AMINOACETONE IN ACUTE INTERMITTENT PORPHYRIA

660

methicillin had failed, in 6 cases chloramphenicol, in 6 cases erythromycin, and in 1 case novobiocin. The longest period of continuous treatment was twenty-four days (total dose 36 g.), but one patient had two courses of twenty-one days’ each (total 68 g.). For the 21 cases the number of treatment-days was 289. I shall not discuss synergism but merely state the relevant facts: In 1 case, after

days’ treatment with fusidic acid and phenoxymethyl penicillin, the staphylococcus, which had been penicillin-resistant before treatment, was found to be penicillinseven

sensitive. In another case where there had been no response to fusidic acid alone, phenoxymethyl penicillin was added, and in five days the phage-type had changed to phage-type 75/77. In a third case, after ten days with fusidic acid and penicillin V 125 mg. t.d.s., the phage-type changed to 52A/79. In all these cases with continued treatment the organism was completely eliminated.

In these 21

the average length of stay was 29-3 comparable series of 34 cases in 1961, before fusidic acid was used, the average stay was 44-8 days. This represents an increase in bed-turnover of 35%. With other antibiotics one can usually say that if five to seven days’ intensive therapy has not produced results, longer courses will seldom be effective; but our experience suggests that in intractable cases it is worth continuing combined therapy with fusidic acid and phenoxymethyl penicillin for at least three to four weeks. Despite long courses of treatment, such as twenty-one days on each of two occasions, there has been no evidence of emergence of resistant strains of staphylococci. Bacteriological investigations with fusidic acid (Barber and Waterworth 1962, Newman et al. 1962) suggest that resistance may be acquired fairly easily; but we have not encountered it-possibly because our patients had phenoxymethyl penicillin as well as fusidic acid. The fact that all the patients were isolated in separate cubicles must also have helped to prevent emergence of resistant strains (Blowers 1961). The fact that all these 21 cases were completely cleared of staphylococcal infection, and that each case was an end case " and not the beginning of a further series of ward cross-infections, is strong evidence cases

days, whereas in

a

"

in favour of ruthless isolation of any such infections immediately. As Allan Gray et al. (1962) says, " no infection however trivial clinically should be ignored ".

Summary The sodium salt of fusidic acid (’ Fucidin ’), combined with phenoxymethyl penicillin, has proved extremely effective in clearing resistant staphylococci-in this case epidemic phage-type 80-from a variety of lesions. In some cases this combination has been successful after failure of a reasonable course of other antibiotics. In 21 cases receiving a full dose of fusidic acid (500 mg. three times a day for a total of 289 patient-days) no sideeffects were seen. Nor did resistant strains emerge. There is some further evidence of a synergistic action between fusidic acid and phenoxymethyl penicillin

(Waterworth 1963). I am indebted to Dr. Austin Darragh, of Leo Laboratories, for supplies of ’Fucidin’; and to Dr. John Anderson, medical superintendent, for permission to publish. REFERENCES Allan

Gray, J. D., Chandler, G. N., Roberts, J. G., Lambert, R. A. (1962) Lancet, ii, 1251. Barber, M., Waterworth, P. M. (1962) ibid. i, 931. (1963) Clin. Med. 70, 5. Blowers, R. (1961) J. clin. Path. 14, 18. Newman, R. L., Bhat, K. M., Hackney, R., Robinson, C., Stewart, G. T. (1962) Brit. med. J. ii, 1645. -

AMINOACETONE IN ACUTE INTERMITTENT PORPHYRIA DONALD P. TSCHUDY

FREDERICK H. WELLAND

M.D. Columbia

M.D. Harvard

ANNIE COLLINS

GEORGE HUNTER B.S.S. Carolina State Morgan From the Metabolism Service, National Cancer Institute, National Institutes of Health, Bethesda, 14, Maryland, U.S.A. B.S.

AMINOACETONE can be formed enzymatically by the condensation of glycine and acetyl coenzyme A (Gibson et al. 1958, Kikuchi et al. 1959, Urata and Granick 1961) or by the oxidation of the hydroxyl group of L-threonine (Elliot 1959, Neuberger and Tait 1960, Urata and Granick 1961). This substance is found in the urine of normal individuals (Mauzerall and Granick 1956), but has been reported absent from the urine of patients with acute intermittent porphyria as well as from the urine of animals with experimentally induced porphyria (De Matteis 1962, De Matteis and Rimington 1962). We describe here the presence of a substance with the properties of aminoacetone in the urine of patients with acute intermittent porphyria. Methods Aminoacetone nitrosation of

hydrochloride was synthesised by the ethylacetoacetate to form oximinoacetone (Charrier 1907), followed by reduction with stannous chloride and hydrochloric acid. (Calculated for aminoacetone hydrochloride : C, 32-89; H, 7-36; N, 12.79. Found: C, 32-51; H, 7-96; N, 12-17.) The aminoketone formed a pyrrole in reaction with acetylacetone under the conditions described by Mauzerall and Granick (1956). The Rp of this pyrrole in butanol-ammonia (upper layer) was 0-93 as previously described (Urata and Granick 1963) and in butanol-acetic acid (upper layer) the Rp was 0-98. The pyrrole formed from the reaction of 8-aminolevulinic acid (A.L.A.) with acetylacetone can be separated from the pyrrole formed from condensation of aminoacetone with acetylacetone, because the former pyrrole can be extracted into ether under certain conditions, whereas the latter pyrrole remains in the aqueous phase (Mauzerall and Granick 1956, Urata and Granick 1961, 1963). This method was used to separate these two pyrroles in the urine of patients with acute intermittent porphyria. 5 or 10 ml. of urine from a 24-hour collection was adjusted to a pH of 4-6 with an equal volume of buffer. 0-25 ml. acetylacetone was added, and the mixheated to 100°C for 10 minutes. The pH was then adjusted to 7-0 with sodium hydroxide, and the solution was extracted with two volumes of ether. The ether was evaporated gently in warm water or allowed to evaporate overnight in the hood. Evaporation under reduced pressure was found to reduce the yield of pyrrole derived from aminoacetone. The residue was dissolved in 2 ml. water, and an equal volume of modified Ehrlich’s reagent (Mauzerall and Granick 1956) in 2 M perchloric acid was added.

acetate

ture was

After 15 minutes, the optical density of the pink solution was determined at 556 [L in aBeckman DU ’ spectrophotometer. When synthetic aminoacetone was converted to the pyrrole by a similar procedure, the molar extinction coefficient, based on initial concentration of aminoacetone, was 7.2 x 104. This value was used to calculate the amount of aminoacetone in the urine. Porphobilinogen (P.B.G.) and 8-amiuolevulimc acid (A.L.A.) were determined by the chromatographic method of Mauzerall and Granick (1956). Paper chromatography of aminoacetone was performed with the butanol-ammonia and butanol/acetic-acid systems of Mauzerall and Granick

-

(1956). Five

patients

with authentic

acute

intermittent

porphyria

661 AMINOACETONE EXCRETION IN ACUTE INTERMITTENT PORPHYRIA

were

All patients were excreting excessive and A.L.A. at the time, as shown in the table.

investigated.

amounts

of

P.B.G.

accompanying

during the same

Discussion

Results

A compound with the chromatographic and solubility characteristics of the pyrrole derived from aminoacetone by reaction with acetylacetone was shown to be present in the urine of four patients with acute intermittent

porphyria (one

of 25 g. glycine orally, the urine collected time interval contained 0-213 mg.

was

not

chromatographed).

Urinary

aminoketones were converted to their corresponding pyrroles by the reaction with acetylacetone, followed by ether extraction at pH 7-0. When the ether was removed, paper chromatography, with the butanol-ammonia and butanol/acetic-acid systems of Mauzerall and Granick (1956), was performed on the residue. Only one pink spot was present after colour development with Ehrlich’s reagent. The spot had an RF of 0-91-0-93 in butanolammonia, corresponding to that produced when synthetic aminoacetone was used for this procedure (Urata and Granick 1963). This system widely separates the pyrrole derived from A.L.A. (RF 0-2) from that derived from aminoacetone (RF 0-93) (Mauzerall and Granick 1956). Evidence was obtained that the ether extract was free of the pyrrole derived from A.L.A., because no spot with the RF corresponding to this pyrrole was seen. But occasionally, when significant amounts of water were carried with the ether layer, a barely discernible spot corresponding to A.L.A. could be observed.

Paper chromatography of the ether residue from patients with acute intermittent porphyria with the butanol/acetic-acid system produced a spot with RF 0-98, again corresponding to that obtained from synthetic aminoacetone. Thus, in contrast to previous reports (De Matteis 1962, De Matteis and Rimington 1962), when the urine of porphyric patients was reacted with acetylacetone a pyrrole was produced which extracted into ether. This pyrrole had the same RF values in two different solvent systems as that derived from synthetic aminoacetone. The pyrrole derived from 8-aminolevulinic acid was not present in measurable quantity in the ether extract, nor was porphobilinogen. No significant differences are seen in the amount of aminoacetone excreted per 24 hours in five patients with acute intermittent porphyria and three non-porphyric patients (see table). The amount of P.B.G. and A.L.A. excreted by the porphyric patients on the same day is also shown in the table. Aminoacetone is

seen to

represent a small fraction of the total aminoketone excretion in acute intermittent porphyria. The effect of glycine administration orally on aminoacetone excretion was studied in case 4. On a constant dietary intake an 8-hour control urine contained 0-065 mg. aminoacetone, whereas the next day, after administration

The enzymatic defect which causes the increased excretion of A.L.A. and porphobilinogen in acute intermittent porphyria is unknown. From the known metabolic pathways related to the production and utilisation of porphyrin precursors it is possible to postulate enzymatic defects which could lead to excessive production or underutilisation of these precursors. Examination of some of these pathways in experimental porphyria suggested defective glycine oxidation (Tschudy et al. 1962). One of the paths by which glycine might be oxidised is via aminoacetone as postulated by Elliot (1959). Aminoacetone could be converted to methylglyoxal, which would be converted to D-lactic acid by glyoxalase and thence to pyruvate by the D-lactate-dehydrogenase system. Absence of aminoacetone from the urine of patients with acute intermittent porphyria would be compatible with the possibility that a defect in aminoacetone formation from glycine could lead to increased production of porphyrin precursors, and at the same time explain the decreased oxidation of glycine to carbon dioxide as observed in experimental porphyria (Tschudy et al. 1962). But, for several reasons, this does not appear to be the case. First, our data strongly suggest that aminoacetone is not absent from the urine of patients with acute intermittent porphyria. Granick and Urata (1963) have demonstrated the presence of aminoacetone in the urine in experimental porphyria. Secondly, mitochondria from the livers of rats made porphyric by allylisopropylacetamide are not defective in producing aminoacetone from glycine and acetyl coenzyme A (nor from L-threonine) (Tschudy et al.,

unpublished). Decreased availability of acetyl coenzyme A in acute and experimental porphyria has been postulated (De Matteis 1962, De Matteis and Rimington 1962). Previous studies of the oxidation of glucose, pyruvate, and acetate revealed these to be normal in experimental porphyria (Tschudy et al. 1962), suggesting that the enzymatic apparatus for the generation of acetyl coenzyme A from carbohydrate was not disturbed in this disorder. Thus, at present, defective aminoacetone synthesis cannot be regarded as the fundamental defect in acute intermittent porphyria.

Summary a compound which has of aminoacetone has been shown to be present in the urine of patients with acute intermittent porphyria. No great quantitative differences were observed between porphyrics and non-porphyrics in the excretion of this compound.

Contrary

the

to

previous reports,

properties

References overleaf

662

SYSTEMIC LUPUS ERYTHEMATOSUS FOLLOWING THYMECTOMY FOR MYASTHENIA GRAVIS

Report of Two Cases DONATO ALARCÓN-SEGOVIA FELLOW IN

M.D. Mexico MEDICINE, MAYO FOUNDATION

RICHARD F. GALBRAITH FELLOW IN

M.D. St. Louis NEUROLOGY, MAYO

FOUNDATION

JORGE E. MALDONADO FELLOW IN

M.D. Bogotá MEDICINE, MAYO FOUNDATION

FRANK M. HOWARD, Jr. M.D. Pennsylvania

the masseters on chewing, and in the summer of 1954 she began to have diplopia. Shortly before her examination at the Mayo Clinic, ptosis of the right eye developed. On physical examination, nasal speech and weakness of the soft palate were noted. A neostigmine methyl sulphate test resulted in improvement. There was no radiological evidence of thymic enlargement. However, thymectomy was carried out uneventfully on Nov. 10, 1954, with removal of a hyperplastic thymus weighing 13 g. On microscopy, germinal centres were found in the parenchyma of the thymus (fig. 1). Although the patient did not obtain relief immediately after the operation, she later showed great improvement. She needed neostigmine only when she had a severe cold or during profuse menstruation. Returning to the Mayo Clinic in March, 1961, she reported that in March, 1960, she had begun to have migratory arthralgia

OF THE SECTION OF NEUROLOGY

MAYO CLINIC AND MAYO

FOUNDATION, ROCHESTER, MINNESOTA, U.S.A.

IT is now recognised that autoimmune phenomena play a part in at least some of the manifestations of systemic lupus erythematosus. That autoimmunity is responsible also for the pathogenesis of myasthenia gravis was postulated by Simpson (1960). The association of myasthenia gravis with pathological conditions of the thymus is well established (Castleman 1955). Although the role of the thymus in the development of myasthenia remains unknown, the recent recognition of the immunological significance of the thymus (Burnet 1962, Miller 1963) suggests that linkage of the thymus gland to myasthenia gravis may be through immune mechanisms. Burnet (1962) ascribed considerable importance to the thymus as the possible site of origin of the " forbidden clones " which he proposes as responsible for autoimmune disease. He also suggested that thymectomy at an early stage in autoimmune disease might prevent further adverse developments. We present here two cases in which treatment of myasthenia by thymectomy was followed by systemic lupus erythematosus, associated in one of them with chronic ulcerative colitis and hepatic cirrhosis. The latter case is also included in a review of systemic lupus erythematosus in patients with chronic ulcerative colitis (Alarcón-Segovia et al. 1963).

Fig. 1 (case 1)-Thymus. (Hwmatoxylin and eosin ;

Observe the two germinal centres. x

115.)

without redness or swelling of joints and that she had been treated with salicylates. In May, 1960, angioneurotic oedema and painful urticaria had developed, and they had persisted until October, 1960, when she started taking prednisone. This medication had continued up to the time of returning to the Clinic, when the amounts of both salicylates and prednisone were gradually reduced and finally discontinued. At the time of Report of Cases her admission, arthralgia had recurred, and she was found to Case 1.—A 32-year-old woman was seen at the Mayo Clinic have disseminated urticaria. ’The right 3rd and 4th proximal in October, 1954. She had had nephritis (Bright’s disease) at interphalangeal joints were swollen and tender. Other joints 5 years of age but since then had enjoyed good health until 1952, were tender and there was slight effusion in the left knee. when she began to have difficulty in talking, a tight feeling in Urinalysis demonstrated minimal proteinuria and microthe throat, and a nasal voice. The episodes lasted five to ten haematuria. Studies of peripheral blood showed rouleaux minutes and were precipitated by nervous tension. During the formation and slight leucopenia (4800 leucocytes per mm.) with spring of 1953, she had noticed weakness of the right hand, normal differential count. The erythrocyte-sedimentation rate difficulty in swallowing, and nasal regurgitation. In October, was 33 mm. in the first hour (Westergren). Two tests for lupus 1953, she had been in hospital elsewhere. A diagnosis of erythematosus and the flocculation test for rheumatoid factor myasthenia gravis was made and she was treated with neo- gave negative results. Values for blood-urea and serumstigmine bromide. In December, 1953, she noticed weakness of glutamic-oxalacetic-transaminase were normal. One of the serological tests for syphilis (the v.D.R.L.) was weakly reactive; DR. TSCHUDY AND OTHERS: REFERENCES the results of the Kahn and Wassermann tests were + + + and Charrier, G. (1907) Chem. Zbl. 78, 1231. the Hinton test was non-reactive. The electrophoretic fractions De Matteis, F. (1962) Pamninerva med. 4, 368. of serum protein were within normal range. An X-ray of the Rimington, C. (1962) Lancet, i, 1332. thorax showed thickened pleura at the leftbase. H. Lond. W. 1051. (1959) Nature, 183, Elliot, The patient was thought to have systemic lupus erythemaGibson, K. D., Laver, W. G., Neuberger, A. (1958) Biochem. J. 70, 71. Granick, S., Urata, G. (1963) J. biol. Chem. 238, 821. tosus ; but this could not be established at the time, and she was Kikuchi, G., Kumar, A., Shemin, D. (1959) Fed. Proc. 18, 259. on treatment with chloroquine phosphate and dismissed Mauzerall, D., Granick, S. (1956) J. biol. Chem. 219, 435. neostigmine bromide. Neuberger, A., Tait, G. H. (1960) Biochim. biophys. Acta, 41, 164. Tschudy, D. P., Rose, J., Hellman, E., Collins, A., Rechcigl, M., Jr. (1962) She returned to the Mayo Clinic in October, 1961, because of Metabolism, 11, 1287. fatigue and"recurrence of arthralgia and urticaria. She gave a Urata, G., Granick, S. (1961) Biochem. biophys. Res. Commun. 4, 96. biol. Chem. history of fibrositis " and she was found to have synovial (1963) J. 238, 811. —

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