INHIBITION BY METHIONINE OF THE TOXICITY OF CRYSTALS FROM AGENISED ZEIN OBSERVATIONS ON LEUCONOSTOC MESENTEROIDES

1130 The criteria for the selection of cases for treatment with vitamin E and of the controls are detailed. The literature of (x-tocopherol therapy is vast and highly The results were assessed after three months’ treatment controversial. The situation has been further complicated by testing the ability to walk. Patients who before by the appearance of articles in the popular press hailing treatment had been forced to halt at less than 500 yards vitamin E as a substance which to all intents and purposes were classed as improved " if after treatment they is a universal remedy. could walk more than 880 yards. In November, 1946, the Council of the College of PhysiOf 41 cases in the treated group 34 were improved; cians and Surgeons of Ontario considered claims made of 25 cases in the control group 5 were improved. A " by the Shute brothers for the usefulness of vitamin-E test shows that the results may be classed as highly in The of the disease. conclusion cardiovascular therapy significant." council was the equivalent of the old Scottish verdict of Points in connexion with the use of vitamin E, " not proven," with about the same damning result including the necessity for high dosage and long (Noble 1947). But, whatever the findings about the treatment, are discussed. treatment of cardiovascular conditions in general, it is For permission to publish these cases I wish to thank difficult to explain the improvement in certain cases Prof. A. M. Boyd, professor of surgery in the University of cited by Vogelsang et al. (1947, 1948) as being due to Manchester and director of the neurovascular clinic at the causes other than vitamin-E therapy. Manchester Royal Infirmary. A complete review of the arguments which have been advanced for and against (x-tocopherol therapy is not REFERENCES feasible here. Certain points, however, demand mention. Adamstone, F. B. (1931) J. Morph. 52, 47. The most important of these is that, to be effective, Boyd, A. M., Ratcliffe, A. H., Jepson, R. P., James, G. W. H. (1949) J. Bone Jt Surg. 31B, 325. vitamin E must be given in large doses and for a long Fisher, R. A., Yates, F. (1948) Statistical Tables. London. time. Both from published reports and from a study of Hamilton, W. F. (1947) Howell’s Textbook of Physiology. Philadelphia. the cases treated from the neurovascular clinic it seems Krogh, A. (1922) The Anatomy and Physiology of Capillaries. that the optimum dosage is about 400 mg. daily. This New Haven. Levy, H., Boas, E. P. (1948) Ann. intern. Med. 28, 1117. should be given in the form of x-tocopherol, the biological Lewis, T., Pickering, G. W., Rothschild, P. (1929) Heart, 15, 359. K. E. (1943) in R. S. Harris and K. V. Thimann’s Vitamins activity of the &bgr;- and y-tocopherols being considerably Mason, and Hormones. New York ; vol. II. less (llason 1943). One of the most striking observations Noble, R. T. (1947) Modern Medicine of Canada, 2, 55. in connexion with vitamin-E therapy is the delay before Shute, E. V. (1942) J. Obstet. Gynœc. 49, 482. Vogelsang, A. B., Skelton, F. R., Shute, W. E. (1948) Surg. In the patient begins to notice any improvement. Gynec. Obstet. 86, 1. obliterative vascular disease this delay is from four to Vogelsang, A. B., Shute, E. V., Shute, W. E. (1947) Med. Rec., N.Y. 160, 21, 91, 163, 230, 279. eight weeks, the average being about six weeks. Negative (1948) Ibid, 161, 83. evidence of the effectiveness of vitamin-E therapy with doses of less than 400 mg. daily for periods shorter than INHIBITION BY METHIONINE OF THE three months must therefore be considered inadmissible. of small success with doses or Conversely, reports TOXICITY OF CRYSTALS FROM treatment for a short time must also be suspect. AGENISED ZEIN In diseases of the circulatory system the number of variables involved may be large and the assessment of OBSERVATIONS ON LEUCONOSTOC MESENTEROIDES In reporting 13 cases of cardioprogress hazardous. J. G. HEATHCOTE vascular lesions Levy and Boas (1948) stress the difficulty B.Sc. Mane., A.R.I.C. of estimating the improvement. Even when, as in patients with intermittent claudication, a yardstick of From the Research Association of British Flour-Millers, Cereals Research Station, St. Albans improvement-albeit an artificial one-is available it must be used with care. Thus it would be possible to THE isolation agenised zein of a crystalline present a series of type-i cases in which 100% success substance which isfrom toxic to rabbits and dogs was reported was obtained ; but other substances are equally successful recently from these laboratories (Bentley et al. 1949). in this type. A series of type-III cases judged by the It was shown further (Heathcote 1949a) that these walking test would be a complete failure. It should be are markedly toxic to the bacterium, Leuconostoc crystals certain that in to observed, however, type-III patients mesenteroides P 60, even at concentrations as low as whom vitamin E was given, though their ability to walk 5 ml. of g. per liquid medium, the bacterial cells being did not increase sufficiently to be considered as " and acid production substantially reduced. agglutinated a for the better was considerable improved," change Still smaller concentrations-e.g., 1-5 tg. per ml.noticed in the nutrition of the feet. milder effects resembling the antagonistic " produced Of the 7 not improved " patients in this series one, of certain chemicals on acid-producing bacteria ,effects who had originally been diagnosed as having diffuse obliterative arteritis, later had a secondary popliteal EFFECT OF ADDING METHIONINE TO CULTURE MEDIUM ON TOXICITY OF CRYSTALS TO L. mesenteroides thrombosis, which would be sufficient to explain the lack of improvement. Examination of the case-records of EXPERIJBiENT I the other 6 did not reveal any common factor which might suggest that vitamin-E therapy would not succeed. From the economic point of view it must be admitted that oc-tocopherol is expensive. Presumably therefore it should be used only where there is a reasonable hope of success that could not be achieved by other means. Even in these cases, however, to give inadequate doses for short periods is just as wasteful as to use the substance DISCUSSION

’

"

x2

—

—

.

haphazardly. SUMMARY

Cases of obliterative vascular disease leading to intermittent claudication were selected from the records of the neurovascular clinic at the Manchester Royal Infirmary.

—

—

1131 Heathcote 1949b), which are believed to be due to an unbalanced mixture of amino-acids. In preliminary work on the mode of action of the toxic crystals on this bacterium relatively large doses of various individual amino-acids, including those known to be -essential for its growth, were added to the culture medium. The amino-acids examined were : dl-alanine,

(Snell 1945,

I-arginine monohydrochloride, dl-aspartic acid, l-cystine, dl-cysteine, glycine, l-glutamic acid hydrochloride, sodium glutamate, l-histidine monohydrochloride, l-hydroxyproline, dl-isoleucine, dl-leucine, dl-lysine, dl-methionine, dl- phenylalanine, dl-proline, dl-serine, dl-tbreonine, dltryptophane, l-tyrosine, and dl-valine. Of these amino-acids, methionine alone showed any The addition of sufficient effect on the toxicity. methionine to the medium nullified the toxic effect and restored acid production as illustrated in the accompanying table. From these and other experiments it appears that the toxic substance formed by the action of nitrogen trichloride on zein is absorbed by the bacteria in similar manner to methionine but that when methionine is present in excess it is preferentially absorbed. Consequently, it is not unlikely that the toxic substance is similar to methionine in chemical composition, in which case it is based on the methionyl residue of the zein molecule. More detailed evidence implicating methionine as the essential reactant is contained in a further paper from these laboratories (Nature, Lond. in the press). Since nitrogen trichloride can act as an oxidising agent, the simple oxidation products of methionine,methionine sulphoxide and methionine sulphone-were tested for toxicity towards L. mesenteroides P 60. The sulphone was found to be more toxic than the sulphoxide but the toxicity of both compounds was negligible compared with that of the crystals isolated from agenised zein. The crystals cannot, therefore, be identified with either of these two compounds. This result is in keeping with the comparative harmlessness of oxidised peptone (Lyman et al. 1947), containing methionine sulphone, when it is used as a medium for microbiological assays, with L. mesenteroides P 60 as the test organism. Further evidence suggesting a relationship between the unknown substance and methionine is derived from methionine assays on hydrolysates of the crystals prepared from agenised zein. Hydrolysis with 2-5N HCl in the autoclave at 15 lb. pressure for 6, 12, and 24 hours, though causing some reduction in toxicity, did not completely destroy it. Treatment with 6N HCl for 68 hours at 110°C, however, completely removed all toxicity. From a hydrolysate of crystals (0-5 mg.) prepared in this way the excess acid was removed on a water bath and the hydrolysate was neutralised and made to volume. The solution was tested in the usual manner (Heathcote 1949a) to ensure the complete absence of toxicity towards L. mesenteroides P 60 and was then assayed for methionine using the same organism and a supplemented medium of oxidised peptone (’ Difco’). The result of the microbiological assay indicated a methioninereplacement value equivalent to 0-8% of l-methionine. Paper chromatograms on the hydrolysed material failed to reveal the presence of free methionine. The inference therefore is that the hydrolysis produces some substance possessing only partial methionine-replacement value for the organism. This work supports the conclusion in an earlier paper from these laboratories (Bentley et al. 1948): that it is the methionine portion of a protein molecule which gives it toxic properties when it is treated with nitrogen trichloride. SUMMARY

The addition of methionine arrests the toxic effect

on

Leuconostoc mesenteroides P 60 of the crystals isolated from

agenised

zein.

This

finding

is in

keeping

with the

view that it is the methionine portion of the protein molecule which is essential for the production of the toxic substance. REFERENCES

Bentley, H. R., Booth, R. G., Greer, E. N., Heathcote, J. G., Hutchinson, J. B., Moran, T. (1948) Nature, Lond. 161, 126. McDermott, E. E., Pace, J., Whitehead, J. K., Moran, T. (1949) Ibid, 164, 438. Heathcote, J. G. (1949a) Ibid, p. 439. (1949b) J. gen. Microbiol. 3, 392. Lyman, C. M., Moseley, O., Wood, S., Hale, F. (1947) Arch. Biochem. —

—

10, 427. Snell, E. E. (1945) Advances in Protein Chemistry. New York; vol. II, p. 101.

EOSINOPHILIC LEUKÆMIA AND FAMILIAL EOSINOPHILIA TWO ILLUSTRATIVE CASES

J. D. GRAY M.D. McGill PATHOLOGIST, AND ROYAL EYE

BELGRAVE HOSPITAL FOR CHILDREN COLLEGE HOSPITAL LONDON

GROUP),

HOSPITAL (KING’S

SYDNEY SHAW M.B. Lond. ASSISTANT CLINICAL

PATHOLOGIST, SCHOOL,

HOSPITAL MEDICAL

CHARING CROSS LONDON

and Plum (1939), reviewing eosinophilic leukaemia, summarised 14 published cases, since when a further 9 cases have been described : Thomsen

Drennan and Biggart (1930) described a case of eosinophilic response to an irritant in a boy, aged 15 years, whose leucocytes numbered 61,000-73,000 per c.mm. (eosinophils

28-70%). Stephens (1935) described a case of acute eosinophilic leukaemia, in a girl, aged 17, with 130,000 leucocytes per c.mm. (eosinophils 68%), and Reye (1940) another in a boy, aged 51/2 years, with 160,000 leucocytes per c.mm. (eosinophils 72%). Lepak (1940) described eosinophilic leukaemia, possibly complicated by tuberculosis, in a man, aged 41, with 950040,300 leucocytes per e.mm. (eosinophils 15-30%). Bass (1941) reported the case of a boy, aged 61/a years, with 9400-51,000 leucocytes per c.mm. (eosinophils 29-70%). Goehl (1942) reported a case of eosinophilic leukaemia in a male, aged 18, with 7900-190,000 leucocytes per c.mm. (eosinophils 5-88%). Fenner (1943) reported one in a male, aged 19, with 56,00063,000 leucocytes per c.mm. (eosinophils 55%). Friedman et al. (1944) reported one in a boy, aged 11 years, with 126,000 leucocytes per c.mm. (eosinophils 82%). Hodgson et al. (1945) reported a case of myeloid leukaemia, probably eosinophilic, in a woman, aged 44, with 46,000 leucocytes per c.mm. (eosinophils 43%). Sex and Age.-Of the 23 cases 18 have been in males. The age-incidence ranges from 1 year 4 months to 55 years, the distribution being 6 cases in the age-group 1-14 years, 4 in that of 15-20 years, 2 in that of 25-35 years, and 9 in that of 40 years or more; in 2 cases the age was not given. Duration.-The duration of the illness, where stated, was in 5 cases more than two years, and in 13 cases was from a few days to fourteen months, suggesting that eosinophilic leukaemia tends to run an acute rather than a chronic course. Eosinophilia.-The percentage of eosinophils ranged from 41 to 70, with an average of 63. The white-cell count fluctuated from normal to more than 200,000 per c.mm., the average being about 76,000. Liver, Spleen, and Lymph-glands.-Enlargement of the liver, spleen, and lymph-glands was not constant. Any combination might be present or absent at any time of the disease. In nearly every published case attention was drawn to the maturity of the eosinophils in the peripheral circulation, the coarseness and heaping of the granules, and the vacuolation. Eosinophil and metamyelocytes, myelocytes, myeloblasts, and normoblasts were met with in the serial counts in all the

neutrophil