Ultrasensitive detection of trypsin in serum via nanochannel device.

A highly sensitive trypsin sensing system in serum was developed by using an anodic alumina oxide (AAO)-based, trypsin substrate-decorated hybrid ion permeation membrane. Owing to the trypsin-triggered peptide hydrolyzation reaction, the surface electrical feature of the peptide-decorated hybrid ion membrane changed. The electric double layer effect reduces the effective ion current diameter in the AAO nano unit, so that the ion current rectification ratio will be enhanced, realizing the quantitative detection of trypsin. The lowest detection concentration can be achieved as low as 0.1 pM. This method is no need for sample pre-preparation, easy to operate, highly sensitive, and also applicable to other enzyme evaluation systems by changing corresponding substrates. This study provides a new idea for selective measurements of proteases in complex biological samples.

In Vitro and In Vivo Evaluation of Casein as a Drug Carrier for Enzymatically Triggered Dissolution Enhancement from Solid Dispersions.

Due to its unique properties, such as biodegradability, biocompatibility, high amphiphilic property, and micelle formation, casein (CS) has been increasingly studied for drug delivery. We used CS as a drug carrier in solid dispersions (SDs) and evaluated the effect of its degradation by trypsin on drug dissolution from the dispersions. SDs of CS and mefenamic acid (MA) were prepared by physical mixing, kneading, and coprecipitation methods. In comparison to pure MA, the dispersions were evaluated for drug-protein interaction, loss of drug crystalinity, and drug morphology by differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared spectroscopy, and scanning electron microscopy. Drug dissolution from the dispersions was evaluated in simulated intestinal fluid as enzyme free and trypsin-enriched media. Furthermore, in vivo drug absorption of MA from CS-MA coprecipitate was evaluated in rats, in comparison with a reference SD of polyethylene glycol and MA (PEG-MA SD). Relative to other CS preparations, CS-MA coprecipitate showed the highest loss of drug crystallinity, drug micronization, and CS-MA interaction. CS remarkably enhanced the dissolution rate and extent of MA from the physical and kneaded mixtures. However, the highest dissolution enhancement was obtained when MA was coprecipitated with CS. Trypsin that can hydrolyze CS during dissolution resulted in further enhancement of MA dissolution from the physical and kneaded mixtures. However, a corresponding retardation effect was obtained for the coprecipitate. In correlation with in vitro drug release, CS-MA coprecipitate also showed significantly higher MA bioavailability in rats than PEG-MA SD.

Proteomic studies with a novel nano-magnetic chelating system to capture metalloproteins and its application in the preliminary study of monocyte and macrophage sub-secretome.

A new chelating chromatography method was developed based in the use of magnetic iron oxide nanoparticles functionalized with EDTA-TMS ((N-(trimethoxysilylpropyl)ethylenediaminetriacetate trisodium salt). These particles combine a high surface area, biocompatibility and magnetic removal from solution, with the chelating affinity towards metal ions. The particles were used to selectively capture metallo-dependant proteins in secretome obtained from human monocytes and mouse macrophages. Secreted metallo-dependant proteins are highly important sources of information since they are involved in several pathological processes. The identification of secreted proteins involved in these processes is highly important for diagnosis or monitoring the progression of a disease. In this multiple-approach study it was possible to not only selectively capture several secreted metallo-dependant proteins, but also to significantly avoid masking proteins such as the highly abundant albumin form the fetal bovine serum used to supplement the cell culture medium. Overall, the magnetic nanoparticle-based chelating chromatography method developed here has proved to be a sensitive, low cost, and a quick tool for sample treatment in order to selectively enrich metalloproteins while overcoming the contamination of highly abundant proteins.

Label-Free real-time quantification of enzyme levels by interferometric spectroscopy combined with gelatin-modified nanoporous anodic alumina photonic films.

Herein, we present an interferometric sensor based on the combination of chemically functionalized nanoporous anodic alumina photonic films (NAA-PFs) and reflectometric interference spectroscopy (RIfS) aimed to detect trace levels of enzymes by selective digestion of gelatin. The fabrication and sensing performance of the proposed sensor were characterized in real-time by estimating the changes in effective optical thickness (i.e., sensing principle) of gelatin-modified NAA-PFs (i.e., sensing element) during enzymatic digestion. The working range (WR), sensitivity (S), low limit of detection (LLoD), and linearity (R(2)) of this enzymatic sensor were established by a series of experiments with different concentrations of gelatin (i.e., specific chemical sensing element) and trypsin (i.e., analyte), a model protease enzyme with relevant implications as a biomarker in the diagnosis of several diseases. The chemical selectivity of the sensor was demonstrated by comparison of gelatin digestion by other nonspecific enzyme models such as chymotrypsin and horseradish peroxidase. Furthermore, the role of the chemical sensing element (i.e., gelatin) was assessed by using hemoglobin instead of gelatin. Finally, we demonstrated that this sensor can be readily used to establish the kinetic parameters of enzymatic reactions. The obtained results revealed that the presented sensor has a promising potential to be used as a point-of-care system for fast detection of gastrointestinal diseases at early stages.

Real-time fluorometric turn-on assay for protease activity and inhibitor screening with a benzoperylene probe.

A real-time fluorescence turn-on strategy for protease activity and inhibitor screening has been developed. A negatively charged benzo[ghi]perylene derivative (probe 1) was employed. Protamine is a cationic protein which can induce aggregation of probe 1 via strong electrostatic and hydrophobic interactions. The fluorescence of probe 1 was efficiently quenched. In the presence of a protease, protamine was enzymatically hydrolyzed and probe 1 de-aggregated. The recovery of the probe 1 monomer fluorescence could be detected. The protease activity could be monitored in real-time. In addition, upon addition of a protease inhibitor, the protease-catalyzed hydrolysis was inhibited, which led to a decreased fluorescence recovery. The fluorometric assay thus could also be employed for screening protease inhibitors.

Conformational changes of bovine beta-trypsin and trypsinogen induced by divalent ions: an energy-dispersive X-ray diffraction and functional study.

The radius of gyration (R(g)) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique as a function of Ca(2+) or SO(4)(2-) concentration; these results have been supplemented with measurements of association equilibrium constants of Ca(2+) to its binding site(s) on both serine proteases and some of their adducts (with an effector and/or an inhibitor). As a whole, all information reported in the present work demonstrates that: (i) the strains exerted by different ions on these proteases produce diverse structural modifications; and (ii) at least in the case of Ca(2+), the changes in R(g) can be ascribed to the direct interaction of the binding site(s) on the protein matrix with the cation.

[Exoproteinases of the oomycete Phytophthora infestans]. / Ekzoproteinazy oomitseta Phytophthora infestans.

When grown in a medium containing heat-stable potato tuber proteins, the oomycete Phytophthora infestans (Mont.) de Bary produces a set of exoproteinases active at neutral and mildly basic pH values. These extracellular proteinases have been shown by SDS-PAGE with the presence of gelatin to include at least six components differing in molecular weight. Inhibitory analysis and study of the effects of the enzymes on various synthetic substrates show that the culture liquid of P. infestans contains mainly serine proteinases specific to trypsin and subtilisin and metalloproteinases. Their activity is suppressed by proteinase-inhibitor proteins from potato tubers. It is suggested that P. infestans exoproteinases may be the metabolic target for natural proteinase inhibitors from potato.

Influence of intraduodenally infused olive and coconut oil on postprandial exocrine pancreatic secretions of growing pigs.

The effect of dietary vegetable oils differing in fatty acid composition that were infused directly into the duodenum on exocrine pancreatic secretions in pigs has not previously been studied. The objective of the present study was to determine the acute response of the exocrine pancreas to vegetable oils with various fatty acid profiles under prandial conditions. Six growing pigs (BW 13.2 kg) were surgically prepared with pancreatic duct catheters and duodenal reentrant T-cannulas. The animals were fed twice a day (1000 and 1600) a commercial weaner diet at a rate of 2% of BW. Beginning with the morning feeding, olive oil, coconut oil, or saline as a control were infused in boluses every 5 min in total 0.1% of BW over a period of 1 h directly into the duodenum according to a 3 x 3 Latin square design. Pancreatic juice was collected over a period of 4 h, beginning 1 h preprandially (0900) until 3 h postprandially (1300). A time effect was observed after the infusion of olive oil on the volume of secretion, on protein contents and outputs, as well as on lipase contents and outputs and on colipase contents. The infusion of saline and coconut oil changed the runs of the curves for lipase and colipase outputs. No time x treatment interactions were observed regarding volume of secretion, protein contents and outputs, trypsin contents and outputs, and lipase outputs. The runs of the curves for lipase contents were different between the olive oil and saline treatment and between the olive oil and coconut oil treatment. The runs of the curves for the olive oil and saline treatment differed from each other regarding colipase contents. Pooled values of colipase outputs were elevated after coconut oil treatment, and a positive correlation between trypsin and colipase contents was found. Under prandial conditions, the exocrine pancreas responds differently in its acute secretion to different vegetable oils due to the differences in the fatty acid profiles.

Some nutritional properties of the seeds of three Mucuna species.

The seeds of Mucuna nivea, M. pruriens and M. utilis showed ash 4.3-5.1%, oil 4.9-5.5%, protein 25.9-27.5%, L-dopa 3.6-4.2%, trypsin 28.5-39.7 mg/g and chymotrypsin inhibitor activity 19.3-24.6 mg/g. The trypsin and chymotrypsin inhibitor activity increased in pod hull and seeds while the amount of protein increased in seeds and decreased in pod hull with maturity. The essential amino acid profile was comparable to the FAO pattern (lysine 6.0-6.4%). The fatty acid composition had total unsaturated acids 51.9-55.9%, but were poor in oil contents.

The effect of faecal enema on five microflora-associated characteristics in patients with antibiotic-associated diarrhoea.

BACKGROUND: Patients with antibiotic-associated diarrhoea (AAD) show significant disturbances in short-chain fatty acid pattern. In the present study five more microflora-associated characteristics (MACs) were investigated before and after administration of an enema containing faecal microflora from a healthy person on a Western diet. METHODS: The functions of the microflora were determined with gas chromatography, electrophoresis, and spectrophotometry. RESULTS: The conversion of cholesterol to coprostanol and the concentration of urobilinogen and trypsin were significantly reduced in comparison with healthy persons. The pattern of mucin was altered, but beta-aspartylglycine remained the same as in healthy persons. Enema treatment influenced these functions to different extents. CONCLUSION: Most MACs were significantly disturbed in patients with AAD. Administration of a human faecal enema modified these changes and relieved diarrhoea, usually within 4 days.

In vitro rat pancreatic digestive enzyme activities and raw and heated glandless cottonseed and soybean flours.

Higher nitrogen and lipid digestibilities have been obtained with diets containing cottonseed flour rather than soybean flour. To explain these results, in vitro studies were carried out to compare the effects of raw and heated glandless (without gossypol) cottonseed flours versus soybean flours on pancreatic digestive enzyme activities. These effects were compared with those obtained without addition of flour in standard assays. Apparent lipase (lipase colipase dependent) and potential lipase (lipase with saturating amounts of colipase), colipase, phospholipase A2, amylase, trypsin and chymotrypsin activities were measured on specific substrates. Phospholipase A2 and amylase activities were enhanced, while chymotrypsin activity was diminished with both raw and heated flours. Compared with raw and heated soybean flours, raw and heated cottonseed flours promoted higher potential lipase, chymotrypsin, trypsin and lipase activities. Heat treatment of cottonseed flour enhanced apparent lipase, colipase, chymotrypsin, trypsin activities and diminished potential lipase, phospholipase A2 and amylase activities. When soybean flour was heated, apparent lipase, phospholipase A2, chymotrypsin, trypsin and amylase activities were raised while those of potential lipase were decreased. Our findings show that in vitro raw or heated cottonseed flours affect less digestive enzymes than raw or heated soybean flours, apparent lipase activity excepted. Moreover, only chymotrypsin activities were seriously lowered with both flours, especially with raw soybean flour. Hypotheses are suggested to account for the differences in alterations.

Expression of acinar and ductal products in Capan-1 cells growing in synthetic serum and serum-free media.

BACKGROUND: Capan-1 is a human pancreatic adenocarcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum. METHODS: Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis. RESULTS: Capan-1, PANC-1, IBF-CP3, and MDAAmp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process. CONCLUSION: The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.

Human urinary trypsin inhibitor, urinastatin, prevents pancreatic injuries induced by pancreaticobiliary duct obstruction with cerulein stimulation and systemic hypotension in the rat.

OBJECTIVE: The protective effects of human urinary trypsin inhibitor against pancreatic injuries in multifactor-related experimental model of acute pancreatitis were evaluated. DESIGN: Experimental study. MATERIALS AND METHODS: Acute pancreatitis was induced by short-termed (1-hour) pancreatico-biliary duct obstruction with cerulein stimulation (30 minutes; 0.2 microgram/kg per hour) and systemic hypotension (30 minutes; 30% reduction of mean arterial pressure) in rats. In this model, the protective effects of UTI against pancreatic injuries were evaluated at a dose of 10,000 U/kg per hour. RESULTS: In this model, significant increases in portal serum amylase, cathepsin B and malate dehydrogenase levels were observed as compared with the control rats. The redistribution of cathepsin B from the lysosomal to the zymogen fraction and activation of trypsinogen were also observed. Moreover, the increased lysosomal and mitochondrial fragility as well as impaired pancreatic adenylate energy metabolism were noted. The therapeutic administration of human urinary trypsin inhibitor had significant protective effects against these pancreatic injuries. Furthermore, the combined prophylactic and therapeutic administration of human urinary trypsin inhibitor had more significant protective effects than only therapeutic treatment. CONCLUSIONS: These results suggest the importance of timing and of selecting a pertinent protease inhibitor, such as urinary trypsin inhibitor, in the treatment of pancreatitis.

Somatostatin attenuates ischemic intestinal injury.

Pancreatic-derived proteases play a central role in the pathogenesis of ischemic intestinal injury. We postulated that exocrine blockade by pretreatment with a long-acting somatostatin analogue, octreotide acetate, would attenuate ischemic mucosal injury. Sprague-Dawley rats received subcutaneous octreotide (10 micrograms/kg/d) for 6 days by means of surgically implanted infusion ports. In a group of sham control rats, splanchnic blood flow (portal vein Doppler measurement) and duodenal trypsin activity (p-toluene sulfonyl-L-arginine methyl ester assay) were determined. In a separate experiment, pretreated animals were subjected to 60 minutes of superior mesenteric artery ischemia alone or followed by 30 minutes of reperfusion. Gross extent of hemorrhagic necrosis and microscopic injury (rank analysis) were assessed by a blinded observer. Pretreatment with octreotide reduced intraluminal duodenal trypsin activity by 46% without affecting portal blood flow. However, octreotide pretreatment significantly attenuated the microscopic depth of injury during ischemia and the extent of gross injury during reperfusion. It appears that somatostatin may have an adjuvant role in the prevention or progression of intestinal ischemic injury.

Protease activity and autodigestion (autolysis) assays using Coomassie blue dye binding.

A new proteolytic assay is described involving Coomassie blue. Under specified conditions, the amount of Coomassie-stained casein protein hydrolyzed by several proteases was proportional to the amount of protease. Coomassie dye reaction was used directly to determine the change in protein concentration of the substrate casein during proteolysis by three proteases: stem bromelain, papain, and trypsin. This method can be used with 0.1- to 0.5-micrograms quantities of protease. The dye reagent was used directly on the protease protein in order to obtain an assay of autodigestion. Autodigestion of bromelain at 50 and 25 degrees C was followed by measuring the amount of residual protease protein with time.

Analysis of trypsin inhibitors and lectins in white kidney beans (Phaseolus vulgaris, var. Processor) in a combined method.

Buffered saline extraction, affinity chromatography, and Folin-BSA protein assay were used consecutively to provide a combined method for analysis of trypsin inhibitors and lectins in white kidney beans (Phaseolus vulgaris, var. Processor). The method was tested by following the decrease of both antinutritional factors by germination of the beans for 7 days at 20 degrees C. Repeatability coefficients of variation were 2-7.4% for the trypsin inhibitors and 2.2-10% for the lectins. After 7 days of germination, trypsin inhibitors and lectins were reduced by 72 and 92%, respectively.

Inhibition of cholecystokinin-stimulated pancreaticobiliary output in man by the cholecystokinin receptor antagonist MK-329.

MK-329 (formerly L-364,718) is a new nonpeptide antagonist for the peripheral (type-A) cholecystokinin (CCK) receptor, which has proved effective in blocking the actions of both exogenous and endogenous CCK in several species. To evaluate the effect of MK-329 on CCK-stimulated pancreaticobiliary output in man, six normal subjects received 10 mg MK-329 or placebo orally in a randomized, crossover fashion, before a background intravenous infusion of secretin (5 pmol/kg/h) and two doses of CCK-8 (approximately 15 and 40 pmol/kg/h, each for 1 h). Gastric and duodenal juice were aspirated separately via two double-lumen tubes, with 51Cr-ethylene-diaminetetraacetic acid as a duodenal marker. After placebo treatment the background infusion of secretin produced maximum plasma concentrations of secretin similar to postprandial values, averaging about 5 pM. After placebo treatment the low dose CCK-8 infusion (15 pmol/kg/h) increased circulating CCK concentrations from basal levels of 1.8 +/- 0.2 pM to levels similar to those observed postprandially, averaging 9.2 +/- 1.3 pM, and the high dose of CCK-8 (40 pmol/kg/h) induced supraphysiologic levels of CCK, averaging 23.4 +/- 3.2 pM. Plasma concentrations of secretin and CCK were not significantly different during MK-329 treatment. As expected, infusion of CCK-8 at both doses stimulated pancreatic exocrine secretion and gallbladder contraction in placebo controls, as indicated by increases in the output of trypsin, amylase, bicarbonate, and bilirubin. Whereas MK-329 did not significantly reduce basal pancreatic secretion, the integrated incremental output of trypsin, amylase, and bicarbonate in response to stimulation with the low (physiologic) CCK dose was inhibited by 74% (p less than 0.01), 89% (NS), and 75% (p less than 0.05), respectively. Basal bilirubin output was virtually abolished after treatment with MK-329, and the response to the low dose of CCK was reduced by 98% (p less than 0.01), indicating almost complete inhibition of gallbladder contraction at physiologic circulating concentrations of CCK. It is concluded that MK-329 is an orally active antagonist of CCK-stimulated pancreaticobiliary output in man and could thus be utilized to explore the physiologic regulation of the exocrine pancreas and gallbladder by CCK.

Dietary fiber supplementation: effect on exocrine pancreatic secretion in man.

The effect of a dietary fiber supplementation program (20 g/d) on exocrine pancreatic gland secretion was evaluated in six healthy male subjects who underwent quantitative assessment of pancreatic enzyme secretion both before and after 4 wk of dietary fiber supplementation. A duodenal perfusion technique was used to quantify the concentrations and output of pancreatic enzymes after ingestion of a standard test meal. Samples were aspirated from the ligament of Trietz and analyzed for pH, total protein, amylase, trypsin, and lipase activity. No significant changes were observed in duodenal flow rate pH, total protein, amylase, or trypsin concentrations and outputs after fiber supplementation. A marked increase in mean (+/- SEM) lipase concentration (U/mL) and output (kU/min) in both the resting and postprandial states was seen, reaching statistical significance (p less than 0.05) at 120 min postprandial. These data suggest that in man, a 4-wk dietary fiber supplementation program can modulate pancreatic lipase secretion.

Endocrine pancreatic function during atrophy of the exocrine gland.

Serum levels of glucose, insulin, and gastric inhibitory polypeptide (GIP) in response to intraduodenal and intravenous glucose loads have been examined in rats with exocrine pancreatic atrophy induced by feeding them a copper-deficient diet supplemented with D-penicillamine for 10-12 weeks. Whereas pancreatic weight and protein and trypsin content were drastically reduced as compared with pair-fed controls, insulin content was not significantly different between experimental (1.10 U) and control (1.40 U) rats. The intravenous glucose infusion was given in a dose (1.2 g/kg/h) simulating the glucose concentrations observed in response to an intraduodenal glucose load of 2.0 g/kg body weight. Both basal and stimulated insulin concentrations were lower in experimental animals as compared with controls. However, if the relative insulin response is considered, insulin secretion was almost identical in experimental and control animals. Both groups released significantly more insulin after intraduodenal glucose load than after intravenous glucose application. It is concluded that the entero-insular axis is intact in rats with exocrine pancreatic atrophy.

[In vitro studies of pancreatic enzyme substitution]. / In-vitro-Untersuchungen zur Pankreasenzymsubstitution.

In-vitro activity of 14 commercial pancreatin preparations, commonly used in the Federal Republic of Germany, were tested. All had been declared by their manufacturers to contain more than 6000 FIP (Fédération International Pharmaceutique) units of lipase and to be acid resistant. The declared lipase and amylase amounts were found to be present in 11 of the 14 preparations. Three of the 14 preparations, said to be acid resistant were found not to be so in buffer with falling pH values between 4.0 and 2.5, so that there occurred an, at times marked, loss of enzyme activity. Most noticeable was the poor solubility of most preparations at pH 6.6. Only three of the 14 liberated their total enzyme content within 60 minutes, as they should for theoretical reasons, based on the relatively short duodeno-cecal transit time.