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INTESTINAL EXPRESSION AND FUNCTIONAL ANALYSIS OF ORGANIC CATION / L-CARNITINE TRANSPORTER (OCTN) IN CROHN’S DISEASE
M Girardin, I Qureshi, S Dionne, R Battat, A Bitton, P Charlebois, G Wild, K Waschke, EG Seidman
McGill IBD Research Group, McGill University Health Centre, Montreal, Quebec
BACKGROUND: Recent genetic studies implicate the IBD5 locus (5q31) as a risk factor for Crohn’s Disease (CD). This gene codes for the organic cation/carnitine transporters (OCTN1, 2 & 3) that transport carnitine, an essential cofactor for long-chain fatty acid oxidation, as well as many important organic cations. Two variants in the OCTN cluster have been reported, forming a haplotype associated with susceptibility to CD. These variants were shown to be associated with modified transporter functions of OCTN1 and 2 in vitro, using fibroblast cultures. OCTN1 is a multispecific and bidirectional organic cation transporter, Na+-independent and pH-dependent, with a low affinity for carnitine. OCTN2 is a Na+-dependent transporter with a high affinity for carnitine. No study has yet investigated the functional aspects of intestinal OCTNs, including transport of carnitine in inflammatory bowel disease (IBD).
PATIENTS & METHODS: We collected intestinal tissue from endoscopic biopsies (n=42) and surgical resections (n=12) in consenting IBD patients (n=28) as well as normal controls (n=18 & 8, respectively). OCTN protein levels were assayed by Western Blot analysis using intestinal biopsy homogenates. Functional analyses were performed utilizing brush border membrane (BBM) vesicles isolated from the intestinal resections. Carnitine transport was measured across the apical membrane using H3 radiolabeled substrate.
RESULTS: Western blots confirmed the presence of intestinal OCTN1 (50 KDa) and OCTN2 (70 KDa) in both IBD and control patients’ tissue. Using BBM vesicles, we observed very rapid, Na+ dependent transport of carnitine across the apical intestinal border (5-10 sec). Our preliminary findings revealed similar amounts of carnitine transport between the CD and the control patient’s tissue samples: 0.13 vs 0.10 [L-H3Carnitine pmol/mg protein/min].
CONCLUSIONS: These studies show, for the first time, the presence of OCTN transporters in intestinal tissue from IBD patients. Although transport of carnitine does not appear to be altered in CD vs. Controls, our preliminary results require further study in a larger cohort of patients. Moreover, genetic analyses of common mutations in the OCTN genes are also currently pending. These studies provide encouraging preliminary results that will permit us to compare OCTN phenotypic or functional attributes with the corresponding genotypic assessment in CD and control patients.
Supported by an Innovations Grant from the CCFC