Retinol esterification by microsomes from the mucosa of human small intestine. Evidence for acyl-coenzyme A retinol acyltransferase activity

Abstract

The mechanism of the intestinal esterification of retinol has been obscure. Recently, an acyl-Coenzyme A (CoA):retinol acyltransferase (ARAT) was found in rat intestinal microsomes, and experiments were therefore conducted to determine whether a corresponding enzyme exists in human small intestine. When microsomes were incubated with [3H]retinol and palmitoyl-CoA, or retinol and [1-14C]palmitoyl-CoA, radioactive retinyl palmitate was formed as identified by alumina column chromatography and reverse-phase high-pressure liquid chromatography. Heating the microsomes for 30 min at 60°C resulted in loss of activity. The esterification was negligible without exogenous acyl-CoA and markedly stimulated by palmitoyl-, oleoyl-, and stearoyl-CoA in concentrations up to 20 μM. The acyl-CoA was successfully replaced by an acyl-CoA generating system, but not by unactivated palmitate (2.5-200 μM). The assay was dependent on the presence of albumin with optimum activity at 2-10 mg/ml. The optimal retinol concentration was 20-30 μM and pH ~7.4. The esterifying activity was completely inhibited by 8 mM of taurocholate and to 90% by 1 mM of 5,5'-dithiobis(2-nitrobenzoic acid). Activity was found throughout the small intestine. In jejunum the rate of retinol esterification was: 3.44 ± 2.24 nmol [3H]retinyl ester formed.mg microsomal protein-1.min-1 (mean ± SD, n = 12). The corresponding activity in whole homogenates of biopsies were 1.17 ± 0.28 (n = 8). It is concluded that human small intestine contains a microsomal acyl-CoA:retinol acyltransferase. Due to its high activity in vitro this enzyme is likely to be responsible for the intestinal esterification of retinol.