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Reduced bile output with chronic enteral and parenteral infusion of amino acids, glucose, and fat emulsion in rabbits.

To assess the effect of chronic administration of amino acids, glucose, and fat on hepatic excretory function, bile flow and bile salt secretion were directly measured in adult rabbits alimented either intravenously or intragastrically. Five groups of animals were studied after 9-11 days on different nutritional regimes: the first, controls, received 154 mM NaCl intravenously and rabbit chow ad libitum; the second, 2.5% amino acid-10% glucose-10% fat emulsion intravenously; the third, the same nutrients intragastrically; the fourth, reduced intake of rabbit chow to match the weight change in the intragastrically fed animals; the fifth was allowed rabbit chow ad libitum and then received the nutrient solutions intragastrically only during bile collection. Bile was collected directly at laparotomy from the common bile duct during three 1-h periods: a basal period when no exogenous bile salt was infused, then with the addition of 1, and finally 2 mumol/min/kg of glycodeoxycholic acid, the main bile acid of rabbits. During the 3-h experiment either saline (control) or the nutrient solution was administered by the respective route. Chronic administration of the nutrient solutions, whether intravenously or intragastrically, significantly (p less than 0.05) reduced bile flow and bile salt secretion compared with controls and the rabbits on a reduced chow intake. The chronic intravenous route had a more pronounced cholestatic effect than the intragastric route. Acute intragastric nutrient infusion had no effect. The decreased bile output in the chronic groups resulted from a reduction in both bile salt secretion and bile salt-independent flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

The relative safety of MRI contrast agent in acute necrotizing pancreatitis.

OBJECTIVE: To validate the safety of gadolinium-diethylenetriamine pentaacetic acid (GD-DTPA) by measuring its effect on pancreatic capillary perfusion and acinar injury in acute pancreatitis. BACKGROUND: Contrast-enhanced computed tomography (CECT) is proposed as a gold standard for early evaluation of acute necrotizing pancreatitis. However, iodinated contrast media used for CECT have been shown in these circumstances to reduce pancreatic capillary flow and increase necrosis and mortality. Recent reports suggest that post-GD MRI provides images comparable to CECT in the assessment of severe acute pancreatitis. METHODS: Necrotizing pancreatitis was induced in 14 Wistar rats by intraductal glycodeoxycholic acid (10 mM/L) and intravenous caerulein (5 microg/kg/h) over 6 hours. Intravital microscopic quantitation of pancreatic capillary blood flow was performed using fluorescein isothiocyanate-labeled erythrocytes after induction of pancreatitis and 30 and 60 minutes after an intravenous bolus of either Ringer's solution or GD-DTPA (0.2 mL/kg). RESULTS: The two study groups were comparable with regard to mean arterial pressure, heart rate, arterial blood gases, hematocrit, amylase, lipase, and trypsinogen activation peptide production throughout the experiment. GD-DTPA did not reduce capillary flow (1.93 +/- 0.05 nL/capillary/min) compared to animals infused with Ringer's solution (1.90 +/- 0.06 nL/capillary/min). CONCLUSIONS: Intravenous injection of GD-DTPA does not further impair pancreatic microcirculation or increase acinar injury in acute necrotizing pancreatitis. Because of this advantage over CT contrast medium, further development of MRI as a staging tool in acute pancreatitis seems desirable.

Animals↗

Serum bile acid levels in protracted diarrhea of infancy.

Significant elevations in two glycine-conjugated serum bile acid levels (cholic and chenodeoxycholic) were detected in a majority of infants with intractable diarrhea of infancy. In contrast, children with chronic inflammatory bowel disease had values of serum bile acids within the normal range. Although intravenous alimentation and constant-infusion elemental diet may alter hepatic function, serum bile acid levels were also elevated in other infants with intractable diarrhea not treated by these methods. We hypothesize that endotoxemia or other unknown mechanisms together with therapy are exerting a detrimental effect on hepatic function.

Bile Acids and Salts↗

Decreased inflammation and improved survival with recombinant human activated protein C treatment in experimental acute pancreatitis.

HYPOTHESIS: Drotrecogin alfa (activated), the pharmacologic form of activated protein C and the first Food and Drug Administration-approved drug for treatment of severe sepsis, is beneficial in experimental acute pancreatitis (AP). DESIGN: Animal study. SETTING: Laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Mild (intravenous cerulein) or severe (intravenous cerulein plus intraductal glycodeoxycholic acid) AP was induced in 72 rats, and coagulation evaluated. Rats with severe AP were randomized to treatment with drotrecogin alfa (activated), 100 microg/kg per hour, or isotonic sodium chloride. MAIN OUTCOME MEASURES: Histologic scoring of pancreatic necrosis, inflammation of the pancreas and lung (measured by myeloperoxidase concentration), coagulation measures, and 24-hour survival. RESULTS: Severe consumptive coagulopathy, hemoconcentration, and leukocytosis were observed 6 hours after induction of severe AP, but not in mild AP. Treatment of AP with drotrecogin did not worsen coagulation measures. Although the degree of pancreatic necrosis was comparable in treated and untreated animals with severe AP, drotrecogin significantly reduced myeloperoxidase levels in the pancreas (P = .009) and lungs (P = .03). The 24-hour survival in severe AP was markedly improved in animals treated with drotrecogin (86% vs 38%; P = .05). CONCLUSIONS: Animals with severe AP have severe consumptive coagulopathy, but administration of drotrecogin alfa (activated), 100 microg/kg per hour, does not worsen coagulation abnormalities. Drotrecogin treatment reduces inflammation in the pancreas and lungs and significantly improves survival. These results encourage clinical investigation of drotrecogin in the treatment of severe AP.

Animals↗

Pancreatic ascites as a powerful inducer of inflammatory cytokines. The role of known vs unknown factors.

OBJECTIVES: To determine if pancreatic ascites will induce interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF-alpha) production outside the pancreas and examine the possible components responsible. DESIGN: Severe pancreatitis was induced in rats (n = 30) by pancreatic duct infusion with 4% glycodeoxycholic acid; pancreatic ascites was collected 18 hours later. In vitro studies used quiescent murine splenic or pulmonary macrophages (10(5)/mL) which were exposed to media alone (control), trypsin, chymotrypsin, cathepsin-B, 20% ascites (vol/vol), 50% ascites, or endotoxin (lipopolysaccharide, 10 micrograms/mL, positive control) for 4 hours. Subsequently, pancreatic ascites was cultured for bacteria and assayed for endotoxin and cytokines (interleukin 1, interleukin 6, interleukin 8, TNF-alpha, or interferon gamma). The experiments were then repeated using 20% and 50% ascites that was sterile and cytokine-free (SCF ascites). In vivo studies used 100% (n = 8) or 50% (n = 12) SCF ascites or normal rat serum (control, n = 12) for a 10-second pulmonary lavage (100 microL) in adult mice, with lungs collected at 6 hours for cytokine gene analysis. SETTING: Surgical basic science research laboratory. MAIN OUTCOME MEASURES: Interleukin 1 beta and TNF-alpha gene induction was assessed by quantitative competitive reverse-transcription polymerase chain reaction and cytokine protein production was determined by enzyme-linked immunosorbent assay. RESULTS: Macrophages responded to untested and SCF ascites in a dose-dependent fashion, with a multifold increase in both IL-1 beta and TNF-alpha messenger RNA (mRNA) and protein, which was often more potent than lipopolysaccharide. Expression of IL-1 beta and TNF-alpha mRNA could not be induced by trypsin, chymotrypsin, or cathepsin-B. All animals undergoing lavage with 100% SCF ascites died within 2 hours, while those undergoing lavage with 50% SCF ascites showed a multifold increase in pulmonary IL-1 beta and TNF-alpha mRNA. CONCLUSIONS: Pancreatic ascites contains factors that are capable of inducing IL-1 beta and TNF-alpha production in vitro and in vivo. This effect cannot be reproduced by activated digestive enzymes and is propagated despite the absence of known inducers of cytokines such as bacteria, endotoxin, or other inflammatory cytokines.

Animals↗

The effect of bile salts on the pancreatic duct mucosal barrier.

The effects of various bile salts on the permeability of the pancreatic duct in cats have been investigated. The sodium salts of cholic, glycocholic, taurocholic, chenodeoxycholic, glycochenodeoxycholic, deoxycholic and glycodeoxycholic acids were perfused through the main duct in concentrations similar to those found in the duodenal contents (15-42 mM). HCO3- and Cl- fluxes were determined before and after perfusion. The unconjugated dihydroxy bile salts increased permeability to these anions to a greater degree than the trihydroxy bile salt, but all were damaging to the duct when they were perfused at an alkaline pH (8.4-9.1). The glycine conjugates of the bile salts were as damaging as the unconjugated form, but 42 mM taurocholate was more damaging than 42 mM glycocholate. When 25 mM glycocholate was perfused at pH 2.4 there was potentiation of the damage produced during perfusion of glycocholate at pH 8.7. Sterile cat bile did not damage the duct, but infected bile did. The ability of bile salts to increase the permeability of the pancreatic ducts must be considered as a possible aetiological factor in the pathogenesis of inflammatory and malignant diseases of the pancreas.

Animals↗

The use of negative ion thermospray liquid chromatography/tandem mass spectrometry for the determination of bile acids and their glycine conjugates.

A study was carried out on the negative ion thermospray liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry of a group of bile acids and their glycine conjugates. The liquid chromatographic/tandem mass spectrometric experiments were performed using low-energy collisions on a triple-quadrupole mass spectrometer. It was found that relatively high collision energies and collision gas pressures were required to produce fragmentation and that some unusual fragments were produced.

Chenodeoxycholic Acid↗

The effect of gabexate mesilate on pancreatic and hepatic microcirculation in acute experimental pancreatitis in rats.

Microcirculatory derangements are important early features in many organs during the process of acute pancreatitis. However, dynamic evaluation of these factors has been difficult. Antiprotease has long been used for the treatment of acute pancreatitis, although its effects and mechanism have not been fully elucidated. The involvement of proteases and microcirculatory derangement early in the course of acute pancreatitis are the main concern of this study. A severe acute pancreatitis model in male Sprague-Dawley rats (225-275 g) was established by adding caerulein (15 microg/kg/ hr) in intravenous infusion fluid and intraductal injection of 0.1 ml glycodeoxycholic acid (5 mM). Gabexate mesilate [GM; ethyl-4-(6-guanidinohexanoyloxy)benzoate methanesulfonate], a synthetic antiprotease, was infused intravenously in doses of 0.01, 0.1, 1, and 10 mg as a therapeutic intervention in this model. Pathology hematocrit, serum amylase level, and glutamic-oxaloacetic transaminase (GOT) levels were used to confirm the severity of disease and effect of therapy. In vivo microscopic technique was used as a investigating tool in this study of microcirculatory derangement in pancreas and liver, 8 hr after induction of acute pancreatitis. GM can significantly improve pathologic criteria and changes of serum amylase levels in the range of 1-10 mg/kg/hr. The severity of changes of hematocrit and GOT was significantly lessened with GM in the range of 0.1-10 mg/kg/hr. This agent also could improve the microcirculatory environment in pancreas and liver after induction of acute pancreatitis according to the parameters, such as flow velocity and rolling leukocyte phenomenon, in the range of 1-10 mg/kg/hr. According to our observation, severity of hyperpermeability had not changed with the treatment of GM. These results indicated the beneficial effects of GM on pancreatic and hepatic microcirculation early in the course of acute pancreatitis. The beneficial effects were noted in serum parameters and hematocrit. The importance of protease activation and remote organ dysfunction is emphasized in the course of acute pancreatitis from this study.

Acute Disease↗

Pancreatic capillary blood flow in an improved model of necrotizing pancreatitis in the rat.

INTRODUCTION: The development of acute pancreatitis is characterized by profound changes in pancreatic microcirculation. Using in vivo microscopy with fluorescent-labeled erythrocytes as tracers we studied changes in pancreatic microcirculation in an improved rat model of necrotizing pancreatitis (NP) in comparison to edematous pancreatitis (EP) and healthy controls. METHODS: Twenty-one male Wistar rats had their pancreatae exteriorized in a temperature-controlled immersion chamber followed by intravenous administration of fluorescent-labeled autologous erythrocytes. EP was induced by intraductal saline and intravenous caerulein (5 microg/kg/h) for 6 h (n = 7) and NP by controlled intraductal infusion of glycodeoxycholic acid (10 mmol/L) followed by intravenous caerulein (n = 7). Control animals received intraductal and intravenous saline (n = 7). The determination of pancreatic microcirculation was performed before as well as 1, 3, and 6 h after intraductal infusion by correlating the number of passing labeled erythrocytes/capillary/min with their concentration per microliter of arterial blood. RESULTS: Pancreatic capillary flow in control animals remained constant over the 6-h observation period. Pancreatic capillary flow in the EP group rapidly increased to 188% of baseline after 3 h and remained significantly elevated throughout the experiments (P = 0.0001). In contrast, pancreatic capillary flow decreased significantly in the group suffering NP with values 46.7% of baseline after 6 h (P = 0.0001). Complete capillary stasis developed in 38% of investigated capillaries in the NP group compared to 0-1% in both other groups (P = 0.0001). CONCLUSION: Pancreatic microcirculation in mild edematous pancreatitis is significantly increased while the evolution of necrotizing pancreatitis in the model studied herein is characterized by a dramatic reduction in pancreatic capillary flow in conjunction with areas of capillary stasis. These results underline the pathophysiologic relevance of the model and of therapeutic measures aimed at an improvement of pancreatic microcirculation in clinical necrotizing pancreatitis.

Animals↗

Postnatal development of hepatic bile formation in the rabbit.

To investigate postnatal maturation of hepatic bile formation, bile output was measured in four groups of rabbits: suckling infants at ages 10-14, 18-22, and 26-30 days, and adults. Bile output was collected directly from the common duct during three 1-hr periods: a basal period followed by intravenous infusion of 1 and then 2 mumol/min/kg of glycodeoxycholic acid. [14C]Erythritol and [3H]inulin clearances measured canalicular bile flow and biliary permeability. Under basal conditions and with the exogenous bile acid, bile flow and bile salt secretion were lowest in 10- to 14-day-old infants and showed a gradual increase with increasing age. Bile salt-dependent flow, the linear increase in flow relative to bile salt secretion, was higher in the 10- to 14- and 17- to 22-day-old compared to the adult and 25- to 30-day-old groups. The ratio of chloride to bile salt secreted was also higher in the two younger groups. Bile salt-independent flow at theoretical zero bile salt secretion was absent in the younger groups, but evident in the adult and 25- to 30-day-old rabbits. Canalicular flow estimated by erythritol clearance was linearly related to bile salt secretion. Inulin clearance relative to erythritol clearance was higher in the 10- to 14-day-old infants than the adults. Thus, bile flow and bile salt secretion are reduced in the young infant but rise to near adult levels at the time of weaning, 25-30 days in the rabbit. The increase in flow results from increased bile salt secretion and the appearance of bile salt-independent flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

High performance liquid-chromatographic analysis of individual bile acids: free, glycine- and taurine-conjugated bile acids.

High performance liquid-chromatographic analyses of individual bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid), free and conjugated with glycine and taurine, are described. The analyses of free and glycine-conjugated bile acids are based on esterification of carboxyl group of bile acids with O-(p-nitrobenzyl)-N, N'-diisopropylisourea (PNBDI). Moreover, ursodeoxycholic acid, hyocholic acid, hyodeoxycholic acid and 3beta-hydroxy-5-cholenoic acid also are able to analyse by this method. These bile acids in biological sample were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine- and taurine-conjugated bile acids. After the separation, free and glycine-conjugated bile acids were esterified with PNBDI directly. Because taurine-conjugated bile acids are unable to be esterified with PNBDI, these bile acids were hydrolyzed by NaOH in order to make free bile acids, and then they were esterified. Because the p-nitrobenzyl ester of bile acids has characteristic ultraviolet absorption, these compounds were separated to individual bile acids by high performance liquid-chromatography, and detected by an UV-detector. An analysis of individual bile acids in human bile was demonstrated.

Bile Acids and Salts↗

Morphometric characteristics and homogeneity of a new model of acute pancreatitis in the rat.

Extreme maldistribution and immediate establishment of severe cellular injury are typical features of traditional bile salt models of acute pancreatitis; both factors complicate assessment and interpretation of therapeutic benefits in trials of experimental therapy. Even more important, both are indications not of the desired induction of pancreatitis but rather of local injury by barotrauma and noxious chemicals. This study contrasts the severity and regional variability of cellular injury in traditional high-dose bile salt models with that seen in a new preparation employing the combination of intravenous (iv) caerulein (CAE) and intraductal (id) low-dose glycodeoxycholic acid (GDOC). Thirty-six male Sprague-Dawley rats (350-450 g) were induced with (group A) high-dose GDOC id (34 mmol/L), low-dose GDOC id (10 mmol/L) (group B), or low-dose GDOC id combined with caerulein iv for 6 h (group C). The regional distribution of histopathologic injury within the pancreas was assessed in 20 fields/organ by two pathologists unaware of the induction technique used. High-dose GDOC id (group A) resulted in extremely heterogenous distribution of injury for all variables (edema, p = 0.001; acinar necrosis, p = 0.0001; inflammation, p = 0.0001; and hemorrhage p = 0.001). The lesions were confined to the head of the pancreas, which showed large areas of necrosis involving entire lobules, whereas adjacent areas were unaffected. Low-dose GDOC id (group B) was more homogenously distributed, but the injury was mild and regional variability (edema, p = 0.0001; acinar necrosis, p less than 0.04; inflammation, p = 0.0001; and hemorrhage p less than 0.05) was still demonstrable. In contrast, low-dose GDOC id combined with CAE iv (group C) produced moderately severe pancreatitis, which equally affected all areas of the gland. There were no geographical differences in acinar necrosis or inflammation. This feature of the new model provides a desirable prerequisite for accurate and reproducible assessment of histopathology in studies aimed at detecting effects of therapy. We suggest that it replace traditional id bile salt infusion models.

Acute Disease↗

Glutamine stabilizes intestinal permeability and reduces pancreatic infection in acute experimental pancreatitis.

Intestinal barrier failure and subsequent translocation of bacteria from the gut play a decisive role in the development of systemic infections in severe acute pancreatitis. Glutamine (GLN) has been shown to stabilize gut barrier function and to reduce bacterial translocation in various experimental settings. The aim of this study was to evaluate whether GLN reduces gut permeability and bacterial infection in a model of acute necrotizing pancreatitis. Acute necrotizing pancreatitis was induced in 50 rats under sterile conditions by intraductal infusion of glycodeoxycholic acid and intravenous infusion of cerulein. Six hours after the induction of pancreatitis, animals were randomly assigned to one of two groups: standard total parental nutrition (TPN) or TPN combined with GLN (0.5 g/kg(-1)/day(-1)). After 96 hours, the animals were killed. The pancreas was prepared for bacteriologic examination, and the ascending colon was mounted in a Ussing chamber for determination of transmucosal resistance and mannitol flux as indicators of intestinal permeability. Transmucosal resistance was 31% higher in the animals treated with GLN- supplemented TPN compared to the animals given standard TPN. Mannitol flux through the epithelium was decreased by 40%. The prevalence of pancreatic infections was 33% in animals given GLN-enriched TPN as compared to 86% in animals receiving standard TPN (P < 0.05). Adding GLN to standard TPN not only reduces the permeability of the colon but decreases pancreatic infections in acute necrotizing pancreatitis in the rat. This confirms previous reports that GLN decreases bacterial translocation by stabilizing the intestinal mucosal barrier. The present findings provide the first evidence suggesting that stabilizing the intestinal barrier can reduce the prevalence of pancreatic infection in acute pancreatitis and that GLN may be useful in preventing septic complications in clinical pancreatitis.

Acute Disease↗

Gut macromolecular permeability in pancreatitis correlates with severity of disease in rats.

BACKGROUND: Increased intestinal macromolecular permeability could allow absorption of substances from the bowel into the systemic circulation and contribute to multiple organ system failure. METHODS: Mild, intermediate, and severe grades of pancreatitis were induced in rats using intravenous caerulein and intraductal glycodeoxycholic acid. [14C]polyethylene glycol (molecular weight, 3350 daltons; 1.1 microCi/142 mg) was instilled into the distal duodenum. At 24 hours, the animals were killed, ascitic fluid was collected for trypsinogen activation peptide measurement, and pancreatic specimens were collected and scored for based on the degree of necrosis, inflammation, and hemorrhage. RESULTS: Gut permeability to polyethylene glycol 3350 (PEG 3350) was increased in animals with early experimental pancreatitis (5.4% +/- 1.2%, n = 20) when compared with control animals (1.8% +/- 0.2%; n = 6) (P = 0.0005). Furthermore, intestinal macromolecular permeability to PEG 3350 correlated with severity of disease as predicted by the method of induction of pancreatitis (P = 0.0003), the histological findings (P = 0.0002), and total ascitic trypsinogen activation peptides content (P = 0.029). CONCLUSIONS: Increased gut permeability in experimental pancreatitis can be correlated with pancreatitis severity.

Animals↗

Application of capillary electrophoresis of characterizing interactions between drugs and bile salts. Part I.

Capillary electrophoresis offers a new way of characterizing interactions between different bile salts and drugs. The observed interactions were characterized with modified model functions known from affinity capillary electrophoresis (ACE) an micellar electrokinetic capillary electrophoresis (MECC). The methodical background of both methods is the change of the ionic mobility of the drug caused by partition between phases and aggregation with the bile salt molecules, respectively. This phenomenon is described by two different physicochemical models. A parameter estimation was carried out in order to obtain the partition coefficients KP as well as constants for the aggregate formation KA. Furthermore, an expression about the specific molar volume of the micelles and stoichiometric coefficients can be given.

Atenolol↗

The synthesis of glycine conjugated 3-oxo-bile acids.

The glycine conjugates of the 3-oxo-derivatives of lithocholic, deoxycholic, chenodeoxycholic and cholic acids have been synthesized from their respective parent bile acid using established procedures. The reaction sequence involved oxidation using either chromic acid or silver carbonate--Celite followed by a conjugation step employing the peptide coupling reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. The final products were obtained in both high yield and purity for use as reference compounds in the analysis of conjugated bile acids in bile and faecal samples by high performance liquid chromatography. Analysis of the prepared 3-oxo-glycobile acids by fast atom bombardment negative ion mass spectrometry, infrared spectroscopy and nuclear magnetic resonance proton spectroscopy is also discussed along with the thin-layer chromatographic properties.

Chemical Phenomena↗

Bile acids are able to reduce blood pressure by attenuating the vascular reactivity in spontaneously hypertensive rats.

Effects of the synthetic bile acids on blood pressure were examined in spontaneously hypertensive rats. Continuous intravenous administration of the bile acids at the rate of 1 mg/min for 20 min significantly lowered the blood pressure by 12 mmHg. In order to examine its blood pressure lowering mechanism, the isolated mesenteric arterial perfusion system was employed. Bile acids in the perfusate inhibited vascular reactivity to norepinephrine and KCl in a dose-dependent manner. This inhibitory action diminished as the concentration of potassium in the perfusate decreased. When the perfusate was free from potassium, its inhibitory action completely disappeared. These results in vivo and in vitro studies strongly suggest that bile acids act directly on the vascular beds and attenuate vascular response to norepinephrine.

Animals↗