Revista
Brasileira
de
Hematologia
e
Hemoterapia
Brazilian
Journal
of
Hematology
and
Hemotherapy
w w w . r b h h . o r g
Original
article
Intestinal
permeability
in
leukemic
patients
prior
to
chemotherapy
Juliana
Brovini
Leite
∗,
Eduardo
Garcia
Vilela,
Henrique
Oswaldo
da
Gama
Torres,
Maria
de
Lourdes
de
Abreu
Ferrari,
Aloísio
Sales
da
Cunha
UniversidadeFederaldeMinasGerais(UFMG),JuizdeFora,MG,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received4February2014 Accepted13June2014 Availableonline17July2014
Keywords:
Leukemia Lactulose Mannitol
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b
s
t
r
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t
Objective:The objective ofthis study wastoevaluate the intestinal barrier functionin
leukemiapatientsbeforethestartofthechemotherapywithanintestinalpermeabilitytest usinglactuloseandmannitolasmarkers.
Methods:Thestudyenrolled20patientsdiagnosedwithleukemia(acuteandchronic).Ten
healthyvolunteerswerealsosubmittedtothetestasacontrolgroup.
Results:The medianlactulose/mannitolratiowas0.019fortheLeukemiaPatientGroup,
whereas in healthycontrolsthemedian was0.009(p-value=0.244).The median lactu-lose/mannitolratioinacuteleukemiapatientswas0.034givingap-valueof0.069when comparedtohealthycontrols.Thissamecomparisonwasmadebetweenacutemyeloid leukemiapatientsandhealthycontrolswithap-valueof0.149.Therewasnosignificant differenceintheintestinalpermeabilitybetweenacuteandchronicleukemiapatients(p -value=0.098).
Conclusion: Theintestinalbarrierfunctionmeasuredusingtheintestinalpermeabilitytest
wassimilarinleukemic patientsoverallandhealthycontrols,buta tendencytowarda differentpatternwasfoundintheintestinalbarrierfunctionofacuteleukemiapatients.
©2014Associac¸ãoBrasileiradeHematologia,HemoterapiaeTerapiaCelular.Published byElsevierEditoraLtda.Allrightsreserved.
Introduction
Leukemiasarediseasescharacterizedbyneoplastic prolifera-tionthataffectthebonemarrowandinhibithematopoiesis, causing abnormalities in peripheral blood and sometimes infiltratingnon-hematopoietictissues.1 Thegastrointestinal
∗ Correspondingauthorat:UniversidadeFederaldeMinasGerais(UFMG),Av.ProfessorAlfredoBalena,190,SantaEfigênia,30130-100Belo
Horizonte,MG,Brazil.
E-mailaddress:[email protected](J.B.Leite).
tract may be affected, either by leukemic infiltration or by therapy-associated complications.2 Leukemia cell
infil-tration may occur in any segment of the gastrointestinal tract andmaycause stomatitis,gingivitisorgum hypertro-phy,oropharyngeal dysphagiaandtheformationofmasses in the esophagus, stomach, small intestines and colon which, inturn, are associated toobstruction, hemorrhage,
http://dx.doi.org/10.1016/j.bjhh.2014.07.007
intussusceptionor enterocolitis.In thesmallgut, leukemia infiltrationmayreducethe integrityofthemucosalbarrier, allowing antigen permeation and reduction of absorption area.3 Theinvolvementofthis organ ismostoftenseen in
acutemyeloidleukemia(AML).2,4
These changes in the intestinal barrier can be studied through intestinal permeability; the most commonly used markersaresugarssuchaslactuloseandmannitol,sincethey donotrequiretheuseofradioactivetechniques.5–7
Theintestinalpermeabilitytest(TL/M)isausefulmethod
toevaluatetheintegrityoftheintestinalmucosainany con-ditionwhich would cause theloosening oftightjunctions, includingthoseaffectingthesmallgut,suchasinCrohn’sand celiacdiseases anddiseases whichareassociatedtoa sec-ondaryinfiltrationofthisorgansuchasleukemia.8Byusing
thismethod,itisalsopossibletostudymucositissecondary totheuseofchemotherapeuticagents.9Changesin
intesti-nalpermeability,detectedbeforechemotherapyinleukemia patients and their eventualclinical consequences, such as a greater antigen permeation, may contribute to elucidate pathophysiologicalmechanismsinvolvedinthecontextofthe diseaseanditstherapy,andpossibly,comeupwithmore spe-cificsolutionstoproblemsrelatedtothesechanges,suchas infectionandgraftversushostdisease(inthecaseofmarrow transplantation).
Objective
Theaimofthestudy wastoevaluatethe intestinalbarrier functioninleukemicpatientspriortochemotherapy,by test-ing intestinalpermeability bythedeterminationofurinary lactuloseandmannitolconcentrationsbyhighperformance liquidchromatography(HPLC).
Methods
Patients
BetweenApril2010andSeptember2011,thisstudyenrolled 20 patients aged 18 years or above, of both genders, with initialdiagnoses ofAML, chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL) and chronic lympho-cyticleukemia(CLL)admittedintheHematologyOutpatient ClinicandEmergencyDepartmentoftheHospitaldasClínicas daUniversidadeFederaldeMinasGerais,beforeundergoing chemotherapyinduction.TheuseoftheTL/M didnotresult
in any change in medical management. Tenover 18-year-oldhealthyvolunteers,ofbothgenders,alsounderwentthe
TL/M.
Patientsdiagnosedwithboweldisease,cirrhosis, conges-tiveheart failure, nephroticsyndrome, thyroid diseases or diabetesmellitus,diseasesthatcouldinterferewith absorp-tion or flow of water and solutes and/or gastrointestinal motilitywereexcludedfromtheresearchaswerepatientswho drankalcoholicbeverageswithinthreedaysandtook non-steroidalanti-inflammatorydrugs(NSAIDs)withinsevendays priortourinecollection.
Methods
The study was approved by the Research Ethics Commit-tee of the Universidade Federal de Minas Gerais (ETIC 0079.0.203.000-11). Allparticipantssignedaninformed con-senttermbeforethestudywasinitiated.
Diagnosisofleukemiawasconfirmedbymyelogram,bone marrowbiopsyandcytogeneticorgeneticstudieswhen nec-essary.
InordertoperformtheTL/M,patientsfastedforeighthours.
Subsequently, theywere instructed to eliminateany resid-ualurineanda120mLiso-osmolarsolutioncontaining6.25g of lactulose(95%) (Sigma–Aldrich, Missouri, USA) and 3.0g of mannitol (PA) (Sigma–Aldrich, Missouri, USA) dilutedin waterwas given.Fasting wasmaintained forthefollowing two hours.All urine volumewas collected duringaperiod offivehours.Subsequently,theurinewashomogenizedand thetotalvolumewasrecorded.Aliquotsof50mLwerestored inlabeledinsealedflasksafteradding10mgofthimerosal (Synth,Diadema,Brazil)toinhibitbacterialgrowth.Samples werefilteredusingamilliporefilter(0.22m)(Millipore, Biller-ica,USA),andtheion-exchangeresinandthematerialwere storedinproperlylabeledcryotubesat−20◦C.
Themannitolandlactuloseconcentrationsweremeasured intheurineusingHPLCequipment(Schimadzu®,Japan)
com-prisinganinjectionpump,anautoinjector,acontrollerwith software that allowsreadings to beinterpreted ata work-station, and a refractive index gauge. Fifty microliters of urinewereintroducedafterthawingusingtheautoinjector.To achievebetterseparationfromothersubstancesintheurine, lactuloseandmannitolwerereadusingtwodifferentcolumns utilizingtwodistinctmobilephases.APhenomenexH+
col-umn(Phenomenex,USA)withamobilephaseofpuremilli-Q sonicatedwaterataflowof0.6mL/minwasusedtoseparate themannitolandaSupelcogelNH2 column(SigmaAldrich,
Bellefonte, USA) withamobile phase ofa solutionof ace-tonitrile and milli-Qsonicatedwater(ratioof75/25)witha flowof1.0mL/minwasemployedtoseparatethelactulose.A SupelcogelH+precolumn(SigmaAldrich,Bellefonte,USA)was
thesameforbothreadings.Differentamplitudesofthewaves generatedbythesolutioncontaininglactuloseandmannitol were capturedatthe workstation,generatinggraphsinthe formofcurves,whichwerethenrecorded.Analyseswere car-riedoutatroomtemperature.
Totestreproducibilityandtostandardizemeasurements, solutionsoflactulosewerepreparedatknownconcentrations of0.1g/L,0.2g/L,0.4g/Land0.8g/L,asweresolutionsof man-nitolatconcentrationsof0.625g/L,1.25g/Land2.5g/Landa simplelinearregressionwasperformedinordertoobtaina straightlineequationforboth.
Bycorrectingfortheurinevolume,theamountexcreted was obtainedfor lactulose and mannitol, which was then dividedbytheamountingestedtocalculateanexcreted per-centageofeachsugar.Thepercentageoflactulosewasdivided bythepercentageofmannitol inordertoobtain the lactu-lose/mannitolexcretionratio(TL/M).
Table1–Intestinalpermeabilitytestbetweenleukemiapatientsandhealthyvolunteers.
Mean% Standarddeviation Median%(range) p-Value
Percentagelactulose
Healthyvolunteers 0.14 0.14 0.09(0.02–0.48) 0.311
Leukemiapatients 0.27 0.26 0.23(0.01–0.97)
Percentagemannitol
Healthyvolunteers 11.44 4.34 11.60(4.70–18.0) 0.403
Leukemiapatients 9.78 5.37 10.55(0.60–21.70)
TL/M
Healthyvolunteers 0.012 0.010 0.009(0.001–0.027) 0.244
Leukemiapatients 0.061 0.115 0.019(0.001–0.488)
TL/M:lactulose/mannitolratio.
Table2–Intestinalpermeabilitytestbetweenacuteleukemiapatientsandhealthyvolunteers.
n Mean% Standarddeviation Median%(range) p-Value
Percentagelactulose
Healthyvolunteers 10 0.14 0.14 0.09(0.02–0.48) 0.170
Leukemiapatients 16 0.31 0.27 0.26(0.01–0.97)
Percentagemannitol
Healthyvolunteers 10 11.44 4.34 11.60(4.70–18.0) 0.215
Acuteleukemiapatients 16 8.97 5.07 8.75(0.60–16.80)
TL/M
Healthyvolunteers 10 0.012 0.010 0.009(0.001–0.027) 0.069
Acuteleukemiapatients 16 0.074 0.126 0.034(0.001–0.483)
TL/M:lactulose/mannitolratio.
datadidnothaveaGaussiandistribution,theMann–Whitney
test was used to compare medians. An alpha error of 5%
(p-value<0.05)wasconsideredthethresholdforstatistical sig-nificance.
Results
Initially, 26 patients withsuspected diagnoses ofleukemia beforethebeginningofthetreatmentwereinvitedtotakepart inthestudy.Aftertheresultsoftheconfirmatorytests,two patientswereexcludedastheywerediagnosedwith myelofi-brosis.Fourotherpatientsrefusedtotakepartintheresearch. Thus,20patients,ninemales(45%)and11females(55%),with
confirmeddiagnosisofleukemiaparticipatedintheresearch. Agesrangedfrom18to81years,withameanof47.2years. Sixteenpatients(80%)hadacute(11AMLandfiveALL)and four(20%)hadchronicleukemia(threeCMLandoneCLL).
Gastrointestinalmanifestationssuchasnauseas,vomits,
abdominalpainordiscomfortanddiarrheawerepresentin
eight(40%)patients,sevenwithAMLandonewithCML.Fever waspresentinnine(45%)patients,sixwithAML,onewithALL
andtwowithCML.
ThemeanTL/Minleukemiapatients,calculatedfromthe
relationshipbetweenlactuloseandmannitolexcretionrates, was0.061±0.115andthemedianwas0.019(0.001to0.483)and themeanTL/Minhealthyvolunteerswas0.012±0.010andthe
medianwas0.009(0.001–0.027)(Table1).
Table3–Intestinalpermeabilitytestbetweenacutemyeloidleukemiapatientsandhealthyvolunteers.
n Mean% Standarddeviation Median%(range) p-Value
Percentagelactulose
Healthyvolunteers 10 0.14 0.14 0.09(0.02–0.48) 0.204
AMLpatients 11 0.33 0.30 0.29(0.01–0.97)
Percentagemannitol
Healthyvolunteers 10 11.44 4.34 11.60(4.70–18.0) 0.397
AMLpatients 11 9.66 4.99 9.90(0.60–16.80)
TL/M
Healthyvolunteers 10 0.012 0.010 0.009(0.001–0.027) 0.149
AMLpatients 11 0.077 0.138 0.042(0.001–0.483)
Table4–Intestinalpermeabilitytestbetweenacuteleukemiapatientsandchronicleukemiapatients.
n Mean% Standarddeviation Median%(range) p-Value
Percentagelactulose
Acuteleukemia 16 0.30 0.27 0.26(0.01–0.97) 0.275
Chronicleukemia 4 0.14 0.19 0.07(0.01–0.42)
Percentagemannitol
Acuteleukemia 16 8.97 5.07 8.75(0.60–16.80) 0.187
Chronicleukemia 4 13.0 6.09 11.40(7.50–21.70)
TL/M
Acuteleukemia 16 0.074 0.126 0.034(0.001–0.483) 0.098
Chronicleukemia 4 0.008 0.008 0.007(0.001–0.019)
TL/M:lactulose/mannitolratio.
Table5–Intestinalpermeabilitytestbetweenacutemyeloidleukemiapatientsandchronicleukemiapatients.
n Mean% Standarddeviation Median%(range) p-Value
Percentagelactulose
AMLpatients 11 0.33 0.30 0.29(0.01–0.97) 0.269
Chronicleukemia 4 0.14 0.19 0.07(0.01–0.42)
Percentagemannitol
AMLpatients 11 9.66 4.99 9.90(0.60–16.80) 0.297
Chronicleukemia 4 13.0 6.09 11.40(7.50–21.70)
TL/M
AMLpatients 11 0.077 0.139 0.042(0.001–0.483) 0.192
Chronicleukemia 4 0.008 0.008 0.007(0.001–0.019)
TL/M:lactulose/mannitolratio.
ComparisonsoftheTL/Mbetweenacuteleukemiapatients
and healthy volunteers, and between AML patients and
healthy volunteers were also performed. In patients with
acuteleukemia,themedianTL/Mwas0.034(0.001–0.483)
giv-ing a p-value of0.069 comparedto the median ofhealthy
volunteers(Table2).OncomparingthemedianTL/MofAML
patients(0.042)andthemedianofhealthyvolunteers(0.009) thep-valuewas0.149(Table3).
The TL/M was also compared between acute leukemia
and chronic leukemia patients. Themedian TL/M foracute
leukemiapatientswas0.034,whereasitwas0.007forchronic leukemiapatients(p-values=0.098)(Table4).ThemedianTL/M
was0.042and0.007forAMLandchronicleukemiapatients, respectively(p-value=0.192)(Table5).
Leukemiapatientswithgastrointestinalsymptomsorfever didnotpresentdifferentTL/Mvaluesfromtheleukemia
sub-groupswithoutthesemanifestations.
Discussion
Inthisstudy,leukemiapatientshadhighermedianTL/Mvalues
(0.019)whencomparedtothemedianofhealthyvolunteers (0.009),however,thedifferencewasnotstatisticallysignificant (p-value=0.244).Thisfindingmaybeexplainedbythesmall samplesizeandbythewiderangeofresultsfoundinleukemia patients(0.001–0.483).However,inthissample,someleukemia patientsdidnothavechangesinthefunctionofthe intesti-nalbarrier, whichisperfectlyunderstandable, sincenotall leukemiapatientshaveinfiltrationofthesmallintestinalwall. Itisassumedthattherearedifferencesbetweensubgroupsof
leukemiaandthismayinvolveagreaterorlessernumberof complications,primarilythoseassociatedtosepsisresulting fromgreaterpermeationofantigensinpatientswithhigher
TL/Mvalues.10In1998,Sundströmetal.11compared
intesti-nalpermeabilityinAMLpatientsbeforechemotherapywith resultsobtainedfromhealthvolunteersandobservedhigher values inthe first group, however, somepatients also had theirintestinalbarrierfunctionpreserved.11Inthisstudy,the
medianoftheTL/MofAMLpatientsbeforebeginningthe
treat-ment (0.043) was significantly higher (p-value=0.02) when comparedtothemedianofhealthyvolunteers(0.025).Bow andMeddings9alsoevaluatedtheintestinalbarrierfunction
inAMLpatientsbeforetheinductionofchemotherapyand foundthattheTL/Mwasalsohigherbeforebeginningthe
ther-apy(0.03)comparedtothemeanreferencescorementioned bytheauthors(TL/M<0.028).9
The median TL/M of acute leukemia patients was 0.034
whereas itwas0.009inhealthy volunteers(p-value=0.069). Althoughthedifferencewasnotstatisticallysignificant,itis possiblethatthereisatendencyfortheintestinalbarrierto bedifferentbetweenthe twogroups.Thiscomparisonwas alsoperformedexclusivelyamongAMLpatients.Themedian
TL/M in AML patients was also higher (0.042) compared to
AMLsaremoreassociatedtointestinalinjurythanother typesofleukemia.2,4 Inthisstudy,themedianT
L/Mofacute
leukemiapatientswas0.034.Whencomparedtothemedian ofthegroupofchronicleukemiapatients(medianTL/M=0.007;
range:0.001–0.019),thep-valuewas0.098.However,onecan also infer that there is a tendency to behave differently betweenthetwogroupsofpatients.AllTL/Mresultsinchronic
leukemiawerewithintherangeofthehealthycontrols.The medianTL/M in AMLpatients was even higher (0.042), but
notsignificantlydifferentcomparedtotheresultsofchronic leukemiapatients(p-value=0.192);thismayalsohavebeen influencedbythewiderangeofresultsofthefirstgroup.
Althoughtherearefewstudiesintheliteratureanalyzing intestinalchangesassociatedtoleukemiasusingtheTL/Min
adultsbeforechemotherapy,itispossibletosupposethata subgroupofacute leukemia patientsmay have higher val-uescomparedtohealthyindividuals.Althoughmoststudies haveaimedto showthe damagecausedbychemotherapy, theintestinalbarrierfunctionislikelytohavealreadybeen impaired and so further changes may add to the lesions caused by chemotherapeutic agents. Evidence of changes inintestinalpermeabilityindicating changesofthe intesti-nalmucosalbarrierinasubsetofleukemia patientsbefore chemotherapymay correlatewith prognosis and so future investigationsshouldtrytoidentifyinterventionstominimize theseeffects.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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