HEPATIC δ-AMINOLÆVULINIC ACID SYNTHETASE IN AN ATTACK OF HEREDITARY COPROPORPHYRIA AND DURING REMISSION

560 response of a suppressed or atrophied adrenal to normal. 5,11 The speed of the adrenal response to corticotrophin is to some extent a measure of previous corticotrophin stimulation, while the extent of the response is a measure of adrenal size and activity. In this way the rate of response of the adrenal to corticoused to assess the pre-existing trophin may be " " drive to the adrenal. Thus the corticotrophin rate of response distinguishes primary hypoadrenalism (no response) from secondary hypoadrenalism (slow response) and from normality. Various tests on this principle have been proposed. One of the most accurate methods uses prolonged intravenous corticotrophin infusions.12 Most such tests are cumbersome ; and we consider that our proposed method, usually involving merely three venepunctures in 24 hours, is simple enough to be used in any hospital or in general practice. However, some cases will

require repeated injections to prove recoverability of the adrenal, and to disprove Addison’s disease when profound secondary hypoadrenalism is present. Such assessment of adrenal reserve may be important in steroid-treated patients-for example, when surgery is contemplated. The insulin hypoglycxmia test has been used to test the whole hypothalamopituitary-adrenal axis in this context, with fairly reliable results. 13 However, it is a much more elaborThe simpler stimulation test with depot ate test. tetracosactrin may well prove to give the same information about the immediate adrenal treated patients.

reserve

in steroid-

The " standard " synacthen test, 2,8 in which plasmacortisol is measured before and 30 or 60 minutes after intramuscular injection of short-acting tetracosactrin, usually demonstrates Addison’s disease satisfactorily. A normal response is often taken to be a rise of 100% We found the mean or more in plasma-cortisol. be 30-5 to plasma-cortisol g. per 100 ml. in 8 normal minutes 60 after intramuscular injection of subjects 0-25 mg. of short-acting synacthen. However, this test is unsatisfactory in differential diagnosis between primary and secondary hypoadrenalism, for its duration is too short to reveal the sluggish adrenal e The proresponse in secondary hypoadrenalism. longed action of synacthen depot results in a greater increment of plasma-cortisol in normal subjects, and distinguishes between primary and secondary hypoadrenalism in many cases. It may thus be less likely to produce doubtful or intermediate responses than the " standard " synacthen test, and so it is a valuable screening-test of adrenal function. We do not consider urinary free cortisol estimation to offer practical advantages over plasma-cortisol estimation in our proposed test for diagnostic purposes, although we have found both in this test and in the " standard " synacthen test that the increase in urinary free cortisol excretion is relatively greater than the increase in plasma-cortisol. However, urinary free cortisol is a useful measure of the biological efficacy The rate of urinary free of adrenal stimulation. cortisol excretion is more or less proportional to the non-protein-bound (biologically active) concentration of cortisol prevailing in plasma. 14 Urinary free cortisol excretion over a period is thus a convenient

of the tissue exposure to unbound plasmacortisol (concentration x time). It appears that with normal adrenals 1 mg. of synacthen depot increases this tissue exposure to cortisol about 30-fold during the first 24 hours after injection or about 20-fold over the 48 hours following injection. Used in this way, urinary free cortisol estimation may be a more realistic indicator than total plasma-cortisol of the true biological effect of administered corticotrophin, and perhaps deserves wider application.

measure

A. G.-T. is in receipt of a research grant from the Calouste Gulbenkian Foundation, and R. F. is in receipt of a grant from the Medical Research Council. Our thanks are also due to Dr. T. B. Binns and to Ciba Laboratories for a grant for expenses. REFERENCES

Landon, J., James, V. H. T., Cryer, R. J., Wynn, V., Frankland, A. W.J. clin. Endocr. 1964, 24, 1206. 2. Wood, J. B., Frankland, A. W., James, V. H. T., Landon, J. Lancet, 1965, i, 243. 3. Besser, G. M., Butler, P. W. P., Plumpton, F. S. Br. med. J. 1967, iv, 391. 4. Nuki, G., Shepherd, J., Downie, W. W., Dick, W. C., Hainsworth, I. R. Lancet, 1969, i, 188. 5. Sandberg, A. A., Eik-Nes, K., Migeon, C. J., Koepf, G. F.J. Lab. clin. Med. 1957, 50, 286. 6. Beardwell, C. G., Burke, C. W., Cope, C. L. J. Endocr. 1968, 42, 1.

79.

7. De Moor, P., Heirwegh, K., Heremans, J. F., Declerck-Raskin, M. J. clin. Invest. 1962, 41, 816. 8. Greig, W. R., Jasani, M. K., Boyle, J. A., Maxwell, J. D. Mem. Soc. Endocr. 1968, 17, 175. 9. El-Shaboury, A. H. Br. med. J. 1968, iii, 653. 10. Nelson, J. K., Neill, D. W., Montgomery, D. A. D., MacKay, J. S., Sheridan, B., Weaver, J. A. ibid. 1968, i, 557. 11. Krusius, F.-E., Oka, M. Ann. rheum. Dis. 1958, 17, 184. 12. Rose, L. I., Williams, G. H., Jagger, P. I., Lauler, D. P. Ann. intern. Med. 1970, 73, 49. 13. Plumpton, F. S., Besser, G. M. Brit. J. Surg. 1969, 56, 216. 14. Beisel, W. R., Cos, J. J., Horton, R., Chao, P. Y., Forsham, P. H. J. clin. Endocr. 1964, 24, 887.

HEPATIC &dgr;-AMINOLÆVULINIC ACID SYNTHETASE IN AN ATTACK OF HEREDITARY COPROPORPHYRIA AND DURING REMISSION

A. J. G. PEARSON

NEIL MCINTYRE

Department of Medicine, Royal Free Hospital (North Western Branch), London N.W.3 D. J. ALLAN

Department of Chemical Pathology, Mount Vernon Hospital, Northwood, Middlesex S. CRASKE G. M. L. WEST Hollowav Sanatorium,Virginia Water, Surrey M. A. D. BEATTIE A. GOLDBERG J. PAXTON

R. MOORE

Medical Research Council Group in Iron Metabolism and Porphyrin Metabolism, Stobhill General Hospital, Glasgow

patients with hereditary coproporphyria have been investigated, two an acute attack and two during remission. In during all four, urinary and fæcal coproporphyrin levels were significantly raised. In one of the patients studied during an attack, hepatic &dgr;-aminolævulinic acid (A.L.A.) synthetase activity was high, but it was normal in a patient in remission at a time when urinary A.L.A. and porphobilinogen levels were also normal. Blood A.L.A. Summary

Four

561 also elevated in two patients who were studied during an attack. In both of these patients excessive urinary excretion of 17-oxosteroid was also found, while in the two patients in remission urinary 17-oxosteroid excretion was within the normal

dehydrase activity

was

range.

Introduction ACUTE intermittent porphyria

(A.i.p.), porphyria variegata, and hereditary coproporphyria are disorders of porphyrin metabolism which are inherited as mendelian autosomal dominants.1 In all three diseases, clinical attacks are episodic and include abdominal pain, constipation, hypertension, and a variety of neurological and mental disturbances. Attacks are often provoked by barbiturates, sulphonamides, and other drugs, and these attacks are accompanied by an excessive urinary excretion of 8-aminolasvulinic acid (A.L.A.) and porphobilinogen (P.B.G.). Despite these similarities, the three diseases can be distinguished by the pattern of fsecal porphyrin excretion. In hereditary coproporphyria, large amounts of coproporphyrin are constantly present in the faeces; in porphyria variegata, fxcal excretion of both coproporphyrin and protoporphyrin is excessive, while in A.I.P. there is only a slight increase in excretion of both of these substances. In both A.I.P. and porphyria variegata there is an excessive activity of hepatic A.L.A. synthetase, 2-5 an enzyme which catalyses the formation of A.L.A. from succinyl CoA and glycine. This is generally considered to be the rate-limiting step in porphyrin biosynthesis.s We present here the results of measurement of hepatic A.L.A. synthetase activity in hereditary coproporphyria. In one patient investigated during an attack activity was high; this confirms the observation of Kaufman and Marver.’ However, in a second patient, A.L.A. synthetase activity during remission was normal. We have also studied the urinary excretion of certain 17-oxosteroids in hereditary coproporphyria, since large amounts of these compounds may be excreted during an attack of A.I.P.a

Case-reports Case1 A 30-year-old housewife with a past history of anorexia nervosa and depression was admitted to hospital in June, 1969. After hysterectomy and appendicectomy, butobarbitone and a variety of other drugs were given; she began to complain of lower abdominal pain, anorexia, and constipation and became increasingly agitated and depressed. She was discharged on July 14, but her mental state deteriorated. She was readmitted on July 22 and

given amylobarbitone, imipramine, and electroconvulsive therapy. Depression improved, but she experienced frequent periods of acute restlessness, confusion, and paranoia. She walked unsteadily, and she had a coarse tremor of arms and legs. On Aug. 7 P.B.G. was found in the urine, and A.I.P. was diagnosed. Barbiturates and other drugs were withdrawn, and treatment with chlorpromazine was started. Abdominal pain, which had been a persistent feature, cleared within 24 hours, but her mental state remained abnormal. After transfer to a general hospital her blood-pressure

was

found to be normal but the pulse-rate was invariably above 90 per minute. Knee jerks became progressively more difficult to elicit, but there was no other objective evidence of neurological disease and there was no reduction in vital capacity. Haematological investigations, blood-urea, and serum-

was

electrolytes were normal. Serum-bilirubin was less than 1-0 mg. per 100 ml.; alkaline phosphatase 8 KingArmstrong units per 100 ml.; aspartate transaminase 110 l.u. per litre; total proteins 6-4 g. per 100 ml. (albumin 4-0, globulin 2-4). The high aspartate transaminase suggested the possibility of structural liver damage, and liver biopsy was done on Sept. 22. Histological examination revealed a normal lobular architecture with slight portal fibrosis. A mononuclear-cell infiltrate was present in the portal tracts and to a lesser degree in the parenchyma; there was a small amount of stainable iron. The findings were compatible with recent liver-cell necrosis, but there was no evidence of chronic liver disease. The liver tissue fluoresced weakly in ultraviolet light. Part of the biopsy specimen was used for measurement of A.L.A. synthetase. Severe constipation delayed faecal porphyrin analysis. When the stools were eventually analysed they were found to contain large amounts of coproporphyrin (table I), and the diagnosis of hereditary coproporphyria was established. The patient’s mental state gradually improved and she was discharged at the end of September. Several members of her family were investigated, and her mother and sister were both found to have a latent form of hereditary coproporphyria with elevated coproporphyrin levels in the urine and faeces. Case 2 A 17-year-old girl was admitted to hospital in April, 1968, with a 5-day history of colicky abdominal pain and diarrhcea. She had previously been healthy and there was no relevant family history. On physical examination, tenderness was elicited in the right iliac fossa. Her appendix was removed using thiopentone sodium to induce ansesthesia. The appendix was normal but it was found that she was 2 months’ pregnant. Post-operatively she vomited and a mild pyrexia developed. Her blood-pressure was 170/100 mm. Hg. 1 month later jaundice appeared, with a

INTERMEDIATES AND HEPATIC AND BLOOD LEVELS OF TABLE I-URINARY AND FaeCAL EXCRETION OF H&M-SYNTHESIS TWO ENZYMES OF THE HaeM BIOSYNTHETIC PATHWAY

*

Means of

duplicate estimations. t

These results

were not

obtained during the

same

admission

as

the hepatic

A.L.A.

synthetase

measurement.

562 bilirubin of 3-8 mg. per 100 ml. and raised transaminases. 3 weeks after the onset of the icterus she developed a bullous eruption on the light exposed parts of her body which lasted for 1 week. At this time excessive amounts of coproporphyrin were found in urine and faeces. She remained well until November, 1968, when she was delivered of a stillborn child. A second pregnancy proceeded uneventfully, and a normal child was born in December, 1969. 1 week after delivery she became acutely ill with abdominal pain and vomiting. The blood-pressure rose to 220/130 mm. Hg and several epileptic seizures occurred. Large amounts of A.L.A. and P.B.G. were found in the urine. After 10 days she improved rapidly and has remained symptom-free. Three members of her family show excessive excretion of fsecal coproporphyrin.

Case 3 A 48-year-old man was originally investigated in 1966 because his sister was severely ill with hereditary coproporphyria. He gave a history of nervousness for a number of years and admitted to excessive intake of alcohol. There was no history of abdominal pain, vomiting, or constipation and his urine had never been dark. The only abnormality on physical examination was slight hepatomegaly. Urine and stool contained grossly elevated levels of coproporphyrin. He has remained symptom-free. Liver biopsy was carried out to establish whether alcoholic liver disease was present. Histological examination revealed normal liver tissue. Case 4 A 26-year-old housewife presented in 1966 during A urinary-tract infection was her second pregnancy. treated with a short course of sulphonamides; the urine became dark and was found to contain increased amounts of P.B.G. Her only other symptom was depression of 2 years’ duration. Quantitative porphyrin analysis showed large amounts of coproporphyrin in both urine and fæces. The child died shortly after birth; the cause of death was not established. She next presented in 1968 with a further attack of depression during the early stages of her third pregnancy. This settled quickly and she gave birth to a normal child. Since that time she has remained in remission. No evidence of hereditary coproporphyria has been found in other members of her family.

Biochemical

Investigations following investigations were done in all four patients: urinary P.B.G., A.L.A., uroporphyrin, coproporphyrin, and 17-oxosteroids; faecal coproporphyrin and protoporphyrin; blood-levels of A.L.A. dehydrase. Two patients (cases 1 and 2) had an acute attack at the time of biochemical investigation. Hepatic A.L.A. synthetase activity was measured in percutaneous liver-biopsy specimens from two patients-during an attack (case 1) and in The

remission (case 3).

Urinary A.L.A. and P.B.G. were measured by the method of Mauzerall and Granick,9 and urinary and faecal porphyrins were measured by the methods of Rimington.1o was assayed by the awl. using a whole-tissue as a substrate. Blood A.L.A. dehydrase activity was assayed by the method of Moore et al.ll Individual urinary 17-oxosteroids were estimated by the method of O’Kelly. 12 The steroid conjugates in a 24-hour specimen of urine were split, the sulphates by solvolysis and the glucuronides by glucuronidase. The free steroids were extracted, separated into groups by thinlayer chromatography, converted to trimethylsilyl ethers, and measured by gas-liquid chromatography.

Hepatic

*

synthetase activity et

Results The results of the measurement of urinary and faecal porphyrins and their precursors are presented in table i. In all four patients, faecal coprophyrins were very high while protoporphyrins were present in normal amounts. Urinary uroporphyrin and coproporphyrin levels were well above normal; they were higher in the two patients in an attack than in the two patients in remission. The figures for porphyrin excretion presented for case 3 were obtained, not at the time of his liver biopsy, but at a previous admission. In the two patients with acute symptoms, urinary A.L.A. and P.B.G. levels were higher than the normal range for our laboratory. In addition, both were found to have elevated levels of blood A.L.A. dehydrase (table i), an enzyme which was not elevated in the two patients in remission. Hepatic A.L.A. synthetase activity was measured in two patients on specimens of liver obtained by percutaneous needle biopsy. In case 1 the A.L.A. synthetase activity during an attack was very high; in case 3 A.L.A. synthetase activity during remission was normal. The results of estimation of the fractionated urinary 17-oxosteroids in these patients are presented in table 11. Only in the two patients in an attack (with elevation of urinary A.L.A. and P.B.G.) were urinary oxosteroid levels elevated. In patient 2, epiandrosterone sulphate and 11-oxyandrosterone glucuronide were elevated, and in patient 3, 11 -hydroxy-xtiocholanolone glucuronide was elevated. Discussion

The results presented in table i support a diagnosis of hereditary coproporphyria in all four patients. Fæcal coproporphyrin excretion was strikingly raised without an accompanying increase in protoporphyrin,

TABLE II-HEREDITARY COPROPORPHYRIA: URINARY

t

A.L.A.

micromethod of Dowdle homogenate with glycine

17-OXOSTEROID EXCRETION

(µG./24 HR.)

These results are the mean values from 7 samples of urine. Normal values were obtained from 17 normal males and 17 normal females.

563

high levels of urinary uroporphyrin and coproporphyrin. In the two patients with symptoms, urinary A.L.A. and P.B.G. levels were also increased. In patient 1, percutaneous liver biopsy was done towards the end of an acute attack; her symptoms were improving rapidly but urinary excretion of A.L.A. and P.B.G. was still abnormal. Hepatic A.L.A. synthetase activity was high. In patient 3, biopsy was done when there were neither symptoms nor elevation of urinary A.L.A. and P.B.G.; hepatic A.L.A. synthetase activity was and there

were

normal. Increase in hepatic A.L.A. synthetase activity has been demonstrated in previous studies of the hereditary hepatic porphyrias. Tschudy and his colleagues were the first to make this observation on liver tissue, obtained soon after death, from a patient with A.I.P., and this finding has been confirmed in other patients with the same condition.3-5 Dowdle et al.4 and 5 also an in hepatic A.L.A. found increase Masuya with in synthetase activity patients porphyria variegata, and Kaufman and Marvermade a similar observation in a patient with hereditary coproporphyria. In four of the above reports it was clear that the high A.L.A. synthetase activity was accompanied by elevation of urinary A.L.A. and P.B.G. levels even though several of the patients were in remission; in the fifth, no details of urinary A.L.A. and P.B.G. excretion were given. Our patient 3 had normal urinary levels of A.L.A. and P.B.G., but hepatic A.L.A. synthetase activity was also normal. We do not know the precise nature of the biochemical defects in A.I.P., porphyria variegata, and hereditary coproporphyria. Tschudy et al. suggested that the biochemical manifestations of A.I.P. could be explained simply by an increase in the activity of hepatic A.L.A. synthetase. On this basis they postulated that the genetic abnormality in A.I.P. might be a defect of a regulator gene which normally controls the synthesis of A.L.A. synthetase. The resemblance of the clinical and biochemical features of A.I.P. to those of porphyria variegata and hereditary coproporphyria suggests that a similar metabolic abnormality must be present in all three. However, it is clear that a single regulator-gene defect with increase in A.L.A. synthetase activity could not account for the differences in the pattern of porphyrin excretion which occur in the three conditions. For this reason the hypothesis put forward by Kaufman and Marverseems to be more attractive. They suggested that the primary abnormality in A.I.P., porphyria variegata, and hereditary coproporphyria is a partial block in haem biosynthesis, the block being at a different point in the pathway in each of the three diseases.7 In the case of hereditary coproporphyria the defect would presumably be in the conversion of coproporphyrinogen ill to protoporphyrinogen by the enzyme coproporphyrinogen oxidase. Since hasm inhibits the synthesis of A.L.A. synthetase, an increase in enzyme activity would result if free hxm levels dropped due to the block in hæm synthesis. Secondary elevation of A.L.A. synthetase could account for the increase in A.L.A. and P.B.G. excretion which occurs in all three diseases, whilst the site of the block would explain the differing patterns of porphyrin excretion. This hypothesis would also allow an explanation for the exacerbation of these diseases which is often seen after the administration of certain drugs. Drugs such

barbiturates induce the synthesis of hepatic cytochrome P-450, a hsem-containing compound which plays an important part in microsomal oxidation of many drugs. 13 Administration of such inducing agents might lead to an intracellular demand for haem which could not be adequately met in the presence of a block in ha:m biosynthesis. Should intracellular hxm levels fall a secondary rise in A.L.A. synthetase activity could then result. In case 3, A.L.A. and P.B.G. excretion and hepatic A.L.A.-synthetase activity were normal. This observation is also explicable on the basis of Kaufman and Marver’s hypothesis, for, if demands were reduced, haem production might be adequate even in the presence of a partial block in its biosynthetic pathway; A.L.A. synthetase activity could then be normal. However, feecal coproporphyrin levels in hereditary coproporphyria are always very high, and it is difficult to see how porphyrin excretion can be so high if the rate of synthesis of A.L.A. is normal. It will be interesting to see whether the lack of elevation of A.L.A. synthetase activity found in this patient can be confirmed in patients with other hereditary hepatic porphyrias at times when A.L.A. and P.B.G. excretion are normal. A.L.A. dehydrase activity was also increased in two patients studied during an attack. This enzyme catalyses the conversion of A.L.A. to P.B.G. The significance of its presence in blood is not known, but it has been shown by other workers that its activity in the liver is increased in the hereditary hepatic porphyrias. 3,7 It would seem likely that A.L.A. dehydrase in blood is a consequence of leakage from hepatocytes and that elevation of the hepatic concentration is associated with a rise in blood activity. The elevation of A.L.A. dehydrase activity in the hereditary hepatic porphyrias is modest compared with the rise in A.L.A. synthetase. Furthermore, the capacity to utilise A.L.A. via A.L.A. dehydrase is not rate-limiting. It is thus unlikely that a rise in A.L.A. dehydrase activity is of major importance in the pathogenesis of these diseases. The increase in its activity may be related to the significant increase in the concentration of its substrate. The spontaneous development (i.e., not drug induced) of many acute episodes in the hereditary hepatic porphyrias led Granick and Kappas 14,15 to postulate that naturally occurring substances might be involved in the regulation of A.L.A. synthetase activity. They showed that a large number of steroid metabolites were potent stimulants of A.L.A. synthetase activity in chick-embryo liver cells; these compounds included

as

dehydroepiandrosterone, epiandrosterone, 11-hydroxySubseætiocholanolone, and 11-oxyandrosterone. and his 8 demonstrated quently, Goldberg colleagues that the excretion of 17-oxosteroids was increased in some patients with A.i.p. both in attacks and during remission; they also found that injection of dehydroepiandrosterone into intact rats led to significant increases in A.L.A. synthetase activity. We measured the urinary excretion of conjugates of the four compounds mentioned above. In the two patients in remission none of the steroids were excreted in excessive amounts; in one of the patients suffering an attack there was an increase in urinary epiandrosterone and 11-oxyandrosterone, while in the other there was a marked elevation of urinary 11-hydroxyxtiocholanolone.

564 The

significance of these findings is not clear; one of patients with increased oxosteroid excretion was given drugs known to provoke acute episodes of coproporphyria, and it is not necessary to invoke an Abnormal oxosteroid oxosteroid as a precipitant. excretion might have been secondary to the acute episode or abnormalities of oxosteroid production might enhance the provocative effect of drugs or play a role in determining the duration of attacks. Although our findings lend general support to the concept of Granick and Kappas, further work is clearly necessary to determine the precise role of steroid metabolites in the precipitation of symptomatic episodes. our two

We thank Dr. Peter Scheuer for interpreting the liver biopsy of patient 1; Dr. A. W. M. Smith and Dr. J. A. A. Hunter, of the Victoria Hospital, Kirkcaldy, supplied the samples and casehistory of patient 2; we thank Dr. J. K. Grant, department of steroid biochemistry, Royal Infirmary, Glasgow, for allowing one of us (J. P.) to use his laboratory. Mr. G. G. Thompson gave valuable technical help.

Requests for reprints should be addressed

to

N. M.

REFERENCES

Goldberg, A., Rimington, C., Lochhead, A. C. Lancet, 1967, i, 632. Tschudy, D. P., Perlroth, M. G., Marver, H. S., Collins, A., Hunter, G., Rechsigl, M. Proc. natn. Acad. Sci. U.S.A. 1965, 1966, 53, 841. 3. Nakao, K., Wada, O., Kitamura, T., Uono, K., Urata, G. Nature, 1966, 210, 838. 4. Dowdle, E. B., Mustard, P., Eales, L. S. Afr. med. J. 1967, 41, 1093. 5. Masuya, T. Acta hœmat. jap. 1969, 32, 519. 6. Tait, G. H. in Porphyrins and Related Compounds (edited by T. W. Goodwin); p. 19. London, 1968. 7. Kaufman, L., Marver, H. New Engl. J. Med. 1970, 283, 954. 8. Goldberg, A., Moore, M. R., Beattie, A. D., Hall, P. E., McCallum, J., Grant, J. K. Lancet, 1969, i, 115. 9. Mauzerall, D., Granick, S. J. biol. Chem. 1956, 219, 435. 10. Rimington, C. Association of Clinical Pathologists Broadsheet, 1961, no. 36. 11. Moore, M. R., Beattie, A. D., Thompson, G. C., Goldberg, A. Clin. Sci. 1971, 40, 81. 12. O’Kelly, D. A. PH.D. thesis, University of Glasgow, 1968. 13. Schmidt, R., Marver, H. S., Hammaker, L. Biochem. Biophys. Res. Commun. 1966, 24, 319. 14. Granick, S., Kappas, A. J. biol. Chem. 1967, 242, 4587. 15. Kappas, A., Granick, S. ibid. 1968, 243, 346. 1. 2.

bility is considered that neoplasms might arise from the interaction with the surrounding tissues of abraded wear particles from total joint replacement prostheses made entirely of cobalt-chromium alloy. We have considerable information on the biochemical interaction of cobalt, which is one component of this alloy, with tissues and body-fluids and on its mode of solution under these conditions. Comparable studies on the finely powdered complete alloy are now in progress.

Previous work 2-4 has shown the carcinogenicity of powdered pure metallic cobalt, cadmium, and nickel when suspended in chick or horse serum and injected into the skeletal muscle of rats. The criticism may be made that rats are very likely to develop tumours with a number of experimentally introduced agents. However, under our conditions, tumours have not arisen with similar injections of powdered metallic zinc or tungsten2 nor with injections of powdered iron, tantalum, chromium, beryllium, manganese, copper, and molybdenum.5 Arsenic has also been implanted but was found to be so toxic that the animals did not survive long enough to allow tumour formation.5 Malignant transplantable and metastasising tumours (including fibrosarcomas, rhabdomyosarcomas, and cellular sarcomas) can arise very quickly after implantation of powdered cobalt, nickel, and cadmium; the earliest tumours appear at about 3 months and the incidence of tumours may be as high as 100% of all animals injected. More commonly the incidence is around 50-75 %. The histogenesis of such tumours in rat skeletal muscle has been described.6 In view of this we sought possible carcinogenic effects of the wear particles formed from total joint replacements made of cobalt-chromium alloy. Such particles are undoubtedly formed with clinical use, since soft-tissue staining is visible in the region of these joint replacements on later exploration or at necropsy. Materials and Methods

CARCINOGENIC PROPERTIES OF WEAR PARTICLES FROM PROSTHESES MADE IN COBALT-CHROMIUM ALLOY

J. C.

Production of Wear Particles A machine has been constructed in which prostheses for the total replacement of the hip and knee may be TABLE I-CHEMICAL COMPOSITION

(%

BY

WEIGHT)

OF ALLOY AND

WEAR PARTICLES

HEATH

Strangeways Research Laboratory, Cambridge M. A. R. FREEMAN

Hospital, London E.1, and Biomechanics Unit, Imperial College of Science and Technology, London S.W.7

London

S. A. V. SWANSON Biomechanics Unit, and

Imperial College of Science

Technology

Particles produced by the working, in Summary a bath of Ringer’s solution, of an artificial total joint made from cobalt-chromium alloy have been shown to be carcinogenic for rat muscle. Introduction

TOTAL joint replacement is becoming clinically commonplace, and it is therefore important to consider the biological and engineering properties of available bearing materials. In this paper the possi-

worked continuously under mechanical, chemical, and thermal conditions simulating those in life. The prostheses were lubricated either with Ringer’s solution or with synovial fluid. In both environments, although to a lesser extent in the latter, detritus from articulating cobalt-chromium alloy surfaces appeared as a black deposit in which the particles ranged down to 0.1 µ in diameter. The apparently larger particles may have been aggregates of smaller particles of this size. The chemical composition of one sample of the parent alloy and of one sample of detritus is shown in table i. Three samples of this debris (samples A, B, and C) were collected and injected into the skeletal muscle of the