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.

[The ways of prophylaxis of the pancreatic injuries occurring on background of cholangitis and choledocholithiasis complicated by obturation jaundice: algorithm for diagnosis and treatment].

The results of treatment of 45 patients, in whom obturation jaundice have occurred on background of purulent cholangitis and biliary calculous disease, were summarized. preoperatively conservative therapy was conducted to the patients, including ozonotherapy during 9 - 10 days (main group) and 2 - 4 days (control group). All the patients were operated on. Conservative therapy, conducted during 9 -- 10 days, have permitted to improve clinical and laboratory indices more rapidly postoperatively, to prognosticate possibilities of the pancreatic gland damage, pancreatitis occurrence, as well as its exacerbation in future.

Pancreatic response in healthy dogs fed diets of various fat compositions.

OBJECTIVE: To indirectly assess the pancreatic response in healthy dogs that were fed diets of different fat compositions with or without supplemental pancreatic enzymes and medium-chain triglycerides (MCTs). ANIMALS: 10 healthy adult dogs. PROCEDURES: Dogs were fed 4 diets once in random order at 1-week intervals; food was withheld from the dogs for > or = 12 hours prior to the feeding of each diet. Diets A and B contained 16% and 5% crude fat, respectively; diet C was composed of diet A with pancreatic enzymes; and diet D was composed of diet B with pancreatic enzymes and MCTs. Serum canine trypsin-like immunoreactivity (cTLI) and canine pancreatic lipase immunoreactivity (cPLI) concentrations were measured before (0 hours) and at 1 to 2 and 6 hours after feeding. Serum gastrin concentration was measured at 0 hours and at 5 to 10 minutes and 1 to 2 hours after feeding. A gastrin assay validation study was performed to confirm accuracy of test results in dogs. Data were analyzed by use of a repeated-measures general ANOVA. RESULTS: Serum cTLI, cPLI, or gastrin concentrations in the dogs did not differ among the different diets fed, among dogs, or over time. When multiple comparisons were analyzed, diet D caused the least amount of measurable pancreatic response, although this difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Results did not indicate a significant effect of dietary fat content or addition of supplemental MCT oil or pancreatic enzymes in diets on serum cTLI, cPLI, or gastrin concentrations in healthy dogs.

[Neonatal cystic fibrosis screening program in the state of Paraná: evaluation 30 months after implementation]. / Programa de triagem neonatal para fibrose cística no estado do Paraná: avaliação após 30 meses de sua implantação.

OBJECTIVES: To present and analyze the results of the National Neonatal Cystic Fibrosis Screening Program in Paraná, 30 months after its implementation. METHODS: This is a descriptive study, with an analysis of the data from the screening of around 98% of all neonates in the period from September 2001 to April 2004, undertaken at the Neonatal Screening Program laboratory of the Fundação Ecumênica de Proteção ao Excepcional do Paraná. Blood samples for the Guthrie test were collected on hospital discharge, ideally between the second and sixth days postpartum, and filter papers were sent for immunoreactive trypsin assay by the immunofluorometric method. Children whose immunoreactive trypsin assay results were > or = 70 ng/ml for two distinct samples during the first 30 days of life, were referred for sweat conductivity testing by the Wescor method. In cases when the result was greater than 50 mMol/l quantitative chlorine and/or sodium in sweat was assayed (iontophoresis with pilocarpine). RESULTS: From a total of 456,982 tests, 4,028 (0.9%) children presented a first immunoreactive trypsin assay above the cutoff point set. Four hundred and seventy-eight of these (12.5%) also had a second blood sample assayed with immunoreactive trypsin above 70 ng/ml and 56 (11.7%) of these were referred to specialized clinics after their sweat conductivity test results were above 50 mMol/l and 48 (0.01% of the total number of children screened) had a diagnosis of cystic fibrosis confirmed. The incidence for the state of Paraná was 1:9,520, although some children have not yet been fully investigated. CONCLUSIONS: Neonatal screening for cystic fibrosis in the State of Paraná, in accordance with Health Ministry directives, was a pioneering initiative for Brazil. Many patients were diagnosed early, even asymptomatic ones, which is a challenge to improving prognosis with this fatal disease.

Programa de triagem neonatal para fibrose cística no estado do Paraná: avaliação após 30 meses de sua implantação / Neonatal cystic fibrosis screening program in the state of Paraná: evaluation 30 months after implementation

OBJETIVOS: Apresentar e analisar os resultados da implantação do Programa Nacional de Triagem Neonatal em Fibrose Cística, no Paraná, após 30 meses do seu início. MÉTODOS: Foi realizado um estudo descritivo com análise dos dados da triagem feita em aproximadamente 98 por cento dos recém-nascidos entre setembro de 2001 e abril de 2004 no laboratório do Serviço de Triagem Neonatal da Fundação Ecumênica de Proteção ao Excepcional do Paraná. As amostras de sangue para o "teste do pezinho" foram coletadas na alta hospitalar, idealmente entre o segundo e sexto dias de vida, com o envio dos papéis-filtro para a dosagem de tripsina imunorreativa por imunofluorimetria. Crianças com valores da dosagem de tripsina imunorreativa > 70 ng/ml em duas amostras distintas, nos primeiros 30 dias de vida, foram submetidas à determinação da condutividade do suor pelo método de Wescor. Com o resultado maior que 50 mMol/l procedeu-se à dosagem quantitativa de cloro e/ou sódio no suor (iontoforese por pilocarpina). RESULTADOS: De 456.982 exames realizados, 4.028 (0,9 por cento) crianças apresentaram a primeira dosagem de tripsina imunorreativa acima do ponto de corte estabelecido. Dessas, 478 (12,5 por cento) tiveram uma segunda amostra de sangue com dosagem de tripsina imunorreativa acima de 70 ng/ml, das quais, após a realização do teste de suor por condutividade, 56 (11,7 por cento) crianças foram encaminhadas para ambulatórios especializados com resultado acima de 50 mMol/l e 48 (0,01 por cento do total de triagens) tiveram o diagnóstico de fibrose cística confirmado. A incidência foi de 1:9.520 em nosso estado, embora algumas crianças ainda não tenham completado a investigação. CONCLUSÕES: A triagem neonatal para fibrose cística no estado do Paraná, de acordo com as normas do Ministério da Saúde, foi uma iniciativa pioneira no Brasil. Realizou-se o diagnóstico precocemente em muitos pacientes, mesmo os assintomáticos, fato que representa um desafio à melhora no prognóstico dessa doença fatal.

Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects.

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.

Evaluation of the long-term pancreatic effects of constitutive nitric oxide synthase inhibition in dogs.

Constitutive and inducible isoforms of nitric oxide synthase (NOS) catalyze the synthesis of nitric oxide (NO) from L-arginine in various tissues and in different pathophysiologic states. Short-term treatment with NOS inhibitors has been associated with pancreatic enzyme elevations and pancreatic acinar cell degeneration; however, long-term pancreatic effects of NOS inhibition are not known. The purpose of this study was to evaluate the subchronic pancreatic effects of L-nitro-arginine (LNA), a compound that preferentially inhibits constitutive NOS isoforms. LNA was administered orally at doses of 10 and 30 mg/kg per day to 6 female dogs/group for 4 weeks. To differentiate whether the pancreatic effects of LNA may be related to its arginine structure, an additional group was given L-arginine (L-Arg) at plasma concentrations similar to the high dose of LNA (30 mg/kg per day). Pancreatic effects were monitored by changes in serum levels of pancreatic enzymes at regular intervals and by microscopic examinations at the end of the study. Both LNA and L-Arg were systematically available throughout the 4-week study period. LNA produced dose-related elevations (1.3-10-fold above concurrent control) in serum levels of pancreatic enzymes (amylase, lipase and trypsin-like immunoreactivity) during the 4-week treatment period with peak elevations occurring during the first week. Histologic assessments of the pancreas conducted at the end of the 4-week dosing period were unremarkable. Additionally, LNA treatment resulted in reduction in heart rate (40%), gastric distension and gastric mucosal erosion and ulceration. No pancreatic, cardiac, or gastric effects were seen with L-Arg, indicating that above effects were likely due to NOS inhibition. Results of this study confirmed previous observations of acute pancreatic alterations following the inhibition of constitutive NOS isoforms. However, these pancreatic alterations appear to be only transient effects as elevations in serum enzymes declined over time and no structural acinar cell damage was seen after continuous treatment with LNA for 4 weeks, suggesting an adaptation to NOS inhibition over time.

Subclinical exocrine pancreatic insufficiency in dogs.

OBJECTIVE: To study progression of autoimmune-mediated atrophic lymphocytic pancreatitis from the subclinical to the clinical phase (exocrine pancreatic insufficiency [EPI]) and determine whether progression of the disease could be halted by treatment with immunosuppressive drugs. DESIGN: Randomized controlled trial. ANIMALS: 20 dogs with subclinical EPI. PROCEDURE: Diagnosis of subclinical EPI was determined on the basis of repeatedly low serum trypsin like-immunoreactivity (TLI) in dogs with no signs of EPI. Laparotomy was performed on 12 dogs with partial acinar atrophy and atrophic lymphocytic pancreatitis. A treatment group (7 dogs) received an immunosuppressive drug (azathioprine) for 9 to 18 months, and a nontreatment group (13) received no medication. RESULTS: During the subclinical phase, serum TLI was repeatedly low (< 5.0 microg/L). Although a few dogs had nonspecific gastrointestinal tract signs, they did not need diet supplementation with enzymes. While receiving immunosuppressive medication, treated dogs had no clinical signs of EPI, but within 2 to 6 months after treatment was stopped, 2 dogs had signs of EPI, and diet supplementation with enzymes was started. Five of the 13 untreated dogs needed diet supplementation with enzymes within 6 to 46 months. During follow-up of 1 to 6 years, 3 of the 7 treated dogs and 8 of the 13 untreated dogs did not need continuous diet supplementation with enzymes. CONCLUSIONS AND CLINICAL RELEVANCE: Progression of atrophic lymphocytic pancreatitis varied widely. The subclinical phase may last for years and sometimes for life. The value of early treatment with an immunosuppressive drug was questionable and, because of the slow natural progression of the disease, cannot be recommended.

Bacterial translocation in the course of acute pancreatitis: beneficial role of nonabsorbable antibiotics and lactitol enemas.

Two-hundred Wistar rats were allocated to 4 groups. The groups, 3 representing our acute pancreatitis model induced by intrabiliary injection of a trypsin/enterokinase mixture, were studied as follows: (A) no treatment; (B) given a daily 30-ml enema with 20 mg/kg rifaximin; (C) given a daily 30-ml enema with 20 mg/kg rifaximin plus lactitol 0.5 g/kg, and (D) given a daily 30-ml enema with warm saline. A further group of healthy rats was given an intrabiliary injection of 0.15 ml saline. Sacrifices were made after 6, 12, 24, 48 and 72 h of observation. Serial blood samples were drawn to measure pancreatic enzymes and endotoxin. At sacrifice, ascites, lymph nodes, pancreas, spleen, portal vein blood, arterial blood and bile were obtained for bacteriological culture. Both enema treatments brought about a significant improvement in survival. Enema treatments did not affect the serum level of pancreatic enzymes. A time-course increase in endotoxin level was observed in untreated rats. However, significantly decreased levels were observed after both enema treatments. Overall, ascites was the sample most frequently infected. Lymph nodes contiguous to the gut were found to be infected more frequently than those close to major vessels. The histological pancreatic damage was of a significantly lesser degree in both enema treatment groups. Virtually all severe necrotico-hemorrhagic pancreatic lesions were associated with bacterial infection. These data suggest that bacterial translocation plays a relevant role in the outcome of experimental necrotizing pancreatitis. Intra-abdominal spread and lymphatics seem to be the pathways most likely involved in such processes. Colonic cleansing by non-absorbable antibiotics and lactitol seems to exert a beneficial effect on the supervening infection of experimental necrotizing pancreatitis.

Effect of thermal treatments on the chemical and biological value of irradiated and non-irradiated cowpea bean (Vigna unguiculata L. Walp) flour.

Chemical and nutritional characteristics of irradiated and nonirradiated cowpea bean at 0.2 kGy submitted to several thermal treatments were studied. The cowpea bean flours irradiated at 0.2 kGy were superior to those non-irradiated concerning digestibility and the flours submitted to microwave oven were superior to those cooked under low pressure, autoclaved or even raw from the nutritional point of view. As to the presence of anti-nutritional factors, the results showed that the variety of cowpea bean under research presented neither trypsin inhibitor nor haemagglutinin activity, however, regarding tannin, a 0.006% content was found and considered negligible.

Pancreatic tissue damage by transcatheter arterial embolization for hepatoma.

We analyzed the serial changes in serum pancreatic enzyme activities by transcatheter arterial embolization (TAE) in 20 hepatoma patients with liver cirrhosis in an attempt to evaluate the incidence of the pancreatic tissue damage by TAE. Serum amylase activities increased in two (10%) cases, elastase 1 levels in six (30%) cases, and trypsin and pancreatic secretory trypsin inhibitor (PSTI) levels in each of five (25%) cases. Consequently, TAE resulted in the elevation of at least more than one serum pancreatic enzyme in eight (40%) of 20 cases, although none had clinical symptoms related to pancreatitis. When the adverse effect on the pancreatic tissue was compared among 6 cases of the superselective TAE and 14 cases of the nonsuperselective TAE, which were performed from the segmental and the nonsegmental hepatic arteries, respectively, the elevation of serum pancreatic enzymes was caused only by nonsuperselective TAE, not by superselective TAE. The volumes of Spongel and Lipiodol used or the injected doses of the anticancer agent mitomycin C were not different between the two groups. These results indicate that TAE for the treatment of hepatoma frequently causes pancreatic tissue damage, and the position of the inserted catheter tip is very important to avoid the pancreatic tissue damage by TAE.

Effect of protease inhibitor FUT-175 on acute hemorrhagic pancreatitis in mice.

The effect of FUT-175, a new synthetic trypsin inhibitor, was evaluated in a lethal model of acute hemorrhagic pancreatitis induced by feeding mice a choline-deficient, ethionine-supplemented diet (CDE diet). When FUT-175 was given prophylactically at doses of 20 mg/kg body wt, the survival rate was significantly improved (p less than 0.05). The serum amylase concentration and trypsin activity in serum were also significantly diminished (p less than 0.05). Prophylactic administration of 5 mg/kg body wt of FUT-175 showed a statistically significant decrease in the serum trypsin activity, but it did not improve the survival rate. Therapeutic administration of FUT-175 showed no favorable effect. FUT-175 showed protective effects on the course of severe acute pancreatitis only when given prophylactically at the beginning of the pancreatitis induced by the CDE diet.

Pancreatic function following partial pancreatectomy and anastomosis of the pancreatic duct to the stomach or duodenum in dogs.

The effects of pancreatic duct anastomosis to stomach (stomach group) or duodenum (duodenal group) on pancreatic function were examined in dogs following two thirds pancreatectomy. Normal fasting blood glucose concentrations were maintained in both groups despite significant reductions in glucose tolerance in the stomach group, and reductions in fasting insulin and insulin peak response in both groups. Pancreatic exocrine function was significantly decreased in both groups, though plasma p-aminobenzoic acid (PABA) concentrations were generally higher in the duodenal group. A correlation was found between plasma trypsin-like immunoreactivity (TLI) and pancreatic weight. These results indicate that anastomosis of the pancreas to bowel can be undertaken with minimal postoperative complications and that the site of the anastomosis influences pancreatic function. They suggest that preservation of more than one third of the pancreas is required for optimal function. The complementary information provided by the PABA and TLI tests suggests their dual application will be clinically useful for the detection and characterisation of naturally occurring pancreatic diseases.

Evaluation of tests of exocrine and endocrine pancreatic function in older patients with cystic fibrosis.

In order to determine the value of noninvasive tests in the analysis of pancreatic function in cystic fibrosis, 14 older cystic fibrosis patients were studied by a set of noninvasive tests of exocrine and endocrine pancreatic function. The tests, comprising trypsin, total amylase, pancreatic isoamylase, lipase, pancreatic polypeptide (PP), glucose and insulin in fasting serum, PP, glucose and insulin in postprandial serum, and p-aminobenzoic acid (PABA) excretion in urine, were compared to fecal fat excretion after discontinuation of pancreatic enzyme supplementation. Eleven of the 14 patients were found to have a fecal fat excretion of more than 7 g/day. Serum levels of trypsin, pancreatic isoamylase and lipase, and the urinary excretion of PABA showed significant negative correlations with fecal fat excretion. Endocrine pancreatic function was abnormal in the majority of patients with fibrocystic disease. Although serum trypsin, postprandial PP, and urinary PABA excretion were the most sensitive tests for severe exocrine pancreatic insufficiency, the differences in sensitivity were rather modest. Therefore, the type of test to be selected for clinical use is mainly dependent upon factors as accessibility, simplicity, patient's acceptability, and costs.

Inhibition of rat endotoxin-induced lethality by BN 52021 and BN 52063, compounds with PAF-acether antagonistic effect and protease-inhibitory activity.

Since proteolytic enzymes play a key role in endotoxaemia and anaphylaxis, diseases in which PAF-acether may be involved, we have investigated the activity of BN 52021 and BN 52063, two potent and specific PAF-acether antagonists, on plasma protease activity in the rat. After oral administration, both drugs dose-dependently decreased plasma trypsin-like activity (PTLA) although they did not show any inhibitory effect in vitro. PAF-acether and endotoxin injected into rats increased PTLA, the increase with endotoxin appearing more slowly but to a higher extent than with PAF-acether. Inhibition of the endotoxin-induced increase in PTLA was obtained with BN 52021, BN 52063 and dexamethasone, whereas aprotinin was ineffective. Since BN 52021 and BN 52063 reduced also the endotoxin-induced lethality in the rat, their antiprotease activity may be a consequence of their PAF-antagonistic effect. Further studies will be required with other PAF antagonists to assess fully this assumption.