Lawrance E. Shinn

Anticatalase Activity of Sulfanilamide and Related Compounds. III. Oxygen Tension and Bacteriostasis in Pneumococcal Cultures

Summary 1. Reduction of the percentage of oxygen in the superambient air of broth cultures of the Type I pneumococcus greatly reduced or prevented bacteriostasis by sulfanilamide. 2. When the oxygen concentration was reduced to 0.04% an actual stimulus of growth by sulfanilamide was found. 3. When the oxygen was further reduced by the presence of pyrogallate or hydrosulfite the inhibition by sulfanilamide reappeared. 4. Hydrogen peroxide was detected only in cultures in equilibrium with atmospheres containing 10% oxygen or more. These concentrations correspond to those permitting effective bacteriostasis and are comparable to those obtainable in the plasma. 5. The lack of inhibition at intermediate concentrations shows that oxygen plays a vital rôle in the action of sulfanilamide. The failure to form peroxide at these same concentrations is taken as evidence that oxygen exerts its influence through the agency of hydrogen peroxide. The stimulus and recurring inhibition at the lowest values is interpreted as evidence of the formation of a toxic reduction compound, possibly a sulfide. The latter type of inhibibition may play a rôle in any bacteriostatic effect against anaerobes.

Therapy of Experimental Staphylococcus Infections with Sulfonamide Compounds

Conclusions Generally speaking, our experience with these drugs in staphylococcic infections is in marked contrast to the decisively favorable results of sulfanilamide in hemolytic streptococcal infections. Disulfanilamide, by virtue of its apparent therapeutic effect over a limited time period has a certain promise, which, however, failed of clinical confirmation in the small number of cases observed. A more diversified experimental trial is merited. Both experimentally and clinically the results with “Di-Septal” in this preliminary study were essentially negative.

Anticatalase Activity of Sulfanilamide and Related Compounds. I. Effect of Ultraviolet Irradiation

Conclusions The ability of sulfanilamide and structurally related compounds to develop color on ultraviolet irradiation appears to be dependent on the presence or absence of substituents in the p-amino and sulfamido groups. A study of the anticatalase properties of sulfanilamide and related compounds indicates a possible correlation between therapeutic activity and the intrinsic anticatalase activity associated with the non-irradiated compound. Neither color production nor the high anticatalase activity developed as a consequence of irradiation appear to be related to therapeutic effectiveness.

Conversion of Sulfanilamide into p-Hydroxylamino-benzene-sulfonamide by Ultraviolet Irradiation

In a previous publication 1 the authors demonstrated that solutions of sulfanilamide and certain related compounds were endowed with antieatalase activity following irradiation with ultraviolet light. It was suggested in a subsequent paper 2 that this activity might result from the formation of either the p-hydroxylamino derivative or free hydroxylamine in the irradiated solutions. Taking hydroxylamine as a standard, it was calculated that the activity found could be explained by the conversion of about 2% of the sulfanilamide in an irradiated 8 mg % solution into the active compound. At that time no evidence was available to show that the assumed derivatives were actually formed under the conditions described. With a method recently developed by Rosenthal and Bauer, 3 it has been possible to demonstrate that solutions of sulfanilamide upon ultraviolet irradiation yield appreciable amounts of a substance which reacts as the p-hydroxylamino derivative. Rosenthal and Bauers method for the estimation of the p-hydroxylamino derivative in the presence of the free amine is based on the fact that in a mixture of the two substances, the amine can be acetylated by acetic anhydride and thus prevented from participating in the diazotization and coupling. The hydroxylamino form is not so affected and can thus be determined colorimetrically. As standards Rosenthal has recommended either p-hydroxylamino-benzene-sulfonamide or p-hydroxylaminobenzoic acid. We have chosen to use the benzoic acid.∗ Analyses were carried out precisely as in the specifications of Rosenthal and Bauer. The compounds were examined in simple, neutral aqueous solutions and in this respect were parallel to the standards. Complications arising from other organic materials which might be present in urine were, of course, not encountered. Sulfanilamide was irradiated by exposing a thin layer of an aqueous solution for 1 min at a distance of 3 in. from an ultraviolet lamp.

Potentiating Influence of Urine on Sulfanilamide's Bacteriostatic Effect on E. coli in vitro.:

In a previous publication 1 one of us (Mellon) reported a poten-tiative effect of sulfanilamide on hemolytic streptococci when the organisms had been exposed to physiological salt solution during the process of dilution preliminary to seeding the test cultures. If this exposure was omitted and dilution in broth substituted, the remarkable bacteriostatic effect of sulfanilamide in low concentrations was not noted. In the presence of normal human serum the effect is enhanced. In other words, a sterilizing effect is obtained from the combined action of these minimal factors which exceeds many times a simple summation effect. Kenny, Johnston, and von Haebler 2 have recently reported a very favorable series of clinical cases of E. coli infection of the urinary tract which were successfully treated with sulfanilamide. They showed that with oral medication of 1.5 gm. a day for 5-7 days sterilization of the urine was obtained during the period of treatment in all of 46 cases of infection with a typical E. coli. The concentration of free sulfanilamide obtained in the urine of treated cases ranged from less than 1:100,000 to 1:1,000. It was shown experimentally that the static or bactericidal action of the compound in vitro was roughly proportional to its concentration. It appeared to us that a potentiation similar to that displayed by saline for hemolytic streptococci might be anticipated in this instance with the culture medium itself (the urine) playing the role of the potentiating agent. Bacteriostatic tests were accordingly made on a strain of E. coli freshly isolated from a case of cystitis which had had no sulfanilamide therapy. Normal pooled urine was adjusted to pH 7.2 and sterilized by Seitz filtration. The broth employed in the following experiments was a 2% proteose-peptone beef infusion of the same pH.

Anticatalase Activity of Sulfanilamide and Related Compounds. II. Relation to Growth Inhibition in Pneumococcus

In a previous publication 1 we demonstrated the anticatalase property of sulfanilamide and showed that this attribute is markedly increased by the exposure of dilute solutions to ultraviolet light. It was suggested that anticatalase activity may play an important rôle in the therapeutic action of sulfanilamide against such organisms as the streptococcus and pneumococcus, a theory originally promulgated by Locke. 2 The hypothetical mechanism may be stated as follows: The growing bacterial cell has the power to convert sulfanilamide, presumably through mild oxidation, into a derivative which is a highly active anticatalase.∗ This reaction produces an accumulation of anticatalase in the immediate vicinity of the cell. The streptococcus and pneumococcus, being active producers of hydrogen peroxide, are able to grow only so long as the peroxide concentration can be kept below a critical level by outward diffusion or destruction. Usually this is accomplished by catalase of which there is ample reserve in the body and in cultures containing blood. However, in the presence of anticatalase, inactivation of catalase takes place in the zone immediately adjacent to the cell with resultant accumulation of hydrogen peroxide to toxic levels. The feasibility of this explanation is enhanced if we recall that catalase must diffuse into the reaction-zone from the outside environment, whereas anticatalase and peroxide, produced by the cell itself, are already present in the critical zone in their maximal concentrations. This report presents experimental evidence in support of the foregoing hypothesis. Ten cc of a 0.2% glucose broth were placed in each of a series of 50 cc Ehrlenmeyer flasks. This amount gave a surface-volume ratio of approximately 1.6. 3 Catalase was supplied through addition of 5 × 10-5 cc of hemolyzed whole rabbit-blood. This amount consistently gave the most clear-cut results with the particular samples of blood and broth used.

Anti-Catalase Activity of Sulfanilamide and Related Compounds. VI. Further Studies on Sulfonhydroxamides

Summary P-Caproylaminobenzenesulfonhydroxamide produces bacteriostasis of pneumococci in blood without the lag characteristic of sulfanilamide. The activity is only about one-eighth of the corresponding bacteriostatic power in broth. That the immediate nature of the effect is due to the hydroxamide group was demonstrated by the use of p-toluenesulfonhydroxamide in which the potentially active p-amino group is absent. The effect of the hydroxamide group is transient in nature. A second period of bacteriostatic activity manifested by p-caproylaminobenzenesulfonhydrox-amide is probably due to the free amino group formed by deacylation of the caproylamino group.

Anticatalase Activity of Sulfanilamide and Related Compounds. V. Bacteriostatic Activity of some Sulfonhydroxamides

Summary The acyl aminobenzenesulfonhydroxamides have strong anti-catalase activity and, when present in broth cultures of the pneumococcus, cause inhibition of growth associated with increased accumulation of hydrogen peroxide. When the caproyl compound is added to growing cultures, inhibition of growth is detectable almost immediately but reaches a maximum only after time for accumulation of peroxide has elapsed. Inhibition by sulfanilamide, on the other hand, is detectable somewhat later and approaches a maximum more slowly.

Anticatalase Activity of Sulfanilamide and Related Compounds. IV. Peroxide Accumulation and Growth Inhibition in Pneumococcus

Summary The effect of sulfanilamide, and other compounds capable of inhibiting catalase, upon growth and accumulation of hydrogen peroxide in broth cultures of the pneumococcus has been subjected to quantitative measurement. The concentration of peroxide per unit of growth was from 2 to 10 times greater in the presence of compounds which caused bacteriostasis than in control cultures.