Absorption kinetics for leucine, cycloleucine and alpha-aminoisobutyric acid by rat small intestine in vivo

The intestinal absorption kinetics of three neutral amino acids, leucine, cycloleucine and alpha-aminoisobutyric acid, has been studied in rat jejunum in vivo, with luminal perfusion during successive periods, by measuring the passive component and the active transport. The mass-transfer coefficients of the passive process, are similar for the three amino acids and increase with the perfusion rate. The transport component, obtained from the difference between total absorption and passive diffusion, shows saturation kinetics and also increases with the perfusion rate. The apparent Michaelis constants, Km, and the maximal transport rates for the three amino acids have been determined. The Km values are greater than those reported for in vitro studies, a result imputable to greater thickness of the unstirred layers in vivo and to the unequal signification of the constant in both conditions. Passive flux has proved to be an important component for in vivo absorption, even at low substrate concentrations (1-5 mM), so that its evaluation cannot be neglected for the calculation ot the kinetic constants of the mediated transport.     

Effects of dietary zinc depletion and food restriction on intestinal transport of cadmium in the rat

The effects of Zn depletion and short-term fasting on intestinal transport of Cd were examined in perfused rat small intestines. The small intestine was isolated with its vascular network intact, then simultaneously perfused from the luminal and vascular sides. A Zn-depleted state that results in marked hypozincemia was produced in some rats by feeding a Zn-deficient diet for 4 days. Uptake of Cd from the luminal perfusate was greater in the Zn-depleted rats, whereas transport of Cd to the vascular perfusate was not affected. Fasting overnight prior to perfusion did not influence Cd transport nor alter the effect of Zn depletion on Cd uptake. The Cd concentration in the soluble fraction of intestinal mucosa from perfused intestines was not different between Zn-depleted and Zn-adequate rats. Gel filtration chromatography of the soluble fraction showed a shift in the distribution of Cd from metallothionein to high molecular weight ligands in intestines from Zn-depleted rats. The decrease in amount of metallothionein-associated Cd corresponded to a decrease of total intestinal metallothionein as measured by the Cd-binding assay. The results suggest association of Cd with intestinal metallothionein did not influence the absorption of Cd under these conditions.     

Jejunal Creatine Absorption: What is the Role of the Basolateral Membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.     

Endogenous prolactin modulated the calcium absorption in the jejunum of suckling rats

Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous prolactin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the 45Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 +/- 0.35 micromol.h-1.cm-1, p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumen-to-plasma and net calcium fluxes in jejunum from 2.07 +/- 0.31 to 1.19 +/- 0.12 and 1.40 +/- 0.35 to 0.88 +/- 0.18 micromol.h-1.cm-1 (p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/L Na+-containing or Na+-free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of 45Ca under luminal sodium free condition was also higher than that of the control condition (2.26% +/- 0.07% vs. 2.01% +/- 0.09% administered dose, p < 0.05). However, 3H-mannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma 45Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol.     

Effect of the pH on intestinal absorption of sugars in vivo.

Influence of the pH on the absorption rate of sugars by rat intestine in vivo has been revised by means of a technique for intestinal lumen perfusion with 1 minute absorption periods. Absorption at pH 2.5, 5, 7, 8.5, and 10 has been comparied in each animal. Absorption rate of D-glucose, D-galactose and D-fructose is highest at pH 7 and decreases at the lower or higher pH values. The pH does not affect the absorption of D-arabinose. The pH effect is attributed to changes in the transport system for sugars.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

Absorption of 5-methyltetrahydrofolate in rat jejunum with intact blood and lymphatic vessels

Transport results from in vitro studies may not be applicable to in vivo situations. In this study, we extended our previous in vitro observations regarding the intestinal transport of 5-methyltetrahydrofolate to in vivo studies in the unanesthetized rat and examined the effect of the unstirred water layer on the absorption process. We used a well defined intestinal perfusion technique. Absorption of 0.5 and 5 microM 5-methyltetrahydrofolate proceeded in a linear manner for 40 min of perfusion at 0.31 and 1.74 nmol/100 cm per min, respectively. Absorption of 0.5 microM 5-methyltetrahydrofolate increased with increasing perfusate flow-rate from 0.5 to 2 to 4 ml/min, indicating an unstirred water layer influence on the absorption rate. Absorption of the substrate was saturable with an apparent Kt of 5.7 microM and Vmax of 3.45 nmol/100 cm per min. Absorption was pH-dependent, and was inhibited by structural analogues. In contrast to the in vitro data, addition of glucose (20 mM) to the perfusate was unnecessary for in vivo absorption to proceed. Unconjugated cholic (5 mM) and deoxycholic (1 mM) acids and the organic anion rose bengal (0.1 mM) inhibited the absorption of 0.5 microM 5-methyltetrahydrofolate when added to the perfusate. Conclusions: the results of previous in vitro studies of 5-methyltetrahydrofolate intestinal transport are applicable to in vivo situations, except that luminal glucose was found to be unnecessary in the latter. The unstirred water layer modulated the absorption of 5-methyltetrahydrofolate, while unconjugated bile acids and rose bengal inhibited it.     

Intestinal absorption by carrier-mediated transports: two-dimensional laminar flow model

The two-dimensional laminar flow model was adapted to the intestinal absorption of drug and biological substances by carrier-mediated transports in the single perfusion experiments and we investigated the effects of the unstirred water layer on the Michaelis constant and the maximum transport velocity. According to the calculated values, the half saturation concentration at the inlet was larger than the true Michaelis constant at the intestinal wall. The apparent values of the Michaelis constant and the maximum transport velocity obtained by the Lineweaver-Burk plots were larger than the true ones, and the relations were not linear. These deviations increased as the ratio of the maximum transport velocity to the Michaelis constant increased and as the perfusion rate decreased. In the concurrent presence of a passive transport, underestimation of the carrier-mediated transport component of the absorption rate (at steady state) was predicted. It is considered to cause the underestimation of the maximum transport velocity. When water was absorbed (or secreted), the absorption rate increased (or decreased) and did not saturate. This two-dimensional laminar flow model would enable us to analyze the experimental data to determine the true values of the Michaelis constant and the maximum transport velocity.     

Jejunal and cecal 3-oxy-methyl-D-glucose absorption in chicken using a perfusion system in vivo

3-oxy-methyl-D-glucose (3-OMG) absorption by jejunum and caecum has been studied in the domestic fowl in vivo, with luminal perfusion, during 5 min periods. The diffusion component was evaluated in the presence of phloridzin (10(-3) M) that inhibits the active transport mechanism. Kd of jejunal and cecal diffusion of the monosaccharide have been calculated, showing a similar value. The Kt and Vmax of 3-OMG absorption were calculated using a graphical method for the two intestinal segments. The caecum showed a lower Kt and Vmax than the jejunum did.     

Role of unstirred layer in intestinal absorption of phenylalanine in vivo

The appearance rate of l- and d-phenylalanine in the venous blood of rat jejunal loops in vivo is increased up to 60% if the intraluminal solution is mixed more efficiently by the simultaneous perfusion of air. The effect decreases as the luminal concentration is increased to 100 mmol/1. Thus, the apparent Michaelis constants are by 50% lower in the case of the reduced unstirred layer (26 to 17 for l- and 9 to 6 mmol/1 for d-phenylalanine).The enhancement of the absorption and the reduction of the Michaelis constants can be attributed to the reduction of the effective unstirred layer thickness by about 400–500 μm.     

Intestinal absorption of hawthorn flavonoids--in vitro, in situ and in vivo correlations

Our previous studies identified hyperoside (HP), isoquercitrin (IQ) and epicatechin (EC) to be the major active flavonoid components of the hawthorn phenolic extract from hawthorn fruits demonstrating inhibitory effect on in vitro Cu(+2)-mediated low density lipoproteins oxidation. Among these three hawthorn flavonoids, EC was the only one detectable in plasma after the oral administration of hawthorn phenolic extract to rats. The present study aims to investigate the intestinal absorption mechanisms of these three hawthorn flavonoids by in vitro Caco-2 monolayer model, rat in situ intestinal perfusion model and in vivo pharmacokinetics studies in rats. In addition, in order to investigate the effect of the co-occurring components in hawthorn phenolic extract on the intestinal absorption of these three major hawthorn flavonoids, intestinal absorption transport profiles of HP, IQ and EC in forms of individual pure compound, mixture of pure compounds and hawthorn phenolic extract were studied and compared. The observations from in vitro Caco-2 monolayer model and in situ intestinal perfusion model indicated that all three studied hawthorn flavonoids have quite limited permeabilities. EC and IQ demonstrated more extensive metabolism in the rat in situ intestinal perfusion model and in vivo study than in Caco-2 monolayer model. Moreover, results from the Caco-2 monolayer model, rat in situ intestinal perfusion model as well as the in vivo pharmacokinetics studies in rats consistently showed that the co-occurring components in hawthorn phenolic extract might not have significant effect on the intestinal absorption of the three major hawthorn flavonoids studied.     

Membrane digestion and transport under physiological conditions: a review of available data

A technique to study membrane digestion and transport in the small intestine under physiological conditions has been developed. The technique is based on a continuous perfusion of a chronically isolated loop of the rat small intestine. Membrane hydrolysis and transport of some nutrients in the rat small intestine in chronic, as well as in acute (in situ) experiments was investigated. The absorption of hexoses and amino acids has been found to be 2.5-4 times higher under physiological conditions than in acute in situ experiments. Both the active transport of glucose released from maltose hydrolysis and the hydrolysis of the latter is increased under physiological conditions. A coupling between the final stages of hydrolysis and the initial stages of transport in chronic experiments was shown to be highly efficient; practically all or nearly all glucose released is being transported without entering the luminal phase. The hydrolysis rate of starch during the perfusion of a small intestinal segment in chronic experiments is many times higher than that in acute experiments or under anaesthesia. The enzymatic and transport activities revealed using a widely accepted technique in situ, the more so, in vitro account for only a small fraction of those which are typical of undisturbed processes under conditions close to the physiological. The levels of functioning of the digestive-transport systems of the small intestine considered as natural levels developed in the process of evolution, actually reflect only residual processes and, in most cases, they account for 1/3 to 1/10 of the true level of an actual physiological process.     

Aluminium-induced impairment of transcellular calcium absorption in the small intestine: calcium uptake and glutathione influence

Aluminium (Al) has been recognised as a cause of bone tissue disorders. The aims of this work were to investigate: (i) whether Al affects calcium (Ca) entry into enterocyte, and (ii) the possibility that the Al effect upon calbindin-D-related Ca transport would be influenced by intestinal glutathione (GSH) levels. In isolated chicken duodenal enterocytes, 100 microM Al lactate produced a decrease in both, the maximum uptake rate and the affinity constant of 45Ca uptake (CaUPT). This effect of Al on CaUPT was concentration-dependent in the micromolar range, showing an inhibitory saturation type phenomenon which appeared to be higher at pH 6.5 than at pH 7.4, and was not modified by the Ca channel activators A23187 and capsaicin. The simultaneous administration of Al (50 mg elemental Al/kg body weight, as AlCl3) and GSH (10 mmol/kg body weight) to rats during 7 days, prevented the inhibitory effects of Al on Ca transport. The protective effect of GSH was accompanied by an increased duodenal calbindin-D9k expression. Experimental depletion of intestinal GSH by means of D,L-buthionin-[S,R] sulfoximine, a gamma-glutamylcystein-synthase inhibitor, given as a single i.p. dose of 2 mmol/kg body weight, enhanced the degree of reduction of Ca absorption ascribed to Al. Our results suggest that Al might interfere Ca uptake by enterocytes through a general effect on cell membrane, and that an oxidative stress state induced by Al would reduce intestine GSH level affecting calbindin-D function and/or synthesis, thus leading to a reduced transcellular Ca absorption in the small intestine.     

Effect of ethanol on glycylsarcosine absorption

Glycylsarcosine (GlySar) absorption by the rat intestine is not altered by acute ethanol administration (luminal perfusion of a 0.7 M ethanol solution) or by chronic consumption of a 15% ethanol solution in drinking water. Both total absorption, per entire rat small intestine, and specific absorption per mg dry weight of mucosa, were unaffected by ethanol. During the absorption of GlySar, glycine, produced by hydrolysis of the peptide in the cytosol of the intestinal cells, appears in the intestinal lumen. During acute ethanol administration the luminal appearance of glycine is decreased probably due to a reduction in intracellular hydrolysis of the dipeptide.     

Kinetics of the mediated transport and the passive net flux of phenylalanine across the rat small intestine in vivo

The kinetics of L-phenylalanine absorption by rat jejunum, in vivo, has been studied with luminal perfusion (0.68 ml/min) during successive periods at different substrate concentrations. The non-saturable passive component, measured by inhibiting the active transport with 60 mM methionine, was a linear function of the substrate concentration with an apparent mass-transfer coefficient of 1.42 nmoles/cm/min/mmoles/l. The transport component, estimated from the difference between total absorption and the passive component, displays saturation kinetics with an apparent transport constant (Km) of 7.5 mM and maximal transport rate (Vmax) of 107 nmoles/cm/min. Active transport seems to be the main component in absorbing phenylalanine proceeding from the digestion of food proteins.     

Study of acute pharmacologic effects of prolactin on calcium and water transport in the rat colon by an in vivo perfusion technique

We investigated the acute effect of intraperitoneally administered prolactin on calcium and water transport in colon of sexually mature female Wistar rats using an in vivo perfusion technique. Test solution containing (in mM) NaCl, 100; KCl, 4.7; MgSO4, 1.2; CaCl2, 20; D-glucose, 11; sodium ferrocyanide (Na4Fe(CN)6), an index of net water transport, 20; and 0.7 (microCi 45CaCl2 (1 Ci = 37 GBq) was perfused througth the 8-cm colonic loop for 60 min at perfusion rates of 0.5 or 1.0 mL x min(-1). Calcium and water transport was also studied under a no flow condition to stimulate the condition often found in the colon by in vivo ligated colonic loop for 30 min. Control results showed no correlation between calcium transport and water flux. Flow of luminal solution at 0.5 and 1.0 mL x min(-1) was found to reverse net calcium absorption from 0.04+/-0.01 nmol x g(-1) dry weight x h(-1) to net calcium secretion of 0.04+/-0.04 and 0.9+/-0.02 nmol x g(-1) dry weight x h(-1), respectively. Neither 0.4, 0.6, nor 1.0 mg x kg(-1) prolactin had any effect on calcium fluxes in the colon. On the other hand, at a perfusion rate of 1 mL x min(-1), 0.4 mg x kg(-1) prolactin significantly decreased net water absorption from 3.86+/-0.90 to 0.88+/-0.64 mL x g(-1) dry weight x h(-1) (P < 0.001), and the higher doses of 0.6 and 1.0 mg x kg(-1) prolactin reversed net water absorption to net water secretion of 2.20+/-0.63 and 2.33+/-0.89 mL x g(-1) dry weight x h(-1), respectively (P < 0.001). The stimulatory effect of prolactin on water transport was completely abolished by reducing the perfusion rate from 1.0 mL x min(-1) to zero. The stimulatory effect of prolactin on water secretion at perfusion rate of 1.0 mL x min(-1) was also abolished when luminal [Na+] was reduced from 180 to 80 mM. We concluded that, unlike in the small intestine, calcium fluxes in the colon are not related to water transport and did not respond at all to prolactin. Water transport, on the other hand, was reversed from net absorption to secretion by prolactin. We propose that this prolactin-induced water secretion is probably mediated by recycling of luminal sodium in the vicinity of tight junctions.     

Regulation of direct transintestinal cholesterol excretion in mice

Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux (TICE) contributes significantly to cholesterol removal in mice. Our aim was to investigate whether the activity of this novel pathway can be influenced by dietary factors. In addition, we studied the role of cholesterol acceptors at the luminal side of the enterocyte. Mice were fed a Western-type diet (0.25% wt/wt cholesterol; 16% wt/wt fat), a high-fat diet (no cholesterol; 24% wt/wt fat), or high-cholesterol diet (2% wt/wt), and TICE was measured by isolated intestinal perfusion. Bile salt-phospholipid mixtures served as cholesterol acceptor. Western-type and high-fat diet increased TICE by 50 and 100%, respectively. In contrast, the high-cholesterol diet did not influence TICE. Intestinal scavenger receptor class B type 1 (Sr-B1) mRNA and protein levels correlated with the rate of TICE. Unexpectedly, although confirming a role for Sr-B1, TICE was significantly increased in Sr-B1-deficient mice. Apart from the long-term effect of diets on TICE, acute effects by luminal cholesterol acceptors were also investigated. The phospholipid content of perfusate was the most important regulator of TICE; bile salt concentration or hydrophobicity of bile salts had little effect. In conclusion, TICE can be manipulated by dietary intervention. Specific dietary modifications might provide means to stimulate TICE and, thereby, to enhance total cholesterol turnover.     

Certain aspects of the mechanism of intestinal transport of sodium, potassium, calcium and magnesium in rats

Intestinal absorption of sodium, potassium, calcium and magnesium was studied in rats by the method of intestinal perfusion using ouabain as an inhibitor of sodium-potassium dependent ATPase. At the same time the activity of ATPase and phosphatase were determined in homogenates of intestinal mucosa. A significant effect on the concentration of the determined ions was demonstrated in the transport of these ions, and also an unquestionable participation of intestinal ATPase in the direction and intensity of this transport. It was found that the multidirectional effect of ouabain on the transport of cations depended on their concentration. In the case of concentrations of cations similar to those in the mean food rations it has been demonstrated that ouabain increased the absorption of sodium, potassium and calcium and inhibited the absorption of magnesium. With a threefold higher ions concentration the absorption of potassium and magnesium was inhibited, without changing the transport of sodium and calcium. The possible explanation of the mechanism of these effects is discussed.     

Use of the paracellular way for the intestinal absorption of sugars

Passive absorption of D-Galactose (in the presence of 0.5 mM phlorizin), 2-deoxy-D-glucose and D-Mannitol by rat jejunum has been measured in vivo by perfusion of an intestinal segment with recirculation, along successive absorption periods of 5 or 10 min duration. In the range of 1 to 40 mM concentrations, the three solutes were absorbed at a very similar rate that varied as a lineal function of the concentrations in the perfusion solution. Absorption of 1 mM solute was not modified by the presence of 40 mM glucose or galactose. Passive absorption kinetics suggests processes of simple diffusion or solvent drag. The use of paracellular way for the passive absorption is supported by the fact that triaminopyrimidine (TAP) and protamine, which decrease the permeability through the tight junctions, also inhibit the absorption, with similar characteristics for both actions: TAP inhibition (53%) is very rapid and can be easily reversed, while that of protamine (30%) requires some time of previous exposure, lasts longer and can be reversed by heparin. The same analogy is shown by two actions that enhance the paracellular permeability: theophylline increases (30%) the passive absorption with lasting effect, while luminal hypertony enhances absorption transitorily. The passive absorption of the assayed solutes could be estimated to take place by the paracellular way in at least 50% and probably 70% or even more. The measure of net fluid fluxes reveals that solute fluxes must be prevailingly explained by simple diffusion, as the solvent drag can only play a very minor role.