To investigate the potential of proton (¹H) nuclear magnetic resonance (NMR) spectroscopy in characterization of different Marsh Grades of villous atrophy in intestinal mucosal biopsies of patients with Celiac disease (CeD).Total 24 patients (mean age 28.3 ± 8.2 years) were recruited. An informed consent was taken and the study was approved by Institute Ethics Committee. All patients were treated according to standard regimen. Diagnosis of CeD was made on the basis of European Society of Pediatric Gastroenterology Hepatology and Nutrition which included combination of clinical manifestations, celiac specific serological markers and histological abnormalities. Modified Marsh-Oberhuber classification was used for histopathological evaluation of biopsies. Grade 0 corresponded to normal histology, while Grade 1 referred to increase of intraepithelial lymphocytes (IEL); Grade 2 corresponded to increased IELs along with crypt hyperplasia. Grade 3 referred to increased IELs along with crypt hyperplasia and variable degrees of villous atrophy. A further semi-quantitative sub-typing of the villous atrophies was performed as follows: Grade 3a denoted mild villous atrophy; Grade 3b denoted moderate villous atrophy while Grade 3c denoted total villous atrophy. Of 24 patients, 4 were with Grade 3a, 8 with Grade 3b and 12 with Grade 3c. Small intestinal mucosal biopsies (5-6 bits) were collected at the time of endoscopy and water soluble metabolites were extracted using perchloric acid extraction. The resultant lyophilized powder was dissolved in deuterium oxide and sodium trimethyl-silyl-[2, 2, 3, 3-H₄] was used as chemical shift reference. ¹H one dimensional and two dimensional total correlation spectroscopy NMR experiments were performed at 700 MHz (Agilent, U.S.A.). Each 1D spectrum was integrated and intensities for various peaks corresponding to different metabolites were determined using VnmrJ software (Agilent, Technologies). Partial least squares-discriminant analysis (PLS-DA) was performed on integrated intensities using Unscrambler 10.2 (CAMO Software, Oslo, Norway). This is the first study B to our knowledge that demonstrated differences in metabolic profile of intestinal biopsy of CeD patients with different Marsh Grades. Comparison of metabolic profile of Grade 3a patients could not be done as number of patients was less. PLS-DA plot showed clear distinction between CeD patients with Grade 3c and Grade 3b (Fig. 1). Statistical models from PLS-DA analysis showed the cumulative explained variance of (R²) of 0.95 and cross validated predictive fraction (Q²) of 0.83. The corresponding loading plot suggested that metabolites; alanine, pyruvate, succinate, creatine and acetoacetate contributed for the distinct clustering of the Grade 3c and Grade 3b patients (Fig. 2). The levels of these metabolites were higher in CeD patients with Grade 3c compared to patients with Grade 3b. In a previous study, significantly higher concentrations of isoleucine, leucine, aspartate, succinate and pyruvate compared to controls were reported¹. Abnormalities in glycolysis, Kreb’s cycle (energy deficiency) and amino acid metabolism contributing to villous atrophy were suggested¹. Thus, higher levels of several metabolites seen in Grade 3c patients compared to Grade 3b indicated a relation between degree of severity of villous atrophy and metabolic abnormalities. Higher levels of pyruvate seen in patients with Grade 3c compared to Grade 3b suggested more impairment in utilization of pyruvate for energy generation. Further, raised alanine level suggested that pyruvate may be converted to alanine by transamination in Grade 3c patients compared to Grade 3b patients. Additionally, higher level of succinate in the Grade 3c patients compared to Grade 3b patients further supported decreased oxidative phosphorylation. Further our results showed higher level of acetoacetate in Grade 3c patients compared to Grade 3b patients suggesting the use ketone bodies as energy fuel. Since, the energy derived through oxidations of ketone bodies was less compared to carbohydrate oxidation; the intestinal mucosa in Grade 3c patients was more energy deprived compared to Grade 3b patients. The energy produced from the oxidation of ketones may not be sufficient for rapid turnover of enterocytes which is required to maintain normal villous length,² thereby leading to severe villous atrophy as seen in Grade 3c patients. Moreover, elevated level of creatine was observed in Grade 3c patients compared to Grade 3b. In general, creatine is degraded by intestinal bacteria, thus, elevated creatine level may be because of reduced bacterial degradation suggesting that imbalance of gut microflora was more pronounced in Grade 3c patients. These metabolic abnormalities may probably contribute to severe villous atrophy seen in Grade3c patients. Present NMR metabonomics study provides an insight into the biochemical changes associated with the different mucosal histological grades. Metabolites like pyruvate, alanine and succinate may have potential to serve as biomarker/s for assessment of villous abnormality.