Comparative assessment of the resistance of the unstirred water layer to solute transport between two different intestinal perfusion systems

The resistance of the unstirred water layer to solute transport was estimated in two different intestinal single-pass perfusion systems for a comparative study, using D-glucose as a model compound. One is a well established perfusion system in anesthetized rats as a standard (system A). The other is the one in unanesthetized rats for comparison (system B). It was demonstrated that in system B as well as in system A the resistance of the unstirred water layer to D-glucose transport should be taken into account and this resistance, accordingly, the effective thickness of the unstirred water layer (delta) which is assumed to be in proportion to its resistance, could be described as a function of the perfusion rate by using a film model. The delta decreased with increasing perfusion rate and was larger in system A than in system B at each perfusion rate; 785 microns in system A versus 319 microns in system B at the perfusion rate of 0.16 ml/min and 337 microns versus 184 micron at that of 2.95 ml/min. Thus in system B the effective thickness, accordingly, the resistance, of the unstirred water layer was reduced to about 50% of that in system A, but the resistance of the unstirred water layer could still account for 85% of the total resistance at the maximum as far as D-glucose absorption was concerned, while 93% in system A. These results suggest that, compared with perfusion experiments in anesthetized rats (system A), the resistance of the unstirred water layer is reduced but cannot be left out of consideration even if perfusion experiments are performed in unanesthetized rats (system B). And the lower resistance of the unstirred water layer in system B was attributed to a turbulent flow in contrary to a laminar flow in system A.     

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

As part or a systematic study of alcoholism and thiamine absorption, the effect of diet-induced thiamine deficiency and the role of the unstirred water layer on 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 μM) was greater in the thiamine-deficient rats thatn in the controls. In contrast, uptake rates for high thiamine concentrations (20 and 50 μM) were similar in both groups. While $\text{1J}{}_{\max }$ was unaltered, $\text{1K}{}_{\mathrm{m}}$ 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 $\text{1J}{}_{\max }$ and $\text{1K}{}_{\mathrm{m}}$ under unstirred and st irred conditions. Without stirring, there was no difference in $\text{1J}{}_{\max }$ between the two groups. In contrast, both $\text{1K}{}_{\mathrm{m}}$ and the water layer were reduced in the thiamine-deficient rats. With stirring, $\text{1J}{}_{\max }$ was not affected, but both $\text{1K}{}_{\mathrm{m}}$ 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 pathological 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.     

Experimental demonstration of the effect of the unstirred water layer on the kinetic constants of the membrane transport ofd-glucose in rabbit jejunum

Summary The rate of active transport of a probe molecule into the intestinal mucosal cells is determined by the rate of movement of the solute molecule across two barriers, the unstirred water layer and the microvillus membrane of the epithelial cell. Previously a theoretical equation has been derived which describedJ _d, the velocity of unidirectional flux, as a function of the characteristics of the transport carrier in the membrane and of the resistance of the overlying unstirred water layer (UWL). The predictions of these equations have been tested experimentally by studying the effect of the rate of stirring of the bulk phase on thein vitro uptake ofd-glucose by rabbit jejunum. These studies demonstrated that, first, alterations in the UWL have a profound effect on the magnitude of the apparent affinity constant, xK _m ^* , of the active transport process. Second, at bulk phase concentrations in excess of theK _m the passive component of the experimentally determined flux rate becomes of such magnitude as to introduce significant error into the estimate of both the maximal transport rate,J _d ^m , and the trueK _m. Third, as a result of the UWL, the use of double-reciprocal plots to determineJ _d ^m andK _m leads to the overestimation of these constants. Finally, failure to account for the UWL leads to important quantitative errors describing a number of the characteristics of the transport process: these include an underestimation of the Q₁₀ and the effect of sodium ion on the active transport of glucose in the jejunum. The results confirm that the kinetic characteristics of the uptake of an actively transported molecule are a complex function of the resistance of both the UWL and the mucosal cell membrane, and this transport process can be adequately described by a newly-derived equation. It is apparent that there are serious limitations in the interpretation of much of the previously published data dealing with active transport processes in the intestine, since these studies failed to account for the effect of the UWL.     

Limitations of the Eadie-Hofstee plot to estimate kinetic parameters of intestinal transport in the presence of an unstirred water layer

Summary In the presence of an intestinal unstirred water layer, the relationship between substrate concentration (C ₁) and unidirectional flux (J _d) is not described by the equation for a rectangular hyperbole. Accordingly, transformations of the Michaelis-Menten equation may not necessarily be linear and may lead to serious errors in the estimation of the affinity constant (K _m) and maximal transport rate (J _d ^m ) of carrier-mediated processes. An equation has previously been derived which describedJ _d under conditions of varying effective thickness or surface area of the unstirred water layer, the free diffusion coefficient of the probe molecule, and the distribution of transport sites along the villus. These theoretical curves have been analyzed by using the Eadie-Hofstee transformation (J _d vs. J_d/C₁) of the Michaelis-Menten equation. Use of this plot leads to serious discrepancies between the true and apparent affinity constants and between true and apparent maximal transport rates. These differences are further magnified by failure to correct for the contribution of passive permeation. The Eadie-Hofstee plot is of use, however, to infer certain qualitative characteristics of active transport processes, such as the variation in affinity constants and overlying resistance of the unstirred water layer at different sites along the villus and to predict the adequacy of the correction for the contribution of passive permeation.Abbreviations Used in the Text C ₁ Concentration of the probe molecule in the bulk phase - C ₂ Concentration of the probe molecule at the aqueous-membrane interface - d Effective thickness of UWL - D Free diffusion coefficient - d ⁿ d atn th segment of the villus - f _n Proportion of total carrier transport sites present on each segment of villus - J Unidirectional flux of probe molecule, uncorrected for surface area - J _d Unidirectional flux of probe molecule determined experimentally, corrected for surface area - J _d ^m Maximal transport rate, corrected for surface area - J _d ^m* Apparent maximal transport rate - J ^m Maximal transport rate, uncorrected for surface area - K _m Michaelis constant (true affinity constant) - K _m ^* Apparent affinity constant - K _m ⁿ K _m atnth segment of the villus - n The perpendicular height of the villus was divided into ten equal segments numberedn ₁ ton ₁₀ - p Passive permeability coefficient - S _m Functional surface area of the membrane - S _w Effective surface of UWL - S _w ⁿ S _w atnth segment of the villus - UWL Intestinal unstirred water layer     

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.     

Aging: its influence on the intestinal unstirred water layer thickness, surface area, and resistance in the unanesthetized rat

The intestinal absorption of some nutrients changes with aging. As the unstirred water layer (UWL) is an important rate limiting step in the absorption of nutrients in general and of lipid soluble nutrients in particular, we investigated possible changes in the UWL dimensions in the aging rat in vivo. We measured the thickness (d) of the UWL using rapid changes in the luminal sodium concentration to induce changes in the transmucosal potential differences. We assessed the surface area (Sw) and resistance (d/SwD) of the UWL at varying flow rates by using increasingly lipophilic medium chain saturated alcohols as probes. At high UWL resistance, d decreased from 318 to 268 microns between 1 and 29 months of age. As the animals aged, Sw changed from 114 to 106 cm2/100 cm and from 262 to 214 cm2/100 cm at low and high flow rates, respectively, using dodecanol as a probe. The resistance of the UWL (d/SwD) remained relatively stable at all ages studied. These experiments demonstrate that age-related changes in absorption are dependent on the aqueous diffusion coefficient and degree of lipid solubility of the specific nutrients. At low UWL resistance, absorption of compounds with higher diffusion coefficients and greater aqueous solubility is decreased with aging. In contrast, previous studies have demonstrated that the absorption of nutrients with low diffusion coefficients and high lipid solubility increases with aging especially when the resistance of the UWL is high.     

The effect of auxiliary conditions on intestinal unstirred layer diffusion modelled by numerical simulation.

Estimation of intestinal unstirred layer thickness usually involves inducing transmural potential difference changes by altering the content of the solution used to perfuse the small intestine. Osmotically active solutes, such as mannitol, when added to the luminal solution diffuse across the unstirred water layer (UWL) and induce osmotically dependent changes in potential difference. As an alternative procedure, the sodium ion in the luminal fluid can be replaced by another ion. As the sodium ion diffuses out of the UWL, the change in concentration next to the intestinal membrane alters the transmural potential difference. In both cases, UWL thickness is calculated from the time course of the potential difference changes, using a solution to the diffusion equation. The diffusion equation solution which allows the calculation of intestinal unstirred layer thickness was examined by simulation, using the method of numerical solutions. This process readily allows examination of the time course of diffusion under various imposed circumstances. The existing model for diffusion across the unstirred layer is based on auxiliary conditions which are unlikely to be fulfilled in the same intestine. The present simulation additionally incorporated the effects of membrane permeability, fluid absorption and less than instantaneous bulk phase concentration change. Simulation indicated that changes within the physiologically relevant range in the chosen auxiliary conditions (with the real unstirred layer length kept constant) can alter estimates of the apparent half-time. Consequently, changes in parameters unassociated with the unstirred layer would be misconstrued as alterations in unstirred layer thickness.     

A method for estimating the diffusion resistance of the unstirred layer of microorganisms

A general enzyme-mediated flux equation which incorporates diffusion resistance was derived. By fitting the model to phosphate flux data from a blue-green alga, Anacystis nidulans, diffusion resistance, the half-saturation constant and maximal uptake velocity were estimated. Diffusion resistance for this organism was estimated to be 29 s / cm with a relative precision of about 10%. The parameters were also estimated by graphical analysis of a Woolf plot, but this method was less precise and tended to yield biased estimates.     

Measurement of the effective thickness of the mucosal unstirred layer in Necturus gallbladder epithelium

The effective thickness of the unstirred fluid layer (USL) adjacent to an epithelial barrier can be estimated from the time course for the accumulation or depletion of a solute at the membrane surface. In 1985 we reported an unstirred layer thickness of approximately 70 microns for Necturus gallbladder epithelium. In our earlier studies the delay caused by noninstantaneous bulk solution mixing was not taken into account and thus the USL thickness was systematically overestimated. In the present studies we describe an analysis of the time course of solute arrival at the membrane surface that takes into account noninstantaneous bulk solution mixing. We also describe a simple technique to monitor the accumulation or depletion of a solute at the membrane surface. The time course for the change in the concentration of either tetramethylammonium (TMA+) or tetrabutylammonium (TBA+) upon elevation of bulk solution concentration is sensed at the membrane surface with an ion-sensitive microelectrode. Because of the high selectivity of the ion-sensitive resin for TMA+ or TBA+ over other monovalent cations in the solution (Na+ and K+), a low concentration (1-2 mM) of the probe can be used. By measuring the time course of the arrival of first one probe and then the other, under identical superfusion conditions, sufficient information is obtained to eliminate multiple fits to the data, obtained when only one probe is used. Neglecting bulk solution mixing caused an error greater than 50% in estimated apparent USL thickness. The effective thickness of the USL depends critically upon chamber geometry, flow rate, and the position of superfusion and suction pipettes. Under our experimental conditions the effective USL at the mucosal surface of Necturus gallbladder epithelium was approximately 40 microns.     

The determination of the kinetic parameters of a carrier mediated transport process in the presence of an unstirred water layer

A procedure is described, based on the Eadie-Hofstee plot, from which it is possible to determine the Michaelis constant and the maximum velocity of a membrane bound transport process that is separated from the substrate in the bulk solution by an unstirred layer. This can be done without knowing the magnitude of the latter provided that its effective thickness can be varied by altering the rate of stirring. Further, if the affinity of the carrier for the substrate is sufficiently large, then, it is possible to determine the rate constant and the effective concentration of the membrane bound carrier.     

Kinetic and chemical studies on the isomerization of monosaccharides in N-methylmorpholine-N-oxide (NMMO) under Lyocell conditions

The Lyocell process is a modern and environmentally fully compatible industrial fiber-making technology. Cellulosic pulp is dissolved without chemical derivatization in a melt of N-methylmorpholine-N-oxide monohydrate (NMMO). In the present work, the reactions of monosaccharides under Lyocell conditions were investigated in detail, using capillary zone electrophoresis as the analytical technique to clarify the composition of reaction mixtures and to follow the kinetics. Under Lyocell conditions, xylose and glucose undergo two competitive reactions: rapid conversion to nonreducing products, and complete isomerization involving the whole carbohydrate backbone, via ketose intermediates. Sugar acids are present in minor amounts only, as demonstrated by employing isotopically labeled material for NMR techniques.     

Dependence of the apparent thickness of the unstirred layer at the intestinal mucosa on nutrient concentration

The thickness of the intestinal unstirred layer determined with glycine is shown to increase from 247 ± 12 to 319 ± 14 μm (p < 0.001) as its concentration is raised from 5 to 40 mM; with glucose raised from 10 to 40 mM, the apparent value increases from 316 ± 13 to 380 ± 26 μm, because at higher concentrations the nutrients penetrate deeper to the bases of intestinal villi. The weak positive correlation between the apparent layer thickness (assayed with glucose, maltose, sucrose, alanine, leucine, and Gly-Ala in rats) and the parameter characterizing the rate of nutrient uptake (maximal short-circuit current response to nutrient) contradicts the assertion that the transporters are distributed uniformly along the villus height but because of high absorption in the upper part the nutrients do not reach the deeper transporters. It appears that the transporter distribution along the villi is nonuniform. The described method may serve to study the topography of nutrient transporters.     

A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer.

A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.     

Determination of kinetic parameters of a carrier-mediated transport in the perfused intestine by two-dimensional laminar flow model: effects of the unstirred water layer

The kinetic parameters of a carrier-mediated transport for D-glucose and for taurocholate were determined from rat in situ intestinal single perfusion experiments. The true parameters were obtained by the two-dimensional laminar flow model, in which the solute concentration at the aqueous-intestinal membrane interface can be calculated numerically without assuming the aqueous diffusion layer, discriminating the effects of the unstirred water layer. The true Michaelis constant was 4.5 mM for D-glucose and 1.5 mM for taurocholate. The true maximal transport velocity was 3.4 nmol/s per cm2 for D-glucose and 0.29 nmol/s per cm2 for taurocholate. The apparent Michaelis constant was raised by the factor of 6.6 for D-glucose and 3.6 for taurocholate due to the effects of the unstirred water layer. The maximal transport velocity was relatively unaffected by the unstirred water layer in both compounds. The values of the effective (operational) thickness of the unstirred water layer were compatible with those reported previously by employing various experimental methods. The kinetic parameters obtained in vitro everted sacs, for comparison, almost coincided with the true ones in situ. Therefore, the two-dimensional laminar flow model is shown to be valid not only for determining the kinetic parameters of a carrier-mediated transport in situ but also for predicting the absorption rate in situ from the uptake rate in vitro.     

Intestinal uptake of fatty acids and cholesterol in four animals species and man: role of unstirred water layer and bile salt micelle

The uptake (Jd) of fatty acids (FA), fatty alcohols (Alc) and cholesterol (C) into the jejunum of rats (R, Rattus norvegicus), rabbits (RAB, Oryctolagus cuniculus), guinea pigs (GB, Cavia porcellus), and hamsters (H, Mesocricetus auratus) was assessed in vitro. Using jejunal discs the Jd of Alc was H greater than R = GP greater than RAB, the Jd of FA was H-RAB greater than R greater than GP, but the Jd of C was R greater than H greater than RAB greater than GP. The Jd of FA was quantitatively and qualitatively different when using jejunal biopsies; in man the Jd of FA into biopsies was greater than in the other animal species, but there was no difference in Jd of FA into normal human jejunal biopsies and those showing severe abnormalities in villus architecture. There are marked species differences in the passive permeability properties of the jejunum and in the effective resistance of the overlying unstirred water layer, but these differences do not explain the species variations in the uptake of cholesterol.     

Equilibrium constants under physiological conditions for the condensation of acetaldehyde with tetrahydrofolic acid

The nonenzymatic reaction of ethanol-derived CH₃CHO with tissue constituents continues to be of interest as a potential mechanism underlying the toxicity of alcohol. The current study has focused on the spontaneous condensation of CH₃CHO with H₄folate under physiological conditions (38 °C, pH 7.0, I = 0.25 M). Computer analysis of uv spectral changes with increasing CH₃CHO concentrations demonstrated the presence of at least two different adducts. The observed equilibrium constant (K_obs) for the formation of the first adduct is 91 ± 2 m^?1 (121 ± 2 m^?1 at 25 °C), a value which is unaffected by variations in ionic strength (0.06–1.0 m) or by free [Mg²⁺] up to 5 mm. The NMR spectrum is compatible with the structure: 5,10-CH₃CH-H₄folate analogous to the naturally occurring 5,10-CH₂-H₄folate. The formation of the latter compound from HCHO and H₄folate, however, is much more favorable under the same conditions [K_obs = 3.0 ± 0.2 × 10⁴ M^?1 (38 °C), 3.6 ± 0.1 × 10⁴ M^?1 (25 °C)]. At the levels of CH₃CHO which accumulate during ethanol metabolism in vivo only a small fraction of the H₄folate will exist as the CH₃CHO derivative, yet it may ultimately be the ratio of free CH₃CHO to free HCHO in tissue which determines the physiological importance of the CH₃CHO adduct. Other adduct(s) of CH₃CHO with H₄folate are observed at very high levels of CH₃CHO but are unlikely to be of physiological significance.     

Binding constants of phenylalanine for the four mononucleotides

Summary Earlier work has shown that several properties of amino acids correlate directly with properties of their anticodonic nucleotides. Furthermore, in precipitation studies with thermal proteinoids and homopolyribonucleotides, an anticodonic preference was displayed between Lys-rich, Pro-rich and Gly-rich thermal proteinoids and their anticodonic polyribonucleotides. However, Phe-rich thermal proteinoid displayed a preference for its codonic nucleotide, poly U. This inconsistency seemed to be explained by a folding in of the hydrophobic residues of Phe causing the proteinoid to appear more hydrophilic. The present work used nuclear magnetic resonance techniques to resolve a limited question: To which of the four nucleotides does Phe bind most strongly? The results show quite clearly that Phe binds most strongly to its anticodonic nucleotide, AMP.     

Quantitative cleavage of the N-glycosidic bond under the normal conditions of methanolysis used for the analysis of glycoprotein monosaccharides

The most common method used for the liberation of monosaccharides from glycoprotein N-glycans involves anhydrous methanolysis because it liberates almost quantitatively monosaccharides as O-methylglycosides, which are resistant to further degradation. However, it is generally assumed that this method does not cleave quantitatively the N-glycosidic bonds. This paper demonstrates that classical methanolysis conditions quantitatively cleave the N-glycosidic bond (96%), liberating glucosamine (and not its O-methylglycosides) and other minor reaction products which were identified. Because other N-acetyl-d-glucosamine (GlcNAc) residues are quantitatively liberated as the O-methylglycosides of glucosamine, the GlcNAc residue involved in the N-glycosidic bond is separated from the others using gas chromatography of heptafluorobutyrate derivatives.