Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when Nα-blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions.

Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans.

Gastric lipase (HGL) contributes significantly to fat digestion. However, little is known about its neurohormonal regulation in humans. We studied the role of CCK and cholinergic mechanisms in the postprandial regulation of HGL and pancreatic lipase (HPL) secretion in six healthy subjects. Gastric emptying of a mixed meal and outputs of HGL, pepsin, acid, and HPL were determined with a double-indicator technique. Three experiments were performed in random order: intravenous infusion of 1) placebo, 2) low-dose atropine (5 micrograms.kg-.h-1), and 3) the CCK-A receptor antagonist loxiglumide (22 mumol.kg-.h-1). Atropine decreased postprandial outputs of HGL, pepsin, gastric acid, and HPL (P < 0.03) while slowing gastric emptying (P < 0.05). Loxiglumide markedly increased the secretion of HGL, pepsin, and acid while distinctly reducing HPL outputs and accelerating gastric emptying (P < 0.03). Plasma CCK and gastrin levels increased during loxiglumide infusion (P < 0.03). Atropine enhanced gastrin but not CCK release. Postprandial HGL, pepsin, and acid secretion are under positive cholinergic but negative CCK control, whereas HPL is stimulated by cholinergic and CCK mechanisms. We conclude that CCK and cholinergic mechanisms have an important role in the coordination of HGL and HPL secretion to optimize digestion of dietary lipids in humans.

Mate Tea Inhibits In Vitro Pancreatic Lipase Activity and Has Hypolipidemic Effect on High-fat Diet-induced Obese Mice

The inhibitory effects of mate tea (MT), a beverage produced with leaves from Ilex paraguariensis, in vitro lipase activity and on obesity in obese mice models were examined. For the in vitro experiment, porcine and human pancreatic lipase (PL) activities were determined by measuring the rate of release of oleic acid from hydrolysis of olive oil emulsified with taurocholate, phospholipids, gum arabic, or polyvinyl alcohol. For the in vivo experiments, animals were fed with a standard diet (SD, n = 10) or high-fat diet (HFD, n = 30) for 16 weeks. After the first 8 weeks on the HFD, the animals were treated with 1 and 2 g/kg of body weight of MT. The time course of the body weight and obesity-related biochemical parameters were evaluated. The results showed that MT inhibited both porcine and human PL (half-maximal inhibitory concentration = 1.5 mg MT/ml) and induced a strong inhibition of the porcine lipase activity in the hydrolysis of substrate emulsified with taurocholate + phosphatidylcholine (PC) (83 +/- 3.8%) or PC alone (62 +/- 4.3%). MT suppressed the increases in body weight (P < 0.05) and decreased the serum triglycerides and low-density lipoprotein (LDL)-cholesterol concentrations at both doses (from 190.3 +/- 5.7 to 135.0 +/- 8.9 mg/dl, from 189.1 +/- 7.3 to 129.3 +/- 17.6 mg/dl; P < 0.05, respectively) after they had been increased by the HFD. The liver lipid content was also decreased by the diet containing MT (from 132.6 +/- 3.9 to 95.6 +/- 6.1 mg/g of tissue; P < 0.05). These results suggest that MT could be a potentially therapeutic alternative in the treatment of obesity caused by a HFD.

Prospective Evaluation of Laparoscopic Pancreatic Biopsies in 11 Healthy Cats

BackgroundDefinitive diagnosis of feline pancreatic disease is dependent on histologic examination of biopsies.HypothesisLaparoscopic punch biopsy of the pancreas does not significantly affect pancreatic health or clinical status of healthy cats, and provides an adequate biopsy sample for histopathology.AnimalsEleven healthy female domestic shorthair cats.MethodsEffects of laparoscopic pancreatic visualization alone in 5 cats compared with laparoscopic pancreatic visualization and punch biopsy in 6 cats were studied. Temperature, pulse, and respiratory rate, physical examination, and daily caloric intake were evaluated for 1 week before and 1 week after the procedure. Pain scores (simple descriptive score and dynamic interactive visual assessment score) were evaluated hourly during the 1st 6 hours postprocedure. Complete blood cell counts, serum biochemical profiles, serum feline pancreatic lipase immunoreactivity, and urine specific gravity were evaluated before the procedure and at 6, 24, and 72 hours postprocedure. One month postprocedure, during sterilization, the pancreas was reassessed visually in all cats, and microscopically in the biopsy group.ResultsFor all variables evaluated, there were no significant differences between biopsy and control cats. Re-evaluation of the pancreatic biopsy site 1 month later documented a normal tissue response to biopsy. The laparoscopic punch biopsy forceps provided high-quality pancreatic biopsy samples with an average size of 5 mm x 4 mm on 2-dimensional cut section.Conclusions and Clinical ImportanceLaparoscopic pancreatic biopsy is a useful and safe technique in healthy cats.

Evaluation of different natural ingredients as satiety inductors

Dissertation to obtain a Master’s Degree in Chemical and Biochemical Engineering

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Effects of prolonged ethanol intake and malnutrition on rat pancreas

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.

Évaluation gastro-intestinale chez des chiens présentant un comportement de léchage excessif de surface

L’objectif de cette étude était de démontrer que le léchage excessif de surface (LES) chez le chien représente un signe clinique d’un trouble digestif sous-jacent plutôt qu’un trouble obsessionnel compulsif. Vingt chiens présentés pour LES (groupe L) ont été divisés en 2 sous-groupes de 10 chiens chacun : L0, sans, et LD, avec des signes cliniques digestifs concomitants. Dix chiens en santé ont été assignés à un groupe contrôle (groupe C). Une évaluation comportementale complète, un examen physique et neurologique ont été réalisés avant un bilan diagnostic gastro-intestinal (GI) complet (hématologie, biochimie, analyse urinaire, mesure des acides biliaires pré et post-prandiaux et de l’immunoréactivité spécifique de la lipase pancréatique canine, flottaison fécale au sulfate de zinc, culture de selles, échographie abdominale et endoscopie GI haute avec prise de biopsies). En fonction des résultats, un interniste recommandait un traitement approprié. Les chiens étaient suivis pendant 90 jours durant lesquels le comportement de léchage était enregistré. Des troubles GI ont été identifiés chez 14/20 chiens du groupe L. Ces troubles GI sous-jacents incluaient une infiltration éosinophilique du tractus GI, une infiltration lymphoplasmocytaire du tractus GI, un retard de vidange gastrique, un syndrome du côlon irritable, une pancréatite chronique, un corps étranger gastrique et une giardiose. Une amélioration >50% en fréquence ou en durée par rapport au comportement de léchage initial a été observée chez une majorité de chiens (56%). La moitié des chiens ont complètement cessé le LES. En dehors du LES, il n’y avait pas de différence significative de comportement (p.ex. anxiété), entre les chiens L et les chiens C. Les troubles GI doivent être considérés dans le diagnostic différentiel du LES chez le chien.

Studies on fish lipases

Lipids constitute a significant portion of the biomass of earth and lipolytic enzymes play a very important role in lipid turn over. Apart from their biological significance, lipolytic enzymes are also very important in the fields of nutrition, food technology, medicine and preparative and analytical lipid biochemistry. Recent developments in the study of proteins and enzymes have largely benefited the study of lipolytic enzymes, that some of these enzymes were isolated in pure form. Even today there is a continuous search for new and potent sources of these lipolytic enzymes. The zest for elucidating the structure and mechanism of action of the enzymes obtained in pure form for biochemist still remains unabated. The literature shows no record of such an effort for the study of lipases from marine sources. The fact that many fishes like oil sardine, mackerel, cat fish, seer etc. contains large amounts of lipid shows the possibility of the existence of lipases in significant amounts necessitating their exhaustive study. Such a study will, not only provide alternate sources for lipase but also will provide methods to curb lipolysis and the resultant rancidity and off flavor development in fish and fishery products.

Emulsifier in broiler diets containing different fat sources

This present study aimed at evaluating the effect of the addition of an emulsifier to diets containing soybean oil, poultry fat or their blend, on the performance, carcass traits, serum lipid levels, pancreatic lipase concentration and nutrient digestibility of broilers. A randomized block design was applied using a 3 x 2 factorial arrangement, with three fat sources (soybean oil, poultry fat, and a blend of 50% soybean oil and 50% poultry fat) and the addition or not of an emulsifier. In experiment I, broiler performance, carcass traits, serum cholesterol, HDL, and triglyceride levels, and pancreatic lipase activity in 42-day-old broilers were evaluated. In experiment II, dry matter (DM), ether extract (EE), crude protein (CP) and crude fiber (CF) coefficients of digestibility were analyzed. Broilers fed the diet containing soybean oil and emulsifier presented higher body weight, weight gain and better feed conversion ratio. When birds were fed poultry fat and the fat blend (soybean oil and poultry fat) and the emulsifier was added to the diets, pancreatic lipase concentration increased. It was concluded that the use of soybean oil, poultry fat and their blend does no in the diet does not influence the performance, carcass traits, or serum cholesterol, HDL and triglyceride levels of 42-day-old broilers. The addition of emulsifiers to diets containing poultry fat improves ether extract digestibility and increases the production and secretion of pancreatic lipase.

Energy and oil levels in broiler starter diets

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of broiler breeder age on pancreas enzymes activity and digestive tract weight of embryos and chicks

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Atividade de enzimas digestivas de Lithobates catesbeianus durante o desenvolvimento larval

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)