Selma H. Rutenburg

HISTOCHEMICAL DEMONSTRATION OF β-d-GLUCURONIDASE WITH A SYNTHETIC SUBSTRATE

The synthesis of a substrate, 6-bromo-2-naphthyl β-d-glucopyruronoside, is described, and the procedure for the histochemical demonstrations of β-d-glucuronidase activity and experiments to determine the reliability of the results of localization of enzymatic activity are given. The results in normal tissues of the rat are presented. Greatest concentration of enzymatic activity was seen in liver, spleen, epithelium of gastrointestinal tract, uterus, thyroid, and in the white matter of the nervous system.

HISTOCHEMICAL DEMONSTRATION OF β-D-GALACTOSIDASE IN THE RAT

A histochemical technic for demonstration of β-d-galactosidase in formalin fixed sections has been presented. Enzymatic activity was best demonstrated in frozen sections of the kidney, liver, gastrointestinal tract, testis, epididymis and vas deferens of the rat and to a lesser extent in salivary gland, pancreas, thyroid, trachea, lung, spleen and lymphoid tissue. Leucocytes stained intensely. Enzymatic activity was largely present in the cytoplasm of epithelial cells. Cell nuclei, myocardium, connective tissue and erythrocytes were inactive.

THE HISTOCHEMICAL DEMONSTRATION OF α-d-GLUCOSIDASE IN MAMMALIAN TISSUES

A histochemical method for the demonstration of α-d-glucosidase is presented. The procedure and the problems encountered in its development are discussed. Surveys of mammalian tissues showed that this enzyme is generally demonstrable by this method only in the mucosa of the duodenum and upper jejunum and in the kidney cortex. Starvation resulted in decreased enzymatic activity in the intestinal mucosa, whereas increased activity was observed after the ingestion of food.

Hepatic Clearance of Endotoxin absorbed from the Intestine.

Summary Data are presented showing that endotoxins absorbed from the intestine are present in the blood, and in much higher incidence in portal than in systemic vein blood. The incidence of endotoxemia in portal vein blood varies with the size of the intraintestinal pool of endotoxin. A reduced capacity of the R.E. system in the liver for extracting endotoxin allows endotoxin in the portal vein blood to traverse the liver and enter the general circulation.

Detoxification of Endotoxin by Splenic Extracts.

Summary A fraction which rapidly detoxifies bacterial endotoxin has been prepared from normal dog spleen. The detoxifying system is thermolabile, precipitable with ammonium sulfate, completely inactivated by trypsin and by tetraethyl pyrophosphate. These findings suggest that the active component is a protein with the properties of an esterase type of enzyme.

A method for the colorimetric determination of α-d-glucosidase with a chromogenic substrate☆

Abstract A method has been developed for the assay of α- d -glucosidase activity in mammalian tissue and serum with 6-bromo-2-naphthyl α- d -glucopyranoside as substrate. The effects of the type and concentration of buffer, pH, incubation time, and concentration of tissue and substrate on the rate of enzymic hydrolysis are presented. The tissues of the rat were more active than those of the dog. The highest enzymic activity in both animals was found in the kidney and small intestine.

Fractionation of an Esterase from Calf Spleen Implicated in the Detoxification of Bacterial Endotoxin

Discussion and Summary The use of the two successive fractionation procedures resulted in the preparation of an esterase-rich fraction which was capable of detoxifying endotoxin. The partial inhibition of esterase activity—but not detoxifying power—by DFP or TEPP indicated that the final Sephadex Fraction II contained a cholinesterase-type enzyme not involved in the detoxification reaction. The concentration of DFP employed was sufficient to have inhibited the esterolytic activity of proteases such as trypsin or chymotrypsin, if present. Consequently, the bulk of the enzyme in Fraction II which was active in the detoxification of endotoxin is considered for the present to be an esterase of the nonspecific, carboxylic type. Most of the esterase activity present in DEAE-cellulose Fractions I and III (Fig. 1) was inhibited by DFP and these fractions possessed only a weak capacity to inactivate endotoxin. Whereas the spleen enzyme is considered to be a nonspecific esterase of the type found in normal serum, it differs from the latter in some important respects. The degradation and inactivation of endotoxin in serum or plasma was reported to occur as a two step reaction requiring two different esterases, one of which was a lipoprotein esterase (4). Although not yet established, it appears that in the spleen fraction only one esterase functions in the degradation and inactivation of endotoxin. The spleen esterase is not associated with a lipoprotein and no lipoprotein was detected in the isolated fraction. The spleen esterase also differs from the plasma esterases in chromatographic behavior and in heat stability.

Host Resistance to Bacteria in Hemorrhagic Shock V. Mobilization of Phagocytes.

Summary and Conclusion When the peritoneal cavity of the rabbit recovering from mild shock in response to transfusion is challenged by an irritant fluid, the cellular response as compared to that in a normal rabbit is extremely poor. The failure to mobilize polymorphonuclear leucocytes is, for the most part, not due to defective hemodynamics, but to a disturbance of vascular permeability to migrating leucocytes. The failure to supply additional macrophages at the site of the irritation appears to be due to a defect in the cellular processes involved in the conversion of their precursors. The presence of a substantial intracellular and extracellular bacterial population in the peritoneal fluid of the shocked animal, and the absence of bacteria in the peritoneal fluid of the unshocked animal, is further evidence of the loss of resistance to bacteria in the shocked animal, and of its persistence after restoration of a normal hemodynamic status.

Resistance to Bacteria in Hemorrhagic Shock. VII. Demonstration of Leucotoxin in Plasma of Shocked Rabbit.

Summary and Conclusions 1) Normal macrophages from the peritoneal cavity of the normal and shocked rabbit, and granulocytes from the irritated peritoneal cavity of the normal rabbit show no significant morphologic injury after immersion in normal rabbit plasma for one hour at 37°C. But after immersion in plasma from rabbits in hemorrhagic shock, a considerable percent of the cells show morphologic injury. A large number of granulocytes show changes similar to those characteristic of the L.E. cells. These changes are reversible if the shock plasma is replaced by normal plasma. 2) Shock plasma produces a severe depression of the phagocytic index; the percent of bacteria ingested is some 40% less than in normal plasma, and the percent of cells containing bacteria is some 30% less than in normal plasma. Shock plasma also reduces the bacteriostatic power of the phagocytes. The granulocyte in this respect appears to be damaged more severely than the macrophage. The extent of the injury produced by the plasma from rabbits in advanced shock appears to be no greater than that produced by the plasma of rabbits after two hours of shock, i.e. while they are still responsive to transfusion. The injurious property of plasma of the reversibly shocked rabbit persists, with undiminished potency, for at least four hours after transfusion. 3) It is concluded that a leucotoxin develops in the blood of the rabbit in hemorrhagic shock, and that this leucotoxin severely impairs the antibacterial potential of the animal.

On the Nature of Tolerance of Endotoxin.

Tolerance of endotoxin has been found to be related to the pinocytic and phagocytic capacity of the reticulo-endothelial system (1–4). Recently the plasma of the tolerant animal has been found to contain a transferable factor that suppresses pyrogenicity and increases the rate of phagocytosis in the recipient (5–7). Such data, though doubtless of considerable significance, do not sufficiently explain what it is that constitutes increased tolerance. We have reported the presence in normal spleen of a protein with an esterase-like behavior (8) which is capable of rapidly degrading endotoxin(9), as indicated by tests: a, for toxicity of endotoxin for the 10-day-old chick embryo, and for the pertussis-treated mouse (10), and b, for the capacity of endotoxin to fix complement on exposure to specific antibody.† That this protein can alter the endotoxin molecule was also demonstrated by its ability to block 2 phage receptor sites on the endotoxin molecule,‡ and by production of multiple precipitation bands, instead of a single one, on Ouchterlony plates. The validity of these tests for assessing the presence or absence of potency in this protein fraction of spleen was affirmed by the finding that the results by more than one of the above 5 methods on any given test sample, though not quantitatively comparable, have nearly always been in the same direction.