Analytical subcellular fractionation of jejunal biopsy specimens: enzyme activities, organelle pathology and response to gluten withdrawal in patients with coeliac disease.

Abstract

1. Portions of jejunal biopsy specimens from patients with coeliac disease were homogenized in isotonic sucrose solution and subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. 2. The activities and distribution of marker enzymes for the principal subcellular organelles were determined by highly sensitive enzymic micro-assays employing fluorigenic and radiolabelled substrates. 3. In untreated coeliac disease the brush border showed decreased activities of five marker enzymes, which were reduced to up to one-eighth of control values. On sucrose density gradients the distinct brush-border peak at density 1.21 was replaced by a broad peak at density 1.16. 4. Two out of three lysosomal enzymes showed increased activities and there was evidence of enhanced fragility of both lysosomes and peroxisomes. 5. There were reduced activities of both mitochondrial and cytosol malate dehydrogenase, with increased activities of cytosol lactate dehydrogenase. 6. The endoplasmic reticulum and basal-lateral membranes were relatively unaffected except that the equilibrium densities were slightly increased and decreased respectively. 7. The patients were treated by gluten withdrawal and subsequent biopsies examined as above. Biopsies from patients treated for 2–4 weeks and who had not yet achieved a morphological improvement showed a partial return to normal of the pathological organelles. 8. Treated patients with partial villus atrophy showed progressively improved enzyme activities and a partial reappearance of the brush-border peak on the sucrose density gradients. 9. Patients whose intestinal mucosa had returned to morphological normality after prolonged gluten withdrawal showed a persistent defect in the brush-border enzyme activities. This was particularly striking for β-glucosidase, which was persistently deficient from the brush-border although approximately one-third of the total activity of this enzyme is normally found in this organelle. 10. The properties and enzyme activities of lysosomes, peroxisomes, endoplasmic reticulum, mitochondria and cytosol were essentially normal in the biopsies from fully treated patients.