Comparison of fecal pyruvate kinase isoform M 2 and calprotectin in assessment of pediatric inflammatory bowel disease severity and activity

1Child & Mother Specialist Hospital in Poznan, Poznań, Poland; 2Poznan University of Medical Sciences, Department of Pediatric Gastroenterology & Metabolic Diseases, Poznań, Poland; 3Nicolaus Copernicus University, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Department of Pediatric Endoscopy and Gastrointestinal Function Testing, Bydgoszcz, Poland; 4Medical University of Silesia, Department of Paediatrics, Katowice, Poland; 5Medical University of Gdansk, Department of Pediatrics, Pediatric Gastroenterology, Hepatology and Nutrition, Gdańsk, Poland; 6Poznan University of Medical Sciences, Department of Informatics and Statistics, Poznań, Poland; 7Poznan University of Medical Sciences, Poznań, Poland; 8Poznan University of Medical Sciences, Department of Paediatric Surgery, Traumatology and Urology, Poznań, Poland; 9Poznan University of Medical Sciences, Department of General, Gastroenterological and Endocrinological Surgery, Poznań, Poland; 10Institute of Human Genetics, Polish Academy of Sciences, Poznań, Poland


INTRODUCTION
Embryonic M2 isoform of pyruvate kinase (M2-PK) is a cytosolic enzyme catalyzing a phosphate group transfer in glycolysis (Walkowiak et al., 2005).Its tetrameric and dimeric forms are present in proliferating cells of many tissues, including leukocytes (Gupta & Bamezai, 2010).Upon leukocyte destruction in the gastrointestinal tract, the protein is released to the fecal stream (Walkowiak et al., 2005).M2-PK is stable in stools, which increases the potential value of fecal M2-PK concentration assessment both in intestinal inflammation and cancer.We postulated that fecal M2-PK could serve as a biomarker of inflammation in pediatric inflammatory bowel diseases (IBD) (Czub et al., 2007).Other groups concentrated on M2-PK utility in cancer diagnostics (Gupta & Bamezai, 2010).
Calprotectin is a calcium and zinc binding protein belonging to S100 protein family.It constitutes more than 40% of neutrophil cytosol proteins and exhibits bacteriostatic activity (Yui et al., 2003).It has been shown that fecal calprotectin (FC) concentration correlates with severity of inflammatory bowel disease endoscopic presentation (Aomatsu et al., 2011;D'Haens et al., 2012;Önal et al., 2012).It is also known that fecal calprotectin may be used to predict IBD relapses (Lasson et al., 2013;Mao et al., 2012).Although calprotectin's sensitivity and specificity in adult IBD is 93% and 96% respectively, it was demonstrated that in children the specificity is significantly lower (van Rheenen et al., 2010).
To date two studies comparing M2-PK and calprotectin were published.In 2008 Shastri et al. described fecal M2-PK and FC concentrations in a population of 276 adult patients with IBD, concluding that M2-PK had inferior specificity (Shastri et al., 2008).In 2010 Turner et al. investigated fecal M2-PK, FC, lactoferrin and S100A12 protein in children with severe ulcerative colitis (Turner et al., 2010).They showed that only M2-PK had constructive and predictive validity, while other markers failed to meet this criterion.Therefore the aim of this study was to compare the value of M2-PK and calprotectin in assessment of pediatric IBD activity.

MATERIALS AND METHODS
One hundred fifty-six children were enrolled for the study, including 75 with ulcerative colitis (UC), 46 with Crohn's disease (CD) and 35 healthy controls (HC).The diagnosis of IBD was based on the physical examination, endoscopic, histologic and radiologic evaluations, as well as supplementary laboratory tests (Kornbluth et al., 2004;Turner et al., 2007;Van Assche et al., 2010).Disease severity was described using Truelove-Witts (TW) scores in children with ulcerative colitis (Mahadevan et al., 2002) (Kobelska-Dubiel et al., 2007) and The Pediatric Crohn's Disease Activity Index (PCDAI) in Crohn's disease patients (Hyams et al., 1991).Whether disease was active or in remission was determined on basis of an extended set of criteria including endoscopy, magnetic resonance enterography, ultrasonography and biomarkers of inflammation in the blood, depending on the case studied and the clinical context.Group characteristics are presented in Table 1.Median TW score was 1.0 (interquartile range [IQR]: 0-5), median PCDAI score was 42.5 (7-60).
The study was conducted in four tertiary care centres and one secondary care centre.Fresh stool samples were initially stored at 4 degrees Celsius and at -70 degrees Celsius after transfer to the laboratory.Dimeric M2-PK concentration was assessed using commercially available sandwich ELISA with monoclonal antibodies (ScheBo Biotech, Giesen, Germany) and expressed in U/g.Although manufacturer-suggested cut-off value was 4 U/g, results for cut-off set at 5 U/g were analysed as well to increase the test specificity.FC levels were determined employing PhiCal ELISA Test (Calpro, Lysaker, Norway).Cut-off concentration for FC was 15 μg/mL.Both analyses were performed in the same stool specimens.All samples were assessed by the same observer who was unaware of group allocation.
Statistical analysis was carried out using STATISTICA data analysis software system v. 10 (StatSoft, Inc., Tulsa, United States of America) and Analyse-it v. 2.30 (Analyse-it Software, Leeds, United Kingdom).Sensitivity, specificity and area under receiver operating characteristic (ROC) curve were calculated.The areas under the receiver operating characteristic curves (AUC) for M2-PK and FC in different settings were compared.These comparisons were made after adjusting M2-PK cut-off level so that M2-PK specificity equaled that of FC at standard cut-off value of 15.M2-PK and FC performance was compared with the use of Whitney-Mann U-test.The level of significance was set at p<0.05.The data are presented as median (IQR), unless stated otherwise.
Parents of all patients, and patients at least 16 years old have expressed their written, informed consent to participation in the study.The study project received a positive opinion of the Bioethical Committee at Poznan University of Medical Sciences (decision 1740/04).

RESULTS
Fecal concentrations of M2-PK and FC in groups are shown in Table 2.The highest M2-PK concentration was 1849.0U/g, while the highest FC concentration was 556.0 μg/mL, both in active, severe UC.Highest concentrations of M2-PK and FC in patients with CD was 770.4 U/g (mild CD in remission) and 456.0 μg/mL (active, severe CD), respectively.
Fecal concentrations of M2-PK and FC in subgroups of children with IBD depending on disease activity status and disease severity are shown in Table 3. and Table 4.

DISCUSSION
This is the first study to directly compare M2-PK and FC in assessment of severity and activity of pediatric IBD.It presents a response to questions that arose after we found that M2-PK is a marker of pediatric IBD activity (Czub et al., 2007).The main concern was to determine whether M2-PK could improve IBD diagnostics in children.
The data obtained lend support to the notion that M2-PK is inferior to FC in IBD diagnostics.This was clearly visible in two areas: identification of UC patients among HS and recognition of UC patients in remission among HS.Both were expressed in marked AUC differences.With regard to CD, the AUC differences were smaller, yet significant.Given the high sensitivity and good specificity of FC, M2-PK would present no added value in CD (Henderson et al., 2013).
In general, observations regarding FC that were made in this study are in line with what was previously described (Bunn et al., 2001;Komraus et al., 2012;Kostakis et al., 2013).They seem to contradict the conclusions drawn by Shaoul et al. who found no correlation between clinical IBD activity and FC concentrations (Shaoul et al., 2012) and to agree with a report by D'Haens et al. in which a correlation between clinical score in UC and FC was described (D'Haens et al., 2012).
One of the limitations of this study is that it investigated M2-PK and FC in the context of disease severity and activity without detailed analysis of response to treatment in a longer period of observation.This was rendered impossible by the settings.
The future directions of research should include questions, such as: how fecal markers could be used to decrease the number of endoscopic investigations without compromising diagnosis (Aomatsu et al., 2011;van Rheenen et al., 2010), to predict relapses in patients that do not exhibit symptoms (Kallel et al., 2011;Mao et al., 2012), and to predict the course of disease in general (Lasson et al., 2013;Önal et al., 2012).
In conclusion, these data show that in children, calprotectin reflects IBD severity and activity better than M2-PK.