Hamid M. Said

Biotin transport in the human intestine: Site of maximum transport and effect of pH*

Previous studies from our laboratory have characterized the transport process of biotin across the brush border membrane and the basolateral membrane of the human intestine. In this study we further characterized biotin transport in the human intestine by examining the vitamins transport process in different areas of the small intestine (duodenum, jejunum, and ileum) and the effect of pH on the transport process using a brush border membrane vesicle technique. In all areas examined, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear and lower in the presence of a choline gradient (out greater than in). Transport of biotin by the Na+-dependent process (i.e., the carrier-mediated process) was found to be higher in the duodenum than the jejunum, which was in turn higher than that in the ileum. This decrease in biotin transport distally was found to be due to a decrease in the Vmax of the transport process of the vitamin with no changes in the apparent Km. This indicates that the number (i.e., the density) of transport carriers for biotin decreases distally. In the presence of a Na+ gradient (out greater than in), decreasing incubation buffer pH from 8.0 to 5.5 (intravesicular pH was 7.4) was found to cause an increase in biotin transport. This increase was found to be due to the acidic buffer pH (i.e., not due to the pH gradient imposed across the membrane) and occurred through an increase in the transport of the vitamin by the nonmediated process. These results demonstrate that the proximal part of the small intestine is the site of maximum transport of biotin in humans. Furthermore, variation in incubation medium pH affects biotin transport through changes in the substrate transport by the nonmediated process.

Biotin transport in basolateral membrane vesicles of human intestine

The characteristics of the exit process of biotin from the enterocyte, i.e., transport across the basolateral membrane, was determined using an enriched basolateral membrane vesicle preparation of human intestine. Purity and suitability of basolateral membrane vesicles for transport studies was confirmed by enzymatic and functional criteria. Orientation of human basolateral membrane vesicles was determined by [3H]ouabain binding studies and was found to be 64% inside-out vesicles and the rest right-side-out vesicles and membrane sheets. Osmolarity studies indicated that the uptake of biotin by these vesicles represents transport into the intravesicular compartment, with little binding to membrane surfaces. The rate of biotin transport was linear for approximately 40 s but decreased thereafter. Transport of biotin was (a) Na+-independent, (b) saturable as a function of concentration, with an apparent KM of 1.1 microM and Vmax of 0.9 pmol/mg protein.15 s, (c) inhibited by structural analogues (desthiobiotin and biotin methyl ester) and related compounds (thioctic acid and thioctic amide), and (d) stimulated by inducing a positive intravesicular electrical potential. These studies are the first to demonstrate the existence of a carrier-mediated transport system for biotin in the basolateral membrane of human intestine.

Ontogenesis of the intestinal transport of biotin in the rat

Developmental aspects of the intestinal transport of biotin were examined in suckling (16 day old) and weanling (24 day old) rats using the everted sac technique. The results were compared with those of adult rats previously reported by us using the same intestinal preparation. Transport of biotin was linear for 20 min of incubation in all age groups. Transport of biotin was significantly (p less than 0.05) lower in the jejunum than the ileum of suckling rats but was not significantly different in the jejunum and the ileum of weanling rats. In adult rats, biotin transport was significantly (p less than 0.01) higher in the jejunum than the ileum. In all age groups, transport of biotin in the jejunum was saturable at low concentrations (less than 10 microM) but linear at high concentrations. The apparent Km and Vmax of the saturable process showed a progressive increase from suckling to weanling to adult rats (apparent Km of 0.63, 2.49, and 3.37 microM; Vmax of 18.3, 44.7, and 124.4 pmol/g.min, respectively). On the other hand, the rate of transport by the nonsaturable process showed a progressive decrease with maturation (143.8, 111.6, and 87.5 pmol/g.min for suckling, weanling, and adult rats, respectively). Transport of biotin in suckling and weanling rats was similar to that of adult rats in that it was Na+-, energy-, and temperature-dependent and inhibited by structural analogues. These results demonstrate that biotin transport undergoes clear maturational changes. These changes include a decrease in the affinity and an increase in the activity (and/or the numbers) of the transport carrier, a decrease in the rate of transport by the nonsaturable process, and a change in the preferential site of transport.

Transport of glycyl-L-proline in intestinal brush-border membrane vesicles of the suckling rat: characteristics and maturation

Transport of the dipeptide glycine-L-proline (Gly-L-Pro) in the developing intestine of suckling rats and its subsequent maturation in adult rats was examined using the brush-border membrane vesicles (BBMV) technique. Uptake of Gly-L-Pro by BBMV was mainly the result of transport into the intravesicular space with little binding to membrane surfaces. Transport of Gly-L-Pro in BBMV of suckling rats was: (1) Na+ independent; (2) pH dependent with maximum uptake at an incubation buffer pH of 5.0; (3) saturable as a function of concentration (apparent Km = 21.5 +/- 7.9 mM, Vmax = 8.6 +/- 1.5 nmol/mg protein per 10 s); (4) inhibited by other di- and tripeptides; and (5) stimulated and inhibited by inducing a negative and positive intravesicular membrane electrical potential, respectively. Similarly, transport of Gly-L-Pro in intestinal BBMV of adult rats was saturable as a function of concentration (apparent Km = 17.4 +/- 8.6 mM, Vmax = 9.1 +/- 2.1 nmol/mg protein per 10 s) and was stimulated and inhibited by inducing a relatively negative and positive intravesicular membrane potential, respectively. No difference in the transport kinetic parameters of Gly-L-Pro was observed in suckling and adult rats, indicating a similar activity (and/or number) and affinity of the transport carrier in the two age groups. These results demonstrate that the transport of Gly-L-Pro is by a carrier-mediated process which is fully developed at the suckling period. Furthermore, the process is H+-dependent but not Na+-dependent, electrogenic and most probably occurs by a Gly-L-Pro/H+ cotransport mechanism.

Effect of human milk folate binding protein on folate intestinal transport

The present study examined the effect of human milk folate binding protein (FBP) on the intestinal transport of 5-methyltetrahydrofolate (5-CH3H4PteGlu). This was performed by examining the transport of radiolabeled 5-CH3H4PteGlu bound to FBP using everted sacs of rat intestine. In the jejunum at pH 6, transport of 27 nM bound 5-CH3H4PteGlu was linear with time for 30 min of incubation. Transport of 13 nM bound 5-CH3H4PteGlu was higher in the jejunum than in the ileum at both pH 6 (2.1 +/- 0.3 and 0.36 +/- 0.03 pmol/g wet wt/25 min, respectively) and pH 8 (1.9 +/- 0.3 and 0.32 +/- 0.02 pmol/g wet wt/25 min, respectively). In the jejunum, transport of 13 nM bound 5-CH3H4PteGlu at pH 6 was less than transport of an equimolar concentration of free 5-CH3H4PteGlu (2.1 +/- 0.3 and 5.1 +/- 0.5 pmol/g wet wt/25 min, respectively) but was similar at pH 8 (1.9 +/- 0.3 and 2.47 +/- 0.3 pmol/g wet wt/25 min, respectively). In the ileum transport of bound and free 5-CH3H4PteGlu was similar at pH 6 (0.36 +/- 0.03) and 0.41 +/- 0.06 pmol/g wet wt/25 min, respectively) and pH 8 (0.32 +/- 0.02 and 0.43 +/- 0.1 pmol/g wet wt/25 min, respectively). The transport process of bound 5-CH3H4PteGlu in the jejunum was energy, temperature, and Na+ dependent, but not pH dependent, and was competitively inhibited by sulfasalazine. Ninety-two percent of the transport substrate that appeared in the serosal compartment following incubation with bound 5-CH3H4PteGlu was found to be free (unbound) 5-CH3H4PteGlu. These results show that human milk FBP decreases the rate of transport of 5-CH3H4PteGlu in the jejunum and suggest that FBP-bound 5-CH3H4PteGlu may utilize the same transport system as free 5-CH3H4PteGlu. The results also suggest a role for human milk FBP in regulating the nutritional bioavailability of folate.

Effect of Epidermal Growth Factor and Artificial Feeding in Suckling Rats

ABSTRACT. To evaluate the effects of epidermal growth factor (EGF) in dietary milk, a new method of delivering an artificial (EGF-deficient) formula was developed using 42 rat pups, 1–14 days of age. In a second study the effect of EGF was evaluated in suckling rats from 3–11 days of age: group 1, mother-fed; group 2, mother-fed plus daily injections of EGF (0.1 µg/g body weight); group 3, artificial milk fed with added EGF (62 ng/ml); and group 4, artificial milk fed without EGF. Each group consists of nine rats. In group 2 there was premature eye opening and tooth eruption and a significant reduction in body weight and weight of liver, kidney, thyroid, and thymus but an increase in length of the intestine and weights of stomach, pancreas, lung, and adrenal (p<0.04), when compared to group 1. Both groups 3 and 4 showed premature tooth eruption and eye opening, and their body weights and most organ weights were similar to group 2; exceptions were a smaller stomach, thyroid, thymus, lung, and adrenal, which were similar to those in group 1. In addition, intestinal length in groups 3 and 4 were similar to the mother-fed EGF-treated pups (group 2). There was no difference in intestinal length between the artificially fed pups, whether or not they received oral EGF. These findings demonstrate a new and effective technique of artificial feeding and suggest that the increase in intestinal length caused by injections of EGF (0.1 µg/g body weight) can also be induced by feeding an artificial milk with or without physiologic levels of EGF.

Intestinal glucose transport in suckling rats fed artificial milk with and without added epidermal growth factor

ABSTRACT: Edpidermal growth factor (EGF), a potent mitogen present in milk, is postulated to play an important physiologic role in the ontogeny of the intestine. An artificial rat milk, free of EGF, was developed and fed to newborn rats in an effort to evaluate the physiologic role of EGF on intestinal glucose transport. Comparisons were made between mother-fed (group 1) and artificially fed pups (group 2). Group 1 was divided into two subgroups to receive either the EGF vehicle (group 1-A) or twice daily subcutaneous injections (0.1 μg/g body weight/day) of EGF (group 1-B). Group 2 was divided into four subgroups: formula without EGF (group 2-A), formula with 62 ng/ml of EGF (group 2-B), formula with 200 ng/ml EGF (group 2-C), and formula without EGF but added anti-EGF IgG (40 ng/ml) plus subcutaneous injections of 1 μg anti-EGF twice daily (group 2-D). Treatment regimens were initiated on day 3 and assays performed on day 11 of life. Glucose transport was measured using gut “sheet” preparations in all but group 2-C, which utilized brush border membrane preparations. Group 1-B showed a significant (p < 0.03) increase in gut length (47.5 ±1.2 versus 42 ± 0.9 cm) and an increase (p < 0.03) in both Vmax(10.2 ± 0.5 versus 7.2 ± 0.3 nM/mg tissue) and Km (2.74 ± 0.49 versus 1.20 ± 0.19 mM) compared to group 1-A. All pups in group 2 showed a significant (p < 0.03) increase in intestinal length compared to group 1-A, but no difference was seen between group 1-B and all subsets of group 2. Glucose uptake was similarly affected with the exception that only the Vmax was significantly increased by tube feeding. The suggested increase in glucose carriers resulting from EGF was further supported by the finding that only the large oral or systemic dose of EGF (groups 1-B and 2-C) caused a significant (p < 0.01) increase in the 1-minute accumulation (“overshoot”) of glucose in the brush border membrane. Whereas pharmacologic doses of EGF caused premature development of intestinal glucose transport and increased gut length, artificial feeding alone caused a similar increase in intestinal length and Vmax for glucose transport.

A dual, concentration-dependent transport system for riboflavin in rat intestine in vitro

Abstract The intestinal mucosal-to-serosal transport of riboflavin was examined using rat everted sacs. Maximum transport of 0.5 μM riboflavin occurred in the proximal small intestine. Transport of low (0.5 μM) and high (3.6 μM) concentrations of riboflavin, was linear with time for 30 min incubation and occurred at a rate of 1.55 and 3.59 pmole/g tissue wet wt/min, respectively. Transport of 0.5 μM riboflavin was pH-dependent, being maximal at pH 6.5–7.5 but decreased at lower pH. The transport process of riboflavin involves two systems: 1) an active, carrier-mediated system and 2) difussion. The active system is characterized by: a) saturation with an apparent K t of 0.38 μM; b) Na + -dependence; c) energy-dependence; d) temperature-dependence; e) inhibition by structural analogue and f) accumulation against a concentration gradient. The difussion system is characterized by: a) linear increase in the rate of transport as a functio of increasing mucosal concentration of riboflavin above 1 μM; b) Na + -independence; c) energy-independence and d) inability to accumulate the substrte against a concentration gradient. Conclusion: the intestinal transport of low concentrations of riboflavin occurs by an active, carrie-mediated system while transport of high concentratins occurs by simple diffusion.

Developmental Maturation of D-Glucose Active Transport System in Rat Intestine

Developmental changes in the D-glucose-active transport system were examined in jejunum of rats of different age groups (3, 5, 7, 11, 13, 17, 25 and 90 days old) using the intestinal sheet technique. Transport of D-glucose by the active system was determined by subtracting the passive component, as determined by uptake of L-3H-glucose from the total uptake. The transport rate of 0.1 and 1 mM D-glucose in 13- and 90-day-old rats was linear with time for 12 min of incubation, but decreased thereafter. Kinetic parameters of the active transport system of D-glucose were determined by examining the transport of D-glucose after 12 min of incubation as a function of concentration. Vmax of the active transport system was highest in 3-day-old rats and decreased with age reaching the adult value by day 7 after birth. Kt of the active transport system was the same in 3- 5- and 7-day-old rats, but increased progressively with age reaching the adult value by day 25 after birth. These data demonstrate that transport of D-glucose by the active system is highest during the early days of life, but decreases thereafter. It also suggests that this enhancement is most probably due to greater activity and/or number, and affinity of the transporting carriers of D-glucose.

Maturational changes in glutamine transport by rat jejunal brush border membrane vesicles

ABSTRACT: The ontogeny of glutamine uptake by jejunal brush border membrane vesicles was studied in suckling and weanling rats and compared with the data obtained from previous studies done on adult rats in our laboratory. Glutamine uptake represented transport into the intravesicular space rather than mere binding into the membrane as evident by osmolality study. The process of glutamine uptake was temperature dependent suggesting a carriermediated process with a pH optimum at 7.0. Glutamine uptake was driven by Na+ and K+ gradient in both suckling and weanling rats. Both processes exhibited saturation kinetics and were inhibited by other neutral amino acids suggesting the presence of Na+-dependent neutral brush border system and Na+-independent (L)-like system. The Vmaxof Na+-dependent and Na+-independent processes were significantly greater in suckling rats with Vmax of 4.9 ± 0.36 nmol·mg protein-1.7 s-1 compared to weanling rats with Vmax of 2.4 ± 0.2 nmol·mg protein-1. 7 s-1 and adult rats with Vmax of 0.70 nmol·mg protein-1 -7 s-1. The greater Vmax in suckling rats is also evident when the kinetic parameters are analyzed by subtracting the sodium-dependent uptake values from the sodium-independent values. Vmax of 1.59 ±0.3 and 0.76 ± 0.01 nmol·mg protein-17 s-1 in suckling and weanling rats, respectively, p < 0.01. Km values were not different at 2.5 ± 0.6 and 3.5 ± 0.6 mM, respectively). The data suggest that the activity and/or the number of transporters are greater during the period of active growth and development. We conclude that glutamine transport in brush border membrane vesicles undergoes age-dependent changes with greater maximal capacity to transport glutamine in the suckling period.