280 also observed in one patient with cirrhosis of the liver and stainable hepatocellular iron and in three relatives of patients with idiopathic haemochromatosis who were given venesection therapy because more than 50% of hepatocytes in sections of a percutaneous liver biopsy specimen contained stainable iron (see figure). Three of our patients (A, C, D) drank generous amounts of beer before treatment but did not drink alcoholic beverages to excess during phlebotomy. In the three relatives with normal serum-ferritin concentrations, 1 ng/ml of serum-ferritin corresponded to approximately 20 mg (16-26) of storage iron calculated from either the initial ferritin value or from the rate of decline in the serum-ferritin concentration. The relationship between the initial ferritin concentration and the estimated size of iron stores was less precise in the patients with a moderate to marked increase in serum-ferritin. Three patients with ferritin values of 1200 ng/ml, 2116 ng/ml, and 1250 ng/ml had estimated body iron stores of 5 g, 5 g, and 16 g respectively. The rate of decline of serumferritin concentration was about the same in five of the patients with idiopathic hxmochromatosis: a fall of 1 ng/ml of serum-ferritin corresponded to approximately 2-5 mg (1-3-4-6) of storage iron. The rate of fall of serum-ferritin in the remaining patient (A) with idiopathic hsemochromatosis was slow until iron stores were nearly exhausted. Leyland and coworkers do not refer to the method used for their analysis of serum-ferritin nor to its precision for known sera assayed concurrently with patients’ sera. They also fail to indicate whether sera from individual patients were assayed with the same set of standards. It is also important to know whether sera from patients were analysed regularly at more than one dilution. Green et al. have alluded to the potential pitfall of the high-dose hook effect in the serum-ferritin assay -sera from patients with severe iron-storage disease mistakenly appearing to have low serum concentrations by a twosite immunoradiometric assay unless they are assayed at high dilutions. Further information on the precision of the technique is required before the results reported by Leyland et al. can be confidently ascribed to biological variations in serum-ferritin concentration during venesection. was

excess

Department of Medicine, University Hospital, and University of Western Ontario, London, Ontario, Canada N6A 5C1

L. S. VALBERG D. MACKINNON J. W. D. MCDONALD

DEMONSTRATION OF DAPSONE IN URINE AND SERUM BY ELISA INHIBITION

S!R,—Conventional methods for demonstrating dapsone are either insensitive or not practicable for general use in leprosy endemic areas. We have developed an immunoassay that is both simple and sensitive. , Diazotised dapsone was conjugated to bovine serum albumin, and 10 mg of conjugate emulsified in Freund’s complete adjuvant was injected into rabbits. Booster injections of 10 mg of conjugate in Freund’s incomplete adjuvant were given after 3 weeks. Anti-dapsone antibody was demonstrated by microscale enzyme-linked immunosorbent assay (micro-ELISA). We used diazotised dapsone/horseshoe-crab haemocyanin conjugate (D.D.S-H.C.H.) to coat the wells of polystyrene microtitre trays, and horseradish-peroxidase-conjugated anti-rabbit IgG antiserum (Institut Pasteur, Paris). Sera positive for anti-dapsone antibody were identified by adding a solution of 5-aminosalicylic acid/H202 and looking for. development of a brown colour. Addition of as little as 0.1fLg dapsone to each well inhibited positive reactions by two doubling dilutions. 10 pg sulphadiazine, a structural analogue of dapsone, did not inhibit the ELISA response to the same extent. A test for inhibition of ELISA (ELISIT) was then developed to detect dapsone in urine and sera from leprosy patients. Urine was collected from 44 leprosy patients in Kenya, 40 of whom were supposed to be taking 300 mg of the drug once a week, whilst the other 4 were taking lower doses. The urine was collected 2 days after a scheduled dose. It was preserved by adding hydrochloric acid. Before testing the urine its pH was adjusted to 7.2. In addition urine taken 6 h after the intake of 300 mg dapsone was obtained from one Dutch volunteer. Urine from 16 healthy individuals not taking dapsone were also tested. Sera of 3 leprosy patients taking 100 mg daily and one volunteer whose blood-sample was taken 6 h after a single dose of 300 mg dapsone were examined too. Controlsera from 4 healthy people not taking dapsone were treated in the same way. The wells of the tray were first coated (30 min, 37 °C) with 100 1 of carbonate-buffered solution (pH 9-6) containing 0.2 fLg D.D.S.-H.C.H. After the normal washing cycle, 50 ,1 of the samples under test were added. The samples were diluted in P.B.s./tween/H.c.H. and 50 }jd of 1/500 diluted anti-dapsone rabbit serum was added. After incubation, washing, conjugate -

HYPERGLYCÆMIC EFFECT OF SYNTHETIC SALMON CALCITONIN

SIR,-Dr Gattereau and his colleagues’ have reported that

single subcutaneous injection of 100 M.R.C. units of synthetic salmon calcitonin resulted in hypergtycsemia, and they suggest that long-term administration of calcitonin may lead to diabetes. We have treated over forty patients with Paget’s disease of bone for up to 8 years with daily injections of synthetic human calcitonin and none has clinical diabetes. Ziegler et al.2 reported that the impairment of glucose metabolism seen after an acute injection of calcitonin was not borne out in the longer term. It is very unlikely that calcitonin treatment will cause diabetes. a

Endocrine Unit,

Royal Postgraduate Medical School and Hammersmith Hospital, London W12 0HS

4.

IMOGEN M. A. EVANS

G. F. JOPLIN IAIN MACINTYRE

Green, R. and others. Blood, 1977, 50, 545.

Crosby, W. H., Conrad, M. E., Wheby, M. S. ibid. 1963, 22, 429. 1. Gattereau, A., Bielmann, P., Durivage, J., Larochelle, P. Lancet, 1977, ii, 5.

1076. 2.

Ziegler, R., Holz, G., Raue, F., Minne, H., Delling, G. in Human Calcitonin and Paget’s Disease (edited by I. MacIntyre); p. 167. 1977.

Demonstration of dapsone by ELISIT. (1) and (2) A-H standard dapsone solutions of 10, 3, 1, 0.3, 0-1, 0.03, 0-01, and 0.003 fig/ml. (3)-(7) A-H and (8) A-D: urine from Kenyan leprosy patients, 1/10. (8) E-H: urine from Dutch volunteer, 1/10, 1/50, 1/250, and 1/1250. (9) and (10) A-H: urine from 16 healthy persons, 1/10. (11) A-F: sera from 3 Dutch leprosy patients (A and B, C and D, E and F duplicates), 1/100. (11) G and H: serum from Dutch volunteer (duplicates), 1/100. (12) A-H: sera from 4 healthy persons (A and B, C and D, E and F, G and H duplicates), 1/100.

281

addition, and so on the tests were read (see figure) and yielded the following results: (1) Dapsone solutions containing 0.3 fLg/ml can be detected

by ELISIT. (2) A positive result for 1 of the Kenyan urine samples may be questionable (well B4). In 2 other samples (C3 and B6) the quantity of dapsone is very low (around 0-3 g/ml. 6 other samples contained not more than 1 fLg/ml (A6, 8, C6, F5-7). The remaining 35 Kenyan urine samples all contained more than 1 fJ-g/ml. (3) 6 h after

a

single dose of 300 mg dapsone, urine diluted

1/250 was positive.

(4) Positive and negative sera can be clearly distinguished when diluted 1/100. The urine samples were used to compare ELISA inhibition with two conventional spectrophotometric procedures.l.2 Out of 10 samples, negative on one or both spectrophotometric procedures, 9 were positive and 1 equivocal on ELISA inhibition

retrospectively. Criteria have been laid down for such a study in cardiac patients,’ and these would serve as a better basis for the measurement of morbidity than those used by Walesby et al. The literature on surgical nutrition is bedevilled with apocryphal tales, and workers must not perpetuate these by failing to define terms of reference. The paper by Walesby et al. is based on a small number of patients with inadequate information about the statistical methods, and no satisfactory objective evidence to support the statement that "nutritionally depleted patients" undergoing cardiac surgery are "at greater risk from postoperative morbidity". Department of Surgery, University of Newcastle upon Tyne, Royal Victorial Infirmary, Newcastle upon Tyne NE1 4LP

A. J. RICH P. D. WRIGHT

testing. Sera from patients taking dapsone, which would be expected contain 1-3 fLg/mPwere dapsone positive by ELISIT even when diluted a hundredfold. The sensitivity of ELISIT and its simplicity may be of practical value in leprosy-endemic countries with limited laboratory services.

SOMATOSTATIN-TOLBUTAMIDE TEST IN HYPERINSULINISM DUE TO BETA-CELL HYPERPLASIA

to

Technical details of the micro-ELIsA (not given here) may be had from H. H. We thank Dr F. van Knapen and his colleagues in the National Institute of Public Health, Bilthoven, Netherlands, for their help, and the Netherlands Leprosy Relief Association for financial sup-

port. H. HUIKESHOVEN

J. E. LANDHEER Department of Tropical Hygiene, Royal Tropical Institute, Amsterdam, Netherlands W.H.O. International Immunology Training and Research Centre, Amsterdam

A. C. V. DENDEREN M. VLASMAN D. L. LEIKER P. K. DAS

O.L GOLDRING K. W. PONDMAN

SIR,-In normal subjects somatostatin significantly inhibits insulin secretion after glucose, glucagon, and tolbutamide stimulation .2 Lorenzi et al. studying four patients with hyperinsulinism (two adenomas, one carcinoma, and another undetermined) found that somatostatin did not inhibit insulin secretion induced by tolbutamide. We have studied a patient with hyperinsulinism due to pancreatic beta-cell hyperplasia. Before pancreatectomy, a tolbutamide (1 g intravenously) stimulation test was done, before Abel, R. M., Fischer, J. E., Mortimer, B. J., Barnett, G. O., Austen, W. G. Archs Surg. 1976, 111, 45. 2. Efendic, S., Luft, R. Claro, A. Acta endocr. 1976, 81, 743. 3. Lorenzi, M., Gerich, J. H., Karam, J. H. and Forsham, P. H. J. clin. Endocr. Metab. 1975, 40, 1121. 1.

NUTRITION AND OPEN HEART SURGERY

SIR,-Mr Walesby and his colleagues (Jan. 14, p. 76) have used two regression equations based on body weight for the calculation of predicted total-body potassium (T.B.K.). Their use of such formulae is open to criticism on two grounds. The formular may give a falsely high value of predicted T.B.K. in the presence of fluid retention which, as Walesby et al. state, is frequently found in the group of patients they studied. Secondly, the formulae are based on data derived from normal adults. There is no evidence that the equations remain valid when used for undernourished patients-indeed one might expect normal homceostatic mechanisms to break down in patients with cardiac failure, the so-called "sick-cell syndrome". The validity of using T.B.K. as a single criterion of undernutrition is also questionable, and Walesby et al. present no other evidence to support their statement that four of their patients were nutritionally depleted or that the remainder were normally nourished. Absence of clinical signs is no guide since these signs, as Walesby et al. state, may be masked by fluid retention. Walesby et al. also fall into the trap of equating duration of hospital stay with morbidity. Delay in discharging a patient may be related to matters completely unconnected with fitness for discharge. Home circumstances, unwilling relatives, or other delays in the hospital or social services may unduly prolong admission, and such incidents are difficult to recognise 1. Bratton, A. C., Marshall, E. K. J. biol. Chem. 1939, 128, 537. 2. Ellard, G. A., Gammon, P. T., Helmy, H. S., Rees, R. J. W. Am. J. trop.

Med. Hyg. 1974, 23, 464. 3. Powell, R. D., De Gowin, R. L., Bennet Eppes, R., McNamara, J. V., Carson, P. E. Int. J. Lepr. 1967, 35, 590.

Time Imn) Insulin and glucose response to tolbutamide stimulation before and after somatostatin infusion. T=Tolbutamide only. ST=Somatostatin-tolbutamide. Tolbutamide give at 10 min, somatostatin

(by infusion) over 0-40 min.