Jan Slabý

Synthetic substrates in the histochemical demonstration of intestinal disaccharidases.

SummaryThe splitting of 6-Br-2-naphthyl-, α-naphthyl-, and 4-Cl-5-Br-3-indolyl-glycosides which proved useful for the assessment of cytological localization of intestinal enzymes in previous studies was investigated using isolated human and rat intestinal disaccharidases as a source of enzyme activities.Previous findings based on histochemical studies were confirmed and extended. 6-Br-2naphthylα-D-glucoside is cleaved by glucoamylase and sucrase-isomaltase. The participatio of trehalase in splitting of this substrate is very low and can be neglected. The mentioned α-glucosidases are responsible for the brush border staining of enterocytes with this substrate when unfixed cold microtome sections are used. Even when a differential heat inactivation of sucrase-isomaltase and of glucoamylase occurs during paraffin embedding (so that the staining in paraffin sections is due mostly to glucoamylase) the use of natural substrates is desirable for a more precise assessment of sucrase-isomaltase activity (but without the possibility of a correct localization).4-Cl-5-Br-3-indolyl-β-D-fucoside is the substrate of choice for the demonstration of lactase. Even when this substrate is split also by “hetero-β-galactosidase” and by acid (lysosomal) β-galactosidase these activities do not disturb the histochemical demonstration of lactase. If however some doubts arise, the inhibition with p-Cl-mercuribenzoate (2 · 10−4 M) is to be emloyed (lactase activity is not inhibited). Due to a low Km and a high Vmax of indolyl-fucoside and due to its extreme stability in solution (which enables to use the substrate solution repeatidly) this substrate is suitable in routine practice even though it is expensive. α-naphthyl- and 4-Cl-5-Br-3-indolyl-β-D-glucosides are split by lactase and β-glucosidase. Due to the fact that the mutual delineation of these activities is not easy and that Km an Vmax for lactase are not so favourable as in the case of fucoside these substrates are not recommended for the assessment of lactase.6-Br-2-naphthyl-β-D-glucoside is the substrate of choice for the histochemical studies concerned with “hetero-β-galactosidase” and 4-Cl-5-Br-3-indolyl-β-D-galactoside for acid β-galactosidase.

Effective peritoneal therapy of acute pancreatitis in the rat with glutaryl-trialanin-ethylamide: a novel inhibitor of pancreatic elastase.

The six hour peritoneal lavage with glutaryl-trialanin-ethylamide, a low molecular competitive inhibitor of pancreatic elastase (IC50-8 mumol/l), effectively suppresses the evolution of taurocholate induced acute pancreatitis in the rat. The lavage alone is followed by a marked decrease of fat necrosis and amylase and lipase activity in serum. The area of pancreatic haemorrhage was significantly reduced only after the lavage solution was supplemented with Glt-Ala3-NHEt. The effect was not enhanced by a bolus injection of the inhibitor before starting the lavage. The combination of Glt-Ala3-NHEt with aprotinin or nafamstate mesilate produced only marginal greater benefit. The effect of Glt-Ala3-NHEt on pancreatic haemorrhage is time and dose related even with delayed onset of the lavage. Animals treated with peritoneal lavage without Get-Ala3-NHEt lived longer than controls (p less than 0.05), but by 60 hours the survival rate of both groups was almost the same (76 v 74%). All animals lavaged with Glt-Ala3-NHEt survived 120 hours and the difference in the survival rate between this and both remaining groups was significant (100% v 76% v 74% - p less than 0.05). The results were considered favourable and preliminary clinical trials of Glt-Ala3-NHEt in subjects with acute pancreatitis justified.

Histochemical demonstration of the intestinal hetero-?-galactosidase (glucosidase)

SummaryThe histochemical demonstration of hetero-β-galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with β-D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of α-naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.6-Br-2-naphthyl-β-D-glucoside is the most specific substrate for hetero-β-galactosidase. It is not split by lactase and acid β-galactosidase. Only lysosomal β-glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero-β-galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.The simultaneous azocoupling method with α-naphthyl-β-D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero-β-galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero-β-galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal β-glucosidase does not seem to interfere.Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero-β-galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero-β-galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal β-galactosidase and possibly of lysosomal β-glucosidase (depending on the glycoside used) is always to be considered.Hetero-β-galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunners glands the activity was lower. The occurrence of a low activity of hetero-β-galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero-β-galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.

Elastolytic activity of human duodenal contents

Elastolytic activity of human duodenal contents was determined using the new chromogenic substrate succinyl-trialanine-p-nitroanilide (Suc-Ala3-NAp). The mean output values after pancreatic stimulation with pancreozymin and secretin were significantly higher in controls than in subjects with impairment of other secretory values (volume, bicarbonate, amylase, lipase). Agar gel electrophoresis and chromatography on DEAE-Sephadex revealed one to two fractions which differed in mobility (cathodic and anodic fraction), elution with different NaCl concentrations (0.15 M, cathodic fraction; 0.3 M, anodic fraction), and in behaviour towards synthetic and natural substrate (Suc-Ala3-NAp) and elastin-Congo Red). The cathodic fraction cleaved both substrates, whereas the anodic fraction cleaved only Suc-Ala3-NAp. After trypsin and enterokinase treatment the anodic fraction behaved as the cathodic fraction on DEAE-Sephadex chromatography. The molecular weights (Sephadex G-100) and the Michaelis constants (Suc-Ala3-NAp) of both fractions were identical (24 500; 0.45 X 10(-3) M). These fractions represent probably diffenent activation forms of pancreatic elastase.

Isolation and analysis of peptidic fragments of α-gliadin using reversed-phase high-performance liquid chromatography

Summary Peptidic fragments of α-gliadin were obtained by peptic-tryptic-pancreatic (PTP) digestion of the α-gliadin fraction isolated by ion-exchange chromatography on a sulphopropyl-Sephadex C-50 column. The proteolytic digest was fractionated by ultrafiltration into three subfractions, PTPa 1 –PTPa 3 . The subfraction PTPa 2 was then analysed and individual peaks were separated using reversed-phase high-performance liquid chromatography (RP-HPLC) using a gradient of acetonitrile in 0.1% trifluoroacetic acid and a Separon SGX-C 18 sorbent. A 100-mg amount of the PTPa 2 subfraction was separated in a single analysis by preparative RP-HPLC and twenty peaks were obtained for further characterization. The molecular mass in range 300–3000 was established for individual peptidic fragments by gel-permeation chromatography on a TSK-G2000 SW column.

New low-molecular inhibitors of pancreatic elastase with possible in vivo application: Alkylamides of N-acylated tripeptides

Out of a series of alkylamides of N-acylated tripeptides, Glt-(Ala)2-Pro-NH-Et and Glt-(Ala)3-NH-Pr were found to be potent inhibitors of porcine and human pancreatic elastase, and because they are free of toxic groups they might be considered for in vivo application.

Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes

The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general fomula Sc-Ala2-X-pNA, Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.