Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 1328Abstract. –Background and Objectives:Calprotectin is a protein especially expressed in neutrophil cytosol. In the la st few yea rs, Feca l calprotectin (FC) turned out to be a direct mark-er of ga strointestina l infla mma tion. Beca use of the simplicity of the method, it has been studied in several gastroenterologic diseases but no da-ta a re a va ila ble a bout its concentra tion in chil-dren with Sma ll Intestina l Ba cteria l Overgrowth (SIBO), a complex and not well known condition defined by an excessive germs proliferation, es-pecially anaerobic, in the small bowel, and char-acterized by dyspeptic and malabsorption symp-toms. The aim of this study was to evaluate FC va lues in children with SIBO, compa ring to healthy subjects, in order to clarify if an inflam-matory process coexists with SIBO.Materials and Methods:We enrolled fifty-eight children affected by SIBO, as diagnosed by Lactu-lose Breath Test (LBT). They were assessed for FC values on stool samples. We compared them with a control population of 60 healthy children.Results:In SIBO pa tients, a media n va lue of 36.0 mg/kg and a mean value ± SD of 43.0 ± 31.6mg/kg were calculated, while in healthy controls the median value was 29.5 mg/kg and the mean va lue ± SD wa s 35.7 ± 20.7 mg/kg, showing no sta tistica lly significa nt differences between the two groups (p = 0.07).Conclusions:FC va lues a re nega tive in chil-dren a ffected by SIBO, not differing from those obtained in healthy children, suggesting that no subclinica l intestina l infla mma tion involving neutrophils occurs in patients with higher prolif-eration of bacteria in the small bowel. The pres-ence of high FC levels in children affected by SI-BO might not be caused by bacterial overgrowth itself and, in this case, another cause should be investigated.Key Words:Fecal calprotectin, Small intestinal bacterial over-growth, Children, Lactulose breath test.Corresponding Author:Claudia Fantacci, MD; e-mail: claudiafantacci@yahoo.itIntroductionCalprotectin is a 36 kDa Calcium and Zincum binding protein which belongs to the S100 pro-tein family 1and it is mapped on the gene q12-q21 on chromosome 12. The S100 protein family is composed by about twenty proteins which are expressed in various cell types and have in com-mon the skill to bind Calcium ion, a second mes-senger which activates their function. S100 pro-tein family is involved in the complicated mecha-nisms of the intracellular transduction, than it takes part in the regulation of various processes such as protein phosphorylation, transcription,cell differentiation, cell cycle regulation, cell growth and proliferation, cell motility, inflamma-tory and immune response regulation 3. In partic-ular, Calprotectin has been described for the first time in 1980 by Fagerhol et al.4, who isolated it from leukocytes and named it “L1 protein”. Af-terwards, it has been found in cells, tissues and fluids in all parts of human body 5, but its pecu-liarity is to be expecially expressed in neutrophil cytosol. In fact, here its concentration is estimat-ed at 5-15 mg/ml and it constitutes about 5% of total proteins in neutrophil granulocytes 4,6. This entails that in inflammatory reactions, with neu-trophil activation and death, Calprotectin is re-leased and then its concentration in body fluids increases, constituting a marker of those inflam-matory processes in which neutrophil granulo-cytes are involved 7-9. Consequently, concerning gastrointestinal diseases, when there is an in-flammatory process in gastrointestinal mucosa,Calprotectin is released in the gut lumen and then it can be retrieved in feces 10-12. When, in 1992,Røseth et al.13described the method for the ex-traction and the assessment of Calprotectin in fe-ces, to quantify its concentrations in various gas-2011; 15: 1328-1335Fecal Calprotectin concentration in children affected by SIBOC. FUNDARÒ, C. FANTACCI, V . ANSUINI, V . GIORGIO, S. FILONI, F . BARBARO*, A. GASBARRINI*, C. ROSSI**Department of Pediatric; *Department of Gastroenterology and **Clinical Chemistry Laboratory,School of Medicine, Catholic University of the Sacred Heart, Gemelli Hospital, Rome (Italy)trointestinal diseases became possible. Because of the simplicity of the method, in the last few years fecal Calprotectin (FC) has been evaluated in various gastrointestinal disorders14-17and has emerged as a sensible and useful marker of gas-trointestinal inflammation, becoming an impor-tant aid in clinical practice.Small Intestinal Bacterial Overgrowth (SIBO) is a qualitative and quantitative variation of intesti-nal flora characterized by an excessive germs pro-liferation, especially anaerobic, in the small bow-el, exceeding 105Colony Forming Unit (CFU) of organisms per ml of intestinal juice18. This disor-der is not actually well known, and for explaining its pathogenesis several factors have been thought to be involved. A number of conditions which can compromise the delicate equilibrium of the gas-trointestinal tract have been supposed to play a role, such as intestinal dismotility (diabetic neu-ropathy, scleroderma, accelerated gastric empty-ing, chronic renal failure), gastrointestinal anato-my changes (gastric atrophy, small bowel divertic-ulosis, intestinal stenosis, gut surgery, resection of the ileocecal valve), hypo or achlorydria, ageing, immunodeficiency and malnutrition19. With regard to clinical aspects, patients affected by SIBO can suffer from dyspeptic and malabsorption symp-toms, such as bloating, meteorism, abdominal dis-comfort or pain, flatulence, diarrhea, steatorrhea, weight loss and anaemia20. The diagnosis of SIBO can be assessed with different methods. The gold standard is the culture of upper intestinal aspirate but it is an invasive and difficult to perform tech-nique, which requires an expert staff18. Today, one of the most used is the Lactulose Breath Test (LBT)21, which is a more simple and less invasive and expensive methodic. LBT is characterized by high sensitivity and specificity22.At present, no data are available about FC con-centrations in children with SIBO.This prospective study was designed to evalu-ate FC concentrations in children affected by SI-BO, comparing them to a group of healthy con-trols, in order to clarify if an inflammatory process coexists with SIBO.Materials and MethodsWe evaluated fifty-eight consecutive children with SIBO as assessed by LBT. They were re-ferred to the Pediatric Gastroenterology Outpa-tients Unit of Catholic University of the Sacred Heart, Gemelli Hospital of Rome between April 1st2008 and September 1st2009.Children who took Non Steroidal Anti-Inflam-matory Drugs (NSAIDs), antibiotics, gastric acidity inhibitors or drugs influencing gut motili-ty within the previous 2 months were excluded. Children who were affected by other gastroin-testinal disorders, respiratory or urinary infec-tions, or chronic diseases such as rheumatoid arthritis, diabetes, thyroid diseases, connective tissue diseases, or had a history of intestinal surgery were excluded. Children who had nasal or menstrual bleeding in the last three weeks were excluded too.The control population included sixty healthy children, without SIBO (as assessed by negative LBT). They were referred to our General Pedi-atrics Outpatients Unit for routine medical care. All patients affected by SIBO and all healthy controls were assessed for F C values after stool sample measurements.All children were clinically evaluated at three and six months of follow-up.All patients and control subjects were enrolled with parents informed consent, according to the Ethics Committee of our University.Hydrogen/Methane Lactulose Breath TestHydrogen (H2)/methane (CH4) Lactulose Breath Test (LBT) was performed under standard conditions. No patients had received laxatives in the 30 days preceding the test. Subjects were asked to have a carbohydrate-restricted dinner on the day before the test and to fast for at least 12hours to minimize basal H2excretion. On the day of testing, patients received a mouthwash with 20 mL of chlorhexidine 0.05%. Physical exercise was not allowed for 30 minutes before and dur-ing the test. End-alveolar breath samples were collected immediately before lactulose ingestion (lactulose 10 g in solution 20 mL). Samples were taken every 15 minutes for 4 hours with a 2 bag system, consisting of a mouthpiece, a T-valve, and 2 collapsible bags; the first one collects dead space air, the second one collects alveolar air. The breath sample was aspirated from this bag into a 20 mL plastic syringe. Samples were ana-lyzed immediately for H2and CH4with a model DP Quintron gas chromatograph (Quintron In-strument Company, Milwaukee, WI, USA). The results were expressed as parts per million. A normal LBT was defined as the absence of anearly rise in H2or CH4excretion of more of 20 parts per million within the first 90 minutes.1329Fecal Calprotectin concentration in children affected by SIBO1330FC Measurement and RangesOne hundred-eighteen stool samples were col-lected, using a disposable plastic test tube. Speci-mens were returned to the laboratory within 48hours of defecation. The weight of the samples necessary for the test was 40-120 mg. This little amount was collected with a specific device and then diluted with a buffer solution containing cit-rate and urea in a weight per volume ratio 1:50(20 µl of stool sample in 980 µl of buffer solu-tion). If necessary, a second dilution 1:250 (200µl of the first diluted solution in 800 µl of buffer solution) could be performed for very concentrat-ed stool samples. After this procedure, the sam-ple was mixed for 30 seconds by a vortex method, homogenized for 25 minutes and then one milliliter of the homogenate was centrifuged for 20 minutes. The supernatant was collected and kept refrigerated at –20°C. Within seven days, the samples were thawed at room tempera-ture and then Calprotectin concentration was ac-tually measured by the quantitative ELISA test Calprest ®(Eurospital Spa, Trieste, Italy).Laboratory ranges were expressed as mg of Calprotectin/kg of feces. The linearity of the method was 15-500 mg/kg.On the basis of data available in literature concerning the F C cut-off value in the pediatric age, a negative FC concen-tration was defined by a FC value lower than 100mg/kg, while a positive FC concentration was de-fined by a F C value equal or higher than 100mg/kg 16,23.Statistical AnalysisThe statistical analysis was performed with ANOV A test. Student’s t -test was used for data analysis. A p value <0,05 has been considered statistically significant. All results have been pre-sented as median and mean ± standard deviation (SD), or as absolute count numbers when appro-priate.ResultsThe results were reported on Tables I, II and Figures 1, 2.Fifty-eight children affected by SIBO and six-ty healthy subjects were evaluated.The number of males/females was 39/19 in the group of patients affected by SIBO and 36/24 in the group of healthy controls. The age range of the children in the two groups was respectivelyC. Fundarò, C. Fantacci, V . Ansuini, V . Giorgio, S. Filoni, F . Barbaro, A. Gasbarrini, C. Rossi52-202 months and 52-211 months, with a mean age of 121.8 ± 38.9 months and 126.8 ± 46.9months. Concerning demographic data, a p value of 0.26 was calculated, demonstrating that no statistically significant differences for sex and age were observed between the two groups.F ifty-six (96.6%) patients affected by SIBO and sixty (100%) healthy children had a negative F C value. In particular, the range of F C values obtained in the two groups was <15-159 mg/kg and <15-89 mg/kg respectively. In the group of patients affected by SIBO, a median value of 36.0 mg/kg and a mean value ± SD of 43.0 ±31.6 mg/kg were calculated, while in the group of healthy controls the median value was 29.5mg/kg and the mean value ± SD was 35.7 ± 20.7mg/kg. Evaluating these results obtained in the two groups, a p value of 0.07 was calculated,suggesting that no statistically significant differ-ences came out between FC concentrations in pa-tients affected by SIBO in comparison with healthy children.DiscussionFor the first time, our case control study shows that F C levels in children affected by SIBO are not statistically different from those obtained in healthy controls. Our findings are similar to those pointed out by Montalto et al 24, who per-Table I.Demographic characteristics of patients affected by SIBO and healthy controls.*p = 0.26.Table II.Fecal Calprotectin values obtained in patients af-fected by SIBO and in healthy controls.*p = 0.07.1331Fecal Calprotectin concentration in children affected by SIBOformed the only study available in literature about the correlation between SIBO and FC con-centrations. Their study was carried out on an adult population: they evaluated 40 patients af-fected by SIBO and 40 controls, demonstrating no statistically significant differences in FC con-centrations between the two groups.In the last few years, the importance of F C measurement in the management of gastrointesti-nal disorders is becoming more and more evi-dent, and it is settling as an useful marker of gas-trointestinal inflammation which can support the clinical practice 8.In fact, FC concentration increases in a num-ber of organic gastroenterologic conditions such as colorectal cancer, NSAIDs enteropathy, al-choolic enteropathy, active inflammatory bowel diseases (IBD), acute gastroenteritis, allergic col-itis and gastro-esophageal reflux disease 8,25-28.This happens because it is released from neu-trophils in gut lumen during gastrointestinal in-flammation, then it binds Ca 2+, becoming resis-tant against heat and proteolysis. Consequently, it is eliminated intact in feces and there it can re-main stable at room temperature for about 7days 10,11. This allows to measure it by means of a simple and non invasive laboratory test, which requires a little amount of feces. These character-istics make F C measurement a convenient labo-ratory test, easy to be performed by patients, es-pecially in the pediatric age.Furthermore, supporting data that FC can con-stitute a direct marker of those gastrointestinal inflammatory processes in which neutrophils are involved, some studies which compared FC mea-surement with invasive techniques have shown interesting results.Røseth et al 29investigated the correlation be-tween the faecal excretion of the granulocyte marker protein and that of 111-Indium-labelled granulocytes in patients with IBD. In fact, faecal excretion of 111-Indium-labelled neutrophilic granulocytes has been suggested as the gold stan-dard of disease activity, but it is a complex and expensive method which expose patients to ion-izing irradiation. The results obtained in this study suggested that FC reflects the granulocyte migration through the gut wall in patients with IBD and hence could be used as a simple, inex-pensive alternative to the 111-indium technique.Limburg et al 30evaluated 110 subjects with chronic diarrhea who were referred for colonoscopy and observed that increased FC lev-els were significantly associated with the colono-scopic and histological findings of colorectal in-flammation.A recent metanalysis has analyzed 30 prospec-tive studies which compared F C levels against the histological diagnosis in patients with diag-nosis of IBD. It evaluated F C concentrations of 5983 adults and children and demonstrated that FC has a sensitivity of 95% and a specificity of 91% in IBD diagnosis. The same metanalysis shows that the diagnostic precision in childhoodFigure 2.Fecal Calprotectin values obtained in the group of patients affected by SIBO and in healthy controls.F e c a l c a l p r o t e c t i n (m g /k g )Patients ControlsFigure parison between the age of the patients af-fected by SIBO and healthy controls.A g e (m o n t h s )Patients Controlspopulation is higher than in the adult popula-tion23. F urthermore, F C values of children with IBDs in remission turn into normal ranges be-coming non statistically different from those of healthy children16,31, while they increase again in relapses, preceding clinical symptoms32-36. Moreover, FC values in functional symptoms have been demonstrated to be not statistically different from controls16, and this is true in children affected by IBD too. So, FC can help in distinguishing functional pains from relapses in a child affected by IBD, and this is very im-portant for these subjects because they present with an increased frequency bowel movements, urgency and abdominal cramping, and these symptoms can be mistakenly interpreted as a flare-up37.Concerning literature which has examined FC levels specifically in the pediatric age, a remark-able study is that of Berni Canani et al16, who en-rolled 281 children assessed for gastrointestinal symptoms. Among these subjects, those of them affected by a disease characterized by gastroin-testinal mucosal inflammation, such as Crohn’s disease (38 children), ulcerative colitis (45 chil-dren) had increased FC concentrations, while 44 children suffering from functional gastrointesti-nal disorders (F GIDs) showed normal values. Therefore, they pointed out that FC is a sensitive but not disease specific marker to easily detect inflammation throughout the whole gastrointesti-nal tract and may help in identifying an organic disease and in the differential diagnosis of func-tional bowel disorders.All these results impact on clinical practice be-cause suggest that several invasive diagnostic techniques can be avoided, and this is even more important in Pediatrics38.SIBO is a condition characterized by an exces-sive germs proliferation, especially anaerobic, in the small bowel (more than 105CF U/ml of in-testinal juice)18, liable to antibiotic treatment, which improves gastrointestinal symptoms39. Generally, in the intestinal tract there are 103-104CF U/ml of bacteria such Enterococcus and Lactobacillus, and there are a number of factors which permit to restrain bacterial overgrowth. Among these, there are anatomical and function-al factors (such as gastric acidity, ileocecal valve continence, gall and pancreatic secretions and their antibacterial activity), mechanical factors (the peristalsis) and factors which inhibit bacteri-al adhesion to the epithelium (gastric mucus, se-cretory IgA and epithelial desquamation)40,41.Moreover, gut micloflora plays a crucial role in the development of intestinal defences: the colonization with diverse intestinal microbes, in fact, is necessary for the synthesis and the secre-tion of polymeric immunoglobulin A and the generation of a balanced T Helper cell response. By studying germ-free animals, it results that neither function exists in the germ-free state, but rapidly develops after germ colonization42.In-testinal bacteria maintain “a physiological in-flammation” in the human gut which is efficient-ly protective and necessary to have an appropri-ate local immune response, while a disregulation of the mucosal immune response can switch a “controlled” toward an “uncontrolled”intestinal inflammation, paving the way to pathology43. Therefore, when intestinal bacteria exceed, this label equilibrium can be broken. The presence of a higher bacterial number in the small bowel causes a premature and abnormal deconjugation of the bile acids, determining a larger jejunal re-absorption and secondary lipid malabsorption. Moreover, contaminant bacteria can cause a di-rect damage on entherocytes because of their adesivity on epithelial surface and because of their competition with entherocytes for the link with the complex vitamin B12 – intrinsic factor. This results in a reduction of the vitamin B12 ab-sorption. Even if some type of bacteria can pro-duce the vitamin theirselves, finally the subject has reduced levels of bio-availability of vitamin B12 and can have malabsorption symptoms40. Otherwise, patients affected by SIBO often suffer from a nebulouse symptomatology charac-terized by diarrhea, flatulence, abdominal pain or discomfort. Underlying these symptoms, there is the glucidic malabsorption, which causes an ac-centuated fermentation and then higher produc-tion of water, short chain fatty acids and gas such as carbon dioxide, hydrogen and methane44. Whether the presence of SIBO leads to small intestinal mucosal changes is not well known. There are some investigations about the histolog-ical changes caused by SIBO in animal models, where changes of villus and crypt architecture and an increase in chronic inflammatory cells number – mostly lymphocytes of the lamina pro-pria – have been shown45-47.Recently, a retrospective study has been per-formed on 122 subjects who underwent upper gastrointestinal endoscopic examination because of gastrointestinal symptoms. Among these pa-tients, 67 was affected by SIBO (as assessed by duodenal aspirate culture >105CF U/ml), whileC. Fundarò, C. Fantacci, V. Ansuini, V. Giorgio, S. Filoni, F. Barbaro, A. Gasbarrini, C. Rossi133255 had a negative culture (<105CF U/ml) and they were considered controls. F rom these duo-denal biopsy has emerged one feature significant-ly more frequent in SIBO than in controls, which was villous blunting to crypt ratio (<3:1)48.SIBO seems, also, to determine a higher level of IgA in the proximal small intestine particular-ly when the overgrowth is caused by colonic type bacteria 49. Nevertheless, no study performed on patients with SIBO about direct parameters that indicates the number of leucocytes neutrophils in the gut wall are available. Montalto et al 24have published about F C concentrations in adults af-fected by SIBO considering it as an indirect para-meter of intestinal inflammation. Their results suggested that no inflammatory changes involv-ing neutrophils occurs in SIBO. On the edge of this finding, we have evaluated for the first time F C concentrations in a pediatric population af-fected by SIBO, comparing this values with healthy controls. No statistically significant dif-ferences have been found between cases and con-trol subjects (p = 0.07), according to the findings observed in adults. This confirms the hypothesis that no subclinical intestinal inflammation in-volving neutrophils occurs in patients with high-er proliferation of bacteria in the small bowel.The presence of high FC levels in children affect-ed by SIBO might not be caused by bacterial overgrowth itself and, in this case, another cause should be investigated.In conclusion, our study demonstrates for the first time that F ecal Calprotectin values do not increase in children affected by SIBO. Our re-sults are similar to the findings obtained in adults, supporting the hypothesis that no subclin-ical intestinal inflammation involving neutrophils occurs in SIBO.References1)F AGERHOL MK.Nomenclature for proteins: is Calpro-tectin a proper name for the elusive myelomonocyt-ic protein? 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