Non invasive biomarker for assessment of villous abnormalities in patients with

celiac disease and other enteropathies: An alternative to mucosal biopsies

Alka Kumari1, Anil K Verma1,4, P Das2, G Jindal1, S Prakash1, B Nayak1, S Datta Gupta2, Lalit kumar3, Vineet Ahuja1, Govind K Makharia1
1
Department of Gastroenterology & Human Nutrition, 2Department of Pathology,
Department of Medical Oncology, BR Ambedkar Institute Rotary Cancer Center;
3

All India Institute of Medical Sciences, New Delhi, India.

Celiac Disease Research Laboratory, Department of Pediatrics, Università Politecnica delle Marche, Ancona, Italy
4

Background Methods
 Villous atrophy is hallmark of celiac disease (CeD). Study design: Single center case control study
Study period: June 2013 to May 2016
 Demonstration of villous abnormalities is a invasive procedure.
 There is a need for reliable non-invasive biomarker(s), which can Patients recruitment
demonstrate presence of villous atrophy.
 Such a biomarker(s) will be valuable not only in the diagnosis but Healthy control Celiac disease Other enteropathies
Disease control
monitoring of the disease. (HC) (n=209) (DC) (n=103) (CeD) (n=110) (OE) (n=46)
 We have shown earlier CYP3A4 expression and its function, as a
•Invitation for participation
biomarker of villous abnormality. Moron Blood •Inclusion/Exclusion criteria Blood Blood
B et.al, Am J Gastroenterol 2013 •Recruitment Duodenal Biopsy Duodenal Biopsy
3 months
(n=67) •Collection of samples
Follow-up
(n=43) 6 months (n=18) 6 months
GFD treatment
Aim Blood Blood
Blood Blood
Duodenal Biopsy Duodenal Biopsy
 To explore a non-invasive biomarker(s) for the assessment of villous Duodenal Biopsy
abnormalities in patients with celiac disease & other enteropathies.
Storage of samples at -800C
(Blood & Duodenal biopsy)
Markers in blood Markers in mucosal biopsy  To confirm reliability of above markers in a model where there is cyclical
changes in enteropathy (patients receiving myeloablative therapy for
Levels of citrulline in plasma – HPLC Expression of P5CS in mucosal biopsy – IHC hematopoietic stem cell transplantation (HSCTs) (Fig: 4A, B & C)
(Fig:1A) (Fig:1B)
 Only blood samples were collected at different time-points i.e. before
Levels of plasma I-FABP – ELISA Expression of I-FABP gene –qPCR (Fig: 2C)
and after transplantation (-7, -5, -1, 0, +7, +15 and +28th day)
(Fig:2A) Localization of I-FABP – IHC (Fig:2B)
Levels of serum Reg1α – ELISA Expression of reg1α gene –qPCR (Fig: 3C) Statistical analysis:
(Fig:3A) Localization of reg1α - IHC (Fig:3B)
 Statistical analysis was preformed by IBM SPSS version 20

Results
1. A).Levels of citrulline (marker of enterocyte’s synthetic function) in plasma & B). Localization of P5CS (rate limiting enzyme 4.Cyclical variations in the levels
of citrulline synthesis) in mucosal biopsies of controls, patients with CeD (both before and after GFD) and other enteropathies of citrulline & I-FABP in patients
receiving high-dose
Exp. Exp. chemotherapy for bone marrow
1. A
P=0.001
P=0.001
B) Exp.
transplantation (BMT)
P=0.001
P=0.001

Disease control CeD-BASE CeD-GFD Other enteropathies

2. A):I-FABP (marker of enterocyte damage) levels in plasma B). Localization in tissues & C). mRNA expression in
Sequential fall and rise in levels of plasma
tissues of controls, patients with CeD (both before and after GFD) and other enteropathies citrulline, changes similar to that seen in total
leucocyte counts, suggesting cyclical
2. A)
v
P=0.001 C) P=0.001 enterocyte injudy and and recovery of
P=0.001 B) Exp. Exp. Exp. P=0.64
P=0.26 enterocytes
P=0.06

Disease control CeD-BASE CeD-GFD

3. A):Reg1α (marker of enterocyte regeneration) levels in serum B). Localization & C). mRNA expression in mucosal
biopsies of controls, patients with CeD (both before and after GFD) and other enteropathies
Cyclical changes in levels of citrulline
3. A) B) C) P=1.0 correlates with total leukocyte count
Exp. Exp. Exp. P=0.01
P=0.04

Disease control CeD-BASE CeD-GFD

HC=Healthy control, DC= Disease control, CeD-BASE= CeD at baseline, CeD-GFD= CeD after gluten free diet, OE= Other enteropathies, No definite pattern in the levels of I-FABP
TLCs= Total leukocyte counts, Exp.= Expression, I-FABP= Intestinal fatty acid binding protein, Reg1α= Regenerating gene 1α

Conclusions
The consistent changes in all above experimental groups in the level of plasma citrulline suggest that citrulline is a reliable
marker for estimation of enterocyte mass for diagnosis and monitoring of villous abnormalities.
Authors declare no conflict of interest