Amino acid absorption in jejunum of rats in vivo--a kinetic comparison of distal resection effects

Jejunal absorption of leucine and cycloleucine by sham and 50% distal resected rats in vivo was studied by measuring the passive component and the active transport. After 5 months postresection the total amino acid absorption was increased. The mass-transfer coefficients of the passive process (obtained in presence of methionine) were higher in remnant jejunum than that in control rats, whereas the active transport remained unaltered after resection. When the kinetic constants of the saturable and non-saturable components were corrected for the unstirred water layer effects, the "real KD" increased in the resected group, whilst similar values for the "real Km and Jmax" were obtained.     

A simple apparatus for performing short-time (1--2 seconds) uptake measurements in small volumes; its application to D-glucose transport studies in brush border vesicles from rabbit jejunum and ileum

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10--20 microliter. Using this technique the kinetic parameters Km and V of D-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the Km for D-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.     

Effects on intestinal nutrient uptake and brush border membrane enzymes in response to atherogenic diet in rhesus monkeys

Transport of nutrients and kinetic parameters (Vmax and Km) of brush border membrane (BBM) enzymes were studied in duodenum, jejunum, and ileum from atherogenic diet-fed monkeys. The Km remained unaltered while feeding of atherogenic diet resulted in higher Vmax of sucrase, maltase, and alkaline phosphatase and lower Vmax of gamma-glutamyltranspeptidase and leucine-aminopeptidase compared to controls. Na+-dependent D-glucose transport was higher in duodenum and jejunum and unaltered in ileum. In contrast to D-glucose transport, the transport of amino acids was decreased in all three intestinal segments from atherogenic diet-fed monkeys.     

Kinetic parameters of maltose hydrolysis and glucose intake in the rat small intestine in a chronic experiment

"True" (corrected for the influence of the pre-epithelial layer) kinetic constants of maltose hydrolysis (Km and Vmax) and Glucose active transport (Kt and Jmax) in the isolated loop of the rat small intestine in chronic experiments were determined using a new mathematical approach. The Km (4.260.25 mM) does not differ from that, obtained in in vitro experiments on the homogenates of mucous membrane taken from the same intestinal loops, and the Vmax (0.72 +/- 0.07 mol/(min.cm)) is 1.7 times lower than that in in vitro experiments. The Kt and Jmax values are 3.18 +/- 0.68 mM and 0.73 +/- 0.07 mol/(min.cm), resp. The estimated values of Km, Kt and Vmax are in accordance with the corresponding published data, whereas the Jmax is several times higher than the value generally believed on the basis of acute experiments in vivo. A high level of glucose absorption in the small intestine of unanesthetized animals is achieved mainly due to a high permeability of the pre-epithelial layer and a high capacity of the active transport as a major mechanism of glucose absorption in the small intestine under normal conditions.     

ATP-dependent transport of organic anions into isolated basolateral membrane vesicles from rat intestine

The mechanism for the cellular extrusion of organic anions across the intestinal basolateral membrane was examined using isolated membrane vesicles from rat jejunum, ileum, and colon. It was found that 17beta-estradiol 17beta-D-glucuronide (E217betaG) is taken up in an ATP-dependent manner into the basolateral membrane vesicles (BLMVs) but not into the brush-border or microsomal counterparts. The ATP-dependent uptake of E217betaG into BLMVs from jejunum and ileum was described by a single component with a Km value of 23.5 and 8.31 microM, respectively, whereas that into the BLMVs from colon was described by assuming the presence of high (Km=0.82 microM)- and low-affinity (Km=35.4 microM) components. Taurocholate, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole glucuronide and taurolithocholate sulfate, but not leukotriene C4, were significantly taken up by the BLMVs. In addition to such substrate specificity, the inhibitor sensitivity of the ATP-dependent transport in BLMVs was similar to that of rat multidrug resistance-associated protein 3 (Mrp3), which is located on the basolateral membrane of enterocytes. Together with the fact that the rank order of the extent of the expression of Mrp3 (jejunum < ileum < colon) is in parallel with that of the extent of the transport of ligands, these results suggest that the ATP-dependent uptake of organic anions into isolated intestinal BLMVs is at least partly mediated by Mrp3.     

Thiamine transport in thiamine-deficient rats. Role of the unstirred water layer.

As part of a systematic study of alcoholism and thiamine absorption, the effect of diet-induced thiamine deficiency and the role of the unstirred water layer on the thiamine transport were investigated. Using 3H-labeled dextran as a marker of adherent mucosal volume, jejunal uptake of 14C-labeled thiamine hydrochloride was measured, in vitro, in thiamine-deficient rats and pair-fed controls. Uptake of low thiamine concentrations (0.2 and 0.5 muM) was greater in the thiamine-deficient rats than in the controls. In contrast, uptake rates for high thiamine concentrations (20 and 50 muM) were similar in both groups. While Jmax was unaltered, Km was decreased in thiamine deficiency, suggesting a decrease in unstirred water layer thickness. Accordingly, the thickness of the water layer was measured in both groups of animals and correlated with Jmax and Km under unstirred and stirred conditions. Without stirring, there was no difference in Jmax between the two groups. In contrast, both Km and the water layer were reduced in the thiamine-deficient rats. With stirring, Jmax was not affected, but both Km and the water layer thickness were reduced to similar values in both groups. Reversal of thiamine deficiency resulted in the return of thiamine uptake and the unstirred water layer thickness to control values. These data support the concept of a dual system of thiamine transport and emphasize the role of the unstirred water layer as an important determinant of transport kinetics not only under physiologic situations but also in diet-induced rat thiamine deficiency, a model for a clinical patholigical state. The decrease in the unstirred water layer thickness in thiamine deficiency may be also viewed as a possible adaptive mechanism to facilitate absorption of meager supplies of thiamine.     

Na+, Li+ and Cl− transport by brush border membranes from rabbit jejunum

Na+, Li+, K+, Rb+, Br-, Cl- and SO4(2-) transport were studied in brush border membrane vesicles isolated from rabbit jejunum. Li+ uptakes were measured by flameless atomic absorption spectroscopy, and all others were measured using isotopic flux and liquid scintillation counting. All uptakes were performed with a rapid filtration procedure. A method is presented for separating various components of ion uptake: 1) passive diffusion, 2) mediated transport and 3) binding. It was concluded that a Na+/H+ exchange mechanism exists in the jejunal brush border. The exchanger was inhibited with 300 microM amiloride or harmaline. The kinetic parameters for sodium transport by this mechanism depend on the pH of the intravesicular solution. The application of a pH gradient (pHin = 5.5, pHout = 7.5) causes an increase in Jmax (50 to 125 pmol/mg protein . sec) with no change in Kt (congruent to 4.5 nM). Competition experiments show that other monovalent cations, e.g. Li+ and NH4+, share the Na+/H+ exchanger. This was confirmed with direct measurements of Li+ uptakes. Saturable uptake mechanisms were also observed for K+, Rb+ and SO4(2-), but not for Br-. The Jmax for K+ and Rb+ are similar to the Jmax for Na+, suggesting that they may share a transporter. The SO4(2-) system appears to be a Na+/SO4(2-) cotransport system. There does not appear to be either a Cl-/OH- transport mechanism of the type observed in ileum or a specific Na+/Cl- symporter.     

Catechol-O-methyltransferase and aromatic L-amino acid decarboxylase activities in human gastrointestinal tissues

Human gastrointestinal samples from the corpus, antrum, bulbus, jejunum and ileum were assayed for soluble and membrane-bound catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activity in vitro. The mean soluble COMT activities with 3,4-dihydroxybenzoic acid (DBA) and 3,4-dihydroxyphenylalanine (L-DOPA) as substrate were 70-242 and 70-174 pmol/min mg, respectively. The membrane-bound COMT activities ranged from 33 to 60 pmol/min mg in the different parts of the intestine. The AADC activities, measured with L-DOPA as the substrate, increased from 114 pmol/min mg in the corpus to 3488 pmol/min mg in the jejunum. The affinity of the soluble COMT was approximately 20 times higher for DBA (Km 15-19 microM) than for L-DOPA (Km 300-600 microM). The Km-values for L-DOPA of AADC and COMT were of the same order of magnitude. The specific COMT inhibitors, nitecapone and OR-611, effectively inhibited in vitro the human intestinal COMT activity. Nanomolar concentrations caused 50% inhibition with both DBA and L-DOPA as substrate.     

Distribution of peptide YY in the gastrointestinal tract of the rat, dog, and monkey

The objective of this study was to characterize the distribution and concentration of peptide YY (PYY) in the gastrointestinal tract of the rat, dog, and monkey. In the rat, the greatest concentration of PYY was detected in the ileum and colon. The concentrations of PYY in the ileum and colon were 72 +/- 49 and 768 +/- 180 ng/g tissue, respectively. In the dog, PYY was found primarily in extracts of the mucosal layer of the ileum and colon, with smaller amounts in the distal jejunum. The concentration of PYY in the mucosal layers of the canine distal jejunum was 113 +/- 25 ng/g tissue, proximal jejunum 302 +/- 56, mid jejunum 507 +/- 151, distal ileum 691 +/- 184, and colon 1706 +/- 774 ng/g tissue. In the monkey gastrointestinal tract, PYY was detected predominantly in mucosal extracts of the jejunum, ileum, and colon. The concentration of PYY in the mucosal layer extract of the jejunum was 92 +/- 23, ileum 615 +/- 127, and colon 1013 +/- 243 ng/g tissue.     

Milk protein digestion and lysosomal proteinases of the ileal mucosa in young rats]

The proteolytic activity of lysosomal and pancreatic proteinases was studied in the chyme and tissue homogenates of the jejunum and ileum of 12- and 30-day rats in order to elucidate whether lysosomal proteinases of the mucous membrane of the ileum participate in cavitary digestion. During milk feeding the proteolytic activity of acid (lysosomal) proteinases in the ileum was 3 times greater than in the jejunum, which has been demonstrated both for the mucous membrane and the contents of these parts of the small intestine. In rats on definitive nutrition the activity of acid proteinases from the jejunum and ileum remained almost unchanged both in the mucous membrane and in the contents. The data obtained also indicate that pancreatic proteinase absorbed from the chyme of the small intestine may participate in parietal digestion.     

Characterization of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells.

The unstirred water layer has been shown to lead to an underestimation of apparent Km (Km(app)) values for active transport processes in intestinal whole tissue preparations. Isolated cells offer several potential advantages in the study of transport processes including a decreased diffusion layer of water adjacent to their absorptive membranes. Initial studies in cells isolated from rat intestine involving measurements of CO2 and lactate production and O2 consumption showed that overall metabolic pathways were functioning. Next, unidirectional uptake rates of bile acids across the isolated cell membrane were determined following correction for extracellular fluid contamination with a non-absorbable marker. Using epithelial cells isolated from jejunum P(app) for eight bile acid monomers varied from 24.9 (taurocholate) to 1563 (deoxycholate) nmol/min/100 mg protein/mM. From these data the incremental free energy changes for the addition of a hydroxyl, glycine and taurine group to the bile acid molecule were calculated to be 982, 1040 and 1464 cal/mol, respectively, values similar to those obtained after correction for unstirred water layer resistance in whole tissue preparations. Following subtraction of the passive component in isolated ileal cells complete kinetic curves for taurocholate and taurodeoxycholate yielded V(app) values of 109 and 70 nmol/min per 100 mg, respectively. Km(app) values of 0.24 mM (taurocholate) and 0.10 mM (taurodeoxycholate) are lower than usually recorded in whole tissue. Bile acid uptake into cells from ileum, but not jejunum, was affected by temperature, metabolic and competitive inhibition. These studies indicate that isolated epithelial cells are a metabolically viable, relatively purified intestinal preparation which discriminates between active and passive transport processes for bile acids under conditions where unstirred water layer artifacts are minimized.     

Effects of thyroxine and dexamethasone on kinetic characteristics of the small intestine enzymes in rat pups following a low-protein diet in female rats during lactation

It is shown, that the value of Km for maltase, alkaline phosphatase, aminopeptidase M and glycyl-L-leucinedipeptidase, prepared from the jejunum and ileum of 10-day rat litter in membrane and soluble forms in most cases differed but a little in control animals and the rat litter whose mothers in the period of lactation had a diet with 2.5-fold reduced content of protein, and did not change under action of injected thyroxin and dexamethasone. It may be assumed that in the given experimental conditions each of the investigated digestive hydrolases in membrane and soluble forms represents the same enzyme. In conditions of the protein insufficiency in lactating females diet and under action of exogene hormones, apparently, no significant changes occur in structure of synthesised enzymes.     

Distribution, purification and characterization of rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase

Rat intestinal UDPgalactose: N-acetylglucosaminyl(beta 1----4)galactosyltransferase activity was studied as to its intestinal and villus-to-crypt distribution, and then purified and characterized. Rapid UDPgalactose hydrolysis was noted in the duodenum and jejunum; little to no breakdown was detected in the distal ileum, cecum and proximal colon. Product analysis suggested that UDPgalactose hydrolysis was due to nucleotide-sugar pyrophosphatase and galactose-1-phosphate phosphatase activities; ileum appeared to have little of the first activity and none of the latter. An aboral gradient of galactosyltransferase activity was noted, activity being 3-4-fold higher in the ileum, cecum and proximal colon. Total homogenate exogenous acceptor galactosyltransferase activities showed no villus-crypt differences but activity measured with intact isolated cells demonstrated higher activity with crypt cells; this was particularly evident in the ileum. Galactosyltransferase activity was purified from ileal-colonic mucosa. An over 4000-fold purification with 75 percent yield was achieved. Only one band of approx. 70-75 kDa was noted on sodium dodecyl sulfate polyacrylamide electrophoresis. As with other eukaryotic galactosyltransferase activities, there was an absolute requirement for Mn2+; the concentration required for half maximal activity was only 2.5 microM and higher concentrations did not inhibit. The Km for UDPgalactose was 30 microM.     

Fluid secretory responses to enterotoxin STa and 8-bromo-cyclic GMP in fed and nutrionally-deprived gerbils: jejunum, ileum and colon in vivo

Fluid transport was measured gravimetrically in vivo in the jejunum, ileum and colon of fed, fasting (four days) and undernourished (50 % of control food intake for 21 days) gerbils (Gerbillus cheesmani). The effects of luminal enterotoxin Escherichia coli STa (50 ng/ml) and luminal 8-bromo-cyclic GMP (cGMP 1 mM) on fluid transport across jejunum, ileum and colon were also assessed. Fasting and undernourishment reversed the normal basal fluid absorption measured in fed ileum and colon into secretion. Neither fasting nor undernourishment had any effect on jejunal basal fluid absorption. In jejuna, ilea and colons of fed animals as well as in jejuna from fasting and undernourished gerbils STa (50 ng) reversed the normal absorptive "tone" to secretion but it had no significant effects on fluid secretion in either the ileum or colon from fasted gerbils. STa increased significantly the fluid secretion in ileum from undernourished gerbils. Luminal cGMP had no effect on basal absorptive tone in the jejunum of fed and fasted gerbils, but reversed absorption into secretion in the jejuna from undernourished gerbils. In the ilea taken from fed animals the small basal absorption was reversed to secretion by luminal cGMP. Although cGMP produced no significant changes in fluid secretion in the ilea taken from fasted gerbils, yet it caused a significant increase in those from undernourished gerbils. In the colon taken from fed animals cGMP decreased the basal fluid absorption significantly, but it had no significant effect on fluid secretion in the colon of fasted or undernourished gerbils. We conclude that fasting and undernourishment have no significant effects on fluid transport across the gerbil jejunum but reversed basal absorption in the fed ileum and colon into secretion. cGMP mimic the effects of STa in the jejunum taken from undernourished gerbils, in the ileum obtained under the three nutritional states and in the colon taken from fasting animals.     

The small intestine can both absorb and glucuronidate luminal flavonoids.

We have studied the perfusion of the jejunum and ileum in an isolated rat intestine model with flavonoids and hydroxycinnamates and the influence of glycosylation on the subsequent metabolism. Flavone and flavonol glucosides and their corresponding aglycones are glucuronidated during transfer across the rat jejunum and ileum and this glucuronidation occurs without the need for gut microflora. Furthermore, this suggests the presence of glycosidases as well as UDP-glucuronyl transferase in the jejunum. In contrast, quercetin-3-glucoside and rutin are mainly absorbed unmetabolised. The results suggest that the more highly reducing phenolics are absorbed predominantly as glucuronides (96.5%+/-4.6) of the amount absorbed, whereas monophenolic hydroxycinnamates and monophenolic B-ring flavonoids are less predisposed to glucuronidation and higher levels of aglycone (88.1%+/-10.1) are detected on absorption through both the jejunum and ileum.     

Effect of early antibiotic intervention on specific bacterial communities and immune parameters in the small intestine of growing pigs fed different protein level diets

Antibiotics are designed to affect gut microbiota and subsequently gut homeostasis. However, limited information exists about short- and long-term effects of early antibiotic intervention (EAI) on gut homeostasis (especially for the small intestine) of pigs following antibiotic withdrawal. We investigated the impact of EAI on specific bacterial communities, microbial metabolites and mucosal immune parameters in the small intestine of later-growth-stage pigs fed with diets differing in CP levels. Eighteen litters of piglets were fed creep feed with or without antibiotics from day 7 to day 42. At day 42, pigs within each group were offered a normal- or low-CP diet. Five pigs per group were slaughtered at days 77 and 120. At day 77, EAI increased Enterobacteriaceae counts in the jejunum and ileum and decreased Bifidobacterium counts in the jejunum and ileum (P < 0.05). Moreover, tryptamine, putrescine, secretory immunoglobulin (Ig) A and IgG concentrations in the ileum and interleukin-10 (IL-10) mRNA and protein levels in the jejunum and ileum were decreased in pigs with EAI (P < 0.05). At day 120, EAI only suppressed Clostridium cluster XIVa counts in the jejunum and ileum (P < 0.05). These results suggest that EAI has a short-term effect on specific bacterial communities, amino acid decarboxylation and mucosal immune parameters in the small intestine (particularly in the ileum). At days 77 and 120, feeding a low-CP diet affected Bifidobacterium, Clostridium cluster IV, Clostridium cluster XIVa and Enterobacteriaceae counts in the jejunum or ileum (P < 0.05). Moreover, feeding a low-CP diet increased the concentrations of Igs in the jejunum and decreased pro-inflammatory cytokines levels in the jejunum and ileum (P < 0.05). At day 120, feeding a low-CP diet increased short-chain fatty acid concentrations, reduced ammonia and spermidine concentrations and up-regulated genes related to barrier function in the jejunum and ileum (P < 0.05). These results suggest that feeding a low-CP diet changes specific bacterial communities and intestinal metabolite concentrations and modifies mucosal immune parameters. These findings contribute to our understanding on the duration of the impact of EAI on gut homeostasis and may provide basis data for nutritional modification in young pigs after antibiotic treatment.     

Proteolytic and peptidase activities of the jejunum and ileum of the rat during postnatal development

1. Proteolytic (substrate nitrocasein), tripeptidase (substrate glycylglycylgly-cine) and aminopeptidase (substrate leucyl-beta-naphthylamide) activities were studied in homogenates of jejunal and ileal mucosa of 7-, 10-, 14-, 21-, 35- and 60-day-old rats. 2. Proteolytic activity was practically the same in jejunum of 7-, 10-, 14- and 21-day-old rats, but after day 21 a significant increase was observed. The activity of the ileum changed very little during postnatal development and was always higher than that of the jejunum. 3. Tripeptidase activity was low in the jejunum of 7- and 14-day-old rats, an increase was observed between day 14 and 21, but later no substantial changes were found. There were no changes in the ileum. The activity in the jejunum of 7- and 14-day-old rats was lower than in the ileum, but later the jejunum was more active than the ileum. 4. Aminopeptidase activity had a similar developmental pattern to tripeptidase activity. A low activity was found in the jejunum of 7- and 14-day-old rats, the maximum was in 21-day-old rats and then a decrease was observed, though values for 60-day-old rats were still higher than for 7- and 10-day-old rats. The activity in the ileum was practically the same in all age groups studied except in 14- and 21-day-old rats, where a transient peak was observed.     

Postnatal development of intestinal bile salt transport. Relationship to membrane physico-chemical changes

The postnatal development of intestinal bile salt transport in the rat was examined using the villus technique. Jejunal uptake of taurocholate was linear with respect to incubation concentration at all study ages. Ileal uptake was linear with taurocholate concentration during the first 2 postnatal weeks; a curvilinear relationship indicating the presence of saturable transport appeared during the third week. With the appearance of ileal active transport at age 3 weeks, the Km (app) was constant at 0.49 mM, 0.59 mM, and 0.50 mM in 3-week, 4-week, and adult animals, respectively. The V(app) was 14.65 nmol X mg-1 (dry wt) X min-1 at 3 weeks and declined with age to 11.40 and 10.51 nmol X mg-1 (dry wt) X min-1 in 4-week and adult animals, respectively. The role of physico-chemical changes in the microvillus membrane in the development of ileal active transport was examined. With increasing postnatal age, microvillus membrane cholesterol content rose while the phospholipid content remained unchanged in both ileum and jejunum. Corresponding rises in the cholesterol/phospholipid ratio were observed in both sites. Simultaneously, the microvillus membrane fatty acid composition was changing from predominantly saturated to unsaturated species in both ileum and jejunum. The microvillus membrane fluorescence anisotropy (r) increased with postnatal age in jejunum when measured at 25 degrees C and 37 degrees C and ileum when measured at 25 degrees C; however, no change was noted in ileum when measured at 37 degrees C. Ileal active bile salt transport develops during the third postnatal week, and is associated with concurrent changes in membrane lipid composition and fluidity when measured at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane permeability parameters for some amino acids and beta-lactam antibiotics: application of the boundary layer approach

The boundary layer approach to analyzing the results of the perfused intestinal segment method of measuring membrane permeabilities is applied to the amino acids; leucine, valine, phenylalanine, lysine and aspartic acid and the beta-lactam antibiotics, cephalexin and penicillin V. The analysis indicates that in determining the membrane parameters, Pw vs. Cw data are preferable to using Jss = PwCw vs. Cw data. It is further shown that the carrier permeability, Pc* = Jmax*/Km, may be the most significant parameter to consider since luminal amino acid or drug concentration may generally be below the Km value. A comparison of P*c values for the beta-lactams with results for passively absorbed compounds indicates that the cephalosporins would be expected to be well absorbed orally based on the perfusion results. This suggests that this approach may be useful in estimating oral drug absorption for compounds that are absorbed passively as well as by a carrier-mediated mechanism.     

Studies on inositolphosphatase in rat small intestine

The possibility that inositolphosphatase differs from other intestinal phosphatases was tested by comparing several enzymatic characteristics of phosphatase activities of rat intestinal homogenate acting on various specific substrates. Optimum pH and temperature, Km, Vmax, heat stability, inhibition and metal ion requirement studies suggest that inositolphosphatase differs from phytase and p-nitrophenylphosphatase. Furthermore, we found that inositolphosphatase activity was about 2 times higher in duodenum and jejunum than ileum. It sedimented (90-100%) with a high-speed particulate fraction of mucosal homogenate; 42% of the activity was separated with the brush border membrane isolated from mucosal homogenate. Partial separation by gel filtration on Sephadex G200 and chromatography on phenyl Sepharose CL 4B provided additional evidence to suggest that inositolphosphatase and phytase are different enzymes.