Properties of different pancreatin preparations used in pancreatic exocrine insufficiency.

BACKGROUND: Pancreatic enzyme preparations are a life-saving substitution for a pivotal physiological function of the entire organism that is impaired in chronic pancreatitis, cystic fibrosis and other diseases with exocrine pancreatic insufficiency. Pancreatic enzyme preparations, generically called pancreatin, are not alike. Rather, they present a broad variety of pancreatin composition. AIM: The properties of a set of commercially available pancreatin preparations were investigated in light of the physiological tasks such enzymes must fulfill during the normal digestive process. METHODS: Measurements of size, surface, acid resistance, release of enzymes, pharmacokinetics and batch consistency were undertaken. RESULTS: Although all pancreatin preparations contain the declared lipase units and are acid-stable, a wide variation was observed in the particle size (pyloric passage), specific surface area and release kinetics of lipase activity at pH 6 (duodenum). CONCLUSION: At present, available pancreatin preparations vary widely with respect to investigated parameters, which may have consequences for facilitating optimal digestion.

Enzyme content and acid stability of enteric-coated pancreatic enzyme products in vitro.

OBJECTIVES: Pancreatic enzymes are prescribed routinely for pancreatic insufficiency. In the current health care environment, drug substitution is commonly performed although there is no proof of therapeutic or bioequivalence for these products. The purpose of this in vitro, prospective study was to evaluate the enzyme contents and dissolution of various capsules of pancreatic enzyme using current United States Pharmacopoeia (USP) methodology. METHODS: Nine different pancreatic enzyme products were purchased on the market and supplied to Irvine Analytical Laboratories (IAL) (Irvine, CA). All test products were maintained in the laboratory environment, at room temperature, throughout the testing period by IAL. USP procedures for assay and dissolution testing of pancrelipase delayed-release capsules, as described in the latest USP supplement were observed during product testing, including determination of amylase, lipase, and protease activity. In addition, a point assay with measurement of lipase after dissolution in simulated gastric fluid pH of 1.0 for 1 hour and then dissolution in pH 6 phosphate buffer for 30 minutes performed in accordance with USP guidelines. RESULTS: Assay results of amylase, protease, and lipase from the 9 tested products are within USP specified limits. The percentage of label claim for these enzymes was higher than depicted in their label except for one drug batch. However, the percentage of lipase activity after dissolution varied with 2 of 3 batches of 1 drug not dissolving, and 1 batch of another drug, revealing only 8% lipase activity in the USP dissolution test. CONCLUSION: While assay of pancreatic enzymes reveal they were equal to their USP claims regarding their enzyme content, not all pancreatic enzyme replacements are equal in their release of lipase activity according to USP requirements. The findings maybe clinically seen with therapeutic failures of enzyme products. The FDA has recently decreed that all pancreatic enzyme products will require an approved NDA as differences in pharmaceutical quality have been identified in this product. Thus, it is considered that substitution of these products maybe questionable. Things are seldom what they seem- not all pancreatic enzyme replacements are equal. Further studies are warranted to investigate dissolution characteristics.

Mobilization of xanthine oxidase from the gastrointestinal tract in acute pancreatitis.

BACKGROUND: Xanthine oxidoreductase has been proposed to play a role in the development of local and systemic effects of acute pancreatitis. Under physiologic conditions, the enzyme exists mainly as xanthine dehydrogenase (XDH) but can be converted by proteolytic cleavage to its superoxide-generating form xanthine oxidase (XOD). In addition to its intracellular location XDH/XOD is also associated to the polysaccharide chains of proteoglycans on the external endothelial cell membrane. In the early stages of acute pancreatitis, this enzyme seems to be arising from its mobilization from the gastrointestinal endothelial cell surface. Taking into account the ability of alpha-amylase to hydrolyze the internal alpha-1,4 linkages of polysaccharides, we wanted to elucidate the involvement of alpha-amylase in XDH/XOD mobilization from the gastrointestinal endothelial cell surface and the relevance of the ascitic fluid (AF) as the source of alpha-amylase in experimental acute pancreatitis. METHODS: Acute pancreatitis was induced in male Wistar rats by intraductal administration of 5% sodium taurocholate. In another experimental group 3000 U/Kg alpha-amylase was i.v. administered. The concentrations of XDH, XOD and alpha-amylase in plasma and AF and myeloperoxidase (MPO) in lung have been evaluated. In additional experiments, the effect of peritoneal lavage and the absorption of alpha-amylase present in the AF by an isolated intestine have been determined. RESULTS: Similar increase in XDH+XOD activity in plasma was observed after induction of acute pancreatitis and after i.v. administration of alpha-amylase. Nevertheless, the conversion from XDH to XOD was only observed in the pancreatitis group. Lung inflammation measured as MPO activity was observed only in the pancreatitis group. In addition peritoneal lavage prevented the increase in alpha-amylase and XDH+XOD in plasma after induction of pancreatitis. Finally, it was observed that alpha-amylase is absorbed from the AF by the intestine. CONCLUSIONS: During the early stages of acute pancreatitis, alpha-amylase absorbed from AF through the gastrointestinal tract could interfere with the binding of XDH/XOD attached to glycoproteins of the endothelial cells. Proteolytic enzymes convert XDH into its oxidase form promoting an increase in circulating XOD that has been reported to be one of the mechanisms involved in the triggering of the systemic inflammatory process.

Cyclodextrin glycosyltransferase may be the only starch-degrading enzyme in Bacillus macerans.

Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source. In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch. At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells. Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B. macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid.

Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini.

Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism.

Peritoneal absorption of pancreatic enzymes in dogs.

To elucidate peritoneal absorption of pancreatic enzymes, plasma levels of amylase, lipase, and trypsinogen were measured after the intraperitoneal injection of 10 ml human pancreatic juice in dogs. Plasma pancreatic amylase, lipase, and trypsinogen were determined using the immunoassay specific to the corresponding human pancreatic enzyme to exclude cross-reaction with the endogenous enzyme activities of the canine plasma. Plasma immunoreactivity of human amylase persistently rose during 24 h after the injection, whereas elevation of plasma amylase enzyme activity become significant only at 24 h. Increase of plasma lipase was not remarkable. Significant increase of the immunoreactivity was observed only at 24 h but there was no significant increase of the enzyme activity during this period. Plasma trypsinogen immunoreactivity peaked at 2 h remained significantly elevated during 24 h. The transperitoneal absorption of pancreatic enzymes in dogs was confirmed using intraperitoneal injection of human pancreatic juice combined with immunoassay specific to human pancreatic enzymes.

Stimulation of rat parotid secretion by cAMP analogues that synergistically activate the type II isoenzyme of the cAMP-dependent protein kinase.

Beta-adrenergic-stimulated parotid secretion is believed to be mediated by activation of the cAMP-dependent protein kinase (PK-A). However, the relative roles of the type I and II PK-A isoenzymes are still unclear. Combinations of site-selective, lipophilic cAMP analogues that synergistically activate each PK-A were used to investigate this problem. The selectivity of synergistic activation with these combinations was verified with the partially purified parotid PK-A isoenzymes, using kemptide as a substrate. Synergism in activation of PK-AII was only seen with 8-thiomethyl cAMP (8-TM) and N-6-benzoyl cyclic AMP (N6B), while PK-AI was only synergistically activated by 8-(6-aminohexyl) amino cyclic AMP (AHA) and N6B. Additive activation of each isoenzyme was observed for the combination of 8-TM and AHA. Rates of amylase secretion from dispersed parotid acini in response to secretagogues were determined with a coupled enzyme assay for amylase activity, which was adapted for use in a microplate reader. Cells were stimulated to secrete during 30 min with different doses (0.1-1.0 mM) and combinations of the cyclic nucleotide analogues. Alone, N6B was most effective in stimulating amylase secretion. The basal amylase secretory rate was stimulated by these secretagogues (0.44 mM) to the following extent: 53-fold (N6B), 8-fold (8-AHA), 2-fold (8-TM). In combination at a series of concentrations, only 8-TM + N6B produced synergistic stimulation of secretion, while AHA + N6B and 8-TM + AHA did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of 45Ca uptake and amylase release by cationic amino acids in parotid cells.

L-arginine, L-ornithine, L-lysine and L-histidine (each 10 mM) stimulated amylase release from rat parotid cells. The secretory response to the cationic amino acids was suppressed in the absence of extracellular Ca2+ and, at physiological Ca2+ concentration, coincided with stimulation of 45Ca net uptake by the parotid cells. All cationic amino acids also accumulated inside the parotid cells. Nevertheless, the concept that the stimulation of amylase release is merely attributable to depolarization of the plasma membrane, secondary to the accumulation of these positively charged amino acids in the parotid cells, is questioned in view of both the inverse correlation found between their secretory effects and degree of ionization and the knowledge that parotid cells are electrically inexcitable.

Pancreatic enzyme activity in pregnancy.

Serum amylase activity and the amylase:creatinine clearance ratio (Cam:Ccr%) are two of the most commonly used indicators for the diagnosis of pancreatitis. However, published data on the effect of pregnancy on these indicators are conflicting. Furthermore, there are no published data on the effect of pregnancy on serum lipase activity, which is considered one of the most sensitive and specific indicators of pancreatitis. A study was undertaken to determine the effect of pregnancy and gestational age on serum amylase, serum lipase and Cam:Ccr% levels and to establish a baseline of normal values for use in the diagnosis of pancreatitis in pregnant women. Serum amylase, serum lipase and Cam:Ccr% levels were determined on a sample population consisting of 175 pregnant women with gestational ages ranging from 5 to 40 weeks and on a control group of 44 reproductive-age, nonpregnant women. The study results indicated that there is no significant difference in serum amylase, serum lipase and Cam:Ccr% levels between pregnant and nonpregnant women. Cam:Ccr% showed a small but statistically significant increase in the third trimester of pregnancy.

Changes in microfilament distribution during amylase exocytosis in rat parotid salivary glands in vitro.

In parotid acini, microfilaments were predominantly localized at the luminal border when visualized with the fluorescent probe, rhodamine-phalloidin. There was no fluorescence in the cytoplasm. When acini were stimulated by isoprenaline, the membranes of the secretory granules appeared to become undercoated by microfilaments immediately after fusion with the luminal membrane. The intensity of fluorescence was correlated with the extent of enlargement of the lumina. These findings indicate that the microfilament system (actin-myosin contractile system) may play a role in regulating the movement of the luminal plasma membrane rather than in the control of transport and access of secretory granules to the lumen.

Effect of cytochalasin D on acinar cell structure and secretion in rat parotid salivary glands in vitro.

Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.

In vivo and in vitro studies of microsphere pancreatic supplements.

Over the past 5 years, the Leeds Regional Cystic Fibrosis (CF) Unit has provided comprehensive annual assessments of CF patients that include dietary assessments and fat absorption studies. Enteric-coated microsphere pancreatic enzyme preparations (microsphere preparations) were compared to conventional enzymes as therapeutic agents for these patients. Presently in the U.K., two microsphere preparations are licensed for use and a further two products are likely to receive licenses in the near future. The success of these preparations is dependent on the ability of the microsphere coating to resist dissolution until the pH exceeds approximately 5.5 and thus prevent inactivation of lipase in the acid environment of the stomach. A study comparing Pancrex V Forte, a conventional enzyme preparation, to three microsphere preparations, Pancrease, Creon, and pancreatin Merck, confirmed the superiority of Pancrease and Creon over Pancrex V Forte and pancreatin Merck with regard to control of symptoms, and nitrogen and fat absorption. Because of differences in the physical characteristics of various microsphere preparations, the dissolution rates of Pancrease, Creon, and pancreatin Merck were compared in vitro. In aqueous buffers, striking differences among the preparations were seen at pH 5.5; whereas only 25% of available lipase was released from Creon, both Pancrease and pancreatin Merck show almost complete dissolution at this pH. Only at pH 6.5 and above do all three preparations show complete dissolution. In duodenal juice, as in aqueous buffers, lipase release from Creon takes place at a lower rate than with the other two preparations until pH 6.0 or higher is attained.(ABSTRACT TRUNCATED AT 250 WORDS)