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The role of natural and synthetic zeolites as feed additives on the prevention and/or the treatment of certain farmanimal diseases:A reviewD.Papaioannou,P.D.Katsoulos,N.Panousis *,H.KaratziasClinic of Productive Animal Medicine,School of Veterinary Medicine,Aristotle University of Thessaloniki,54124Thessaloniki,GreeceReceived 4April 2005;received in revised form 12May 2005;accepted 12May 2005Available online 28June 2005AbstractThe present review comments on the role of the use of zeolites as feed additives on the prevention and/or the treatment of certain farm animal diseases.Both natural and synthetic zeolites have been used in animal nutrition mainly to improve performance traits and,based on their fundamental physicochemical properties,they were also tested and found to be eﬃcacious in the prevention of ammonia and heavy metal toxicities,poisonings as well as radioactive elements uptake and metabolic skeletal defects.During the last decade,their utilization as mycotoxin-binding adsorbents has been a topic of considerable interest and many published research data indicate their potential eﬃcacy against diﬀerent types of mycotoxins either as a primary material or after speciﬁc modiﬁcations related to their surface properties.Ingested zeolites are involved in many biochemical processes through ion exchange,adsorption and catalysis.Recent ﬁndings support their role in the prevention of certain metabolic diseases in dairy cows,as well as their shifting eﬀect on nitrogen excretion from urine to faeces in monogastric animals,which results in lower aerial ammonia concentration in the conﬁnement facilities.Moreover,new evidence provide insights into potential mechanisms involved in zeolites supporting eﬀect on animals suﬀered from gastrointestinal disturbances,including intestinal parasite infections.All the proposed mechanisms of zeolites Õeﬀects are summarized in the present review and possible focus topics for further research in selected areas are suggested.Ó2005Elsevier Inc.All rights reserved.Keywords:Zeolites;Animal health;Prevention;Treatment1.IntroductionZeolites are crystalline,hydrated aluminosilicates of alkali and alkaline earth cations,consisting of three-dimensional frameworks of SiO 4À4and AlO 5À4tetrahedra linked through the shared oxygen atoms.Both natural and synthetic zeolites are porous materials,character-ized by the ability to lose and gain water reversibly,to adsorb molecules of appropriate cross-sectional diame-ter (adsorption property,or acting as molecular sieves)and to exchange their constituent cations without majorchange of their structure (ion-exchange property)[1,2].The exploitation of these properties underlies the use of zeolites in a wide range of industrial and agricultural applications and particularly in animal nutrition since mid-1960s [3].Many researchers have proved that the dietary inclu-sion of zeolites improves average daily gain and/or feed conversion in pigs [1,4–13],calves [1,7],sheep [7,14–16],and broilers [17–19].Zeolites also enhance the reproduc-tive performance of sows [1,20,21],increase the milk yield of dairy cows [22,23]and the egg production of laying hens [24,25]and have beneﬁcial eﬀects on egg weight and the interior egg characteristics [24–26].How-ever,the extent of performance enhancement eﬀects is related to the type of the used zeolite,its purity and1387-1811/$-see front matter Ó2005Elsevier Inc.All rights reserved.doi:10.1016/j.micromeso.2005.05.030*Corresponding author.Tel.:+302310994501;fax:+302310994550.E-mail address:panousis@vet.auth.gr (N.Panousis)./locate/micromesoMicroporous and Mesoporous Materials 84(2005)161–170physicochemical properties,as well as to the supplemen-tation level used in the diets.Besides,the particle size of the zeolitic material,crystallite size and the degree of aggregation,as well as the porosity of individual parti-cles determine the access of ingestaﬂuids to the zeolitic surface during passage across gastrointestinal tract and strongly aﬀect its ion exchange,adsorption and catalytic properties.Table1summarizes the possible mechanisms by which zeolites may exert their performance promot-ing properties in farm animals.Apart from the positive eﬀects on animalsÕperfor-mance,dietary supplementation of zeolites appears to represent an eﬃcacious,complementary,supportive strategy in the prevention of certain diseases and the improvement of animalsÕhealth status.The aim of the present review article is to address the data concerning the inﬂuence of the in-feed inclusion of natural and syn-thetic zeolites on certain diseases of farm animals,to summarize the proposed mechanisms of zeolitesÕeﬀects and to suggest possible focus topics for further research in selected areas.2.Ameliorative eﬀect on the consequences of mycotoxicosesIn the recent years,high incidence rates of contami-nation of cereal grains and animal feed with mycotoxins are reported worldwide[38].One of the latest ap-proaches to this global concern has been the use of nutritionally inert adsorbents in the diet that sequester mycotoxins,thus reducing intestinal absorption and, additionally,avoiding toxic eﬀects for livestock and the carry-over of toxin compounds to animal products. For this purpose,phyllosilicates such as hydrated sodium calcium aluminosilicates(HSCAS)and benton-ite––which consist of layered crystalline structures and possess similar physicochemical properties with zeo-lites––have beenﬁrst used successfully in poultry,pig, sheep,cattle and laboratory animals[33,34,39–47].Apart from phyllosilicates,the use of zeolites has shown,lately,very promising results as well.In general, adsorption process on binders is strongly related to charge distribution,pore dimensions and accessible sur-face area of the adsorbent,as well as to polarity,solubil-ity and molecular dimensions of the certain mycotoxin which is to be adsorbed[46].Molecular sizes of aﬂatox-ins range from5.18A˚(B1and B2)to6.50A˚(G1and G2) and only zeolites with entry channels wide enough to permit the diﬀusion of aﬂatoxin molecules to the intra-crystalline structure are capable of demonstrating a clear sequestering eﬀect.Clinoptilolite,a natural zeolite,has high adsorption indexes in vitro,more than80%,for aﬂatoxins B1[48,49]and G2[48]and the adsorption pro-cess begins with a fast reaction whereby most of the toxin is adsorbed within theﬁrst few minutes[48].On the contrary,Lemke et al.[50]conducted a variety of in vitro adsorption studies and reported a limited degree of clinoptilolite ability to bind aﬂatoxin B1eﬀectively. According to them,adsorbent materials should be checked through a multi-tiered system of in vitro tests in order potential interactive factors,such as intestinal physicochemical variables and feed components,to be precluded.Indeed,a previous in vitro study had demon-strated average aﬂatoxin retention in natural zeolites of 60%,but also a nitrogen compound-related adsorbent eﬀectiveness,when liquid media quality parameters had been taken into account[51].The in vivo eﬃcacy of zeolites to ameliorate the consequences of aﬂatoxico-sis,mainly in poultry,has also been veriﬁed in many cases(Table2).In the case of phyllosilicates,the results of in vitro mycotoxin–clay interaction tests suggest the existence of areas of heterogenous adsorption aﬃnities onto sur-face,the presence of diﬀerent adsorption mechanisms or both[57].Nevertheless,the formation of strong bonds by chemisorption and the interaction of b-car-bonyl group of aﬂatoxin B1with the uncoordinated edge site aluminum ions in these adsorbents have been suggested as the binding mechanism which interprets their well-established high aﬃnity for aﬂatoxin B1 [39,58,59].Although the exact binding mechanisms of zeolites on aﬂatoxins have not been determined the pos-sibility to act through similar with phyllosilicates mech-anism cannot be precluded and should be investigated.As far as other mycotoxins are concerned,mineral adsorbents exert a lower eﬃcacy against mycotoxins containing less polar functional groups,which are required for eﬃcient chemisorption on hydrophilic negatively charged mineral surfaces to occur.This limi-tation can be overcome by the use of chemically modi-ﬁed clays.Modiﬁcations consist of alterations ofTable1Proposed mechanisms involved in animalsÕperformance promoting properties of the dietary use of zeolitesMechanismsAmmonia binding eﬀect Elimination of toxic eﬀects of NHþ4produced by intestinal microbial activity[8,10]Fecal elimination of p-cresol Reduction of the absorption of toxic products of intestinal microbial degradation,such as p-cresol[27] Retarding eﬀect on digesta transit Slower passage rate of digesta through the intestines and more eﬃcient use of nutrients[1,25,28] Enhanced pancreatic enzymes activity Favorable eﬀect on feed components hydrolysis over a wider range of pH,improved energy and protein retention[29,30]Aﬂatoxin sequestering eﬀect Elimination of mycotoxin growth inhibitory eﬀects[31–37]162 D.Papaioannou et al./Microporous and Mesoporous Materials84(2005)161–170surface properties resulting in an increased hydropho-bicity,by exchange of structural charge-balance cations with high molecular weight quaternary amines.In vitro results have veriﬁed the binding eﬃcacy of modiﬁed montmorilonite and clinoptilolite against zearalenone and ochratoxin A[60,61].However,naturally occurring clays also exert a moderate binding eﬃcacy against mycotoxins,other than aﬂatoxins,as evidenced for zearalenone and ochratoxin A by in vitro[57]andﬁeld trials[62,63],as well as for cyclopiazonic acid in exper-iments with broilers[45].Remarkable conclusions related to zeolitesÕeﬃcacy against zearalenone toxicosis have also been drawn by in vivo studies.Feeding zearalenone to rats,Smith[64] demonstrated that the dietary use of a synthetic anion exchange zeolite could alter the faecal and urinary excre-tory patterns of zearalenone due to the elimination of its intestinal absorption.Recently,the dietary use of a clin-optilolite-rich tuﬀwas also eﬀective in decreasing zearal-enone and a-zearalenole excretion in pigs fed diets contaminated with500ppb zearalenone[65].Addition-ally,in aﬁeld-case of zearalenone toxicosis with mean concentrations of660ppb,Papaioannou et al.[20] reported that the supplementation of a clinoptilolite rich tuﬀat the rate of2%in the ration of pregnant sows im-plied a rather protective role against the consequences of zearalenone ingestion,as evidenced by the improvement of indicative performance traits.Similar results were also obtained in swine with the dietary use of a modiﬁed clinoptilolite–healandite rich tuﬀat0.2%and with zea-ralenone concentration exceeding3.5ppm[66].Apart from surface interactions,the in vivo eﬃcacy of mineral adsorbents against zearalenone could also re-sult from other implicating mechanisms.The entero-he-patic circulation of zearalenone and its derivatives in pigs retards their elimination and enhances the duration of adverse eﬀects[67].Whether certain types of zeolites are able to aﬀect the entero-hepatic cycling of zearale-none,thus counteracting the toxic eﬀects of its biological action,is a hypothesis that awaits further research, although there is evidence of Ca-enriched clinoptiloliteÕs high aﬃnity to the bile acids in the intestinal tract[68].3.Supportive eﬀect on diarrhoea syndromeThere is an abundance of published data which indi-cate that the dietary use of natural zeolites reduces the incidence and decreases the severity and the duration of diarrhoea in calves[1,69–72]and pigs[1,5,13,72–75]. The exact mechanism of zeolitesÕeﬀect is not quite clear so far,although there is evidence that the use of zeolites may eliminate various predisposing and/or causative fac-tors which are associated in the culmination of intestinal disturbances in an interactive way.Apart from zeolitesÕretarding eﬀect on intestinal passage rate[1]and their water adsorption property,which leads to the appear-ance of drier and more compact faeces,as in the case of phillipsite[75]or clinoptilolite[76],Vrzgula et al.[71]also proposed that the ameliorative eﬀect on diar-rhoea syndrome of calves might result from either the alteration of metabolic acidosis,through eﬀects on os-motic pressure in the intestinal lumen,or the increased retention of the enteropathogenic Escherichia coli.As far as we know,there is no evidence in the available lit-erature for retention of enteropathogenic E.coli on the outer surface of zeolite particles.However,clinoptilolite and mordenite are capable to adsorb and partially inac-tivate the thermo-labile(LT)E.coli enterotoxin in vitro, thus constricting its attachment to the intestinalTable2In vivo studies concerning the eﬀect of the dietary use of zeolites during aﬂatoxicosesType of zeolite Dietary inclusion rate(%)Animal model ObservationsClinoptilolite1Broilers Growth depression caused by2.5ppm aﬂatoxin(aﬂ)was alleviated by15%[31] Clinoptilolite5Geese Prophylactic eﬀect on growth rate and liver enzymatic activity[32]Mordenite0.5Broilers Reducing eﬀect on toxicity of aﬂ(3.5mg kgÀ1diet)as indicated byweight gain and changes in uric acid and albumin concentrations[33] Clinoptilolite0.5Weaned piglets Growth inhibitory eﬀects and alterations of liver enzyme activity inducedby500ppb aﬂwere prevented[34]Synthetic zeolite0.5Broilers No signiﬁcant eﬀect on biochemical or haematological indexes whenadministered simultaneously with2.5mg aﬂkgÀ1diet[52]Clinoptilolite0.5Pregnant rats No eﬀect on maternal and developmental toxicities of aﬂ(2mg kgÀ1body weight)[53]Clinoptilolite5Quail chicks Growth inhibitory eﬀects of2mg kgÀ1diet diminished by70%[35] Clinoptilolite 1.5Broilers Growth inhibitory eﬀects of100ppb aﬂdiminished over a studyperiod of42days[36]Clinoptilolite 1.5–2.5Broilers Adverse eﬀects of2.5mg kgÀ1diet on biochemical and haematologicalproﬁles were reduced[54]Zeolite NaA1Broilers Protection against growth inhibitory eﬀects of2.5mg kgÀ1diet[37] Clinoptilolite 1.5–2.5Broilers Moderate to signiﬁcant decrease of incidence and severity of certaintarget-organs degenerative changes induced by2.5mg aﬂkgÀ1diet[55] Clinoptilolite2Laying hens Signiﬁcant decrease in liver mycotoxin concentration and liver weightduring aﬂatoxicosis caused by2.5mg kgÀ1diet[56]D.Papaioannou et al./Microporous and Mesoporous Materials84(2005)161–170163cell-membrane receptors[77].Furthermore,the adsorp-tion capacity of clinoptilolite and mordenite has been proved to be higher than94%for virions of bovine rota-virus and coronavirus,although infectivity level of zeolite–virus complex seems to remain unchanged[78]. Interactions among virions and the outer surface of adsorbent particles have been proposed,since the former have dimensions considerably larger(60–80nm and 60–220nm for rota-and coronavirus particles,respec-tively)than the entry channels of the aforementioned zeolites.In a more recent study of Rodrigues-Fuentes et al.[68],dealing with the development and the properties of an anti-diarrhoeic drug for humans based on clinop-tilolite,zeolite had no eﬀect on rate of passage of inges-ta,neither acted as a water adsorbent.Instead,they proposed that the anti-diarrhoeic eﬀect of clinoptilolite is due to the adsorption of(i)bile acids,‘‘one of the endogenic causes of diarrhoea’’,(ii)aﬂatoxin B1,‘‘a mycotoxin that produces severe toxicity in animals and humans’’and(iii)glucose,‘‘whose high content in intes-tinalﬂuid acts as an irritant factor and whose transport through the intestinal cells is reversed during diarrhoea’’.Concerning the newborn animals,the administration of zeolites appears to reduce the incidence of diarrhoea through the enhancement of passive immunity,as they increase the net absorption of colostrum immunoglobu-lins in calves[71,79,80]and pigs[81].Intestinal hypersensitivity to feed antigens or the mal-absorption syndrome,induced by a low enzyme activity, can both predispose to post-weaning infectious enteritis in pigs.According to Papaioannou et al.[13],clinoptil-olite has the ability to adsorb dietary substances,which may result in intestinal hypersensitivity phenomena[82], or to support the maintenance and even the restoration of the digestive enzyme activity in newly weaned piglets. This should also be evaluated in future studies as an additional explanation for clinoptilolitesÕminimising ef-fect on diarrhoea syndrome.4.Prevention of metabolic diseases in dairy cowsMilk fever and ketosis are of the most common metabolic diseases in high producing dairy cows.In the recent years,a number of experiments have been con-ducted in order to control these diseases using zeolites as feed additives.The results of these experiments are very promising but further investigation is required to deﬁne the exact mechanisms of zeolitesÕaction.k feverInitially,a series of experiments has been conducted in order to study the potential use of synthetic zeolite A for the prevention of milk fever in dairy cows.The objective of these experiments was to reduce the bio-availability of dietary Ca in the gastrointestinal tract by the administration of synthetic zeolite A,based on the evidence that one of the best ways to prevent milk fever is to feed cows with low calcium diets during the dry period[83–87].The results obtained were satisfac-tory as the administration of synthetic zeolite A,either as an oral drench or supplemented to the total mixed ra-tion,during the dry period reduced the bioavailability of dietary Ca and eﬃciently protected against milk fever, by stimulating Ca-homeostatic mechanisms prior to par-turition[88–93].Furthermore,Thilsing-Hansen et al.[92]proposed that the best ratio zeolite/Ca for the pre-vention of milk fever was10–20and that zeolite had the same eﬃciency either administrated for the last4 or2weeks of the dry period.More recently,Katsoulos et al.[94]showed that clin-optilolite was eﬀective in the prevention of milk fever as well.The incidence of milk fever was signiﬁcantly lower in cows that were receiving a concentrate supplemented with clinoptilolite at the level of2.5%(5.9%)during the last month of the dry period and the onset of lactation compared to the animals in the control group(38.9%), which were not receiving clinoptilolite,whereas was not signiﬁcantly diﬀerent than those that were receiving 1.25%clinoptilolite(17.6%)with the concentrates at the same period.The authors suggested that clinoptilolite might have had similar eﬀect with zeolite A in activating Ca homeostatic mechanisms prior to parturition.As a consequence,the animals receiving2.5%clinoptilolite responded faster and more eﬃciently in the drop of serum Ca observed at the day of calving and did not show any clinical signs of milk fever the following days. However,the exact mechanism for this positive eﬀect of clinoptilolite is currently unknown and should be fur-ther investigated.4.2.KetosisThe best strategy to prevent ketosis in dairy cows is to improve the energy uptake both in the dry period and the onset of lactation[95].According to Katsoulos et al.[23],the use of clinoptilolite has been shown to be eﬀective in improving the energy balance at this critical period as they observed that feeding dairy cows on a diet supplemented with clinoptilolite at the level of2.5%of the concentrate feed,resulted in signiﬁcantly lower incidence of ketosis(5.9%)during theﬁrst month after parturition,compared to the control group(38.9%) and the group of the animals receiving a concentrate supplemented with1.25%clinoptilolite(35.3%).These researchers suggested that clinoptilolite improved the energy status of the cows,either via prepartum enhance-ment of propionate production in rumen or through the improvement of the post-ruminal digestion of starch.164 D.Papaioannou et al./Microporous and Mesoporous Materials84(2005)161–1705.Protective role in intoxications and poisonings5.1.Ammonia toxicityWhite and Ohlrogge[96]ﬁrst stated that ammonium ions formed by the enzyme decomposition of non-pro-tein nitrogen were immediately ion exchanged into the zeolite structure and held there for several hours until released by the regenerative action of Na+,entering the rumen in saliva during the after-feeding fermenta-tion period.From both in vitro and in vivo experimentsthey found that up to15%of the NHþ4in the rumencould be taken up by the zeolite.These observations were the causation for the conduction of many experi-ments in order to determine the inﬂuence of zeoliteson rumen NHþ4concentration and their potential usefor the counteraction of the toxic eﬀects of urea inclu-sion in ruminantsÕrations.Hemken et al.[97]showed that supplementation of 6%clinoptilolite,in the ration of dairy cows containing urea,signiﬁcantly reduced rumen NH3concentration. The same trend was observed by the dietary addition of5%clinoptilolite in steers[98]and lambs[99].Fur-thermore,clinoptilolite was eﬀective in reducing rumen ammonia concentration even when no urea was present in the ration of steers receiving a high concentrate diet, and that this reduction was linearly associated to the percentage of clinoptilolite inclusion[100].Nestorov [101]referred that simultaneous administration of clin-optilolite and urea in sheep protects rumenﬂora from toxic eﬀects of ammonia by inhibiting the reduction of microbiota population.In contrast to the former observations,Bergero et al.[102]and Bosi et al.[103]found that daily administration of250g or200g of clinoptilolite, respectively,in dairy cows did not aﬀect rumen NHþ4 concentration.The same result had the dietary inclu-sion of2%synthetic zeolite A in dairy cows ration [104]and5%clinoptilolite in steers receiving a high roughage diet[98].The binding of NHþ4to zeolites has been noted in pigsas well,and many researchers suggested this action as the possible mechanism for the observed improved per-formance of the animals receiving zeolites.There are evi-dences that clinoptilolite elevates nitrogen excretion in feces[8,105]and reduces the ammonia concentration in blood serum[8,10,106],when supplemented to the basal diets of pigs.Furthermore,Pond et al.[10]and Yannakopoulos et al.[12]found that clinoptilolite reduced the weight of the organs involved in the metab-olism of ammonia(liver and kidneys),as the conse-quence of the reduced ammonia concentration in the gastrointestinal tract.Such observations result fromthe direct binding of NHþ4to zeolites,as clinoptilolitehas no adverse eﬀect on the ureolytic bacteria of the large intestine and urease activity[107]anophosphate poisoningThe dietary use of clinoptilolite appears to be eﬀective in the prevention of organophosphates poisoning. Experiments in sheep have shown that the oral adminis-tration of clinoptilolite at the dose of2g/kg of body weight,earlier or simultaneously with an organophos-phate(VX),partially protects from poisoning by inhib-iting the decrease in cholinesterase activity[108]and by protecting rumenﬂora[109].The protective eﬀect of clinoptilolite on cholinesterase activity has been ob-served in mice receiving higher doses of organophos-phates as well[110,111].5.3.Heavy metal toxicity and adsorption of radioactive elementsZeolites,due to their high ion-exchange capacity, have been used eﬀectively for the prevention of heavy metal toxicity in animals.Pond et al.[112]found that clinoptilolite protects growing mice from lead(Pb)tox-icity when added to their ration in such quantities that the ratio clinoptilolite/Pb to be10/1.According to Pond et al.[113],similar protection is provided in swine as well.The selectivity of clinoptilolite for cadmium(Cd) and Pb has been studied in vitro in order to be investi-gated whether its use reduces the levels of these elements in rumen and abomasalﬂuid.The experiments showed that clinoptilolite bent the91%of Pb and the99%of Cd in rumenﬂuid within24h,and in the abomasalﬂuid the94%of Pb within less than1h[114].The toxic eﬀects of long-term ingestion of Cd(100ppm CdCl2)on female rats and their progeny were not diminished by the simul-taneous feeding of a clinoptilolite-rich tuﬀat5%in the diet[115].Adversely,the eﬃcacy of clinoptilolite against Cd toxicity has been proved in pigs by the same authors who observed that3%clinoptilolite supplementation prevented the cadmium-induced iron deﬁcient anemia in growing swine that were receiving150ppm CdCl2 [116].The results of these experiments suggest the feasi-bility of using zeolites and mainly clinoptilolite as a feed additive in the prevention of certain types of heavy metal intoxications in farm animals or in aquatic biolog-ical systems,as is the case in the study of Jain[117], where is ascertained the capacity of zeolite to enhance the removal of Pb from water,thus decreasing its avail-ability to the teleostﬁsh Heteropneustes fossilis.Apart from heavy metals,zeolites can also bind radioactive elements,thus being suggested as a means of altering their uptake and excretion from the body. Zeolitic matrix exchanges radio-nuclides in the gastroin-testinal tract and is excreted by normal processes,there-by eliminating radioactive elementsÕassimilation into the body.Arnek and Forsberg[118]proved the selectiv-ity of some natural zeolites such as clinoptilolite, chabazite and modernite for cesium and GomonajD.Papaioannou et al./Microporous and Mesoporous Materials84(2005)161–170165et al.[119]the selectivity of clinoptilolite for strodium and zirconium.Phillippo et al.[120]showed that the dietary use of clinoptilolite may constitute a simple and cost-eﬀective method for minimizing the adsorption of radioactive cesium by sheep grazing contaminated pastures,although there might be no eﬀect on cesium already been built-up in the body due to a previous exposure.Furthermore,Forsberg et al.[121]observed that the administration of mordenite in sheep and goats increased the excretion of cesium with feces and reduced its accumulation in tissues.On the other hand,Rachu-bik and Kowalski[122]demonstrated that synthetic zeolite-enriched diets exerted an inconsistent pattern of radiostrontium assimilation in the bone tissue and liver or kidneys of rats intragastrically dosed with an aqueous solution of90SrCl2.5.4.Copper toxicityIvan et al.[123]observed that the inclusion of ben-tonite,a phyllosilicate,in the ration of sheep at the rate of0.5%signiﬁcantly reduced the Cu concentration in liver and suggested the use of this material in order to prevent copper poisoning.In contrast,clinoptilolite does not seem to be eﬀective,as Pond[124]found that the addition of2%clinoptilolite to the basal diet of sheep containing10or20ppm Cu did not protect against the toxic signs of Cu and increased the mortality in lambs fed the diet with20ppm Cu.A lack of any ef-fect on liver Cu accumulation was also found in growing pigs which were on diets supplemented with0.5%syn-thetic zeolite A and250ppm Cu[125].Clinoptilolite was expected to exchange Cu in the lumen of the intes-tine,thereby decreasing the toxicity of excess Cu for sheep,whose tolerance for Cu is low compared with that of other food animals.However,such action was not observed probably due to a shift in ion-exchange relativeto Cu and NHþ4or to some other complex interaction,which resulted in a net increase in Cu available for absorption from the intestinal tract[124].The optimum ratio of clinoptilolite to Cu in the diet for reducing the intestinal absorption of the latter has not been deter-mined,but such information,according to Pond[126], is needed in order to establish appropriate levels of die-tary clinoptilolite supplementation.6.Impact on parasite infectionsConsidering the potential eﬃcacy of zeolites against parasite infections,the results of the experimentsﬁrst conducted in rats were encouraging for their use in other animal species as well.According to Wells and McHugh [127],the administration of clinoptilolite at the rate of 10%of a conventional diet facilitated the removal of parasites from the intestinal lumen of rats infected with the nematode Nippostrongylus brasiliensis.Furthermore, Wells and Kilduﬀ[128]observed a more accelerated intestinal a-D-glucosidase and aminopeptidase activity restitution in rats fed a commercial diet supplemented with clinoptilolite(5%)and recovering from N.brasilien-sis infection.Conﬁrming the observations in rats,Deli-giannis et al.[129]recently proved the eﬃcacy of clinoptilolite against parasite infections in growing lambs.They showed that feeding lambs,primarily infected with a single dose of gastrointestinal(GI) nematodes,with a concentrate mixture containing3% clinoptilolite signiﬁcantly decreased their total worm burden and faecal egg counts per capita fecundity and demonstrated that clinoptilolite supplementation re-duced the establishment of GI nematodes and resulted in a good performance of the animals.Interestingly,zeolites have also been tested as anthel-mintic loaded carriers,through retarding drug release and prolonging its therapeutic action.Sustained-release mechanism implies a slow desorption of the drug mole-cules from the external surface and the internal zeolitic cavities,as they are progressively replaced by host pro-teins and water molecules,respectively,during the intes-tinal transport of the drug-zeolite compound.Promising results were obtained,atﬁrst,as regards tetramisole-loaded synthetic zeolite Y[130]and recently,pyrantel-and fenbendazole-loaded synthetic zeolite Y in rats infested with N.brasiliensis and dichlorvos-loaded zeo-lite Y in pigs infected with Ascaris suum[131].In the case of tetramisole and dichlorvos,anthelmintic mole-cules are small enough toﬁt through the entry channels of zeolite Y(windows of7.4A˚),while fenbendazole loading requires an initial partial dealumination of the zeolitic carrier and large pyrantel molecules allow only outer surface loading to occur.7.Prevention of metabolic skeletal defectsThe dietary inclusion of synthetic zeolite A(at the rates of0.75%or1.5%)in broilers which are on a diet with inadequate or marginal levels of calcium results in an increase of bone ash content along with a reduc-tion of rachitic lesions[132].Accordingly,the incorpora-tion of zeolite A in the same diets at1%exerts a clear beneﬁcial eﬀect in reducing the incidence of tibial dys-chondroplasia[132–134].Although tibial dyschondro-plasia is a metabolic cartilage disease which represents the endpoint of several mechanisms,the incidence is in-creased when high dietary levels of phosphorus are used [135]or when dietary calcium is lower than0.85%[136]. Similarly,the beneﬁcial eﬀect of zeolite A is inconsistent and largely depends on the dietary level of calcium. According to Watkins and Southern[137],the dietary use of0.75%zeolite A in broilers is accompanied by alterations in mineral absorption and tissue distribution,166 D.Papaioannou et al./Microporous and Mesoporous Materials84(2005)161–170。

Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants Foods, and Dietary Supplements

Cellular Antioxidant Activity(CAA)Assay forAssessing Antioxidants,Foods,and DietarySupplementsK ELLY L.W OLFE†AND R UI H AI L IU*,‡,†Department of Food Science and Institute of Comparative and Environmental Toxicology,CornellUniversity,Ithaca,New York14853-7201A cellular antioxidant activity(CAA)assay for quantifying the antioxidant activity of phytochemicals, food extracts,and dietary supplements has been developed.Dichloroﬂuorescin is a probe that is trapped within cells and is easily oxidized toﬂuorescent dichloroﬂuorescein(DCF).The method measures the ability of compounds to prevent the formation of DCF by2,2′-azobis(2-amidinopropane) dihydrochloride(ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2cells.The decrease in cellularﬂuorescence when compared to the control cells indicates the antioxidant capacity of the compounds.The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay,and the results were expressed in micromoles of quercetin equivalents per100µmol of phytochemical or micromoles of quercetin equivalents per100g of fresh fruit. Quercetin had the highest CAA value,followed by kaempferol,epigallocatechin gallate(EGCG), myricetin,and luteolin among the pure compounds tested.Among the selected fruits tested,blueberry had the highest CAA value,followed by cranberry>apple)red grape>green grape.The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake,metabolism,and location of antioxidant compounds within cells.KEYWORDS:Antioxidant;antioxidant activity;antioxidant activity assay;dietary supplements;free radicals;fruits;phenolics;phytochemicals;vegetablesINTRODUCTIONHeart disease and cancer are the two leading causes of death in the United States(1),and oxidative stress is thought to be an important contributing factor in their development.Oxidative stress is an imbalance between the production of reactive oxygen species(ROS)and antioxidant defense and may lead to oxidative damage(2,3).It can result from a deﬁciency in antioxidant defense mechanisms,or from an increase in ROS,due to exposure to elevated ROS levels,the presence of toxins metabolized to ROS,or excessive activation of ROS systems, such as those mediated by chronic infection and inﬂammation (4).In addition to endogenously produced antioxidants and enzymes that catalyze the metabolism of ROS,ROS can be scavenged by exogenously obtained antioxidants,such as phenolics,carotenoids,and vitamins found in fruits and vegetables.Fruits and vegetables are excellent sources of phenolic compounds(5,6).Consumption of these compounds from dietary plant sources may increase protective antioxidants in the body and help combat cardiovascular diseases and cancer, as supported by epidemiological studies(7–13).The2005 Dietary Guidelines for Americans recommends consumption of at least four servings of fruits andﬁve servings of vegetables per day based on a dietary requirement of2000kcal(14). The measurement of antioxidant activity is an important screening method to compare the oxidation/reduction poten-tials of fruits and vegetables and their phytochemicals in various systems.Many chemistry methods are currently in wide use,including the oxygen radical absorbance capacity (ORAC)(15),total radical-trapping antioxidant parameter (TRAP)(16,17),Trolox equivalent antioxidant capacity (TEAC)(18),total oxyradical scavenging capacity(TOSC) (19),and the peroxyl radical scavenging capacity(PSC) assays,the latter of which was recently developed by our laboratory(20).The ferric reducing/antioxidant power(FRAP) assay(21)and the DPPH free radical method(22)measure the ability of antioxidants to reduce ferric iron and2,2-diphenylpicrylhydrazyl,respectively.Despite wide usage of these chemical antioxidant activity assays,their ability to predict in vivo activity is questioned for a number of reasons.Some are performed at nonphysiological pH and temperature,and none of them take into account the*Address correspondence to this author at the Department of FoodScience,Stocking Hall,Cornell University,Ithaca,NY14853-7201[telephone(607)255-6235;fax(607)254-4868;e-mail RL23@].†Department of Food Science.‡Institute of Comparative and Environmental Toxicology.8896J.Agric.Food Chem.2007,55,8896–890710.1021/jf0715166CCC:$37.00 2007American Chemical SocietyPublished on Web09/29/2007bioavailability,uptake,and metabolism of the antioxidant compounds(23).The protocols often do not include the appropriate biological substrates to be protected,relevant types of oxidants encountered,or the partitioning of compounds between the water and lipid phases and the inﬂuence of interfacial behavior(24).Biological systems are much more complex than the simple chemical mixtures employed,and antioxidant compounds may operate via multiple mechanisms (25).The different efﬁcacies of compounds in the various assays attest to the functional variation.The best measures are from animal models and human studies;however,these are expensive and time-consuming and not suitable for initial antioxidant screening of foods and dietary supplements(23).Cell culture models provide an approach that is cost-effective,relatively fast, and address some issues of uptake,distribution,and metabolism. Therefore,there is an urgent need to develop a cell-based antioxidant activity assay to screen foods,phytochemicals,anddietary supplements for potential biological activity.The objective of this research was to develop a quantiﬁable cellular antioxidant activity(CAA)assay,which would serve as more suitable method to measure antioxidant activity than the currently used“test tube”chemistry methods.We believe that this model better represents the complexity of biological systems than the popular chemistry antioxidant activity assays and is an important tool for screening foods,phytochemicals, and dietary supplements for potential biological activity. MATERIALS AND METHODSChemicals.Folin–Ciocalteu reagent,2′,7′-dichloroﬂuorescin di-acetate(DCFH-DA),ethanol,glutaraldehyde,methylene blue, ascorbic acid,caffeic acid,(+)-catechin,(-)-epicatechin,(-)-epigallocatechin gallate(EGCG),ferulic acid,kaempferol,luteolin, myricetin,phloretin,quercetin dihydrate,resveratrol,and taxifolin were purchased from Sigma-Aldrich,Inc.(St.Louis,MO).Gallic acid was obtained from ICN Biomedicals,Inc.(Aurora,OH). Dimethyl sulfoxide and acetic acid were obtained from Fisher Scientiﬁc(Pittsburgh,PA),and2,2′-azobis(2-amidinopropane) dihydrochloride(ABAP)was purchased from Wako Chemicals USA, Inc.(Richmond,VA).Sodium carbonate,acetone,and methanol were obtained from Mallinckrodt Baker,Inc.(Phillipsburg,NJ).The HepG2cells were obtained from the American Type Culture Collection(ATCC)(Rockville,MD).Williams’Medium E(WME) and Hanks’Balanced Salt Solution(HBSS)were purchased from Gibco Life Technologies(Grand Island,NY).Fetal bovine serum (FBS)was obtained from Atlanta Biologicals(Lawrenceville, GA).Fruit Samples.Wild blueberries were obtained from the Wild Blueberry Association of North America(Orono,ME).Red Delicious apples were obtained from Cornell Orchards(Ithaca,NY).Green and red seedless table grapes and frozen cranberries were purchased at a local supermarket(Ithaca,NY).Fruit Extractions.Extracts were obtained from the fruits using80% acetone,as described previously(6).Determination of Total Phenolic Content.The total phenolic contents of the fruit extracts were determined using the Folin–Ciocalteu colorimetric method(26),as modiﬁed by our laboratory(27,28).Results were expressed as mean micromoles of gallic acid equivalents(GAE) per100g of fresh fruit(SD for three replicates.Preparation of Chemical and Fruit Sample Solutions.A20mM stock solution of DCFH-DA in methanol was prepared,aliquoted,and stored at-20°C.A200mM ABAP stock solution was prepared,and aliquots were stored at-40°C.Working phytochemical and fruit extract solutions were prepared just prior to use.Caffeic acid,(+)-catechin, EGCG,(-)-epicatechin,ferulic acid,gallic acid,kaempferol,myricetin, phloretin,resveratrol,and taxifolin were dissolved in ethanol,luteolin was dissolved in methanol,and quercetin was dissolved in dimethyl sulfoxide before further dilution in treatment medium(WME with2 mM L-glutamine and10mM Hepes).Fruit extracts were diluted in treatment medium.Final treatment solutions contained<2%solvent, and there was no cytotoxicity to HepG2cells at those concen-trations.Cell Culture.HepG2cells were grown in growth medium(WME supplemented with5%FBS,10mM Hepes,2mM L-glutamine,5µg/ mL insulin,0.05µg/mL hydrocortisone,50units/mL penicillin,50µg/ mL streptomycin,and100µg/mL gentamicin)and were maintained at 37°C and5%CO2as described previously(29,30).Cells used in this study were between passages12and35.Cytotoxicity.Cytotoxicity was measured using the method of Oliver et al.(31)with modiﬁcations by our laboratory(32).HepG2 cells were seeded at4×104/well on a96-well plate in100µL of growth medium and incubated for24h at37°C.The medium was removed,and the cells were washed with PBS.Treatments of fruit extracts or antioxidant compounds in100µL of treatment medium (WME supplemented with2mM L-glutamine and10mM Hepes) were applied to the cells,and the plates were incubated at37°C for24h.The treatment medium was removed,and the cells were washed with PBS.A volume of50µL/well methylene blue staining solution(98%HBSS,0.67%glutaraldehyde,0.6%methylene blue) was applied to each well,and the plate was incubated at37°C for 1h.The dye was removed,and the plate was immersed in fresh deionized water three times,or until the water was clear.The water was tapped out of the wells,and the plate was allowed to air-dry brieﬂy before100µL of elution solution(49%PBS,50%ethanol, 1%acetic acid)was added to each well.The microplate was placed on a bench-top shaker for20min to allow uniform elution.The absorbance was read at570nm with blank subtraction using the MRX II DYNEX spectrophotometer(DYNEX Inc.,Chantilly,VA). Concentrations of pure compounds or fruit extracts that decreased the absorbance by>10%when compared to the control were considered to be cytotoxic.CAA of Pure Phytochemicals and Fruit Extracts(Figure2). Human hepatocellular carcinoma HepG2cells were seeded at a density of6×104/well on a96-well microplate in100µL of growth medium/well.The outside wells of the plate were not used as there was much more variation from them than from the inner wells. Twenty-four hours after seeding,the growth medium was removed and the wells were washed with PBS.Triplicate wells were treated for1h with100µL of pure phytochemical compounds or fruit extracts plus25µM DCFH-DA dissolved in treatment medium. When a PBS wash was utilized,wells were then washed with100µL of PBS.Then600µM ABAP was applied to the cells in100µL of HBSS,and the96-well microplate was placed into a Fluoroskan Ascent FL plate-reader(ThermoLabsystems,Franklin,MA)at37°C.Emission at538nm was measured with excitation at485nm every5min for1h.Each plate included triplicate control and blank wells:control wells contained cells treated with DCFH-DA and oxidant;blank wells contained cells treated with dye and HBSS without oxidant.Quantiﬁcation of CAA.After blank subtraction from theﬂuo-rescence readings,the area under the curve ofﬂuorescenceversus Figure1.Total phenolic contents of selected fruits(mean(SD,n) 3).Bars with different letters are signiﬁcantly different(p<0.05).Cellular Antioxidant Activity Assay J.Agric.Food Chem.,Vol.55,No.22,20078897time was integrated to calculate the CAA value at each concentration of pure phytochemical compound or fruit extract as follows:CAA unit )100-(∫SA ⁄∫CA )×100where ∫SA is the integrated area under the sample ﬂuorescence versus time curve and ∫CA is the integrated area from the control curve.The median effective dose (EC 50)was determined for the pure phytochemical compounds and fruit extracts from the median effect plot of log (f a /f u )versus log (dose),where f a is the fraction affected and f u is the fraction unaffected by the treatment.To quantify intraexperimental variation,the EC 50values were stated as mean (SD for triplicate sets of data obtained from the same experiment.Interexperimental variation was obtained for some representative pure phytochemical compounds and fruit extracts by averaging the ﬂuorescence values from triplicate wells in each trial to obtain one EC 50value per experiment and calculating the mean (SD for at least four trials.In each experiment,quercetin was used as a standard,and cellular antioxidant activities for pure phytochemical compounds were expressed as micromoles of quercetin equivalents (QE)per 100µmol of compound,whereas for fruit extracts they were expressed as micromoles of QE per 100g of fruit.To compare the antioxidant quality of different fruits,CAA was also calculated as micromoles of QE per 100µmol of total phenolics.Statistical Analyses.All results were presented as mean (parisons between two means were performed using unpaired Student’s t tests.When there were more than two means,differences were detected by ANOVA followed by multiple comparisons using Fisher’s least signiﬁcant difference test.Differences were considered to be signiﬁcant when p <0.05.RESULTSTotal Phenolic Contents of Fruit Extracts.To characterize the fruit extracts used in the cellular antioxidant activity assay,the total phenolic contents of the fruits were quantiﬁed (Figure 1).Blueberry contained the most phenolics with 2609(28µmol of GAE/100g of fresh fruit,followed by cranberry (1554(134µmol of GAE/100g),red grape (1443(72µmol of GAE/100g),green grape (994(56µmol of GAE/100g),and apple (916(41µmol of GAE/100g).CAA.The proposed principle of the CAA assay is shown in Figure 2.On the basis of the optimization trials (data not shown),a concentration of 25µM DCFH-DA was used because lower levels did not yield consistent ﬂuorescence measurements and higher concentrations decreased the sensitivity of the assay.ABAP caused oxidation of DCFH-DA in a dose–response manner up to a dose of 2mM (data not shown).The treatment level of 600µM was chosen because it yielded adequate ﬂuorescence readings while inducing a reasonable level of oxidation that could be inhibited by many phytochemicals and fruit extracts.The kinetics of DCFH oxidation in HepG2cells by peroxyl radicals generated from ABAP is shown in Figure 3.The increase in ﬂuorescence from DCF formation was inhibited by pure phytochemical compounds and fruit extracts in a dose-dependent manner,as demonstrated by the curves generated from cells treated with quercetin (Figure 3A ,B ),gallic acid (Figure 3C ,D ),and blueberry extracts (Figure 3E ,F ).Inhibition of oxidation was seen when no PBS wash was done between antioxidant and ABAP treatments (Figure 3A ,C ,E )and when a PBS wash was performed (Figure 3B ,D ,F ).To calculate the EC 50,the dose–response curve from the ratio of the area under the curve of the sample to that of the control and the median effect curve were plotted for each sample.The dose–response curves and median effect plots generated from the data presented from quercetin and blueberry extracts in Figure 3are shown in Figures 4and 5,respectively.The EC 50is the concentration at which f a /f u )1(i.e.,CAA unit )50),as calculated from the linear regression of the median effect curve.The EC 50values of CAA for pure phytochemical compounds and fruit extracts are listed in Table 1along with theircytotoxicFigure 2.Method and proposed principle of the cellular antioxidant activity (CAA)assay.Cells were pretreated with antioxidant compounds or fruitextracts and DCFH-DA.The antioxidants bound to the cell membrane and/or passed through the membrane to enter the cell.DCFH-DA diffused into the cell where cellular esterases cleaved the diacetate moiety to form the more polar DCFH,which was trapped within the cell.Cells were treated with ABAP,which was able to diffuse into cells.ABAP spontaneously decomposed to form peroxyl radicals.These peroxyl radicals attacked the cell membrane to produce more radicals and oxidized the intracellular DCFH to the ﬂuorescent DCF.Antioxidants prevented oxidation of DCFH and membrane lipids and reduced the formation of DCF.8898J.Agric.Food Chem.,Vol.55,No.22,2007Wolfe and Liuconcentrations.The values presented are from triplicate samples in the same experiment,and the coefﬁcient of variation (CV)represents intraexperimental variation.When more than one experiment was performed for the sample,representative results from one trial were presented.In the protocol involving no PBS wash between antioxidant and ABAP treatments (no PBS wash)for the pure phytochemical compounds,quercetin was the most efﬁcacious antioxidant,followed by kaempferol,EGCG,myrice-tin,luteolin,gallic acid,ascorbic acid,caffeic acid,and catechin (Table 1).Epicatechin and ferulic acid had low activity within the doses tested,and their EC 50values could not be calculated.Phloretin,resveratrol,and taxifolin had activity only at doses much higher than their cytotoxic concentrations.For those experiments including a PBS wash between treatments,the order of efﬁcacies was similar to that obtained from the no PBS wash protocol,except that the CAA values from ascorbic acid and catechin were low and the EC 50values of CAA could not be calculated (Table 1).Quercetin,kaempferol,and luteolin had slightly higher EC 50concentra-tions when a PBS wash was done between treatments (p <0.05).Myricetin had similar EC 50values in each of the two protocols (p >0.05),as did EGCG (p >0.05).GallicacidFigure 3.Peroxyl radical-induced oxidation of DCFH to DCF in HepG2cells and the inhibition of oxidation by quercetin (A ,B ),gallic acid (C ,D ),andblueberry extracts (E ,F )over time,using the protocol involving no PBS wash between antioxidant and ABAP treatments (A ,C ,E )and the protocol with a PBS wash (B ,D ,F ),to remove antioxidants in the medium not associated with cells.The curves shown in each graph are from a single experiment (mean (SD,n )3).Cellular Antioxidant Activity Assay J.Agric.Food Chem.,Vol.55,No.22,20078899and caffeic acid had much lower activity when a PBS wash was performed compared to that in the protocol without a PBS wash (p <0.05).The intraexperimental coefﬁcient of variation (CV)for the pure compounds was under 10%when a PBS wash was utilized and modestly higher when a PBS wash was not done (Table 1).The EC 50values of CAA for the fruit extracts are presented in Table 1.Blueberry was the most effective at inhibiting peroxyl radical-induced DCFH oxidation,followed by cranberry,apple,red grape,and green grape.The order of efﬁcacy was the same with or without a PBS wash between fruit extracts and ABAP treatments.The fruit extracts all had lower EC 50values in the no PBS protocol than in the PBS wash protocol (p <0.05).The intraexperimental CV ranged from 2.59to 16.0%,with the majority of trials yielding a CV of <10%(Table 1).The relationship between EC 50values and total phenolic contents of fruit extracts was examined.When no PBS wash was employed between treatments,the EC 50values for CAA were not signiﬁcantly correlated to total phenolic contents in fruits (R 2)0.450;p )0.215).EC 50values were weakly correlated to total phenolic contents (R 2)0.830;p )0.032)in fruits when a PBS wash was done.The reproducibility of EC 50values of CAA from similar experiments performed on different days (interexperimental variation)was evaluated for representative compounds tested using the protocols with and without a PBS wash (Tables 2and 3).For pure compounds with no PBS wash performed between treatments,the CV values of interexperimental variation for quercetin and gallic acid were 6and 17%,respectively (Table 2).The CV of the protocol without a PBS wash interexperimental variation for blueberry was 22%(Table 2).When a PBS wash was done between antioxidant and oxidant treatments,the CV values for quercetin,gallic acid,and blueberry extracts were 11.3,11.4,and 7.56%,respectively (Table 3).The EC 50values were converted to CAA values,expressed as micromoles of QE per 100µmol of compound for pure antioxidant compounds (Figure 6)and micromoles of QE per 100g of fresh fruit for fruit extracts (Figure 7).When no PBS wash was done between antioxidant and ABAP treatments,quercetin had the highest CAA value (p <0.05),followed by kaempferol (75.3(4.7µmol of QE/100µmol),EGCG (42.2(3.1),myricetin (36.8(3.8),and luteolin (22.6(0.2),which were all signiﬁcantly different (p <0.05).The CAA values for gallic acid,ascorbic acid,and caffeic acid were not signiﬁcantly different (9.08(0.95,8.84( 1.18,and 5.59(0.70,respectively)(p >0.05),and catechin’s CAA value was similar to caffeic acid’s at 2.03(0.24µmol of QE/100µmol (p >0.05).When the HepG2cells were washed with PBS between treatments,the order of activity was nearly the same:quercetin >kaempferol (81.1(2.7µmol of QE/100µmol)>myricetin (33.1(1.0))EGCG (32.3(0.9)>luteolin (22.2(1.0)>gallic acid (1.53(0.12))caffeic acid (0.997(0.074)at a signiﬁcance level of p <0.05.For the fruit extracts in the group without a PBS wash,blueberry had the highest CAA value (171(12µmol ofQE/Figure 4.Dose–response curves for inhibition of peroxyl radical-induced DCFH oxidation by quercetin (A ,B )and blueberry extracts (C ,D )without aPBS wash between treatments in the protocol involving no PBS wash between antioxidant and ABAP treatments (A ,C )and the protocol with a PBS wash (B ,D ).The curves shown are each from a single experiment (mean (SD,n )3).8900J.Agric.Food Chem.,Vol.55,No.22,2007Wolfe and Liu100g)(p <0.05).The remaining fruits had activity in the order of cranberry (52.1(1.3)>apple (28.1(4.1))red grape (24.1(1.7)>green grape (9.39(0.49)at a signiﬁcance level of p <0.05.Again,in the PBS wash protocol,blueberryhadFigure 5.Median effect plots for inhibition of peroxyl radical-induced DCFH oxidation by quercetin (A ,B )and blueberry extracts (C ,D )in the protocolinvolving no PBS wash between antioxidant and ABAP treatments (A ,C )and the protocol with a PBS wash (B ,D ).The curves shown are from a single experiment (n )3).Table 1.EC 50Values for the Inhibition of Peroxyl Radical-Induced DCFH Oxidation by Selected Pure Phytochemical Compounds and Fruits (Mean (SD,n )3)and Their Cytotoxic Concentrationsno PBS washPBS washcompound EC 50(µM)CV (%)EC 50(µM)CV (%)cytotoxicity a (µM)quercetin b 5.92(0.07 1.18 5.09(0.19 3.65>20kaempferol b 7.85(0.51 6.53 6.31(0.21 3.3430EGCG 14.0(1.07.3915.8(0.4 2.81>100myricetin 16.1(1.7010.615.4(0.5 2.96200luteolin b 26.1(0.26 1.0123.1(1.0 4.4820gallic acid b 65.4(7.311.1335(267.81>500ascorbic acid 67.5(9.414.0>500>500caffeic acid b 95.3(15.316.1525(387.25>500catechin 292(3211.0>500>500epicatechin >200>600>500ferulic acid >250>500>500phloretin >25>2525resveratrol >40>4040taxifolin>150>150150no PBS washPBS washfruit EC 50(mg/mL)CV (%)EC 50(mg/mL)CV (%)cytotoxicity a (mg/mL)blueberry b 3.440(0.239 6.9410.81(0.44 4.0960cranberry b 11.31(0.29 2.5936.17(1.20 3.3160apple b21.31(3.3416.038.60(3.268.45>100red grape b 24.49(1.737.0542.33(2.22 5.23>100green grape b62.89(3.195.0753.01(3.125.89>100aDose at which the cell number is reduced by >10%after 24h of treatment.b EC 50values for no PBS wash and PBS wash are signiﬁcantly different (p <0.05).Cellular Antioxidant Activity Assay J.Agric.Food Chem.,Vol.55,No.22,20078901the greatest activity(47(1.9µmol of QE/100g)(p<0.05), followed by cranberry(14.2(0.5)(p<0.05).The CAA value of apple(13.3(1.1)was not signiﬁcantly different from that of cranberry(p>0.05),and the CAA value of red grape(12.1 (0.6)was similar to that of apple(p>0.05).Green grapehad the lowest CAA value(9.67(0.57)(p<0.05)when a PBS wash was performed between treatments.To compare the antioxidant quality of different fruits,CAA values can be expressed as micromoles of QE per100µmol of total phenolics(Table4).This value makes it possible to compare the antioxidant quality of the total phytochemicals in whole foods compared to pure phytochemical compounds.In the protocol without a PBS wash,blueberry exhibited the highest antioxidant quality(8.70(0.09µmol of QE/100µmol of total phenolics)(p<0.05),followed by similar values from cranberry (3.36(0.09)and apple(3.07(0.45)and then red grape(1.67 (0.12)and green grape(1.04(0.05).These values arecomparable to the activities of100µmol of gallic acid,ascorbic acid,caffeic acid,and catechin(Figure6).When a PBS wash was done,the antioxidant quality values were1.82(0.07µmol of QE/100µmol of total phenolics for blueberry,1.45(0.12 for apple,0.973(0.057for green grape,0.914(0.030for cranberry,and0.839(0.044for red grape,comparable to the efﬁcacies of100µmol of gallic acid or caffeic acid(Figure6). There were no signiﬁcant differences between the antioxidant qualities of cranberry and green grape or cranberry and red grape (p>0.05)in the PBS wash protocol.DISCUSSIONPrinciple of the CAA Assay.We have developed a much-needed method to measure antioxidant activity in cell culture. As indicated at the First International Congress on Antioxidant Methods,there is an urgent requirement for more appropriate methods to evaluate the antioxidant activity of dietary supple-ments,phytochemicals,and foods than the chemistry methods in common usage(23).The CAA assay addresses this need for a biologically relevant protocol.In this method(Figure2)the probe,DCFH-DA,is taken up by HepG2human hepatocarci-noma cells and deacetylated to DCFH.Peroxyl radicals gener-ated from ABAP lead to the oxidation of DCFH toﬂuorescent DCF,and the level ofﬂuorescence measured upon excitation is proportional to the level of oxidation.Pure phytochemical compounds and fruit extracts quench peroxyl radicals and inhibitTable2.Variation among EC50Values for Representative Pure Phytochemical Compounds and Fruit Extracts Obtained When No PBS Wash Was Performed between Antioxidant and ABAP Treatments(Mean (SD)intraexperimental interexperimental compound trial EC50a(µM)CV(%)EC50(µM)CV(%) quercetin1 5.92(0.07 1.182 6.07(0.25 4.203 6.21(0.24 3.864 5.28(0.23 4.405 5.98(0.18 3.01 5.89(0.36 6.06 gallic acid165.4(7.311.13251.3(3.0 5.77377.3(4.9 6.36463.3(2.7 4.1964.3(10.716.6intraexperimental interexperimental fruit trial EC50a(mg/mL)CV(%)EC50(mg/mL)CV(%) blueberry1 3.44(0.24 6.942 3.49(0.3911.173 3.83(0.297.564 2.20(0.167.395 2.60(0.06 2.13 3.11(0.6822.0a n)3.Table3.Variation among EC50Values for Representative Pure Phytochemical Compounds and Fruit Extracts Obtained When a PBS Wash Was Performed between Antioxidant and ABAP Treatments(Mean (SD)intraexperimental interexperimental compound trial EC50a(µM)CV(%)EC50(µM)CV(%) quercetin1 5.55(0.09 1.602 4.48(0.18 3.943 5.40(0.20 3.734 5.09(0.19 3.655 5.06(0.19 3.77 5.12(0.5811.3 gallic acid1289(12 4.052270(3211.783350(21 6.144347(3710.605335(267.81318(3611.4intraexperimental interexperimental fruit trial EC50a(mg/mL)CV(%)EC50(mg/mL)CV(%) blueberry111.2(1.211.08210.3(0.87.95311.6(0.4 3.4549.52(0.48 5.05510.8(0.4 4.0910.7(0.87.56a n)3.Figure6.Cellular antioxidant activity(CAA)of selected pure phytochemical compounds(mean(SD,n)3).Bars with different letters are signiﬁcantly different(p<0.05).8902J.Agric.Food Chem.,Vol.55,No.22,2007Wolfe and Liu。

CNAS国际认证有效性论证-中英文对照版

CNAS国际认可论证IAF是国际认可论坛IAF是国际认可论坛(International Accreditation Forum)的缩写，它是由有关国家认可机构参加的多边合作组织，成立于1993年1月，现有成员30多个，中国合格评定国家认可委员会(CNAS)是其成员单位之一，中国也是17个发起国之一。

其主要目标是协调各国认证制度，通过统一规范各成员国的审核员资格要求、培训准则及质量体系认证机构的评定和认证程序，使其在技术运作上保持一致，从而确保有效的国际互认，它在世界上包括两大组织，分别是欧洲认证认可组织(EAC)和太平洋认可合作组织(PAC)。

以认可项目等效性为基础，国际认可论坛多边承认协议签约的认可机构批准的认可，使组织持有在世界的某一地区已被认可的认证证书能在世界的任何地区被承认。

因此，被IAF多边承认协议签约认可机构认可的认证机构在管理体系、产品、服务、人员和其它类似的符合性评审项目所颁发的认证证书在国际贸易等领域均能得到世界各国承认与信任。

欧洲认证认可组织EAC欧洲认证认可组织EAC 始建于1991年，目的是在欧洲建立一个区域性的认可制度，实现对各成员国的认证机构能力的相互承认，从而达到对认证证书的相互承认。

EAC 的组成成员主要来自欧洲经济共同体和欧洲自由贸易联盟成员国的国家认可机构。

在各通过同行评定的加盟成员国认可机构间签署多边承认协议。

同行评定是指由各认可机构派代表组成联合审核组，依据ISO/CAS_CO226、ISO/CA8CO227及EN45012和 ISO/IEC48号指南对其他认可机构进行能力评审。

已签署协议的有：FINAS芬兰、RVC荷兰、NA挪威、SWEDAC瑞典、SAS瑞士、NACCB英国。

太平洋认可合作组织太平洋认可合作组织（Pacific Accreditation Cooperation, 英文缩写PAC），是由亚太经济合作组织（APEC）成员经济体的认证机构的认可机构或类似合格评定机构的认可机构及利益相关方组成的协会。

药学英语Unit 14Text B 注释及译文

Text B How Does FDA Approve New Drugs?Under current law, all new drugs need proof that they are effective, as well as safe, before they can be approved for marketing. But it’s important to realize that no drug is absolutely safe. There is always some risk of an adverse reaction. It’s when the benefits outweigh the risks that FDA considers a drug safe enough to approve.1.outweigh [a u tˊweɪ] vt. 在重量上超过；在重要性或价值方面超过根据现行法规，对于所有新药，只有经确认安全、有效，才会被批准上市。

但是，我们应该意识到没有所谓绝对安全的药物，认识到这一点是很重要的。

任何药物都可能有不良反应。

因此，只有认定某种药物的益处大于其风险时，FDA 才会认为某种药物足够安全，可批准其上市。

In fact, it was only 25 years ago that U.S. drug law first embraced the idea of risk vs. benefit that now the key to new drugs approval. Providing evidence of safety before marketing was first required by Federal Food, Drug and Cosmetic Act in 1938, but not until the Drug Amendments of 1962 did firms also have to show a drug’s effectiveness before marketing. Before any drug gets on the market today, FDA decides—as quickly as a thorough evaluation allows—whether the studies submitted by the drug’s sponsor(usually the manufacturer) show it to be safe and effective for its intend use.1.embrace [im'breis] vt. 包括; 包含；接受；信奉, 皈依vt. & vi.拥抱n.拥抱, 怀抱2.cosmetic [kɔz'metik]n.化妆品adj.化妆用的; 美容的；装点门面的Federal Food, Drug and Cosmetic Act联邦食品、药品和化妆法案3.Drug Amendments [ə'mendmənts] 药品法修正案4.thorough ['θʌrəu] adj.彻底的；十足的；考虑周到的5.sponsor ['spɔnsə] vt.赞助, 发起, 主办n. 申办者事实上，直到25年前美国药品管理法才刚刚引入“风险-益处评估”的概念。

英语作文阅卷仲裁阈值

Grading is an essential component of the educational process, providing valuable feedback to students and serving as a measure of their academic progress. However, the subjectivity inherent in essay grading has long been a contentious issue, with concerns over inconsistencies and biases affecting the fairness and accuracy of the process. To address this challenge, the concept of an "essay grading arbitration threshold" has emerged as a potential solution, aiming to establish a more objective and equitable system for evaluating written assignments.The essay grading arbitration threshold refers to a predetermined range or margin within which essay scores are considered acceptable without further review or intervention. This threshold is typically established based on statistical analysis, expert consensus, and a thorough understanding of the grading rubric and assessment criteria. By defining a range of acceptable scores, the arbitration threshold seeks to minimize the impact of individual grader biases and inconsistencies, ensuring that students' work is evaluated fairly and consistently.The implementation of an essay grading arbitration threshold involves several key steps. First, a panel of experienced and qualified graders is assembled to independently evaluate a representative sample of student essays. Their scores are thenanalyzed to identify patterns, outliers, and areas of disagreement. Based on this analysis, a range of acceptable scores is determined, taking into account factors such as the complexity of the assignment, the rubric's criteria, and the expected level of student performance.Once the arbitration threshold is established, it is applied to the grading process. Essays that receive scores within the predetermined range are considered final, without further review. However, essays that fall outside of the threshold are flagged for a second round of grading by an additional grader or a panel of graders. This additional review helps to ensure that any outlier scores are carefully scrutinized and that the final grade assigned is fair and accurate.The implementation of an essay grading arbitration threshold offers several potential benefits. Firstly, it promotes consistency and fairness in the grading process by reducing the impact of individual biases and inconsistencies. By establishing a range of acceptable scores, the system recognizes that grading is a subjective process and that some variation in scores is expected. This acknowledgment helps to minimize the risk of unfair grading practices that can disadvantage certain students or groups.Secondly, the arbitration threshold can enhance the efficiency of the grading process by reducing the need for extensive re-grading or third-party reviews. Essays that fall within the acceptable range can be graded more quickly, freeing up resources for more thorough reviews of outlier cases. This efficiency can be particularly valuable in large-scale assessments or high-stakes testing scenarios, where timely and accurate grading is crucial.Furthermore, the use of an essay grading arbitration threshold can foster greater transparency and accountability in the grading process. By establishing clear criteria and thresholds, the system provides a framework for consistent and defensible grading decisions. This transparency can help to build trust among students, educators, and stakeholders, as well as facilitate constructive feedback and continuous improvement of the grading process.However, it is important to note that the implementation of an essay grading arbitration threshold is not without challenges. Determining the appropriate threshold range requires careful analysis and collaboration among subject matter experts, educators, and statisticians. Additionally, the system must be regularly reviewed and adjusted to account for changes incurricular requirements, assessment rubrics, and student populations.In conclusion, the essay grading arbitration threshold represents a promising approach to addressing the inherent subjectivity and potential biases in essay grading. By establishing a predetermined range of acceptable scores and implementing a systematic review process for outlier cases, this system aims to promote fairness, consistency, and efficiency in the evaluation of written assignments. While challenges exist in its implementation and ongoing maintenance, the potential benefits of this approach warrant further exploration and refinement as educators and policymakers strive to create more equitable and effective assessment practices.。

老年急性缺血性脑卒中患者神经功能缺损加重的危险因素

老年急性缺血性脑卒中患者神经功能缺损加重的危险因素任行龙，聂贝贝，马丽霞，孙冬丽郑州大学第一附属医院神经ICU ，河南郑州450000【摘要】目的探讨老年急性缺血性脑卒中(AIS)患者发病1周内神经功能缺损加重的危险因素。

方法选取2020年6月至2022年8月郑州大学第一附属医院收治的135例老年AIS 患者作为研究对象，依据发病1周内神经功能缺损程度分为加重组38例和非加重组97例。

收集并比较两组患者的一般资料、血清学指标[总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、超敏C 反应蛋白(hs-CRP)、基质金属蛋白酶-9(MMP -9)、脂蛋白相关磷脂酶A2(Lp-PLA2)]。

采用单因素和二元Logistic 回归分析老年AIS 患者发病1周内神经功能缺损加重的危险因素。

结果单因素分析结果显示，加重组患者的年龄、缺血性脑卒中史占比、入院时美国国立卫生研究院卒中评分量表(NIHSS)评分、血清TG 、LDL-C 、hs-CRP 、MMP -9、Lp-PLA2水平明显高于非加重组，差异均有统计学意义(P <0.05)；经二元Logistic 回归分析结果显示，高龄、既往缺血性脑卒中史、较高入院时NIHSS 评分及血清TG 、LDL-C 、hs-CRP 、MMP -9、Lp-PLA2水平升高均为老年AIS 患者发病1周内神经功能缺损加重的独立危险因素(P <0.05)。

结论临床应针对上述老年AIS 患者发病1周内神经功能缺损加重的危险因素进行早期干预，以降低老年AIS 患者发病1周内神经功能缺损加重的风险，改善老年AIS 患者的预后。

【关键词】老年；急性缺血性脑卒中；神经功能缺损；危险因素【中图分类号】R743.3【文献标识码】A【文章编号】1003—6350（2023）24—3544—04Risk factors analysis of aggravation of neurological deficit in elderly patients with acute ischemic stroke.REN Xing-long,NIE Bei-bei,MA Li-xia,SUN Dong-li.Neurological ICU,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,Henan,CHINA【Abstract 】ObjectiveTo analyze the risk factors of aggravation of neurological deficits within one week afteronset in elderly patients with acute ischemic stroke (AIS).MethodsA total of 135elderly patients with AIS admittedto the First Affiliated Hospital of Zhengzhou University from June 2020to August 2022were selected as the study sub-jects.They were divided into an exacerbation group (n =38)and a non-exacerbation group (n =97)based on the degree of neurological deficit within one week after onset.The general information and serological indicators [total cholesterol (TC),triglyceride (TG),high-density lipoprotein cholesterol (HDL-C),low-density lipoprotein cholesterol (LDL-C),high-sensitivity C-reactive protein (hs-CRP),matrix metalloproteinase -9(MMP -9),lipoprotein-associated phospholi-pase A2(Lp-PLA2)]of the two groups were collected and compared.Single-factor and binary logistic regression were used to analyze the risk factors for aggravation of neurological deficits in elderly AIS patients within one week after on-set.ResultsThe results of univariate analysis showed that age,proportion of patients with ischemic stroke history,andNIHSS score,serum TG,LDL-C,hs-CRP,MMP -9,Lp-PLA2levels at admission in the exacerbation group were signifi-cantly higher than those in the non-exacerbation group (P <0.05).The results of binary logistic regression analysis showed that advanced age,previous history of ischemic stroke,higher NIHSS score at admission,and elevated serum TG,LDL-C,hs-CRP,MMP -9,and Lp-PLA2levels were all independent risk factors for aggravation of neurological def-icit within one week after onset in elderly patients with AIS (P <0.05).ConclusionEarly clinical intervention shouldbe carried out according to the above risk factors for aggravation of neurological deficits within one week after onset in elderly patients with AIS,so as to reduce the risk of aggravation and improve the prognosis of the patients.【Key words 】Elderly;Acute ischemic stroke;Neurological deficit;Risk factors ·论著·doi:10.3969/j.issn.1003-6350.2023.24.010基金项目：河南省科学技术厅重点研发与推广专项(科技攻关)(编号：202102310490)。

微生物外文翻译之三

2. Growth on PAHs as sole carbon sources
Microbial degradation of PAHs and other hydrophobic substrates is believed to be limited by the amounts dissolved in the water phase (Ogram et al., 1985; Rijnaarts et al., 1990; Volkering et al., 1992; Volkering et al., 1993; Harms and Bosma, 1997; Bosma et al., 1997), with sorbed, crystalline, and non-aqueous phase liquid (NAPL)-dissolved PAHs being unavailable to PAH-degrading organisms. Bioavailability is considered a dynamic process, determined by the rate of substrate mass-transfer to microbial cells relative to their intrinsic catabolic activity (Bosma et al., 1997; Harms and Bosma, 1997). It has been described by a bioavailability number, Bn, (Koch, 1990; Bosma et al., 1997), which is a measure of a microorganism’s substrate degradation eﬃciency in a given environment. Bn is deﬁned as the capacity of an organism’s or a population’s environment to provide a chemical, divided by the capacity of the organism or population to transform that chemical. At high mass transfer rates, the overall biodegradation rate is controlled by the metabolic activity of the bacteria (Bn > 1), i.e. by both the speciﬁc activity of the cells and the population density. At Bn ¼ 1, the biodegradation rate is equally controlled by the physical transport and the microbial activity. When the transport of the substrate decreases or the bacterial population grows, the mass transfer becomes the factor that limits the biodegradation (Bn ! 1).

批判质疑能力量化评估作文

批判质疑能力量化评估作文英文回答：Critical Considerations in Quantifying Argumentative Writing:Validity, Reliability, and Generalizability:Quantifying argumentative writing faces challenges in ensuring the validity, reliability, and generalizability of assessments. The selection of criteria for evaluation, the subjectivity of raters, and the limited scope of quantifiable aspects of writing can lead to concerns about the accuracy and consistency of scoring.Reductionism and Oversimplification:Quantifying argumentative writing runs the risk of reducing its complexity to a set of numerical measures, potentially oversimplifying the multifaceted nature of thepersuasive process. By focusing on quantifiable elements, important qualitative aspects, such as the writer's voice, rhetorical strategies, and impact on the reader, may be overlooked or devalued.Contextual Factors and Individuality:Quantified assessments often fail to account for contextual factors that influence argumentation, such as the audience, purpose, and rhetorical situation. This can lead to unfair or inaccurate evaluations that overlook the unique strengths and weaknesses of individual writers.Impact on Writing Instruction:A focus on quantifying argumentative writing can have unintended consequences for writing instruction. By emphasizing measurable criteria, educators may prioritize these aspects at the expense of fostering creativity, critical thinking, and the development of a nuanced understanding of argumentation.Limitations of Current Practices:Current methods for quantifying argumentative writing often rely on superficial measures, such as word count or sentence length, which provide limited insight into the quality of the writing. More sophisticated approaches are needed that capture the complexity of argumentation.中文回答：论证性写作量化评估的批判。

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The National Agriculture Statistics Service (NASS) has developed a generalized edit and imputation system called AGGIES (Todaro,1999) that solves simultaneous linear-inequality edits using Chernikova-type algorithms for determining the minimum number of fields to change so that a record satisfies the edits. These algorithms can simultaneously deal with a large number of mathematical constraints. Since both AGGIES and StEPS are written in SAS, a successful application of the ACES data with the AGGIES software would provide strong justification for including all or part of the AGGIES system in StEPS. The objective of this study was to determine the feasibility of replacing all or part of the ACES current manual editing system with an automated editing software while still maintaining (or even improving) data quality. To do this, we conducted a feasibility and an evaluation study using 1996 ACES data with the AGGIES software package. The feasibility study examined whether AGGIES can be used to perform ACES editing. The evaluation study then examined the quality of the edit results. Willimack et al (2000) demonstrates that a large portion of the current ACES analysts’ editing strategy does not take a Fellegi-Holt (simultaneous solution) approach. Thus, we use test data with simulated errors to perform the evaluation. ACES does not perform item imputation: instead, they use deterministic (logical) edits to replace missing values or obtain new information directly from the companies. Consequently, this study did not include any evaluation of imputation methods. This paper provides our results, along with our recommendation. Section 2 provides background on ACES. Section 3 describes the features of AGGIES. Section 4 presents the results of our feasibility study, including detailed descriptions of edits, data groups, and edit groups. Section 5 presents our evaluation study results. Section 6 provides a discussion of our results. We make a few concluding remarks in Section 7. 2. Annual Capital Expenditures Survey The Annual Capital Expenditures Survey (ACES) collects information about the nature and level of capital expenditures in non-farm companies, organizations, and associations in the United States. The data collected is used to prepare national measures of capital spending and to formulate fiscal and monetary policy. Different forms are mailed to the companies depending on whether they are an employer company or non-employer company. Nonemployer companies respond with a short form questionnaire (ACE-2 form). ACE-2 form responses have a high percentage of UAAs (Undeliverable as addressed), out of scope, and nonresponse (Caldwell, 1999.) Employer companies respond with a long form questionnaire (ACE-1 form). Most of the analyst review concentrates on ACE-1 form responses. This report discusses only employer companies responding with the ACE-1 form. The ACE-1 form respondents report fixed assets and capital expenditures for the calendar year in all subsidiaries and divisions for all operations within the United States. The ACE-1 form collects data for total capital expenditures in various items of the questionnaire. Total capital expenditures is first reported in survey Item 1. Total capital expenditures is also collected in survey 2
Report Issued: 11/29/2000
Hale Waihona Puke Results of Evaluation of AGGIES for ACES Maria Garcia and Katherine J. Thompson ABSTRACT The U. S. Census Bureau’s Annual Capital Expenditures Survey (ACES) collects data about domestic capital expenditures in non-farm businesses operating within the United States. Analysts manually edit the ACES data using a specified set of editing rules. Although individual edits are straightforward, the hierarchical combination of edits are complicated with several nested levels of simultaneous balance requirements. We investigate the feasibility of replacing the current ACES editing procedures with an automated system based on National Agricultural Statistics Service's generalized edit and imputation system (AGGIES). The AGGIES system solves simultaneous linear-inequality edits using Chernikova-type algorithms for determining the minimum number of fields to change so that a record satisfies all edits. These algorithms can simultaneously deal with a large number of mathematical constraints and have been successfully applied in Statistics Canada's Generalized Edit and Imputation System and Statistics Netherlands' CherryPI system. Key Words: data editing, Fellegi-Holt model, error localization 1. Introduction Data collected using the Annual Capital Expenditures survey (ACES) is the primary source of information about domestic capital expenditures within the United States for federal agencies, private industry organizations, and academic researchers. ACES is a mail-out/mail back survey of companies, collecting detailed data for several related aggregate variables. The current ACES edit system automatically handles certain types of item non-response, then generates lists of edit-failing records which are manually corrected by analysts. Aside from item non-response, there is no automatic resolution of edit-failing records. In the near future, ACES will move from its current processing system to the U.S. Census Bureau’s re-engineered post-data-collection system, the Standardized Economic Processing System (StEPS). The StEPS system is composed of integrated SAS modules that perform several survey processing activities, including data editing, imputation, and estimation (Ahmed and Tasky, 1999). The StEPS editing modules have been used successfully by several other economic surveys at the U.S. Census Bureau. However, none of these surveys collect items that must satisfy several simultaneous ratio edit and balance requirements. Consequently, the StEPS edit software requires some enhancements to correctly automatically edit the ACES data. 1