Stanley P. Kramer

THE HISTOCHEMICAL DEMONSTRATION OF PANCREATIC-LIKE LIPASE AND COMPARISON WITH THE DISTRIBUTION OF ESTERASE.

A new histochemical method for lipase was developed using naphthol-AS nonanoate as a substrate and sodium taurocholate as an activator. In a comparison with esterase, pancreatic-like lipase activity was studied in various tissues. As a result, it was disclosed that the pancreatic acinar cells show the highest activation with sodium taurocholate, and lipase activity is located in the region immediately surrounding the zymogen granules and not within the granules themselves. Maximum activity of esterase and lipase differed among various tissues of the rat. Esterase activity is most intense in liver and kidney followed by pancreas. Pancreatic-like lipase activity is most striking in pancreas, less so in the liver and in the kidney the activity is rather weak. Because of the exceptional esterase activity of macrophages, mast cells and pericytes of the cerebral capillaries, pancreatic-like lipase activity should be ascribed to them with caution and only in terms of the difference between reaction without and with sodium taurocholate.

Human pancreatic lipase study with bile salt activation and substrates from a homologous series of naphthyl alkanoates

Abstract Because sodium cholate proved to be a more reliable activator than sodium taurocholate for human serum lipase assay, a series of α-naphthyl alkanoates (of 8-, 9-, 10-, and 14-carbon acids), β-naphthyl alkanoates (of 8–16-carbon acids) and β-naphthyl alkenoates (of 2-nonenoic and 2-decenoic acids) were prepared and were studied for their sensitivity toward pancreatic lipase with cholate and taurocholate activation and in the presence of human serum. Maximal sensitivity to pancreatic lipase was found both in the α- and β-naphthyl nonanoates, suggesting that esters from acids of this chain length best meet the structural requirements for substrates for pancreatic lipase. Cholate proved a more reliable activator then taurocholate of the β-esters. β-Naphthyl nonanoate (βC9) was singled out for special study. Saturation of the enzyme (2 μg. pancreas/ml.) occurred at a substrate concentration of 0.2–0.25 mg./ml. leading to zero-order kinetics and hydrolysis rates directly proportional to enzyme concentration. Maximal incubation temperature for measurement of human pancreatic lipase in human serum was 37.5 °C. while that for pancreas alone was 45 °. Serum was observed to be a noncompetitive inhibitor of βC9. Some sera had more antilipase than others. Studies are now in progress on the use of βC9 for lipase assay. These studies may lead to better substrates and methods for histochemistry and for assay of human serum lipase activity.

DEVELOPMENT OF A CLINICALLY USEFUL COLORIMETRIC METHOD FOR SERUM LIPASE.

Summary Sodium cholate has proved to be a more reliable activator of human pancreatic lipase in human serum than the unsatisfactory sodium taurocholate, which has been useful in the measurement of dog serum lipase. Since both activators work well on human pancreas alone, human serum plays a role in determining the ability to detect human pancreatic lipase with either activator. A method for the determination of human serum lipase which takes advantage of this species difference has been developed. This new method utilizes well buffered sodium cholate as a reliable activator and β-naphthyl myristate as a substrate. The advantage of the myristate over the laurate ester of β-naphtyl is the slower rate of hydrolysis of the former by serum esterase. As little as 0.2 μg. of a pooled extract of human pancreas added to 0.2 ml. of human serum can be detected. Lipase activity can be demonstrated in normal human serum, with an upper limit of 265 Klett units or 75 lipase units, and with a mean of 155 Klett units or 43 lipase units. A survey of serum lipase in patients suspected of having pancreatitis was performed using a one hour and a five hour incubation method. Elevation of both lipase and amylase activity in serum correlated with the presence of early acute pancreatitis. Although correlation with amylase activity was closer, false positive amylase activity was detected in some patients by means of the lipase assay. Marked elevations in amylase were almost always accompanied by elevations in lipase. Although the five hour test is more reliable, the one hour test may serve as a useful adjunct in emergencies, since a positive one hour test consistently predicted a positive five hour test.

Assay of trypsin activity of human serum with a chromogenic substrate, CBZ-diglycyl-l-arginyl-2-naphthylamide hydrochloride (GGANA)

Abstract Recent development of a very sensitive chromogenic substrate (GGANA) for trypsin and reports of proteins (P) in serum which form inhibitor-resistant active complexes (PT) with trypsin (T) have prompted an investigation of trypsin-like activity of serum. GGANA was sensitive to T and to normal human serum but was not affected noticeably by proteolytic enzymes of serum once considered trypsin-like (that is, thrombin, fibrinolysin, and kallikrein). Though serum inhibited T to some degree, GGANA could be used to measure small quantities of T added to serum (as little as 0.05 μg./ml.). The rate of inhibition of the tryptic activity, however, was inversely proportional to the concentration of the T in the serum. Presumably this phenomenon occurs because the fraction of T that can be bound by the small quantity of high molecular weight protective protein (P) to form the active complex PT in the presence of inhibitors (I) is closer to 100% in a large excess of serum which can provide an effective concentration of P. In the absence of an effective concentration of P some of the T can be bound by I to form inactive IT, PT is irreversibly inactivated by acid while IT apparently gains some activity on acid treatment followed by neutralization. Since normal serum irreversibly loses its activity on acid treatment, it probably contains PT (endogenous). How PT, P, I, and IT activity of serum changes with acute pancreatitis would be difficult to predict. The serum trypsin-like activity of 171 normal individuals averaged 391 Klett units per 30 minutes with an upper limit of 785 and a lower limit of −4. The activity found for four cases of acute pancreatitis averaged −19. The completed study of the variation of PT in serum in pancreatitis using the chromogenic peptide, GGANA, as a substrate will be reported later.

Preliminary study of an esterase-susceptible water-soluble agent (S-73) for regional chemotherapy

This is a preliminary report on the experimental and clinical use of a new alkylating agent (S‐73) which is water soluble, stable to hydrolysis for hours, but with esterase‐susceptible linkages, which results in inactivation of the agent by esterase in the blood. S‐73 is a potent cytotoxic agent when injected near its target but much larger doses are required to produce cytotoxic effects when injected farther away from it, because of rapid inactivation and dilution. In no instance is bone marrow depressed.

Enzyme alterable alkylating agents X: Experimental study of an esterase-susceptible water-soluble agent (S-73) for Regional Chemotherapy☆

Abstract Although circumvention of the problem of systemic toxicity during regional infusions was shown to be possible with S-182, an alkylating agent with a very short half-life, practical use of this drug was hampered by its lack of water solubility and damage to normal tissue near the injection site. Studies were undertaken to find an agent without such shortcomings and ethylene N-2-chloroethyl-N-methylglycine, diester (S-73), a stable water-soluble, bifunctional alkylating agent with esterase-susceptible bonds was studied further. This compound should be rapidly detoxified by esterases in blood and epithelial tissue. In dogs, low doses of S-73 (0.27 μm./kg.) injected into a branch of the mesenteric artery, about 10 cm. from the intestine, damaged the intestinal epithelium but no damage occurred when injected 20 cm. away. HN2 (0.06 μm./kg.) unlike S-73, did not lose its cytotoxic effect when injected twice as far from its target. High doses of S-192 (a monofunctional mustard which could result from the cleavage of S-73 by esterase) were innocuous when injected 10 cm. from the intestine. Bone marrow depression occurred only with high doses of S-73 (17.6 μm./kg.) injected into the left ventricle but not through the femoral vein. The significance of this observation is enhanced by the observation that 2.6 μm. per kilogram of HN2 by either route depresses bone marrow. In vitro studies, however, show that S-73 is not rapidly detoxified in blood, and suggest that much of the compound may be in the uncyclized form which is incapable of alkylating at the time it reaches the target. Nevertheless, in vivo studies reveal that considerable alkylation has occurred at the target. Furthermore, rapid detoxification of S-73 may be inferred by the higher doses required to effect regional damage, the relationship of its cytotoxic effect to distance from the target, and the lack of local effect of its presumed metabolite. Since very high doses of S-192 caused damage in the lungs of dogs, it is not surprising that equivalently high doses of S-73 are also damaging to lung. Preliminary clinical trials [2] in advanced cancer patients have shown that high doses of S-73 have not produced damage to either bone marrow or lung. Mucosal damage on intramesenteric arterial injection and spinal cord damage via the lower aorta in dogs as well as preliminary clinical observations have shown that S-73 is a potent cytotoxic agent when injected near a limited target. On the other hand high doses in a larger tumor-bearing area produced minimal damage to local tissue with no significant systemic effects. Whether clinical cancer can be specifically and significantly damaged with this new agent is in the process of investigation.

Species-dependent pleomorphic behavior of pancreatic tryptic activity☆

Abstract Homogenates of canine and human pancreas enriched with trypsin of endogenous origin only were obtained when such homogenates were incubated at 0°C. with aliquots of bovine trypsin (Tryptar) rendered insoluble by mechanical entrapment inside the lattices of polyacrylamide gel. Insoluble bovine trypsin was able to activate pancreatic trypsinogen and overcome pancreatic trypsin inhibitor prior to its removal by centrifugation. In addition, it was observed that some human pancreases readily developed tryptic activity in the usually long interval between death and autopsy, and that others generated substantial activity after homogenization and incubation at 0°C. In contrast, homogenates of canine pancreas required the addition of exogenous trypsin for conversion of trypsinogen to trypsin. When the generated tryptic activity of pancreatic homogenates was compared with that of purified bovine trypsin, human trypsin differed from bovine and canine trypsin in its substantially lower inhibition by ovomucoid and soybean trypsin inhibitor. The ease of conversion of human trypsinogen to trypsin, the stability of the latter, its lower sensitivity to trypsin inhibitor, and variation in content of the inhibitor are undoubtedly factors in the etiology of pancreatitis, and they may explain the significant rate of occurrence of this disease in man.