SOCIETY OF PUBLIC ANALYSTS

866 TABLE

I-POTENTIATION

OF SULPHATHIAZOLE GLUTAMINE

ACTION

BY

solution is therefore this acid.

possibly

due to the presence of

CONCLUSIONS

The difference between the stability of glutamine and that of the substance causing the L effect in lab-lemco, and the fact that glutamine probably only occurs in lab-lemco to the extent of 0-4% (Rees 1951) is evidence that glutamine is not alone responsible for the potentiating effect of lab-lemco. SUMMARY

Glutamine potentiates the bacteriofrenic action of sulphathiazole, rendering it bactericidal against Bact. coli growing in a synthetic medium. This activity is reduced on heating. TT = Heavy turbidity. Three control cultures containing 16 mg. sulphathiazole per 100 ml. but no glutamine, contained 24, 38, and 44 surviving organisms per

loopful.

for sulphathiazole-potentiating activity was serially diluted in 5 ml. amounts of synthetic medium containing 16 mg. sulphathiazole per 100ml. The synthetic medium used was Bigger’s (1950) modification of Gale’s (1947) medium, which contains - only inorganic salts, ammonia, and glucose. The test cultures were inoculated by adding to each culture bottle a drop (Dreyer pipette) of a 1/10,000 dilution in buffered saline solution of a 24-hour synthetic medium culture of Bact. coli strain no. 18. After incubation, the number of viable organisms in each culture was estimated by spreading a 5 mm. loopful of the culture over the surface of an agar slope and counting the resulting colonies. RESULTS

The

I wish to thank the Medical Research Council of Ireland for a grant which made this work possible, and Prof. F. S. Stewart under whose direction I have been working. Messrs. L. Light & Co. Ltd., provided 1 g. of glutamine. REFERENCES

Bigger, J. W. (1950) Lancet, ii, 46. Ware, G. C. (1950) Ibid, p. 427. Gale, E. F. (1947) The Chemical Activities of Bacteria. London, Rees, H. G. (1951) Unpublished personal communication. Vickery, H. B., Pucher, G. W., Clark, H. E., Chibnall, A. C., Westall, R. G. (1935) Biochem. J. 29, 2710. -

Medical Societies SOCIETY OF PUBLIC ANALYSTS Evaluation of Chemotherapeutic Substances THE biological methods group of the Society of Public Analysts and other Analytical Chemists held a symposium on Oct. 26-27 on the Evaluation of Chemotherapeutic

Substances.

experiments were designed to compare the Dr. G. M. FINDLAY, the chairman, in opening the sulphathiazole potentiating activities of lab-lemco and discussion, emphasised the need for rapid and accurate of glutamine before and after heating. 1 % solutions of labmethods of estimating the chemotherapeutic activities lemco and of glutamine were sterilised by filtration through of the many hundreds of compounds which are now Seitz EK filters. A portion of each sterile solution was being prepared for the specific treatment of the parasitic heated in an autoclave for 20 minutes at a pressure of infections of man and his domestic animals. The final 15 lb. per sq. in. For testing, dilutions were prepared and crucial tests can obviously be carried out only in in synthetic medium so as to contain 1/1000 of the man and his domestic animals, but preliminary laboratory substance under test and 16 mg. of sulphathiazole per tests are essential both for determining relative efficiency 100 ml. and for the accurate assay of substances already found Table i shows the potentiating effect produced by to be effective. Improved methods are required to glutamine on the normally bacteriofrenic action of evaluate anthelmintics, fungicides, and compounds sulphathiazole. It also shows that this activity is active against viruses. substantially reduced by heating. Neither the heated Dr. W. L. M. PERRY dealt with the general problems nor the unheated preparations of glutamine show any involved in planning large-scale collaborative antibiotic inhibitory effect on the growth of Bact. coli strain no. 18 assays to ensure adequate international standardisation. in synthetic medium not containing sulphathiazole. He discussed the relationship between _technique and Table n compares the L activity of lab-lemco before the " over-accurate" assay, the homogeneity results, and after heating, and shows no similar reduction in of estimates, and the validity of the combination of activity. results. At present few laboratories use exactly the same COMMENT technique for antibiotic assays. Dr. J. UNGAR described the various types of in-vitro Vickery et al. (1935) have shown that glutamine is almost completely converted to pyrrolidonecarboxylic and in-vivo tests which have to be carried out before a acid and ammonia when heated in aqueous solution, compound can be used in man. Acute systemic and local and the apparent activity of the heated glutamine toxicities, as well as chronic toxicities, must be ascertained in several species of laboratory animals. The TABLE II-COMPARISON OF THE SULPHATHIAZOLE-POTENTIATING absorption and distribution in the tissues, normal and ACTIVITY OF HEATED AND UNHEATED LAB-LEMCO inflamed, and the mode of excretion are important, as

Thuee control cultures containing 16 mg. sulphathia,zole per. 100 ml. but no lab-lemco, contained 47. 61, and 83 surviving organisms per

loopful.

also is the likelihood of sensitisation and the interaction with other drugs. These remarks were illustrated by reference to ’Estopen,’ a penicillin ester which is concentrated in the inflamed lung. Dr. J. H. HUMPHREY pointed out that, apart from good experimental design, the most important aim in assay methods involving diffusion of antibiotics into seeded agar plates is to obtain a sharp edge to the inhibition zones and a good slope. Penicillin and streptomycin give sharp edges with suitable organisms, probably because the uptake or destruction of these antibiotics

867 near

the

With the

zone

edge gives

newer

a

steep

antibiotics the

concentration gradient. less sharp. The

factors determining slope are largely physical. Prof. J. M. ROBSON described the use of the rabbit and mouse eye for determining antituberculous action ; one great advantage of the method is its safety for all those performing the experiments. Mr. A. R. --NIARTI--,, PH.D., discussed the methods used in employing mice for screening antituberculous compounds ; and he described the relation of the chick-embryo test to the mouse test. Miss L. DICKINSON gave details - of antiviral tests carried out in the developing chick embryo and in mice. Comparable results have been obtained in mice and chick embryos with the Nigg pneumonitis virus. Dr. L. G. GoODWiN described the use of weanling rats for the evaluation of amcebicidal drugs, illustrating his remarks with a coloured film. The use of the infected rat provides a method which fulfils the essentials for a satisfactory amcebicidal test, for the infection is reproducible without fail and untreated controls show a very constant lesion ; known remedies for the disease in man produce a recognisable effect ; the test takes only a short time ; and the infecting agent is derived from man and produces the same type of lesion in the rat as in man. The only difficulty in the use of the rat is the presence of an amœba—Entamœba muris—which is not affected by amcebicidal drugs ; and special precautions must be taken to prevent infection of the rat colony with E. muris. Dr. J. D. FULTON described various media used for the growth of pathogenic amcebae in vitro and their possibilities for providing methods for testing amoehicidal drugs. He examined the question whether these amoebicidal drugs act directly on the amoebae or indirectly on the bacterial flora which provides the pabulum for the amœbæ. He also referred to the possibility of using Trypanosoma cruzi in place of bacteria for the nutrition of the amcebae. LOURIE pointed out that refined statistical methods are invaluable for biological standardisation, but in the search for new compounds of clinical value they are useless if inappropriate experimental procedures In Africa for the control of trypanosomiasis are used. mass treatment is giving way to mass chemoprophylaxis, and new compounds are being devised for this purpose. There is therefore urgent need for a simple and rapid method of screening compounds in regard to their prophylactic activity. There is difficulty in standardising trypanocides of the melaminyl series by chemical or physical means. Standard preparations of these substances are required, and batches intended for use in man should be biologically tested in relation to these standards. Mr. D. G. DAVEY, PH.D., described the various laboratory tests employed for evaluating antimalarial compounds in the light of the fact that, except possibly in the case of Plasmodium malariœ, none of the malarial parasites in man has been established in laboratory animals. The tests used are unlikely to pass over any compound which is of potential value in man. Dr. ANN BiSHOP pointed out that quinine gives rise to little or no drug resistance, and with pamaquin only a very slight increase can be induced. Proguanil, however, readily induces resistance in P. gallinaceun; strains treated with sulphadiazine or sulphanilamide became resistant to these and to proguanil. Metachloridine also induces a high degree of drug resistance in malaria parasites. In testing for ability to produce drug resistance experimental strains of the parasite must be maintained in a state of acute infection. Results from tests on latent infections are not comparable. Dr. F. HAWKING urged that when a compound has once been shown to be effective in the laboratory it should be tested as soon as possible in man, in order to bring to light toxic reactions which cannot be elicited in laboratory animals.

Dr. E. M.

New Inventions

zones are

AN APPARATUS FOR THE ACCURATE MEASUREMENT OF INTRAVENOUS INFUSIONS THE siphon-burette illustrated here was designed to facilitate accurate measurement of the amount of procaine given by continuous intravenous infusion during operations on the heart. It has also proved useful when giving anaesthetic drugs, such as thiupentone by slow infusion, and in blood-transfusion. In cardiac cases the circulation is easily overloaded by intravenous fluid, and an adequate blood-level of procaine can be maintained onlv by infusing a concentrated solution slowly. The standard drop-counting method of estimating the rate of an infusion is too inaccurate, and the calculation of milligrammes perr minute is tedious when rates such as F 1 ml. per minute, or less, have to be measured. With this burette the fall of the fluid level from one ml. calibration to the next is readily timed and great accuracy in dosage is obtained. Thee apparatus consists of a glass bulb (A) drawn at its lower end to form a narrow burette (B) of 10-15 ml. capacity and calibrated. A partition (C) separates the bulb from the

it pass a siphon tube (D) and a pressure-

equalising tube (E). A side tube (F) is attached above the level of E. Procaine solution from the reservoir bottle drips into bulb A until it reaches the top of tube D, when it rapidly siphons and tills the burette. As the level in the burette falls, its rate can be very accurately timed as the meniscus passes the calibrations. By the time the level in the burette reaches the 10 ml. mark, the bulb has refilled and again siphons into the burette. The apparatus is a closed system ; no more liquid can enter the bulb than leaves the burette ; and the flow is controlled by the one gate clip (G) placed near the needle as in a standard giving set. Before the venepuncture is performed, all air is expelled from the tubing below the burette, and this is achieved by allowing it to siphon a few times and raising and lowering the rubber tube. When the air has been expelled it is important to adjust the levels so that the burette is filled to the 0 mark each time the bulb siphons. If the burette is underfilled, the meniscus may disappear into the rubber tubing before siphoning occurs. This is adjusted by closing the clip at G and cautiously opening the side tube F to allow liquid to run into the bulb. Similarly if the burette is overfilled, the side tube is opened after closing the clip H on the reservoir tubing Once and fluid is allowed to run from the burette. adjusted, it will fill to the same level indefinitely. The apparatus, which is made ofPyrex ’ glass, can be sterilised by autoclaving. My thanks are due to Mr. Humphries of Messrs. John BelI & Croyden, Wigmore Street, London, W.1, for his advjce The apparatus is and skill in producing the final design. obtainable from this firm. B. A. SELLICK, M.B., D.A. Middlesex Hospital, Consultant Anæsthetist. London, W.1.