Lipopolysaccharide Concentrations During Superflux Dialysis Using Unfiltered Bicarbonate DialysateA NNE VAN T ELLINGEN,*M URIEL P.C.G ROOTEMAN,‡R ONALD P RONK,†J ENNY VAN L OON,§M ARC G.V ERVLOET,‡P IET M.TER W EE,‡AND M ENSO J.N UBÉ*In the present report,the design of a new dialysate delivery system to produce low to moderately contaminated dialysate is described.In addition,thefirst data on bacterial counts and lipopolysaccharide(LPS)concentrations in both the dialysate and the blood during hemodialysis(HD)with superflux dia-lyzers are presented.In this prospective study,37patients were randomized into two consecutive periods of12weeks to HD with a highflux polysulfon(PS),a superflux PS,a superflux cellulosic tri-acetate(CTA)or a superflux CTA di-alyzer withfiltered dialysate(CTA f),resulting in74periods in which measurements were obtained.Filtered dialysate showed significantly lower bacterial counts,if compared with nonfiltered dialysate(p<0.001).As for LPS,marked differ-ences were not observed betweenfiltered and nonfiltered dialysate,whereas mean plasma LPS concentrations were below the value of the dialysate at all time points(p<0.001). Plasma LPS concentrations decreased significantly during HD with all four modalities(F60:t00.032؎0.005,t1800.026؎0.009endotoxin units(EU)/ml,p؍0.001;F500S,t00.031؎0.004,t1800.027؎0.005EU/ml,p؍0.001;Tricea150G:t0 0.032؎0.004,t1800.025؎0.005EU/ml,p<0.001;and Tricea150G f:t00.034؎0.007,t1800.025؎0.006EU/ml,p <0.001).During HD with highly permeable dialyzers and moderately contaminated dialysate,plasma LPS concentra-tions decreased significantly,irrespective of the material used (PS or CTA),theflux characteristics of the devices(highflux or superflux),or the presence of a bacterialfilter.ASAIO Journal2002;48:383–388.B ack transport of bacterially derived products from dialysate fluid to the circulation has been correlated with both acute and long-term complications in hemodialysis(HD)patients.Ac-cording to recent literature,acute pyrogenic reactions such as fever,chills,and hypotension occur in approximately0.5–5 per1,000HD sessions.1–3In addition,nausea,myalgia,head-ache,and sleepiness have been attributed to the use of non-sterile dialysate.4It has been suggested that the repeated trans-membrane passage of bacterial fragments activate peripheral blood mononuclear cells(PBMC),resulting in the intermittent secretion of a variety of proinflammatory cytokines,including interleukin-1␣(IL-1␣),interleukin-6(IL-6),and tumor necrosis factor␣(TNF␣).5Due to this repetitive stimulation,a chronic microinflammatory state is induced,which may contribute to the development of long-term HD related complications such as dialysis related amyloidosis,1,6,7malnutrition,8,9accelerated atherosclerosis,10and increased mortality.11Several in vitro studies suggest that sterile dialysate is crucial to prevent back transport of these cytokine-inducing substances. Therefore,it seems mandatory that water used for dialysis treatment meet stringent microbiological and nonpyrogenic criteria.However,these in vitro experiments were performed under highly nonphysiologic conditions.12In many of these studies lipopolysaccharide(LPS)challenge levels were500to 1,000-fold greater than the maximum accepted dose in clinical HD.13In fact,data from controlled clinical studies are lacking and evidence based guidelines are not available.Hence,it may not be surprising that official standards to prepare dialysis fluid and thefinal dialysate itself,vary markedly(Table1).The strictest standard is met in the European Pharmacopoeia,with limits of100colony forming units per milliliter(CFU/ml)for bacteria and0.25endotoxin units(EU)/ml for LPS in dialysis water.14Higher bacterial counts are accepted by the American Association of Medical Instrumentation(AAMI),with levels up to200CFU/ml in the dialysis water and up to2,000CFU/ml in thefinal dialysate.15As for the maximal LPS concentration in dialysisfluid,no recommendations are given.In clinical prac-tice,several studies have shown that many HD centers world-wide do not comply with the above prescriptions.16–18A survey of Greek centers shows that the total bacterial counts in dialysate exceeded the AAMI standards in36.3%.19Further-more,the situation is potentially aggravated by the widespread use of liquid bicarbonate concentrate,in conjunction with highflux membranes,predisposing to bacterial growth and backfiltration of bacterial fragments.20Therefore,even more stringent criteria for the purity of the dialysisfluid have been recently formulated,e.g.,Ͻ0.1CFU/ml for bacterial growth and0.03–0.05EU/ml for the presence of endotoxins.21,22 Recently,dialyzers with superior permeability for middle and large molecular weight uremic toxins,23termed superflux dialyzers,were introduced in our center.As we moved to a new center,a new dialysate delivery system based on current recommendations,with a few modifications,24–26was con-structed.The present report describes both the design and the effectiveness of this water treatment system to produce low to moderately contaminated dialysate without the need for a bacterialfilter.In addition,bacterial counts and LPS concen-trations in both the dialysate and blood were compared be-tween groups of patients undergoing long-term dialysis treat-ment with two types of superflux dialyzers(polysulfon[PS]andFrom the*Department of Nephrology,and†Department of Pharma-cology,Medical Centre Alkmaar,Alkmaar;‡Department of Nephrol-ogy,VU Medical Centre,Amsterdam;§Department of Clinical Chem-istry,Leiden University Medical Centre,Leiden,The Netherlands.Submitted for consideration April2001;accepted for publication inrevised form December2001.Reprint requests:Anne van Tellingen,Department of Nephrology,Medical Centre Alkmaar,Wilhelminalaan12,1815JD Alkmaar.ASAIO Journal2002383cellulosic tri-acetate[CTA])and a group of chronic HD pa-tients undergoing HD treatment with a standard highflux dialyzer(PS).In the case of superflux CTA,standard dialysate was compared with ultra-pure dialysate.Materials and MethodsStudy DesignBefore participating in the study,all patients were dialyzed three times a week using a highflux PS dialyzer.Thirty-seven patients were randomized in two successive periods to HD with either a highflux PS(F60),a superflux PS(F500S),a superflux CTA(Tricea150G)or a superflux CTA dialyzer with filtered dialysate(Tricea150G f)for12consecutive weeks, resulting in74periods in which measurements were obtained. To avoid carry-over effects,a washout period of1month was instituted.Blood samples were taken at the end of each study period from the afferent line before dialysis(t0)and from the efferent line180min(t180)afterward.Samples were analyzed for limulus amebocyte lysate(LAL)reactivity.Dialysate sam-ples were taken at t180from the outlet port of the dialysis machine and were cultured for bacterial growth and analyzed for LAL reactivity.Dialysis Procedure and MaterialsThe dialysis sessions lasted3to5hours,depending upon the individual prescription of the patient.Onlyfirst-use dialyzers were used.Characteristics of the three dialyzers used in this study(PS:F60and F500S,Fresenius,Bad Homburg,Ger-many;CTA:Tricea150G,Baxter,Osaka,Japan)are depicted in Table2.As for the superflux dialyzers,these devices have been designed to maximize convective transport by increasing the pressure drop along thefibers of the membrane.Further-more,the pore size and/or distribution of the pores influences the permeability.These characteristics result in a better clear-ance of middle and high molecular weight substances,as measured by2-microglobulin(J.Vienken,personal commu-nication).Filtered dialysate was obtained by the interposition of a bacterial PSfilter(SPS600,Fresenius,Bad Homburg, Germany).27According to the individual needs of the patients, bloodflow and ultrafiltration(UF)rates were kept constant between200and250ml/min and between300and1,000 ml/h,respectively.Isolated UF was not performed.Bicarbon-ate powder(BiBag)was used for preparation of the dialysate. Dialysateflow was500ml/min.Anticoagulation was achieved by dalteparin with an initial dose of2,500to6,000IU,fol-lowed by an extra dose of500to1,000IU during the dialysis treatment,if necessary.Individual conditions(bloodflow,UF, dalteparin dose)were kept stable throughout the study period. Water Treatment SystemThe water system(Figure1)is supplied with drinking water from the community waterworks piping grid.Two consecutive coarsefilters(100m and10m)and two microfilters(5and 1m)are installed to remove suspended particles.Next,water hardness is sharply reduced from13°to2°German Hardness by two parallel connected softeners.Downstream from the softener,a100L buffer tank separates the water from the main water supply and a high pressure pump delivers water from the tank to the reverse osmosis system(RO).The RO input pressure is14.6bar(212PSI).The RO,represented in Figure1as one membrane,actually consists of four semipermeable mem-branes(molecular size exclusion,90Da)serially connected. The permeate is conducted to the distribution loop and the amount of water that is not used directly by the dialysis ma-chines is recycled and passes once more through RO by means of the pressure pumping device.This repeated purification results in a water recovery rate of75%,a rejection rate forTable1.Microbial Standards for Dialysis Water and Dialysis Fluids*StandardDialysis Water Dialysis FluidViable Counts†(CFU/ml)Endotoxin‡(EU/ml)Viable Counts(CFU/ml)Endotoxin(EU/ml)European Pharmacopoeia(1997)Յ100Յ0.25n.s.n.s.German Dialysis Standard(1993)Ͻ200n.s.Ͻ2000sterility requested Pyrogen free requested Japanese Society for Dialysis Therapy(1995)n.s.n.s.Ͻ100Ͻ0.25 Swedish Pharmacopoeia(1997)Ͻ100Ͻ0.25Ͻ100Ͻ0.25 Canadian Standard Association(1986)Յ200Ͻ1ng/ml n.s.n.s.AAMI Standard USA(1996)Յ200n.s.Յ2000n.s.*See Van der Linde.33n.s.,not specified.†CFU(colony forming units)/ml is defined as the number of colonies formed on agar plates by viable organisms calculated for1ml of test solution.‡Endotoxin concentrations are based on in vitro measurement using the limulus amoebocyte lysate(LAL)test.One endotoxin unit(EU)is the LAL reactivity of0.1ng of the United States Pharmacopoeia reference standard endotoxin EC-5from E.coli0113:H10:K,that is,1ng equals10EU,with that standard.Table2.Membrane CharacteristicsF60F500S Tricea 150GInner lumen(m)200155200 Wall thickness(m)403515UF coefficient(ml/mm Hg/h)40300(H2O)29Surface area(m2) 1.3 1.2 1.5Clearance(ml/min)2-microglobulin(MW,11.8kDa)3865–80Not knownSieving coefficient2-microglobulin0.650.90.8UF,ultrafiltration;MW,molecular weight.384VAN TELLINGEN ET AL.bacteria of 99%,for organic substances of 99%,and for inor-ganic ions of 95–98%(Filmtec 4°Tapwater RO elements,USF Aquapur B.V.,Zoetermeer,The Netherlands).The distribution loop is built of PEX (polyethylene,crosslinked),is free of dead zones,and consists of stainless steel quick-couplings fitted directly to the loop without faucets.The RO water produced enters the distribution loop at a pres-sure of 2bar (29PSI)and flows with a speed of 1.2m/s.A black sleeve shields the piping system from light.To prevent bacterial contamination of the water treatment system,filters are replaced every 6months or sooner when the pressure drop exceeds 0.5bar.Alarms are triggered when the water hardness exceeds 0.5°German Hardness,the reject rate of the RO drops below 90%,and the conductivity pre RO and post RO exceeds 20S/stly,samples for bacterial de-termination are taken monthly from the RO water distribution loop itself,as well as from the outlet of each dialysis machine.Analytic MethodsMicrobiologic Evaluation of the Dialysate.Dialysate sam-ples were drawn into sterile tubes.Total plate counts were performed on Reasoners 2A-agar (R 2A)(Difco Laboratories,Detroit,MI)after 24hours of incubation at 37°C and 7days at 21°C.Isolation of pathogens was performed on McConkey agar (Becton-Dickinson,Germany)after 48hours of incuba-tion at 37°C.Identi fication of microorganisms was achieved using Analytical Pro file Index identi fication (Analytical Pro file Index system S.A.,Montalieu Verceu,France).Endotoxin Assay.Blood samples for LPS determinations were collected at t 0and t 180in heparinized and pyrogen-sterile Endo Tubes (Chromogenix Instrumentation Laboratory Spa,Milano,Italy).After centrifugation (10min,190g ),samples were stored at Ϫ20°C until determination.LPS concentrations in platelet rich plasma (PRP)were quanti fied by an end-point chromogenic method based on the activation of clotting fac-tors in LAL (Bio Whittaker,Boehringer Ingelheim Bioproducts,Verviers,France)and subsequent conversion of chromogenic substrate by the activated clotting factors.The chromogenic end-product was measured bichromatically at 405–490nm.As LPS in plasma is partly bound to lipoprotein-binding protein (LBP),phospholipid transfer protein (PLTP),or various lipopro-tein fractions,including LDL and VLDL,28–30all plasma sam-ples were diluted 10times and heated for 15minutes at 75°C to overcome inhibition/enhancement by these proteins.The highest concentration of the standard curve was 1.2EU/ml PRP.Aliquots of all plasma samples were spiked with 0.024EU/ml LPS.Mean ϮSD recovery at t 0was 72.7%Ϯ14.3%,whereas the mean ϮSD recovery at t 180was 67.2%Ϯ15.6%.Sample recoveries (50–100%)were below the value of the recoveries of healthy volunteers (100%).The latter observation may be due to a rise in acute phase proteins in HD patients,associated with an increased LPS binding,which is then not completed inhibited by the dilution/heating procedure as de-scribed above.Data at t 180were corrected for changes in hematocrit (Ht):corrected value t180ϭ(Ht t0/Ht t180)ϫvalue t180.Dialysate samples for LPS determinations were collected at t 180in pyrogen-sterile FALCON 2063polypropylene tubes (Becton Dickinson,Franklin Lakes,USA).LPS activity in dia-lysate was quanti fied by a kinetic chromogenic method based on the LAL assay (Bio Whittaker,Wakersville,USA).Standard series of puri fied Escherichia coli 055:B5LPS were made in the range of 0.005to 50.0EU/ml.Inhibition and interference testing was performed on each sample by an endotoxin spike.To overcome inhibition/enhancement,all dialysate samples were diluted 10times.All determinations were performed in duplicate,and recoveries of spikes between 50and 150%were accepted;limit of determination was 0.05EU/ml.Statistical AnalysisData are expressed as mean (ϮSD)or median and range when appropriate.Analysis was performed with the Statistical Package for Social Sciences/PC ϩsoftware system using anal-ysis of variance,and paired and unpaired t-tests to study the differences between groups.Differences were considered sta-tistically signi ficant at p Ͻ0.05.ResultsDialysate CulturesBacterial culture results from the dialysate are shown in Table 3.Gram positive organisms were cultured in 8of 74cases,whereas 7of 8also showed gram negative organisms.When comparing filtered with non filtered dialysate,a signi fi-cant difference was observed (filtered dialysate 0[0–3]CFU/ml;non filtered dialysate 26[0–310]CFU/ml,p Ͻ0.001).Pseudomonas species were not isolated.LPS Content of DialysateThe LPS concentrations in dialysate during HD with filtered and non filtered dialysate are shown in Table 3.Marked differ-ences were not found between filtered and non filtered dialy-sate (filtered dialysate 0.051Ϯ0.0052EU/ml,non filtered dialysate 0.051Ϯ0.0048EU/ml,p ϭnot signi ficant[ns]).Figure 1.The water treatment system consists of the following elements.Four consecutive filters (a and b)remove suspended particles.Two parallel connected softeners (c)reduce water hard-ness.A high pressure pump (e)delivers water from the storage tank (d)to the reverse osmosis system (RO [g]).The permeate is con-ducted to the distribution loop (h),whereas the reject (f)consists of bacteria and (in)organic substances.The water that is not used directly by the dialysis machines passes once more through the RO by means of the pressure pumping device (see text).a,Pre filters;b,micro filters;c,softeners;d,storage tank;e,high pressure pump;f,reject;g,RO system;h,distribution loop;i,dialysis machines.385LIPOPOLYSACCHARIDE DURING SUPERFLUX DIALYSISLPS Content of PlasmaAt baseline,all groups showed comparable LPS concentra-tions (F 60,0.032Ϯ0.005EU/ml;F 500S,0.031Ϯ0.004EU/ml;Tricea 150G,0.032Ϯ0.004EU/ml;and Tricea 150G,0.034Ϯ0.007EU/ml,p ϭns).Both at the beginning and end of the dialysis session,mean LPS concentrations in plasma were signi ficantly below the values in the dialysate (plasma,t 00.032Ϯ0.005and t 1800.028Ϯ0.006EU/ml,respectively;dialysate,0.051Ϯ0.005EU/ml,p Ͻ0.001).During HD,a signi ficant decrease was observed in all four modalities (F 60:t 0mean,0.032Ϯ0.005,t 180mean,0.026Ϯ0.009EU/ml,p ϭ0.001;F 500S:t 0mean,0.031Ϯ0.004,t 180mean,0.027Ϯ0.005EU/ml,p ϭ0.001;Tricea 150G:t 0mean,0.032Ϯ0.004,t 180mean,0.025Ϯ0.005EU/ml,p Ͻ0.001;and Tricea 150G f :t 0mean,0.034Ϯ0.007,t 180mean,0.025Ϯ0.006EU/ml,p Ͻ0.001).A “borderline ”signi ficant difference was observed between PS and CTA dialysis (PS Ϫ18.8%,CTA Ϫ24.2%,p ϭ0.06)(Figure 2).Correlation Between Dialysate Cultures,LPS Content of Dialysate,and PlasmaNo correlation was found between the dialysate cultures and the LPS concentrations in the dialysate (r ϭϪ0.035,p ϭns).Furthermore,marked correlations were not found between dialysate cultures and plasma LPS concentrations,nor between the dialysate and plasma LPS concentrations (r ϭϪ0.06,p ϭns,respectively,r ϭϪ0.02,p ϭns).DiscussionThe data presented in this report clearly show that the water treatment system used in our center produces low to moder-ately contaminated dialysate without the need for expensive,disposable bacterial filters.31,32With respect to the water de-livery system,several factors may be responsible for the excel-lent bacterial quality of the dialysate.21,24,33–35To prevent bacterial growth and bio-film formation,dead spaces in the distribution loop are completely avoided,whereas a water flow with a speed of 1.2m/s is provided.In addition,the distribution loop itself is built of PEX,which is an attractive alternative to stainless steel,having a smooth inert surface resistant to biologic erosion.21,26Furthermore,to provide op-timal puri fication of the incoming water,the RO system con-sists of four serially connected membranes,while in contrast to many other reports,19,36the storage tank is placed before the RO system.This arrangement is aimed at preventing bacterial growth due to stagnancy of the puri fied permeate downstream of the RO system.Finally,the use of dry powder bicarbonate cartridges improved the microbiological quality of the dialysate.24,26Plasma LPS concentrations were signi ficantly below the value in the dialysate,both before and after HD.Remarkably,during HD with all four modalities,plasma LPS concentrations decreased signi ficantly.As we did not measure the rate of back transport,these findings suggest that either back transport of LPS did not occur at all or plasma clearance of LPS was greater than back transport and/or clearance of LPS at the dialyzer.The mechanism that may account for the decrease of LPS during HD has not been elucidated.As the concentration of LPS in the plasma was below the level of the dialysate,diffu-sive removal of LPS seems highly unlikely.A bactericidal/permeability increasing protein (BPI)dependent mechanism may in fluence the biologic response to LPS,as BPI increases during HD.37,38However,the stimulation of BPI during HD in a recently published study showed comparable data between CTA and PS devices.38Finally,reliable data about the adsorp-tive capacity and/or the convective transport of membranes for LPS are lacking.Our findings are interesting for several reasons.First,dialyz-ers with superior permeability for harmful middle and large molecular weight substances,such as leptin,39,402-micro-globulin,41uremic toxins involved in extrarenal homocysteine metabolism,42and advanced glycation end-products (AGEs),43appear to be safe and do not permit the transfer of LAL positive material from the dialysate to the blood,at least under the conditions found in our center.Therefore,our data shed some doubt on the clinical relevance of the induction of a microin-flammatory state due to the transfer of LAL positive bacterial fragments during both high flux and super flux bicarbonate HDTable 3.Dialysate Cultures and LipopolysaccharideConcentrationsCharacteristic Non filtered Dialysate (n ϭ57)Filtered Dialysate (n ϭ17)Dialysate culture (CFU/ml)26(0–310)*0(0–3)*†Gram stain (number)Positive 1Negative431Positive and negative 7None418LPS (EU/ml)0.051(Ͻ0.05–0.086)0.051(Ͻ0.05–0.071)‡CFU,colony forming unit;LPS,lipopolysaccharide;EU,endotoxin unit.*Median (range).†p Ͻ0.001,filtered versus non filtered dialysate.‡p ϭ0.47,filtered versus non filtereddialysate.Figure 2.Plasma lipopolysaccharide (LPS)concentrations during 12weeks of hemodialysis.LPS concentrations decreased signi fi-cantly during dialysis with all four dialyzers.A “borderline ”signi fi-cant difference was observed between high flux polysulfon (F 500S and F 60)and cellulosic tri-acetate (Tricea 150G f and 150G)dialysis (p ϭ0.06).386VAN TELLINGEN ET AL.and low to moderate levels of dialysate contamination.Sec-ond,surprisingly,all dialyzers used in this study reduced the LPS concentrations in the plasma.In this respect,it is of note that mild endotoxemia is not confined to dialysis patients but has been described recently in apparently healthy persons44or may originate from chronic infections,smoking,gut barrier dysfunction,and chronic heart failure.45,46It has been well documented that most chronic HD patients exhibit multiorgan diseases.It is intriguing to speculate that,HD with biocompat-ible highflux and superflux dialyzers,such as PS and CTA, instead of worsening,may improve the microinflammatory state that is induced by the endotoxemia accompanying these comorbid conditions.Of note,the higher levels of cultured micro-organisms in the nonfiltered modalities did not result in higher LPS concentra-tions.However,it has been suggested that not only LAL pos-itive substances but also other bacterially derived fragments, such as exotoxins and outer membrane proteins(peptidogly-cans,muramyl-peptides),may permeate the membrane of the dialyzer and activate mononuclear cells.13Like LPS,these cytokine-inducing substances may intermittently elicit the se-cretion of a variety of proinflammatory cytokines,resulting in a chronic microinflammatory state.However,preliminary data on plasma levels of IL-1,IL-6,TNF-␣,and various acute phase proteins(C-reactive protein,prealbumin,and lipopro-tein[a])showed no marked changes during either a single dialysis session or during long-term HD.47,48To summarize,the construction of a new water delivery system resulted in the production of moderately contaminated dialysisfluid,without the need for a bacterialfilter.Almost all LPS measurements were within the reference range,whereas during HD with both highflux and superflux dialyzers these values were lower in the plasma than in the dialysate,before as well as after HD.Remarkably,as demonstrated in this study, concomitant use of highly permeable dialyzers and moder-ately contaminated dialysate resulted in a significant decrease of LPS concentrations during HD,irrespective of the material used(PS or CTA),theflux characteristics of the devices(high flux or superflux),or the presence of a bacterialfilter.AcknowledgmentWe wish to thank Professor A.Sturk and J.Duits for their critical reading of the manuscript and the staff and patients of the dialysis department for their indispensable support and enthusiasm.Finally, we thank Stichting Diafoon,Baxter B.V.,and Fresenius Medical Care B.V.forfinancial support.References1.Baz M,Durand C,Ragon A,et al:Using ultrapure water inhemodialysis delays carpal tunnel syndrome.Int J Artif 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