
==== Front
Rev Assoc Med Bras (1992)
Rev Assoc Med Bras (1992)
ramb
Revista da Associação Médica Brasileira
0104-4230
1806-9282
Associação Médica Brasileira

00614
10.1590/1806-9282.20240704
Original Article
Can serum M30 levels be utilized as an activation marker in patients with ulcerative colitis?
https://orcid.org/0000-0003-0040-1982
Binicier Omer Burcak 1 *Conceptualization Data curation Formal Analysis Writing – original draft
https://orcid.org/0000-0001-8830-0371
Sarı Sevil Ozer 1 Data curation
https://orcid.org/0000-0001-5918-6178
Pakoz Zehra Betul 2 Conceptualization Data curation Formal Analysis Writing – original draft
https://orcid.org/0000-0002-1483-997X
Basok Banu Isbilen 3 Formal Analysis Writing – original draft
1 University of Health Sciences Turkey, Izmir Faculty of Medicine, Department of Gastroenterology – İzmir, Turkey.
2 Katip Celebi University, Ataturk Training and Research Hospital, Department of Gastroenterology – İzmir, Turkey.
3 University of Health Sciences Turkey, Izmir Faculty of Medicine, Department of Medical Biochemistry – İzmir, Turkey.
*Corresponding author: binicieromer@yahoo.com
Conflicts of interest: the authors declare there is no conflicts of interest.

13 9 2024
2024
70 9 e2024070417 5 2024
17 6 2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
SUMMARY

OBJECTIVE:

Ascertainment of disease activation is an important component of therapeutic decisions in ulcerative colitis patients and may present certain clinical challenges. The objective of this study was to determine serum levels of the M30 fragment of cytokeratin 18 and its utility as an activation marker in patients with ulcerative colitis, who are known to have increased apoptosis.

METHODS:

A total of 60 ulcerative colitis (30 active and 30 remission) patients aged over 18 years and 29 healthy individuals as controls were included in the study. M30, C-reactive protein, and mean platelet volume were evaluated in all participants and compared between ulcerative colitis patients and controls, as well as between those with active disease or remission.

RESULTS:

Although ulcerative colitis patients with active disease had higher M30 levels than those in remission, the difference was not statistically significant (p=0.085). The mean M30 levels tended to increase with increasing extent of involvement, although the differences were not significant (p=0.065). The comparison of C-reactive protein and mean platelet volume according to the site of involvement, however, showed significant differences (p=0.02 and 0.004, respectively). M30 did not show significant correlations with C-reactive protein, mean platelet volume, and Mayo Score (p=0.0834, 0.768, and 0.401, respectively).

CONCLUSIONS:

Our results suggest that, in contrast to C-reactive protein and mean platelet volume, M30 levels do not have a significant role as an activation marker in ulcerative colitis patients. Thus, we believe that M30 may not represent an appropriate marker to be used for this purpose.

KEYWORDS:

Ulcerative colitis
M30
Apoptosis
Inflammation
Cytokeratin-18
==== Body
pmcINTRODUCTION

Ulcerative colitis (UC) is an inflammatory bowel condition that affects the colonic mucosa and is clinically characterized by activations/remissions during the course of the disease 1 . It may lead to proctitis, left-sided colitis, or extensive colonic involvement. Although genetic and environmental factors have been implicated in its etiology, the exact cause remains unknown 2,3 . An increased number of T lymphocytes in the colonic mucosa represents the fundamental histologic finding, and apoptosis occurring in association with impaired T cell functions plays an important role in its pathogenesis 4 . T lymphocytes increase the expression of Fas-ligands, and increased apoptosis is observed in colonic cells with Fas expression. At the same time, increased Fas-ligation results in the activation and migration of inflammatory cells, leading to augmented apoptotic injury in colonic epithelial cells as an important pathogenetic mechanism 5 .

Ascertainment of disease activation is an important component of therapeutic decisions in UC patients and may present certain clinical challenges, including the invasive nature of colonoscopy, radiation exposure, and the inability of current activation markers to arrive at an accurate differential diagnosis 6,7 . Therefore, the identification of non-invasive and practical methods that will allow detecting UC activation represents an interesting research area 8,9 .

Cytokeratins are an epithelial-specific subgroup of intermediate filament proteins that are mainly involved in protecting cells from apoptosis and necrosis 10 . Increased serum levels of cytokeratins are a marker for apoptosis 11 . Until now, the M30 fragment of cytokeratin 18 has been found to be associated with disease severity and prognosis in a number of different conditions, including hepatocellular carcinoma, gastric carcinoma, non-alcoholic fatty liver disease, and hepatitis B 12-15 .

The objective of this study was to determine serum levels of the M30 fragment of cytokeratin 18 and its utility as an activation marker in patients with UC, who are known to have increased apoptosis.

METHODS

Patient selection: A total of 60 UC patients aged over 18 years treated with a diagnosis of UC in our unit between December 2018 and May 2019 were included. Also, 29 healthy ­individuals served as controls. The diagnosis was based on clinical, ­endoscopic, and histopathological findings.

Study design: Patients were categorized into two groups, namely, those who had active disease based on clinical and endoscopic findings and those who were in remission. The disease activation was assessed using the Mayo Score, which rated stool frequency, rectal bleeding, mucosal appearance, and Physician's Global Assessment on a 0–3-point scale, yielding a total Mayo Score between 0 and 12 16 . The site of involvement was grouped into three, namely, proctitis, left colitis, and extensive colitis. Also recorded were the ­medications used for UC treatment. M30, C-reactive protein (CRP), and mean platelet volume (MPV) were evaluated in all ­participants and compared between UC patients and controls, as well as between those with active disease or ­remission. The ­correlations between M30 and CRP, MPV, and Mayo Score were analyzed.

Exclusion criteria: Age under 18 years, history of intestinal ­surgery, presence of active infection, coexistent hepatic and/or renal failure, presence of chronic disorders, pregnancy, and lactation.

Laboratory methods: After an overnight fast, venous blood ­samples were collected into plain blood collection tubes (BD Vacutainer® SST II Advance Tube, 5 mL, 13×100 mm, United States). Serum ­samples were separated from cellular fragments by centrifugation for 10 min at 1,500 g within 1 h after blood sampling. Serum samples were aliquoted and stored at -80°C until further analysis.

Serum M30 concentrations were determined by a ­commercial kit employing a quantitative sandwich enzyme ­immunoassay technique (Human Cytokeratin 18-M30, SinoGeneClon Biotech Co. Ltd., Hangzhou, China).

STATISTICAL ANALYSIS

Statistical analysis of the study was done using SPSS 25.0 (IBM Statistical Package for Social Sciences software version 25). Continuous variables were expressed as a ­mean±standard ­deviation and categorical variables as a percentage. The chi-square test was used to compare categorical values, and the Mann-Whitney U test was used to compare continuous ­variables between groups. Receiver operating characteristic (ROC) ­analysis was performed to calculate the cutoff values. An area under curve (AUC), positive predictive value (PPV), and negative predictive value (NPV) were obtained. Correlations between M30, CRP, and Mayo Score were determined using Spearmen's rho test. A p-value less than 0.05 was considered statistically significant.

Ethical considerations: The study protocol was approved by the local ethics committee (approval no: 2019/14-37). All patients and controls provided written informed consent for study participation.

RESULTS

Of the 60 UC (30 active, 30 remission) patients and 29 healthy controls, 47 (52.8%) were male and 42 (47.2%) were female. Women comprised 40 and 62.1% of the patient and control groups, respectively. There was a significant gender difference between patient and control subjects (p=0.005). The mean age was 45.1±13.8 years (range: 18–71 years) in patients diagnosed with UC patients and 46.0±15.4 years (range: 18–81 years) among controls, with no significant difference (p=0.366). Patients with active disease or in remission were ­comparable in terms of the site of involvement, disease duration, and treatments received (p=0.148, 0.450, and 0.196, respectively).

Ulcerative colitis patients and controls had comparable M30 levels (p=0.132). Although UC patients with active ­disease had higher M30 levels than those in remission, the difference was not statistically significant (p=0.085). However, MPV levels were significantly different between patients with active disease or in remission (p=0.01). Also, significant differences in CRP were found both between UC patients and controls and between UC patients who had active disease and were in remission (p=0.03, and<0.001, respectively). Table 1 shows a comparison of UC patients with active disease or in remission with respect to laboratory parameters.

Table 1 Comparison of ulcerative colitis patients with active disease or in remission versus control subjects.

	Active (n=30)	Remission (n=30)	Controls (n=29)	p	
M30, pg/mL (mean±SD)	605.34±794.63	307.56±116.85	360.53±96.47	0.132*
0.085**	
CRP, mg/dL (mean±SD)	4.99±4.79	0.45±0.44	0.33±0.4	0.03*
<0.001**	
MPV, fL (mean±SD)	8.19±1.23	8.8±0.95	8.54±0.75	0.01*
0.806**	
SD: standard deviation; CRP: C-reactive protein; MPV: mean platelet volume.

* Comparison for active disease-remission.

** Comparison between UC patients and controls.

Also, M30, CRP, and MPV differences according to the site of involvement were also examined. The mean M30 ­levels tended to increase with increasing extent of involvement, although the differences were not significant (p=0.065). The comparison of CRP and MPV according to the site of involvement, however, showed significant differences (p=0.02 and 0.004, respectively). Table 2 summarizes M30, CRP, and MPV according to the site of involvement.

Table 2 M30, C-reactive protein, and mean platelet volume levels according to the site of disease involvement.

	Rectum (n=12)	Left colitis (n=25)	Extensive colitis (n=23)	p	
M30, pg/mL (mean±SD)	272.7±79.9	475.4±490.9	531.9±787.4	0.065	
CRP, mg/dL (mean±SD)	0.78±0.94	1.50±1.94	5.06±5.53	0.02	
MPV (fL) (mean±SD)	8.31±0.76	9.1±1.12	7.98±1.03	0.004	
CRP: C-reactive protein; MPV: mean platelet volume; SD: standard deviation.

Correlations between M30, CRP, MPV, and Mayo Score were evaluated. Accordingly, M30 did not show significant correlations with CRP, MPV, and Mayo Score (p=0.0834, 0.768, and 0.401, respectively). However, there was a significant correlation between MPV and CRP (p=0.036). Similarly, Mayo Score and CRP were significantly correlated (p<0.001).

A ROC curve analysis was carried out to examine the ­efficiency of M30, CRP, and MPV in assessing the disease ­activity in UC patients. This analysis showed that while MPV and CRP had significant efficiency, M30 had no statistically ­significant role in showing the disease activity (p=0.014, ≤0.001, and =0.132, respectively) (Figure 1).

Figure 1 Mean and confidence interval for scores in question 4, according to observer group.

DISCUSSION

Non-invasive methods for ascertaining UC activation have been an interesting research area. Obviously, such non-­invasive ­methods should ideally be practical and reliable, as well as ­represent a viable alternative for endoscopic procedures. Currently, CRP and calprotectin are commonly used for this purpose 17 . On the contrary, CRP is frequently elevated in many other acute conditions, such as infections, limiting its utility in UC activation assessments 18 . Reduced MPV is another parameter used to evaluate UC activation although similar to CRP, MPV ­levels may also be diminished in certain infectious, rheumatoid, or cardiac conditions 19,20 .

Cytokeratin 18 is synthesized in epithelial cells and is involved in many cellular processes such as apoptosis, mitosis, and stress response. Serum levels of the M30 fraction of cytokeratin 18 have been investigated in hepatic, lung, kidney, and many other cancer types, with some studies reporting significant results 21 . M30, which is a substrate for caspase 3, is a marker of apoptosis. M30 serum levels are increased due to the loss of membrane integrity following cell death and can be detected using the ELISA method 16,22 . These observations suggested that M30 may also represent an appropriate marker for assessing the disease activity in UC since apoptosis plays a major role in the pathogenesis of UC. Apoptosis is increased mainly due to increased activity of TNF, interleukins, and interferons. Also, inflammation destroys the integrity of the mucosal barrier in patients with acute UC activation 23 .

Drug therapy targeted at reducing apoptosis in colonic cells is associated with mucosal healing in UC patients 24 . Furthermore, endoscopic activation and microscopic ­inflammation are strongly linked with epithelial apoptosis 25 . Thus, an assumption was made that M30 could assist in differentiating active UC patients and patients in remission. In contrast with CRP levels, UC patients and control subjects did not differ significantly in M30 levels in the current study. Again, in contrast with CRP and MPV, UC patients with active disease or in remission were not significantly different in terms of M30 levels as well. We also failed to find any correlations between M30, CRP, MPV, and Mayo Score. A ROC curve analysis suggested a significant association between UC disease activity and MPV and CRP, while no such associations were observed for M30.

In a 2012 study by Aktaş et al., patients with active UC had significantly higher M30 levels as compared to those in remission, supporting the role of increased apoptotic activity in subjects with active disease 26 . Although mean M30 levels were highest in patients with active disease and lowest in controls, the differences were not statistically significant in the current study. Again, patients in remission and with active disease did not differ significantly in this respect.

Another hypothesis that was tested was that increased length of disease involvement would be associated with increasing levels of M30. In a study by Ueyama et al., increased FasL expression was found in patients with active extensive colitis and in those with left-sided involvement, while those with rectal involvement only had no such increase, suggesting that patients with proctitis may have less apoptotic activity 5 . Thus, we assumed that M30 levels would be higher with increasing disease extent, due to increased inflammation and apoptosis. Despite a trend toward increased M30 levels with increasing extent of the disease as compared to those with rectal involvement only, these differences did not reach statistical significance. Conversely, a significant association of CRP and MPV with the site of involvement was found.

The most important limitation of our study is the small sample size. Studies with larger sample sizes would certainly provide more meaningful results. Also, there was a significant gender difference between patients and controls. Treatments administered for UC may have an effect on the measured levels of the parameters investigated. Again, no histological assessment of the inflammation and apoptosis was performed. Another limitation was that we did not correlate M30 levels with fecal calprotectin. Fecal calprotectin cannot be used routinely in our country yet since only a few laboratories can perform the test.

CONCLUSION

Our results suggest that, in contrast to CRP and MPV, M30 levels do not have a significant role as an activation marker in UC patients. Thus, we believe that M30 may not represent an appropriate marker to be used for this purpose. However, based on certain trends observed in this study as well as the published data, we recommend further studies with larger sample sizes.

Funding: none.
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REFERENCES

1 Ordás I Eckmann L Talamini M Baumgart DC Sandborn WJ Ulcerative colitis Lancet 2012 380 9853 1606 1619 10.1016/S0140-6736(12)60150-0 22914296
2 Orholm M Munkholm P Langholz E Nielsen OH Sørensen TI Binder V Familial occurrence of inflammatory bowel disease N Engl J Med 1991 324 2 84 88 10.1056/NEJM199101103240203 1984188
3 López-Serrano P Pérez-Calle JL Pérez-Fernández MT Fernández-Font JM Boixeda Miguel D Fernández-Rodríguez CM Environmental risk factors in inflammatory bowel diseases. Investigating the hygiene hypothesis: a Spanish case-control study Scand J Gastroenterol 2010 45 12 1464 1471 10.3109/00365521.2010.510575 20704469
4 Sturm A Souza HS Fiocchi C Mucosal T cell proliferation and apoptosis in inflammatory bowel disease Curr Drug Targets 2008 9 5 381 387 10.2174/138945008784221198 18473766
5 Ueyama H Kiyohara T Sawada N Isozaki K Kitamura S Kondo S High Fas ligand expression on lymphocytes in lesions of ulcerative colitis Gut 1998 43 1 48 55 10.1136/gut.43.1.48 9771405
6 Chatu S Subramanian V Pollok RC Meta-analysis: diagnostic medical radiation exposure in inflammatory bowel disease Aliment Pharmacol Ther 2012 35 5 529 539 10.1111/j.1365-2036.2011.04975.x 22239831
7 Vrabie R Kane S Noninvasive markers of disease activity in inflammatory bowel disease Gastroenterol Hepatol (N Y) 2014 10 9 576 584 27551251
8 Smith LA Gaya DR Utility of faecal calprotectin analysis in adult inflammatory bowel disease World J Gastroenterol 2012 18 46 6782 6789 10.3748/wjg.v18.i46.6782 23239916
9 Manolakis AC Kapsoritakis AN Tiaka EK Potamianos SP Calprotectin, calgranulin C, and other members of the s100 protein family in inflammatory bowel disease Dig Dis Sci 2011 56 6 1601 1611 10.1007/s10620-010-1494-9 21203903
10 Ku NO Strnad P Bantel H Omary MB Keratins: biomarkers and modulators of apoptotic and necrotic cell death in the liver Hepatology 2016 64 3 966 976 10.1002/hep.28493 26853542
11 Oshima RG Apoptosis and keratin intermediate filaments Cell Death Differ 2002 9 5 486 492 10.1038/sj.cdd.4400988 11973607
12 Elalfy H Besheer T Arafa MM El-Hussiny MA Latif MA Alsayed SAM Caspase-cleaved cytokeratin 18 fragment M30 as a potential biomarker of macrovascular invasion in hepatocellular carcinoma J Gastrointest Cancer 2018 49 3 260 267 10.1007/s12029-017-9937-6 28361205
13 Huang Y Yang L Lin Y Chang X Wu H Chen Y Prognostic value of non-invasive serum Cytokeratin 18 detection in gastrointestinal cancer: a meta-analysis J Cancer 2019 10 20 4814 4823 10.7150/jca.31408 31598152
14 Xue L Lu X He J Zhang T Wu X Zhang Y Serum CK 18-M30 reflect liver pathological severity during NAFLD progression in a rat model Pathol Res Pract 2018 214 11 1778 1786 10.1016/j.prp.2018.08.016 30149902
15 Yilmaz B Aktas B Altinbas A Ginis Z Ozturk G Ekiz F The role of M30 in predicting the severity of liver fibrosis and inflammation in chronic hepatitis B patients Hepat Mon 2016 16 9 e35640 10.5812/hepatmon.35640 27822256
16 Feldstein AE Wieckowska A Lopez AR Liu YC Zein NN McCullough AJ Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: a multicenter validation study Hepatology 2009 50 4 1072 1078 10.1002/hep.23050 19585618
17 Vermeire S Assche G Rutgeerts P. C-reactive protein as a marker for inflammatory bowel disease Inflamm Bowel Dis 2004 10 5 661 665 10.1097/00054725-200409000-00026 15472532
18 Vermeire S Assche G Rutgeerts P Laboratory markers in IBD: useful, magic, or unnecessary toys? Gut 2006 55 3 426 431 10.1136/gut.2005.069476 16474109
19 Yüksel O Helvaci K Başar O Köklü S Caner S Helvaci N An overlooked indicator of disease activity in ulcerative colitis: mean platelet volume Platelets 2009 20 4 277 281 10.1080/09537100902856781 19459134
20 Korniluk A Koper-Lenkiewicz OM Kamińska J Kemona H Dymicka-Piekarska V Mean platelet volume (MPV): new perspectives for an old marker in the course and prognosis of inflammatory conditions Mediators Inflamm 2019 2019 9213074 9213074 10.1155/2019/9213074 31148950
21 Oshima RG Baribault H Caulín C Oncogenic regulation and function of keratins 8 and 18 Cancer Metastasis Rev 1996 15 4 445 471 10.1007/BF00054012 9034603
22 Kramer G Erdal H Mertens HJ Nap M Mauermann J Steiner G Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18 Cancer Res 2004 64 5 1751 1756 10.1158/0008-5472.can-03-2455 14996736
23 Edelblum KL Yan F Yamaoka T Polk DB Regulation of apoptosis during homeostasis and disease in the intestinal epithelium Inflamm Bowel Dis 2006 12 5 413 424 10.1097/01.MIB.0000217334.30689.3e 16670531
24 Che L Li Y Song R Qin C Hao W Wang B Anti-inflammatory and anti-apoptosis activity of taraxasterol in ulcerative colitis in vitro and in vivo Exp Ther Med 2019 18 3 1745 1751 10.3892/etm.2019.7736 31410133
25 Seidelin JB Nielsen OH Epithelial apoptosis: cause or consequence of ulcerative colitis? Scand J Gastroenterol 2009 44 12 1429 1434 19958058
26 Aktaş B Altınbaş A Başar O Yılmaz B Ekiz F Giniş Z Serum m 30 levels reflects ulcerative colitis activity Inflamm Bowel Dis 2013 19 11 2400 2403 10.1097/MIB.0b013e3182a0ea38 23945185
