H. Rinderknecht

A new ultrasensitive method for the determination of proteolytic activity

Abstract An ultrasensitive method for the colorimetric determination of proteolytic activity in biological fluids is described. It is based on an insoluble substrate derived from hide powder labeled covalently with Remazolbrilliant Blue. All of six proteases tested: trypsin, chymotrypsin, elastase, collagenase, fibrinolysin and pepsin can be determined at concentrations of 50–100 ng/ml incubation mixture. Trypsin, fibrinolysin and elastase can still be estimated at concentrations as low as 1–2 ng/ml assay mixture. In contrast to other insoluble, non-covalent protein-dye complexes in use for the determination of proteolytic activity, the new chromogenic substrates are not affected by the presence of albumin.

Studies of Pure Pancreatic Secretions in Chronic Alcoholic Subjects without Pancreatic Insufficiency

To determine the role alcohol might play in altering pancreatic function, we have examined pure pancreatic juice, obtained by endoscopic cannulation of the pancreatic duct, from a group of 10 chronic alcoholic subjects without history or clinical or laboratory evidence of pancreatic disease and compared the results with those obtained from 15 healthy, non-alcoholic subjects. These findings confirm observations in experimental animals made by others and support the hypothesis that chronic alcohol abuse may damage the pancreas via a sequence of events involving protein hypersecretion. Increase in concentration was not uniform for all proteins measured. Unexpectedly, chronic alcoholics exhibited a highly significant elevation (two- to three-fold over normal) in trypsinogen, in contrast to statistically insignificant increases of other zymogens and trypsin inhibitor. The strikingly increased ratio of trypsinogen to trypsin inhibitor observed in all our alcoholic patients may indicate a weakening of the defence mechanism provided by the trypsin inhibitor against premature intraductal activation of zymogens and explain the predisposition of these patients to pancreatitis.

New methods for the determination of elastase.

Abstract Highly sensitive fluorimetric and colorimetric methods for estimation of pancreatic elastase have been developed. Two covalently labeled substrates, Fluorescein-elastin and Remazolbrilliant Blue-elastin, were prepared for this purpose. Pancreatic elastase has been assayed selectively in the presence of trypsin and chymotrypsin. The potentiating effect of these enzymes on the activity of elastase is described. The influence of this synergism on elastase determinations and its significance in acute pancreatic disease is discussed.

Determination of trypsin and chymotrypsin with remazolbrilliant blue-hide

Abstract The determination of trypsin and chymotrypsin with hide-substrates covalently labeled with Remazolbrilliant Blue R is described. Specific assay conditions and kinetic data for these two enzymes are presented and improvements in the preparation of Remazolbrilliant Blue-hide (RBB-hide) are reported. RBB-hide, in contrast to the synthetic substrate N- benzoyl- dl -arginine p- nitroanilide (BAPNA), is almost completely resistant to the action of α2-macroglobulin-bound trypsin in the presence of serum. Conversely, BAPNA appears to be resistant to the trypsin -α1- antitrypsin complex, but RBB-hide is hydrolyzed to a small extent. These findings are of significance in the study of tryptic activity of plasma, ascites, pleural effusions, etc. in various pathological conditions.

Pancreatic secretion after secretin and cholecystokinin stimulation in chronic alcoholics with and without cirrhosis

We have studied the volume, protein concentration, total protein, and chymotrypsin and trypsin outputs in pure pancreatic juice (PPJ) following endoscopic cannulation of the pancreatic duct in 11 male and 2 female patients with advanced alcoholic cirrhosis (AC). Results were compared to those obtained from 21 nonalcoholic volunteers (NAV) and 26 chronic alcoholic (CA) patients without cirrhosis. Intravenous stimulation with secretin followed 10 min later by intravenous cholecystokinin-pancreozymin (CCK-PZ) resulted in highly significant increases in volumes during both phases of pancreatic stimulation in AC compared to NAV and CA. Protein concentration and total output during secretin stimulation was not different among the three groups. During CCK-PZ stimulation, CA exhibited a significant elevation in protein concentration and total output compared to NAV and AC. Although total chymotrypsin output was lower in secretin-stimulated CA than other groups, no other differences between the groups were observed in either of the hormone-stimulation phases. Marked elevations in trypsin output were observed in secretin-stimulated AC and in CCK-PZ-stimulated AC and CA. The high PPJ volume and the relatively low protein concentration observed in AC may effect a washout phenomenon resulting in a decreased tendency for ductal protein precipitation in these patients.

A new automated method for the determination of serum α-amylase

Abstract A simple automated method for the determination of serum α-amylase is described. It is based on a fluorescent starch substrate, possesses high sensitivity, linearity up to at least 1000 Somogyi units and permits processing of 30 samples per hour.

Determination of elastolytic activity in blood of normal subjects and patients with acute pancreatitis

Abstract A new, sensitive fluorometric method for the determination of elastolytic activity in plasma and other biological fluids is described. It is based on an elastin substrate labelled with the fluorescent dimethylaminonaphthalene-sulfonyl radical. Assay of elastolytic activity in plasma of 30 normal subjects gave values corresponding to approximately 2 μg of elastase/ml. Analogous determinations in a series of 30 patients with acute pancreatitis demonstrated greatly reduced levels (mean: 0.6 μg/ml) or absence of plasma elastolytic activity. The possible significance of these findings is discussed.

Clinical evaluation of an a-amylase assay with insoluble starch labeled with Remazolbrilliant Blue (amylopectin-azure)

Summary 1. A simple, rapid and highly reproducible amylase assay for diagnostic use is presented. 2. The procedure is based on an insoluble starch labeled with Remazolbrilliant Blue which has been reported previously. 3. Detailed methodology and kinetic studies with the new substrate are described. 4. A clinical evaluation of the new method was made by comparing it with two other widely used procedures in concurrent determinations of serum and urinary amylase of normal subjects and patients with acute pancreatitis. 5. Statistical analysis of the results demonstrates the diagnostic validity of the new assay.

Ribonuclease C and pancreatic secretory proteins in the peripheral circulation before and after pancreatectomy for pancreatic cancer

Increases in the activities of human circulatory ribonucleases have been associated with malignancies and other pathological conditions. To elucidate the relationship between circulatory ribonuclease and pancreatic malignancy, we have determined the activities in serum of ribonucleases selective for polycytidylic acid (ribonuclease C) and of α-amylase before and after surgery for pancreatic cancer and for several malignant and nonmalignant disorders unrelated to the pancreas. Prior to pancreatectomy, the activity of ribonuclease C in the serum of three pancreatic cancer patients was within the range of values obtained for healthy subjects. Unexpectedly, ribonuclease C began to increase within 2 hr after surgery, and amounts of ribonuclease C more than threefold higher than preoperative levels were found for at least 3 weeks after pancreatectomy. Variations in the serum concentrations of urea nitrogen and creatinine followed time courses which differed from those of ribonuclease C; thus the increases in the activity of ribonuclease C could not be attributed to renal impairment. The concentrations of α-amylase and trypsinogen in the blood of two of the pancreatic cancer patients were considerably higher than those of healthy subjects and declined rapidly to subnormal levels after pancreatectomy. Only a small amount of α-amylase and no trypsinogen were found in the blood of the third patient both before and after surgery, and samples of pure pancreatic juice obtained preoperatively after sequential intravenous administration of secretin and cholecystokinin-pancreozymin were negative for α-amylase and three proteolytic zymogens; nevertheless, the presurgical level of ribonuclease C in his serum was within the normal range. For all of the patients treated for nonpancreatic disorders, the concentrations of ribonuclease C and α-amylase in serum were not significantly altered by surgery. The rate and ultimate extent of the decrease in circulatory α-amylase following pancreatectomy indicate that ≈-80% of the α-amylase in blood is pancreatic in origin and that the half-life of pancreatic α-amylase in the peripheral circulation ≈-15 hr. From the highly significant increases in circulatory ribonuclease C which followed pancreatectomy for pancreatic cancer, we conclude that the exocrine pancreas is neither the sole nor the principal source of ribonuclease C in the peripheral circulation.

An elastase inhibitor from canine mandibular gland

Ein Inhibitor der Enzyms-Elastase wird beschrieben und dessen biologische Bedeutung sowie die eventuelle therapeutische Verwendung diskutiert.