Processing Industries. ter derivatives was developed (29). By using negative-ion. chemical ionization, selected-ion-monitoring detection, a

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1 APPLIED AND ENVIRONMENTAL MIROBIOLOGY, My 199, p /9/ $2./ opyright X 199, Amerin Soiety for Miroiology Vol. 56, No. 5 omprison of the Limulus Ameoyte Lyste Test nd Gs hromtogrphy-mss Spetrometry for Mesuring Lipopolyshrides (Endotoxins) in Airorne Dust from Poultry- Proessing Industries ANDERS SONESSON,1* LENNART LARSSON,2 ANDREJS SHUTZ,3 LARS HAGMAR,3 AND TORGNY HALLBERG2 Deprtment of Tehnil Anlytil hemistry, hemil enter, Lund University, S-221 Lund,1 Deprtment of Medil Miroiology, Lund University Hospitl, S Lund,2 nd Deprtment of Ouptionl nd Environmentl Mediine, Lund University Hospitl, S Lund,3 Sweden Reeived 27 Novemer 1989/Aepted 3 Jnury 199 The lipopolyshride (endotoxin) ontent in irorne dust smples from three different poultry slughterhouses ws determined with oth the hromogeni Limulus meoyte lyste ssy nd gs hromtogrphymss spetrometry nlysis of lipopolyshride-derived 3-hydroxy ftty ids. Grm-negtive ell wlls were lso mesured y using two-dimensionl gs hromtogrphy/eletron-pture nlysis of diminopimeli id originting from the peptidoglyn. The orreltion etween the results of the Limulus ssy nd those of gs hromtogrphy-mss spetrometry for determintion of the lipopolyshride ontent in the dust smples ws poor, wheres good orreltion ws otined etween lipopolyshride nd diminopimeli id onentrtions with the gs hromtogrphi methods. The results suggest tht it is predominntly ellwll-dissoited lipopolyshrides tht re mesured with the Limulus ssy, wheres the gs hromtogrphi methods llow determintion of totl onentrtions of lipopolyshride, inluding Limulus-intive lipopolyshride, grm-negtive ells, nd ellulr deris. Workers in griulturl environments re exposed to wide vriety of irorne orgni dusts ontining different toxi produts of miroil origin (15, 16, 22). The development of respirtory diseses, e.g., llergi lveolitis, hs een ssoited with inhltion of irorne miroorgnisms (22). For exmple, lipopolyshrides (LPS; endotoxins) of grm-negtive teri hve een proposed s mjor ustive gents of lung disorders mong griulture workers (9). Inhltion of irorne LPS hs lso een ssoited with suh symptoms s ough, hedhe, dirrhe, nd fever (15). An irorne LPS onentrtion of.1 to.2,ug/m3 hs een suggested s ritil level for overshift derese in lung funtion (23). The LPS ontent of orgni dust is usully determined with the Limulus meoyte lyste (LAL) test. Dusts olleted on filters re extrted with pyrogen-free wter, nd the extrts re then nlyzed (1, 5, 8, 15, 23). The presene of LPS in the extrts indues geltion of the LAL. However, it is known tht severl sustnes inluding peptidoglyn (from teril ell wlls), dextrns, ertin proteins, nd polynuleotides lso tivte the LAL retion (17, 37), wheres ertin other sustnes, e.g. eletrolytes, hormones, nd ntiiotis, my inhiit the test (35). An lterntive pproh to mesuring LPS is sed on the detetion of speifi struturl omponents (iomrkers) of the LPS moleule (1). Lipid A, the lipid omponent of LPS nd responsile for the endotoxi effets of the moleule (2), ontins ertin molr frtion of 3-hydroxy ftty ids (38). These ftty ids my serve s mrkers for the mount of LPS. A method utilizing gs hromtogrphy (G)-mss spetrometry (MS) nlysis of 3-hydroxy ftty ids in LPS s 3--pentfluoroenzoyl (PFBO)-methyl es- * orresponding uthor ter derivtives ws developed (29). By using negtive-ion hemil ioniztion, seleted-ion-monitoring detetion, detetion limit of 1 ng of LPS of Esherihi oli per ml in queous solutions ws otined (29). In the present study, hromogeni LAL test nd the G-MS method were used in omprtive study to mesure LPS in irorne dust smpled in poultry slughterhouses. In ddition, we mesured diminopimeli id (DAP), whih hs een shown to e useful mrker of peptidoglyn in grm-negtive teri (33). The DAP nlyses were performed with two-dimensionl G with eletron-pture detetion (ED) (28). It ws found tht it is predominntly ell-envelope-dissoited LPS tht re mesured with the LAL ssy, wheres the G methods mesure the totl mounts of grm-negtive ells, deris, nd free LPS in the smple. MATERIALS AND METHODS hemils nd solutions. Anlytil-grde DAP ws otined from Sigm hemil o., St. Louis, Mo.; stok solutions were prepred in.1 M hydrohlori id nd stored t 4. Heptfluoroutyri nhydride nd PFBOhloride were purhsed from Fluk, Buhs, Switzerlnd; etyl hloride ws from E. Merk AG, Drmstdt, Federl Repuli of Germny; isoutyl lohol ws from Jnssen, Beerse, Belgium; nd phenol-wter-extrted LPS of E. oli 55:B5 ws from Sigm. All solvents used were of nlytil regent grde nd purhsed from My & Bker, Dgenhm, United Kingdom. The 3-hydroxy ftty id stndrds 3-hydroxynonnoi id (3-OH-9:) nd 3-hydroxytetrdenoi id (3-OH-14:) were from our lortory olletion of stndrds, nd 3-hydroxyhexdenoi id (3-OH-16:) ws generous gift from Erik Jntzen, Sttens Institutt for Folkehelse, Oslo, Norwy. 3-Hydroxydode- Downloded from on August 27, 217 y guest

2 1272 SONESSON ET AL. noi id (3-OH-12:) nd 3-hydroxyotdenoi id (3-OH-18:) were isolted from lyophilized ells of Pseudomons eruginos nd Helioter pylori (oth linil isoltes), respetively. The 4 M hydrohlori id in methnol ws prepred y dding 3 ml of etyl hloride to 75 ml of methnol, nd the 3 M hydrohlori id in isoutyl lohol ws prepred y dding 2 ml of etyl hloride to 8 ml of isoutyl lohol. All glsswre were heted t 4 for 1 h efore use. The test tues used hd Teflon-lined srew ps. Bteril suspensions nd LPS stndrds. A strin of E. oli, isolted from linil smple, ws used to prepre teril suspensions of different onentrtions (in glss tues). The onentrtion of the stok suspension ws 1.3 x 18 FU/ml s determined y vile ount. Two 1-ml smples were tken from eh suspension nd lyophilized, one smple eing sujeted to LAL ssy (polypropylene tues) nd the other to G-MS nlysis (glss tues). The E. oli 55:B5 LPS ws used for onstruting the stndrd urve (1 to 2, ng) in queous solution for use with the G-MS method. The 3-OH-14: ontent of the LPS ws determined y G s trifluoroetyl-methyl ester (7, 26). LPS stndrd preprtions were lso nlyzed with the LAL ssy. Dust smples. Airorne dust smples from three different poultry slughterhouses were olleted on onditioned preweighed memrne ellulose ette filters (37-mm dimeter,.8-jim pore size; Millipore orp., Bedford, Mss.) y mens of personl smplers (2 to 3 liters/min; sell AF 123) t rething zone level. Smpling periods were of 2 to 3 h in durtion. After determintion of the totl erosol mss, the filters were eluted in 1 ml of pyrogen-free wter for 6 min (23). Smples, 1 ml, were sujeted to the LAL ssy, wheres the remining volumes were trnsferred to glss tues nd lyophilized for G-MS nd G-ED nlyses (see elow). Extrts of lnk filters were lso nlyzed. In ddition, five polyvinyl hloride filters (37-mm dimeter,.8-jim pore size; Nulepore orp., Plesnton, lif.) were lso used. These filters were ut in hlf fter smpling. One-hlf ws eluted in wter s ove nd sujeted to the LAL ssy, nd the other ws diretly sujeted to id hydrolysis for G-MS nlysis (see elow). LAL ssy. A hromogeni LAL ssy (otest; Kivitrum, Molndl, Sweden) (6, 23) ws performed y Rgnr Rylnder nd ollegues, Deprtment of Environmentl Hygiene, University of Gothenurg, Gothenurg, Sweden. Smple preprtion. The freeze-dried filter extrts, with the internl stndrd (35 ng of 3-OH-9:) dded, were heted in 1 ml of 4 M methnoli hydrohlori id t 1 for 18 h. After ooling, 1 ml of wter nd 1.5 ml of hexne were dded, nd the smple ws extrted. The hexne phse ws trnsferred to new tue nd evported under redued pressure, nd the methnoli phse ws sved for DAP nlysis. The methyl esters in the evported hexne phse were redissolved in.1 ml of etonitrile nd 2 RI of PFBO-hloride ws dded, the mixture then eing heted t 15 for 1 h. After ooling,.5 ml of heptne nd 1 ml of 1 M phosphte uffer (ph 7.) were dded. The tue ws shken nd entrifuged (out 1, x g), nd the orgni phse ws evported. This proedure ws modifition of method desried erlier (27). The smple ws mde up with heptne prior to G-MS nlysis. For mesurement of DAP, the queous methnoli solution ws lyophilized nd the residue ws sujeted to hydrolysis in 6 M hydrohlori id t 15 for 6 h. The id ws removed in lyophilizer. The roxyl groups of the APPL. ENVIRON. MIROBIOL. mino id were esterified y heting in 3 M isoutnoli hydrohlori id, nd the mino groups were ylted with heptfluoroutyri nhydride s desried elsewhere (27). The preprtion ws mde up with ethyl ette prior to nlysis with G-ED. The yield of DAP otined in nlyzing stndrd suspensions of E. oli ells (18 FU) y this modified method ws ompred with tht of n erlierdesried method (27). G. A model 416 G (rlo Er, Rodno, Itly) equipped with flme ioniztion detetor, n ll-glss splitless injetion system, nd fused-sili pillry olumn (3 m y.32-mm inside dimeter) oted with ross-linked SE-52 (film thikness,.2 jim; J&W, Folsom, lif.) ws used to determine the mount of 3-OH-14: in the E. oli LPS stndrd nd the yield in the PFBO derivtiztion step. The temperture of the injetor ws 28 nd tht of the detetor ws 3. The initil temperture of the olumn ws 8, progrmmed to inrese y 8 /min (strting 1 min fter injetion) to finl temperture of 28. Injetions were mde in the splitless mode, the split vlve eing opened 1 min fter injetion. Hydrogen served s the rrier gs t flow rte of 1 ml/min. An SP427 Integrtor (Spetr Physis, Sn Jose, lif.) ws used for integrtion of pek res. G-ED. A model 37 (Vrin, Los Altos, lif.) G equipped with 63Ni ED operting in the frequeny-pulsed mode nd with MUSI (multiple swithing intelligent ontroller) olumn-swithing system (hrompk, Middelurg, The Netherlnds) ws used for the mesurement of DAP. A fused-sili wide-ore olumn (1 m y.53-mm inside dimeter) with ross-linked P-Sil-8 (hrompk) s the sttionry phse (film thikness, 6.4 jim) ws used s preolumn tthed to flme ioniztion detetor, nd fused-sili pillry olumn (25 m y.22-mm inside dimeter) oted with ross-linked SE-3 (film thikness,.2 jum; SGE, Ringwood, Austrli) served s the nlytil olumn tthed to the ED. The nitrogen rrier gs flow rte ws 4 ml/min through the preolumn nd 1 ml/min through the nlytil olumn. The mkeup gs (nitrogen) flow rte through the ED ws 15 ml/min. Injetions were mde on-olumn. The temperture of the injetor ws 25 nd tht of the detetors ws 35. The initil temperture of the preolumn, 14, ws progrmmed to inrese y 1 /min to finl temperture of 26. The old trp, hilled to -7 with liquid ron dioxide, ws heted to 25 upon reinjetion of the trpped frtions into the nlytil olumn, whih ws kept t onstnt temperture of 26. Dt hndling ws proessed with the hrompk ontrol nd integrtion system, using n IBM PS/2 model 3 nd hrompk BD 7 printer plotter. G-MS. An R1-1 qudrupole G-MS system (Riermg, Rueil-Mlmison, Frne) ws used. The G used ws rlo Er model 416 equipped with n ll-glss splitless injetor nd with fused-sili pillry olumn (25 m y.22-mm inside dimeter) oted with ross-linked SE-52 (film thikness,.2 jim). Helium, t n inlet pressure of.8 kg/m2, served s rrier gs. The temperture of the injetor ws 25, tht of the interfe etween the G nd the ion soure ws 25, nd tht of the ion soure ws 12. The olumn temperture ws initilly held t 14 nd fter 1 min ws inresed to 26 y 1 /min. The split vlve ws opened 1 min fter injetion. The isoutne regent gs in hemil ioniztion t.7 torr (purity, >99.95%) ws ionized with eletrons t n energy of 93 ev. The mnul integrtion fility in the MS system stndrd softwre ws used for pek integrtion. Downloded from on August 27, 217 y guest

3 VOL. 56, 199 LAL AND G-MS TO MEASURE LPS IN AIRBORNE DUST 1273 TABLE 1. Anlysis of n E. oli 55:B5 LPS stndrd preprtion with the hromogeni LAL test nd with G-MS LPS LPS (ng) y: smples (ng) LAL G-MS 2, 1,27 2,2 1, The dt represent one representtive experiment. Lyophilized in polypropylene tues. I Lyophilized in glss tues. LPS ws lulted from the mount of 3-OH-14:; the LPS stndrd used ontined 2% (wt/wt) 3-OH-14:. The hydroxy ftty id derivtives were nlyzed with seleted-ion monitoring, using omintions of different ion-monitoring sets hrteristi of the different derivtives. The time events nd ions were 11 min, m/z (3-OH-9:); 1 min, m/z (3-OH-1:); 2 min, m/z (3-OH-12:); 2 min, mlz (3-OH-14:); 2 min, mlz 48.4 (3-OH-16:); nd 2 min, mlz 58.5 (3-OH-18:), orresponding to the moleulr rdil nions of the 3-O-PFBO-methyl derivtives of the different ids (27). RESULTS DAP nlysis. A lirtion urve for DAP ws prepred with stndrd preprtions in the rnge 1 to 2, ng. A liner urve with the eqution y = 1.4x + 22 (r2 =.92) ws otined, nd the detetion limit ws estimted to e pproximtely 5 pg (injeted mount) t signl/noise rtio of 3:1. Anlysis of the methnoli phse fter methnolysis of stndrd suspensions of E. oli ells gve pproximtely the sme yields of DAP (91%; stndrd devition = 7%; n = 6) s did diret hydrolysis of the sme teril ellulr onentrtions, using 6 M hydrohlori id (27). LPS nlysis. The method used for prepring 3-O-PFBOmethyl esters ws modifition of n erlier method (27). Use of 4 M methnoli hydrohlori id methnolysis t 1 for 18 h hs een found to quntittively relese mide-linked 3-hydroxy ftty ids of LPS (26). Further, y inresing the onentrtion of the PFBO-hloride, the yield in the PFBO derivtiztion step ws inresed from 65 to 9% (stndrd devition = 4%; n = 6), s determined y summtion of pek res from the 3-O-PFBO-methyl esters nd 3-hydroxymethyl esters (with free hydroxyl group) of 3-OH-9: nd 3-OH-14:, respetively. As expeted, the 3-O-PFBO-methyl esters ll produed moleulr rdil ions s the se pek in negtive ion hemil ioniztion MS (27), ions whih were susequently used in the seleted-ion-monitoring nlyses. The eqution of the stndrd urve for E. oli LPS ws y = 1.6 x 1-5X x 1-5 (r2 =.994) over the rnge 1 to 2, ng, nd the detetion limit ws 1 ng/ml. The E. oli LPS stndrd ws found to ontin 2% (wt/wt) of 3-OH-14:, whih is in greement with the results of previous studies (19). The E. oli LPS stndrd ws lso nlyzed with the LAL ssy. The endotoxin stndrd of the otest (LAL) gve LPS vlues pproximtely hlf the 1,- nd 2,-ng mounts, s ompred with the G-MS method (Tle 1). For the freeze-dried preprtions ontining 1 to 5 ng of LPS, however, the LAL ssy gve onsiderly lower vlues, proly due to ineffetive extrtion of the LPS dhered to the polypropylene tue wll (see Disussion). TABLE 2. Determintion of LPS in queous solutions ontining vrious mounts of E. oli ells Vile LPS (ng) y: ells LAL (ng). G-MS (ng).d 1.3 x 18 31, 289,6 4.4 x 17 11, 91,2 8.8 x 16 2, 16,1 1.8 x ,2 3.5 x 1' Numer of ells y vile ount. The dt represent one representtive experiment. Lyophilized in polypropylene tues. d Lyophilized in glss tues. LPS in E. oli smples. The LPS ontent of lyophilized preprtions ontining vrying mounts of E. oli ells is presented in Tle 2. lerly, derese in LPS vlues follows derese in teril ell ount. The quotients etween the G-MS vlues nd the LAL vlues vried etween 8 nd 11 (exept for the lowest vlue). The preision of the LAL ssy (7 ng, oeffiient of vrition, 4.7%; n = 5) ws similr to tht of G-MS (1,36 ng; oeffiient of vrition, 6.6%; n = 5) when queous solutions ontining 16 vile E. oli ells were nlyzed. The higher vlues for LPS ontent/onentrtion of ells, otined with the LAL ssy in the preision study, my hve een due to disruption of ells during pipetting steps in the preprtion of the solutions, thus lierting LPS into the solution (see Disussion). Dust smples. Results from the nlyses of irorne dusts, olleted in the three poultry slughterhouses (S-H1, S-H2, nd S-H3), re summrized in Tle 3. The highest vlues for dust, LPS, nd DAP were otined in smples from S-Hi. The orreltion etween onentrtions of dust nd LPS ontent y the LAL ssy ws high in smples from S-H2 (r2 =.92) nd from S-H3 (r2 =.9) ut lower in those from S-Hi (r2 =.2) (Fig. 1); with G-MS, low orreltion ws otined for smples from ll three plnts studied (Fig. 1). The DAP vlues, inditing onentrtions of grm-negtive ell wlls, were lso poorly orrelted with the dust levels (Fig. 2). onsequently, no good liner reltionship ws otined etween the G-MS method nd the LAL ssy for mesurement of LPS in the dust smples studied (Fig. 3). A good liner reltionship ws otined, however, in smples from ll three plnts etween LPS s mesured y G-MS nd DAP vlues, wheres the reltionship etween LPS vlues otined with LAL nd DAP vlues ws poorer (Fig. 4). The onentrtions of irorne LPS were slightly higher when lulted from the DAP vlues thn when lulted from the mounts of 3-hydroxy ftty ids (Tle 3). A vriety of 3-hydroxy ftty ids were found in the dust smples nlyzed y G-MS. Most undnt were 3-OH- 1: nd 3-OH-12:, whih re mjor onstituents of LPS, e.g., of Pseudomons nd Ainetoter spp. (38). Other 3-hydroxy ftty ids found were 3-OH-14: (present in most LPS, espeilly of memers of the fmily Enteroteriee [38]) nd 3-OH-16: nd 3-OH-18: (e.g., found in LPS of Frnisell nd Agroterium spp. [38]), present in vrying mounts ut sent from some smples. No 3- hydroxy ftty ids or DAP ws found in extrts of lnk filters. Extrts of ellulose ette filters hve een shown to e intive in the LAL test (5, 15). ulture studies of dust smples, desried in Hgmr et l. (L. Hgmr, A. Shutz, T. Hllerg, nd A. Sjoholm, Int. Arh. Environ. Helth, in Downloded from on August 27, 217 y guest

4 1274 SONESSON ET AL. APPL. ENVIRON. MIROBIOL. Prmeter TABLE 3. LPS nd grm-negtive ells present in irorne dust smpled from three poultry slughterhouses (S-H) S-Hi (25) S-H2 (17) S-H3 (19) GM Rnge GM Rnge GM Rnge Totl dust mg mg/m LPS (LAL) ng/mgd ng/m , LPS (G-MS)e ng/mgd , , ,22 ng/m3 7,6 2-64,4 1,9 2-6,5 1,36 9-5,83 DAP nglmgd , ng/m3 1,36 7-4, , ,8 ells (DAPWf 18/mgd /m LPS (DAP)9 ng/mg 1, ,22 1, , 1, ,92 ng/m3 11, , 2, ,2 2, ,4 Numer in prentheses is numer of smples. GM, Geometri men. LPS determined with the LAL ssy. d Present in totl dust. e LPS determined with G-MS seleted-ion montoring. f Grm-negtive ells lulted from the onentrtion of DAP ssuming tht 1 ng of DAP orresponds to 2.9 x 16 ells (33). g LPS lulted from the mount of DAP, ssuming tht 1 ng of DAP orresponds to 8.7 ng of LPS (28). press) showed most of the vile irorne teri to elong to grm-positive speies (minly ogulse-negtive stphylooi), whih were 1- to 1-fold more undnt thn grm-negtive speies found (Esherihi, Proteus, nd Ainetoter spp.). Totl irorne teri ontent ws 4 x 15 to 4 x 16 FU/m3, wheres the numer of grmnegtive ells ording to DAP vlues (Tle 3), thus inluding vile nd nonvile ells plus ellulr deris, ws 18 to 19 ells per m3. It is possile tht the 3-OH-16: nd 3-OH-18: deteted derived from nonvile ells nd deris. Additionl miroil dt s well s results from helth studies of the slughterhouse workers hve een reported elsewhere y Hgmr et l. (in press). In studies in whih polyvinyl hloride filters were used, LPS vlues y G-MS were 12 to 15 times those otined with LAL (Tle 4), inditing tht LPS olleted on the filters were not quntittively relesed into the wter extrt used in the LAL ssy. DISUSSION The presene of LPS-ontining irorne dust in the working environment hs een ssoited with vriety of helth hzrds nd lung diseses. Byssinosis mong otton workers hs een relted to the presene of grm-negtive teri nd LPS in otton dust (3, 21, 23, 24). In severl studies, high levels of LPS hve een reported in poultry nd hog onfinement uildings, s well s in poultry proessing plnts, nd onstitute potentil helth hzrds (5, 8, 15, 16). Thus, relile nd seletive methods of mesuring irorne LPS re needed to e le to relte LPS onentrtions to ouptionl helth effets. Hitherto, the LPS ontent in irorne dust hs generlly een mesured with the LAL test, n ssy in whih lipid A tivtes the lotting sde in lyste from meoytes of Limulus polyphemus (3). The moleulr environment of lipid A is ritil for the tivtion of this iologil effet (12). Most of the ell wll LPS is intive in the test sine lipid A is not exposed to the Limulus enzymes t the surfe of the outer memrne (14), nd thus it is predominntly ell-wll-dissoited LPS tht is mesured. As the tivity of the lyste my vry oth etween different ommeril preprtions nd etween different thes, stndrdiztion is ruil feture of nlysis (18). The G-MS method, on the other hnd, mesures speifi struturl omponents of lipid A. Hene, the totl mount of LPS in smple is determined eqully effiiently regrdless of whether the LPS is present in ell-wll-dissoited or ssoited stte or tthed to other orgni moleules. This ws refleted in our nlyses of the E. oli suspensions: we otined lrger vlues for LPS with G-MS thn with LAL (Tle 2). Similr findings hve een reported for meningool strins without exessive relese of LPS (2). Munford nd Hll (13) mesured LPS-ontining frgments of outer memrnes y oth the LAL ssy nd rdioimmunossy (mesuring the mount of the LPS polyshride prt) nd found the rdioimmunossy vlues to e s muh s 1 times greter thn those otined with LAL. The irorne dust olleted on the filters in this study ws proly of heterogeni origin, ontining prtiles of skin, fethers, fees, vrious fiers, et. The orreltion etween the mounts of dust nd LPS s determined y the LAL ssy ws high in the two plnts with lower dust levels (men vlues, 3.4 nd 1.9 mg/m3) ut lower t S-Hi, where the onentrtion of irorne dust ws higher (men vlue, 7.9 Downloded from on August 27, 217 y guest

5 VOL. 56, 199 LAL AND G-MS TO MEASURE LPS IN AIRBORNE DUST 1275 T 2 /) n.. -g GO-MS y = x R^2 =.588 y = * x R^2 = LAL.21 -MS 1 5 D 34 3 y = x R^2 = ) -j '& 4- Y) D * 2 - LAL Dust mg/3 l G-MS y = x RA2 =.158 * LAL y = x RA2 =.919 In G-MS u ~~ ~~~* O. El Il I Dust mg/m3 G-MS y = x RA2 =.487 * LAL y = x R2.94 I G-MS Qr #._L n - - I LAL Dust mg/rn3 FIG. 1. orreltion etween totl irorne dust nd LPS s mesured y G-MS nd LAL in () SH-1, () SH-2, nd () SH-3. mg/m3). Thelin et l. (32) lso reported good orreltion etween dust nd LPS (LAL) in smples from poultry frm. In the present study, the orreltion ws poor etween dust nd LPS (G-MS), s it ws etween dust nd DAP, lthough the men vlues (nnogrms per milligrms of totl dust) t the different plnts were similr (Tle 3). Tht LPS vlues otined with G-MS were higher thn those otined with LAL reflets the different LPS sttes eing nlyzed with the two methods. The LPS vlues otined with G-MS were lulted v 2 m 1i Z m 12 L y= x R^2= Dust mg/m Dust mg/rn3 y = x RA Dust mg/rn3 FIG. 2. orreltion etween totl irorne dust nd DAP mesured y G-ED in () SH-1, () SH-2, nd () SH-3. ssuming tht the vlue of 3-OH-14:, the sum of 3-OH-1: nd 3-OH-12: vlues, nd the sum of 3-OH-16: nd 3- OH-18: vlues eh onstituted 2%o (wt/wt) of LPS. Usully 4 mol of 3-hydroxy ftty ids is present in lipid A, orresponding to pproximtely 15 to 3o (wt/wt) of LPS (7), nd the 3-OH-14: in the E. oli LPS stndrd ws in tht rnge in our study (i.e., 2%, wt/wt). Tht these pproximtions were pplile ws supported y the good orreltion etween LPS (G-MS) nd DAP (Fig. 4). lultion of LPS vlues from the DAP vlue gve mounts whih were pproximtely twie the LPS vlues otined with G-MS,. A Downloded from on August 27, 217 y guest

6 1276 SONESSON ET AL. APPL. ENVIRON. MIROBIOL. I 2 - ) o - h o (W) 1 ~~~~~ y = x RA2 =.167 I 2 m o n -J J- 1. G-MS y = x R^2 =.757 * LAL y = x R^2 =.159 G-MS ~~~~~ ~~~~~ 8 n L 6 6 O 4 ) 2 6 o 5' - 4' 6 OD ' A. ' LAL LPS nq/rn3 y = x FRA2 =.226 m 1 y = x R^2 =.32 2 LAL LPS n/m LAL LPS ng/m3 FIG. 3. orreltion etween the LAL ssy nd the G-MS method for mesurement of LPS in irorne dust in () SH-1, () SH-2, nd () SH-3. though the differenes were not sttistilly signifint (Student's t test; P >.1). The G-MS method hs erlier een used in study of few dust smples from poultry onfinement uildings (27); in tht study, no 3-hydroxy ftty ids were found, lthough high levels of DAP were deteted (thus suggesting the possile presene of DAP-ontining grmpositive teri [25]). The method used here for determining the ontent of LPS in irorne dust is sed on the ssumption tht the LPS olleted on the filters is relesed into the wter quntittively. This ssumption is doutful, however, s it is well o -j ' Q * v LAL-,-LAL DAP nq/rn3 G-MS y = x R^2 =.835 * LAL y = e-2x RA2 = DAP ng/rn3 G-MS = y x R^2 =.625 * LAL y = e-2x R^2 =.437 /EI El G-MS LAXL A-F- -r DAP ng/m3 FIG. 4. orreltion etween DAP onentrtions s determined y G-ED nd LPS onentrtions s determined y oth G-MS nd the LAL ssy in () SH-1, () SH-2, nd () SH-3. known tht LPS my dhere very strongly to oth polr nd nonpolr surfes (36). This my explin the low reovery in the present study when smples ontining smll mounts of LPS (<5 ng) nd teril ells (<16 FU) were extrted from polypropylene tues for nlysis with LAL (Tles 1 nd 2). Morris et l. (11) reported lrge differenes in reproduiility etween the use of old nd wrm wter when extrting LPS from otton. An pproh suitle for the G-MS method might e diret hydrolysis of the filter. ellulose ette filters re not suitle, however, s the E Downloded from on August 27, 217 y guest

7 VOL. 56, 199 LAL AND G-MS TO MEASURE LPS IN AIRBORNE DUST 1277 TABLE 4. Determintion of LPS olleted on polyvinyl hloride filters with the hromogeni LAL test nd with G-MS Smple LAL LPS (ng/m3) y: G-MS 1 <.l 9,39 2 <.1 2,75 3 < ,45 Detetion limit of the LAL ssy, i.e.,.1 ng/m3. filter is degrded y the id solution. The polyvinyl hloride filters used withstnd the idi onditions during hydrolysis, nd the results indite tht only low proportions of the LPS olleted on the filters re extrted into the wter. By using diret hydrolysis of polyvinyl hloride filters followed y G-MS, the totl mount of mteril olleted on the filter is ville for study. Studies hve shown LPS vlues otined with the LAL ssy to orrelte with the pyrogeniity of its lipid A, ut not with other toxi (i.e., endotoxi) effets of lipid A (31). Severl sustnes of miroil origin hve een shown to tivte the LAL test nonspeifilly (17, 37), nd other sustnes hve een found to intivte the ssy (35). The G-MS method mesures the totl mount of LPS in smple, even when LPS is present in iologilly intive form. However, it my e possile tht LAL-intive LPS, e.g., present in ellulr deris, eomes iologilly tive when inhled into the lung. It is known lso tht peptidoglyn my use ertin endotoxi effets (4, 34), nd severl studies hve reported the mjority of irorne vile teril ells in poultry plnts to e grm positive (1, 5, 8; Hgmr et l., in press). G-MS nlysis of suh iomrkers s D-lnine, DAP, nd 3-hydroxy ftty ids provides useful informtion of the mounts of grm-positive nd grm-negtive ells nd LPS in irorne dusts for evluting the potentil risk of endotoxi effets of the working environment. AKNOWLEDGMENTS We re indeted to Rgnr Rylnder nd ollegues, University of Gothenurg, Gothenurg, Sweden, for nlyses of the filter smples with the LAL test, nd to Gorn Odhm nd Anders Vleur, Lortory of Eologil hemistry, Lund University, for providing G-MS nd for ssistne with mss spetrometry. 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