Transport of beta-lactoglobulin across rabbit ileum in vitro

Abstract

Intestinal transepithelial transport constitutes a major limiting step in the transfer of food protein antigens to the blood. This transport was studied in isolated rabbit ileum in Ussing chamber in vitro for the milk protein antigen beta-lactoglobulin (beta-Lg). The transepithelial passage of beta-Lg was measured by enzyme-linked immunosorbent assay (ELISA) and radiolabeled protein transfer and compared with that of the nonmetabolizable marker polyethylene glycol (PEG)-4000. When 1 mg/ml of beta-[14C]Lg or [3H]PEG was added to the mucosal side of the tissue, the total uptake, measured as the transfer of radiolabeled material across the ileum, was significantly higher for beta-Lg than for PEG (5.46 +/- 1.75 vs. 1.43 +/- 0.26 micrograms.h-1.cm-2). Measured by ELISA, 6-9% of the total amount of beta-Lg transported was absorbed in an intact antigenic form. This transport of intact beta-Lg was inhibited by the metabolic inhibitors 50 mM 2-deoxyglucose and 1 mM azide added simultaneously, was reduced by the microtubule assembly inhibitor 0.05 mM colchicine, and was enhanced by 20 mM ammonia, which inhibits lysosomal proteolytic activity. These results indicate that beta-Lg is efficiently absorbed by the intestinal mucosa of adult animals, partly in intact antigenic form and that beta-Lg transport is probably transcellular, as observed for other proteins. The finding that beta-Lg is absorbed in intact antigenic form agrees with other reports implying that beta-Lg is the main factor responsible for milk protein immunoreactivity and intolerance.

In vitro transport of β-lactoglobulin across the jejunum of lactose-fed rats

“…The jejunum (beginning 10 cm distal to the Treitz ligament) was removed, rinsed with cold saline, and mounted in the Ussing chamber, as previously described (Li et al, 1989). (Madara, 1989 (Cornell et al, 1971;Warshaw et al, 1974;Heyman et al, 1982;Marcon-Genty et al, 1989). In the present study, the rate of j3-LG absorption through rat jejunum in vitro, as determined by transfer of radioactive material, was slightly higher than that previously reported for rabbit ileum (Marcon-Genty et al, 1989).…”

“…(Madara, 1989 (Cornell et al, 1971;Warshaw et al, 1974;Heyman et al, 1982;Marcon-Genty et al, 1989). In the present study, the rate of j3-LG absorption through rat jejunum in vitro, as determined by transfer of radioactive material, was slightly higher than that previously reported for rabbit ileum (Marcon-Genty et al, 1989). The nature of the absorbed material provides information on the intracellular processing which occurs during protein transport (Walker at al, 1972;Stern and Walker, 1984 (Armbrecht and Wasserman, 1976;Andrieux et al, 1982;Debiec et al, 1985), steroid absorption (iritani and Wells, 1966) and epithelial cell turnover (Meslin et al, 1981 ).…”

“…In vitro experiments have led to the conclusion that an uptake of intact !-LG occurs in adult small intestine (Stern and Walker, 1984;Marcon-Genty et al, 1989). Up to 0.43 Ilgo h -1 ocm-2 antigenic P-LG crossed rabbit ileum mounted in the Ussing chamber (Marcon-Genty et al, 1989), and using isolated ileal loop in situ, up to 2.03 ng-mi-I of antigenic (3-LG was recovered in the portal blood per cm 2 of ileal mucosa (Gotteland et al, 1989).…”

“…Both histologic (Cornell et al, 1971;Walker et al, 1972) and metabolic (Heyman et al, 1982;Marcon-Genty et al, 1989) evidence has shown that protein transport across the epithelium is mainly transcellular and proceeds by endocytosis, intracellular transport and exocytosis through the basolateral membrane. Therefore, it has been suggested that modifications in enterocyte membrane composition may affect protein absorption by the gut (Udall and Walker, 1982).…”

Transport of β-lactoglobulin and α-lactalbumin in enterocyte-like Caco-2 cells

“…For both (3-Lg and a-La, a quantitative apical-to-basal transcytosis of the proteins was quite evident. It has previously been suggested that microtubules are implicated in this transport (Marcon-Genty et al, 1989;Caillard and Tome, 1992;Caillard and Tome, 1994).…”