Three-dimensional structure of lung elastin demonstrated by scanning electron microscopy/stereo-pair images.

The aim of this study was to expose the inflated 3-D structure of lung elastin. Formic acid digestion followed by freeze-drying unveiled the lamellar framework. The 3-D structure of elastin was well preserved within the alveolar septa and ducts, as demonstrated by scanning electron microscopy/stereo-pair photography. Elastin fibers are seen in the alveolar septa, which are continuous with the lamellae. The removal of collagen fibers and cells by formic acid was visualised as a function of time: The optimum was 48 hours. Transverse sections still retained some collagen fibrils and partially digested cells in addition to elastin as shown by transmission electron microscopy (TEM). Formic acid digestion followed by critical point drying caused damage to the lamellar structures and they appeared to collapse. Sodium hydroxide digestion combined with freeze-drying did not preserve the 3-D lamellar structure of elastin, but converted it into flat ribbonlike bands. The main structures remaining following alkali treatment were identified by TEM as collagen fibrils well preserved in their original locations.

Pancreatic atrophy: a new model using serial intra-peritoneal injections of L-arginine.

No simple rat model for chronic pancreatitis exists at present. A single dose of arginine has recently been shown to induce acute pancreatitis in rats. This study was designed to assess whether serial injections of arginine would induce reproducible chronic pancreatic damage. Forty rats received an intra-peritoneal injection of 500 mg per 100 g body weight of L-arginine followed by three injections of 250 mg per 100 g over 10 days. The rats were killed 24 h after each injection and at intervals of up to 6 months. Serum amylase levels were increased in the acute phase only. Examination of the pancreas at 24 h showed a severe oedematous pancreatitis. By day 5 there was up to 90% acinar destruction with adipose tissue replacement, although ductal, vascular, and islet cells appeared undamaged. These changes were present 6 months after injection. This is proposed as a new, simple, and reproducible method of inducing chronic pancreatic damage in the rat.

Temporal relationship of acinar and microvascular changes in caerulein-induced pancreatitis.

A study in rats investigated the temporal relationship between acinar cell changes and alterations in the local microvasculature in oedematous pancreatitis produced by administration of caerulein 5 micrograms kg-1 h-1. Samples were taken from experimental and control animals after 15 min, 30 min, 1 h and 2 h of caerulein infusion. Transmission electron microscopy showed ultrastructural acinar cell changes after 15 min whereas the earliest microvascular changes were seen after 30 min. Ultrastructural alterations in the acinar cells thus preceded local microvascular changes. Microvascular distortion appears to be a consequence and not a cause of pancreatitis in the caerulein model.

Microvasculature of the pancreas, liver, and kidney in cerulein-induced pancreatitis.

Edematous pancreatitis was induced in 12 male Sprague-Dawley rats using supramaximal doses of the cholecystokinin analogue cerulein (5 micrograms/kg per hour). The microvasculature of the pancreas, liver, and kidney was examined using scanning electron microscopy of microvascular corrosion casts in 12 test animals and four controls at intervals of 30 minutes, 1 hour, 2 hours, and 4 hours. Distortion of the pancreatic and hepatic microvasculature was seen as early as 30 minutes and progressed during the study period. The renal vasculature remained normal throughout. Light microscopic analysis revealed no morphologic abnormalities in the walls of the pancreatic, hepatic, or renal microvasculature. This study demonstrates that cerulein-induced pancreatitis is associated with marked distortion of the pancreatic and hepatic microvasculature; the abnormalities start early in the disease and progress during the study period.

An alkali digestion method to expose connective tissue fibers: a scanning electron microscopy study of rat lung.

Alkali digestion has been used to remove cellular elements of tissues thus exposing the underlying connective tissue framework. We studied the action of this severe alkali treatment on the delicate tissues of rat lung. The lungs of male Sprague-Dawley rats were perfused with saline to remove blood and then inflated by fixation through the airways at 20 cm pressure. Sections of lung 2 x 5 x 5 mm were immersed in 2.5 M NaOH at 25 degrees C for 6 h, 16 h, 24 h, 48 h, and 72 h. The alkali was changed daily. Tissues were washed to neutral with water (24 h), treated with tannic acid (1%, 3h), post-fixed with osmic acid (1%, 3 h) and processed for SEM. At 6 h, epithelial cells started to peel off the alveolar surface. At 16 h the digestion process was well advanced. At 48 h the cells were completely removed revealing the lattice network of connective tissue fibers within the alveolar surface. The method allows the complete removal of cellular elements of the lung while retaining the very fine 3D structure of the connective tissue matrix.

Pulmonary microvasculature in experimental acute haemorrhagic and oedematous pancreatitis.

The pulmonary microvasculature was examined in two experimental models of acute pancreatitis by scanning electron microscopy of microvascular corrosion casts. Haemorrhagic pancreatitis was induced in eight male Sprague-Dawley rats using an intraductal injection of 5 per cent sodium taurocholate. Oedematous pancreatitis was induced in seven male Sprague-Dawley rats using an intravenous infusion of supramaximal doses of caerulein (5 micrograms/kg per hour). The pulmonary vessels were cast using a polymer resin and the cast studied by scanning electron microscopy at 3 and 12 h in those with haemorrhagic and at 1 and 4 h in those with oedematous pancreatitis. Vascular abnormalities were present in both models at the initial study time with abruptly terminating vessels being more prominent in the caerulein model. At the later times, however, the abnormalities in the sodium taurocholate model were much more severe, with a substantial loss of vascular density, tortuosity and abrupt terminations of those vessels present. Microvascular abnormalities may be responsible for some of the pulmonary changes seen in oedematous and haemorrhagic pancreatitis.

Free radical inhibition and serial chemiluminescence in evolving experimental pancreatitis.

Oxygen free radical activity and inhibition were examined in experimental pancreatitis. Twenty-five rats were randomized to five groups: controls received intravenous saline, to simulate pancreatitis one group received intravenous caerulein (5 micrograms kg-1 h-1), and three groups received sodium taurocholate via the pancreatic duct (0.2 ml, 5 per cent), either alone, following allopurinol or immediately before superoxide dismutase. Chemiluminescence (a phenomenon based on the emission of light during chemical reactions and which is dependent on oxygen free radical activity) was used as an index of oxygen free radical activity and was measured in tissue samples at 5-min intervals following induction of pancreatitis. The control mean(s.e.m.) serum amylase level 1 h after induction of pancreatitis was 635(13) units. It was significantly elevated in caerulein-induced pancreatitis, 1833(118) units (P less than 0.05) and exceeded 3000 units in all taurocholate-infused animals. Mean(s.e.m.) chemiluminescence ranged from 44 (8) mV 100 mg-1 at time zero to 404(113) mV 100 mg-1 at 1 h in controls. In caerulein-induced pancreatitis mean(s.e.m.) chemiluminescence peaked at 20 min (1399(239) mV 100 mg-1, P less than 0.02) and in taurocholate-induced pancreatitis at 15 min (2316(95) mV 100 mg-1, P less than 0.004). Superoxide dismutase significantly reduced chemiluminescence and hyperamylasaemia in taurocholate groups. Increasing oxygen free radical activity paralleled evolving pancreatitis. Superoxide dismutase may have a therapeutic role in pancreatitis.

The skeletal framework of human kidney and renal cell carcinoma. A scanning electron microscopic study.

The three dimensional architecture of the connective tissue framework of normal human kidney and three renal cell carcinomas was studied. A sodium hydroxide maceration technique was used to remove the cellular elements thus exposing the underlying connective tissue structures. The collagen fibrillar network was visualized using the scanning electron microscope. In normal kidney the fibres were fine, and smooth, and corresponded to the shapes of the original parenchymal constituents. The fibres of the kidney tumours were coarse in nature and irregularly distributed. The technique provides a rapid method for studying connective tissue fibres in normal and diseased tissue. The three dimensional architecture thus exposed enhances our knowledge of tumour stroma.

Suitability of control materials in the differential inhibition assay for human pancreatic and salivary amylase.

We investigated the behavior of 26 quality-control sera with the inhibitor method for differential amylase (EC 3.2.1.1) assay. We also studied the sensitivity to the wheat-derived inhibitor of pancreatic amylases from 10 different animals in comparison with human pancreatic and salivary amylase. The results indicate that only control materials containing human amylases can be measured accurately. The animal amylases (bovine, equine, porcine) used in many quality control sera are relatively insensitive to the inhibitor as compared with human pancreatic and salivary amylase.

Problems associated with the radioimmunoassay of serum trypsin.

A commercial trypsin radioimmunoassay (RIA) kit was used for its ability to measure trypsin bound to the serum protease inhibitors, alpha 2 macroglobulin (alpha 2 M) and alpha 1 anti-trypsin (alpha 1 AT). Only 20% of trypsin bound to alpha 2 M and 70% bound to alpha 1 AT was detected by the assay system. Recovery of trypsin added to human serum varied from 0 to 20%. Standard curves prepared from purified human cationic trypsin did not exhibit parallelism with the kit standard curves. Inclusion of horse serum in the standard solutions improved the parallelism observed. Immunoreactive trypsin (IRT) levels obtained for serum samples were found to vary considerably depending on the standard curve used to calculate the assay results. Lower IRT levels were observed when trypsin standards prepared in the absence of horse serum were used as reference.

Isolation and characterization of four inhibitors from wheat flour which display differential inhibition specificities for human salivary and human pancreatic alpha-amylases.

Four alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors were isolated from an albumin fraction of wheat flour by ion-exchange and gel-filtration chromatography. The purified inhibitors were characterized according to their electrophoretic mobilities, molecular weights, carbohydrate, content, sulphydryl content, susceptibility to proteolytic digestion and specificities in inhibiting human salivary and pancreatic alpha-amylases. The properties of these inhibitors ae compared to similar proteins isolated by other workers.

Interaction of human alpha-amylases with inhibitors from wheat flour.

The interaction of four purified alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) inhibitors with human salivary and pancreatic alpha-amylases was investigated. The inhibitory activity of the four proteins towards salivary alpha-amylase was significantly increased by pre-incubation of the enzyme with inhibitor before adding substrate. This effect was not observed with the inhibition of pancreatic alpha-amylase by inhibitors 1 and 2. Inhibition of both amylases was affected to different degrees by incubating starch with inhibitor prior to the addition of enzyme. Maltose, at concentrations which only slightly affected amylase activity, prevented the inhibition of both enzymes by all four inhibitors. Gel filtration studies on salivary amylase-inhibitor mixtures showed the formation of EI complexes on a mol-to-mol ratio. A similar complex between pancreatic alpha-amylase and inhibitor 4 was observed, though complex formation between pancreatic alpha-amylase and the other inhibitors was not clearly demonstrated.

Renal clearance of pancreatic and salivary amylase relative to creatinine in patients with chronic renal insufficiency.

Pancreatic and salivary amylase/creatinine clearance ratios in patients with various degrees of renal impairment were compared with those obtained for control subjects. In chronic renal insufficiency (mean GFR 30 ml/min +/- 15 SD; n = 13) the clearance ratios for pancreatic (mean 3.5 +/- 1.85 SD) and salivary (mean 2.3 +/- 1.3 SD) amylase were significantly higher (P less than 0.05) than those in controls. Corresponding control values (n = 26) were 2.64 +/- 0.86 (pancreatic) and 1.64 +/- 0.95 (salivary). Three patients showed values above the normal limit. In the diabetic group (mean GFR 41 ml/min +/- 22 SD; n = 10) salivary amylase/creatinine clearance ratios (mean 2.36 +/- 1.55 SD) were significantly higher than in controls (P less than 0.05). Three patients showed raised values. Pancreatic amylase clearance was raised in only one of these patients. Three patients with terminal disease (mean GFR 10 ml/min) showed markedly raised (two- to threefold) clearance ratios for both salivary and pancreatic amylase. Of a total of 26 patients, eight had increased total amylase/creatinine clearance ratios. Pancreatic amylase/creatinine clearance was increased in seven patients, while nine patients showed raised salivary amylase/creatinine ratios. Patients with raised clearance ratios did not have clinical evidence of pancreatitis. We suggest that, in the presence of impaired renal function, a high amylase/creatinine clearance ratio need not be indicative of pancreatic disease.

Activity of serum alpha-amylases in cystic fibrosis.

A new method which uses a differential inhibitor to measure pancreatic and salivary type alpha-amylases (EC 3.2.1.1) was applied to serum samples from 46 cystic fibrosis (CF) patients (age range 4-14 years) and 50 controls of the same age group. The levels of pancreatic type amylase were lower in the CF patients (median 26.5 I.U./1) than the controls (median 81.5) (P less than 0.001). The results for salivary-type enzyme, however, did not support the previously reported finding of higher than usual levels in CF patients. This discrepancy is probably due to differences in analytical methods. It is felt that this procedure will be of value in the investigation of patients for cystic fibrosis and other pancreatic disorders.

Pancreatic and salivary amylase/creatinine clearance ratios in normal subjects and in patients with chronic pancreatitis.

The clearance of pancreatic and salivary amylase relative to creatinine was measured in 26 control subjects and 22 patients with chronic pancreatitis. Control values for pancreatic amylase clearance (+/- SD) were 2.64 +/- 0.86% compared with 1.54 +/- 0.95% for salivary amylase. In chronic pancreatitis, pancreatic amylase clearance ratios were significantly higher than controls (P less than 0.0005, mean 4.09 +/- 1.63 SD). The difference in clearance rate of salivary amylase did not reach a level of significance when compared with the control group. Twelve of the 22 patients showed pancreatic amylase clearance values above the normal limit of 4.4, while only five were abnormal when the clearance of total amylase was measured. The patients also showed statistically higher (P less than 0.0005) levels of serum salivary amylase when compared with 69 control sera. No such difference was found for the pancreatic component of serum amylase. Comparison of beta2-microglobulin clearance values showed no statistical difference between patients and controls.

Sphincteric mechanism of the main pancreatic duct in the dog. An experimental model of the isolated sphincteric preparation.

This study describes an in vitro experimental model of the sphincter at the lower end of the main pancreatic duct in the dog. This model, employing a drop counter to measure the drop rate, monitors the perfusion rate of Tyrode through the sphincter. Acetylcholine (ACh), employed to establish the sensitivity and viability of the isolated sphincter, produced a contraction of the sphincter which was concentration-response related. Saline, as a control, atropine and hexamethonium did not affect the drop rate. Atropine (20 microgram) partially abolished the contraction produced by 50 microgram of ACh; hexamethonium (50 microgram) did not have an effect. Caerulein 50 microgram produced a relaxation of the sphincter; adrenaline 50 microgram a contraction. Both sympathetic and parasympathetic mechanisms seem to play a role in the regulation of sphincteric activity of the main pancreatic duct in dogs.