George A. Emerson

The Laxative Principle in Prunes

No satisfactory study of the widely recognized cathartic effect of the French or Santa Clara variety of prune has ever been reported as far as we know. The purgative action appears in foodstuffs derived from prunes as well as in the dried prune itself. The apparent thermostability of the evacuant principle may be the factor leading Sollmann 1 and others to include prunes with the colloid and emollient laxatives which act “mainly by retaining water in the intestine by imbibition, thus modifying the bulk and consistency of the feces, so that these are more easily expelled.” Water-soluble substances in prunes have previously been held to play only a subsidiary role in catharsis; Sollmann 1 explains their action thus: “in fruits (prunes....., etc.) this colloid action is supported by the organic acids and sugars.” We studied the cathartic efficacy of the soluble compounds present in dried prunes in an attempt to isolate the active substance responsible. Since such an active principle is present in very high dilution, routine chemical tests yield little information. The color reactions described by Tumin Katti and Beal, Fuller, Warren, and others 2 for anthraquinone derivatives which act through an irritant effect, are negative according to Mrak and Smith 3 for all prune extracts tested, indicating the absence of emodin-like compounds. The FeCl3 test for phenolic groups is positive. Three methods were used to evaluate the potency of various extracts. In spite of Sollmanns criticism 1 of the Magnus technique 4 with isolated gut, it is a convenient one to determine whether or not activity is present in materials tested. Preliminary tests were made by this technique on various prune preparations and the results so obtained were in accord with those found in intact animals by either Machts 5 or Eddys 6 method. Actions of prune extracts on isolated rabbit, guinea pig, dog and cat jejunum or duodenum in either Sollmann and Rademaekers, 7 or Sollmann and von Oettingens 8 solutions are summarized in Table I. The individual responses were confirmed at least in triplicate, and the tabulated results represent some 200 separate experiments. The water extracts were prepared according to the directions of Mrak and Cruess. 9 All other extracts were thoroughly freed of solvent by prolonged evaporation in vacuo and dissolved or suspended in water before being added to the bath. None of the extracts was sufficiently acidic or basic to influence the observed effect. Summary. As tested by the techniques of Magnus, Macht, and Eddy, the laxative properties of prunes are not due entirely to colloidal or emollient effects in the intestine, as has been previously supposed, but would seem to depend also on the presence of an agent soluble in water, alcohol and pyridine. This active principle in prunes which is broken down by strong hydrolysis has otherwise certain chemical and physiological properties similar to di-hydroxy-phenyl isatin and to caffeic and chlorogenic acid.

Acute Toxicity of Trypan Blue, Gentian Violet and Brilliant Green.∗

Difficulties encountered in administering sufficient amounts of the usual chaulmoogra oil derivatives uniformly to effect a cure in leprous subjects have led to the attempted application of other forms of therapy. Thus, recent clinical developments 1 , 2 again have aroused interest in the efficacy of certain non-chaulmoogry (Link Missing 1) drugs, synthetic dyes in particular, against leprosy. In the course of examining the possible antileprotic activity of certain such compounds by a standardized technique, 3 determination of their toxicity in the test animals was a necessary preliminary. As trypan blue, gentian violet, and brilliant green have been used in an empirical manner in the clinic before a critical pharmacological evaluation of their worth has been made, it is advisable to present the results of this toxicity study on experimental animals, before toxic doses may unavoidably be given to human leprous subjects. The intravenous dosage of these dyes, as reputedly used by Ryrie 1 and Ryles, 2 is within the possible lethal range as estimated from experimental animals. While these workers do not give the sources of the dyes used by them, nor their standards of purity, so that it cannot be judged whether or not their dyes are comparable in purity to those used by us, it is significant that Ryrie 1 admits the occurrence of dangerously severe acute toxic effects in his patients. Previously reported toxicity data on these 3 dyes are so inadequate and variant that a recent encyclopedic work on chemotherapy 4 omits all reference to their toxicity. Discrepancies in some cases may be explained by variations in purity and by the use of old solutions. Photodynamic action is not a significant factor in the toxicity of these dyes as far as we could determine. We will later make a more extended report surveying the therapeutic effectiveness as well as the chronic and acute toxicity of these agents. Such a study is indicated in view of enthusiastic endorsement of dye therapy in leprosy on grounds that it is as good as treatment with hydnocarpus oil derivatives, safe, cheap and not unduly painful.

Quantitative Colorimetric Estimation of Morphine in Biological Fluids by the Iodoxybenzoate Method.

Summary The qualitative iodoxybenzoate test of Leake has been found suitable for quantitative adaptation to the estimation of morphine. Methods for determining morphine in biological fluids and a rapid method of accurately checking the morphine content of morphine hydrochloride are suggested. Development of a satisfactorily accurate quantitative application of the usual color reactions for morphine has been retarded because of their general lack of (a) proper selectivity for this single opium alkaloid, (b) permanence of hue produced or (c) sufficient sensitivity. In addition such reactions are in most instances dependent to an undesirable degree on the temperature and pH of the test solution and the presence of commonly occurring contaminants. This situation is not surprising since most of these reactions are supposedly due to the formation of complex unstable chromogenic substances. A more ideal quantitative test should be the stoichiometric conversion of morphine to a simple colored form. Leake 1 suggested that such an effect could be obtained for many substances by reacting their free phenolic groups with an aqueous solution of ammonium iodoxybenzoate, the oxidative powers of which had been studied in another regard by Loevenhart 2 and others. Morphine rapidly develops the same color when treated with this agent as is produced by slow spontaneous oxidation of morphine solutions in air, and it was recommended 1 that this reaction be used not only as a qualitative test to differentiate morphine from codeine, heroin, dionin and apomorphine, but also as the basis of a quantitative colorimetric estimation. We have found that when 25 to 50 mg. of powdered ammonium iodoxybenzoate are added to 5 to 10 cc. of 0.005 M to 0.02 M aqueous solutions of morphine salts sufficient agreement with Beers Law holds so that colorimeter readings yield results accurate to ±3% if the comparison with a standard is made after 30 minutes are allowed for full development of the color. Spectroscopic examination of color absorption by the treated solutions reveals that in concentrations higher than 0.03 M extinction extends further toward the yellow band than with the lower concentrations, which is represented grossly by a change in shade from orange to garnet and considerable deviation in colorimetric measurements unless a suitable filter is used.

Some Effects of Ether on Bioluminescence in the Lampyrid, Photuris pennsylvanica

In normal anesthetic concentrations ether has no direct chemical action on the dehydrogenase system involved in carbohydrate metabolism in mammalian brain, but through humoral effects ether anesthesia in the intact animal has a marked action indirectly on the rate of autoxidation of subsequently excised surviving brain, due presumably to the limiting of available carbohydrate. 1 The present paper demonstrates a striking corollary in the Lampyridae, for while ether has little action directly on the dehydrogenase system having to do with bioluminescence, it does exert various indirect effects in the intact insect which yield again quite unexpected results. The light-organ of Photuris pennsylvanica generally flashes spontaneously at irregular intermittent intervals. The characteristics of the flash have been studied photoelectrically by Snell 2 and the mechanism of control by Gerretsen, Snell and others, 2 it appears agreed that the flash is under nervous control. Access of adequate oxygen to the light-producing areas, which is a requisite for luminescence, is regulated by supposedly innervated 3 muscular tissue which normally between flashes almost completely occludes the entrance to tracheoles leading to the light-organ. Flashes may be artificially elicited through single shocks or short tetanic electrical stimulation, and the so-called pseudo-flash by rapidly raising the oxygen tension in an environment in which the oxygen concentration is low. Both artificial methods were found to be lacking in uniformity and to have potentialities of irreversibly injuring the flies, in confirmation of Snells 2 work. Since no regular frequency of flashing can be induced in captured flies, it was obvious that little significant data could be obtained using flashing as a criterion for comparison of treated flies with controls. Creightons 3 observation, that injection of minute amounts of epinephrine hydrochloride into the fly results in an action on the occluding musculature of the tracheoles to permit the constant access of oxygen and a resultant constant bright glow of the light-organ, afforded an opportunity of preparing specimens for observation of the direct effects of gaseous agents on the luciferin-luciferase system in situ.

Mechanism of the Emetic Action of the Chaulmoogrates.

Summary Evidence is presented indicating that the emetic effecr of the chaulmoogrates is central. The action of Cannabis, atropine anti morphine in aliolishing the emetic response in dogs and cats is reported.

Lipolytic Activity of Rat Lepromata During Treatment with Various Antileprotics.

We previously discussed the distribution of lipase in tissues of normal and experimentally infected leprous rats, 1 as observed by us preliminary to a study of changes in lipolytic activity during treatment of infected animals. Our subsequent observations demonstrating that there is no significant change in lipolytic activity of tissues of leprous animals during treatment are reported elsewhere. 2 This present report is concerned with changes in the lipase content of the leproma during intensive “plancha” treatment 3 with various antileprotic drugs. The lipolytic activity of 60 untreated lepromata and 36 treated lepromata was estimated by Loevenharts method 4 as used by us previously, and was found to be 0.16±0.03% and 0.15±0.04% respectively. Approximately half the material in both cases was obtained by biopsy and the other half from animals dying or sacrificed. It is apparent from these determinations that there is no tendency during treatment for the lipolytic activity of lepromatous subcutaneous tissue to approach the value of 0.83±0.07% found by us for normal subcutaneous tissue of rats with “early stage” leprosy. No correlation was found between apparent lipase content of lepromata and type of antileprotic drug used. The average lipolytic activity of lepromata of rats treated over 6 months with maximum tolerated doses of the more important drugs considered was: ethyl chaulmoograte, 0.15±0.03%; “Alepol,” 0.15±0.05%; ethyl di-n-heptyl acetate,† 0.11±0.04%; sodium dihydrochaulmoogryl p-phenetidine sulfonate, 0.12±0.05%; sodium chaulmoogryl p-phenetidine sulfonate, 0.13±0.02%. Two possible explanations of the cause of the low lipolytic activity of leprous tissues were examined experimentally.

Effect of Chaulmoogric Acid Derivatives on Lipolytic Activity in vitro.

Following the demonstration by Shaw-Mackenzie 1 that sodium gynocardate specifically activates pancreatic lipase in vitro by some 100% under the experimental conditions employed, Rogers 2 advanced the hypothesis that the mode of action of chaulmoogrates in leprosy therapy consists of a similar activation in vivo which would facilitate humoral destruction of the fatty capsule of Mycobacterium leprae. Significant differences were found by Rogers in the ethyl-butyrase activity of sera, shipped on ice from India, of treated and untreated lepers, the former approximating normal. Neill and Dewar 3 have been unable, however, to confirm these observations in a more extensive study of fresh leprous sera. We have previously studied 4 by Loevenharts method 5 the in vivo effects of certain antileprotic drugs on the ethyl-butyrase activity of tissues of rats experimentally infected with a standard strain of rat leprosy supplied by Dr. E. L. Walker. No reversion of the lipolytic activity to normal was brought about by any of the chaulmoogrates studied. Preliminary in vitro ethyl-butyrase studies on liver bries, in which 1 cc. of a 1% solution of the test agent was added to the Loevenhart reaction mixture, revealed an inhibition rather than an activation, varying to some extent with the type of chaulmoograte used. For the following drugsf averages of 5 determinations expressed as per cent of original activity lost, were: sodium gynocar-date, 60%; alepol, 40%; sodium chaulmoogryl-p-phenetidine sulfonate, 90%; sodium dihydrochaulmoogryl-p-phenetidine sulfonate, 90%; sodium chaulmoogryl glycinate, 65%; hydroxymercuriethoxy chaulmoogric anhydride 6 (saturated), 10%; and sodium dichaul-moogryl-β-glycerophosphate, 15%. Addition of an only slightly soluble salt of a weak acid (sodium gynocardate) to an enzyme-substrate system results in considerable inaccuracy in the determination of the true end-point on titration unless careful comparison with a color standard is made, but the magnitude of the activating effect observed by Shaw-Mackenzie indicated a more significant factor was involved. It occurred to us that since in Shaw-Mackenzies experiments sodium gynocardate was added to a glycerol pancreatic extract and the hydrolysis of the olive oil substrate allowed to proceed for 18 to 24 hours before titration, considerable enzymatic synthesis of chaulmoogryl glycer-ides must also take place in this time, resulting in the removal of weaker acids (chaulmoogric and hydnocarpic) with replacement by stronger acids from the glycerides of the olive oil substrate, thus leading to an apparent higher acid value on titration. Summary. In addition to the previously demonstrated lack of in vivo lipase activation by the antileprotic chaulmoogrates, it is shown that the apparent in vitro activation is not real, but is more likely an artefact due to the disturbance of equilibria brought about by the introduction of a salt of a weak acid into an enzyme reaction system containing large amounts of free glycerol. If the chaul-moogrates exert any indirect action in leprosy therapy besides their direct bactericidal action, it is improbable that the fat-splitting ferment is involved.

Lipolytic Activity of Rat Tissues in Experimental Leprosy.

Walker and Sweeney 1 suggested that an indirect or physiological method of therapeutic action of chaulmoogra oil in leprosy might be stimulation of non-specific lipolytic activity of the tissues which would attack the fatty capsule of acid-fast bacilli. We have undertaken the study of the lipolytic activity of leprous tissue in experimental rat leprosy in comparison with that of normal tissues of the same animal, and the determination of whether or not the observed ratios may be altered by treatment. We decided to ascertain first, however, what effect the presence of leprosy in the body might have on the tissue distribution of lipase in comparison with that noted in non-infected normal animals. Our observations on this point are reported herewith. Estimations of the lipolytic activity of representative tissues were made by Loevenharts method 2 on 8 healthy adult female rats and on 22 female leprous rats. The latter had been inoculated with a buffered saline suspension of finely ground rat leprous tissue from 140 to 194 days previously by Dr. E. L. Walker and Miss M. A. Sweeney. The source of this material was spontaneous leprosy in wild rats obtained from the U. S. P. H. Plague Laboratory in San Francisco, transferred about every 6 months through the Burlin-game strain of albino rats for 10 generations without apparent alteration in virulence. Definitely palpable lesions were present at the time of death. The 8 healthy rats and half the leprous rats were killed by a blow on the head while the remainder of the leprous rats died from pneumonia. Tissues were taken at death in all cases, and particular care was used to grind them thoroughly. Estimations on individual tissues were made in triplicate and were in agreement within the limits of error of titration with a micro-burette, that is, 0.005 ce.