Research ArticleMarkers of Perioperative Bowel Complications inColorectal Surgery Patients
Radomiacuter Hyšpler1 Alena Tichaacute1 Milan Kaška2 Lenka Caloudkovaacute3
Lenka Pliacuteškovaacute3 Eduard Havel2 and Zdenjk Zadaacutek1
1Department of Research and Development University Hospital Hradec Kralove 500 05 Hradec Kralove Czech Republic2Department of Surgery University Hospital Hradec Kralove 500 05 Hradec Kralove Czech Republic3Department of Clinical Chemistry University Hospital Hradec Kralove 500 05 Hradec Kralove Czech Republic
Correspondence should be addressed to Radomır Hyspler rhysplerlfhkcunicz
Received 25 September 2015 Accepted 26 November 2015
Academic Editor Carlo Chiarla
Copyright copy 2015 Radomır Hyspler et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Colorectal cancer is a clinical condition whose treatment often involves intestinal resection Such treatment frequently results intwo major gastrointestinal complications after surgery anastomotic leakage and prolonged ileus Anastomotic leakage is a seriouscomplication which more often than not is diagnosed late to date C-reactive protein is the only available diagnostic marker Amonocentric prospective open case-control study was performed in patients (119899 = 117) undergoing colorectal surgery Intestinalfatty acid binding protein (i-FABP) citrulline D-lactate exhaled hydrogen Escherichia coli genomic DNA and ischemia modifiedalbumin (IMA) were determined preoperatively postoperatively and on the following four consecutive days Bacterial DNA wasnot detected in any sample and i-FABP and D-lactate lacked any distinct potential to detect postoperative bowel complicationsExhaled breath hydrogen content showed unacceptably low sensitivity However citrulline turned out to be a specific marker forprolonged ileus on postoperative days 3-4 Using a cut-off value of 20120583molL a sensitivity and specificity of sim75 was achievedon postoperative day 4 IMA was found to be an efficient predictor of anastomosis leak by calculating the difference betweenpreoperative and postoperative values This test had 100 sensitivity and 80 specificity and 100 negative and 20 positivepredictive value
1 Introduction
Colorectal cancer is the fourth most common cause ofdeath by cancer worldwide whose main form of treatmentconsists of intestinal resection Perioperative gastrointestinalcomplications or dysfunctions are frequent [1 2] In mostcases they are clinically suspected [3 4] to be the result ofsepsis ileus feeding intolerance diarrhea digestive bleedingor intestinal ischemia Evaluating the small bowel conditionmay in some instances be difficult for several reasons suchas its deep intra-abdominal location nonspecific symptomsand a lack of suitable blood plasma markers
Two major types of gastrointestinal complications arecommon after colorectal surgery anastomotic leakage(dehiscence) and prolonged ileus Of particular concernare the complications by anastomotic dehiscence occurring
mainly after rectal surgery It is often diagnosed late due toa low index of suspicion based on clinical and conventionallaboratory practice [5] often leading to the development ofseptic complications and reoperation Elevated C-reactiveprotein levels have been found as an ldquoearlyrdquo indicator ofanastomotic leakage from the second to fourth postoperativedays However it offers rather poor sensitivity and specific-ity (70ndash80) [6] and usually reflects an already-triggeredinflammatory reaction
As correctly stated by Steele et al [3] the major con-tributing factor to the viability and integrity of an anastomosisprocedure is adequate blood flow suggesting that anasto-mosis ischemia is probably the leading cause of dehiscenceRecently a new test for ischemia-modified albumin (IMA)detection was developed using copper binding assay [7]Thismodified assay offers several advantages over the older and
Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 428535 7 pageshttpdxdoiorg1011552015428535
2 Disease Markers
widely used cobalt binding assay [8] The copper bindingassay possesses improved sensitivity on the albuminrsquos N-terminal aminoacid residuesrsquo capacity for binding transitionmetal ions IMA has been tested as a potential marker ofbowel ischemia as detected by cobalt binding assay withsome level of efficiency [9 10] It is sensitive enough to detectsmall areas of bowel ischemia during hernia incarceration inanimal experiments [11]
Intestinal ischemia also causes epithelial destructionwhich has been associated with an elevated plasmatic con-centration of i-FABP (Intestinal Fatty Acid Binding Protein)a protein expressed in the cytosol of enterocytes and one ofthe proposed markers of enterocyte necrosis [12] The majordisadvantage of i-FABP as a highly organ specific protein isthe desquamation of enterocytes into the lumen when theyare damaged and the consequent limited absorption of theprotein into the bodyThis is probably the reason for the poorsensitivity of the analysis described in availablemeta-analyses[12]
Plasmatic D-lactate concentration has been proposed asa sensitive marker in detecting gut failure and endotoxemialikely due to an impaired intestinal barrier function [13]It is normally produced in the fermentative organs of thegastrointestinal tract (cecum colon) Several pathogenicbacteria produce D-lactate including Bacteroides fragilisEscherichia coli Klebsiella pneumoniae and Staphylococcusaureus Gut ischemia results in elevated D-lactate levels[13] a condition also found in peristaltic disorders suchas blind loop syndrome A similar test the quantization ofhydrogen found in exhaled breath as a marker of Klebsiellaand Staphylococcus overgrowth in the bowel lumen has alsobeen attempted [14]
Gastrointestinal dysfunction due to a prolonged ileushas been associated with a worse prognosis in postoperativepatients particularly when persisting formore than one week[15] According to the European Society for Clinical NutritionandMetabolism recommendations [16] prolonged postoper-ative ileus is a type I intestinal failure and of spontaneouslyresolving nature Currently enhanced recovery techniquesare available although a suitable laboratory marker is stilllacking
Citrulline is an 120572-amino acid synthesized mainly fromglutamine by small bowel enterocytes [17] Plasmatic cit-rulline concentration (normally within 20ndash40120583molL) isdetermined by the balance between gut citrulline synthesisand kidney citrulline degradation It has been previouslydemonstrated that plasmatic citrulline concentration is asimple and reliable biomarker of enterocyte mass in patientswith chronic small bowel pathologies It can be correlatedwith the severity and extent of villous atrophy in both patientswith normal small bowel length or villous atrophy associatedsmall bowel disease [18]
On a different subject bacterial translocation is definedas the escape of viable indigenous bacteria from the gastroin-testinal (GI) tract into the mesenteric lymph nodes liverspleen and bloodstream In this regard PCR analysis hasa higher sensitivity than blood or mesenteric lymph nodecultures in assessing bacterial translocation (most commonly
E coli) from the intestine during an early postabdominalsurgery stage [19]
The present study aims to develop and validate suitablereadily available and relatively noninvasive biomarkers forthe diagnosis and monitoring of postoperative gastrointesti-nal complications where early diagnosis is of utmost impor-tance Due to the unavailability or nonexistence of otherdiagnostic methods laboratory tests from blood samplesare the definitive candidates as diagnostic tools of smallbowel damage evaluation Ischemia-modified albumin waspreliminarily found by us as a very promising predictor ofanastomosis leakage already showing positive results in aslittle as two hours after the operation On the other handcitrulline could be used as a suitable monitoring marker ofprolonged postoperative ileus As this manuscript is beingwritten any of these findings have not yet been published
2 Patients and Methods
A monocentric prospective open case-control study wasperformed in patients undergoing colorectal surgery Thestudy focused primarily on the identification of plasmaticbiomarkers of bowel postoperative complications All proce-dures were approved by the Ethics Committee (Ref number201107S45P) according to the Declaration of Helsinki (June1964 and later amended) Eligible patients were properlyinformed of the studyrsquos aim and methods in both verbal andwritten form and a willing informed consent was obtained
The inclusion criteria encompassed elective large bowelsurgery for malignant disease involving one resection ofthe pathological bowel segment and one anastomosis andthe absence of any other primary bowel disease (such asCrohn disease or ulcerative colitis) severe nephropathy orhepatopathy The mean time duration of operation was of160 plusmn 65min and operational trauma did not vary signif-icantly within individual cases
The study protocol consisted of blood sample collection(8mL of peripheral blood into a BDVacutainer with dipotas-sium EDTA) preoperatively (day minus1) early postoperatively(120plusmn30min after the end of the operation day 0) and dailypostoperatively for four consecutive days (6-7 am days 1ndash4) Blood samples were afterwards analyzed by the methodsfurther described
Intestinal fatty acid binding protein (i-FABP)was analyzedusing the Human i-FABP ELISA kit (Hycult Biotech Nether-lands) according to the manufacturerrsquos instructions
Citrulline plasmatic concentration was analyzed usinghigh performance liquid chromatography with the additionof fluorescence detection (Shimadzu LC-10A ShimadzuJapan) after plasma ultrafiltration (Amicon 10 kDa MerckGermany) and derivatization using o-phthalyldialdehydeand mercaptopropionic acid [20] The fluorescence detectorwas set at excitationemission wavelengths of 235340 nmChromatographic separation was carried out in a MerckLichroCART 250x4 Lichrospher RP-18e 5-micron column
D-Lactate plasmatic concentration was determined by anin-house developed protocol [21] using deproteination byultrafiltration (Amicon 30 kDa Merck Germany) followedby the coupled reaction of D-lactate dehydrogenase to
Disease Markers 3
reduce nicotinamide adenine dinucleotide and D-glutamatepyruvate transaminase The reagents were obtained from aD-lactate assay kit manufactured byMegazyme International(Wicklow Ireland) The end-point concentration of nicoti-namide adenine dinucleotide was determined by measuringabsorbance at 340 nm
Hydrogen concentration from exhaled breath was evalu-ated using a Gastro+ analyzer (Bedfont Scientific Ltd UK)The instrument was calibrated and validated according tomanufacturerrsquos instructions using the original gas mixture
E coli genomic DNA content was analyzed in 1mLof plasma by QIAamp DNA Mini Kit (Qiagen HildenGermany) Primers were designed targeting the uidA gene(beta-glucuronidase gene specific for E coli) and amplifiedby nested PCR [22] Negative results were confirmed bytwo methods of quantitative DNA determination Firstlyreal-time PCR was carried out using primers targetingthe sfmD gene area coding ldquoa putative outer membraneexport usher proteinrdquo [23] Secondly in-house primers andprobe (TaqMan format) from E coli 16S ribosomal RNAgene (GenBank Accession number J018591) were designedas follows sense primer 51015840-GGTAGAATTCCAGGTGTA-31015840 antisense primer 51015840-GGGTATCTAATCCTGTTTG-31015840and probe FAM-TGAGCGTCAGTCTTCGTCCA-BHQ1TaqDNA inhibition was checked in all samples with negativeresults Increased PCR sensitivity by preamplification waschecked with no apparent influence on the results obtained
Plasmatic concentation of Ischemia modified albumin wasdetermined by copper binding assay as previously described[7] using a microplate reader Infinite 200 PRO (TecanSwitzerland) The protein fraction was purified from inter-fering low-molecular weight substances by ultrafiltration(Amicon 30 kDa Merck Germany) followed by double washwith phosphate buffered saline and then mixed with copper(II) chloride for 10 minutes The residual unbound copperwas determined by fluorescence quenching of lucifer yellowdye
3 Results
Operation trauma had a significant effect on all the param-eters tested Out of 117 enrolled patients (Table 1) compli-cation cases were recorded in 25 (214) patients (Table 2)Twelve complication cases (103) were directly related tothe patientsrsquo bowel with prolonged ileus and anastomosisleakage complications being predominant The complicationrate was found to be dependent upon tumor location Of thenumber of patients with colon tumor (119899 = 50) only 2 (1case) suffered from prolonged ileus and another 2 (1 case)suffered from anastomosis leak however the patients withrectal tumor (119899 = 67) showed a higher rate of postoperativecomplications 6 (4 cases) suffered fromprolonged ileus and6 (4 cases) from anastomosis leak
The results obtained from the analysis of potentialmarkers are shown with their statistical significance andcompared to baseline values in Table 3
Genomic DNA from Escherichia coli was not detectedin any sample despite great effort dedicated to improve thesensitivity of the analysis
Table 1 Study group description (number of cases are shown if nototherwise stated)
Characteristic Number of casesTotal number of patients 117Sex 67 m 50 fAge (years) 66 plusmn 95Body mass index (kgsdotmminus2) 269 plusmn 48ASA physical status classification 81 (II) 35 (III) 1 (IV)Localisation of tumor 50 colon 67 rectumOperation duration (min) 160 plusmn 65Hypertension comorbidity 71Diabetes comorbidity 34Coronary artery diseasecomorbidity 15
Obesity 29Neoadjuvant chemoradiotherapy 39Intensive care unit stay (days) 16 plusmn 14Hospital stay (days) 105 plusmn 67First postop gas passage (days) 18 plusmn 10First postop stool passage (days) 28 plusmn 16
The plasma concentration level of i-FABP was found tobe increased at early stages of the analysis (day 0) only todecrease significantly at a later postoperative period (day3-day 4) This marker was unable to discriminate betweenthe studied bowel complications despite it being an organspecific protein D-Lactate plasmatic concentration howeverwas found in significantly elevated levels throughout thestudied postoperative period and did not return to baselinevalues even after four days This marker though also lackedthe capacity to further distinguish between the studiedbowel complications Hydrogen content from exhaled breathanother analyte related to bacterial metabolism was alsofound in increased levels within the postoperative periodIts higher concentrations were correlated with an elevatedincidence of postoperative complications A preselected cut-off value of 10 ppm showed sensitivityspecificity rates of417886 for bowel complications and 357949 for
4 Disease Markers
Table 3 Laboratory markers
Test Day minus1 Day 0 Day 1 Day 2 Day 3 Day 4
Hydrogen (ppm) ND 1 (0 1)max 14
1 (075 2)max 12
1 (1 3)max 33lowast
1 (1 4)max 28lowast
1 (1 3)max 29lowast
i-FABP (pgmL) 417(232 748)
583lowastlowast(318 965)
462(292 762)
367lowast(162 563)
269lowastlowast(163 432)
284lowastlowast(143 519)
D-Lactate (120583molL) 334(263 397)
902lowastlowast(780 102)
112lowastlowast(791 139)
956lowastlowast(469 137)
483lowastlowast(326 738)
358lowastlowast(284 487)
Citrulline (120583molL) 340(281 437)
276lowastlowast(226 354)
202lowastlowast(153 251)
211lowastlowast(160 249)
228lowastlowast(185 293)
249lowastlowast(191 320)
IMA (F unitsg albumin) 799(653 892)
740(498 881) ND ND ND ND
lowast119901 lt 005 against baseline value lowastlowast119901 lt 0001 against baseline value
Data are presented as median (25th 75th percentile)ND not determined
Prolonged ileus not presentProlonged ileus present
Plas
mat
ic ci
trul
line (120583
mol
L)
0
10
20
30
40
50
minus1 1 2 3 40Postoperative time (days)
Figure 1 Time-profile of citrulline concentration in plasma
extrabowel complications An increased cut-off value of20 ppm resulted in sensitivity rates of 333 and 286 andspecificity of 981 and 98 respectively
Citrulline plasmatic levels were significantly decreasedduring the postoperative period reflecting a diminishedenterocyte metabolic activity Citrulline however turned outto be a specific marker for prolonged ileus on postoperativedays 3-4 (Figure 1) A cut-off value of 20120583molL achievedsensitivity and specificity rates of 75 and 76 respectivelyin the diagnosis of prolonged ileus on postoperative day 4(Figure 2) At first glance plasmatic IMAconcentration levelsdid not seem to deviate significantly from preoperative topostoperative periods within the analyzed patientsrsquo groupand due to its relatively short half-life we considered itunnecessary to further analyze its plasmatic level at laterstages of the postoperative period Regardless plasmaticIMA concentration difference values (postoperative minuspreoperative) were calculated seeking to eliminate the knownlarge biological scatter and surprisingly we found IMA
0
01
02
03
04
05
06
07
08
09
1
Sens
itivi
ty (t
rue p
ositi
ves)
01 02 03 04 05 06 07 08 09 10
1 minus specificity (false positives)
Citrulline 4IMA difference in all patientsIMA difference in rectal cancer
Figure 2 ROC curves of citrulline versus prolonged ileus (in allpatients) and IMAdifference versus anastomosis leak (in all patientsand in colorectal cancer patients)
to be an efficient predictor of anastomosis leak (Figure 3119901 = 0017) (ROC curves for IMA as a predictor ofanastomosis leak are presented in Figure 2) Assuming adifference of 270 fluorescent units per gram of albumin as acut-off value the test had 100 sensitivity and 80 specificitymeaning a 100 rate of negative predictive value and 20positive predictive value
4 Discussion
The results obtained in the present study demonstrate someof the existent pitfalls in the interpretation of bowel diseasedstates as given by the sole analysis of a biomarkersrsquo changepatterns The bowel specific markers i-FABP and D-lactatewhich are significantly increased in mesenteric ischemia
proved to be clinically useless in the prediction ormonitoringof postoperative complications Concerning i-FABP the lackof diagnostic efficiency could be attributed to an insufficientamount of enterocytes undergoing perioperative necrosisalthough the postoperative time-profile could also be con-sidered partly at fault since the significant decreased levelsof plasmatic i-FABP were probably caused by a diminishedenterocyte turnover during the selected time period for theanalysis
D-Lactate predominantly a byproduct of gut florametabolism was found in increased levels during the postop-erative period most likely due to unbalanced bowel micro-biota populations caused by prophylactic antibiotics andpreoperative bowel preparation Exhaled breath hydrogencontent another microbial byproduct exhibited a betteroutcome in the detection of perioperative complications fur-ther assessing a defined role for bowel microbiota metabolicbyproductsrsquo as a diagnostic tool of postoperative complica-tions Its apparent usefulness notwithstanding the proposedmethod suffers from too low sensitivity to be of any clinicalvalue
The most disappointing results were obtained from theE coli genomic DNA detection test Despite optimisticreferences [19] the protocol as employed by our group didnotmanage to detect any E coli genomicDNA in the patientsrsquoplasmaThe sensitivity of the test has been determined in sim10DNA copies per milliliter of plasma It is rather possible thatthe operational procedure for colorectal cancer according tolocal standards does not cause significant bacteremia
Of all the included and tested markers only two werefound of any clinical value citrulline and IMA Citrulline wasdefined as a suitable marker of postoperative prolonged ileusbecause its plasmatic concentration reflects the metabolicand peristaltic activity of the bowel Decreased basal valuesof plasmatic citrulline concentration were found in all theanalyzed patients reflecting a ldquophysiologicalrdquo postoperativeileus status Afterwards these concentration values wereincreased in the absence of prolonged postoperative ileusand remained low in its continued presence Therefore
we suggest that plasmatic citrulline concentration could beused clinically even without the knowledge of basal valuesIts reference interval ranges from 20 to 40120583molL with20120583molL as a suitable cut-off value for prolonged ileustesting Citrulline is usually analyzed as part of the amino acidspectrum by high performance liquid chromatography thusrepresenting a major drawback in its clinical use Citrullinecontent analysis is only available in large hospitals andsame-day laboratory response is hard to achieve if notunfeasible Also as prolonged ileus is commonly a self-resolving condition [16] there is little clinical demand fora molecular diagnostic biomarker Regardless citrulline stillmay be useful as a potential marker in research studiesdealing with preoperative or postoperative changes enablingthe quantitative estimation of prolonged postoperative ileusseverity
The major finding of this study was the clinical value ofIMA in diagnosing postoperative complications of colorectalsurgery Anastomosis leakage is a potentially disastrous con-dition which often leads to sepsis and postponed adjuvantchemotherapeutic treatment therefore compromising thesurvival rate of the patients C-reactive protein has beenpreviously identified as the optimal marker available yet itcan only be used until the fourth postoperative day with aset cut-off value of 150mgL [24] However it is a markerindicating an already ongoing inflammatory response thusan earliermarker is needfully sought after [2 3 24]Themajorrole of ischemia in anastomosis leak has been acknowledged[2] but a suitable early marker has not yet been identifiedCobalt-based IMA assays were used as early as the year 2000[8] and for approximately ten years after that as a marker ofcoronary events but it could not claim superiority over otherpreviously well-established cardiomarkers and its clinical usehas recently vanished Its characteristics as a sensitive andnonspecific organmarker along with an improved and quan-tifiable detection method by copper-binding assays make ituseful in the early diagnosis of perioperative bowel damageAs it is known from cobalt-based assays IMA suffers fromlarge interindividual variability which may be overcome bypre- and postoperative determination Also there is room forpotential overall improvement of the assay as well Copperbinding to albumin molecules is known to be pH dependent[25] The developed assay (CuBA [7]) omitted any bufferingdespite the tendency of serum pH towards the alkaline range(approx pH 8) due to CO
2loss ex vivo In alkaline solution
albumin binds Cu(2+) ions not only to N-terminal site butalso to the metal binding site [25] which would lead to ahigher biological variability in the test This concerning issuewas solved by implementing a double protein wash of plasmaproteins with phosphate buffered saline using ultrafiltration
The theoretical outcome of the CuBA test as currentlydeveloped is outstanding [7] though still in need of validationin human clinical samples and it could also benefit fromfurther analytical improvements Some additional advantagesof the test lie in its simplicity and low economical costNevertheless these significant results should be indepen-dently assayed and confirmed on a higher number of patientsfurther verifying sensitivity specificity negative and positivepredictive values and accuracy of the test
6 Disease Markers
5 Conclusions
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
widely used cobalt binding assay [8] The copper bindingassay possesses improved sensitivity on the albuminrsquos N-terminal aminoacid residuesrsquo capacity for binding transitionmetal ions IMA has been tested as a potential marker ofbowel ischemia as detected by cobalt binding assay withsome level of efficiency [9 10] It is sensitive enough to detectsmall areas of bowel ischemia during hernia incarceration inanimal experiments [11]
Intestinal ischemia also causes epithelial destructionwhich has been associated with an elevated plasmatic con-centration of i-FABP (Intestinal Fatty Acid Binding Protein)a protein expressed in the cytosol of enterocytes and one ofthe proposed markers of enterocyte necrosis [12] The majordisadvantage of i-FABP as a highly organ specific protein isthe desquamation of enterocytes into the lumen when theyare damaged and the consequent limited absorption of theprotein into the bodyThis is probably the reason for the poorsensitivity of the analysis described in availablemeta-analyses[12]
Plasmatic D-lactate concentration has been proposed asa sensitive marker in detecting gut failure and endotoxemialikely due to an impaired intestinal barrier function [13]It is normally produced in the fermentative organs of thegastrointestinal tract (cecum colon) Several pathogenicbacteria produce D-lactate including Bacteroides fragilisEscherichia coli Klebsiella pneumoniae and Staphylococcusaureus Gut ischemia results in elevated D-lactate levels[13] a condition also found in peristaltic disorders suchas blind loop syndrome A similar test the quantization ofhydrogen found in exhaled breath as a marker of Klebsiellaand Staphylococcus overgrowth in the bowel lumen has alsobeen attempted [14]
Gastrointestinal dysfunction due to a prolonged ileushas been associated with a worse prognosis in postoperativepatients particularly when persisting formore than one week[15] According to the European Society for Clinical NutritionandMetabolism recommendations [16] prolonged postoper-ative ileus is a type I intestinal failure and of spontaneouslyresolving nature Currently enhanced recovery techniquesare available although a suitable laboratory marker is stilllacking
Citrulline is an 120572-amino acid synthesized mainly fromglutamine by small bowel enterocytes [17] Plasmatic cit-rulline concentration (normally within 20ndash40120583molL) isdetermined by the balance between gut citrulline synthesisand kidney citrulline degradation It has been previouslydemonstrated that plasmatic citrulline concentration is asimple and reliable biomarker of enterocyte mass in patientswith chronic small bowel pathologies It can be correlatedwith the severity and extent of villous atrophy in both patientswith normal small bowel length or villous atrophy associatedsmall bowel disease [18]
On a different subject bacterial translocation is definedas the escape of viable indigenous bacteria from the gastroin-testinal (GI) tract into the mesenteric lymph nodes liverspleen and bloodstream In this regard PCR analysis hasa higher sensitivity than blood or mesenteric lymph nodecultures in assessing bacterial translocation (most commonly
E coli) from the intestine during an early postabdominalsurgery stage [19]
The present study aims to develop and validate suitablereadily available and relatively noninvasive biomarkers forthe diagnosis and monitoring of postoperative gastrointesti-nal complications where early diagnosis is of utmost impor-tance Due to the unavailability or nonexistence of otherdiagnostic methods laboratory tests from blood samplesare the definitive candidates as diagnostic tools of smallbowel damage evaluation Ischemia-modified albumin waspreliminarily found by us as a very promising predictor ofanastomosis leakage already showing positive results in aslittle as two hours after the operation On the other handcitrulline could be used as a suitable monitoring marker ofprolonged postoperative ileus As this manuscript is beingwritten any of these findings have not yet been published
2 Patients and Methods
A monocentric prospective open case-control study wasperformed in patients undergoing colorectal surgery Thestudy focused primarily on the identification of plasmaticbiomarkers of bowel postoperative complications All proce-dures were approved by the Ethics Committee (Ref number201107S45P) according to the Declaration of Helsinki (June1964 and later amended) Eligible patients were properlyinformed of the studyrsquos aim and methods in both verbal andwritten form and a willing informed consent was obtained
The inclusion criteria encompassed elective large bowelsurgery for malignant disease involving one resection ofthe pathological bowel segment and one anastomosis andthe absence of any other primary bowel disease (such asCrohn disease or ulcerative colitis) severe nephropathy orhepatopathy The mean time duration of operation was of160 plusmn 65min and operational trauma did not vary signif-icantly within individual cases
The study protocol consisted of blood sample collection(8mL of peripheral blood into a BDVacutainer with dipotas-sium EDTA) preoperatively (day minus1) early postoperatively(120plusmn30min after the end of the operation day 0) and dailypostoperatively for four consecutive days (6-7 am days 1ndash4) Blood samples were afterwards analyzed by the methodsfurther described
Intestinal fatty acid binding protein (i-FABP)was analyzedusing the Human i-FABP ELISA kit (Hycult Biotech Nether-lands) according to the manufacturerrsquos instructions
Citrulline plasmatic concentration was analyzed usinghigh performance liquid chromatography with the additionof fluorescence detection (Shimadzu LC-10A ShimadzuJapan) after plasma ultrafiltration (Amicon 10 kDa MerckGermany) and derivatization using o-phthalyldialdehydeand mercaptopropionic acid [20] The fluorescence detectorwas set at excitationemission wavelengths of 235340 nmChromatographic separation was carried out in a MerckLichroCART 250x4 Lichrospher RP-18e 5-micron column
D-Lactate plasmatic concentration was determined by anin-house developed protocol [21] using deproteination byultrafiltration (Amicon 30 kDa Merck Germany) followedby the coupled reaction of D-lactate dehydrogenase to
Disease Markers 3
reduce nicotinamide adenine dinucleotide and D-glutamatepyruvate transaminase The reagents were obtained from aD-lactate assay kit manufactured byMegazyme International(Wicklow Ireland) The end-point concentration of nicoti-namide adenine dinucleotide was determined by measuringabsorbance at 340 nm
Hydrogen concentration from exhaled breath was evalu-ated using a Gastro+ analyzer (Bedfont Scientific Ltd UK)The instrument was calibrated and validated according tomanufacturerrsquos instructions using the original gas mixture
E coli genomic DNA content was analyzed in 1mLof plasma by QIAamp DNA Mini Kit (Qiagen HildenGermany) Primers were designed targeting the uidA gene(beta-glucuronidase gene specific for E coli) and amplifiedby nested PCR [22] Negative results were confirmed bytwo methods of quantitative DNA determination Firstlyreal-time PCR was carried out using primers targetingthe sfmD gene area coding ldquoa putative outer membraneexport usher proteinrdquo [23] Secondly in-house primers andprobe (TaqMan format) from E coli 16S ribosomal RNAgene (GenBank Accession number J018591) were designedas follows sense primer 51015840-GGTAGAATTCCAGGTGTA-31015840 antisense primer 51015840-GGGTATCTAATCCTGTTTG-31015840and probe FAM-TGAGCGTCAGTCTTCGTCCA-BHQ1TaqDNA inhibition was checked in all samples with negativeresults Increased PCR sensitivity by preamplification waschecked with no apparent influence on the results obtained
Plasmatic concentation of Ischemia modified albumin wasdetermined by copper binding assay as previously described[7] using a microplate reader Infinite 200 PRO (TecanSwitzerland) The protein fraction was purified from inter-fering low-molecular weight substances by ultrafiltration(Amicon 30 kDa Merck Germany) followed by double washwith phosphate buffered saline and then mixed with copper(II) chloride for 10 minutes The residual unbound copperwas determined by fluorescence quenching of lucifer yellowdye
3 Results
Operation trauma had a significant effect on all the param-eters tested Out of 117 enrolled patients (Table 1) compli-cation cases were recorded in 25 (214) patients (Table 2)Twelve complication cases (103) were directly related tothe patientsrsquo bowel with prolonged ileus and anastomosisleakage complications being predominant The complicationrate was found to be dependent upon tumor location Of thenumber of patients with colon tumor (119899 = 50) only 2 (1case) suffered from prolonged ileus and another 2 (1 case)suffered from anastomosis leak however the patients withrectal tumor (119899 = 67) showed a higher rate of postoperativecomplications 6 (4 cases) suffered fromprolonged ileus and6 (4 cases) from anastomosis leak
The results obtained from the analysis of potentialmarkers are shown with their statistical significance andcompared to baseline values in Table 3
Genomic DNA from Escherichia coli was not detectedin any sample despite great effort dedicated to improve thesensitivity of the analysis
Table 1 Study group description (number of cases are shown if nototherwise stated)
Characteristic Number of casesTotal number of patients 117Sex 67 m 50 fAge (years) 66 plusmn 95Body mass index (kgsdotmminus2) 269 plusmn 48ASA physical status classification 81 (II) 35 (III) 1 (IV)Localisation of tumor 50 colon 67 rectumOperation duration (min) 160 plusmn 65Hypertension comorbidity 71Diabetes comorbidity 34Coronary artery diseasecomorbidity 15
Obesity 29Neoadjuvant chemoradiotherapy 39Intensive care unit stay (days) 16 plusmn 14Hospital stay (days) 105 plusmn 67First postop gas passage (days) 18 plusmn 10First postop stool passage (days) 28 plusmn 16
The plasma concentration level of i-FABP was found tobe increased at early stages of the analysis (day 0) only todecrease significantly at a later postoperative period (day3-day 4) This marker was unable to discriminate betweenthe studied bowel complications despite it being an organspecific protein D-Lactate plasmatic concentration howeverwas found in significantly elevated levels throughout thestudied postoperative period and did not return to baselinevalues even after four days This marker though also lackedthe capacity to further distinguish between the studiedbowel complications Hydrogen content from exhaled breathanother analyte related to bacterial metabolism was alsofound in increased levels within the postoperative periodIts higher concentrations were correlated with an elevatedincidence of postoperative complications A preselected cut-off value of 10 ppm showed sensitivityspecificity rates of417886 for bowel complications and 357949 for
4 Disease Markers
Table 3 Laboratory markers
Test Day minus1 Day 0 Day 1 Day 2 Day 3 Day 4
Hydrogen (ppm) ND 1 (0 1)max 14
1 (075 2)max 12
1 (1 3)max 33lowast
1 (1 4)max 28lowast
1 (1 3)max 29lowast
i-FABP (pgmL) 417(232 748)
583lowastlowast(318 965)
462(292 762)
367lowast(162 563)
269lowastlowast(163 432)
284lowastlowast(143 519)
D-Lactate (120583molL) 334(263 397)
902lowastlowast(780 102)
112lowastlowast(791 139)
956lowastlowast(469 137)
483lowastlowast(326 738)
358lowastlowast(284 487)
Citrulline (120583molL) 340(281 437)
276lowastlowast(226 354)
202lowastlowast(153 251)
211lowastlowast(160 249)
228lowastlowast(185 293)
249lowastlowast(191 320)
IMA (F unitsg albumin) 799(653 892)
740(498 881) ND ND ND ND
lowast119901 lt 005 against baseline value lowastlowast119901 lt 0001 against baseline value
Data are presented as median (25th 75th percentile)ND not determined
Prolonged ileus not presentProlonged ileus present
Plas
mat
ic ci
trul
line (120583
mol
L)
0
10
20
30
40
50
minus1 1 2 3 40Postoperative time (days)
Figure 1 Time-profile of citrulline concentration in plasma
extrabowel complications An increased cut-off value of20 ppm resulted in sensitivity rates of 333 and 286 andspecificity of 981 and 98 respectively
Citrulline plasmatic levels were significantly decreasedduring the postoperative period reflecting a diminishedenterocyte metabolic activity Citrulline however turned outto be a specific marker for prolonged ileus on postoperativedays 3-4 (Figure 1) A cut-off value of 20120583molL achievedsensitivity and specificity rates of 75 and 76 respectivelyin the diagnosis of prolonged ileus on postoperative day 4(Figure 2) At first glance plasmatic IMAconcentration levelsdid not seem to deviate significantly from preoperative topostoperative periods within the analyzed patientsrsquo groupand due to its relatively short half-life we considered itunnecessary to further analyze its plasmatic level at laterstages of the postoperative period Regardless plasmaticIMA concentration difference values (postoperative minuspreoperative) were calculated seeking to eliminate the knownlarge biological scatter and surprisingly we found IMA
0
01
02
03
04
05
06
07
08
09
1
Sens
itivi
ty (t
rue p
ositi
ves)
01 02 03 04 05 06 07 08 09 10
1 minus specificity (false positives)
Citrulline 4IMA difference in all patientsIMA difference in rectal cancer
Figure 2 ROC curves of citrulline versus prolonged ileus (in allpatients) and IMAdifference versus anastomosis leak (in all patientsand in colorectal cancer patients)
to be an efficient predictor of anastomosis leak (Figure 3119901 = 0017) (ROC curves for IMA as a predictor ofanastomosis leak are presented in Figure 2) Assuming adifference of 270 fluorescent units per gram of albumin as acut-off value the test had 100 sensitivity and 80 specificitymeaning a 100 rate of negative predictive value and 20positive predictive value
4 Discussion
The results obtained in the present study demonstrate someof the existent pitfalls in the interpretation of bowel diseasedstates as given by the sole analysis of a biomarkersrsquo changepatterns The bowel specific markers i-FABP and D-lactatewhich are significantly increased in mesenteric ischemia
proved to be clinically useless in the prediction ormonitoringof postoperative complications Concerning i-FABP the lackof diagnostic efficiency could be attributed to an insufficientamount of enterocytes undergoing perioperative necrosisalthough the postoperative time-profile could also be con-sidered partly at fault since the significant decreased levelsof plasmatic i-FABP were probably caused by a diminishedenterocyte turnover during the selected time period for theanalysis
D-Lactate predominantly a byproduct of gut florametabolism was found in increased levels during the postop-erative period most likely due to unbalanced bowel micro-biota populations caused by prophylactic antibiotics andpreoperative bowel preparation Exhaled breath hydrogencontent another microbial byproduct exhibited a betteroutcome in the detection of perioperative complications fur-ther assessing a defined role for bowel microbiota metabolicbyproductsrsquo as a diagnostic tool of postoperative complica-tions Its apparent usefulness notwithstanding the proposedmethod suffers from too low sensitivity to be of any clinicalvalue
The most disappointing results were obtained from theE coli genomic DNA detection test Despite optimisticreferences [19] the protocol as employed by our group didnotmanage to detect any E coli genomicDNA in the patientsrsquoplasmaThe sensitivity of the test has been determined in sim10DNA copies per milliliter of plasma It is rather possible thatthe operational procedure for colorectal cancer according tolocal standards does not cause significant bacteremia
Of all the included and tested markers only two werefound of any clinical value citrulline and IMA Citrulline wasdefined as a suitable marker of postoperative prolonged ileusbecause its plasmatic concentration reflects the metabolicand peristaltic activity of the bowel Decreased basal valuesof plasmatic citrulline concentration were found in all theanalyzed patients reflecting a ldquophysiologicalrdquo postoperativeileus status Afterwards these concentration values wereincreased in the absence of prolonged postoperative ileusand remained low in its continued presence Therefore
we suggest that plasmatic citrulline concentration could beused clinically even without the knowledge of basal valuesIts reference interval ranges from 20 to 40120583molL with20120583molL as a suitable cut-off value for prolonged ileustesting Citrulline is usually analyzed as part of the amino acidspectrum by high performance liquid chromatography thusrepresenting a major drawback in its clinical use Citrullinecontent analysis is only available in large hospitals andsame-day laboratory response is hard to achieve if notunfeasible Also as prolonged ileus is commonly a self-resolving condition [16] there is little clinical demand fora molecular diagnostic biomarker Regardless citrulline stillmay be useful as a potential marker in research studiesdealing with preoperative or postoperative changes enablingthe quantitative estimation of prolonged postoperative ileusseverity
The major finding of this study was the clinical value ofIMA in diagnosing postoperative complications of colorectalsurgery Anastomosis leakage is a potentially disastrous con-dition which often leads to sepsis and postponed adjuvantchemotherapeutic treatment therefore compromising thesurvival rate of the patients C-reactive protein has beenpreviously identified as the optimal marker available yet itcan only be used until the fourth postoperative day with aset cut-off value of 150mgL [24] However it is a markerindicating an already ongoing inflammatory response thusan earliermarker is needfully sought after [2 3 24]Themajorrole of ischemia in anastomosis leak has been acknowledged[2] but a suitable early marker has not yet been identifiedCobalt-based IMA assays were used as early as the year 2000[8] and for approximately ten years after that as a marker ofcoronary events but it could not claim superiority over otherpreviously well-established cardiomarkers and its clinical usehas recently vanished Its characteristics as a sensitive andnonspecific organmarker along with an improved and quan-tifiable detection method by copper-binding assays make ituseful in the early diagnosis of perioperative bowel damageAs it is known from cobalt-based assays IMA suffers fromlarge interindividual variability which may be overcome bypre- and postoperative determination Also there is room forpotential overall improvement of the assay as well Copperbinding to albumin molecules is known to be pH dependent[25] The developed assay (CuBA [7]) omitted any bufferingdespite the tendency of serum pH towards the alkaline range(approx pH 8) due to CO
2loss ex vivo In alkaline solution
albumin binds Cu(2+) ions not only to N-terminal site butalso to the metal binding site [25] which would lead to ahigher biological variability in the test This concerning issuewas solved by implementing a double protein wash of plasmaproteins with phosphate buffered saline using ultrafiltration
The theoretical outcome of the CuBA test as currentlydeveloped is outstanding [7] though still in need of validationin human clinical samples and it could also benefit fromfurther analytical improvements Some additional advantagesof the test lie in its simplicity and low economical costNevertheless these significant results should be indepen-dently assayed and confirmed on a higher number of patientsfurther verifying sensitivity specificity negative and positivepredictive values and accuracy of the test
6 Disease Markers
5 Conclusions
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
reduce nicotinamide adenine dinucleotide and D-glutamatepyruvate transaminase The reagents were obtained from aD-lactate assay kit manufactured byMegazyme International(Wicklow Ireland) The end-point concentration of nicoti-namide adenine dinucleotide was determined by measuringabsorbance at 340 nm
Hydrogen concentration from exhaled breath was evalu-ated using a Gastro+ analyzer (Bedfont Scientific Ltd UK)The instrument was calibrated and validated according tomanufacturerrsquos instructions using the original gas mixture
E coli genomic DNA content was analyzed in 1mLof plasma by QIAamp DNA Mini Kit (Qiagen HildenGermany) Primers were designed targeting the uidA gene(beta-glucuronidase gene specific for E coli) and amplifiedby nested PCR [22] Negative results were confirmed bytwo methods of quantitative DNA determination Firstlyreal-time PCR was carried out using primers targetingthe sfmD gene area coding ldquoa putative outer membraneexport usher proteinrdquo [23] Secondly in-house primers andprobe (TaqMan format) from E coli 16S ribosomal RNAgene (GenBank Accession number J018591) were designedas follows sense primer 51015840-GGTAGAATTCCAGGTGTA-31015840 antisense primer 51015840-GGGTATCTAATCCTGTTTG-31015840and probe FAM-TGAGCGTCAGTCTTCGTCCA-BHQ1TaqDNA inhibition was checked in all samples with negativeresults Increased PCR sensitivity by preamplification waschecked with no apparent influence on the results obtained
Plasmatic concentation of Ischemia modified albumin wasdetermined by copper binding assay as previously described[7] using a microplate reader Infinite 200 PRO (TecanSwitzerland) The protein fraction was purified from inter-fering low-molecular weight substances by ultrafiltration(Amicon 30 kDa Merck Germany) followed by double washwith phosphate buffered saline and then mixed with copper(II) chloride for 10 minutes The residual unbound copperwas determined by fluorescence quenching of lucifer yellowdye
3 Results
Operation trauma had a significant effect on all the param-eters tested Out of 117 enrolled patients (Table 1) compli-cation cases were recorded in 25 (214) patients (Table 2)Twelve complication cases (103) were directly related tothe patientsrsquo bowel with prolonged ileus and anastomosisleakage complications being predominant The complicationrate was found to be dependent upon tumor location Of thenumber of patients with colon tumor (119899 = 50) only 2 (1case) suffered from prolonged ileus and another 2 (1 case)suffered from anastomosis leak however the patients withrectal tumor (119899 = 67) showed a higher rate of postoperativecomplications 6 (4 cases) suffered fromprolonged ileus and6 (4 cases) from anastomosis leak
The results obtained from the analysis of potentialmarkers are shown with their statistical significance andcompared to baseline values in Table 3
Genomic DNA from Escherichia coli was not detectedin any sample despite great effort dedicated to improve thesensitivity of the analysis
Table 1 Study group description (number of cases are shown if nototherwise stated)
Characteristic Number of casesTotal number of patients 117Sex 67 m 50 fAge (years) 66 plusmn 95Body mass index (kgsdotmminus2) 269 plusmn 48ASA physical status classification 81 (II) 35 (III) 1 (IV)Localisation of tumor 50 colon 67 rectumOperation duration (min) 160 plusmn 65Hypertension comorbidity 71Diabetes comorbidity 34Coronary artery diseasecomorbidity 15
Obesity 29Neoadjuvant chemoradiotherapy 39Intensive care unit stay (days) 16 plusmn 14Hospital stay (days) 105 plusmn 67First postop gas passage (days) 18 plusmn 10First postop stool passage (days) 28 plusmn 16
The plasma concentration level of i-FABP was found tobe increased at early stages of the analysis (day 0) only todecrease significantly at a later postoperative period (day3-day 4) This marker was unable to discriminate betweenthe studied bowel complications despite it being an organspecific protein D-Lactate plasmatic concentration howeverwas found in significantly elevated levels throughout thestudied postoperative period and did not return to baselinevalues even after four days This marker though also lackedthe capacity to further distinguish between the studiedbowel complications Hydrogen content from exhaled breathanother analyte related to bacterial metabolism was alsofound in increased levels within the postoperative periodIts higher concentrations were correlated with an elevatedincidence of postoperative complications A preselected cut-off value of 10 ppm showed sensitivityspecificity rates of417886 for bowel complications and 357949 for
4 Disease Markers
Table 3 Laboratory markers
Test Day minus1 Day 0 Day 1 Day 2 Day 3 Day 4
Hydrogen (ppm) ND 1 (0 1)max 14
1 (075 2)max 12
1 (1 3)max 33lowast
1 (1 4)max 28lowast
1 (1 3)max 29lowast
i-FABP (pgmL) 417(232 748)
583lowastlowast(318 965)
462(292 762)
367lowast(162 563)
269lowastlowast(163 432)
284lowastlowast(143 519)
D-Lactate (120583molL) 334(263 397)
902lowastlowast(780 102)
112lowastlowast(791 139)
956lowastlowast(469 137)
483lowastlowast(326 738)
358lowastlowast(284 487)
Citrulline (120583molL) 340(281 437)
276lowastlowast(226 354)
202lowastlowast(153 251)
211lowastlowast(160 249)
228lowastlowast(185 293)
249lowastlowast(191 320)
IMA (F unitsg albumin) 799(653 892)
740(498 881) ND ND ND ND
lowast119901 lt 005 against baseline value lowastlowast119901 lt 0001 against baseline value
Data are presented as median (25th 75th percentile)ND not determined
Prolonged ileus not presentProlonged ileus present
Plas
mat
ic ci
trul
line (120583
mol
L)
0
10
20
30
40
50
minus1 1 2 3 40Postoperative time (days)
Figure 1 Time-profile of citrulline concentration in plasma
extrabowel complications An increased cut-off value of20 ppm resulted in sensitivity rates of 333 and 286 andspecificity of 981 and 98 respectively
Citrulline plasmatic levels were significantly decreasedduring the postoperative period reflecting a diminishedenterocyte metabolic activity Citrulline however turned outto be a specific marker for prolonged ileus on postoperativedays 3-4 (Figure 1) A cut-off value of 20120583molL achievedsensitivity and specificity rates of 75 and 76 respectivelyin the diagnosis of prolonged ileus on postoperative day 4(Figure 2) At first glance plasmatic IMAconcentration levelsdid not seem to deviate significantly from preoperative topostoperative periods within the analyzed patientsrsquo groupand due to its relatively short half-life we considered itunnecessary to further analyze its plasmatic level at laterstages of the postoperative period Regardless plasmaticIMA concentration difference values (postoperative minuspreoperative) were calculated seeking to eliminate the knownlarge biological scatter and surprisingly we found IMA
0
01
02
03
04
05
06
07
08
09
1
Sens
itivi
ty (t
rue p
ositi
ves)
01 02 03 04 05 06 07 08 09 10
1 minus specificity (false positives)
Citrulline 4IMA difference in all patientsIMA difference in rectal cancer
Figure 2 ROC curves of citrulline versus prolonged ileus (in allpatients) and IMAdifference versus anastomosis leak (in all patientsand in colorectal cancer patients)
to be an efficient predictor of anastomosis leak (Figure 3119901 = 0017) (ROC curves for IMA as a predictor ofanastomosis leak are presented in Figure 2) Assuming adifference of 270 fluorescent units per gram of albumin as acut-off value the test had 100 sensitivity and 80 specificitymeaning a 100 rate of negative predictive value and 20positive predictive value
4 Discussion
The results obtained in the present study demonstrate someof the existent pitfalls in the interpretation of bowel diseasedstates as given by the sole analysis of a biomarkersrsquo changepatterns The bowel specific markers i-FABP and D-lactatewhich are significantly increased in mesenteric ischemia
proved to be clinically useless in the prediction ormonitoringof postoperative complications Concerning i-FABP the lackof diagnostic efficiency could be attributed to an insufficientamount of enterocytes undergoing perioperative necrosisalthough the postoperative time-profile could also be con-sidered partly at fault since the significant decreased levelsof plasmatic i-FABP were probably caused by a diminishedenterocyte turnover during the selected time period for theanalysis
D-Lactate predominantly a byproduct of gut florametabolism was found in increased levels during the postop-erative period most likely due to unbalanced bowel micro-biota populations caused by prophylactic antibiotics andpreoperative bowel preparation Exhaled breath hydrogencontent another microbial byproduct exhibited a betteroutcome in the detection of perioperative complications fur-ther assessing a defined role for bowel microbiota metabolicbyproductsrsquo as a diagnostic tool of postoperative complica-tions Its apparent usefulness notwithstanding the proposedmethod suffers from too low sensitivity to be of any clinicalvalue
The most disappointing results were obtained from theE coli genomic DNA detection test Despite optimisticreferences [19] the protocol as employed by our group didnotmanage to detect any E coli genomicDNA in the patientsrsquoplasmaThe sensitivity of the test has been determined in sim10DNA copies per milliliter of plasma It is rather possible thatthe operational procedure for colorectal cancer according tolocal standards does not cause significant bacteremia
Of all the included and tested markers only two werefound of any clinical value citrulline and IMA Citrulline wasdefined as a suitable marker of postoperative prolonged ileusbecause its plasmatic concentration reflects the metabolicand peristaltic activity of the bowel Decreased basal valuesof plasmatic citrulline concentration were found in all theanalyzed patients reflecting a ldquophysiologicalrdquo postoperativeileus status Afterwards these concentration values wereincreased in the absence of prolonged postoperative ileusand remained low in its continued presence Therefore
we suggest that plasmatic citrulline concentration could beused clinically even without the knowledge of basal valuesIts reference interval ranges from 20 to 40120583molL with20120583molL as a suitable cut-off value for prolonged ileustesting Citrulline is usually analyzed as part of the amino acidspectrum by high performance liquid chromatography thusrepresenting a major drawback in its clinical use Citrullinecontent analysis is only available in large hospitals andsame-day laboratory response is hard to achieve if notunfeasible Also as prolonged ileus is commonly a self-resolving condition [16] there is little clinical demand fora molecular diagnostic biomarker Regardless citrulline stillmay be useful as a potential marker in research studiesdealing with preoperative or postoperative changes enablingthe quantitative estimation of prolonged postoperative ileusseverity
The major finding of this study was the clinical value ofIMA in diagnosing postoperative complications of colorectalsurgery Anastomosis leakage is a potentially disastrous con-dition which often leads to sepsis and postponed adjuvantchemotherapeutic treatment therefore compromising thesurvival rate of the patients C-reactive protein has beenpreviously identified as the optimal marker available yet itcan only be used until the fourth postoperative day with aset cut-off value of 150mgL [24] However it is a markerindicating an already ongoing inflammatory response thusan earliermarker is needfully sought after [2 3 24]Themajorrole of ischemia in anastomosis leak has been acknowledged[2] but a suitable early marker has not yet been identifiedCobalt-based IMA assays were used as early as the year 2000[8] and for approximately ten years after that as a marker ofcoronary events but it could not claim superiority over otherpreviously well-established cardiomarkers and its clinical usehas recently vanished Its characteristics as a sensitive andnonspecific organmarker along with an improved and quan-tifiable detection method by copper-binding assays make ituseful in the early diagnosis of perioperative bowel damageAs it is known from cobalt-based assays IMA suffers fromlarge interindividual variability which may be overcome bypre- and postoperative determination Also there is room forpotential overall improvement of the assay as well Copperbinding to albumin molecules is known to be pH dependent[25] The developed assay (CuBA [7]) omitted any bufferingdespite the tendency of serum pH towards the alkaline range(approx pH 8) due to CO
2loss ex vivo In alkaline solution
albumin binds Cu(2+) ions not only to N-terminal site butalso to the metal binding site [25] which would lead to ahigher biological variability in the test This concerning issuewas solved by implementing a double protein wash of plasmaproteins with phosphate buffered saline using ultrafiltration
The theoretical outcome of the CuBA test as currentlydeveloped is outstanding [7] though still in need of validationin human clinical samples and it could also benefit fromfurther analytical improvements Some additional advantagesof the test lie in its simplicity and low economical costNevertheless these significant results should be indepen-dently assayed and confirmed on a higher number of patientsfurther verifying sensitivity specificity negative and positivepredictive values and accuracy of the test
6 Disease Markers
5 Conclusions
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
lowast119901 lt 005 against baseline value lowastlowast119901 lt 0001 against baseline value
Data are presented as median (25th 75th percentile)ND not determined
Prolonged ileus not presentProlonged ileus present
Plas
mat
ic ci
trul
line (120583
mol
L)
0
10
20
30
40
50
minus1 1 2 3 40Postoperative time (days)
Figure 1 Time-profile of citrulline concentration in plasma
extrabowel complications An increased cut-off value of20 ppm resulted in sensitivity rates of 333 and 286 andspecificity of 981 and 98 respectively
Citrulline plasmatic levels were significantly decreasedduring the postoperative period reflecting a diminishedenterocyte metabolic activity Citrulline however turned outto be a specific marker for prolonged ileus on postoperativedays 3-4 (Figure 1) A cut-off value of 20120583molL achievedsensitivity and specificity rates of 75 and 76 respectivelyin the diagnosis of prolonged ileus on postoperative day 4(Figure 2) At first glance plasmatic IMAconcentration levelsdid not seem to deviate significantly from preoperative topostoperative periods within the analyzed patientsrsquo groupand due to its relatively short half-life we considered itunnecessary to further analyze its plasmatic level at laterstages of the postoperative period Regardless plasmaticIMA concentration difference values (postoperative minuspreoperative) were calculated seeking to eliminate the knownlarge biological scatter and surprisingly we found IMA
0
01
02
03
04
05
06
07
08
09
1
Sens
itivi
ty (t
rue p
ositi
ves)
01 02 03 04 05 06 07 08 09 10
1 minus specificity (false positives)
Citrulline 4IMA difference in all patientsIMA difference in rectal cancer
Figure 2 ROC curves of citrulline versus prolonged ileus (in allpatients) and IMAdifference versus anastomosis leak (in all patientsand in colorectal cancer patients)
to be an efficient predictor of anastomosis leak (Figure 3119901 = 0017) (ROC curves for IMA as a predictor ofanastomosis leak are presented in Figure 2) Assuming adifference of 270 fluorescent units per gram of albumin as acut-off value the test had 100 sensitivity and 80 specificitymeaning a 100 rate of negative predictive value and 20positive predictive value
4 Discussion
The results obtained in the present study demonstrate someof the existent pitfalls in the interpretation of bowel diseasedstates as given by the sole analysis of a biomarkersrsquo changepatterns The bowel specific markers i-FABP and D-lactatewhich are significantly increased in mesenteric ischemia
proved to be clinically useless in the prediction ormonitoringof postoperative complications Concerning i-FABP the lackof diagnostic efficiency could be attributed to an insufficientamount of enterocytes undergoing perioperative necrosisalthough the postoperative time-profile could also be con-sidered partly at fault since the significant decreased levelsof plasmatic i-FABP were probably caused by a diminishedenterocyte turnover during the selected time period for theanalysis
D-Lactate predominantly a byproduct of gut florametabolism was found in increased levels during the postop-erative period most likely due to unbalanced bowel micro-biota populations caused by prophylactic antibiotics andpreoperative bowel preparation Exhaled breath hydrogencontent another microbial byproduct exhibited a betteroutcome in the detection of perioperative complications fur-ther assessing a defined role for bowel microbiota metabolicbyproductsrsquo as a diagnostic tool of postoperative complica-tions Its apparent usefulness notwithstanding the proposedmethod suffers from too low sensitivity to be of any clinicalvalue
The most disappointing results were obtained from theE coli genomic DNA detection test Despite optimisticreferences [19] the protocol as employed by our group didnotmanage to detect any E coli genomicDNA in the patientsrsquoplasmaThe sensitivity of the test has been determined in sim10DNA copies per milliliter of plasma It is rather possible thatthe operational procedure for colorectal cancer according tolocal standards does not cause significant bacteremia
Of all the included and tested markers only two werefound of any clinical value citrulline and IMA Citrulline wasdefined as a suitable marker of postoperative prolonged ileusbecause its plasmatic concentration reflects the metabolicand peristaltic activity of the bowel Decreased basal valuesof plasmatic citrulline concentration were found in all theanalyzed patients reflecting a ldquophysiologicalrdquo postoperativeileus status Afterwards these concentration values wereincreased in the absence of prolonged postoperative ileusand remained low in its continued presence Therefore
we suggest that plasmatic citrulline concentration could beused clinically even without the knowledge of basal valuesIts reference interval ranges from 20 to 40120583molL with20120583molL as a suitable cut-off value for prolonged ileustesting Citrulline is usually analyzed as part of the amino acidspectrum by high performance liquid chromatography thusrepresenting a major drawback in its clinical use Citrullinecontent analysis is only available in large hospitals andsame-day laboratory response is hard to achieve if notunfeasible Also as prolonged ileus is commonly a self-resolving condition [16] there is little clinical demand fora molecular diagnostic biomarker Regardless citrulline stillmay be useful as a potential marker in research studiesdealing with preoperative or postoperative changes enablingthe quantitative estimation of prolonged postoperative ileusseverity
The major finding of this study was the clinical value ofIMA in diagnosing postoperative complications of colorectalsurgery Anastomosis leakage is a potentially disastrous con-dition which often leads to sepsis and postponed adjuvantchemotherapeutic treatment therefore compromising thesurvival rate of the patients C-reactive protein has beenpreviously identified as the optimal marker available yet itcan only be used until the fourth postoperative day with aset cut-off value of 150mgL [24] However it is a markerindicating an already ongoing inflammatory response thusan earliermarker is needfully sought after [2 3 24]Themajorrole of ischemia in anastomosis leak has been acknowledged[2] but a suitable early marker has not yet been identifiedCobalt-based IMA assays were used as early as the year 2000[8] and for approximately ten years after that as a marker ofcoronary events but it could not claim superiority over otherpreviously well-established cardiomarkers and its clinical usehas recently vanished Its characteristics as a sensitive andnonspecific organmarker along with an improved and quan-tifiable detection method by copper-binding assays make ituseful in the early diagnosis of perioperative bowel damageAs it is known from cobalt-based assays IMA suffers fromlarge interindividual variability which may be overcome bypre- and postoperative determination Also there is room forpotential overall improvement of the assay as well Copperbinding to albumin molecules is known to be pH dependent[25] The developed assay (CuBA [7]) omitted any bufferingdespite the tendency of serum pH towards the alkaline range(approx pH 8) due to CO
2loss ex vivo In alkaline solution
albumin binds Cu(2+) ions not only to N-terminal site butalso to the metal binding site [25] which would lead to ahigher biological variability in the test This concerning issuewas solved by implementing a double protein wash of plasmaproteins with phosphate buffered saline using ultrafiltration
The theoretical outcome of the CuBA test as currentlydeveloped is outstanding [7] though still in need of validationin human clinical samples and it could also benefit fromfurther analytical improvements Some additional advantagesof the test lie in its simplicity and low economical costNevertheless these significant results should be indepen-dently assayed and confirmed on a higher number of patientsfurther verifying sensitivity specificity negative and positivepredictive values and accuracy of the test
6 Disease Markers
5 Conclusions
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
proved to be clinically useless in the prediction ormonitoringof postoperative complications Concerning i-FABP the lackof diagnostic efficiency could be attributed to an insufficientamount of enterocytes undergoing perioperative necrosisalthough the postoperative time-profile could also be con-sidered partly at fault since the significant decreased levelsof plasmatic i-FABP were probably caused by a diminishedenterocyte turnover during the selected time period for theanalysis
D-Lactate predominantly a byproduct of gut florametabolism was found in increased levels during the postop-erative period most likely due to unbalanced bowel micro-biota populations caused by prophylactic antibiotics andpreoperative bowel preparation Exhaled breath hydrogencontent another microbial byproduct exhibited a betteroutcome in the detection of perioperative complications fur-ther assessing a defined role for bowel microbiota metabolicbyproductsrsquo as a diagnostic tool of postoperative complica-tions Its apparent usefulness notwithstanding the proposedmethod suffers from too low sensitivity to be of any clinicalvalue
The most disappointing results were obtained from theE coli genomic DNA detection test Despite optimisticreferences [19] the protocol as employed by our group didnotmanage to detect any E coli genomicDNA in the patientsrsquoplasmaThe sensitivity of the test has been determined in sim10DNA copies per milliliter of plasma It is rather possible thatthe operational procedure for colorectal cancer according tolocal standards does not cause significant bacteremia
Of all the included and tested markers only two werefound of any clinical value citrulline and IMA Citrulline wasdefined as a suitable marker of postoperative prolonged ileusbecause its plasmatic concentration reflects the metabolicand peristaltic activity of the bowel Decreased basal valuesof plasmatic citrulline concentration were found in all theanalyzed patients reflecting a ldquophysiologicalrdquo postoperativeileus status Afterwards these concentration values wereincreased in the absence of prolonged postoperative ileusand remained low in its continued presence Therefore
we suggest that plasmatic citrulline concentration could beused clinically even without the knowledge of basal valuesIts reference interval ranges from 20 to 40120583molL with20120583molL as a suitable cut-off value for prolonged ileustesting Citrulline is usually analyzed as part of the amino acidspectrum by high performance liquid chromatography thusrepresenting a major drawback in its clinical use Citrullinecontent analysis is only available in large hospitals andsame-day laboratory response is hard to achieve if notunfeasible Also as prolonged ileus is commonly a self-resolving condition [16] there is little clinical demand fora molecular diagnostic biomarker Regardless citrulline stillmay be useful as a potential marker in research studiesdealing with preoperative or postoperative changes enablingthe quantitative estimation of prolonged postoperative ileusseverity
The major finding of this study was the clinical value ofIMA in diagnosing postoperative complications of colorectalsurgery Anastomosis leakage is a potentially disastrous con-dition which often leads to sepsis and postponed adjuvantchemotherapeutic treatment therefore compromising thesurvival rate of the patients C-reactive protein has beenpreviously identified as the optimal marker available yet itcan only be used until the fourth postoperative day with aset cut-off value of 150mgL [24] However it is a markerindicating an already ongoing inflammatory response thusan earliermarker is needfully sought after [2 3 24]Themajorrole of ischemia in anastomosis leak has been acknowledged[2] but a suitable early marker has not yet been identifiedCobalt-based IMA assays were used as early as the year 2000[8] and for approximately ten years after that as a marker ofcoronary events but it could not claim superiority over otherpreviously well-established cardiomarkers and its clinical usehas recently vanished Its characteristics as a sensitive andnonspecific organmarker along with an improved and quan-tifiable detection method by copper-binding assays make ituseful in the early diagnosis of perioperative bowel damageAs it is known from cobalt-based assays IMA suffers fromlarge interindividual variability which may be overcome bypre- and postoperative determination Also there is room forpotential overall improvement of the assay as well Copperbinding to albumin molecules is known to be pH dependent[25] The developed assay (CuBA [7]) omitted any bufferingdespite the tendency of serum pH towards the alkaline range(approx pH 8) due to CO
2loss ex vivo In alkaline solution
albumin binds Cu(2+) ions not only to N-terminal site butalso to the metal binding site [25] which would lead to ahigher biological variability in the test This concerning issuewas solved by implementing a double protein wash of plasmaproteins with phosphate buffered saline using ultrafiltration
The theoretical outcome of the CuBA test as currentlydeveloped is outstanding [7] though still in need of validationin human clinical samples and it could also benefit fromfurther analytical improvements Some additional advantagesof the test lie in its simplicity and low economical costNevertheless these significant results should be indepen-dently assayed and confirmed on a higher number of patientsfurther verifying sensitivity specificity negative and positivepredictive values and accuracy of the test
6 Disease Markers
5 Conclusions
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
The present study achieved the novel identification of twoclinically useful marker tests for oncological colorectalsurgery complications The 120572-amino acid citrulline may wellbe of clinical assistance in distinguishing between prolongedileus and postoperative metabolic ldquostunningrdquo of the smallbowel On the other hand ischaemia modified albumin hasbeen proved as an effective predictor of anastomosis leakagewhich could be useful in the early detection of this mostserious periperative complication common amongst rectalcancer patients
Disclosure
The funding organization had no role in the experimentaldesign data collection and analysis decision to publish orthe preparation of the paper
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The present study was funded by the Czech RepublicrsquosMinistry of Health Grant no NT13 536-412 (to RadomırHyspler) The authors thank Dr Ian McColl and Dr DanielDiaz for their excellent language assistance and proofreadingthe paper
References
[1] B Andersson D Ansari M Norden J Nilsson and RAndersson ldquoSurgical stress response after colorectal resectionrdquoInternational Surgery vol 98 no 4 pp 292ndash299 2013
[2] J L A van Vugt K W Reisinger J P M Derikx D Boermaand J H M B Stoot ldquoImproving the outcomes in oncologicalcolorectal surgeryrdquo World Journal of Gastroenterology vol 20no 35 pp 12445ndash12457 2014
[3] S R Steele J Bleier B Champagne et al ldquoImproving outcomesand cost-effectiveness of colorectal surgeryrdquo Journal of Gastroin-testinal Surgery vol 18 no 11 pp 1944ndash1956 2014
[4] A Reintam P Parm R Kitus H Kern and J StarkopfldquoGastrointestinal symptoms in intensive care patientsrdquo ActaAnaesthesiologica Scandinavica vol 53 no 3 pp 318ndash324 2009
[5] N Hyman T L Manchester T Osler B Burns and P ACataldo ldquoAnastomotic leaks after intestinal anastomosis itrsquoslater than you thinkrdquoAnnals of Surgery vol 245 no 2 pp 254ndash258 2007
[6] J J Platt M L Ramanathan R A Crosbie et al ldquoC-reactiveprotein as a predictor of postoperative infective complicationsafter curative resection in patients with colorectal cancerrdquoAnnals of Surgical Oncology vol 19 no 13 pp 4168ndash4177 2012
[7] J E Eom E Lee K H Jeon et al ldquoDevelopment of an albumincopper binding (ACuB) assay to detect ischemia modifiedalbuminrdquo Analytical Sciences vol 30 no 10 pp 985ndash990 2014
[8] D Bar-Or E Lau and J V Winkler ldquoA novel assay for cobalt-albumin binding and its potential as a marker for myocar-dial ischemiamdasha preliminary reportrdquo Journal of EmergencyMedicine vol 19 no 4 pp 311ndash315 2000
[9] A Gunduz S Turkmen S Turedi et al ldquoTime-dependentvariations in ischemia-modified albumin levels in mesentericischemiardquoAcademic EmergencyMedicine vol 16 no 6 pp 539ndash543 2009
[10] Z D Dundar B Cander M Gul K U Karabulut andS Girisgin ldquoSerum ischemia-modified albumin levels in anexperimental acute mesenteric ischemia modelrdquo AcademicEmergency Medicine vol 17 no 11 pp 1233ndash1238 2010
[11] H Kadioglu D Omur S Bozkurt et al ldquoIschemia modifiedalbumin can predict necrosis at incarcerated herniasrdquo DiseaseMarkers vol 35 no 6 pp 807ndash810 2013
[12] N J Evennett M S Petrov A Mittal and J A WindsorldquoSystematic review and pooled estimates for the diagnosticaccuracy of serological markers for intestinal ischemiardquo WorldJournal of Surgery vol 33 no 7 pp 1374ndash1383 2009
[13] P Ruan Z Gong and Q Zhang ldquoChanges in plasma D(-)-lactate diamine oxidase and endotoxin in patients with livercirrhosisrdquo Hepatobiliary amp Pancreatic Diseases Internationalvol 3 no 1 pp 58ndash61 2004
[14] T Kanazuru E F Sato K Nagata et al ldquoRole of hydrogengeneration by Klebsiella pneumoniae in the oral cavityrdquo TheJournal of Microbiology vol 48 no 6 pp 778ndash783 2010
[15] Z Moghadamyeghaneh G S Hwang M H Hanna et al ldquoRiskfactors for prolonged ileus following colon surgeryrdquo SurgicalEndoscopy 2015
[16] L Pironi J Arends J Baxter et al ldquoESPEN endorsed recom-mendations Definition and classification of intestinal failure inadultsrdquo Clinical Nutrition vol 34 no 2 pp 171ndash180 2015
[17] E Curis I Nicolis CMoinard et al ldquoAlmost all about citrullinein mammalsrdquo Amino Acids vol 29 no 3 pp 177ndash205 2005
[18] P Crenn C Coudray-Lucas F Thuillier L Cynober and BMessing ldquoPostabsorptive plasma citrulline concentration is amarker of absorptive enterocyte mass and intestinal failure inhumansrdquo Gastroenterology vol 119 no 6 pp 1496ndash1505 2000
[19] Z Qiao Z Li J Li L Lu Y Lv and J Li ldquoBacterial translo-cation and change in intestinal permeability in patients afterabdominal surgeryrdquo Journal of Huazhong University of Scienceand Technology Medical Science vol 29 no 4 pp 486ndash4912009
[20] D C Turnell and J D H Cooper ldquoRapid assay for amino acidsin serum or urine by pre-column derivatization and reversed-phase liquid chromatographyrdquo Clinical Chemistry vol 28 no 3pp 527ndash531 1982
[21] R Hyspler A Ticha I Svobodova and Z Zadak ldquoSpectropho-tometric method for D-lactate determination in blood plasmaand urinerdquoKlinicka Biochemie aMetabolismus vol 23 no 4 pp193ndash197 2015
[22] D Juck J Ingram M Prevost J Coallier and C Greer ldquoNestedPCR protocol for the rapid detection of Escherichia coli inpotable waterrdquo Canadian Journal of Microbiology vol 42 no8 pp 862ndash866 1996
[23] E Kaclıkova D Pangallo K Oravcova H Drahovska andT Kuchta ldquoQuantification of Escherichia coli by kinetic 51015840-nuclease polymerase chain reaction (real-time PCR) orientedto sfmD generdquo Letters in Applied Microbiology vol 41 no 2 pp132ndash135 2005
Disease Markers 7
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
[24] F D McDermott A Heeney M E Kelly R J Steele G LCarlson and D C Winter ldquoSystematic review of preoperativeintraoperative and postoperative risk factors for colorectalanastomotic leaksrdquo British Journal of Surgery vol 102 no 5 pp462ndash479 2015
[25] M Sokołowska K Pawlas and W Bal ldquoEffect of commonbuffers and heterocyclic ligands on the binding of Cu(II) at themultimetal binding site in human serum albuminrdquoBioinorganicChemistry andApplications vol 2010 Article ID 725153 7 pages2010
