Age-related faecal calprotectin, lactoferrin and tumour M2-PK concentrations in healthy volunteers

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1 Original Article Age-related faecal calprotectin, lactoferrin and tumour M-PK concentrations in healthy volunteers Shilpa Joshi, Stephen J Lewis, Siobhan Creanor and Ruth M Ayling Department of Clinical Biochemistry; Department of Gastroenterology, Derriford Hospital, Plymouth PL6 8DH; Department of Biostatistics and Epidemiology, University of Plymouth, Tamar Science Park, Plymouth PL6 8BX, UK Corresponding author: Ruth M Ayling. ruthayling@clinicalbiochemistry.org.uk Editorial comment: Readers should be aware that the content of this research paper has changed substantially from the version that was originally published on-line on 9 September 9. A reappraisal of the data has resulted in substantial changes to the originally quoted reference ranges. One author of the originally posted article has disassociated herself from this final version of the paper. Abstract Objective: Measurement of the faecal markers calprotectin, lactoferrin and tumour M-PK has been reported to be useful in the diagnosis and management of a range of gastrointestinal disorders in both children and adults. The aim of this study was to investigate the requirement for age-related reference ranges. Methods: Faecal samples were obtained from healthy subjects and analysis of calprotectin, lactoferrin and tumour M-PK performed using commercially available enzyme-linked immunosorbent assay. Results: In the healthy subjects median concentrations were as follows: for calprotectin 9 y, mg/g, 59 y, mg/g and 6 y, 7 mg/g; for lactoferrin 9 y,. mg/g, y,.5 mg/g; and for tumour M-PK all subjects, U/mL. Significant differences between age groups for different markers resulted in the following age-related reference ranges: calprotectin 9 y,,66 mg/g, 59 y,,5 mg/g, 6 y,, mg/g; lactoferrin 9 y,,9 mg/g, y,.6 mg/g. Conclusion: In healthy individuals, we found there to be variation in the faecal inflammatory markers calprotectin and lactoferrin with age. For both calprotectin and lactoferrin children aged 9 y had significantly higher concentrations than subjects aged y. For calprotectin but not lactoferrin, adults 6 years had a higher concentration than those aged 59 y. There was no change with age in the metabolomic marker faecal tumour M-PK in healthy subjects. The knowledge of age-related reference ranges in healthy subjects is important to fully interpret changes in gastrointestinal disease. Ann Clin Biochem ; 7: DOI:.58/acb.9.96 Introduction The gold standard for the diagnosis of intestinal inflammation is endoscopy and biopsy. However, this is invasive for the patient, uses considerable resources and carries a degree of clinical risk. The measurement of faecal proteins is a non-invasive method of investigating bowel pathology and provides a surrogate marker of inflammation with many potential advantages over endoscopy and biopsy. Such proteins have the potential to be used as screening tools in patients developing new bowel symptoms and in predicting relapse in those with established disease. Calprotectin is a calcium and zinc binding protein that makes up about 6% of neutrophil cytosolic protein; its concentration in faeces is about six times that of plasma. Early studies showed calprotectin to be raised in patients with inflammatory bowel disease and to be correlated with endoscopic and histological evidence of inflammation. It has since been shown to be useful for the diagnosis of inflammatory bowel disease, 5 to predict its severity in adults 6 and children, 7 to predict relapse 8 and to monitor response to treatment. 9 Lactoferrin is an iron binding protein that is synthesized and stored in the secondary granules of polymorphonuclear neutrophils. It has been shown to be useful in identifying intestinal inflammation and to monitor treatment in inflammatory bowel disease. In patients with lower gastrointestinal symptoms, faecal calprotectin and lactoferrin have been found to be equally recommendable. Lactoferrin is more stable than calprotectin and may be the marker of choice where delay in transit to the laboratory is anticipated. Tumour M-PK is the dimeric isoform of pyruvate kinase, which is upregulated in proliferating tissues. 5 Its measurement in faeces has been investigated as a biomarker in colorectal malignancy 6 9 and has been found to be more sensitive and specific than guaiac-based faecal occult Annals of Clinical Biochemistry ; 7: 59 6

2 6 Annals of Clinical Biochemistry Volume 7 May blood tests with an overall sensitivity of 77.9% and specificity of 7. 8.%. It has been recommended as a tool for general population screening, although is not yet widely used. All three proteins can be measured using standard enzyme-linked immunosorbent assay (ELISA) techniques. The use of faecal markers in clinical practice has led to their application to a wide spectrum of patients in many age groups. The aim of this study was to investigate age-related variation in faecal concentration of calprotectin, lactoferrin and tumour M-PK. Materials and methods Subjects Faecal samples were obtained from healthy subjects (7 female and 6 male) ranging in age from to 86 y, recruited from hospital staff, families and friends. Prior to inclusion in the study, the subjects or their parents completed a health questionnaire to exclude significant intercurrent illness, preexisting bowel conditions or medications with the potential to affect the gastrointestinal tract. Statistical analysis Statistical analysis was performed using Minitab software and the Analyse-it software for Excel, version 7., (Leeds, UK). The subjects were stratified into five age bands 9 (n ¼ 7), 9 (n ¼ 5), 9 (n ¼ ), 59 (n ¼ 7) and 6 y (n ¼ ). The data were not normally distributed. The Kruskal Wallis test was used to examine for evidence of statistically significant differences between the age groups for each marker, based on the logged data, at the 5% significance level. A follow-up multiple comparisons procedure, based on the Mann Whitney test with a Bonferroni correction, was then used to identify which pairs of age groups significantly differed. For statistical purposes samples below the limit of detection of the assay were treated as zero. Reference ranges were calculated from the logged data as the 97.5th centile. Ethical approval Ethical approval for the study was obtained from the Cornwall and Devon Research Ethics Committee. Methods Faecal samples were obtained using a proprietary stool collection device (Protocult, Hemelab, Rochester, MN, USA) and delivered to the laboratory by the patient the same day or posted to arrive the next day. On receipt, samples were frozen at 8C prior to analysis, which was performed within six months. All samples were tested for occult blood prior to measurement of faecal protein analysis and found to be negative. For the measurement of calprotectin, mg of faeces were extracted in 5 ml of extraction buffer. The supernatant was analysed by an ELISA (Phical, Calpro, Lysaker, Norway) using wells coated with polyclonal rabbit antibody and immunoaffinity-purified alkaline phosphatase-labelled anticalprotectin, with detection of the enzyme reaction with a substrate at 5 nm. Lactoferrin was measured by extracting 5 mg of faeces in 5 ml of extraction buffer and analysing the supernatant with an ELISA (IBD-Scan, Techlab, Blacksburg, VA, USA) using wells coated with polyclonal antibody and a horseradish peroxidaseconjugated polyclonal antilactoferrin rabbit antibody. The enzyme reaction was detected with tetramethylbenzidine and peroxide at 5 nm. Tumour M-PK was quantified by ELISA (ScheBo Biotech, Giessen, Germany). One hundred milligram of faeces was extracted in ml of extraction buffer. The supernatant was analysed by an ELISA, which uses wells coated with monoclonal antibody and a second biotinylated monoclonal antibody to tumour M-PK with detection using a streptavidin peroxidase conjugate and photometric measurement of the enzyme reaction with tetramethylbenzidine at 5 nm. The limits of detection of the assays were calprotectin, mg/g, lactoferrin,. mg/g and for tumour M-PK, U/mL. Results Faecal samples were obtained from healthy subjects (7 females and 6 males) ranging in age from to 86 y. All samples tested negative for faecal occult blood. The faecal calprotectin, lactoferrin and tumour M-PK concentrations for the healthy subjects, according to age, are shown in Figure as logged data with the descriptive statistics of the non-transformed data in Table. Six additional subjects were excluded from the study on the basis of information in the questionnaire: history of irritable bowel syndrome (), awaiting colonoscopy (), taking non-steroidal anti-inflammatory drugs () and possible gastritis (). All other subjects were assumed to be healthy. Analysis was not performed for calprotectin in three cases and for tumour M-PK in two cases due to insufficient sample. The results of the Kruskal Wallis tests indicated that there was evidence of differences between age groups for each marker (calprotectin: P ¼.; lactoferrin: P,.; tumour M-PK: P ¼.). The follow-up multiple comparisons showed that there was insufficient evidence to support any significant differences between the 9, 9 and 59 y age groups, for any of the markers (all corrected P..9). However, for calprotectin there was evidence that, on average, the 9 y age group was significantly higher than both the 9 y age group (corrected P ¼.) and the 59 y age group (corrected P ¼.). Similarly, for lactoferrin, there was evidence to support that, on average, the 9 y age group was significantly higher than each of the 9, 59 and 6 y age groups (corrected P-values of.,. and., respectively). If the statistical differences between groups were ignored for lactoferrin, a reference range derived from all subjects would be 7.7 mg/g.

3 Joshi et al. Faecal calprotectin, lactoferrin and tumour M-PK in healthy volunteers 6 (a) 6 log e (Calprotectin + ) (b) log e (Lactoferrin + ) (c) log e (Tumour M-PK + ) Figure (a) Faecal calprotectin, (b) faecal lactoferrin and (c) faecal tumour M-PK results in healthy subjects shown as log-transformed data for each age band For tumour M-PK, the follow-up multiple comparisons indicated no difference between age groups. The suggested age-related reference ranges for calprotectin, lactoferrin and tumour M-PK are shown in Table. Discussion Calprotectin, lactoferrin and tumour M-PK are useful faecal markers and they are easily measured. The manufacturers quote reference ranges of,5 mg/g faeces for calprotectin and,7.5 mg/g for lactoferrin. These are comparable to the ranges we obtained for our largest calprotectin group Table Descriptive statistics for calprotectin, lactoferrin and tumour M-PK showing number of results (n), mean, standard deviation (SD) median and range for each age band n Mean SD Median Range Calprotectin mg/g mg/g mg/g mg/g Lactoferrin mg/g mg/g mg/g mg/g , , , , ,. 5.9 Tumour M-PK U/mL U/mL U/mL U/mL , , , , , 9. and for lactoferrin with differences between groups disregarded. Previous studies of calprotectin and lactoferrin have included measurements in healthy subjects (Table ), but no previous study has collected samples from healthy subjects and provided age-related references ranges determined from statistical principles. One of these studies 5 looked at subjects aged 5 7 y and found an increase in faecal calprotectin with age. There is much less information on faecal lactoferrin in healthy controls. A study in 56 subjects found a median concentration of. mg/g (range..55) and a more recent study (n ¼ ) showed a median of. mg/g (range.8.). Previous studies of tumour M-PK have included measurement in controls (Table ). Our results suggest that separate reference ranges are required for calprotectin and lactoferrin in children aged 9 y and that for calprotectin a separate reference range is also required in those 6 y. There are changes in both cellular and humoral immunity with age 6 and age-related changes in inflammatory cells in the gastrointestinal mucosa. 7 Other causes for the observed changes could include alterations in gut flora and changes in diet and lifestyle differences with age. 5 Faecal calprotectin concentrations are known to be considerably elevated in infants 8 and evidence on the effect of diet is conflicting. Lactoferrin is present in breast milk and is found in the faeces of breast-fed infants. 9 However, no child included in Table Suggested age-related reference ranges for calprotectin, lactoferrin and tumour M-PK Age (y) Calprotectin Lactoferrin 9s,66,9 59,5 h,:6 6, Tumour M-PK (U/mL) ",9:

4 6 Annals of Clinical Biochemistry Volume 7 May Table Concentrations of faecal calprotectin in healthy subjects taken from previous studies (adapted from Konikoff et al. ). (a) Healthy adults and (b) healthy children Median (a) Healthy adults Range Patients (n) Age (y) (b) Healthy children (mean) Table Concentrations of faecal tumour M-PK in healthy subjects taken from previous studies (U/mL) Median Range Controls (n) , 97 8 our study was being breast-fed to account for the higher lactoferrin concentrations in the youngest age band. The manufacturer s recommended reference range for the metabolomic marker tumour M-PK is, U/mL independent of age. The marker has been shown to be a useful screening tool for colorectal cancer, correlating with more advanced stages of cancer, 9, and has also been suggested to have potential as a marker in inflammatory bowel disease, alone or in conjunction with calprotectin,, so may become more relevant to the paediatric population. Our reference range for tumour M-PK seems higher than we might have expected and we are conducting larger studies to further evaluate our result. In conclusion, we have examined concentrations of faecal calprotectin, lactoferrin and tumour M-PK in samples from healthy subjects aged 86 y. Our findings suggest that there are no age-related changes in faecal tumour M-PK. However, in healthy normal subjects faecal calprotectin is higher in those aged. 9 and 6 y than in 59 y olds and lactoferrin is higher in 9 y olds than in y olds. The appreciation of age-related change is important to enable appropriate interpretation of faecal inflammatory markers in gastrointestinal disease. DECLARATIONS Competing interests: None. Funding: None. Ethical approval: From the Cornwall and Devon LREC (6/Q/). Guarantor: RMA. Contributorship: SJ, SL and RMA contributed to the design and practical aspects of the study. SC analysed the data and all authors were involved with writing of the paper. Editorial comment: Readers should be aware that the content of this research paper has changed substantially from the version that was originally published on-line on 9 September 9. A reappraisal of the data has resulted in substantial changes to the originally quoted reference ranges. One author of the originally posted article has disassociated herself from this final version of the paper. REFERENCES Hommes DW, van Deventer SJ. Endoscopy in inflammatory bowel diseases. Gastroenterology ;6:56 7 Fagerhol MK, Andersson KB, Naess-Andresen CF, Brandtzaeg P, Dale I. Calprotectin (the L leucocyte protein). In: Smith VL, Dedman JR, eds. Stimulus Response Coupling: The Role of Intracellular Calcium-Binding Proteins. Boca Raton, FL, USA: CRC Press Inc, 99:87 Roseth AG, Fagerhol MK, Aadland E, et al. Assessment of the neutrophil dominating protein calprotectin in faeces. A methodological study. Scand J Gastroenterol 99;7:79 8 Roseth AG, Aadland E, Jahsen J, Raknerud N. 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