To investigate the effect of gluten-free diet (GFD) on the metabolic profile of small intestinal mucosa of patients with celiac disease (CeD) using in-vitro proton (¹H) nuclear magnetic resonance (NMR) spectroscopy.The immune response in CeD is known to trigger by the ingestion of gluten and related proteins present in cereals such as wheat, rye and barley. The inflammation of small intestine leads to villous abnormalities affecting the absorption of nutrients in CeD. Currently, lifelong exclusion of gluten from the diet is the only treatment for CeD which has been shown to reduce histological abnormalities in mucosa and improve nutritional status. Thus, it is important to understand the biochemical changes associated with mucosal recovery in CeD. Therefore, in the present study, the metabolic profile of small intestinal mucosal biopsies of patients with CeD prior to and after GFD was investigated using in vitro ¹H NMR spectroscopy and multivariate data analysis methods. Eleven patients (mean age 24.2 + 12.9 years) were recruited for this study. The diagnosis of CeD was made on the basis of European Society of Pediatric Gastroenterology Hepatology and Nutrition. All eleven patients followed strict GFD for six months. An informed consent was taken and the institute Ethics committee approved the study. Small intestinal mucosal biopsy samples (6 bits) were collected at the time of endoscopy from all CeD patients, both before and after GFD. Water soluble metabolites were extracted using perchloric acid extraction method and lyophilized powder obtained was dissolved in deuterium oxide. Sodium sodium trimethyl-silyl-[2, 2, 3, 3-H₄] was used as a standard for chemical shift and quantification of metabolites. One dimensional and two dimensional total correlation spectroscopy NMR experiments were carried out at 700 MHz (Agilent, U.S.A.). Comparison of metabolites in CeD patients before and after GFD were carried out using paired t-test (SPSS 20.0, Inc., Chicago, IL). A p-value <0.05 was considered significant. Partial least squares-discriminant analysis (PLS-DA) was also performed using Unscrambler 10.2 (CAMO Software, Oslo, Norway).To the best of our knowledge, this is the first study that elucidated the effect of GFD on the metabolic profile of intestinal mucosa of patients with CeD. Table 1 presents the concentration of metabolites in the intestinal mucosa of CeD patients before and after GFD. Our data showed that concentrations of glutamate (Glu) and glutamine (Gln) were significantly decreased after GFD. While a significant increase in the concentration of glycrosphosphocholine (GPC) was seen after GFD. PLS-DA of concentration data classified the samples from CeD patients before and after GFD in separate clusters (Fig.1). The model was validated [R² (0.99) and Q² (0.91)] and loading plot indicated that Glu, glycine and allantoin have significantly contributed for the separation of groups (Fig.2). It was reported that Glu and Gln are the primary oxidative fuels used by intestinal epithelial cells for energy generation¹. A significant decrease in the concentration of Glu and Gln in the intestinal mucosa of patients after GFD indicated that these were being utilized for energy generation for the intestinal functions such as rapid renewal of enterocytes and absorption of nutrients. These biochemical changes lead to mucosal recovery in CeD patients. Further, our results showed significant increase in the concentration of GPC after GFD. We have earlier shown that concentration of GPC was significantly lower in treatment naïve CeD patients as compared to disease controls suggesting villous abnormalities². GPC is an important component of cell membrane and is essential for the regulation of cell growth and differentiation. Thus an increase in GPC levels following GFD in CeD patients indicated increase in membrane metabolism required for rapid turnover of enterocytes to maintain the villous length. Further, PLS-DA suggested that Glu, gly and allantoin were significant contributors for separation of CeD patients before and after GFD. Allantoin is a biomarker of oxidative stress; hence its decrease suggested reduced inflammation of mucosa in CeD patients after GFD.Present study demonstrated the biochemical changes associated with villous abnormalities and its recovery after GFD and suggested that Glu, Gln and GPC are important metabolites for maintaining intestinal integrity. The results may have implications in determining alternative biomarker/s for diagnosis and treatment management of CeD.