Mucoadhesive-to-penetrating controllable peptosomes-in-microspheres co-loaded with anti-miR-31 oligonucleotide and Curcumin for targeted colorectal cancer therapy.

Background: Accumulating evidences indicate that nanomedicines greatly decrease the side effects and enhance the efficacy of colorectal cancer (CRC) treatment. In particular, the use of rectal delivery of nanomedicines, with advantages such as fast therapeutic effects and a diminishing hepatic first-pass effect, is currently emerging. Method: We established a CRC targeted delivery system, in which α-lactalbumin peptosomes (PSs) co-loaded with a microRNA (miR)-31 inhibitor (miR-31i) and curcumin (Cur) were encapsuslated in thiolated TEMPO oxidized Konjac glucomannan (sOKGM) microspheres, referred as sOKGM-PS-miR-31i/Cur. The CRC targeting capability, drug release profiles, mucoadhesive-to-penetrating properties and therapeutic efficacy of sOKGM-PS-miR-31i/Cur delivery system were evaluated in colorectal cancer cells and azoxymethane-dextran sodium (AOM-DSS) induced tumor models. Results: sOKGM-PS-miR-31i/Cur delivery system were stable in the harsh gastrointestinal environment after rectal or oral administration; and were also mucoadhesive due to disulfide bond interactions with the colonic mucus layer, resulting in an enhanced drug retention and local bioavailability in the colon. Concomitantly, the released PS-miR-31i/Cur PSs from the microsphere was mucus-penetrating, efficiently passing through the colonic mucus layer, and allowed Cur and miR-31i specifically target to colon tumor cells with the guide of CD133 targeting peptides. Consequently, rectal delivery of sOKGM-PS-miR-31i/Cur microspheres suppressed tumor growth in an azoxymethane-dextran sodium sulfate (AOM-DSS)-induced tumor model. Conclusion: sOKGM-PS-miR-31i/Cur microspheres are effective rectal delivery system with combined advantages of mucoadhesive and mucus-penetrating properties, representing a potent and viable therapeutic approach for CRC.

α-Lactalbumin:Oleic Acid Complex Spontaneously Delivers Oleic Acid to Artificial and Erythrocyte Membranes.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein-fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity.

Dietary protein structure affects endogenous ileal amino acids but not true ileal amino acid digestibility in growing male rats.

BACKGROUND: The amount of endogenous, as opposed to undigested dietary, protein in digesta is a measure of fundamental interest related to gut physiology and function. OBJECTIVE: The objective of this study was to determine whether alimentation with proteins having differing amino acid compositions influenced endogenous ileal amino acids (EIAAs) and true ileal amino acid digestibility (TIAAD) values. METHODS: Male rats (n = 8) were fed a purified diet containing 100 g/kg of 1 of 5 protein hydrolysates, each derived from a different semipurified intact protein source [gelatin, beef muscle (BM), casein, soy protein isolate (SPI), and lactalbumin] devoid of antinutritional factors or fiber. The rats were fed their respective hydrolysate-based diet for 1 d after receiving the same diet but containing the corresponding intact protein source for 7 d. Titanium dioxide was used as an indigestible marker. Ileal digesta were collected after the rats were killed, and EIAAs were determined (precipitate + retentate) after centrifugation and ultrafiltration of the digesta. The TIAAD values of the intact protein sources were determined using EIAA flows based on each protein hydrolysate. RESULTS: Mean EIAA flows differed (P < 0.05) across protein hydrolysates for most amino acids, with the mean ± SEM EIAA flows across amino acids being 262 ± 17, 253 ± 12, 248 ± 18, 226 ± 14, and 191 ± 20 mg/kg dry matter intake for the gelatin, BM, casein, SPI, and lactalbumin hydrolysates, respectively. The only difference (P < 0.05) for the mean EIAA flows across amino acids within each protein hydrolysate was observed between gelatin (262 ± 17 mg/kg) and lactalbumin (191 ± 20 mg/kg) hydrolysates. Except for Trp (P < 0.001) in gelatin and lactalbumin hydrolysates, EIAA flows determined using the casein hydrolysate were not different (P ≥ 0.05) from EIAA flows determined using the other protein hydrolysates. TIAAD values were not generally different (P ≥ 0.05) regardless of the hydrolysate used to determine the EIAA flows. CONCLUSIONS: Protein source affected EIAA flows, although the differences had little effect on TIAAD. Enzyme hydrolyzed casein is a suitable model hydrolysate for determining TIAAD with the enzyme-hydrolyzed protein-ultrafiltration technique.

Spreading of proteins and its effect on adsorption and desorption kinetics.

The kinetics of adsorption of lysozyme and alpha-lactalbumin from aqueous solution on silica and hydrophobized silica has been studied. The initial rate of adsorption of lysozyme at the hydrophilic surface is comparable with the limiting flux. For lysozyme at the hydrophobic surface and alpha-lactalbumin on both surfaces, the rate of adsorption is lower than the limiting flux, but the adsorption proceeds cooperatively, as manifested by an increase in the adsorption rate after the first protein molecules are adsorbed. At the hydrophilic surface, adsorption saturation (reflected in a steady-state value of the adsorbed amount) of both proteins strongly depends on the rate of adsorption, but for the hydrophobic surface no such dependency is observed. It points to structural relaxation ("spreading") of the adsorbed protein molecules, which occurs at the hydrophobic surface faster than at the hydrophilic one. For lysozyme, desorption has been studied as well. It is found that the desorbable fraction decreases after longer residence time of the protein at the interface.

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy.

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.

Sexual dimorphism in the permeability response of coronary microvessels to adenosine.

Gender influences volume regulation via several mechanisms; whether these include microvascular exchange, especially in the heart, is not known. In response to adenosine (Ado), permeability (P(s)) to protein of coronary arterioles of female pigs decreases acutely. Whether Ado induces similar P(s) changes in arterioles from males or whether equivalent responses occur in coronary venules of either sex has not been determined. Hypotheses that 1) basal P(s) properties and 2) P(s) responses to vasoactive stimuli are sex independent were evaluated from measures of P(s) to two hydrophilic proteins, alpha-lactalbumin and porcine serum albumin (PSA), in arterioles and venules isolated from hearts of adult male and female pigs. Consistent with hypothesis 1, basal P(s) values of both microvessel types were independent of sex. Contrary to hypothesis 2, P(s) responses to Ado varied with sex, protein, and vessel type. Confirming earlier studies, Ado induced a approximately 20% decrease in P(s) to both proteins in coronary arterioles from females. In arterioles from males, Ado did not change P(s) for alpha-lactalbumin (P(s)(alpha-lactalb), 3 +/- 13%), whereas P(s) for PSA (P(s)(PSA)) decreased by 27 +/- 8% (P < 0.005). In venules from females, Ado elevated P(s)(PSA) by 44 +/- 20% (P < 0.05), whereas in those from males, Ado reduced P(s)(PSA) by 24 +/- 5% (P < 0.05). The variety of outcomes is consistent with transvascular protein and protein-carried solute flux being regulated by multiple sex-dependent mechanisms in the heart and provides evidence of differences in exchange homeostasis of males and females in health and, likely, disease.

An electrodiffusion-filtration model for effects of endothelial surface glycocalyx on microvessel permeability to macromolecules.

Endothelial surface glycocalyx plays an important role in the regulation of microvessel permeability by possibly changing its charge and configuration. To investigate the mechanisms by which surface properties of the endothelial cells control the changes in microvessel permeability, we extended the electrodiffusion model developed by Fu et al. [Am. J. Physiol. 284, H1240-1250 (2003)], which is for the interendothelial cleft with a negatively charged surface glycocalyx layer, to include the filtration due to hydrostatic and oncotic pressures across the microvessel wall as well as the electrical potential across the glycocalyx layer On the basis of the hypotheses proposed by Curry [Microcirculation 1(1): 11-26 (1994)], the predictions from this electrodiffusion-filtration model provide a good agreement with experimental data for permeability of negatively charged a-lactalbumin summarized in Curry [Microcirculation 1(1), 11-26 (1994)] under various conditions. In addition, we applied this new model to describe the transport of negatively charged macromolecules, bovine serum albumin (BSA), across venular microvessels in frog mesentery. According to the model, the convective component of the albumin transport is greatly diminished by the presence of a negatively charged glycocalyx under both normal and increased permeability conditions.

HAMLET interacts with histones and chromatin in tumor cell nuclei.

HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

Tumor necrosis factor-alpha-induced leukocyte adhesion and microvessel permeability.

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-alpha (3.5 microg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity (L(p)) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled alpha-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-alpha did not affect basal microvessel permeability, but intravenous injection of TNF-alpha caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either L(p) or solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-alpha-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.

Protein adsorption onto auto-assembled polyelectrolyte films.

Surface modification by deposition of ordered protein systems constitutes one of the major objectives of bio-related chemistry and biotechnology. In this respect a concept has recently been reported aimed at fabricating multilayers by the consecutive adsorption of positively and negatively charged polyelectrolytes. We investigate the adsorption processes between polyelectrolyte multilayers and a series of positively and negatively charged proteins. The film buildup and adsorption experiments were followed by Scanning Angle Reflectometry (SAR). We find that proteins strongly interact with the polyelectrolyte film whatever the sign of the charge of both the multilayer and the protein. When charges of the multilayer and the protein are similar, one usually observes the formation of protein monolayers, which can become dense. We also show that when the protein and the multilayer become oppositely charged, the adsorbed amounts are usually larger and the formation of thick protein layers extending up to several times the largest dimension of the protein can be observed. Our results confirm that electrostatic interactions dominate protein/polyelectrolyte multilayer interactions.

Semisolid, self-catalyzed poly(ortho ester)s as controlled-release systems: protein release and protein stability issues.

Semisolid, self-catalyzed poly(ortho ester)s (POEs), are investigated as potential sustained-release systems for proteins. In this study, some factors influencing protein release kinetics and protein instability were evaluated. As model proteins, lysozyme, alpha-lactalbumin, bovine serum albumin, and vascular endothelial growth factor, which were lyophilized from various buffer solutions in the absence and presence of lyoprotectants, were used. For all protein formulations, the release kinetics followed the visually observed polymer dissolution profile. In the absence of any buffers in the protein formulation, the release was continuous. Formulations containing a buffer below pH 7 accelerated POE degradation, resulting in faster protein release. In contrast, a strong buffer capacity at pH 7 reduced the POE degradation and resulted in a biphasic release pattern. Moreover, proteins with a high isoelectric point (pI > 7) appeared to catalyze the POE degradation, and the effect of the buffer strength and pH was much smaller than for proteins with low pI (< 7). In the absence of lyoprotectants, all proteins tested showed an increasing fraction of covalent protein aggregates during the release. Protein formulations containing a lyoprotectant, such as sucrose or trehalose, did not show a significantly increased aggregation, whereas there was a minor influence of the large solid loadings on the release kinetics. In conclusion, this semisolid, self-catalyzed POE showed good promise as a sustained-release matrix for bioactive proteins.

The effects of alpha-lactabumin and whey protein concentrate on dry matter recovery, TCA soluble protein levels, and peptide distribution in the rat gastrointestinal tract.

The effects of two dietary proteins on dry matter recovery, trichloroacetic acid (TCA) soluble protein concentration, and peptide distribution in gastrointestinal contents were investigated in rats trained to consume, in a single 2-hour daily meal, diets containing alpha-lactalbumin (alpha-LA) or whey protein concentrate (WPC) for two weeks. Compared with the WPC diet, the alpha-LA diet emptied faster from the stomach. Dry matter recovery was higher in the stomach contents of rats fed the WPC diet than in those given the alpha-LA diet, but dry matter content in the small intestine was comparable. TCA soluble protein levels in the stomach and the small intestinal contents were also significantly (P < 0.001) higher in rats fed the WPC diet. The concentration of peptides having molecular weights (MW) ranging from 12,500-30,000 daltons (Da) was higher in the stomach contents of rats fed the WPC diet. Conversely, the level of peptides ranging from 5000-12,500 Da was higher in the stomach contents of rats fed the alpha-LA diet. For both diets, the small intestinal contents were characterized by high levels of amino acids and small peptides. These results suggest that the hydrolysis and absorption of alpha-LA is faster than that of WPC.

Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments.

Whereas the glycocalyx of endothelial cells has been shown to influence solute flux from capillary microvessels, little is known about its contribution to the movement of macromolecules across the walls of other microvessels. We evaluated the hypothesis that a glycocalyx contributes resistance to protein flux measured in coronary arterioles. Apparent solute permeability (P(s)) to two proteins of different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum albumin (PSA), was determined in arterioles isolated from the hearts of 43 female Yucatan miniature swine. P(s) was assessed in arterioles with an "intact" glycocalyx under control conditions and again after suffusion with adenosine (Ado, 10(-5) M, n = 42 arterioles, N = 29 pigs). In a second set of experiments (n = 21 arterioles, N = 21 pigs) arteriolar P(s) was determined before and after perfusion with enzyme (pronase or heparinase), which was used to digest the glycocalyx. P(s) was assessed a third time on those microvessels after exposure to Ado. Consistent with the hypothesis, P(s) for PSA (P(PSA)(s)) and P(s) for alpha-lactalb (P(alpha-lactalb)(s)) increased from basal levels following enzyme treatment. Subsequent suffusion with Ado, a significant metabolite known to alter coronary vascular smooth muscle tone and permeability, resulted in a significant reduction of basal P(alpha-lactalb)(s) in both untreated and enzyme-treated arterioles. Furthermore, in untreated arterioles, P(PSA)(s) was unchanged by Ado suffusion, whereas Ado induced a pronounced reduction in P(PSA)(s) of enzyme-treated vessels. These data demonstrate that in intact coronary arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes significantly to net barrier resistance to solute flux.

Adsorption of low molecular weight proteins to hemodialysis membranes: experimental results and simulations.

Adsorption of alpha-lactalbumin and Cytochrome C on different hollow-fiber hemodialysis modules, whose main membrane constituent was polyacrylonitrile (PAN), polymethylmethacrylate (PMMA) or polysulfone (PS), were compared under different pH and flow rate conditions. These proteins were chosen as models of small scarce proteins like beta2-microglobulin from which the patient's blood should be epurated. Influence of pH suggests the importance of electrostatic interactions on a charged membrane since adsorption greatly decreases at pH 10.9 for Cytochrome C (pI = 10.6) and at pH 7.4 for alpha-lactalbumin (pI = 5). The difference in adsorbance between different membranes is most probably due to their different microstructures. However, the chemical nature of the support plays a non-negligible role since PMMA membranes have smaller initial adsorption rates than PAN membranes. This is correlated with the density of membrane surface charge showing the importance of electrostatic interactions. The influence of the wall shear rate on adsorption kinetics is analyzed through numerical simulations, in terms of transport limitation in the liquid phase and interfacial reaction.

The in vitro displacement of adsorbed model antigens from aluminium-containing adjuvants by interstitial proteins.

Vaccines prepared by adsorbing an antigen onto an aluminium-containing adjuvant are usually administered by intramuscular or subcutaneous injection. The vaccine then comes in contact with interstitial fluid which contains proteins. In vitro displacement studies were performed to determine whether antigens, which are adsorbed to aluminium-containing adjuvants, can be displaced by interstitial proteins. It was found that when previously adsorbed model antigens such as lysozyme or myoglobin were exposed to interstitial proteins such as albumin or fibrinogen that extensive displacement occurred. A factorial study of the displacement of myoglobin from aluminium hydroxide adjuvant by albumin was performed. The displacement occurred rapidly with the majority of the displacement occurring in less than 15 min. Whether the concentration of the adsorbed myoglobin was above or below the adsorptive capacity of the aluminium hydroxide adjuvant affected the amount which could be displaced. Less myoglobin was displaced when the concentration was below the adsorptive capacity. The age of the model vaccine (1, 2 or 7 days) prior to exposure to the interstitial protein did not influence the amount of myoglobin that was displaced. The affinity of model antigens and interstitial proteins for aluminium hydroxide or aluminium phosphate adjuvant was characterized by the adsorption coefficient in the Langmuir equation. In every case studied, the protein having the larger adsorption coefficient was able to displace the protein with the smaller adsorption coefficient.

Prediction of permeability-surface area product data by continuous-distribution pore models.

OBJECTIVE: To investigate the ability of continuous-distribution pore models to accurately predict permeability-surface area product (PS) experimental data in skeletal muscle. METHODS: Models having a water-only (WO) pathway and continuous distributions of microvascular transport-pathway sizes were fit to solute reflection-coefficient (sigma) experimental data (approximately 0.5-16 nm Stokes radii) obtained from skeletal muscle to determine optimal parameter values. Without further modification, these models were used to predict experimental PS values obtained from the literature for small solutes ranging in size from NaCl to inulin and for three proteins, alpha-lactalbumin, ovalbumin, and albumin (approximately 0.23-3.7 nm radii). The protein PSs were determined from fluorescent tracer-diffusion curves and a nonlinear model of tracer diffusion in the cat hindlimb preparation. The model's PS predictions were compared to those of a discrete-pore model previously developed and a fiber-matrix (FM) model. RESULTS: A log-normal (LN) continuous pore-size distribution plus WO-pathway model (three free parameters) fit the sigma data to within the 95% confidence intervals of each of eight solutes spanning a 32-fold size range and was nearly as close to the data as was the two discrete-pore plus WO-pathway model (four free parameters). Both models closely described the PS data for nine solutes spanning a 14-fold size range. The fit of a fiber-matrix plus WO-pathway model (three free parameters) to the sigma data was much poorer than for the other models. CONCLUSIONS: The LN and two-discrete-pore models accurately describe sigma and PS experimental data in cat and human skeletal muscle. Therefore, experimental data resulting from complex microvascular transport processes are well characterized by simple pore models.

Test of a two-pathway model for small-solute exchange across the capillary wall.

We previously proposed a two-pathway model for solute and water transport across vascular endothelium (Fu, B. M., R. Tsay, F. E. Curry, and S. Weinbaum. J. Biomech. Eng. 116: 502-513, 1994) that hypothesized the existence of a continuous slit 2 nm wide along tight junction strands within the interendothelial cleft in parallel with 20 x 150-nm breaks in tight junctions. We tested this model by measuring capillary permeability coefficients (P) to a small solute (sodium fluorescein, radius 0.45 nm), assumed to permeate primarily the 2-nm small pore, and an intermediate-sized solute (FITC-alpha-lactalbumin, radius 2.01 nm) excluded from the small pore. Mean values of the paired diffusive permeability coefficients, Psodium fluorescein and PFITC-alpha-lactalbumin, were 34.4 and 2.9 x 10(-6) cm/s, respectively, after corrections for solvent drag and free dye (n = 26). These permeabilities were accounted for by transport through the large-break pathway without the additional capacity of the hypothetical 2-nm pathway. As a further test we examined the relative reductions of Psodium fluorescein and PFITC-alpha-lactalbumin produced by elevated intracellular cAMP. Within 20 min after the introduction of rolipram and forskolin, Psodium fluorescein and PFITC-alpha-lactalbumin decreased to 0.67 and 0.64 times their respective baseline values. These similar responses to permeability decrease were evidence that the two solutes were carried by a common pathway. Combined results in both control and reduced permeability states did not support the hypothesis that a separate pathway across tight junctions is available for solutes with a radius as large as 0.75 nm. If such a pathway is present, then its size must be smaller than that of sodium fluorescein.

Gut permeability to human alpha-lactalbumin, beta-lactoglobulin, mannitol, and lactulose in celiac disease.

Our objective was to examine the permeability of the gut to protein macromolecules and sugar probes and their possible association in celiac disease patients. We studied the permeability to human alpha-lactalbumin, beta-lactoglobulin, mannitol, and lactulose on 46 occasions in 33 celiac disease patients in various phases of the disease; in addition, mannitol and lactulose permeability was studied in 18 healthy controls. Lactalbumin absorption was detected in 19 of 42 patients tested, more often in celiac disease patients with villous atrophy than in those with normal jejunal biopsy (p = 0.01). Higher absorption of lactalbumin was found in patients with subtotal villous atrophy than in those with normal biopsy (p = 0.02). beta-lactoglobulin was found in four of 42 patients tested. Less mannitol was absorbed by patients with either subtotal or partial villous atrophy than by those with normal histology (p = 0.001 and 0.006, respectively). Lactulose recovery was higher in newly diagnosed patients and patients with subtotal villous atrophy than in controls (p = 0.007 and 0.03, respectively). The lactulose/mannitol ratio was higher in newly diagnosed patients and patients with villous atrophy than in controls (p = 0.002 and 0.002, respectively). The correlation between permeability to lactalbumin and mannitol and lactulose was poor. We conclude that permeability to proteins and sugar molecules is abnormal in celiac disease patients with mucosal damage and that they probably reflect different mechanisms of penetration.

ANP increases capillary permeability to protein independent of perfusate protein composition.

In further studying hormonal regulation of microvascular exchange, we tested two hypotheses: 1) atrial natriuretic peptide (ANP) would increase capillary permeability to protein and 2) the actions of ANP on capillary permeability would be independent of the perfusate protein composition. These hypotheses were tested by assessing in situ capillary transport coefficients: hydraulic conductivity (Lp), apparent and diffusive permeabilities (Ps and Psd, respectively) to the protein alpha-lactalbumine, and convective coupling [Lp(1-sigma)] in mesenteric capillaries in cerebrally pithed frogs (Rana pipiens). The transport coefficients were determined in the absence and presence of frog ANP (fANP) during perfusion with either frog plasma or bovine serum albumin (BSA). Control Lp was 1.8-fold greater in vessels perfused with BSA compared with plasma. In the presence of 10 or 100 nM fANP, Lp was increased by approximately 3.5-fold in plasma-perfused vessels and approximately 2-fold in BSA-perfused vessels from their respective controls. Control Ps was 4.2-fold higher in vessels perfused with BSA compared with plasma. Despite the differences in control permeability, the increase in Ps by fANP was of similar magnitude (2- to 3-fold) for both protein perfusates. Analysis of the pressure-dependent alpha-lactalbumin flux suggested that the increase in capillary permeability induced by fANP is consistent with fANP increasing permeability without altering the selectivity of the capillary barrier.