Characterization and heme oxygenase-1 content of extracellular vesicles in human biofluids.

Heme oxygenase-1 (HO-1), a highly inducible stress protein that degrades heme to biliverdin, carbon monoxide, and free ferrous iron, is increased in blood and other biofluids of subjects with various systemic and neurological disorders. HO-1 does not contain an N-terminal signal peptide and the mechanism responsible for its secretion remains unknown. Extracellular vesicles (EVs) are membrane-bound inclusions that transport microRNAs, messenger RNAs, lipids, and proteins among diverse cellular and extracellular compartments. The objective of the current study was to determine whether EVs in human biofluids contain HO-1, and whether the latter may be transported in EVs from brain to periphery. Total, L1 cell adhesion molecule protein (L1CAM)-enriched (neuron-derived), and glutamate aspartate transporter 1 (GLAST)-enriched (astrocyte-derived) EVs were purified from five different human biofluids (saliva [n = 40], plasma [n = 14], serum [n = 10], urine [n = 10], and cerebrospinal fluid [n = 11]) using polymer precipitation and immuno-affinity-based capture methods. L1CAM-enriched, GLAST-enriched, and L1CAM/GLAST-depleted (LGD) EV, along with EV-depleted (EVD), fractions were validated by nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), and western blot. HO-1 was assayed in all fractions using ELISA and western blot. The majority of HO-1 protein was localized to LGD, L1CAM-enriched, and GLAST-enriched EVs of all human biofluids surveyed after adjusting for age and sex, with little HO-1 protein detected in EVD fractions. HO-1 protein in human biofluids is predominantly localized to EV compartments. A substantial proportion of EV HO-1 in peripheral human biofluids is derived from the central nervous system and may contribute to the systemic manifestations of various neurological conditions.

The Food Matrix and the Gastrointestinal Fluids Alter the Features of Silver Nanoparticles.

Silver nanoparticles (AgNPs) are used in the agri-food sector, which can lead to their ingestion. Their interaction with food and their passage through the gastrointestinal tract can alter their properties and influence their fate upon ingestion. Therefore, this study aims at developing an in vitro method to follow the fate of AgNPs in the gastrointestinal tract. After incorporation of AgNPs into a standardized food matrix, a precolonic digestion is simulated and AgNPs are characterized by different techniques. The presence of food influences the AgNPs properties by forming a corona around nanoparticles. Even if the salivary step does not impact significantly the AgNPs, the pH decrease and the digestive enzymes induce the agglomeration of AgNPs during the gastric phase, while the addition of intestinal fluids disintegrates these clusters. AgNPs can thus reach the intestinal cells under nanometric form, although the presence of food and gastrointestinal fluids modifies their properties compared to pristine AgNPs. They can form a corona around the nanoparticles and act as colloidal stabilizer, which can impact the interaction of AgNPs with intestinal epithelium. This study demonstrates the importance of taking the fate of AgNPs in the gastrointestinal tract into account to perform an accurate risk assessment of nanomaterials.

Evaluation of hydrolytic enzyme activities from digestive fluids of Anthonomus grandis (Coleoptera: Curculionidae).

The cotton boll weevil, Anthonomus grandis, is a major pest of cotton crops in South America. In this work, partial biochemical characterizations of (hemi) cellulases and pectinases activities in the digestive system (head- and gut- extracts) of A. grandis were evaluated. Gut extract section from third instar larvae exhibited endoglucanase, xylanase, ß-glucosidase, and pectinase activities. The endoglucanase and xylanase activities were localized in the foregut, whereas ß-glucosidase activity was mainly detected in the hindgut. In addition, no difference in pectinase activity was observed across the gut sections. Thus, A. grandis digestive system is a potentially interesting reservoir for further lignocellulolytic enzymes research.

Analytical validation of an in-house method for adenosine deaminase determination.

BACKGROUND: The adenosine deaminase (ADA) enzyme is a marker of inflammatory processes whose activity can be measured through a colorimetric method developed as an in-house assay. This validation can reduce costs and expand the alternatives for laboratory diagnosis. METHODS: The ADA analysis was achieved through a modified form of Giusti and Galanti's (1984) method. The following parameters were characterized: calibration curve, linearity, analytical sensitivity, limit of detection, limit of quantification, method working range, precision (within-assay and between-assay), bias, total analytical error, and sample stability. The results were statistically evaluated and compared with quality specifications based on biological variations and the performance of commercial tests. RESULTS: The analytical sensitivity and limit of detection (0.013 and 3.0 U/L, respectively) were lower than those of commercial tests. The method's working range was 3.2-100.0 U/L. According to the quality specification, the method showed optimum performance with a bias <3.5%. However, repeatability (2.2% and 1.7% for normal- and high-activity samples, respectively) and reproducibility achieved worse results when compared to commercial tests. The method demonstrated an inappropriate between-assay precision for low enzymatic activity (10.4%) and the minimum and desirable performance for medium (8.8%) and high (5.0%) activities, respectively. It also presented at least a minimum performance (<25%) for the total analytical error of the three analyzed samples. The pleural fluid samples were found to be stable at -20°C for six days. CONCLUSION: The findings show that the in-house method displays an acceptable performance and is capable of generating results comparable to existing commercial tests.

A Rapid and Ultrasensitive Tetraphenylethylene-Based Probe with Aggregation-Induced Emission for Direct Detection of α-Amylase in Human Body Fluids.

α-Amylase plays a key role in the physiological cycle of the human body; its function is constantly explored and used as an important indicator of some related diseases like acute pancreatitis, acute organophosphorus pesticide poisoning, and anxiety or depression. However, currently, including the assay kit, existing methods suffer from low sensitivity and time consumption or are indirect assays that require the aid of a tool enzyme or inhibitor of competitive substrates; hence, they are not suitable for the low activity and nondestructive sensing of α-amylase in body fluids. A rapid, highly sensitive, and simple direct α-amylase determination in human body fluids is still challenging. In this work, an AIEgen-based small molecule α-amylase sensing system was first established. The probe has no emission signal in aqueous media because of its good solubility, but the insoluble AIE residues can be released after hydrolysis by α-amylase, lighting up fluorescence significantly. In this novel sensing system, the detection limit is calculated to be 0.14 U L-1 in MES buffer with a linear range of 0-45.5 U L-1, having been shortened to 3 min of test time and excellent selectivity to α-amylase compared to other proteins. Moreover, our method is successfully employed to demonstrate the applications in acute pancreatitis diagnosis and psychological stress analysis. The acquisition of this AIE-based method not only provides a simple technique for clinical diagnosis of related diseases but also has a promotional value for the food and pharmaceutical industries.

New spectrophotometric assay for assessments of catalase activity in biological samples.

A novel, simple, and accurate colorimetric assay was established for assessments of catalase activity in biological fluids and tissues. H2O2 dissociation rates are directly proportional to catalase activity, and the principle of the present assay is based on reactions of ammonium metavanadate with H2O2 under acidic conditions. The resulting reduction of vanadium (V) to vanadium (III) produces a red-orange peroxovanadium complex with absorbance maxima at 452 nm. Biological samples containing catalase were incubated with 50-mM phosphate buffer solution containing 10-mM H2O2 as a substrate for two min. Subsequently, ammonium metavanadate in sulfuric acid was used as an indicator reagent and was added to reaction mixtures to determine remaining H2O2 concentrations. The precision of the present novel assay was indicated by coefficients of variation of 4.09% within runs and 2.56% between runs. Moreover, in experiments with homogenized red blood cell solutions, peroxovanate and dichromate assays of catalase activities were highly correlated (r = 0.993). In further experiments, we demonstrated application of the peroxovanadate method to assessments of catalase activity in bacterial and liver homogenates. The present method is accurate, simple, rapid, and inexpensive and can be used for routine clinical measurements and scientific investigations.

Two-point measurement of amylase in drainage fluid predicts severe postoperative pancreatic fistula after gastric cancer surgery.

BACKGROUND: Early identification of patients at risk of postoperative pancreatic fistula (POPF) allows appropriate management after gastrectomy. Although some reports have suggested a correlation between POPF and the concentration of amylase in drained abdominal fluid (D-AMY), this has not been proven to impact sufficiently on clinical decision-making. A sustained high level of D-AMY is often assumed to be due to unsatisfactory drainage or excessive pancreatic leakage. We assessed the clinical utility of measuring D-AMY on postoperative day (POD) 1 and POD3 for prediction of POPF. METHODS: Starting in April 2014, 801 patients who underwent radical gastrectomy with prophylactic drain placement were consecutively enrolled. We routinely measured D-AMY on POD1 and POD3, and compared the incidence of problematic POPF and clinical factors including D-AMY. We also attempted to clarify whether such two-point D-AMY measurement was clinically useful for patient management after gastrectomy. RESULTS: Fifty-one of the patients (6.4%) developed Clavien-Dindo grade III or worse POPF. Using D-AMY cutoffs of 2218 IU/L on POD1 and 555 IU/L on POD3, the patients were successfully classified. The highest risk group, in which D-AMY was higher than the cut-off value on both POD1 and POD3, showed a significantly high rate of occurrence (33/105, 31.4%) and high positive likelihood ratio (6.74). Multivariate analysis showed that classification into this highest risk group was an independent risk factor for development of severe POPF (odds ratio 15.2, 95% CI 7.92-29.0). CONCLUSION: Two-point measurement of D-AMY may be an efficient tool for achieving individualized management of POPF following radical gastrectomy.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

The effect of dietary Chlorella vulgaris supplementation on micro-organism community, enzyme activities and fatty acid profile in the rumen liquid of goats.

Microalgae might be considered as an alternative source of fat and/or protein for ruminant's diets. However, changes in populations of ruminal micro-organisms associated with biohydrogenation process, methane and ammonia production in response to microalgae dietary supplementation have not been well characterized. Thus, 16 cross-bred goats were divided into two groups. Each goat of both groups was fed individually with alfalfa hay and concentrates separately. The concentrates of the control group had no microalgae while those of the treated group were supplemented with 10 g lyophilized Chlorella vulgaris/kg concentrate (chlor). On the 30th experimental day, samples of rumen fluid were collected for microbial DNA extraction, fatty acid profile and enzyme activity analyses. The results showed that the chlor diet compared with the control increased significantly the populations of Methanosphaera stadtmanae, Methanobrevibacter ruminantium and Methanogens bacteria and protozoa in the rumen of goats. A significant reduction in the cellulase activity and in the abundance of Ruminococcus albus, and a significant increase in the protease activity and in the abundance of Clostridium sticklandii in the rumen liquid of goats fed with the chlor diet, compared with the control, were found. Chlorella vulgaris supplementation promoted the formation of trans C18:1 , trans-11 C18:1 and monounsaturated fatty acids (MUFA), while the proportions of C18:0 and long-chain fatty acids (LCFA) reduced significantly in the rumen liquid of goats. This shift in ruminal biohydrogenation pathway was accompanied by a significant increase in Butyrivibrio fibrisolvens trans C18:1 -producing bacteria. In conclusion, the supplementation of diets with microalgae needs further investigation because it enhances the populations of methane-producing bacteria and protozoa.

Early dual drainage combining transpapillary endotherapy and percutaneous catheter drainage in patients with pancreatic fistula associated with severe acute pancreatitis.

BACKGROUND: The development of pancreatic fistula (PF) associated with pancreatic necrosis is of great concern in the management of severe acute pancreatitis (SAP). We expected that early recognition and intervention of PF combined with percutaneous catheter drainage (PCD) for pancreatic infection may improve SAP outcomes. METHODS: Fifteen consecutive patients with SAP were enrolled. Whenever feasible, fine-needle aspiration for fluid collection was performed to determine infection and amylase concentration. For infection and PF with amylase-rich fluid, PCD and transpapillary endotherapy (preferably naso-pancreatic drainage) were carried out as soon as possible. PCD was intensively managed by irrigating the sized-up and multiple large bore catheters. RESULTS: Infected fluid collection and PF were both detected in 13 (86.7%) patients. Pancreatic duct (PD) disruption (n = 6) and organ failure (n = 5) occurred exclusively in patients with amylase-rich collection ≥10,000 U/L. The median timing of PCD and endotherapy was 15.5 and 16.5 days, respectively. No serious complications or mortality resulted from intervention procedures other than stent occlusion in one (6.7%) patient. Surgical intervention due to uncontrollable infection and visceral organ injury was avoided. Fistula closure was achieved in 12 (92.3%) of 13 PF patients with a median duration of 45 days. Disease-related mortality occurred in one (6.7%) patient. CONCLUSION: Amylase-rich fluid collection ≥10,000 U/L may be an indication for further endoscopic investigation of PD disruption. Early dual drainage combining pancreatic endotherapy and PCD is feasible and safe, and may improve treatment outcome.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

Impact of bacterial culture positivity of the drainage fluid during the early postoperative period on the development of intra-abdominal abscesses after gastrectomy.

PURPOSE: The aim of this study was to evaluate the impact of positive bacterial cultures of the drainage fluid (D-cultures) during the early postoperative period on the incidence of intra-abdominal abscess formation following gastrectomy. METHODS: From January 2012 to June 2013, we prospectively performed D-cultures on postoperative day (POD) 1 in consecutive gastric cancer patients who underwent gastrectomy. The univariate and multivariate analyses were performed to identify the risk factors for intra-abdominal abscess formation without anastomotic leakage. RESULTS: The rate of positive D-cultures was 6.4 % on POD 1. According to a univariate analysis, the use of combined organ resection (P = 0.011), the drain amylase level on POD 1 (P = 0.016) and the D-culture status on POD 1 (P = 0.004) were found to be significantly associated with the incidence of intra-abdominal abscesses. A multivariate analysis demonstrated that D-culture positivity on POD 1 was the only independent predictor of intra-abdominal abscess formation (P = 0.011). CONCLUSIONS: The present study demonstrated that bacterial culture positivity of drainage fluid during the early postoperative period has a significant impact on the development of intra-abdominal abscesses after gastrectomy.

Body fluid analysis: clinical utility and applicability of published studies to guide interpretation of today's laboratory testing in serous fluids.

Requests for testing various analytes in serous fluids (e.g., pleural, peritoneal, pericardial effusions) are submitted daily to clinical laboratories. Testing of these fluids deviates from assay manufacturers' specifications, as most laboratory assays are optimized for testing blood or urine specimens. These requests add a burden to clinical laboratories, which need to validate assay performance characteristics in these fluids to exclude matrix interferences (given the different composition of body fluids) while maintaining regulatory compliance. Body fluid testing for a number of analytes has been reported in the literature; however, understanding the clinical utility of these analytes is critical because laboratories must address the analytic and clinical validation requirements, while educating clinicians on proper test utilization. In this article, we review the published data to evaluate the clinical utility of testing for numerous analytes in body fluid specimens. We also highlight the pre-analytic and analytic variables that need to be considered when reviewing published studies in body fluid testing. Finally, we provide guidance on how published studies might (or might not) guide interpretation of test results in today's clinical laboratories.

Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers.

Release of EPA and DHA from salmon oil - a comparison of in vitro digestion with human and porcine gastrointestinal enzymes.

In the present study, we hypothesised whether in vitro digestion of salmon oil would release different amounts of PUFA depending on the origin of the lipolytic enzymes used. For this purpose, in vitro digestion of salmon oil (SO) was performed using human duodenal juice (HDJ) or a commercial enzyme preparation consisting of porcine pancreatin and bile (PB). The lipolytic effect was determined by measuring the release of fatty acids (FA) using solid-phase extraction and GC-flame ionisation detection, withdrawing samples every 20 min during digestion. The amount of FA released indicated that a plateau was reached after 80 min with approximately similar amounts of FA detected using both HDJ and PB (379 (sd 18) and 352 (sd 23) mg/g SO, respectively). However, the release of 18 : 2, EPA (20 : 5) and DHA (22 : 6) was significantly different during in vitro digestion. At 80 min, HDJ and PB released 43 and 33% of 18 : 2, 14 and 9% of EPA and 11 and 9% of DHA, respectively. Both enzyme preparations released approximately the same amounts of the other FA analysed. The effect of the addition of bile salts (BS) was significantly different in the two enzyme systems, where porcine pancreatin highly responded to the increase in BS concentration, in contrast to HDJ.

[Effects of zhuhong ointment on MMPs activities and production by HSF].

HuaFu Shengji is the primary traditional Chinese medicine (TCM) therapy for treating chronic skin ulcer. The high activities of the protein enzyme in the wound fluids is one of the main cause of healing delay. In order to investigate the effect of TCM Zhuhong ointment for promoting wound healing. This research focused on its influence on matrix metalloproteinase (MMP) activities in wound fluids with TCM Yang syndromes, directly on the activated MMP-1,2 activities in vitro and on MMP-1,-2,-9 production by HSF. 8 wound fluid samples were collected, which were diagnosed Yang Syndromes in TCM. Wound fluid activities of MMP-2 and MMP-9 were measured by gelatin zymogram assay. MMP-1 and MMP-2 activities in vitro were measured by substrate cleavage. CCK-8 was used to observe the toxicity of Zhuhong ointment on HSF. MMP-1,-2,-9 production by HSF were detected by confocal microscope. Zhuhong ointment from 1 to 25 g x L(-1) obviously inhibited MMP-2 activity in wound fluid. When Zhuhong ointment was over 5 g x L(-1), it showed significantly inhibitory effect on wound fluid MMP-9 activity. In vitro study, when the mercury concentration was 320 mg x L(-1), Zhuhong ointment solution directly inhibited both MMP-1 activity and MMP-2. But mercury concentration from 0.51-2.56 mg x L(-1), it could activate MMP-1 activity, and from 0.51-64 mg x L(-1), activate MMP-2 activity instead. The mercury concentration when Zhuhong ointment saturated in DMEM was 39.6 mg x L(-1). When the mercury concentration was over 1.23 mg x L(-1), Zhuhong ointment showed toxicity to HSF. At 1.23, 0.62, 0.31 mg x L(-1) of mercury concentration, it increased MMP-1 expression by HSF, and at 1.23, 0.62 mg x L(-1), decreased MMP-2 expression. However, at 1.23, 0.62, 0.31 mg x L(-1), it decreased MMP-9 expression. At higher concentration, Zhuhong ointment can inhibit MMP-2, MMP-9 activities in wound fluid with dose-dependent way and show a direct inhibitory effect on activated MMP-1 and MMP-2 in vitro. But at a lower concentration, it showed two-way adjustment, with increased MMP-1, MMP-2 activities and its expression by HSF and decreased MMP-9 activity.

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt.

Stimulation of Na(+)/K(+)-ATPase translocation to the cell surface increases active Na(+) transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na(+)/K(+)-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na(+)/K(+)-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na(+)/K(+)-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na(+)/K(+)-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na(+)/K(+)-ATPase intracellular translocation and thus in alveolar fluid reabsorption.

The effect of smoking on expression of IL-6 and antioxidants in pancreatic fluids and tissues in patients with chronic pancreatitis.

OBJECTIVES: The present study was conducted to ascertain whether cigarette smoke induces oxidative stress in the human pancreas concurrently with inflammation. MATERIALS AND METHODS: The influence of tobacco smoking on the serum level of interleukin-6 (IL-6), on the activities of glutathione peroxidase (GPx) and copper-zinc superoxide dismutase (Cu/Zn SOD) as well as on the metallothionein (MT) level in the pancreatic pseudocyst fluid and its immunohistochemical localization in tissues of non-smoking (n = 9) and smoking (n = 12) patients with diagnosed chronic pancreatitis (CP) was measured. The concentration of interleukin-6 and metallothionein was determined by means of ELISA and the radioisotopic method, respectively. The enzyme activities in the fluid were assayed by the colorimetric method. Samples of tissues of normal pancreas (n = 4) and CP (non-smoking n = 7; smoking n = 12) were verified histopathologically and then IL-6, MT and enzymes were localized by immunohistochemical staining using the monoclonal anti-human antibody. RESULTS: The concentrations of metallothionein and interleukin-6 were significantly higher in smoking patients with CP (as compared with the non-smoking population (p < 0.01; p < 0.001). Interestingly, the ratio of MT/IL-6 in smoking patients with CP was reduced in comparison to non-smoking patients (respectively: 1.1; 5.6). In smoking patients, a significant elevation of the Cu/Zn SOD and GPx activities was revealed as compared with the non-smokers (p < 0.04; p < 0.0007). These studies clearly demonstrate a moderate and strong expression of IL-6 and enzymes in acinar, islet and duct cells of smoking patients. CONCLUSIONS: These observations favor the role of the oxidative stress in the induction of pancreatitis associated with chronic cigarette smoke inhalation.

Effects of dietary protein source on the digestive enzyme activities and electrolyte composition in the small intestinal fluid of chickens.

An experiment was conducted to investigate the effects of dietary protein source on the digestive enzymes and electrolyte composition of digesta fluid in the duodenum, jejunum, and ileum of chickens. A 2 × 3 factorial and completely randomized design that used 2 types of diets that differed only in their protein source [a corn-soybean meal-miscellaneous meal diet (CSMD) and a corn-miscellaneous meal diet (CMD)] and 3 types of cannulated roosters (duodenal, jejunal, and ileal cannulations) was adopted. The experiments included 5 replicates for each of the 6 treatments, and each replicate involved 3 cannulated chickens. The digesta samples were collected for 1 h every 4 h between 09:30 and 18:30 h on d 31, 33, and 35 of the experiment. The amylase, lipase, trypsin, and chymotrypsin activities and the electrolyte composition in the duodenal, jejunal, and ileal fluid were determined. In general, no significant differences between the 2 dietary groups were observed for the mean of duodenal, jejuna, and ileal amylase, trypsin, chymotrypsin, and lipase, respectively. However, the duodenal amylase activity was lower in the CMD group than the CSMD group (P < 0.05), which was probably related to the lower duodenal pH value that was observed in this group (P < 0.01). A higher jejunal Mg(2+) concentration was observed in chickens that were fed the CMD (P < 0.05), whereas the differences in the Na(+), K(+), Cl(-), and Ca(2+) concentrations in the small intestine were not significant between the 2 diets (P > 0.05). In conclusion, the digestive enzymes and electrolytes in the small intestinal fluid of chickens adapted to the protein source of the diet, and each segment of the small intestine displayed different modifications.

Exoglycosidase markers of diseases.

Exoglycosidases are hydrolases involved in lysosomal degradation of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). In tissues and body fluids, a higher exoglycosidase specific activity is found in N-acetyl-ß-hexosaminidase, than ß-glucuronidase, α-L-fucosidase, ß-galactosidase, α-mannosidase and α-glucosidase. Determination of exoglycosidases (especially N-acetyl-ß-hexosaminidase and ß-glucuronidase) in body fluids could be an inexpensive, easy to perform and sensitive test for pathological evaluation, as well as in screening and monitoring many diseases, including alcohol abuse, risk of arteriosclerosis, bacterial infections (e.g. Lyme borreliosis), chronic inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis, asthma, autoimmune hepatitis and primary biliary cirrhosis, as well as cancers.