepG2-cultures grown in DMEM(final volume 5ml).2.5.Detection of patulin by HPLC–UVFive hundred microliter of the samples(supernatants,control samples or negative samples-which contained only bacteria and no toxin)were diluted with500l l mobile phase.The Merck Hitachi(Tokyo,Japan) chromatographic system consisted of a high pressure pump(L-6200A)and an auto sampler(AS-2000A).The column(BischoffChromatography SC-02-100Prontosil C18,100·2.1mm,3l m particles;Leonberg,Germany) was equilibrated with acetonitrile–water(1:99v/v)as mobile phase (Shephard and Legott,2000),40l l of the samples were injected and eluted with aflow rate of0.3ml/min and detected by UV–VIS spectrophotom-eter(L-4250,Merck Hitachi,Tokyo,Japan)at276nm.The calibration curves for PAT were obtained by daily analyses of eight standard solutions(range between25and1000ng/ml).The intra-day reproducibility for1000ng/ml was1.6%whereas the inter-day reproduc-ibility(measured over a period of5days)was1.75%.To determine the sample concentrations,the peak areas were calculated over the calibration curve.By subtracting the sample values from the values of the control samples,the decrease of PAT by LAB(in%)was obtained.2.6.Detection of ochratoxin A by HPLC-FLDTwo hundred microliter of the samples(supernatants,control samples or negative samples-which contained only bacteria and no toxin)were diluted with800l l mobile phase.The Merck Hitachi(Tokyo,Japan) HPLC-System contained a pump system(L-6200A)and an auto sampler (AS-2000A).An ACT column(Advanced Chromatography Technologies ACE3C18,15cm·3.0mm;3l m particles,Aberdeen,Scotland)with a guard column of the same material was used for performing the mea-surements.According to the protocol of Dall’Asta et al.(2004),an ace-tonitrile–ammonia buffer(NH3/NH4Cl,100mM,pH9.8)eluent(20:80v/ v),was used for the measurements.Samples(40l l)were injected and eluted with aflow rate of0.45ml/min and detected with afluorescence detector(F-1000,Merck Hitachi,Tokyo,Japan)at k ex=380and k em=440nm.The calibration curve was obtained by analysing the peak area of OTA standard solutions of six concentrations in the range between50and 1000ng/ml and was measured every day.The relative standard deviation for100ng/ml was1.49%within one day and1.75%over a period of5 days.To determine the concentration of the samples,the peak area was evaluated over the calibration curve.The decrease of OTA by LAB(in%)was determined by subtracting the sample value from the value of the control sample.2.7.MCN assaysThe MCN experiments were carried out according to the protocol of Darroudi and Natarajan(1991).Briefly,the cells were cultivated infilter topflasks(25cm2,Greiner,Kremsmu¨nster,Austria)for2days in CO2 atmosphere(8.0%)at37°C and95.0%relative humidity.Subsequently, the medium was changed and the cells were exposed to different concen-trations of the test compounds or the solvent(DMSO)for24h.Thefinal concentration of DMSO did not exceed1.0%(v/v).To evaluate the fre-quency of the MCN in binucleated cells(BNC),cytochalasin B(final concentration of 3.0l g/ml)was added to the growth medium after treatment and washing.Forfixation,the cells were trypsinized after28h, treated with cold hypertonic KCl solution(5.6g/l)and subsequently air dried preparations were made(Darroudi and Natarajan,1991).For the detection of the MCN in the binucleated cells,the slides were stained with Diff-Quik(Dade Behring Austria GmbH,Vienna,Austria).For each experimental point,three cultures were treated in parallel and1000cells were evaluated for MCN induction.To study the effects of the mycotoxins on cell division,the fractions of binucleated cells(in%)in relation to the number of mono-,tri-and tetranucleated cells were determined.2.8.StatisticsReduction of the mycotoxin concentrations after incubation in liquid medium with the different strains was calculated by Student’s t-tests,by comparison of the results of the individual experiments with the corre-sponding control samples.Correlation of the removal of the two toxins across strains and two concentrations were assessed by Spearmans rank correlation.Statistical analyses of the genotoxicity tests were performed by repe-ated measurements with one-way ANOVA in Prism version5.0(Graph-Pad Software,Inc.,Sandiago,CA;USA),followed by Bonferroni’s multiple comparison test.P-values with P60.05,were considered statis-tically significant.3.Results3.1.Chemical analytical measurementsThe results of the screening trial are summarized in Table1.The strongest decline of OTA(97%)was detected with a Lactobacillus acidophilus(VM20)strain,also two Bifidobacterium longum(LA02,VM14)strains were highly effective and caused a decrease by approximately50%. With PAT,the strongest effect(ca.80%decrease)was seen with a Bifidobacterium animalis(VM12)strain.Interest-ingly,no correlation between the detoxification capacities of the individual strains and the two mycotoxins was found i.e.the correlation coefficient was0.2and the P-value>0.05.In other words,strains which removed OTA were not effective against PAT and vice versa.However, it is notable that two strains,namely L.plantarum(VM 37)and L.curvatus(LA42),caused similar reduction of both toxins.In order to establish optimal conditions for the screen-ing trials,we conducted several experiments in which the pH dependency of the elimination of the mycotoxins was studied.The results of a representative experiment with PAT and OTA are shown in Fig.1.It can be seen that1400S.Fuchs et al./Food and Chemical Toxicology46(2008)1398–1407maximum removal was observed at pH 5.0,which was used in all subsequent experiments;no increase was observed in preliminary experiments with lower pH values (data not shown).It can be seen in Fig.2that the adsorption of the myco-toxins depends strongly on the amount of bacteria in the incubation mixtures.With both toxins,significant effects were seen when the cfu/ml was P 108/ml.The elimination from liquid medium depends also on the concentrations of the mycotoxins (Fig.3a and b).While OTA was removed from the medium even when high doses (up to 500l g/ml)were added,clear effects were seen with PAT only with substantially lower levels (6100l g/ml).To find out if,and to which extent,the viability of the bacteria affects their detoxification properties,we con-ducted comparative experiments with viable and heat inac-tivated cells.We found that the viability of the cells plays an important role as with heat inactivated cells only a mod-erate reduction of PAT (16%)and OTA (11%)was observed (data not shown).Table 1Removal of PAT and OTA from liquid medium by different LAB-strains a LAB-speciesStrainReduction (%)Patulin Ochratoxin A500ng1000ng 500ng 1000ng B.adolescentis LA187±1.095±1.39**7±1.3610±2.02**B.animalis VM1282±3.07**78±1.65**22±0.23**15±1.34**B.animalis LA1711±5.56*15±3.32**8±3.18*6±1.02B.bifidum VM1311±3.61**16±1.43**28±1.68**25±0.90**B.breve LA146±2.433±2.2516±1.25**17±2.64**B.longum VM147±2:914±1.1257±4.60**58±3.39**B.longum LA024±5.377±2.7752±4.61**54±1.32**L.acidophilus VM194±2.596±2.4218±1.71**12±0.37**L.acidophilus VM2021±3.07**18±1.65**97±2.11**96±1.91**L.acidophilus VM0519±4.15**19±2.65**32±2.03**31±1.30**L.brevis VM2116±3.67**16±2.40**4±1.934±1.58L.brevis VM223±4.199±1.63**11±1.49**5±1.82L.caseiVM0111±1.96**14±2.01**31±1.72**28±1.09**L.casei casei VM253±1.623±1.4112±1.90**15±0.44**L.casei casei LA095±1.10**4±2.015±3.172±1.27L.curvatus LA437±1.946±2.8315±1.72**16±2.42**L.curvatusLA4218±3.11*17±1.70**20±2.17**14±1.44**L.delbrueckii ssp.bulg.VM264±3.287±4.2329±3.40**27±2.38**L.delbrueckii ssp.bulg.VM2713±4.41**10±3.10**18±3.25**13±1.46**L.helveticusVM3410±2.49**8±2.40**24±2.18**25±2.80**L.delbrueckii ctis VM327±2.974±1.9434±1.21**34±0.84**L.paraplantarum VM3526±3.45**24±2.00**12±3.23**18±2.55**L.paraplantarum LA0720±3.13**16±2.11**3±4.394±1.15L.pentosus VM3631±3.26**30±2.98**22±7.45**18±7.46**L.plantarum VM3734±1.34**39±3.23**43±2.84**44±1.27**L.plantarum VM028±2.268±2.78**36±2.24**33±1.53**L.rhamnosus VM406±1.966±.3613±3.89**12±2.16**L.rhamnosus VM044±3.245±1.643±1.082±1.27L.slivarius LA016±3.807±1.835±2.239±1.75**S.thermophilusVM4221±2.78**19±1.43**13±.089**12±0.84**aThe incubation mixture contained 109cfu/ml and either 500ng/ml or 1000ng/ml of PAT or OTA,respectively;the mixtures were incubated at 37°C for 4h.Values indicate means ±SD obtained in three independent measurements.Stars indicate statistical significance to results obtained with corre-sponding control samples which contained only the toxins without bacteria (Student’s t -test,*P 60.05,**P 60.01).Fig.1.pH-dependent removal of PAT (white bars)and OTA (grey bars)from liquid medium by Bifidobacterium animalis VM12(in the case of PAT)and Lactobacillus acidophilus VM20(in the case of OTA).The incubation mixtures contained 109cfu/ml and either 1000ng/ml of PAT or OTA respectively;and were incubated at 37°C for 4h.Bars indicate results obtained in three independent measurements.Stars indicate statistical significance in comparison to the results obtained with pH 5.0(Student’s t -test,**P 60.01).S.Fuchs et al./Food and Chemical Toxicology 46(2008)1398–140714013.2.Impact of LAB on mycotoxin induced MCN formation In order to proof that LAB are able to protect human cells against the toxic effects of the two mycotoxins,the impact of the bacteria on MCN induction and on the inhi-bition of cell division by the mycotoxins in human derived hepatoma (HepG2)cells was investigated in a series ofexperiments.As negative control supernatants of incuba-tion mixtures without toxins were used.While for OTA data on acute toxic effects on HepG2cells were available (Ehrlich,2002),this was not the case for PAT.In the case of OTA a significant reduction of BNC was seen already with a concentration of 5l g/ml;with 50l g/ml the division rate declined by approximately 50%(Ehrlich et al.,2002a ).The acute toxicity of PAT was monitored over a broad dose range.The results indi-cate that the critical concentration of PAT lies between 1.0and 2.0l g/ml.After 24h exposure with the former dose,the viability of the cells was not significantly affected,while the higher dose caused a decline by approximately 60%.The results of MCN experiments with different dose lev-els of PAT are depicted in Fig.4a and b.It can be seen that the toxin caused a significant induction of MCN in the range between 0.25and 1.5l g/ml.PAT/ml (0.5l g)induced a 2.8-fold increase of MCN formation over the background level.Results of experiments with OTAareFig.2.Impact of the cell density of the removal of PAT (white bars)and OTA (grey bars)by Bifidobacterium animalis VM12(in the case of PAT)and Lactobacillus acidophilus VM20(in the case of OTA)from liquid medium.The incubation mixtures contained 109cfu/ml and either 1000ng/ml of PAT or OTA,respectively,and were incubated at 37°C for 4h.Bars indicate results obtained in three independent measurements.Stars indicate statistical significance in comparison to the results obtained with a cell density of 109cfu/ml (Student’s t -test,**P 60.01).Fig.3.(a and b)Concentration dependent removal of the two mycotoxins from liquid medium by LAB.The incubation mixtures contained 109cfu/ml and different concentrations of PAT or OTA,respectively,and were incubated at 37°C for 4h.Bars indicate results obtained in three independent measurements.Stars indicate statistical significance in com-parison to the results obtained with 1l g/ml (Student’s t -test,*P 60.05,**P 60.01).Fig.4.(a and b)Impact of PAT on MCN formation (a)and on cell division (b)in HepG2cells.The cells were incubated with different concentrations of PAT for 24h;subsequently the MCN-frequencies were determined after cytochalasin B treatment as described in Section 2.Bars indicate means ±SD of three incubation experiments.In total,1000cells were evaluated per experimental point.Stars indicate statistical signifi-cance in comparison to the results obtained without toxin (ANOVA,***P 60.001).1402S.Fuchs et al./Food and Chemical Toxicology 46(2008)1398–1407not shown as they were similar to the findings reported in previous articles (Ehrlich et al.,2002a;Knasmu ¨ller et al.,2004a ).The impact of pretreatment of the mycotoxins with LAB on MCN-frequencies and cell division rates in HepG2cells is shown in Figs.5and 6.It can be seen that the LAB caused a significant reduction of MCN induction by both toxins.In the case of PAT (Fig.5a),MCN frequencies were decreased by 39%,the effect caused by OTA (Fig.6a)was reduced by 59%at the highest concentrations.Also the inhibition of cell division (i.e.the decline of BNC forma-tion)by the two toxins was reduced significantly (Fig.5and 6b).4.DiscussionIn the present study,it was shown for the first time that LAB are able to remove PAT from liquid medium.Fur-thermore,the findings of earlier investigations were con-firmed,which indicate that the bacteria are able to eliminate OTA (Turbic et al.,2002;Piotrowska and Zak-owska,2005;Skrinjar et al.,1996)which is a potential genotoxic carcinogen (Ehrlich et al.,2002a;Cavin et al.,2007).It was also demonstrated that preincubation of the toxins with LAB reduces the toxic effects of these com-pounds in human derived liver cells (HepG2).As mentioned above,it is known that certain LAB strains are able to detoxify other mycotoxins such as AFB 1(for review see Kabak et al.,2006),fusarium toxins such as deoxynivalenol,nivalenol,T-2toxin,HT-2toxin (El-Nezami et al.,2002c;Niderkorn et al.,2006)and zea-ralenone (El-Nezami et al.,2002b,2004).Also genotoxic carcinogens contained in fried meat such as heterocyclic aromatic amines and polycyclic aromatic hydrocarbons are inactivated by LAB strains (Stidl et al.,2007a;Knasmu ¨ller et al.,2001).Fig.5.(a and b)Impact of selected LAB strains on MCN-formation (a)and inhibition of cell division (b)by PAT.5·109cfu/ml were incubated for 4h with PAT (25,50,or 100l g/ml);subsequently,the HepG2cells were exposed to 50l l of the supernatants for 24h.MCN frequencies and the rates of BNC were determined as described in Section 2.Bars represent means ±SD of three independent experiments.White bars indicate measurements with LAB;grey bars show results of the experiments without LAB treatment.In total,1000cells were evaluated per experi-mental point.Stars indicate statistical significance in comparison to the results obtained with control samples without toxins (ANOVA,**P 60.01,***P 60.001).Fig.6.(a and b)Impact of selected LAB strains on MCN-formation (6a)and inhibition of cell division (b)by OTA.5·109cfu/ml were incubated for 4h with OTA (100,200or 300l g/ml);subsequently,the HepG2cells were exposed to 250l l of the supernatants for 24h.MCN frequencies and the rates of BNC were determined as described in Section 2.Bars represent means ±SD of three independent experiments (one culture/experiment).White bars indicate results of measurements with LAB;grey bars show results obtained without LAB treatment.In total,1000cells were evaluated per experimental point.Stars indicate statistical significance in comparison to the results obtained with control samples without toxins (ANOVA,*P 60.05,**P 60.01,***P 60.001).S.Fuchs et al./Food and Chemical Toxicology 46(2008)1398–14071403。

JMEPEG (2001)10:567–575᭧ASM InternationalKinetics of Strain Aging in Bake Hardening Ultra Low Carbon Steel—a Comparison with Low Carbon SteelA.K.De,S.Vandeputte,andB.C.De Cooman(Submitted 22February 2000)The kinetics of the static strain aging process have been analyzed in a vacuum-degassed ultra low carbon bake hardenable (ULC BH)steel with a total carbon content of 20wt.ppm through measurement of the strength properties.The influence of prestrain and free interstitial carbon content has been studied.The kinetic results were compared with those of a BH low carbon (LC)steel.In the derivation of the time exponent and the activation energy,only the first stage of aging was considered.It was observed that,at all prestrain levels and matrix solute carbon contents,the initial aging process in the ULC steel obeyed the t 2/3kinetic law and the kinetics were not influenced by the changes in dislocation structure due to prestrain and the dissolved carbon content.In comparison,the aging process and the kinetics in the LC steel were found to be significantly influenced by the amount of prestrain.The presence of carbide particles in LC steels can modify the aging kinetics.atoms n t segregating to per unit length of dislocation in time t Keywords internal friction,kinetics,LC BH steel,prestrain,is given bystrain aging,ULC BH steel1.Introductionn t ϭn 03΂␲2΃13΂ADt k T΃23(Eq 1)Bake hardenable (BH)vacuum-degassed ultra low carbon where n 0is the number of solute atoms per unit volume,A is (ULC)steels (C Ͻ50wt.ppm)have recently received increased the interaction energy between a dislocation and solute atom,attention for autobody applications in the automotive industry.and D is the diffusion coefficient of the segregating solute at Compared to low carbon (LC)BH steels (C Ͼ100wt.ppm),the absolute temperature T .With the advance of the aging ULC BH steels have excellent forming properties and an process,Eq 1fails,however,to describe the kinetics,as it does increased strength that is achieved due to the aging during the not consider the solute depletion surrounding the dislocations,paint baking of the final product.These steels can be processed a fact which was also recognized by Cottrell.on hot dip galvanizing/galvannealing lines without an overaging In order to extend the applicability of the model to systems section,which is necessary for LC steels to allow cementite of supersaturated Fe-C solid solutions,Harper modified the precipitation.Bake hardening is essentially a strain aging proc-above equation to allow for the lowering of solute concentration ess resulting from the interaction between interstitial carbon in the matrix surrounding the dislocations as the aging pro-atoms dissolved in the matrix and the dislocations generated ceeds.[6]He assumed that the rate of segregation at any time during forming operation.The kinetics of the process is con-would be proportional to the solute concentration remaining in trolled by the long-range diffusion of interstitial atoms to the solution and obtainedstrain fields of dislocations.Atmospheres of interstitial atoms are formed in the vicinity of the dislocation cores.Further segregation of interstitials to the dislocations results in carbide W ϭn t n 0ϭ1Ϫexp ͫϪ3L ΂␲2΃13΂ADt k T΃23ͬ(Eq 2)precipitation.The most obvious manifestation of the strain aging process is the increase in the yield stress of the material at all solute levels and aging times.[1]Earlier investigations of the where W is the fraction of solute atoms already segregated in strain aging process in Fe-C alloys have established distinctly time t and L is the total length of dislocations per unit volume.the mechanisms and stages of the process.[2–4]With regard to Harper found a good agreement with his experiments for frac-the kinetics of the aging process,it is likely that the entire tions of solute depletion up to 0.90.The limitations to the aging process cannot be described by a single model.The initial Harper model have been reviewed by Baird.[7]As the Harper stage of the aging process was originally described by Cottrell model does not allow for the back diffusion from the core of and Bilby’s kinetic model.[5]According to Cottrell and Bilby,dislocations,it can only be valid for steels with low atmosphere during the atmosphere formation,the total number of solutedensities.In many strain aging studies in the Fe-C system,a generalized form of Harper’s equation has been used to derive the kinetics of the aging process.Equation 2can be rewritten asA.K.De andB.C.De Cooman,Laboratory for Iron and Steelmaking,Ghent University,9052Ghent,Belgium;and S.Vandeputte,OCAS N.V .,Research Centre of the SIDMAR Group,9060Zelzate,Belgium.ln (1ϪW )ϭϪ΂t␶΃nϭϪ(k t )n(Eq 3)Contact e-mail:bruno.decooman@rug.ac.be.where ␶is a temperature-dependent relaxation constant obeying an Arrhenius-type relation from which the activation enthalpy of the aging process can be derived.The kinetic parameters n and ⌬H derived from fitting experimental data to Eq 3have often been used to interpret precipitation mechanisms or to take into account dislocation inhomogeneities.Any deviation in the value of n from 2/3is generally regarded as being associated with a change in the precipitation mechanism of carbon on dislocations or in the matrix.[8–12]In BH ULC steel,a very low amount of carbon is retained in solid solution at the end of processing and the precipitation of iron carbides is unlikely to take place in this type of steel.[13]Moreover,because of the ultra low level of solute content,the C backdiffusion is expected to be insignificant.Therefore,the application of the Harper derivation should give an accurate description of the aging kinetics until the completion of the Fig.1Measurement of increase in yield stress ⌬␴due to strain agingatmosphere formation in ULC steels.Strain aging in ULC BH steel is technologically very important.Presently,attempts are being made to increase the bake hardenability of these steels through retention of more The parameter W has often been equated to the fractional solute carbon in the matrix by increasing the cooling rates after increase in yield stress,⌬␴/⌬␴max ,during the aging process,soaking in a continuous annealing cycle.In this work,the strain where ⌬␴is the increase in yield stress after aging for time t aging results of a BH ULC steel have been examined with and ⌬␴max is the maximum stress increment due to prolonged respect to changes in prestrain and solute carbon content aging.[1]The proportionality between W and ⌬␴/⌬␴max may be resulting from the application of different cooling rates after questioned when the aging process occurs in supersaturated annealing.The kinetic parameters n and ⌬H of the aging process solid solutions,where the strengthening may result from differ-were determined with the analytical models describing atmos-ent concurrent mechanisms,but it probably is a fair approxima-phere formation.The influence of the prestrain and the solute tion in the case of the ULC steels.carbon content on the time exponent n was evaluated.In the derivation of the kinetics,the increase in upper yield strength 2.2Hartley Modeldue to the aging process was taken into account rather than the solute segregation,as the former is the most consistent This is the only model available so far that allows the kinetics manifestation of the atmosphere formation process.This consid-of strain aging to be derived by measuring the changes in yield eration also stems from the fact that a very small amount of stress.Hartley described the increase in yield stress during carbon is required for atmosphere formation even for a highly aging as due only to the reduction of mobile dislocation length,deformed material.Considering the occupancy of one carbon which is proportional to the linear concentration of carbon on atom per atomic plane threaded by the dislocation at atmosphere the dislocations.Hartley proposed the following aging saturation,the amount of carbon required to saturate a disloca-kinetic equation:[14]tion density of ␳(m Ϫ2)in bcc ferrite can be calculated as(␴y Ϫ␴f )1/2(␴y ϩ␴f )ϭ⌬␴␴ϭK 1ϩK 2΂DtT΃2/3(Eq 5)[C]ppm ϭ8.9и10Ϫ15и␳(Eq 4)So,even for a large dislocation density of 1014/m 2,onlywhere ␴y is the upper yield stress after prestraining and aging,about 1ppm carbon is needed to saturate all the dislocations.␴f is the flow stress at the end of prestraining (Fig.1),t is Whereas in the past the C aging has been successfully analyzed the aging time,T is the aging temperature,D is the diffusion by means of internal friction (IF)or resistivity measurements,coefficient,[14]and K 1and K 2are constants for constant test no diagnostic tool is presently available to monitor accurately conditions,The slope S of the ⌬␴/␴versus t 2/3plot is given such extremely low levels of carbon segregation during atmos-by S ϭK 2(D /T )2/3.With D ϭD 0exp [Ϫ(⌬H /R T )],the activa-phere formation.tion energy ⌬H for carbon diffusion therefore can be easily obtained from the plot of ln (ST 2/3)versus 1/T .However,apart from dimensionality consideration,the phys-2.Application of Kinetic Modelsical interpretation of the use of the term 1/2(␴y ϩ␴f )is not clear in Hartley’s derivation.[14]Since in the derivation of Eq 2.1Harper Model5the degree of atmosphere saturation has been taken into account instead of the total fraction of solute segregating to From Eq 3,a plot of ln [Ϫln (1ϪW )]against ln t will result in a straight line with a slope n and a y intersect proportional to the dislocations,it was considered more appropriate to use the term ⌬␴/⌬␴atm for the degree of saturation in the present work,the diffusivity of the interstitial solute.Since k is expressed as k ϭk 0exp [Ϫ(⌬H /R T )],a plot of ln k versus 1/T will give where ⌬␴atm is the maximum increase in yield stress at atmos-phere saturation.It has been observed that the maximumthe activation energy of the aging process.Table1Chemical composition of the steels(in wt.%)during the fast heating(100ЊC/min)of the specimen in aninfrared radiator furnace over a temperature range of20to300 Steel C Mn P S Al Ti NЊC.[16]At40kHz,the Snoek peak occurs at around192ЊC.The inherent advantage associated with this technique is that ULC0.00200.090.0450.00300.04900.00700.0016it has a very high signal-to-noise ratio compared to the conven-LC0.0390.180.0350.00700.0540…0.0044tional torsion pendulum instrument.Hence,a very low amountof interstitials can be traced accurately with this instrument.Anadditional advantage of this technique is that,as the interstitial increase in yield stress⌬␴atm at atmosphere saturation is con-carbon segregates to the dislocations during heating to the peak stant for a prestrain level up to10%and aging temperatures temperature,one measures the actual interstitial C content in up to170ЊC for the ULC steel investigated.[13]the matrix at the paint baking temperature.Therefore,from Eq5,if the time exponent is set as n,thenthe slope of the ln⌬␴/⌬␴atm versus ln t plot will give the valueof n. 4.Results and Discussion4.1Aging Behavior—ULC and LC Steel and Effect of3.Experimental Procedure and MaterialPrestrain3.1Material and Processing Figures2(a)and(c)describe the aging behavior in theprestrained ULC steel with respect to time and temperature.It The material used for the present study was a vacuum-is observed that,at all prestrain levels,the increase in strength degassed ULC BH steel with the composition as given in Tablereaches a distinct saturation plateau after a time characteristic 1.The aging results for a LC BH steel with the compositionof the aging temperature.The strength then remains almost given in Table1were used to compare the kinetics of aging.[15]constant during further aging.Except for a marginal increase The hot-rolled sheets of both the ULC and LC steel werein strength in specimens prestrained2%at higher aging temper-given75%cold reduction in a laboratory cold rolling mill.atures,no second stage of hardening,i.e.,carbide precipitation, After cold rolling,the sheets were annealed in a(Carl-Wezel,can be seen.This suggests that the solute carbon available in Germany)continuous annealing simulator at850ЊC for60sthe matrix of the ULC steel(SC sheets)is sufficient only to with an overaging cycle of180s at400ЊC.The cooling ratecomplete atmosphere formation.The attainment of the satura-from the annealing temperature was10ЊC/s.The annealedtion plateau marks the end of Cottrell atmosphere formation,as sheets were further given a temper rolling reduction of1.3%.studied previously through the changes in yield point elongation Tensile specimens of80mm gage length were prepared(YPE)behavior.[13]Second,at all prestrain levels and tempera-from these sheets and were prestrained2,5,and10%at a straintures of aging,the maximum strength increase at the end of rate of4ϫ10Ϫ4sϪ1and then aged at temperatures betweenthe atmosphere formation,⌬␴atm,was found to beϷ30MPa. 50and170ЊC for different times in a silicone oil bath with aIn comparison,the aging in the LC steel has two distinct temperature control ofϮ1.5ЊC.stages(Fig.2d and e).A significant strength increase is observed For varying the amount of solute carbon content in the matrixin the second stage or the precipitation stage in the LC steel. of the ULC steel,the cold-rolled sheets were cooled from theThe maximum increase in strength decreases with the increase annealing temperature of850ЊC at three different cooling rates:in prestrain.(1)sheets cooled at the rate of10ЊCиsϪ1,(2)sheets cooled atthe rate of50ЊCиsϪ1to room temperature,and(3)sheets waterquenched from the annealing temperature at the rate of 4.2Effect of Cooling Rate550ЊCиsϪ1to room temperature.In the review of the agingFigure3shows the IF spectra observed for the SC,MC, results,these samples are designated as(a)SC(slow cooling,and FC specimens due to different rates of cooling.The carbon 10ЊCиsϪ1),(b)MC(medium cooling,50ЊCиsϪ1),and(c)FCcontent in the matrix increases with the increase in cooling (fast cooling,550ЊCиsϪ1).Tensile specimens prepared fromrate,as reflected in the IF spectra.The solute carbon content these sheets were prestrained5%and then aged at50ЊC forin the rapidly cooled specimens will be slightly higher than different times.what is measured considering the2min heating time neededto reach the peak temperature of measurement.Rapid cooling 3.2Mechanical Testing introduces some dislocations or vacancies in the material,and,hence,there is a possibility of losing some interstitial carbon The increase in yield stress⌬␴was determined as the differ-to the dislocations during the measurement.This effect is ence between the upper yield stress,␴y,after aging for time texpected to be very limited in slowly cooled specimens(Eq4). and the flow stress,␴f,at the end of prestraining based on theThe strain aging results of these specimens for an aging original specimen dimensions(Fig.1).temperature of50ЊC are shown in Fig.4(a)and(b)with The solute carbon content in the SC,MC,and FC specimensrespect to changes in yield stress and YPE.The distinct features was determined by IF measurements using a high frequencyrevealed in the aging results are as follows.piezoelectric ultrasonic composite oscillator.In this technique,the specimen is set to vibrate longitudinally over a piezoelectricoscillator at40kHz at a vibration strain amplitude of10Ϫ7.•With increasing interstitial carbon content,the aging stage The IF due to stress-induced ordering of interstitials is recordednow gradually advances to the second stage of aging andFig.2Increase in the yield stress with time for different aging temperatures for prestrained(a)to(c)ULC and(d)and(e)LC steelsa significant second stage hardening is observed in the FC saturation(as indicated by the maximum in the YPE values)is again30MPa in all the SC,MC,and FC specimens.and MC specimens.•The completion of the first stage of aging or the atmosphere4.3Kinetics of Agingsaturation occurs faster with the increase in carbon content,as revealed in the YPE results(Fig.4b).The aging results of Fig.2(a)to(c)were replotted in termsof Eq3and5and are shown in Fig.5(a)and(b),respectively.•The maximum increase in yield stress⌬␴atm at atmospherein Table2.It is clear from the figures that within the prestrainand temperature range studied,no change in the slopes isobserved.The values of n found through Harrier’s analysisfall within0.65to0.80,which are quite close to that derivedby Cottrell and Bilby for the interaction of the dislocationand the interstitial carbon for which nХ0.66.Values of nfound through Hartley analysis are also close to the valueof0.66.However,relatively higher values obtained throughHarper’s model were due to the neglect of saturation effectsin this model.The analysis of the kinetics through these models suggestsa normal strain aging kinetics(t2/3law),i.e.,carbon segrega-tion to dislocations alone,and that the kinetics is not alteredby the changes in the dislocation density in the ULC steelswithin the range studied.This is important since amplitude-Fig.3IF spectra in annealed ULC specimens cooled at different ratesdependent IF measurements on prestrained specimens demon-strated that prestraining the ULC steel in excess of7.5%results in dislocation structure changes.[17]The TEM observa-presence of cellular dislocation network formation.While thekinetics changes due to such dislocation structure changeshave not been reported so far,but based on Bullough andNewman’s analysis,a time exponent value of0.77had beenreported earlier[18]considering inhomogeneities in dislocationdistribution(in the region of clusters,cell walls,and carbides).The aging results of the LC steel were also analyzedthrough the Harper and Hartley models for comparison ofaging kinetics with those of ULC steel,and the results areshown in Fig.6(a)and(b),respectively.The n values calcu-lated from the data are given in Table2.It is clear that inthis case the amount of prestrain influences the values of nwithin the temperature range examined.The values of n forspecimens prestrained2%derived through the Harper(0.54to0.55)and Hartley(0.46to0.48)models are both lower (a)than those observed for the ULC specimens.The results pointmore toward a t1/2kinetic law.In specimens prestrained5%,words,at higher prestrain,the Cottrell atmosphere formationprocess dominates,whereas at lower prestrain,the n valuesare suggestive of a mechanism of carbon diffusion toward agrowing cementite particle.In a recent work by Kozeschnikand Buchmayr,[19]it was shown that within a prestrain rangeof0to5%there is a change in the precipitation mechanism.They indicated that,at low dislocation density,the precipita-tion of carbide is favored and,at about5%prestrain andmore,the ferrite matrix is depleted by Cottrell atmosphereformation and no carbide particles can form until at least themajority of carbon atoms have diffused to the dislocations.In earlier works,[11]a t1/2kinetic law had been found to beassociated with dislocation locking by carbon atoms at ferrite-cementite interfaces.The LC steel contains many cementite (b)particles[15]and also a higher amount of manganese than the Fig.4Aging behavior in ULC specimens with different cooling rates ULC steel.Therefore,it is likely that the carbide particles with respect to(a)increase in yield stress and(b)YPE can grow during aging and lead to an additional increase inyield stress.[8,20]Leslie[8]demonstrated that,during aging ofan Fe-Mn-C alloy at temperatures between60and100ЊC,precipitation of carbides both in the matrix and the disloca-Both the equations describe the data quite well up to thecompletion of atmosphere saturation at all the prestrain and tions could be observed as Mn shortens the incubation timeFig.5Kinetic analysis of the aging data of prestrained ULC specimens using (a )Harper and (b )Hartley modelsfor the formation of critical nuclei and affects the activity the solute carbon content (within 20wt.ppm),the aging mech-anism does not change.Earlier aging results with higher initial of carbon.The analysis of the aging data for specimens with different carbon content reported abrupt changes in aging kinetics in quenched-in iron alloys which were ascribed to the changes solute carbon contents (Fig.4)is given in Fig.7(a)and (b).The values of n measured from the slopes of these plots are in aging mechanism from nucleation on dislocation only to the nucleation within the matrix and dislocations.[8,9]The given in Table 3.It is interesting to note that the slopes are almost constant for all the specimen groups and are very close nucleation within the matrix is said to be facilitated by the presence of vacancy rings generated due to quenching.[8]Thisto the value of 0.66.This suggests that,even with changingaging process in the ULC and LC steels as a functionof prestrain and temperaturePrestrain,Aging temperature,⌬H,Steel Model%؇C n kcal/molULC Harper21400.6919.61000.70750.76500.7551400.6520.01000.65750.80500.72101400.7320.31000.74750.78500.73Hartley21400.5919.01000.76750.61500.6351400.6519.01000.79750.68500.61101400.8019.71000.71750.64500.66(a)LC Harper2500.5515.75500.6719.21000.80Hartley2500.4616.81000.485500.6421.81000.58was expected in the present case for FC specimens,but thepresent results suggest that,even if such a mechanism ispresent,it does not influence the aging process strongly.Thisis probably due to the fact that even the highest solute carbonin the FC specimen is too low to cause any matrix nucleation.The activation energy for the atmosphere formation processin ULC steel was calculated from Fig.5(a)and(b)using bothHarper and Hartley derivations,and the results are shown inFig.8(a)and(b),respectively,for the two models.The valuesare given in Table2and are in excellent agreement with theactivation energies of18to20.1kcal/mol for diffusion of carbonin bcc iron during strain aging,as published earlier.[6,21]Theactivation energies derived for the LC steel specimens appar-ently show a strong prestrain dependence.At higher prestrain,the activation energy derived(Table2)is close to that fordiffusion of carbon atoms to the dislocations,whereas at lowerprestrain,a much lower activation energy is found.This impliesthat the underlying aging mechanism in the LC steel is not thesame at all prestrains,a fact that was also revealed in the n(b)values(Table2).Fig.6Kinetic analysis of the aging data for prestrained LC specimensusing(a)Harper and(b)Hartley models5.ConclusionsIn the present work,an attempt was made to apply the to the aging results of an ULC and a LC steel,in order tocompare the aging kinetics for these steels and to obtain theavailable analytical models describing kinetics of strain aging(a)(a)(b)Fig.7Kinetic analysis of the aging data of SC,MC,and FC speci-mens using (a )Harper and (b )Hartley modelsTable 3Kinetic parameter n for the strain aging process in the ULC steel as a function of cooling rate(b)Model Prestrain,%Aging temperature,؇C Cooling raten Fig.8Determination of the activation energy of carbon diffusion during strain aging in prestrained ULC specimens using (a )Harper Hartley 55010ЊC иs Ϫ10.63and (b )Hartley model50ЊC иs Ϫ10.60550ЊC иs Ϫ10.63Harper55010ЊC иs Ϫ10.7150ЊC иs Ϫ10.76550ЊC иs Ϫ10.76•The maximum increase in yield stress at atmosphere satura-tion is 30MPa in the ULC steel,and this does not depend on the amount of prestrain or solute content within the range studied.kinetic parameters n and ⌬H .The kinetics were derived through measurement of the increase in yield stress due to aging.The •The activation energy for the atmosphere formation stage following conclusions can be drawn.in the ULC steel has been found to be 19to 20.3kcal/mol,which is in excellent agreement to the activation energy of •The time exponent n evaluated through different kinetic 18to 20.1kcal/mol for diffusion of carbon in bcc iron models suggested that the dislocation pinning by the carbon during strain aging reported previously in the literature.atoms is the dominant mechanism during the strain aging in the ULC BH steel at all prestrain levels and is not affected In the case of the LC BH steel,the dislocation density has by the changes in dislocation structure due to straining.a significant role in determining both the strengthening level after the second stage of aging and the kinetics of the initial •The amount of prestrain up to 10%or the changes in solute carbon content (up to 20wt.ppm)does not influence the aging process.At lower prestrain,the kinetics follows a t 1/2law,and at higher prestrain,the kinetics is governed mainly byaging kinetics in the ULC steel.11.V.T.L.Buono,M.S.Andrade,and B.M.Gonzalez:Metall.Trans.A, the dislocation and carbon atom interaction,which follows a1998,vol.29A,pp.1415-23.t2/3time dependence.12.R.Bullough and R.C.Newman:Proc.R.Soc.,1959,vol.A249,pp.427-40.13.A.K.De,S Vandeputte,and B.C.De Cooman:Scripta Mater.,1999,vol.41,pp.831-37.References14.S.Hartley:Acta Metall.,1966,vol.14,pp.1237-46.15.A.V.Snick,K.Lips,S.Vandeputte,BC De Cooman,and J.Dilewijns: 1.W.C.Leslie:The Physical Metallurgy of Steels,McGraw-Hill,New in Proc.on Processing and Properties,W.Bleck,ed.,Aachen,Ger-York,NY,1982,p.88.many,1998,Modern LC and ULC Sheet Metals for Cold Forming,2.W.Pitsch and K.Lu¨cke:Arch.Eisenhu¨ttenwes.,1956,vol.1,p.45.vol.2,pp.413-24.3.D.V.Wilson and B.Russell:Acta Metall.,1960,vol.8,pp.36-45.16.I.G.Ritchie and Z.Pan:33rd MWSP Conf.Proc.,ISS,Warrendale,4.P.Elsen and H.P.Hougardy:Steel Res.,1993,vol.64,pp.431-36.PA,1992,vol.29,pp.15-25.5.A.H.Cottrell and B.A.Bilby:Proc.Phys.Soc.,1949,vol.A62,pp.17.A.K.De,K.De Blauwe,S.Vandeputte,and B.C.De Cooman:J.49-62.Alloys Compounds,2000,vol.310(1–2),pp.405-10.6.S.Harper:Phys.Rev.,1951,vol.83,pp.709-12.18.J.D.Baird:Iron and Steel,1963,vol.8,pp.400-05.7.J.D.Baird:Iron and Steel,1963,vol.7,pp.368-74.19.E.Kozeschnik and B.Buchmayr:Steel Res.,1997,vol.68(5),pp.8.W.C.Leslie:Acta Metall.,1961,vol.9,pp.1004-22.224-30.9.R.H.Doremus:Trans.AIME,1960,vol.218,pp.596-605.20.T.Obara,K.Sakata,M.Nishida,and T.Irie:Kawasaki Steel TechnicalReport,1985,vol.12,p.25.10.S.I.Neife,E.Pink,and H.P.Stu¨we:Scripta Metall.Mater.,1994,vol.30,pp.361-66.21.C.Wert:Phys.Rev.,1950,vol.79,pp.601-05.。

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Oxidative stress,AGE,and atherosclerosisE Schleicher1and U Friess11Department for Internal Medicine IV,Clinical Chemistry(Central Laboratory),University of Tuebingen,Tuebingen,GermanyNumerous reports on the molecular mechanism of atherogenesis indicate an increase in oxidative stress, formation of advanced glycoxidation end products(AGEs), chronic inflammation,and activated cellular response particularly in diabetic patients.To elucidate the initiating and early accelerating events this review will focus on the molecular causes of the induction of these stress factors,their interactions,and their contribution to atherogenesis. Metabolic factors such as elevated free fatty acids,high glucose levels or AGEs induce reactive oxygen species(ROS) in vascular cells leading to ongoing AGE formation and to gene induction of proinflammatory cytokines.Vice versa, numerous cytokines found elevated in obesity and diabetes may also induce oxidative stress thus a circulus vitious may be initiated and accelerated.Increased production of ROS, mainly from mitochondria and NAD(P)H oxidase,stimulates signaling cascades including protein kinase C and mitogen-activated protein kinase pathway leading to nuclear translocation of transcription factors such as nuclear factor-j B(NF-j B),activator protein1,and specificity protein1. Subsequently,the expression of numerous genes including cytokines is rapidly induced,which,in turn,may act on vascular cells promoting the deleterious effects.From animal models of accelerated atherosclerosis a causal role ofNAD(P)H oxidase and the AGE/RAGE/NF-j B axis to atherogenesis is suggested.Because all factors involved form a highly interwoven network of interactions,the blockade of ROS or AGE formation at different sites may interrupt the vicious cycle.Promising candidate agents are,currently on trial.Most important to clinical practice,a number of drugs commonly used in the treatment of diabetes,hypertension, or cardiovascular disease,such as angiotensin-converting enzyme inhibitors,AT1receptor blockers,3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors(statins),and thiazolidindiones have shown promising‘preventive’intracellular antioxidant activity in addition to their primary pharmacological actions.Kidney International(2007)72,S17–S26;doi:10.1038/sj.ki.5002382 KEYWORDS:atherogenesis;cytokine;endothelial dysfunction;NAD(P)H oxidase;reactive oxygen species(ROS);glycation endproducts(AGE)Atherosclerosis and clinical consequences of macrovascular disease,such as myocardial infarction and stroke,are the major cause of death in the Western world and are increasing in developing countries.Numerous complex and multi-factorial risk factors have been documented.These include: genes,hypertension,diet,smoking,physical activity,hyperli-pidemia,insulin resistance,and chronic hyperglycemia. Particularly type II diabetic patients display accelerated and more advanced atherosclerotic lesions and an increased incidence of cardiovascular and cerebrovascular disease.1 The reason for the aggressive vascular disease associated with risk factors such as diabetes is still unclear.There is a large body of literature on the mechanisms of the development of atherosclerosis.Numerous data suggest the possible involvement of reactive oxygen species(ROS) and advanced glycoxidation end products(AGEs)as early initiating events occurring long before morphological changes are obvious.Therefore,as a working hypothesis and as outlined in Figure1,we propose that the vascular wall can readily cope with physiologically occurring stress factors. However,chronic action of stress factors and metabolic conditions,such as hyperglycemia,dyslipidemia or(satu-rated)free fatty acids(FFAs)elevated in diabetes may induce ROS and/or AGEs which via the receptor for AGE(RAGE) form a vicious cycle of increasingly accumulating AGEs, enhanced formation of ROS,and sustained activation of the RAGE axis thus forming a milieu to perpetuate the injurious effects of chronic inflammation within the vascular wall. Therefore,this review will particularly focus on the causes of accelerated atherosclerosis in diabetic patients because understanding the pathogenesis in this disease may help to elucidate the molecular mechanism in general. CONCEPTS OF THE PATHOGENESIS OF ATHEROSCLEROSIS To describe the initial mechanism(s)of the atherosclerotic process,three major concepts have been developed during the last decades.2In the‘Response to injury’hypothesis Ross and Glomset3proposed endothelial denudation as initial step causing increased endothelial adhesiveness for thrombocytes and leukocytes concomitant with a change of the antic-oagulant to a procoagulant milieu.The adherent thrombo-cytes and leukocytes release mitogens and vasoactive factors (cytokines and growth factors),which in turn lead to migration of smooth muscle cell(SMC)into the intima and to recruitment of monocytes/macrophages into the arterial wall.Upon uptake of low-density lipoproteins(LDLs)&2007International Society of NephrologyCorrespondence:Department for Internal Medicine IV,Clinical Chemistry (Central Laboratory),University of Tuebingen,Hoppe-Seyler-Street3,D-72076 Tuebingen,Germany.E-mail:Erwin.Schleicher@med.uni-tuebingen.dethe macrophages form ‘foam cells’,a hallmark of early arterial lesions.However,the observation that an intact endothelium may cover early atherosclerotic lesions has questioned this hypothesis.Besides,LDL entry rate into the arterial wall is dependent on the location of the arteria rather than on the presence of denuded endothelium.Therefore,the ‘Response to retention ’hypothesis has been introduced.This theory is essentially based on observations showing that after entry of LDL into the subendothelial space LDL is tightly linked to components of the extracellular matrix particularly to negatively charged proteoglycans.The LDL particle may then be modified by various lipolytic and proteolytic enzymes rendering these modified LDLs prone to macro-phage and smooth muscle uptake and subsequent formation of foam cells.On the basis of the observation that oxidatively modified but not native LDL lead to cholesterol accumula-tion in macrophages via the scavenger pathway has prompted the ‘Oxidative modification ’hypothesis.In this theory,Steinberg et al .4proposed that oxidative modification of LDL leads to enhanced uptake of modified LDL and subsequent foam cell formation.Furthermore,the modified LDL supports the recruitment of circulating monocytes.Invading monocytes are cytotoxic to endothelial cells thus ‘injuring’the vessel wall.Together,oxidative stress,post-ribosomal modifications and inflammatory reactions are a common feature in the three hypothesis for describing early atherosclerosis.But none of them explains all features of early events of atherosclerosis or the failures of antioxidative treatments,for example vitamin E,in human subjects.2The progression from initial vascular injury towards the fully developed atherosclerotic plaque is better understood:recruited thrombocytes and leukocytes release cytokines,vasoactive peptides,and growth hormones.This promotes the proliferation and migration of SMCs into the intima.The build-up of lipids within the arterial wall is thought to trigger further inflammatory response.Recruited macrophages release ROS and,in turn,take up oxidized lipoproteins.Together,the continuing process of lipid accumulation,foam cell formation,recruitment of inflammatory cells and releaseof ROS,cytokines,growth factors,and proteolytic enzymes perpetuates the inflammatory cascade within the vessel wall and finally sets the stage for cellular necrosis,fibrosis and further progression towards the fibro-fatty plaque,the characteristic lesion of end-stage atherosclerosis.CLINICAL FEATURES OF EARLY STATES OF VASCULAR INJURY:ENDOTHELIAL DYSFUNCTIONThe earliest clinical determinant of arterial changes is endothelial dysfunction as determined by flow-associated vasodilatation 5and endothelial dysfunction emerges as a key component in atherogenesis.Endothelial dysfunction is clearly associated with insulin resistance which occurs particularly in obese individuals and type II diabetes.6Then,what is the link between obesity and atherosclerosis?Subjects with accumulation of visceral adipose tissue show,as well,increased ectopic lipid accumulation in liver and skeletal muscle as determined by 1H-NMR.Particularly,the liver fat contributes to insulin resistance.The ectopic lipid deposition may lead to enhanced production of inflammatory cytokines (e.g.,via enhanced oxidative stress)in the affected organ in parenchymal cells and/or infiltrated macrophages as has been shown to occur in adipose tissue.7The tissue crosstalk occurring via the vascular system may be the link between obesity and macrovascular disease.Is there any relation between endothelial dysfunction and oxidative stress?In vitro and in vivo studies have demonstrated that hyperglycemia-induced endothelial dysfunction and endothelium-depen-dent vasorelaxation is contrasted by antioxidants such as vitamin C.8Furthermore,many of the agents with beneficial effects on endothelial dysfunction including statins,angio-tensin-converting enzyme inhibitors or angiotensin II type 1receptor blockers exert significant intracellular antioxidant action.9Together,accumulating data suggest a link between endothelial dysfunction and oxidative stress supporting a potentially causal role of ROS in the initial events of atherosclerosis.OXIDATIVE STRESS AND ATHEROSCLEROSIS ROSAlthough the term oxidative stress is widely used a generally accepted definition is lacking.While most researchers consider oxidative stress as an increased production of short-lived,highly reactive,oxygen-derived molecule species (see below),Sies 10describes oxidative stress as a ‘disturbance in the pro-oxidant/antioxidant balance in favor to the pro-oxidant state’.Several ROSs have been suggested to play central roles in vascular physiology and pathophysiology,the most important of which are superoxide anion (O 2KÀ),hydrogen peroxide (H 2O 2),nitric oxide (NO),and peroxynitrite (ONOO À).Although O 2KÀper se can affect vascular functions,it is also pivotal in generating other reactive species.When O 2KÀis produced in concert with NO K,they rapidly react to form the highly reactive molecule ONOO KÀ.ONOO KÀis an important mediator of lipid peroxidation and protein modification including oxidationAccelerated atherosclerosisAtherosclerosisRepair/regenerative responseVascular stressVascular stress + tissue injuryVascular stress + tissue injury+ risk factors (diabetes)Figure 1|Working hypothesis for the accelerated atherosclerosis in diabetes.Reactive ROS and AGEs that react via the RAGE are generated if the vascular wall is stressed.Because all three partners are linked a vicious cycle is induced.This cycle is enhanced by further stress factors occurring in diabetes.E Schleicher and U Friess :Oxidative stress,AGE,and atherosclerosisof LDL.In the absence of immediately accessible NO K,O2KÀis rapidly converted into H2O2by superoxide dismutase, which then is converted into H2O by either catalase, peroxiredoxins,or glutathione peroxidase.H2O2can also react with reduced transition metals,for example Fe2þ,to form the highly reactive hydroxyl radical(OH K),or it can be metabolized by myeloperoxidase to form hypochlorous acid (HOCl).In summary,ROS can react rapidly and unspecifically with a wide variety of biomolecules but their lifetime and correspondingly their radius of action is very limited.Apart from the short lifetime of ROS effective antioxidant defense mechanism(s),particularly through the redox enzymes,have been developed to detoxify these agents.Furthermore,recent reports indicate that hemoxygenase1is involved in ROS elimination.In diabetes,however,antioxidative defense is impaired.11Biomarkers of oxidative stress in the atherosclerotic lesion There is definite evidence of oxidative stress in atherogenesis because numerous lipoxidation end products(ALEs)or glycoxidation end products(AGEs)have been found to be increased in atherosclerotic lesions as compared to unaffected tissue.Such biomarkers originate from a wide variety of vascular tissue constituents,such as lipids,fatty acids,sugars, amino aids,proteins,or DNA(reviewed by Stocker and Keaney2).Different types of fatty acid oxidation products have been identified in human atherosclerotic lesion.Of these,hydroxyl products of linoleic acid(C18:2D9,12)or hydroxyoctadecaenoic acids and hydroxyl products of arachidonic acid(C20:4D5,8,11,14)or hydroxyeicosatetrae-noic acids are the most abundant type of oxidized lipids in atherosclerosis,12mostly present as cholesterol ester.Other important lipoxidation products in human atherosclerotic lesions are F2-isoprostanes,which occur at levels20-to40-fold lower than hydroxylated FFAs and represent radical oxidation products of arachidonic acid.12Moreover,several end products of glycoxidation(AGEs)have been found to accumulate in the atherosclerotic lesion,including hydro-imidazolones,pentosidine,and,most abundantly,N e-carboxymethyllysine(CML)as discussed below in detail.The processes of lipoxidation and glycoxidation are closely interlinked by the production of reactive carbonyl inter-mediates:the oxidation of polyunsaturated fatty acids can lead to the formation of glyoxal,methylglyoxal,malondial-dehyde,or4-hydroxynonenal,which are widely used as markers of lipoxidation.Likewise,the oxidative decomposi-tion of reducing sugars,such as glucose,fructose,ribose,and of glycolysis intermediates gives rise to glyoxal,methylglyox-al,and3-deoxyglucosone.13All of the carbonyl intermediates are prone to react with the N e-lysyl residues to modify proteins posttranslationally.Specific antibodies have been raised against the corresponding modifications,for example against4-hydroxynonenal or malondialdehyde adducts of apolipoprotein B100or against the CML modification and used to study the kinetic and distribution of ALE/AGE modifications in atherosclerosis and other processes,includ-ing diabetes,uremia,and aging.14–17Further evidence for oxidative protein modifications in the atherosclerotic lesion comes from amino-acid analysis. Compared with normal arteries,oxidized lipoproteins and atherosclerotic samples contain higher concentration of several oxidative amino-acid products(i.e.methionine sulfoxide,o-tyrosine,m-tyrosine,3-chloro-tyrosine,3-nitro-tyrosine,hydroxyleucine,hydroxyvaline,and N e-CML.2,18–20 To monitor the action of oxidative stress on DNA,markers as 8-oxo-deoxyguanosine,1,N2-etheno-20-deoxyguanosine,and N-carboxyethylguanosine have begun to emerge.21Sources of ROS generationAs indicated in Table1,virtually all types of vascular cells were found to produce O2KÀand H2O2upon specific stimulation or in response to physical or metabolic stress. In addition to mitochondrial sources,O2KÀcan be generated by several enzymes including the vascular isoforms of NAD(P)H-oxidase(vascular NOX isoforms),‘uncoupled’NO synthase(s),cytochrome P450oxidase,xanthine oxidase, and cyclooxygenase.The most important enzymatic source is thought to be membrane-associated NAD(P)H-oxidase,33,34 which generates intracellular O2KÀand H2O2in vascular SMCs,endothelial cells,andfibroblasts.However,Nishikawa et al.24found that mitochondria are a major source of ROS in high glucose-treated endothelial cells.ROS induction by cytokines and growth factors Numerous mediators,cytokines,and growth factors have been shown to induce O2KÀproduction(Table1).These include angiotensin II,tumor necrosis factor-a,interleukin-1, platelet-derived-,epidermal-and transforming growth factor, and macrophage inducing factor1most of which via the activation of NAD(P)H oxidase.Moreover,ligand-stimulated RAGE activation(see below)results in the generation of ROS, at least in part,also via activation of the NAD(P)H oxidase.22 However,the origin of the cytokines is unclear.Different tissues may produce and secrete inflammatory cytokines,for example by adipose tissue(adipokines),by liver(hepato-kines),by muscle(myokines),and other sources.It is estimated that approximately30%of circulating interleukin-6is derived from adipose tissue.ROS induction by FFAsElevated triglycerides and elevated FFAs(and altered FFA pattern)are an earlyfinding in obese individuals with insulin resistance and diabetic patients often correlating with endothelial dysfunction.35In these patients at high risk for developing vascular disease,elevated FFA plasma levels derive from excessive dietary fat intake and/or increased adipose tissue lipolysis.The abnormally high plasma levels of particularly saturated fatty acids,palmitate and stearate, induce ROS in endothelial and aortic SMCs as determined by electron spin resonance30or redox-sensitive dyes.31,29,32 Because ROS formation could be prevented by NAD(P)HE Schleicher and U Friess:Oxidative stress,AGE,and atherosclerosisoxidase inhibition but not by blockade of xanthine oxidase, NO synthase,or mitochondrial electron transport and because ROS formation is also prevented by inhibition of protein kinase C,these results suggest a fatty acid-induced, protein kinase C-mediated NAD(P)H oxidase activation with subsequent ROS generation.Enhanced ROS formation leads to activation of nuclear factor-k B(NF-k B).Moreover,FFA-induced NF-k B activation has been observed to mediate cytokine gene expression(interleukin-6)in endothelial cells and to induce endothelial apoptosis.36,37NF-k B activation induced by saturated fatty acids has also been shown to occur in human myotubes.38Palmitate-induced ROS generation is also occurring in CHO cells.39,40The authors provide evidence that lipotoxicity is prevented if either the metabo-lism of the saturated FFA or the deposition in triglyceride is stimulated.Molecular mechanism of ROS generation by high glucose levels in cultured vascular cellsGlucose is taken up by cells via facilitate glucose transport by plasma membrane integrated glucose transporters(family of GLUTs).The GLUTs differ in their location and particularly in their biochemical characteristics and regulation.While essentially all cells,including vascular cells,express GLUT1 for basal glucose transport,the insulin-sensitive GLUT4is essentially only expressed in muscle and adipose tissue and therefore responsible for the majority of the insulin-stimulated glucose uptake after a meal.Both GLUTs are characterized by a low K m,approximately2–5m M.41Glucose transport rate is at about50–70%of maximal capacity at 5m M,65–82%at10m M,and73–88%at15m M extracellular glucose concentration(Figure2).Thus a doubled or tripled increase in extracellular glucose levels leads to an enhanced glucose uptake rate of approximately22or33%only.In liver glucose is taken up by the low affinity(K m¼22m M),high capacity GLUT2which is essentially dependent on glucose concentration.Although these data indicate that vascular cells are protected from excess glucose uptake when extracellular concentrations are high,several reports show an high glucose-induced increase in endothelial ROS content by electron spin resonance30or an redox-sensitive dye.31To elucidate the molecular mechanism analogs of D-glucose were used.Although elevated L-glucose concentrations wereTable1|Formation of ROS in vascular cells induced by different stimuli and metabolic factors(modified from Stocker and Keaney2and supplemented)Stimulus Cell type Source ROS Effect(s)Reference Ligands of G-protein coupled receptorsAngiotensin II EC,SMC NOX O2KÀ,H2O2NOÁformation,hypertensionCell hypertrophy,p38activation2RAGE/NF-k B activationAGEs,CML-albumin EC NOX,mitochondrial H2O2Proinflammatory,proadhesive andprocoagulant gene expression(e.g.VCAM-1,tissue factor)22,23Growth factorsPDGF SMC,FB NOX O2KÀ,H2O2Growth,induction of MCP-12EGF FB NOX H2O2Unknown2TGF-b1EC,FB NOX H2O2Growth inhibition2 CytokinesTNF-a EC,FB NOX O2KÀ,H2O2Unknown2IL-1b EC,FB NOX O2KÀ,H2O2Unknown2Physical stressShear stress EC eNOS+/Àunknown OONOÀ,O2KÀJNK activation2 Metabolic conditions(high glucose,elevated FFAs)HG EC,SMC,arterial rings NOX,mitochondrial O2KÀ,ROS Activation of PKC,NF-k BUpregulation NOX subunitsAccumulation of AGEs,sorbitol,inhibited bystatins24–28Palmitate EC NOX ROS Upregulation of NOX subunits,inhibited bypitavastatin29PalmitateÀ/+HG,EC,SMC NOX,mitochondrial ROS PKC activationInvolvment of AMP kinase,inhibited bymetformin,30,31FFAsÀ/+HG EC Unknown O2KÀEnhancement of HG induced O2KÀproduction by saturated4unsaturated FFAs32AGE,advanced glycoxidation end product;CML,carboxymethyllysine;EC,endothelial cell;HG,high glucose;FB,fibroblast;FFAs,free fatty acids;NF-k B,nuclear factor-k B; NOS,endothelial nitric oxide synthase;NOX,NAD(P)H oxidase isoforms;PKC,protein kinase C;RAGE,receptor for AGE;ROS,reactive oxygen species;SMC,smooth muscle cell; TNF-b2,tumor necrosis factor-b2.E Schleicher and U Friess:Oxidative stress,AGE,and atherosclerosisineffective,30m M of the non-metabolizable analogs D -2-deoxy-glucose or D -3-O -methyl-glucose,like D -glucose,induced ROS within 15–30min.The ROS induction is mediated by activation of NAD(P)H oxidase because inhibition of this enzyme completely prevented the high glucose-induced endothelial ROS formation (Schleicher and Klopfer,unpublished results).These results indicate that elevated extracellular glucose concentrations need to enter the cell for ROS generation but need not to be metabolized.According results have been reported by Lee et al .42in mesangial cells,however they found,similar to Nishikawa et al .,24that mitochondria are also an important source of ROS under hyperglycemic conditions.Together,the data indicate that hyperglycemia induces ROS in endothelial cells via enhanced uptake of glucose but not through increased metabolism of glucose although the detailed molecular mechanism remains unclear.Biological effects of ROSThe effects of O 2KÀand H 2O 2on vascular function depend critically on the amount produced.Intracellular H 2O 2has been shown to modulate the activity of kinases and phosphatases in several signaling pathways by the oxidation of regulatory cysteine residues and there is emerging evidence that mammalian cells do produce H 2O 2physiologically to regulate cell proliferation,differentiation,and migration.43When formed in subtoxic amounts,H 2O 2can act as a second messenger modulating such responses as growth of vascular SMCs and fibroblasts or the expression of inflammatory mediators and matrix components.34Higher amounts of ROS can cause oxidative modification of lipids,proteins,and DNA resulting in significant toxicity,DNA damage,and even apoptosis.One major consequence of increased oxidative stress wall is the increased formation of ALEs/AGEs (see also below).Several lines of evidence indicate that these products have awide variety of extra-and intracellular effects to pertubate vascular structure and cellular functions as recently reviewed by Goldin et al .44First,AGEs may alter properties of the major matrix proteins collagen,vitronectin,and laminin through AGE–AGE intermolecular crosslinks resulting in increased stiffness of the vasculature.Second,AGE/ALE modification of LDL and apolipoprotein B100leads to altered LDL metabolism.Adducts of lysine residues with malondialdehyde and 4-hydroxynonenal in apolipoprotein B-100alter LDL properties to become a ‘high uptake’form of LDL,which cannot longer interact with the receptor for native LDL and instead is recognized by scavenger receptor on macrophages.2Likewise,it could be demonstrated that CML-modified LDL,formed upon reaction of glyoxal and LDL,is not taken up via the LDL receptor.45Third,AGE formation on the extracellular matrix has been shown to alter not only vascular structure but also cellular function.Glycation of key molecules in the basal membrane such as laminin and type I and type IV collagen weakens adhesion of endothelial cells.AGE modification of matrix glycoproteins leads to disparities in proliferation,growth,and secretory activity of different vascular cell types,46thus resulting in altered liberation of cytokines and growth factors.The other important consequence of increased oxidative stress appears to be the direct modulation of redox-sensitive transcription factors and signaling pathways.Studies have suggested that the affinity of certain transcription factors,particularly NF-k B and activator protein 1(AP-1),for their cognate DNA binding site can also be directly modified by ROS.34,47Intracellular ROS regulate several classes of genes relevant to vascular pathology,including antioxidant en-zymes,vasoactive substances,cytokines,and growth factors.Although the exact mechanisms are not elucidated yet,many of the corresponding promoters contain NF-k B-and/or AP-1-responsive elements.34,47In endothelial cells it has been shown by the use of various antioxidants that cytokine-induced expression of the vascular cell adhesion molecule-1(VCAM-1),intracellular adhesion molecule-1,and the chemotactic factor monocyte chemoattractant protein-1involves NF-k B in a redox-dependent fashion.34,47GLYCATION,GLYCOXIDATION,AND FORMATION OF AGEs Formation of AGEsThe discovery of hemoglobin HbA1c and its structurally identification has shown that glucose reacts non-enzymati-cally with free amino groups of proteins yielding a Schiff base which,after Amadori rearrangement,forms a rather stable 1-amino-fructose moiety.The term glycation has been coined for this post-ribosomal modification of proteins.Further studies showed that this reaction is not restricted to hemoglobin and that most abundant plasma proteins such as albumin,g -globulin,fibrinogen,48high-density lipopro-tein,and LDL 49are glycated according to the average plasma glucose level of the respective patient.Glycation of plasma proteins depends also on their half-lives.50,51Furthermore,extracellular matrix proteins from lung,aorta,nerve,V max20510********0102030405060708090100Extracellular glucose concentration G ex (m M )R e l a t i v e g l u c o s e t r a n s p o r t r a t e V r e l (%)V max K m = 22 m MK m = 5 m M K m= 2 m MGLUT-2GLUT 1/4Figure 2|Vascular cells are protected from excessive glucose uptake.Vascular cells express GLUTs with K m between 2and 5m M (e.g.GLUT 1and GLUT 4),whereas the K m of GLUT 2,which is expressed in liver and the pancreatic b -cell,is 22m M .The graphs show the dependency of the relative glucose transport rate from glucose concentration.Vertical lines indicate glucose concentrations of 4,7.5,and 15m M (80,135,and 270mg/dl),respectively,to indicate the ranges of daily variations of blood glucose levels in nondiabetic and diabetic subjects.E Schleicher and U Friess :Oxidative stress,AGE,and atherosclerosisglomerular basement membrane,52and lens53are glycated to an extent which correlates to previous hyperglycemia. Although various attempts have been made to show an effect of protein glycation on the respective protein functions most of the studies used an extent of glycation which is far beyond that occurring in(patho)physiological conditions.54 Cerami’s group recognized that glycation(fructosamine moiety)is not stable under physiological conditions yielding fluorescent compounds after several weeks.55Because the products were derived from glycated proteins the term advanced glycoxidation end products(AGE product)was coined.Meanwhile,a number of AGE products have been identified(for review see Thornalley56).Because the AGE intermediates glyoxal and methylglyoxal may also be formed from lipoxidation the term ALE,in analogy to AGE,was suggested by Januszewski et al.57In addition methylglyoxal may derive from glycolysis intermediates.58Although a hyperglycemia/diabetes-associated increase in AGE/ALE in various cells/tissues has been shown in vitro and in experimental animals as(reviewed by Thorpe and Baynes59 and Vlassara and Palace60)and particularly in cultured endothelial cells,61,25there are only a few reports on enhanced accumulation of AGEs in the vessel wall.In an immunohis-tochemical study using a polyclonal antiserum specific for the CML modification,a marked increase of CML accumulation was observed in atherosclerotic plaques and in foam cells. Moreover,staining was more pronounced in diabetic patients.16,62CML forms early on in atherogenesis and dense intracellular CML deposits were found in macrophage-derived foam cells in the stages of intimal thickening and fatty streak lesions.15Schalkwijk et al.63studied CML accumulation in diabetic and nondiabetic heart.Staining with a monoclonal CML-antibody was approximately sixfold higher in hearts from diabetic patients as compared to control hearts.CML deposition was localized in endothelial and SMCs of small intramyocardial arteries that showed no morphological abnormalities.In patients with acute myo-cardial infarction,CML depositions were threefold increased compared with controls in the small intramyocardial blood vessels and predominantly colocalized with activated en-dothelium(E-selectin-positive)both in infarction and noninfarction areas.63From their data the authors concluded that CML might reflect an increased risk for acute myocardial infarction rather than being a result.Noteworthy,AGE/ALE modification is not limited to protein DNA may be modified too.Accordingly,N-carboxyethylguanosine staining was increased in aortas of diabetic and uremic patients.21 BIOLOGICAL EFFECTS OF AGE:ACTIVATION OF THE AGE/ RAGE/NF-j B-AXISPrevious results showed that AGE may form crosslinks between proteins leading to alteredflexibility and digestibility of the collagen matrix in vascular wall and in skin.However, with the characterization of the receptor for AGE(RAGE)it became clear that AGE may exert their biological effects via amplification through a receptor-coupled signaling pathway.Although other receptors for AGE have been described,most available data have been collected with RAGE.64,65This receptorfirst described as binding protein for AGE by A.M. Schmidt66is a multiligand member of the immunoglobulin superfamily.AGEs may interact with endothelial RAGE to activate cellular events such as upregulation of the transcrip-tion factor NF-k B,67activation of NAD(P)H-oxidase22and activation of p38MAP kinase and ERK1/2MAP kinase cascades.Recently S100/calgranulins,a family of calcium-binding polypeptides that accumulate at sites of chronic inflammation,and HMGB1(amphoterin)have been identi-fied as important activators of the RAGE/NF-k B axis.NF-k B is translocated to the nucleus where it increases transcription of RAGE itself and a number of genes relevant to atherogenesis including VCAM-1,intracellular adhesion molecule-1,tissue factor,endothelin-1,and likely,proin-flammatory cytokines including interleukin-1b,interleukin-6,and tumor necrosis factor-a(as reviewed by Goldin44). AGE/RAGE-mediated gene expression involves,at least in part,intracellular ROS formation.Basta et al.23reported that exposure to CML-albumin led to an increased surface expression of VCAM-1in endothelial cells and to intracel-lular ROS formation by a RAGE-dependent mechanism.Both effects were inhibited by specific anti-RAGE antibody and by inhibition of the NAD(P)H-oxidase.The stimulation of RAGE gene expression by RAGE-mediated signaling indicates triggering of a vicious cycle.Once increased ROS formation is started it may be perpetuating or even enhanced if the stimuli are chronically present.Taken together,the production of ROS,AGE,cytokines,and other gene products involved in atherogenesis are interlinked and may be as outlined in the scheme shown in Figure3.LESSONS FROM ANIMALSFirst insights on how ROS and AGE/ALE formation might interfere with cellular stress response and inflammation,thus promoting progression of the atherosclerotic lesion,came from experimental animal models.The ApoEÀ/Àmouse model allows to dissect the contribution of hyperglycemia,of ROS generating enzymes(such as NADPH oxidase)or of the AGE/RAGE/NF-k B axis to atherogenesis and was used by several groups.ApoEÀ/Àmice are hyperlipidemic with markedly elevated cholesterol and triglyceride levels and display initial atherosclerotic lesions including fatty streaks and foam cell formation as early as at8weeks of age. Advanced atherosclerosis is evident at15weeks of age. ApoEÀ/Àmice were additionally rendered diabetic,either by streptozotocin,68representing a type I diabetes equivalent,or by breeding into a db/db background,69,70representing a type II diabetes model,to study the influence of hyperglycemia and diabetes as additional risk factors.In both combined hyperlipidemia/diabetes models development of athero-sclerosis was accelerated and more advanced.Increased lesion complexity of the aortic vessel wall was found to be accompanied by enhanced AGE formation,enhanced RAGE expression and increased levels of the proinflammatoryE Schleicher and U Friess:Oxidative stress,AGE,and atherosclerosis。

Aero-thermal performance improvements of unshrouded turbines through management of tip leakage and injection flowsJie Gao *,Qun Zheng,Zhengyi Zhang,Yuting JiangCollege of Power and Energy Engineering,Harbin Engineering University,Harbin 150001,Chinaa r t i c l e i n f oArticle history:Received 25August 2013Received in revised form 12March 2014Accepted 15March 2014Available online 13April 2014Keywords:Cavity tipTip leakage flow Tip injectionTip clearance height Injection mass flow rate Aero-thermal performancea b s t r a c tThe tip leakage flow not only is responsible for a signi ficant amount of aerodynamic losses in a turbine stage,but also leads to high heat-loads on the tip region.The paper presents a numerical investigation of in fluences of tip injection on aero-thermal performance of the tip leakage flow for both flat tip and cavity tip in an unshrouded turbine rotor,in an attempt to improve the turbine blade tip aero-thermal per-formance by the management of tip leakage and injection flows.The effects studied include the sensi-tivities to the geometrical clearance height and the injection mass flow rate.The results show that,at all tip clearances,tip injection has a good effect on the control of the leakage flow,and it signi ficantly re-duces the sensitivities of turbine performances to the effects of the tip clearance height.With tip in-jection,cavity tip geometry does not play a major role in the turbine performance improvement,but it obtains the good film-cooling performance on the blade tip.Tip clearance height,blade tip geometry and injection mass flow rate have related effects on the blade tip aero-thermal performance,and the turbine blade tip obtains the best aero-thermal performance with an optimum injection mass flow rate.Ó2014Elsevier Ltd.All rights reserved.1.IntroductionTurbomachinery has seen widespread use in the industry (Wu et al.[1],Gomes et al.[2]and so on);however,the tip clearances between rotor tips and the casing wall,which are necessary to prevent rubbing,result in an undesirable loss of ef ficiency,driven by the pressure difference between the blade pressure side (PS)and the suction side (SS).And,tip leakage flow is believed to be detri-mental to the performance of turbomachinery (Booth et al.[3]and Mohamed and Shaaban [4]).The work output of the rotor decreases because the leakage flow passes through the tip gap without being properly turned and expanded.Thus,tip leakage flow acts as an obstruction and leads to loss generation inside the rotor passage.Booth et al.stated that a tip gap of about 1%of the rotor span could cause a loss of 1e 3%on the stage ef ficiency.Moreover,another important consequence is the deterioration of the blade tip surface,due to regions of high local heat transfer in the narrow gap caused by flow separation and reattachment.Metzger and Rued [5]per-formed fundamental studies and showed that the generated tip leakage flow increases in the heat transfer of about 200%near thetip gap.Therefore,it is important to take measures to minimize the impact of tip leakage flow.In previous studies,several different methods were investigated to control the effects of the tip leakage flow.These methods are generally divided into passive control (no energy import)and active control (energy import)from the viewpoint of the energy.Most of the passive control methods reported involve modi fication of the tip surface geometry in an effort to decrease the leakage mass flow rate,including winglets (Zhou et al.[6]),squealer tips (Lee and Chae [7]),tip surface optimization (Maesschalck et al.[8]),casing treatments (Gao et al.[9])and so on.Of these methods reviewed only the cavity tip (double squealer tips)finds actual application in service.Double squealer rims offer the advantages of minimizing contact surface area and partly shielding cavity surface from ther-mal degradation.The other methods presented added cooling re-quirements that may not be easily addressed.Generally the active turbine clearance flow control can be ach-ieved by means of mechanical force,plasma actuator and tip in-jection.In order to reduce the blade tip clearance as far as possible,a first generation mechanically actuated active clearance control (ACC)system has been designed and fabricated (DeCastro and Melcher [10]).The system utilizes independent actuators,a segmented shroud structure,and a clearance measurement feed-back to provide fast and precise active clearance control throughout engine operation.In addition,the control of the tip leakage flow*Corresponding author.Tel.:þ8613895710245.E-mail addresses:gaojie_d@ ,gaojie_d@ (J.Gao).Contents lists available at ScienceDirectEnergyjournal ho me page:www.elsevier.co m/locate/energy/10.1016/j.energy.2014.03.0600360-5442/Ó2014Elsevier Ltd.All rights reserved.Energy 69(2014)648e 660using plasma actuators mounted to a turbine blade tip was inves-tigated in a linear cascade facility by Van Ness et al.[11].The active control results indicated that the actuators led to qualitative changes in the structure of downstream wake profiles;the tip leakageflow can lead to a significant increase in emission and specific fuel consumption in the high-pressure turbine.The tip injection has showed potential success for increasing the aerodynamic performance around the blade tip regions.Chen et al.[12]studied the aerodynamics of tip injection using a two-dimensional model.They concluded that when the coolant is ejected normal to the tip,it is able to block the leakageflow.Li et al.[13]carried out a numerical simulation of the effects of tip injec-tion.They suggested that the jet could obstruct the tip leakageflow and weaken the interaction between tip leakageflow and main passageflow,improving the turbine efficiency by0.41%when the clearance gap is small.Rao and Camci[14e16]conducted an experimental study of a turbine tip desensitization method based on tip injection in a large-scale rotating turbine rig,and took the effect of the injection mass flow rate and the injection location into consideration.It was found that tip injection could cause the tip leakage vortex to be reduced, and its associated losses decreased to the level observed for the case with half the tip gap height.And,the tip injection at81%blade axial chord was most successful in reducing the total pressure deficit in the leakage vortex.It was shown that such information would be useful in optimizing tip injection schemes.Niu and Zang [17e19]performed parametric investigations of tip injection in a high-turning axial turbine linear cascade,including injection circumferential angle and injection location in the blade thickness direction.The results showed that the nearer to the PS corner the injection holes are,and the smaller the injection circumferential angle is,the more tip injection can affect the tip leakageflow.Be-sides,injection chordwise location also plays an important role in the redistribution of the secondaryflow within the cascade pas-sage,and the holes located in the aft part of blade can perform much better than those in the front part.Yet,the effect of the relative blade-casing motion on theflowfield and heat transfer was not investigated here.Based on the above investigations,they[20] investigated the influence of tip injection at design and off-design incidences.It was found that even at off-design conditions,tip in-jection can still perform well in reducing the tip leakage massflow and its associated losses.However,the potential thermal benefits were not investigated in their research.The tip coolant injection has also been used to protect the blade tips from the high gas temperatures.Hohlfeld et al.[21]and Couch et al.[22]conducted experimental and computational in-vestigations on blowing from the dirt purge holes of aflat tip in a linear cascade.They stated that theflow ejected from the dirt purge holes is able to block the tip leakageflow.The obstruction effect by tip injection performed quite well for a small tip gap,but it was not effective for a large tip gap.It should be noted that,the main focus of their research was the thermal performance of the tip.Newton et al.[23]provided detailed measurements of heat transfer condition on the tip of a general cooled turbine blade,and reported that the injection at the location of separation bubble could result in a better cooling benefit.Kwak and Han[24]inves-tigated thefilm cooling of gas turbine blade tips in a linear cascade. Blade tip heat transfer coefficients were found to increase with the tip clearance height increasing.Thefilm cooling from radial holes along the camber line was shown to be more effective at larger clearances and at higher blowing ratios;however,the aerodynamic performance of tip cooling is unknown.Also,Acharya et al.[25] computationally simulated thefilm cooling of turbine blade tips. Thefilm cooling at three different tip clearances was simulated.The tip injection was found to alter the leakage vortex and also to decrease the heat transfer coefficient along the coolant trajectory;film-cooling effectiveness was found to increase slightly with the tip clearance size.Nowadays,some modern,unshrouded turbine blades have some sort of squealer tips.Mhetras et al.[26]investigated the ef-fects of tip injection on heat transfer conditions on a squealer tip. They stated that due to the combined effect of squealer tip and tip injection,significant improvements could be obtained on the cavity floor and inner rim walls,and a larger cavity depth or higher blowing ratios could give higher effectiveness on all tip surfaces, whereas the overall mixing of cooling air and hot leakageflow was rarely mentioned.Han et al.[27,28]investigated thefilm cooling of bothflat tip and cavity tip in a linear cascade.They found that the film cooling ejections on both plane tip and squealer tip dramati-cally affect theflow behaviors at the tip region,and the coolant injected from the holes on the blade PS produced an additional reduction in the blade tip heat transfer coefficient.Besides,the cooled cavity tip is superior to the cooledflat tip in terms of the thermal performance;however,this labyrinth andfilm cooling design can lead to a coolant accumulation in the squealer cavity.In order to allow the accumulated coolant in the cavity to escape,and meanwhile to cool the tip TE(the trailing edge),a piece of rim wall of PS squealer near the TE is cut.The literature mentioned above were focused either on the aerodynamic performance of the tip leakageflow with tip injection or on the thermal performance of cooled blade tips.Only few in-vestigations have been focused on the aero-thermal performance of the tip leakageflow with injection as well as the combined effect of cavity tip and tip injection.The objectives of this paper are to systematically investigate the physics and loss mechanisms of the tip leakageflow with tip injection;to assess the combined effect of cavity tip and tip injection;to compare the aero-thermal perfor-mance of the tip leakageflow offlat and cavity tips with injection and to examine the effects of tip clearance height and injection massflow rate.Numerical methods(similar methods have been successfully used for the study on a small scale horizontal axis wind turbine in Ref.[29])have been employed to make a comprehensive understanding of the aero-thermal performance of the tip leakage flow with injection onflat and cavity tips.2.Tip injection configurationsIn this investigation,results are presented for bothflat and cavity tips with and without injection.The design clearance is1.5%Fig.1.Schematic of blade tip geometry and mesh with injection holes.J.Gao et al./Energy69(2014)648e660649of blade span.Fig.1shows the blade tip geometry with injection holes.For the squealer tip,the depth of the cavity is2%of the blade span,and the width of the shoulder is0.77mm,which forms a narrow rim and a wide cavity.For all injection cases,there are four radial injection holes placed on each blade tip.The injection holes are assumed to be1mm in diameter and20mm in length,and are located at10%,30%,50%and70%blade axial chords.The blade geometry chosen for the investigation is taken from GE-E3first stage highly loaded turbine rotor as shown in Fig.2, which represents a modern gas turbine blade geometry.The rotor blades are twisted,and have a constant axial chord length of 28.7mm and an aspect ratio(span to chord)of1.39.The main blade geometry parameters are shown in Table1,and detailed blade geometry and operating conditions can be found in the NASA report(Timko[30]).3.Numerical techniquesThe numerical investigation presented in the paper was per-formed by using ANSYS CFX11.0(AEA[31]),an available general finite volume based Navier e Stokes solver.The solutions are ob-tained by solving the compressible Reynolds-Averaged Navier e Stokes equations using afinite volume method to discretize the equations.The overall accuracy is of the second order.A standard k e u two-equation model developed by Wilcox[32]is used in the investigation for turbulence closure,and no wall functions are used.The commercial structured grid generation package Autogrid5 (preprocessor to NUMECA)was used in the investigation.Fig.2 shows the representative computational grid of the rotor blade used.The channel was discretized into H-type grids,while the re-gions around the blade surface and the tip gap were discretized into O-type grids to ensure high grid quality.Also,details of the cavity tip mesh can be seen more closely as shown in Fig.1.The tip gap and the cavity were modeled with butterfly mesh topology.There are17nodes in the boundary layer to provide grid-independent results,and there are33cell layers distributed from the blade tip to the casing wall with23points across each squealer.Additionally, 27grid nodes per holes diameter had been ensured everywhere inside the injection holes,with33nodes in the hole length direc-tion.There are over100points from the leading edge(LE)toward the trailing edge(TE)along the blade chord.The average of the dimensionless distance,yþ(of the closest grid point from wall)on the wall is less than1,and the maximum yþon the wall is about3. In order to keep the grids for the cases considered in the investi-gation as similar as possible,the same grid topology is adopted for all the cases considered.The computational domain consisted of a single blade channel with periodic boundary conditions imposed along the circumfer-ential direction.At the inlet,which is placed at one axial chord length upstream of the rotor blade,absolute total pressure,absolute total temperature andflow angle are specified along with the tur-bulence intensity.At the exit,which is placed at a distance equal to two axial chord lengths downstream of the rotor blade,static pressure is specified.At the inlet of injection holes,injection mass flow rate and static temperature(350K)are also predefined.The isothermal wall condition(478.1K,a scaled experimental value)is imposed on the blade tip,and the other walls are assumed to be adiabatic.In addition,the no-slip condition is applied to all walls. Detailed boundary conditions are listed in Table1.Convergence criteria for these computations are based on the reduction of RMS residuals below1Â10À5.All results have been checked for convergence by using isentropic efficiency monitoring, and the solution was said to be converged when the isentropic efficiency was not changed more than0.01%during10successive iterations.Typically,500iterations were necessary for convergence.Grid independence is checked by comparing the solutions on a flat tip blade with injection obtained with1.3million nodes and the solutions obtained with1.6million nodes.From the distribution of the heat transfer coefficient on the blade tip,it is found that results from the simulation with 1.6million nodes are in reasonable agreement with that with1.3million nodes.Besides,there are few changes in the tip leakage loss when increasing the grid nodes from 1.3to1.6million.Thus,the total number of grid nodes for theflat tip with injection is1.6million in the investigation.Likewise,the total number of grid points for other cases ranges from1.4million to1.9Fig.2.3D computational grid and details of the LE,TE and tip grids.Table1Blade geometry andflow conditions.Parameters ValuesTip(outer)diameter732mmBlade number76Axial chord28.7mmInlet Mach number(relative)0.37Absolute total pressure ratio 2.25Absolute inlet angle from axial direction(hub)69degAbsolute inlet angle from axial direction(midspan)74.5degAbsolute inlet angle from axial direction(shroud)78.5degRotational speed8450r/minInlet turbulence intensity5%parison of heat transfer coefficient on the blade tip:(a)experimental result,and(b)predicted result.J.Gao et al./Energy69(2014)648e660650million cells,with the more complex tip geometry requiring more cells.In order to validate the ability of the standard k e u turbulence model to predict the blade tip leakage flow and heat transfer,a linear cascade studied by Azad et al.[33]was calculated.The cases is a no-injection flat tip blade with a 1.5%tip clearance and tur-bulence intensity Tu ¼6.1%.Since Azad ’s linear cascade data are often used for validation,only cascade heat transfer dataareFig.4.Streamlines near the blade tip (a)without and (b)with injection at a tip clearance of1.5%.Fig.5.Velocity magnitude contours near the blade tip (a)without and (b)withinjection.Fig.6.Entropy-increase contours near the blade tip (a)without and (b)with injection at 30%,50%,80%and 110%axial chords.J.Gao et al./Energy 69(2014)648e 660651presented here for the sake of brevity.Fig.3compares the measured and predicted heat transfer coefficient distributions on the blade tip without injection,and it should be noted that the uncertainty estimate in the heat transfer coefficient reported by Azad is7.9%.The regions of high heat transfer coefficient are located along the PS where theflow accelerates into the tip-gap region.The location and the size of the predicted“sweet spot”region,as well as the corresponding heat transfer coefficients are in excellent agreement with the measurements.In view of the experimental uncertainties,the measurements and predictions appear to exhibit quite reasonable agreement with each other,with the magnitude of the peak predicted heat transfer coefficient agreeing with the cor-responding measured value to within12%.Although there is no experimental data available to validate the present CFD results for the injection cases,as nowadays accepted in open literature,the use of a proper grid with a reliable turbulence modeling provides a modeling of satisfactory accuracy.4.Results and discussionAs a by-product of the presentflow and heat transfer compu-tations,it is possible to compute the isentropic efficiency and the possible effect of tip coolant injection on turbine efficiency. Despite40years development of gas turbine cooling technology, there is no general agreement on the most appropriate definition of cooled turbine efficiency;the critical issue is the choice of a hypothetical“ideal”process.Based on the review of the definitions in use,Young and Horlock[34]presented new proposals for overcoming the problems to compute the inlet total pressure and inlet total temperature after mixing.Thus,the isentropic efficiency can be calculated in terms of the natural turbine efficiency formula.The mixing process is defined to be adiabatic and the total temperature after mixing T0m can be calculated from the steady-flow energy equation,written for semi-perfect gases as,ð1ÀfÞZ T0mT01g c pgðT0Þd T0þXif iZ T0mT01c ic pcðT0Þd T0¼0(1)The total pressure after mixing p0w is given by,p0w 01g ¼Yip01c i01g!ðfiR C=RÞ(2)where f i¼m c i/(m gþPm c i),f¼P fi and the summation is over then coolant streams.c pg is specific heat related with the total tem-perature T0.R denotes the specific gas constant,and p0is the total pressure.The subscripts c i,g and m denote the i th coolant stream, mainstream gas and mixed conditions.Because the blade tip surface temperature was set at a constant temperature of478.1K,in order to recover adiabatic conditions,the mixed-out total temperature at the exit must be modified to reflect the loss of energy through heat transfer.The change in mass-averaged normalized total temperature at the exit of the blade row can be computed as follows,D T0;ex¼P ZAðm v T=v n j wÞd Am RePr(3)In the above equation m is dimensionless viscosity and n is the normalized distance to the wall;m is the normalized massflow rate through the blade passage and A is the surface area.The integration is performed over all of the heat transfer surfaces.Thus,the mass-averaged value of the exit total temperature is computed as,T0;ex¼T unmodifiedþD T0;ex(4) Also,the effect of non-adiabatic wall conditions must be taken into account in the calculation of entropy-increase D S.4.1.The physics and loss mechanisms of tip leakageflow with tip injectionFlow patterns over theflat tip with and without injection at a tip clearance of1.5%are shown in Fig.4.It shows that the coolant ejected from the injection holes reduces the overall tip leakageflow that enters the tip gap in two ways.Firstly,the coolant is injected normally into the tip gap and has little momentum in the direction of the tip leakageflow.The tip leakageflow that crosses the PS of the tip gap collides with the coolant over the tip gap.As shown in Fig.4(b),several streamlines suddenly change the direction in the tip gap,which is due to the collision with the leakageflow from upstream.This happens in a region where the leakageflow velocity is locally low as shown in Fig.5.Secondly,the coolant mixes with theflow across the tip gap downstream of the injection holes,and it slows the leakageflow.Also,Fig.4shows that several leakage streamlines turn upstream of the injection holes(called the “reverse leakageflow”)with the effect of the coolant.Additionally, it is found that with tip injection,the leakageflow is turned toward the SS edge of the blade,which can also be proven from Fig.5.Fig.5shows velocity magnitude contours on the blade tip sur-face with and without tip injection.In Fig.5(a),theflow accelera-tion in the tip-gap region can clearly be seen(except in the“sweet spot”LE region),and theflow decelerates as the leakageflow rolls up into a vortex along the SS.With tip injection as shown in Fig.5(b),due to the lower injection velocity,the tip-gap region is characterized by the accelerating leakageflow interspersed by lower velocity coolant streaks.The momentum exchange between the coolant and theflow that enters the tip gap is expected to block and deflect the tip leakageflow.Thus,it can be concluded that the coolant jets are turned toward the blade SS and appear to form a film on the tip surface.It should be noted that the injection results in the extra mixing losses,although it blocks the leakageflow.In order to judge the overall effect of tip injection on the tip leakage losses,entropy-increase contours near the blade tip with and without injection at0.20.40.60.81.01.21.4Efficiency-increaseLeakage-decreaseValue(%)Injectionmass flow rateparison of tip leakage-decrease and efficiency-increase between without and with tip injection forflat and cavity tips.J.Gao et al./Energy69(2014)648e660 65230%,50%,80%and 110%axial chords are shown in Fig.6.The injection flow signi ficantly alters the nature of the leakage vortex,and also alters the SS passage flow in the vicinity of the tip.With tip injection,the most noticeable phenomenon is the dramatic reduction of the tip leakage vortex in both its strength and size;the passage vortex is signi ficantly weakened due to the changed endwall flow fields.Overall,the turbine performance has been signi ficantly improved.Denton [35]indicated that,the leakage loss depends both on the proportional leakage flow and on the magnitude of the velocity difference between the leakage flow and the main flow with which it mixes.The coolant is injected normally into the tip gap,and then it mixes with the flow across the tip gap downstream of the in-jection holes.After exiting the tip gap,they mix with the main flow inside the blade passage,forming the tip leakage vortex.It can be concluded that,the tip leakage flow and the coolant have approx-imately the same magnitude of the velocity difference.Therefore,the overall effect of tip injection on the turbine performance de-pends on the relative magnitude of the injection mass flow and the reduction in the tip leakage mass flow.If the tip injection can reduce the flow which enters the tip gap from the PS by more than the injection mass flow rate,the turbine performance would be improved,and vice versa.The above analyses can also be proven from Fig.7.The tip leakage and injection mass flow rates are reported as percentage of total turbine mass flow.The leakage-decrease values were ob-tained by calculating the leakage difference between with and without tip injection;so did the ef ficiency-increase values.It is shown that,with tip injection,the tip leakage flow decreases dramatically,and the reduction in the tip leakage flow is larger than the injection mass flow rate.The results in the reduction in the mass flow that exits out of the tip gap from the SS,which increases the turbine ef ficiency obviously.In addition,the cavity tip with injection performs worse performance in controlling the tip leakage flow than the flat tip with injection.This means that there is a mutual coupling between the tip injection and the blade tip geometry.bined effects of cavity tip and tip injection on aero-thermal performance of tip leakage flowsFig.8shows the comparison of velocity vectors and Mach number distribution on flat and cavity tips with an injection mass flow rate of 0.4%.The cut plane passes through the center of the second hole from the LE,and it is parallel to the direction of thetipparison of velocity vectors and Mach number distribution on flat and cavity tips with injection mass flow rate of 0.4%at tip clearances of 1%and 2%:(a)without injection,1%,(b)with injection,1%,(c)without injection,2%,and (d)with injection,2%.J.Gao et al./Energy 69(2014)648e 660653leakage flow.As shown in Fig.8(a),for a flat tip the flow in the blade tip region is directed primarily from the PS to the SS,with the leakage flow rolling up into a vortex as it encounters the SS passage flow.For a cavity tip,the flow inside the cavity is quite complex andstrongly three-dimensional.The flow that enters the tip gap from the LE is directed toward the PS rim,and bounces off the rim surface toward the SS eventually exiting the gap near or past mid-chord,rolling up into a vortex that createsblockage.parison of heat transfer coef ficient distribution on flat and cavity tips at tip clearances of 1%and 2%:(a)without injection,1%,(b)with injection,1%,(c)without injection,2%,and (d)with injection,2%.x /hEntropy-increase (J/(kg·K))x /hEntropy-increase (J/(kg·K))Fig.10.Mass-averaged entropy-increase distribution along the span at rotor exit for flat and cavity tips:(a)1%and (b)2%tip clearances.J.Gao et al./Energy 69(2014)648e 660654As shown in Fig.8(b),for a flat tip with injection,the coolant is injected normally into the tip gap and counteracts with the tip leakage flow passing the tip region near the PS.This blocks the tip leakage mass flow and leads to low momentum activity between the injection holes and the SS corner.At the same time,coolant jets are turned toward the SS of the tip gap.For a cavity tip with in-jection,the coolant first mixes with the flow in the cavity,then it enters the tip gap,and finally it exits the tip gap along the SS squealer of the cavity tip.Thus,it can be concluded that due to the collision of the coolant and the flow that enters the tip gap from the PS,the momentum exchange is smaller than that in the case of the cooled flat tip.Besides,it can be seen that,over the PS inlet of the tip gap,the colors indicate that when coolant is used,the velocity magnitude is lower than that in the uncooled case.This shows that the leakage flow across the tip PS is partially blocked by the coolant injection.At the tip SS,the velocity magnitude is also low in regions where the coolant exits the tip gap.With the effect of tip clearance heights,it is shown that,for the small tip clearances,the coolant from the injection holes fills the tip gap.For the large tip clearances,however,there is physically enough space for the jets to impinge on the outer shroud and then reattach to the blade tip.This means that,with the same injection mass flow rate,the coolant creates a proportionally larger blockage at the smaller tip clearances.Also,Hohlfeld et al.[21]and Couch et al.[22]found that the blockage effect of the coolant is more evident at the smaller tip clearances.The comparison of the heat transfer coef ficient distribution on flat and cavity tips with an injection mass flow rate of 0.4%is shown in Fig.9.It can be seen that without tip injection,the high heat transfer coef ficient occurs near the LE,where the passage flow is driven into the tip gap by higher pressure near the stagnation point of the blade.For the cavity tip,the separation vortex has the characteristics of separation and reattachment,occupying about half of the cavity region.The point of reattachment corresponds to high heat transfer coef ficients on the cavity floor.When the coolant is injected from the blade tip surface,the heat transfer coef ficient along the coolant trajectory decreases signi ficantly.Particularly for the cavity tip,the heat transfer coef ficient is lower due to coolant accumulation inside the tip cavity.However,as the tip clearance increases,the lateral spreading of the coolant jets becomes small,caused by the increased peak velocities in the leakage flow path.Additionally,for the flat tip,high heat transfer coef ficients still occur near the LE,where no injection holes are located.However,for the cavity tip,the heat transfer coef ficient near the TE of the blade tip is reduced obviously due to coolant accumulation from upstream holes in the tip cavity.This recon firmed that the leakage flow is turned toward the blade SS edge,which reduces the magnitude of the velocity difference between the leakage flow and the main flow,and then the tip leakage losses.It can also be seen that the effect of coolant injection on heat transfer coef ficients is relatively large for the small tip gaps (s /h ¼1%),with a reduced effect on the large tip gaps (s /h ¼2%).This is because,for the small clearance gap case,the amount of leakage flow is small,and the coolant injection has relatively large effect on heat transfer coef ficients.However,for the large clearance gap case,because the amount of leakage flow is large,the effect of coolant injection on heat transfer coef ficients is less important.The dominating secondary flow phenomena in the rotor passage are the tip leakage vortex and the passage vortex,and there ism L (%)τ/h (%)ξ(%)τ/h (%)ξm i x i n g (%)τ/h (%)ηi s (%)τ/h (%)Fig.11.Effects of tip clearance on (a)tip leakage flow rate,(b)losses inside the tip gap,(c)caused by the mixing when the leakage flow leaves the tip gap,and (d)isentropic ef ficiency for flat and cavity tips with and without injection.J.Gao et al./Energy 69(2014)648e 660655。

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The average molecular kinetic energy, KE, is directly proportional to the absolute temperature , T, and the number of molecules, N, is proportional to the number of moles, n .Substitution of these terms requires the inclusion of a constant since these are proportionalities and not equalities .The required constant can be combined with 2/3 the(also a constant) , and thus
The kinetic theory postulates the following for an ideal gas:
(1) Gases consist of molecules widely separated in space . The total volume of the molecules is negligible in comparison with the volume of the gas as a whole .Certain gases, for example the noble gases, are monoatomic .In discussions of the kinetic theory of gases, the word molecule is used to designate the smallest particle of any gas. (2)Gas molecules are in constant, rapid, straight-line motion colliding with each other and their container.These collisions are perfectly elastic;energy may be transferred from molecule to molecule , but there is no net decrease in kinetic energy .

tpo68三篇托福阅读TOEFL原文译文题目答案译文背景知识阅读-1 (2)原文 (2)译文 (5)题目 (8)答案 (15)背景知识 (17)阅读-2 (20)原文 (20)译文 (24)题目 (27)答案 (35)背景知识 (37)阅读-3 (41)原文 (41)译文 (44)题目 (47)答案 (54)背景知识 (55)阅读-1原文Salt and the Rise of Venice①The city of Venice, on Italy’s coastline, achieved commercial dominance of southern Europe during the Middle Ages largely because of its extensive trade in the valuable commodity of salt. At first, Venice produced its own salt at its Chioggia saltworks. For a time its principal competitor in the region was the town of Cervia, with Venice having the advantage because Chioggia was more productive. But Chioggia produced a fine-grained salt, so when Venetians wanted coarser salt, they had to import it. Then, in the thirteenth century, after a series of floods and storms destroyed about a third of the salt-producing ponds in Chioggia, the Venetians were forced to import even more salt.②That was when the Venetians made an important discovery. More money could be made buying and selling salt than producing it. Beginning in 1281, the government paid merchants a subsidy on salt landed in Venice from other areas. As a result of this assistance, shipping salt to Venice became so profitable that the salt merchants could afford to ship other goods at prices that undersold theircompetitors. Growing fat on the salt subsidy, Venice merchants could afford to send ships to the eastern Mediterranean, where they picked up valuable cargoes of Indian spices and sold them in western Europe at low prices that their non-Venetian competitors could not afford to offer. That meant that Venetians were paying extremely high prices for salt, but they did not mind expensive salt if they could dominate the spice trade and be leaders in the grain trade. When grain harvests failed in Italy, Venice would use its salt income to subsidize grain imports from other parts of the Mediterranean and thereby corner the Italian grain market.③Unlike the Chinese salt monopoly, the Venetian government never owned salt but simply took a profit from regulating its trade. Enriched by its share of sales on high- priced salt, the salt administration could offer loans to finance other trade. Between the fourteenth and sixteenth centuries, a period when Venice was a leading port for grains and spices, 30 to 50 percent of the tonnage of imports to Venice was in salt. All salt had to go through government agencies. The salt administration issued licenses that told merchants not only how much salt they could export but also to where and at what price. The salt administration also maintained Venice’s palatial public buildings andthe complex hydraulic system that prevented the metropolis from washing away. Many of Venice’s grand statues and ornamental buildings were financed by the salt administration.④Venice carefully built its reputation as a reliable supplier, and so contracts with the merchant state were desirable. Venice was able to dictate terms for these contracts. In 1250, when Venice agreed to supply Mantua and Ferrara with salt, the contract stipulated that these cities would not buy salt from anyone else. This became the model for Venetian salt contracts. As Venice became the salt supplier to more and more countries, it needed more and more salt producers from which to buy. Merchants financed by the salt administration went farther into the Mediterranean, buying salt from many distant sources. Wherever they went, they tried to dominate the supply, control the saltworks, and even acquire them if they could.⑤Venice manipulated markets by controlling production. In the late thirteenth century, wishing to raise the world market price, Venice had all saltworks on the Greek island of Crete destroyed, and it banned the local production of salt. The Venetians then brought in all the saltneeded for local consumption, built stores to sell the imported salt, and paid damages to the owners of the saltworks. The policy was designed to control prices and at the same time keep the locals happy. Aiding its ability to ruthlessly manipulate commerce and control territory, Venice maintained the ships of the merchant fleet as a naval reserve and called them into combat when needed. The Venetian fleet patrolled the Adriatic Sea, stopped ships, inspected cargo, and demanded licensing documents to make sure all commercial traffic was conforming with its regulations.译文盐和威尼斯的崛起①位于意大利海岸线上的威尼斯城在中世纪期间在南欧取得了商业主导地位，主要是因为它广泛从事有价值的盐贸易。

EXPECTATION
Gold atoms SHOULD diffuse in a direction so as to (a) smoothen the surface (capillary action) or (b) oppose concentration gradients
OBSERVATION
Growth Kinetics of Pt-NPGL
Deposition may be controlled to within 0.01 mg/cm2 (1 ml) using only room temperature benchtop chemistry. Deposition stops prior to filling of pores. (?!)
Y. Ding, Y.-J. Kim, J. Erlebacher, “"Nanoporous Gold Leaf:"Ancient Technology"/Advanced Material”, Adv. Mat., 16 (2004), 1897.
NPG: The Fundamental Problem
– “Au adatoms” and “electrolyte”
• Examine the free energy of the mixture vs. Au coverage
f (C Au )= u - Ts
Au-Au bond energy Au-“electrolyte” bond energy “electrolyte”-“electrolyte” bond energy ideal entropy of mixing
thermal stability issues

Chemistry and Physics of Lipids 164 (2011) 205–210Contents lists available at ScienceDirectChemistry and Physics ofLipidsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h e m p h y s l ipKinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesiclesKenji Fukuzawa a ,∗,Aya Ouchi b ,Akira Shibata a ,Shin-ichi Nagaoka b ,Kazuo Mukai ba Faculty of Pharmacy,Yasuda Women’s University,6-13-1,Yasuhigashi,Asaminami-Ku,Hiroshima 731-0153,Japan bFaculty of Science,Ehime University,Matsuyama 790-8577,Japana r t i c l e i n f o Article history:Received 20July 2010Received in revised form 14December 2010Accepted 3January 2011Available online 11 January 2011Keywords:Vitamin E Tocopherol Free radical AntioxidantModel membrane Liposomesa b s t r a c tVitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in bio-logical systems.In this study,we measured the reaction rates of vitamin E (␣-,␤-,␥-,␦-tocopherols,TocH)and tocol with aroxyl radical (ArO •)as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry.Egg yolk phosphatidylcholine (EYPC)vesicles were used as a membrane model.EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H 2O =7:3,v/v)that gave the lowest turbidity in samples.The second-order rate constants (k s )for ␣-TocH in MeOH/H 2O solution with EYPC vesicles were apparently 3.45×105M −1s −1,which was about 8times higher than that (4.50×104M −1s −1)in MeOH/H 2O solution without EYPC vesicles.The corrected k s of ␣-TocH in vesicles,which was calculated assuming that the concentration of ␣-TocH was 133times higher in mem-branes of 10mM EYPC vesicles than in the bulk MeOH/H 2O solution,was 2.60×103M −1s −1,which was one-seventeenth that in MeOH/H 2O solution because of the lower mobility of ␣-TocH in mem-branes.Similar analyses were performed for other vitamin E analogues.The k s of vitamin E in membranes increased in the order of tocol <␦-TocH <␥-TocH ∼␤-TocH <␣-TocH.There was not much difference in the ratios of reaction rates in vesicles and MeOH/H 2O solution among vitamin E analogues [k s (vesicle)/k s (MeOH/H 2O)=7.7,10.0,9.5,7.4,and 5.1for ␣-,␤-,␥-,␦-TocH,and tocol,respectively],but their reported ratios in solutions of micelles and ethanol were quite different [k s (micelle)/k s (EtOH)=100,47,41,15,and 6.3for ␣-,␤-,␥-,␦-TocH,and tocol,respectively].These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes,which increased in the order of tocol <␦-TocH <␥-TocH ∼␤-TocH <␣-TocH.© 2011 Elsevier Ireland Ltd. All rights reserved.1.IntroductionThe antioxidant effect of vitamin E has been ascribed to the oxidation of its phenolic hydroxyl group by lipid peroxyl radi-cals (LOO •),producing corresponding tocopheroxyl radicals (Toc •)(reaction (1)).The mechanism involved has been studied exten-sively by several investigators (Burton et al.,1985;Niki,1987;Mukai,1992;Barclay,1993).LOO •+TocH →LOOH +Toc •(1)ArO •can be regarded as a model for active oxygen radicals (LOO •and others).In a previous work (Mukai et al.,1986),we measured the reaction rates (k s )of vitamin E analogues with 2,6-di-t -butyl-4-∗Corresponding author.Tel.:+81828789444;fax:+81828789540.E-mail address:fukuzawa@yasuda-u.ac.jp (K.Fukuzawa).(4-methoxyphenyl)phenoxyl (aroxyl,ArO •)(see Fig.1)in ethanol (reaction (2))by stopped-flow spectrophotometry.ArO •+TocH k s−→ArOH +Toc •(2)It is well known that lipophilic vitamin E is localized in biomem-branes and functions as an efficient inhibitor of membrane lipid peroxidation (Atkinson et al.,2008;Fukuzawa,2008).We previ-ously reported the rate constant of ␣-tocopherol for inactivation of singlet oxygen in membranes of egg yolk phosphatidylcholine (EYPC)liposomes (Fukuzawa et al.,1997;Fukuzawa,2000).How-ever,except for the rates in micellar membranes using Triton X-100(Mukai et al.,2007),the reaction rates (Eq.(2))of these vitamin E analogues in membranes have not been reported.In the present work,we measured the second-order rate constants (k s )in mem-branes for the reactions of ␣-,␤-,␥-,␦-tocopherols,and tocol (see Fig.1)with ArO •radical using stopped-flow spectrophotometry.We used samples of EYPC vesicles with low turbidity as a membrane model that is suitable for measuring by spectrophotometry.0009-3084/$–see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.chemphyslip.2011.01.001206K.Fukuzawa et al./Chemistry and Physics of Lipids164 (2011) 205–210Fig.1.Molecular structures of␣-,␤-,␥-and␦-tocopherol(TocH),tocol,and aroxyl radical(ArO•).2.Experimental procedures2.1.MaterialsThe structures of the molecules studied in this work are shown in Fig.1.The␣-,␤-,␥-,␦-tocopherols and tocol were kindly sup-plied by Eisai Co.Ltd.(Tokyo,Japan).Egg yolk phosphatidylcholine (EYPC)was obtained from Nippon Oil and Fat Co.(Tokyo,Japan). Aroxyl radical(ArO•)was synthesized as previously reported (Rieker and Scheffler,1965).2.2.Sample preparationEYPC vesicles were used as a membrane model.Because samples with low turbidity that maintain the membrane structure are suit-able for measurement of spectra,we prepared the EYPC vesicles in aqueous solutions containing various concentrations of MeOH.As shown in Table1,the turbidity(absorbance at700nm)decreased with increasing MeOH concentration.Possible formation of EYPC vesicles in aqueous methanol solution was confirmed using a con-focal microscope and measuring the vesicle sizes.The sizes of the EYPC vesicles were determined using a particle sizer380ZLS(Par-ticle Sizing Systems Inc.,California)at room temperature.Table2 shows the sizes of EYPC vesicles at1mM and10mM with and with-out0.2mM␣-TocH in70%MeOH.These results indicate that EYPC is likely to form vesicles with membrane structure in the pres-ence of MeOH at concentrations lower than70%(MeOH:H2O=7:3, v/v).We previously reported that EYPC molecules formed the vesi-cles but not micelles in the presence of MeOH at concentrations lower than70%by the method using a color iron-chelate(ferric-xylenol orange),which showed a peak absorbance at610nm when it bound to membranous EYPC but a peak absorbance at590nm in Table2Sizes of EYPC vesicles with and without␣-TocH in aqueous MeOH solution (MeOH:H2O=7:3,v/v).Vesicles a Diameter1mM EYPC310nm0.2mM␣-TocH/1mM EYPC204nm10mM EYPC393nm0.2mM␣-TocH/10mM EYPC302nma The EYPC vesicles were not detected in the presence of MeOH at concentrations higher than75%.the solution without EYPC or with miceller or mono-dispersed EYPC (Fukuzawa et al.,2006,2009).Based on thesefindings,70%MeOH with and without EYPC was used in the stopped-flow spectrophoto-metric study.We prepared ArO•in70%MeOH solution immediately before measurement by stopped-flow spectrophotometry.2.3.Measurements of stopped-flow spectraThe kinetic data were obtained with a Unisoku Model RS-450stopped-flow spectrophotometer by mixing equal volumes of0.1mM ArO•in70%MeOH solution and20mM EYPC vesicles containing vitamin E in70%MeOH solution under a nitrogen atmo-sphere(Mukai et al.,2007).The time between mixing the two solutions and recording thefirst data point(that is,dead time)was 10–20ms.The reaction was monitored by either single wavelength detection or a photo-diode array detector attached to the stopped-flow spectrophotometer.All measurements were performed at 25.0±0.5◦C.Experimental errors in the rate constants(k s)are esti-mated to be about5%and8%in70%MeOH and vesicle solutions, respectively.3.Results and discussionWe used70%MeOH solution with the lowest turbidity of EYPC vesicles in the followed stopped-flow spectrophotometric study. The changes in the absorption spectrum measured during the reac-tion(Eq.(2))are shown in Fig.2.When a70%MeOH solution (MeOH/H2O)of␣-TocH with or without EYPC vesicles was added to the ArO•solution,the ArO•absorption peak disappeared imme-diately,the␣-Toc•peak appeared,and the isosbestic points were observed.Fig.3shows the decay of ArO•measured by following the decrease in absorbance of ArO•at583nm or585nm due to the reac-tion with various concentrations of␣-TocH in MeOH/H2O solution with or without EYPC vesicles,respectively.Absorbance of ArO•at 583nm in70%MeOH solution with EYPC vesicles in the absence of ␣-TocH immediately arrived a plateau after slight decrease(Figs.3 (B)-line(e)and(C)-line(f)),indicating that ArO•scarcely abstract the hydrogen from unsaturated moieties of fatty acid of EYPC vesi-cles(Eq.(3)).The very small initial decrease of583nm may be caused by the reaction of ArO•with EYPC hydroperoxide(PC-OOH) (Eq.(4)),because PC-OOH was reported to be preformed as little as 0.3mol%in purchased EYPC samples(Fukuzawa et al.,2006): ArO•+EYPC→ArOH+EYPC•(3)Table1Effect of MeOH concentration on turbidity(absorption at700nm)of the solutions of EYPC vesicles containing␣-TocH with and without ArO•.MeOH(%)Absorbance at700nm in various concentrations of MeOH2040607080100␣-TocH 1.680.9670.1690.05300.01310.00636␣-TocH+ArO•a 3.01 1.000.1960.06520.02830.0550 [EYPC]=10×10−3M,[␣-TocH]t=0=1.17×10−4M,[ArO•]t=0=∼5×10−5M.a Absorbance at700nm was measured1s after␣-TocH/EYPC solution was mixed with ArO•solution.K.Fukuzawa et al./Chemistry and Physics of Lipids 164 (2011) 205–21020700.20.40.60.8A BA b s o r b a n c eWavelength / nm 00.30.60.91.2A b s o r b a n c eWavelength / nmFig.2.Changes in electronic absorption spectra of ArO •and ␣-Toc •during the reaction of ArO •with ␣-TocH at 25◦C in 70%MeOH solution (A)without and (B)with 10mM EYPC vesicles.The spectra were recorded at 200ms and 10ms intervals without and with EYPC vesicles,respectively.The arrow indicates a decrease in the absorbance of ArO •and an increase in the absorbance of ␣-Toc •.[ArO •]t =0=∼5.0×10−5M and [␣-TocH]t =0=5.0×10−5M.ArO •+PC-OOH →ArOH +PC-OO •(4)The pseudo-first-order rate constants (k obsd )were determined by evaluating the decrease in the absorbance of the ArO •.The ArO •molecules will exist in the liposome membranes because two lipid-soluble tert -butyl groups are substituted in an ArO •molecule.The k obsd of ArO •at 583nm or 585nm was linearly dependent on the concentration of ␣-TocH as shown in Fig.4,and thus the rate equa-tion was expressed as −d[ArO •]/d t =kobsd[ArO •]=k s[˛-TocH][ArO •](5)where k s is the second-order rate constant for oxidation of ␣-TocH by ArO •.The ratio of ␣-TocH to phospholipids in biological membranes is reported to be 1:1000–2000(Gruger and Tappel,1971).In this experiment,we used the ratio of ␣-TocH to EYPC was 1:100–500,which produced samples with low turbidity suit-able for spectrophotometry.From the result shown in Fig.4(A),the second-order rate constant (k s )of ␣-TocH in MeOH/H 2O solution was calculated to be 4.50×104M −1s −1in the absence of EYPC.The k s values of ␣-TocH incorporated into 4mM and 10mM EYPC vesicles in MeOH/H 2O solution obtained from the result shown in Fig.4(B)were apparently 7.20×105M −1s −1and 3.45×105M −1s −1,respectively,which were 16and 7.7times higher than that in the MeOH/H 2O solution without EYPC vesi-cles.We corrected the k s value of ␣-TocH on the basis of itslocal0.10.20.3A b s o r b a n c e (A r OTime / ms0.10.20.30.415010050A b s o r b a n c e (A r OTime / ms0.050.100.150.20ABCA b s o r b a n c e (A r OTime / sFig.3.Time courses of the decrease of absorption at 585nm (A)and 583nm (B and C)associated with the decay of ArO •in 70%MeOH solution at 25◦C during the reac-tion of ArO •with ␣-TocH in the presence of (A)0mM,(B)4mM,and (C)10mM EYPC vesicles,respectively.Concentrations of ␣-TocH were (A)(a)9.52×10−5M,(b)7.14×10−5M,(c)4.76×10−5M,(d)2.38×10−5M;(B)(a)8.30×10−5M,(b)6.22×10−5M,(c)4.15×10−5M,(d)2.07×10−5M,(e)0M;and (C)(a)1.04×10−4M,(b)8.30×10−5M,(c)6.22×10−5M,(d)4.15×10−5M,(e)2.07×10−5M,(f)0M.[ArO •]t =0=∼5.0×10−5M.concentration because ␣-TocH is localized in membranes of EYPC vesicles,where it reacts with free radicals.The corrected k s values of ␣-TocH in 4mM and 10mM EYPC vesicles,which were calcu-lated assuming that the concentrations of ␣-TocH were 333and 133times higher (Fukuzawa et al.,1997),respectively,in membranes208K.Fukuzawa et al./Chemistry and Physics of Lipids164 (2011) 205–210012345ABko b s d/ s -1[α-TocH] / 10-5M20406080ko b s d/ s -1[α-TocH] / 10-5MFig.4.Dependence of pseudo-first-order rate constant (k obsd )on concentration of ␣-TocH at 25◦C in 70%MeOH solution with and without EYPC vesicles.The k obsd values were obtained by analyzing the decrease in the absorbance of ArO •at 585nm or 583nm,as shown in Fig.3.(A)0mM EYPC,(B)( )4mM and (᭹)10mM EYPC vesicles.than in the bulk MeOH/H 2O solution,were 2.17×103M −1s −1and 2.60×103M −1s −1,respectively.These results indicate that the cor-rected k s values in membranes were nearly constant independent of EYPC concentration under our conditions.The corrected k s value of ␣-TocH was one-seventeenth of that in MeOH/H 2O solution without EYPC,which may be due to the decreased mobility of ␣-TocH in membranes.The k s value of TocH analogues in MeOH/H 2O solution with and without EYPC vesicles was obtained by plotting against their concentrations,as shown in Fig.5.The k s values obtained and adjusted to their concentrations in membranes are summarized in Table 3.The k s values in both membranes and MeOH/H 2O solution increased in the following order:Tocol <␦-TocH <␥-TocH ∼␤-TocH <␣-TocH.The TocH analogues that have lower oxidation potentials showed higher reactivity (Table 3).The corrected k s value of ␣-TocH in vesicles was one-tenth of that in micelles,which was reported to be 2.56×104M −1s −1(Mukai et al.,2007).The lower mobility of ␣-TocH in vesicle mem-branes than in micelle membranes would result in a lower k s value of ␣-TocH invesicles.012345AB 1512963ko b s d/ s -1[TocH] / 10-5M15129630ko b s d/ s -1[TocH] / 10-5MFig.5.Dependence of k obsd on concentration of TocH analogues at 25◦C in 70%MeOH solution.(A)No EYPC and (B)10mM EYPC vesicles.[ArO •]t =0=∼5.0×10−5M.(᭹)␣-TocH;( )␤-TocH;( )␥-TocH;( )␦-TocH;( )tocol.The effect of solvent on the reaction rates (k s )of ␣-TocH with ArO •radical was studied in a previous work (Mukai et al.,1993).The k s values are 5.12×103M −1s −1(ethanol),1.44×104M −1s −1(diethyl ether),9.52×104M −1s −1(benzene),and 1.94×105M −1s −1(n -hexane).The k s value of ␣-TocH in n -hexane is 38times the corresponding value in ethanol.When the logarithm of the rate constant (log k s )of ␣-TocH was plotted as a function of the reciprocal of the solvent dielectric constants (1/ε),it gave a straight line.These results indicate that the aroxyl radical-scavenging rate of ␣-TocH depends on the polarity of the solvents,and suggest that the rate increases with a decrease in the polarity of the membrane field where ␣-TocH reacts with free radicals.Recently,we reported the ratios of the reaction rates in ethanol and micelle solutions,k s (micelle)/k s (EtOH),of ␣-,␤-,␥-,␦-TocH and tocol (Table 4)(Mukai et al.,2007).These ratios were explained by the fact that reaction sites of vitamin E analogues depend on hydrophobicity in micelle membranes,which increased in the order of tocol <␦-TocH <␥-TocH ∼␤-TocH <␣-TocH.In the present work,however,the ratios of reaction rates for ␣-,␤-,␥-,␦-TocH,and tocol in MeOH/H 2O and EYPC vesicle solutions,k s (vesicle)/k s (MeOH/H 2O),are quite different from the ratios of their reaction rates in k s (micelle)/k s (EtOH)(Table 4).These results indicate that ␣-TocH reacts with lipophilic ArO •in membrane regions similar to those where ␤-,␥-,and ␦-TocH react but in regions slightly different from where tocol reacts,and that theK.Fukuzawa et al./Chemistry and Physics of Lipids164 (2011) 205–210209Table3Second-order rate constants(k s)for reaction of vitamin E analogues and tocol with ArO•in aqueous MeOH solution(MeOH:H2O=7:3,v/v)with and without EYPC vesicles, and peak oxidation potentials(E P).MeOH:H2O EYPC vesicle(appeared)EYPC vesicle(corrected)a E P(mV)k s(M−1s−1)k s(M−1s−1)k s(M−1s−1)vs SCE ␣-TocH 4.50×104 3.45×105 2.60×103860␤-TocH 3.04×104 3.04×105 2.29×103920␥-TocH 3.02×104 2.88×105 2.17×103930␦-TocH8.04×103 5.95×104 4.49×102990 Tocol 6.27×103 3.20×104 2.42×1021050 [ArO•]=∼5.0×10−5M.a Corrected k s values were obtained by assuming that the concentration of antioxidants in membranes of EYPC(10mM)vesicles were133times higher than in the bulk MeOH/H2O(7:3)phase(Fukuzawa et al.,1997).Table4Second-order rate constants(k s)for reaction of vitamin E analogues and tocol with ArO•in ethanol solution and Triton X-100micelle solution,and ratios of their k s values in ethanol and Triton X-100micelle solutions and in aqueous MeOH solution(MeOH:H2O=7:3,v/v)without and with EYPC vesicles.EtOH Micelle Ratiok s(M−1s−1)k s(M−1s−1)k s(micelle)/k s(EtOH)k s(vesicle)/k s(MeOH/H2O)␣-Tocopherol 5.12×103 5.12×105(2.56×104)a100(5.0)a7.7(1/17)b␤-Tocopherol 2.24×103 1.05×105(5.25×103)a47(2.4)a10(1/13)b␥-Tocopherol 2.42×103 1.00×105(5.00×103)a41(2.1)a9.5(1/14)b␦-Tocopherol 1.00×103 1.49×104(7.45×102)a15(1/1.3)a7.4(1/18)bTocol0.56×103 3.53×103(1.77×102)a 6.3(1/3.2)a 5.1(1/26)b[ArO•]=∼5.0×10−5M.a The k s values in micelles and consequent ratios were corrected(shown in round brackets)by assuming that the concentration of vitamin E in Triton X-100micelles(5.0wt%)was20times higher than that in the bulk water phase(Mukai et al.,2007).b The ratios of the corrected k s values were shown in round brackets.The k s values were corrected by assuming that the concentration of vitamin E in membranes of EYPC (10mM)vesicles was133times higher than in the bulk MeOH/H2O phase(Fukuzawa et al.,1997).Table5The relative rate constants(100k s(TocH)/k s(␣-TocH),%)for the reaction of vitamin E analogues and tocol with ArO•radical in Triton X-100micelles and EYPC vesicles at 25.0◦C,and relative biological activities.100k s(TocH)/k s(␣-TocH)Biological activities bEYPC vesicle Micelle a(A)(B)(C)(D)c ␣-TocH100100100100100100␤-TocH882125–4015–271238␥-TocH8320113–2059␦-TocH17 2.910.3–2–2 Tocol9.30.69(A)Rat fetal resorption,(B)rat haemolysis,(C)chicken muscle dystrophy,and(D)affinity for␣TTP.The values show the relative ratios(%).a Mukai et al.(2007).b Kijima(1992).c Hosomi et al.(1997).reactivities of the regions of their OH groups with ArO•are in the order of␣-TocH>␤-TocH=␥-TocH>␦-TocH>tocol.From these results,it is concluded that the aroxyl radical-scavenging rates(k s) obtained in the vesicle solution reflect(i)the electron-donating capacity of the vitamin E analogues and(ii)the polarity of the field of membranes where they react.The dielectric constants of MeOH(ε=33.0)and H2O(ε=80.16)molecules are larger than that(ε=25.3)of ethanol.Consequently,we expected that the k s value in MeOH/H2O is smaller than that in ethanol,as described above.However,the k s value(4.50×104M−1s−1)of␣-TocH in MeOH/H2O was nine times larger than that(5.12×103M−1s−1) in ethanol.The increase of the k s value in solution including water molecule was elucidated by the sequential proton loss electron transfer(SPLET)mechanism(Foti et al.,2004;Musialik and Litwinienko,2005).The increase of the k s value of␣-TocH in MeOH/H2O observed may be explained by the above SPLET mechanism.However,the detailed reason for the increase of the k s value of␣-TocH in MeOH/H2O is not clear at present.It will be interesting to compare the relative biological activities (Kijima,1992)with the relative ratios of the radical scavenging rate constants(k s)of␣-,␤-,␥-,and␦-tocopherols.The reported biopo-tency ratios of␣-,␤-,␥-,and␦-tocopherols for rat fetal resorption,rat haemolysis,and chicken muscle dystrophy are shown in Table5.␣-Tocopherol transfer protein(␣TPP),which binds vitamin E and enhances its transfer between membranes,plays an important role in determining the plasma vitamin E level.The relative affinities of vitamin E analogues for␣TPP determined by Hosomi et al.(1997) show a good linear relationship to biological activity,especially the rat fetal resorption test(Table5).The biological activity of vitamin E analogues will be determined by two factors:the free-radical-scavenging activities and affinities for␣TPP.The difference in the relative ratio of k s values in the vesicle solution obtained in this experiment is smaller than that in micelle solution(Table5),which has good correlation with the relative biopotency.The low corre-lation of the relative ratio of the k s values in vesicle membranes with their relative biopotency suggest that the affinity of vitamin E analogues for␣TTP,which may determine their plasma levels,is a major determinant of their biological activity.4.ConclusionsThe k s values of TocH analogues in membranes were determined for thefirst time by stopped-flow spectrophotometry.The appar-ent k s values in membranes were8–10times higher than those in210K.Fukuzawa et al./Chemistry and Physics of Lipids164 (2011) 205–210MeOH/H2O but the k s values corrected according their concentra-tions in membranes were13–18times lower than those in the bulk solution.Because most vitamin E molecules locate and function in membranes,our k s values in membranes of EYPC vesicles are useful to evaluate their abilities to scavenge peroxide radicals in biological systems.AcknowledgmentsWe thank Eisai Co.,Ltd.for the generous gifts of␣-,␤-,␥-,␦-tocopherols,and tocol.We also thank Ms.Masumi Sano of the University of Tokushima for her kind help in the measurement of the size of the EYPC vesicles.This work was partially supported by the grant of Yasuda Women’s University.ReferencesAtkinson,J.,Epand,R.F.,Epand,R.M.,2008.Tocopherols and tocotrienols in mem-branes:a critical review.Free Radic.Biol.Med.44,739–764.Barclay,L.R.C.,1993.Model biomembranes:quantitative studies of peroxidation, antioxidant action,partitioning,and oxidative stress.Can.J.Chem.71,1–16. Burton,G.W.,Doba,T.,Gabe,E.J.,Hughes,L.,Lee,F.L.,Prasad,L.,Ingold,K.U.,1985.Autoxidation of biological molecules.4.Maximizing the antioxidant activity of phenols.J.Am.Chem.Soc.107,7053–7065.Foti,M.C.,Daquino,C.,Geraci,C.,2004.Electron-transfer reaction of cinnamic acids and their methyl esters with DPPH•radical in alcoholic .Chem.69,2309–2314.Fukuzawa,K.,Matsuura,K.,Tokumura,A.,Suzuki,A.,Terao,J.,1997.Kinetics and dynamics of singlet oxygen scavenging by␣-tocopherol in phospholipid model membranes.Free Radic.Biol.Med.22,923–930.Fukuzawa,K.,2000.Singlet oxygen scavenging in phospholipid membranes.Method Enzymol.319,101–110.Fukuzawa,K.,Fujitani,A.,Akai,K.,Tokumura,A.,Terao,J.,Gebicki,J.M.,2006.Measurement of phosphatidylcholine hydroperoxides in solution and inintact membranes by the ferric-xylenol orange assay.Anal.Biochem.359, 18–25.Fukuzawa,K.,2008.Dynamics of lipid peroxidation and antioxidation of␣-tocopherol in membranes.J.Nutr.Sci.Vitaminol.54,273–285.Fukuzawa,K.,Shibata, A.,Okamura, C.,Fujiwara,Y.,Akai,K.,Tsuchiya,K., Tokumura,A.,Gebicki,J.M.,2009.Measurement of lipid hydroperoxides by the ferric-xylenol orange method(1)Characteristics of the ferric-xylenol orange/membrane phosphatidylcholine complex.J.Nutr.Sci.Vitaminol.55, 9–14.Gruger Jr.,E.H.,Tappel,A.L.,1971.Reactions of biological posi-tion of biological membranes.Lipids6,147–148.Hosomi,A.,Arita,M.,Sato,Y.,Kiyose,C.,Ueda,T.,Igarashi,O.,Arai,H.,Inoue,K., 1997.Affinity for␣-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs.FEBS Lett.409,105–108.Kijima,S.,1992.Chemistry of vitamin E.In:Mino,M.,Nakamura,H.,Diplock,A.T., Kayden,H.J.(Eds.),Vitamin E.Its Usefulness in Health and Curing Diseases.Japan Scientific Societies Press,Tokyo and Karger,Tokyo,pp.3–11.Mukai,K.,Watanabe,Y.,Uemoto,Y.,Ishizu,K.,1986.Stopped-flow investiga-tion of antioxidant activity of tocopherols.Bull.Chem.Soc.Jpn.59,3113–3116.Mukai,K.,1992.Synthesis and kinetic study of antioxidant and prooxidant actions of vitamin E derivatives.In:Packer,L.,Fuchs,J.(Eds.),Vitamin E in Health and Disease.Chap.8.Marcel Dekker,Inc.,pp.97–119.Mukai,K.,Morimoto,H.,Kikuchi,S.,Nagaoka,S.,1993.Kinetic study of free-radical-scavenging action of biological hydroquinones(reduced form of ubiquinone, vitamin K and tocopherol quinone)in solution.Biochim.Biophys.Acta1157, 313–317.Mukai,K.,Tokunaga,A.,Itoh,S.,Kanesaki,Y.,Ohara,K.,Nagaoka,S.,Abe,K.,2007.Structure–activity relationship of the free-radical-scavenging reaction by vita-min E(␣-,␤-,␥-,␦-tocopherols)and ubiquinol-10:pH dependence of the reaction rates.J.Phys.Chem.B111,652–662.Musialik,M.,Litwinienko,G.,2005.Scavenging of dpph•radicals by vitamin E is accelerated by its partial ionization:the role of sequential proton loss electron .Lett.7,4951–4954.Niki,E.,1987.Antioxidants in relation to lipid peroxidation.Chem.Phys.Lipids44, 227–253.Rieker,A.,Scheffler,K.,1965.Die beteiligung von phenylresten an der aroxylme-somerie.Liebigs Ann.Chem.689,78–91.。

Study of automobiIe tire rolling resistance and testing technoIogy Human activities on the ecological damage to the environment has become a global problem, to reduce fuel consumption, reduce automobile exhaust emissions is energy conservation, prevention of air pollution in an important measure. Vehicle energy consumption is closely related with the tire rolling resistance. On cars or light trucks, the 3.4% 〜6.6% of fuel consumption used to overcome rolling resistance tires; of loaded radial truck tire with the car example, 12.4% 〜14.5% of fuel consumption to overcome the rolling resistance tires . Tire rolling resistance by 10%, fuel-efficient cars will be 1.2 percent, 4 percent savings trucks. To this end,the tire manufacturers have at home and abroad to develop new low-power tires to reduce rolling resistance, saving fuel.Automobile tires in the rolling process, the total vehicle rolling resistance accounts for about 20% of the resistance, if reduced by 10% per tire rolling resistance, lower 2% 〜3% of fuel, then rolling resistance tires to enhance the level of control of vehicle contribution to fuel economy will be significant, but also in a wide range can be achieved. Therefore, how to effectively control the tires rolling resistance is the industry facing a key issue. This article will explore the various angles and analysis as well as tire rolling resistance testing technology.L SummaryIn the tire rolling process, the cycle of changes in the stress and strain lead to energy loss, the formation of tire rolling resistance, also known as the tire hysteresis energy loss. Studies have shown that to overcome tire rolling resistance on fuel consumption of the general accounting for the total fuel consumption of motor vehicles more than 10%. Reduce rolling resistance tires can reduce vehicle energy consumption, so that the car farther away from efficient. Tire rolling resistance is the overall energy consumption of material, equivalent to the tire rolling units of energy loss from the rolling units in addition to its distance, the dimensionless N • m / m, although its equivalent to the dimensionless force, but from the point of view of energy analysis and understanding more convenient and reasonable.Through the measurement of rolling resistance tires can study the best section. However, the results of lab experiments can only make a comparison, the final road test should be used as the basis of the results.Second, research the history ofAs early as age 60 in the 20th century, Beijing Research and Design Institute of Rubber Industry in turn on the drum machine and measured the wire cotton tire cord tire power loss,also measured on the road when the vehicle speed steady traction resistance. At that time, due to restrictions on the use of equipment, the pilot is in its early exploratory phase of long-term. Since the mid-80s, with the accelerated development of China's tire needs, a small number of tire manufacturers from the United States, Japan and Germany with the introduction of the rolling resistance of the switch position test tire drum testing machine, combined with the development of a new type of radial tire and the analysis of foreign samples a number of tire rolling resistance tires test.Inspection. 70s from the 20th century in the United States, Japan and Europe, such as the economically developed countries, in order to solve energy shortages and the deterioration of environmental quality issues and the rolling resistance tires for a large number of experiments and research work. At the same time, tire rolling resistance testing technologies have also made remarkable progress. Beginning in 2004, the U.S. National Research Center on the control of rolling resistance tires to start a new round of extensive research. In 2007, the European Rubber Manufacturers Association also made to the EU to control the level of rolling resistance of the recommendations. Therefore, China will also face the control of rolling resistance.Third, testing technologyAt present, China has established a laboratory test-based, supplemented by the direction of the outdoor experiment. Steady-state conditions in the interior that is a constant load and speed, the tires when driving to reach thermal equilibrium Tire rolling resistance measurement method of standardization has been achieved.Preliminary results show that the simulation of the city of tire rolling resistance condition than under the conditions of steady-state rolling resistance by 26% 〜47% of the difference between the two aroused people's interest in the emergence of a simulation of various operating conditions of automobile tires non-steady-state test. But so far did not see a unified standard test methods or test protocols. Carried out in the outdoor tire rolling resistance test methods are mainly trailer Act, taxiways and three kinds of torque method, in which a wider application of the trailer Act.Fourth, laboratory equipmentLaboratory test equipment, through decades of effort, has appeared in various types of tire rolling resistance testing machine. Their roads in accordance with the form of simulation points, and to have a steel drum two broad categories. Strip-type test machine to simulate the continuous flat surface, is very expensive test equipment tires. At present, it is the mostwidely to drum testing machine, in particular, a diameter of 1.7 meters to the drum. These test equipment measuring tire rolling resistance by way of points, and measuring method, torque law, power law and reduce the rate of four kinds of law. The use of existing equipment to load and torque of law are most welcome. In the past two decades, the accuracy of test equipment greatly enhanced to reduce the double measurement error, and has formed a set of test data to ensure the repeatability of the equipment necessary for accuracy. Has the full realization of Chinas current industrial production equipment is Jiurong Tianjin rolling resistance testing machine, is divided into car and truck tire with two types of tires, and its accuracy in line with the requirements of ISO. As the outdoor test line tire rolling resistance of non-standard test, the test equipment they use is not changed. Association for the Study of the British automobile industry with the use of the pilot housing trailers, all kinds of tires for air resistance in the same test under the conditions created.V. Test Methods1.Indoor and outdoor test testIndoor and outdoor test test test is based on the distinction between the two types of tire place test method. Indoor test of tire rolling resistance testing machine were conducted, and its experimental conditions, although the Department of simulated conditions of use but can be under control, so that good reproducibility of experimental data. Outdoor test rolling resistance tires are used on test vehicles on the road completed, it is true although the experimental conditions, but susceptible to external factors, pilot error, and therefore tire rolling resistance test to test the main indoor and outdoor test Des.2.Steady-state conditions and non-steady-state conditionsSteady-state conditions in the constant refers to the tire load and speed, and traveling in the tire to reach thermal equilibrium when measuring rolling resistance; non-steady-state conditions, it means the tire change with time in the load and speed, and tire temperature in the process of moving measuring rolling resistance. Of course, the tires in the car on the non-steady-state condition is varied. To the total points, tire condition of the car has the city, the suburbs of the car and coach on. However, a breakdown, and another empty, heavy vehicles, drive, driven, acceleration, deceleration, taxiways, brake, turn, etc. and combinations there of. Typical working condition of the tires as the standard test of non-steady-state conditions, needs to be done to investigate and test a large number of research work. So whether it is the latest ISO 18164 or the existing SAEJ 1269, they were under steady-state conditions. In addition, SAEJ 2452 slowdown is a complex experiment, the experimental conditions thanunder the uniform steady-state movement is much more complicated, but it is not completely under non-steady-state experiment. As can accurately predict tire rolling resistance is uniform or slowdown, more tests need to be verified, such comparison tests being investigated. However, the tire manufacturers tire rolling resistance testing machine mostly for the use of steady-state conditions, if used for non-steady-state conditions by the test needs Plus many new features, testing may be a substantial increase in cost.First of all, to deal with the choice of tires to test full consideration, such as the representativeness of the sample, the tire size, type, rated speed, the original production equipment and the type of wheel rim, as well as the technological level of producers and industry status.Secondly, the performance evaluation of the establishment of test methods. Is not any test method can be used to evaluate the performance level, especially the study of test methods, and therefore take into account the actual tire, the combination of existing technology and the means test, set up in line with the experimental detection conditions.Third, laboratory equipment to ensure precision and accuracy of the equipment of the establishment of standards to ensure that test reproducibility and repeatability, such as samples of the same specifications of the rolling resistance tires are consistent and repeatable, with the control tire for the different experiments comparison.Fourth, the establishment of quality control procedures to ensure that the different rolling resistance testing machine test results are consistent and repeatable, the data in these experiments should also include information such as equipment.Fifth, repeat the same tire test, test results are consistent and repeatable.Sixth, tire prices and the relationship between the dynamic resistance.Seventh, most importantly, in the protection of national industries and safeguard the environment and conserve fuel, to resist the flow of low-quality tires to enter the market, the need for serious thinking.A lot of tests in measuring very small load rolling resistance tire rolling resistance is measured the basic characteristics of the tire positioning accuracy, therefore, control precision and accuracy requirements and other equipment is key. According to many years of experience confirm the accuracy of test equipment requirements is necessary, otherwise the test data can not be guaranteed to reach the standards of repeatability and comparability.Lower rolling resistance tires can significantly save energy and protect the environment, with considerable economic and social benefits. Under the conditions of typical use of thetire rolling resistance and fuel consumption the relationship between the steady-state conditions with non-steady-state conditions compared with the torque method, power law and measured by the speed difference in rolling resistance tires will be The next step of our research objectives.Should be established and planned research projects, and used to determine the appropriate control of the rolling resistance or grade; from different points of view to promote and strengthen the grasp of the concept of rolling resistance and applications.探讨汽车轮胎滚动阻力以及测试技术人类活动对生态环境的破坏已成为全球性问题,减少燃料消耗、降低汽车尾气排放量是节约能源、防止大气污染的重要措施。

酒精中毒大鼠小脑损伤的病理改变赵丽1，关继奎1，陈嘉峰2摘要：目的　观察酒精中毒大鼠小脑损伤的病理改变。

方法　Wistar雄性大鼠以50%食用酒精，逐渐增量灌胃4周造出慢性酒精中毒模型。

通过光学显微镜及透射电子显微镜观察酒精中毒后大鼠小脑损伤的病理改变。

结果　酒精中毒组大鼠光学显微镜观察到小脑颗粒细胞及蒲肯野细胞（purkinje cell）均显著减少，细胞变性明显，尤以蒲肯野细胞为著。

电子显微镜观察到小脑毛细血管内皮细胞中细胞器明显减少。

细胞核形状不规则，核仁不明显、且形状不规则。

结论　酒精中毒后引起小脑细胞及其超微结构损伤，为其导致的运动及认知功能障碍提供了基础理论依据。

关键词：酒精中毒；小脑损伤；病理改变中图分类号：R595.6 文献标识码：APathological changes of cerebellar injury in rats with alcoholism　ZHAO Li， GUAN Jikui， CHEN Jiafeng.（The First People's Hospital of Wuhu， Wuhu 241000， China）Abstract：Objective To observe the pathological changes of cerebellar injury in rats with alcoholism.Methods An animal model of chronic alcoholism was established in Wistar male rats by gavage of 50% edible alcohol at increasing doses for 4 weeks. The pathological changes of cerebellar injury in rats after alcoholism were observed using an optical mi⁃croscope and a transmission electron microscope.Results Optical microscopy in rats with alcoholism showed signifi⁃cantly reduced number of granule cells and Purkinje cells in the cerebellum， and significant cell degeneration， especially in Purkinje cells. Electron microscopy showed significantly reduced organelles， irregularly shaped nucleus， and unobvi⁃ous and irregularly shaped nucleolus in cerebellar capillary endothelial cells.Conclusion Alcoholism induces damages to the cerebellar cells and their ultrastructure， providing a basic theoretical basis for the resulting motor and cognitive dys⁃function.Key words：Alcoholism；Cerebellar injury；Pathological change由于食入性酒精饮品摄入导致酒精中毒（alco⁃holism）引发脑损伤的病例在我国明显增多。

ISSN 1674-8484 CN 11-5904/U汽车安全与节能学报, 2015年, 第6卷第1期J Automotive Safety and Energy, 2015, Vol. 6 No. 1Vehicle Sideslip Angle Estimation based on Fusion of Kinematics-Dynamics MethodsGAO Bolin 1,2, XIE Shugang 2, GONG Jinfeng 2（1. School of Mechanical Engineering, Tianjin University, Tianjin 300072, China；2. China Automotive Technology & Research Center, Tianjin 300300, China）Abstract: A novel method of vehicle sideslip angle estimation was proposed based on a fusion of kinematics and dynamics methods to improve the estimation accuracy. A sideslip angle fusion observer (SAFO) was constructed with three local filters to estimate lateral velocities sending preliminary output to a master filter. The master filter fuses the outputs from all local filters to calculate a global sideslip angle estimation result according to driving information and fusion rules. The results show that the SAFO has good estimation accuracy and stability in a long time running with good robust for sensor signal bias. Therefore, the vehicle test data verifies the SAFO performances.Keywords: vehicle safety; sideslip angle estimation; kinematics method; dynamics method; fusion observer;pseudo-integration基于运动学—动力学方法融合的汽车质心侧偏角估计（英文）高博麟1,2，谢书港2，龚进峰2（1. 天津大学机械工程学院，天津 300072，中国；2. 中国汽车技术研究中心，天津 300300，中国）摘 要： 为了提高汽车质心侧偏角估计的准确性，提出了一种新的、基于运动学—动力学方法的融合估计方法。

A systematic review of antibiotic dosing regimens for septicpatients receiving continuous renal replacement therapy:docurrent studies supply sufficient data?A.M.M.Y .Li 1*,C.D.Gomersall 1,G.Choi 1,Q.Tian 1,G.M.Joynt 1and J.Lipman 21Department of Anaesthesia &Intensive Care,The Chinese University of Hong Kong,Prince of Wales Hospital,Shatin,New Territories,Hong Kong;2Burns Trauma Critical Care Research Centre,University of Queensland,Herston QLD 4029,AustraliaReceived 15April 2009;returned 29May 2009;revised 26July 2009;accepted 27July 2009Background :Drug dosing for septic patients with acute renal failure receiving continuous renalreplacement therapy (CRRT)is complicated,and failure to correctly dose may result in either drug tox-icity or treatment failure and development of antibiotic resistance.The aim of this study was to estab-lish an ideal dataset that needs to be reported when presenting pharmacokinetic data for thesepatients and review current literature for completeness of this dataset.Methods :An ideal dataset was established of the parameters that should be reported when calculatinga drug dosing regimen from first principles.A Medline search was performed of relevant literatureproducing 64citations from which completeness of the specified criteria was examined.Results :None of the studies analysed presented the full dataset that we established as necessary.Of concern,basic pharmacokinetic parameters such as volume of distribution (V d )and clearance (CL)were specified in only 79%and 81%of studies,respectively.Conclusions :A large proportion of current studies do not report key information necessary to devise arational dosing regimen for patients with acute renal failure receiving CRRT,and we hope this datasetwill be a useful guide when reporting future pharmacokinetic data.Keywords:antibiotics,drug dosing,pharmacokinetics,renal replacement therapy,sepsisIntroduction Sepsis and acute renal failure (ARF)are two pathological entities that commonly co-exist in patients admitted to intensive care.1,2Early,appropriate antibiotic therapy and source control while enhancing cellular recovery and controlling metabolic complications associated with uraemia remain the most impor-tant aspects of clinical treatment.3–7However,antibiotic dosing in septic patients with ARF can be complicated and may result in either underdosing,causing treatment failure and antibiotic resistance,or overdosing result-ing in drug toxicity.8This is further compounded by the use of renal replacement therapy (RRT)to maintain homeostasis until renal function has sufficiently recovered,which can result in sig-nificant non-renal clearance of antibiotic.9Since the late 1970s the use of continuous renal replacement therapy (CRRT)has become established in many intensive care units (ICUs)as the preferred modality of RRT.CRRT avoids rapid fluid and electrolyte shifts in haemodynamically unstable patients and gives better control of patient fluid balance than tra-ditional thrice-weekly intermittent haemodialysis (IHD).CRRT is usually performed through a venous catheter situated in a large (usually a femoral or internal jugular)vein,either as con-tinuous veno-venous haemofiltration (CVVH),haemodialysis(CVVHD)or a combination of the two:haemodiafiltration (CVVHDF).CVVH uses a predominantly hydrostatic pressure gradient to pump solute across a filter membrane to achieve clearance.Replacement fluid can be added to the circuit either before blood reaches the membrane (pre-dilution)or after passage over the filter membrane (post-dilution).In contrast,CVVHD uses diffusion across a membrane to effect clearance of solute.This is achieved by generating a continuous concentration gradient using counter-current flow of plasma and dialysate fluid,.....................................................................................................................................................................................................................................................................................................................................................................................................................................*Corresponding author.E-mail:alex_li@Journal of Antimicrobial Chemotherapy (2009)64,929–937doi:10.1093/jac/dkp302Advance Access publication 25August 2009.....................................................................................................................................................................................................................................................................................................................................................................................................................................at Anhui Medical University on April 18, 2015/Downloaded frombetween which equilibration occurs.CVVHDF uses a combination of the two above techniques,convection and diffu-sion to clear solute.In order to devise a rational drug dosing regimen it is necess-ary to know the volume of distribution(V d)and the clearance (CL)of the drug concerned.In the case of patients with ARF being treated with CRRT,clearance will depend on a combi-nation of CRRT clearance,residual renal function and non-renal clearance.Both the volume of distribution and the non-renal clearance may be changed by ARF and critical illness.10–14The problem is further exacerbated by considerable variability in the mode and dose of CRRT.Thus,to be useful to clinicians, studies of antibiotic pharmacokinetics in critically ill patients with ARF should report several parameters in addition to the standard pharmacokinetic dataset.Without these parameters, interpretation of studies and their use in deriving a dosing regimen is limited.The aim of this review therefore was to:(i)establish the ideal dataset that needs to be presented to allow for adequate anti-biotic dosage regimen calculation on CRRT;(ii)examine recent publications on antibiotic dosing in patients with ARF receiving CRRT and to establish whether this dataset is complete;and(iii) examine whether sufficient detail on patient case-mix was given to assess applicability of the data to other critically ill patients receiving CRRT for ARF.MethodsEstablishing the ideal dataset requiredA drug dosing regimen can be calculated fromfirst principles:V d will determine initial or loading dose and total clearance(CL tot) will dictate dosing interval.CL tot is determined by both CRRT (CL CRRT)and non-CRRT(CL non-CRRT)clearance(mainly residual renal and hepatic clearance).Thus,depending on whether antibiotic kill characteristics are time or concentration dependent,a specific serum concentration–time profile can be targeted(Figure1).The mode and dose of CRRT in critically ill patients with ARF is not only highly variable but can have significant effects on CL CRRT,and therefore specific data need to be quoted.Table1 shows equations for calculating CRRT clearance depending on the modality of CRRT used.Passage of drug across afilter membrane is essentially independent of drug molecular weight as the pore size of modern membranes vastly exceeds the size of most commonly used antimicrobials.However,protein binding,membrane type,charge and surface area may play a significant role in limiting drug passage across the haemofilter.This can be expressed,depending on whether CVVH or CVVHD is used,as either a sieving coefficient(S c)or a saturation coefficient(S d),calculated as:S c¼½Drug ultrafiltrate ½Drug plasmaS d¼½Drug dialysate ½Drug plasmaWhen using CVVH,the method of dilution needs to be specified as plasma entering the haemofilter in predilution mode will be diluted by replacementfluid,and a correction factor(CF)needs to be used to calculate clearance(CF¼Q b/Q bþQ rep)(Table1).This would require further mention of bloodflow rates(Q b)andfluid replacement rates(Q rep)in addition to basic CVVH data such as S cand ultrafiltrate rate(Q f).Non-CRRT clearance is predominantly determined by residual renal and hepatic clearance,which should therefore be mentioned in some form.Finally,to assess whether thederived dataset is applicable to the patient population one wants toFigure 1.Calculation of antibacterial doses based onfirst principles:non-CRRT clearance is the sum of non-renal clearance plus residual renal clearance.Table1.Equations for calculating CRRT clearance fromfirstprinciplesMode of CRRT Calculation of CRRT clearanceCVVH(post-dilution)CL CVVH(post)¼Q fÂS cCVVH(pre-dilution)CL CVVH(pre)¼Q fÂS cÂQ b/(Q bþQ rep) CVVHD CL CVVHD¼Q dÂS dCVVHDF CL CVVHDF¼(Q fþQ d)ÂS dCL CVVH(post),clearance from continuous veno-venous haemofiltrationusing postfilter haemodilution;Q f,ultrafiltrate rate;S c,sieving coefficient;CL CVVH(pre),clearance from continuous veno-venous haemofiltration usingpre-filter haemodilution;Q b,bloodflow rate;Q rep,predilution replacementrate;CL CVVHD,dialysateflow rate;S d,saturation coefficient;CL CVVHDF, clearance from continuous veno-venous haemodiafiltration.at Anhui Medical University on April 18, 2015/Downloaded fromdose,a comparison of patient demographics such as age,weight and severity of illness would be helpful.Based on the principles discussed above,ideal(Figure2)and minimum datasets were determined by three of the authors. Disagreements were resolved by discussion.The minimum dataset consisted of:antibiotic assayed,dose recommendation,patient age, weight,markers of residual renal function and hepatic impairment, V d,clearance(total,CRRT and non-CRRT)and(depending on modality of CRRT used),method of dilution,ultrafiltrate,blood and dialysateflow rates.Literature reviewWe searched the Medline database from January1986through to February2008for literature with the medical subject headings‘acute renal failure’,‘pharmacokinetics’,‘clearance’,‘dosage’,‘h(a)emofil-tration’,‘h(a)emodialysis’,‘h(a)emodiafiltration’,‘continuous renal replacement therapy’,‘antibiotics’,‘intensive care’and‘critically ill’.All searches were limited to studies of human subjects and antimicrobials commonly used in intensive care.The searches produced a total of64citations,60related to antibacterials15–73and four related to antifungals89–92(see Table S1;available as Supplementary data at JAC Online /).Three investigators reviewed potentially relevant papers for the specified data and any disagreement was resolved by discussion. ResultsThe percentage of studies providing the ideal dataset we speci-fied is shown in Figure3.Of note,none of the studies included the full set of parameters;29%of studies provided the minimum dataset.Drug dataAll studies specified the antibiotic assayed and7%of studies specified a target concentration in terms of MIC.Dose recommendation or modification was suggested in73%of studies.Patient demographicsAll studies specified the number of patients receiving antibiotics and CRRT.Age and patient weight were given in99%and69%Drug dataAntibioticassayedSpecified target concentration Dose recommendationPatient demographicsAge Weight SeverityofillnessNumber ofpatients instudyResidual renalfunctionHepaticfunction Basic pharmacokineticsVolume of distribution(V d)Total, CRRT and non-CRRT clearanceProtein binding/serumalbuminCRRT clearanceMembrane type/surface areaCVVH CVVHD CVVHDF Post-dilutionPre-dilutionS c Ultrafiltration rate (Q f) Blood flow(Q d) Haematocrit(HCT) Predilution replacement rate (Q rep)S cUltrafiltrationrate (Q f)S dDialysate rate(Q d)S c/S dUltrafiltration rate (Q f)andDialysate rate (Q d)orEffluent rate (Q f+Q d)Figure2.Pharmacokinetic parameters required for antibiotic dosage modification in patients receiving CRRT. at Anhui Medical University on April 18, 2015 / Downloaded fromof studies,respectively.Severity of patient’s illness was speci-fied in 80%of studies.Some measure of residual renal functionwas specified in 75%of studies,and a note of hepatic impair-ment or function,either qualitative or quantitative,was noted in 33%of studies.Basic pharmacokinetics V d and CL tot were specified in 79%and 81%of studies,respect-ively.Protein binding or serum albumin was mentioned in 27%of studies.CRRT clearance The most popular method of CRRT was CVVH,which accounted for 41%of studies.CVVHD and CVVHDF were used in 16%and 24%of studies respectively.Other modalitiesof CRRT included continuous arterio-venous haemofiltration(CAVH;4%),continuous arterio-venous haemodialysis,(CAVHD;12%)and high-volume haemofiltration (HVHF;1.3%).A combination of two of the above techniques was used in the remaining CRRT studies.CVVHPre-or post-dilution mode was specified in 58%of studies.S c was calculated in 63%of studies.The ultrafiltration rate was noted in 91%of studies.Where predilution was used,specifica-tion of blood flow rate (Q d )and haematocrit (HCT)(to calculateplasma flow rate)was mentioned in 80%and 6%,respectively(Figure 4).CVVHDDialysate rate and S d were specified in 67%and 42%of studies,respectively.CVVHDFS c /S d and effluent rate (Q f þQ d )were specified in 67%and 89%of studies.DiscussionOur systematic review of the literature indicates that a large pro-portion of studies of antibiotic dosing in critically ill patientsA n t ib i o t ic M ode of C R R T A ge M e m b r a n e /S A U F /ef f l u e n t /d i a l y s a te r a t e D o s er e c o m m e n d at i o n C L t o t S c /S d V d C L C R R T S e v e r i t y o fi ll n e s ss c o r eR e s i d u a l r e n a l f u n c ti o n W e i g ht H e pa t i c f u n ct io n P B (%)/al b u m i n Bloo d fl o wr a t e (m L /m i n )Speci fi e dt a r g e tc o n c n H a e m o t o c r it Com pl eted at a s et10203040506070P ercentag eofstud i es8090100Figure 3.Percentage of studies specifying the required parameters for pharmacokinetic analysis.CRRT,continuous renal replacement therapy;SA,surface area;CL tot ,total clearance;S c /S d ,sieving/saturation coefficient;V d ,volume of distribution;CL CRRT ,CRRT clearance;PB,protein binding.Note effluent/dialysate rate includes ultrafiltrate (UF)rate where CVVH used.at Anhui Medical University on April 18, 2015/Downloaded fromreceiving CRRT do not report key information necessary to devise a rational dosing regimen or to determine the applica-bility of the findings to the reader’s patients.In fact,none of the studies analysed specified the full dataset.There are certain basic pharmacokinetic parameters such as V d and CL tot that are fundamental requirements for all drug dosing.However,these were only specified in 79%and 81%of studies,respectively.The mode and dose of CRRT are highly variable,but are important determinants for antibiotics normally cleared by the kidneys.It is therefore necessary to estimate CRRT clearance in each individual or,at the very least,know whether the mode and dose of CRRT used in a study approximates to the mode and dose being given to the patient.A small but significant pro-portion of studies omitted even basic information such as ultra-filtrate/dialysate rate (Figure 3).CVVH was the most common modality used in the studies analysed,but only 58%of studies specified the method of repla-cing ultrafiltrate (Figure 4).Given the disparity that can arise in drug clearance between pre-and post-dilution CVVH,specifica-tion of pre-dilution where used,with calculation of an appropri-ate correction factor using blood flow rate (Q b )and replacement fluid rate (Q rep )is essential.One can argue that in most patients Q rep will only vary by a small fraction of the blood flow rate (Q b )and therefore changes will probably not significantly affect overall CRRT clearance.Similarly,using haematocrit to estimate plasma flow rate as opposed to blood flow rate (as drug is essen-tially cleared from plasma),should likewise not significantly impact on overall clearance provided consistency exists between studies.However,both these parameters are easily measured and should therefore be included for study accuracy.It is important to know S c or S d if one is to estimate drug clearance in an individual patient (Figure 2).If these data are lacking (12protein binding)can be used as a surrogate 74as protein binding is the principal determinant of S c and S d .Only 78%of studies gave S c or S d .Of those that did not,none measured protein binding.The importance of specifying all these parameters is illus-trated by two studies of meropenem pharmacokinetics during CVVH.16,27Patients were comparable demographically (age,weight and severity of illness).However,the use of different membranes (polyacrylonitrile compared with polysulfone)may have resulted in different S cvalues (0.63+0.252compared with1.09+0.10).In combination with differing ultrafiltrate rates(25–30mL/min compared with 45.8+6.2mL/min)this resultedin vastly different CRRT clearances between the two studies(17.2mL/min compared with 49.7+8.3mL/min),and conse-quently very different dose recommendations for similar patients(500mg twice daily compared with 1g three times a day of meropenem).Non-CRRT clearance may make a major contribution to totaldrug clearance and can be difficult to quantitatively assess.Thetwo main routes of drug elimination are renal and hepatic.Only74%of studies qualified renal excretion to any degree,and the majority only qualitatively.Hepatic clearance is much more dif-ficult to assess,but we believe some mention of hepatic impair-ment,if it exists,should be specified.This may be qualitative inspecifying a degree of liver impairment (for example the pres-ence or absence of cirrhosis)or semi-quantitative such as serum bilirubin or a measure of hepatic perfusion with indocyaninegreen clearance.Renal elimination is to a large extent deter-mined by glomerular filtration rate,with contributions from tubular secretion and reabsorption,but the ability to upregulate clearance is relatively limited.Consequently,hepatic clearance in the presence of renal failure can become the predominant modality of drug elimination in certain cases.15For example,non-renal (hepatic)clearance of meropenem has been shown to increase from 20%of total elimination to .50%in patients with a creatinine clearance rate of ,30mL/min.75Most studies suggested a dose regimen based on study data.Where one was not advocated,levels and further studies were suggested in the majority.Optimal bacterial killing is related to pharmacokinetic targets,which are in turn related to MIC.Thus to determine whether these dose regimens and modifications are appropriate it is necessary to know what pharmacokinetic end-point was targeted and that endpoint should be referenced to MIC.However,this only occurred in 8%of studies.We believe patient demographic data should be reported to allow the reader to judge whether the findings can be applied to his/her patient.Patient age was included in the dataset as it has significant effects on native renal and hepatic clearance causing in vivo drug clearance variations of 20%–40%,76as well as effectingchanges in total body water,plasma protein binding and Vd .77Similarly,weight variation can have significant effects on volume of distribution,and some studies specified V d only in absolute volume,which needs to be corrected for patient weight for comparison.In addition,studies of the optimal intensity of CRRT reference the CRRT dose to weight.78–80Finally we looked for an indication of severity of illness for comparison between ICU populations.Critical illness may have significant pharmacokinetic effects per se ,and clearance alterations and increases in the Vd of aminoglycoside,b -lactam and carbapenemantibiotics in critically ill septic patients have been reported.81–86Although unproved,this seems unlikely to be an all-or-nothing phenomenon but is related to the severity of illness.Our discussion has focused on the data required for dose cal-culation from first principles.While we believe this is the ideal method of prescription,many clinicians will choose to use doseH C Tinp r edi lut i o nQ b i n p r e d i l u t i o n S cc a l c u la t e d U lt r a f i l t r a t i o n ra t eD i lu tio n m e t h o d s p e c i f i e d 010203040506070P er c e n t ageof s t u d i es 8090100CVVH parameters specified Figure 4.Percentage of studies with necessary dataset to calculate clearance with CVVH.S c ,sieving coefficient;Q b ,blood flow rate;HCT,haematocrit. at Anhui Medical University on April 18, 2015/Downloaded fromadjustment equations (Table 2).As can be seen from Table 2the dose adjustment equations require many of the parameters dis-cussed above such as CL CRRT and CL non-CRRT ,S c and CRRT dose.A third alternative is a ‘best guess’technique whereby one could identify studies using the antibiotic in question which have similar patient demographics,modality and dose of CRRT,and then use the advocated dosing regimen of these studies if patients and practice are sufficiently similar to that used in the prescriber’s unit.Again,this would require specification of most of the parameters in our dataset.Conclusions We have presented a set of criteria we think necessary to calcu-late appropriate doses of antibiotics in septic patients receiving CRRT,from first principles.None of the studies of pharmacoki-netic data examined presented the full range that we specified.Clearly some parameters are of greater importance in calculating clearance than others.As non-renal indications for haemofiltra-tion and haemodialysis continue to expand with the concept of blood purification,the use of these parameters in calculating CRRT drug clearance in patients with both impaired and normal hepato-renal function will increasingly become an important issue.We hope our dataset will be a useful guide when deciding which parameters are worthy of inclusion.Funding No specific financial support or substantive assistance was pro-vided in the preparation of this manuscript.There is no ongoing financial support for any of the authors with respect to this article.Transparency declarations None to declare.Supplementary data Table S1is available as Supplementary data at JAC Online (/).References 1.Rahman TM,Treacher D.Management of acute renal failure on the intensive care unit.Clin Med 2002;2:108–13.2.Rangel-Frausto MS,Pittet D,Costigan M et al .The natural history of the systemic inflammatory response syndrome (SIRS).A pro-spective study.JAMA 1995;273:117–23.3.Garnacho-Montero J,Garcia-Garmendia JL,Barrero-Al-modovar A et al .Impact of adequate 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丙烯制丙烯醛动力学研究英语**Abstract**This study aims to investigate the kinetics of the production of acrolein from propylene. The reaction mechanism, rate constants, and activation energy were determined using various experimental techniques. The results obtained provide valuable insights into the optimization of the acrolein production process.**Introduction**Acrolein, a key intermediate in the chemical industry, is widely used in the production of various chemicals such as acrylic acid, acrylates, and methacrylic acid. It is primarily produced from propylene via catalytic oxidation. Understanding the kinetics of this reaction is crucial for optimizing the production process and enhancing the yield of acrolein.**Materials and Methods**In this study, a series of experiments were conducted to investigate the kinetics of the production of acrolein from propylene. The reaction was carried out in a fixed-bedreactor using a suitable catalyst. The reactor temperature, feed composition, and flow rate were carefully controlled to ensure reproducibility of the experiments. The concentration of acrolein in the product stream was analyzed using gas chromatography.The reaction mechanism was proposed based on literature surveys and experimental observations. The rate constants and activation energy were determined using the Arrhenius equation and the experimental data obtained from the experiments.**Results and Discussion**The experimental results showed that the production of acrolein from propylene follows a complex reaction mechanism. The rate constants and activation energy were determined for the various steps involved in the reaction. The activation energy was found to be relatively high, indicating that the reaction is energy-intensive.The effect of reactor temperature on the acrolein yield was also studied. It was observed that increasing the reactor temperature enhances the reaction rate but also leads to increased side reactions, resulting in a decreasein the acrolein yield. Therefore, optimizing the reactor temperature is crucial for maximizing the acrolein yield.**Conclusion**The kinetic study of the production of acrolein from propylene provides valuable insights into the reaction mechanism, rate constants, and activation energy. Theresults obtained in this study can be used to optimize the acrolein production process and enhance the yield. Future work could focus on improving the catalyst's selectivityand stability to further enhance the acrolein yield.**中文内容****丙烯制丙烯醛动力学研究****摘要**本研究旨在探讨由丙烯制备丙烯醛的动力学过程。

Does losing your home mean losing your school?:Effects of foreclosures on the school mobility of children ☆Vicki Been,Ingrid Gould Ellen,Amy Ellen Schwartz ⁎,Leanna Stiefel,Meryle WeinsteinNew York University,NY,United Statesa b s t r a c ta r t i c l e i n f o Article history:Received 26October 2010Received in revised form 4February 2011Accepted 24February 2011Available online 2March 2011Keywords:Foreclosure Mobility SchoolsPublic education Housing instabilityIn the last few years,millions of homes around the country have entered foreclosure,pushing many families out of their homes and potentially forcing their children to move to new schools.Unfortunately,despite considerable attention to the causes and consequences of mortgage defaults,we understand little about the distribution and severity of these impacts on school children.This paper takes a step toward filling that gap through studying how foreclosures in New York City affect the mobility of public school children across schools.A signi ficant body of research suggests that,in general,switching schools is costly for students,though the magnitude of the effect depends critically on the nature of the move and the quality of the origin and destination schools.©2011Elsevier B.V.All rights reserved.1.IntroductionWe use data on students in New York City's public schools to explore the reach of the foreclosure crisis into the city's student population.To begin,we assess how many students have been affected by foreclosures and compare the characteristics of the students living in foreclosed buildings and the schools that they attend to those of students not directly affected by foreclosures.We next examine whether children living in properties entering foreclo-sure are more likely to switch schools than otherwise similar students.Further,we examine whether –and how –the characteristics of their new schools differ from the characteristics of their original schools and assess how those differences compare to the differences between the original and new schools of similar students who switched schools but were not living in buildings entering foreclosure.We focus primarily on elementary and middle school students for whom the link between residential and school location is strongest.To undertake this work,we use a unique data set on New York City,which links student-level academic records to building-level foreclo-sure data.We focus on the 2003–04and 2006–07academic school years,to give a sense of the way in which the gathering foreclosure crisis played out for public school children.The results are intriguing,suggesting that the foreclosure crisis induced affected students to switch schools more often than they would have otherwise,and,on average,to schools offering academically weaker peers.Together these suggest that foreclosures may negatively affect the academic performance of students living in foreclosed buildings and put additional strain on public schools already facing budget cuts and fiscal retrenchment.12.Background and literature reviewA foreclosure notice may result in several different outcomes.First,owners may resolve the foreclosure by paying back the arrearages or by receiving a modi fication from their lender that allows them to keep the property.Second,owners may sell their property and pay off the mortgage debt,assuming that their mortgage debt does not exceed the value of their property or that the bank forgives any difference.Finally,the bank may complete the foreclosure by auctioning the property to a third party or by taking ownership of the property itself (so-called REO,or real estate owned).In New York City,the time between the filing of a Lis Pendens (“LP ”or “foreclosure notice ”)and the auction of the property is typically about 18months.Foreclosures may affect children in a variety of ways.First,if a homeowner is not able to cure or modify,and instead either sellsRegional Science and Urban Economics 41(2011)407–414☆We thank the Open Society Foundations for support for this research,which funded this work as a part of a three-site study including Washington,DC,and Baltimore,MD.We also thank Kathy Pettit,Jennifer Comey,and two anonymous reviewers for helpful comments and Elizabeth Guernsey for excellent research assistance.⁎Corresponding author.E-mail address:amy.schwartz@ (A.E.Schwartz).1According to a 2008article in USA Today,several school districts,responding to a “wave ”of homeless students now living outside the district lines are “having staffers or private investigators check for families using false addresses.Palm Beach County,Fla.,set up an anonymous tip line that residents can call to report families who might be improperly enrolling students ”(Armour,2008).0166-0462/$–see front matter ©2011Elsevier B.V.All rights reserved.doi:10.1016/j.regsciurbeco.2011.02.006Contents lists available at ScienceDirectRegional Science and Urban Economicsj ou r n a l h o m e p a ge :ww w.e l s ev i e r.c o m/l o c a t e /re g e cher house to pay off the mortgage or loses it to the bank,the family will be forced to leave.Similarly,if a family's landlord cannot pay the mortgage and either sells or loses the property to the bank,then the tenants(and their children)may be forced to move to a new home.It is possible that families who move as a result of foreclosure willfind new housing in the same neighborhood and children will be able to stay in the same school,but in many cases, families will end up moving to new,and perhaps more affordable, neighborhood,and transfer their children,especially young chil-dren,to new schools.If the owners resolve the foreclosure through cure or modification, the children may nevertheless still be affected if the families cut back on spending on the children's education and educational activities in order to have more money to pay off the mortgage.Further,whether the family is forced to move or not,the stress a foreclosure notice produces may affect the children's educational performance.The majority of buildings receiving foreclosure notices in New York City have been multifamily buildings,and thus many of the households living in properties receiving foreclosure notices have been renters(Furman Center,2008).The effects of foreclosure on tenants are not as clear,though for a variety of reasons,we expect elevated rates of departure following a foreclosure notice.Until the Congress passed the‘Protecting Tenants at Foreclosure Act of2009,’which allowed tenants to stay in place for90days or the term of their lease,in most circumstances following a foreclosure,many tenants had few protections in the event of a foreclosure(Been and Glashausser,2009).(Note that most of the multifamily properties receiving foreclosure notices in New York City have been2–4unit properties,which are not governed by the city's system of rent regulation.)When landlords sold their properties to pay off their mortgages,anecdotal reports suggest that they often encouraged tenants to leave because they believed their properties would be more marketable without tenants.New owners also sometimes pushed tenants out—and if a foreclosure was completed,banks typically evicted any remaining tenants,due to both liability concerns and a worry that properties would not be as attractive to potential buyers if they had tenants.Further,even when guaranteed the right to stay under federal or state law,tenants may choose to move from buildings receiving foreclosure notices more frequently than from other buildings because owners struggling to pay their mortgage cut back on maintenance and utilities.As tenants leave their homes,they may move to new neighborhoods and school zones.Moves across schools have been shown to be damaging to children's academic performance(Hanushek et al.,2004).School moves may cause problems getting restarted(including difficulties with the subject matter or tensions with classmates)(Alexander et al., 1996;Lash and Kirkpatrick,1994;Mehana and Reynolds,2004; Nelson et al.,1996;Schwartz et al.,2007;Xu et al.,2009).The involuntary moves precipitated by foreclosure may be even more harmful to students as choices may be limited by the urgency of the move.Students accordingly may move to poorer quality schools,with lower quality teachers or peers who are performing less well(Pettit, 2004;Xu et al.,2009).Despite these ways in which foreclosure could theoretically affect students,there has been little research into what happens to households that live in foreclosed properties,either in New York City or around the country,largely because foreclosure records are property-based,and it is rarely possible to identify and follow occupants.Anecdotal evidence indicates that some homeowners leave the property to become renters or move in with relatives and friends.Others end up homeless(Goodman,2009).Their children may have to change schools as a result and move to schools whose quality differs from their original schools.We hope in this paper tofill this gap in the literature regarding children's schooling,drawing on evidence from New York City.3.Data3.1.Student and school dataWe use student-level data from the New York City Department of Education(NYCDOE)for all students enrolled in the City's public schools on October31st of the school year.The dataset identifies each student's birth date,country of birth,race or ethnicity,gender,free and reduced price lunch status,and home language.The data set also includes the student's grade,information on annual school atten-dance,Limited English Proficient(LEP)status,special education status,and standardized test scores.We can link data for individual students across academic years,as long as a student attends a New York City public school.2The student data include information on the school attended, allowing us to link to school-level data,including school demograph-ics(e.g.percentage black,Hispanic,Asian,or white;percentage eligible for free or reduced price lunch),as well as resource data (expenditures,teacher characteristics),average test scores,and attendance.3.2.Foreclosure dataWe use a dataset of parcel level foreclosure starts,or lis pendens (LP)filings,from the Public Data Corporation.This dataset is updated quarterly;we use data from thefirst quarter of2000through the last quarter of2009.The dataset includes all residential parcels that received a notice of foreclosure and the date of each notice.3We linked these data,through the borough-block-lot(BBL)identifier for eachfiling,to street address,property characteristics,and information about the disposition of the property after the LP was issued(in particular,whether the property was transferred in an arms-length sale,sold at auction or retained by the lender as REO,or had an unknown outcome).The property data come from the Primary Land Use Tax Output (PLUTO)file maintained by the New York City Department of City Planning,as well as the City's Automated City Register Information System(ACRIS),and in some cases the Real Property Assessment Data (RPAD),a database of individual tax lots in New York City with characteristics such as area,zoning,and building class.3.3.Matching students to residential parcels in foreclosureTo link students to properties receiving foreclosure notices,the New York City Department of Education(DOE)matched the students' addresses,for the2003–04and2006–07school years,to the addresses of all properties(other than condominiums and coopera-tives4)receiving foreclosure notices.The DOE records a student's address at three different times during each academic year,on October31st,March1st,and June1st.We2For each year,we exclude students who are missing admit/discharge dates,school, or grade codes.This decreases the number of full-time special education students who are frequently assigned to ungraded classrooms.In most cases these students would be dropped because of the absence of testing data and/or due to limitations in the data on schools serving primarily special education students.3For properties that had more than one LP without a transfer of ownership and a second LP within6months of the priorfiling,we consider it to be the same"instance of foreclosure"and trace outcomes based on the date of thefirst LP.LPs that occur more than6months after an earlier LP are included and considered a separate foreclosurefiling.4The Department of Education matched foreclosure notices and students to building addresses that did not include identifiers distinguishing different units within a building.As a result,for foreclosure notices issued to a unit in a multifamily cooperative or condominium building,the match would not have reliably identified which students living in the building were actually living in the unit receiving the foreclosure notice.These represent a small share of the total foreclosure notices, however.408V.Been et al./Regional Science and Urban Economics41(2011)407–414classify students as living in a building entering foreclosure if the building they live in at a given date receives a foreclosure notice between that date and the next date when addresses are recorded.For example,for a student with an address recorded on October31st, 2003,we classified that student as living in a building receiving a foreclosure notice if that building received a foreclosure notice between that date and March1st when the DOE again records each student's address.More specifically,we classified a student as living in a building that received a foreclosure notice if:•The student lived in a property on October31st,2003,and that property received an LP between November1st,2003and February 29th,2004;•The student lived in a property on March1st,2004,and that property received an LP between March1st,2004and May31st, 2004;and•The student lived in a property on June1st,2004,and that property received an LP between June1st,2004and October30th,2004.This definition is conservative for several reasons.First,the count includes only foreclosure notices that are issued after the date on which we know a child's address,in order to ensure that we are not including students whose families move into a building after a foreclosure notice is resolved or completed.Thus,we may miss students who move into a unit after a foreclosure isfiled,but before a foreclosure is resolved.For example,if a property receives an LP in August,and a student moves into that property in September,the student will not be counted as affected,even though the student's family may be moving into a rental unit in the building and the foreclosure may not yet be resolved.The foreclosure notice could still therefore affect the student.Second,we only match students to LPs issued in the3to5months after we capture a student's address,in order to be fairly confident that the student's family was still living in the property at the time the notice was issued,but the New York foreclosure process is considerably longer than3to5months,so we likely are missing some families who live in properties in foreclosure for periods outside our window.To be clear,this designation does not assume that foreclosures only last3to5months.As noted,the foreclosure process is slow in New York City and takes about18months on average.Instead,the windows are used to be conservative and limit the noise that might be created by including the full18-month foreclosure period. Through this matching process,we create a student-level data set that includes a set of variables identifying whether a student lived in a property that entered foreclosure during that academic year, when the foreclosure started,and the characteristics of the property,along with characteristics of the students and their schools.54.Foreclosures in New York CityAlthough New York City was not hit as hard by foreclosures as cities such as Cleveland and Detroit,it has experienced a significant spike in recent years.The number of properties receiving a notice of foreclosure each year more than doubled between2000and2008, with sharp upturns occurring in2005–06,2006–07and again in 2008–09(see Fig.1).In2009,almost21,000properties received a notice of foreclosure.This rise in foreclosures came as housing prices in New York started to fall.For most of the last decade,New York's housing market enjoyed a strong price appreciation.Between1996and2006,for example,prices in the City rose steadily and by2006,prices were on average124%higher than they were in1996,even after controlling for inflation(Furman Center,2009).That price appreciation probably helped some borrowers avoid foreclosures;while many New Yorkers turned to risky high-cost loans tofinance their home purchases,a relatively small share of them ended up in foreclosure through2005, most likely because they were able to refinance their mortgages or sell their homes if their loans became too burdensome.When prices started to fall near the end of the decade,the number of foreclosures began to rise.Our analysis focuses on children living in properties going through the foreclosure process in the2003–04and2006–07school years.Fig.2shows the number and type of properties entering foreclosure during these two school years.Thefigure underscores both the growth in foreclosures during this period,but also the fact that that a large share of the properties receiving notices of foreclosure in New York City has been small multifamily properties with2–4units.Again,because many of the buildings entering foreclosure are multifamily properties,many affected households are renters.Even if all single-family homes are owner-occupied and one unit in each2–4 family building is owner-occupied,more than half of the housing units in properties entering foreclosure in2006–07would have been occupied by ing these assumptions,we estimate that the share of units entering foreclosure that are occupied by renters increased between our two sample years,reaching54%in the school year2006–07.5Note,however,that we do not know the addresses of the students—we simply know whether the address matched a foreclosure notice address.This prevents us from knowing the building type or neighborhood characteristics of the students whose properties do not experience aforeclosure.Fig.1.Properties that received a Lis Pendensfiling(2000–2009).Fig.2.Foreclosures during school study years,2003–04and2006–07.409 V.Been et al./Regional Science and Urban Economics41(2011)407–4145.Characteristics of students living in properties entering foreclosureNot surprisingly,given both the increase in foreclosure starts and the increased share of affected buildings that were multifamily,the number of public school students living in buildings entering fore-closure rose during this period too.In the2003–04school year,we identify a total of12,067students living in properties entering fore-closure.By the2006–07school year,this number had risen to20,453 students,just over half of whom were in elementary and middle school.These students constituted about2%of all public school students.As for their characteristics,Table1shows that28%of the students living in homes that entered foreclosure during the2006–07school year lived in single family homes,which means that their families likely owned the homes that went into foreclosure.6Close to two thirds lived in2–4family homes and about10%lived in larger apartment buildings.The majority of the students living in these multifamily homes are clearly renters(especially as cooperatives and condominiums have been excluded).In2–4family homes,the owner might live in one unit and rent out the others;in larger apartment buildings,all the units are likely to be rental.The share of students living in2–4family homes going through foreclosure grew between 2003–04and2006–07,while the share living in single family homes declined.While we do not have comparable information on the building types of children who did not live in homes going through foreclosure,we suspect that those going through foreclosure are far more likely to live in single-family and2–4family homes,as these buildings enter foreclosure at a much higher rate than larger buildings.Even after excluding cooperative and condominium buildings,half of all housing units in New York City are located in apartment buildings with more thanfive units,but only10%of students living in buildings entering foreclosure lived in such buildings.7Just over half of the students whose buildings had entered foreclosure attended grades1–8in the2006–07school year, roughly one quarter attended high school,10%attended Kinder-garten or pre-Kindergarten,and9%were enrolled in special education or adult education classes.These proportions are roughly the same as those for other students whose buildings did not enter foreclosure.In the2003–04school year,a slightly greater share of students attended elementary and middle school and a smaller share high school.Again,there was little difference between the students who lived in buildings entering foreclosure and those who did not.We do see a difference in poverty rates,with a greater share of students living in properties entering foreclosure eligible for free or reduced price lunch,especially in the later school year.The most striking difference between the students living in properties that entered foreclosure and those that did not was their racial composition.Students whose buildings entered foreclosure were far more likely to be black than other students in the school system:in 2006–07,57%of students living in buildings entering foreclosure,but only33%of all other students,were black.The proportions were almost identical in the earlier school year.Students whose buildings entered foreclosure were significantly less likely to be white or Asian, and perhaps surprisingly,they were also less likely to be Hispanic. Only29%of students whose buildings entered foreclosure were Hispanic in the2006–07school year,as compared to39%of other students.Again,these proportions were almost identical in the later school year.Given the concentration of foreclosures in particular neighbor-hoods in New York City,it is not surprising that the students living in properties that entered foreclosure were not evenly dispersed throughout the City.Rather,they were concentrated in particular schools.Half of the students living in properties going through foreclosure in2003–04attended just14%of all city schools,while75% of those students attended just31%of all schools.The level of concentration stayed roughly similar in2006–07,with half of students living in properties entering foreclosure attending17%of the City's schools.The characteristics of the schools attended by these children were distinct from those of other schools.To illustrate these differences,we arrayed schools for the two sample years by the percentage of their students living in buildings that entered foreclosure and then divided the schools into quartiles based on this percentage.Table2compares the characteristics of the top quarter of schools–with the highest percentage of children living in buildings that entered foreclosure–with the characteristics of the schools in the bottom quarter,and with the characteristics of all public schools.The share of students who are black was far higher in the high foreclosure-concentration schools.In 2006–07,in the quartile of schools with the highest share of students living in foreclosed properties,56%of students were black,compared to just31%of the student population in all NYC public schools.In the high concentration schools,all other racial groups were under-represented.In addition,as compared to other schools,the percentage of student's eligible for free or reduced price lunch(a measure of poverty)was higher in the high foreclosure-concentration schools, while the percentage of students who were limited English proficient (LEP)was lower.Finally,in the schools with the highest shares of students living in buildings that received a foreclosure notice,both reading and math test scores were significantly lower than in other schools.6.Were children living in properties that received foreclosure notices more likely to move to a new school?Our primary question of interest is whether students living in properties that entered foreclosure were more likely than other students to move to a different school.To test this question,we track students to the next school year(i.e.,we follow students originally in the2006–07school year to the2007–08school year),and we group them into one of three categories.Students could remain in theirTable1Characteristics of children living in foreclosed property.2003–042006–07Students living in foreclosed property Students notliving inforeclosedpropertyStudentsliving inforeclosedpropertyStudents notliving inforeclosedpropertyBuilding structure distributionSingle family31%28%2–4units59%63%5+units10%9%Grade distributionGrades1–8,total57%57%53%52%Grades9–12,etc.24%24%27%29%PreK/K10%10%10%11%Special Ed/other10%9%9%8%Eligibility for free/reduced price lunchPercent eligible78%75%89%79%Racial compositionPercent black56%32%57%33%Percent Hispanic30%39%29%39%Percent white9%15%8%14%Percent Asian/other6%13%7%14%Number of students12,0671,068,11520,4531,110,7806Recall that we do not have access to the building type for the students whosebuildings did not enter foreclosure.7We exclude cooperative and condominium units as they are excluded from ourpool of properties in foreclosure.410V.Been et al./Regional Science and Urban Economics41(2011)407–414original school,move to a new school in the New York City public school system,or exit the school system altogether,either because they moved out of New York City or because they enrolled in a private school.As Table 3shows,elementary and middle school children living in properties that had received foreclosure notices during the 2006–07school year were no less likely than other children to stay in their same school the following year.Approximately 84%of children in conventionally ‘non-terminal ’grades (grades 1–4,6and 7)stayed in the same school in the subsequent school year,regardless of whether they lived in a foreclosed property.While the percentage of students who stayed in their schools is much lower for the commonly ‘terminal ’grades (fifth and eighth grades in New York City,which are often the highest in their school),the percentages are again nearly identical for students living in buildings that had entered foreclosure and those who had not.There is,however,a marked difference in moves made by the children who did not stay in their same school.Children living in properties that received foreclosure notices were less likely to exit the New York City school system but more likely to switch to a new school within the City school system.These patterns were nearly identical for the 2003–04school year.To understand these patterns more fully,we use regression analysis to examine whether children living in foreclosed buildings were more likely to switch schools and less likely to exit the public school system,after controlling for race,poverty,gender,grade,and the original school they attended.Speci fically,we estimate the following models:Y ij =β0+β1X i +β2F i +φj +εijð1Þwhere Y ij is a dummy variable indicating whether student i in original school j moved to a new school between school years (or exited the school system in the second set of regressions),X i is a series of student characteristics,including race,poverty,gender,and grade,F i is a dummy variable indicating whether the student lived in a building that entered foreclosure in the initial school year,and φj is a series of school fixed effects,de fined by the student's “origin ”school.In the first model,we include a simple dummy variable to indicate whether the student lived in a building that entered foreclosure during the baseline school year.In the second model,we interact the foreclosure dummy with the property type,while in the third model,we interact the foreclosure dummy with the outcome of foreclosure (sale,foreclosure auction or REO,or unknown outcome).We estimateTable 3Distribution of students by mobility status,2006–2007.Students living in foreclosed propertyStudents not living in foreclosed property Grades 1–4Stayed in same school 84%83%Changed schools 13%10%Exited system 3%7%Grade 5Stayed in same school 23%22%Changed schools 74%71%Exited system 4%8%Grades 6–7Stayed in same school 85%83%Changed schools 12%10%Exited system 3%7%Grade 8Stayed in same school 6%7%Changed schools 92%85%Exited system3%8%Table 2Schools,by quartile of foreclosure incidence.2003–042006–07All schoolsTop quartile Bottom quartile All schools Top quartile Bottom quartile Percent black 33%54%18%31%56%16%Percent Hispanic 40%31%47%40%30%44%Percent white14%9%16%14%5%18%Percent Asian/other13%7%19%14%8%22%%Receiving free or reduced priced lunch 83%87%81%83%89%80%%LEP14%9%19%16%11%21%%Testing pro ficient or advanced on reading 45%41%49%55%50%61%%Testing pro ficient or advanced on math 54%48%58%71%67%79%Number of schools 9602402401085271271Number of students721,387194,294151,663665,496168,037162,434Table 4Regression of moving to a new school between 2006–07and 2007–08with property type and outcome interactions.Model 1Model 2Model 3White −0.015⁎⁎⁎−0.015⁎⁎⁎−0.015⁎⁎⁎(0.002)(0.002)(0.002)Hispanic −0.011⁎⁎⁎−0.011⁎⁎⁎−0.011⁎⁎⁎(0.002)(0.002)(0.002)Asian/Other −0.016⁎⁎⁎−0.016⁎⁎⁎−0.016⁎⁎⁎(0.002)(0.002)(0.002)Female −0.004⁎⁎⁎−0.004⁎⁎⁎−0.004⁎⁎⁎(0.001)(0.001)(0.001)Not poor −0.011⁎⁎⁎−0.011⁎⁎⁎−0.011⁎⁎⁎(0.002)(0.002)(0.002)Foreclosure0.022⁎⁎⁎(0.004)Foreclosure by property type Foreclosure ⁎single family 0.011⁎⁎(0.006)Foreclosure ⁎2–4unit building0.027⁎⁎⁎(0.005)Foreclosure ⁎5or more unit building 0.026⁎(0.011)Foreclosure by outcome Sold 0.012⁎(0.006)Auctioned0.061⁎⁎⁎(0.013)Unknown outcome 0.018⁎⁎⁎(0.004)Constant−0.038⁎⁎⁎−0.038⁎⁎⁎−0.038⁎⁎⁎(0.011)(0.011)(0.011)School fixed effects Yes Yes Yes N543749543749543749Adjusted R-squared0.590.590.59(1)Robust,clustered standard errors in parentheses.(2)Not poor =not eligible for free or reduced price lunch.(3)Grade dummies not shown.⁎p b 0.05.⁎⁎p b 0.01.⁎⁎⁎p b 0.001.411V.Been et al./Regional Science and Urban Economics 41(2011)407–414。