PANCREATIC SOMATOSTATINOMA

666

they should be encouraged to persist with it if it causes some initial abdominal discomfort. This trial was originally intended to include many more patients. The therapeutic value of increasing fibre intake has, however, been clearly demonstrated and it was considered unethical to deprive patients of such

peripheral inhibitory actions, somatostatin immunoreactivity has been localised to gastrointestinal and pancreatic endocrine (D) cells.3-’ All identified islet-cell types seem able to give rise to tumours, producing distinct clinical symptoms. We

treatment.

tin-producing

sponse and even

I am grateful to The Energen Foods Company, Ashford, Kent, for supplying the wheat and bran crispbread; to the consultants of the Radcliffe Infirmary who allowed me to study their patients; to the pharmacy for their help; to the department of medical illustration for the

figures;

and

to

Dr S. C. Truelove for his advice and encourage-

ment.

REFERENCES 1. Painter, N. S., Almeida, A. Z., Colebourne, K. W. Br. med. J. 1972, ii, 137. 2. Plumley, P. F., Francis, B. J. Am. diet. Ass. 1973, 63, 527. 3. Brodribb, A. J. M., Humphreys, D. M. Br. med. J. 1976, i, 425. 4. Findlay, J. M., Smith, A. N., Mitchell, W. D., Anderson, A. J. M., Eastwood, M. A. Lancet, 1974, i, 146. 5. Smith, A. N., Kirwan, W. O., Shariff, S. Proc. R. Soc. Med. 1974, 67, 1041. 6. Avery Jones, F., Godding, E. W. Br. med. J. 1972, ii, 651. 7. Hall, R. C. ibid. 1976, ii, 1076. 8. Søltoft, J., Gudmand-Høyer, E., Krag, B., Kristensen, E., Wulfe, M. R. Lancet,

1976, i, 270.

PANCREATIC SOMATOSTATINOMA Clinical Features and Physiological Implications L.-I. LARSSON M. A. HIRSCH HOLST S. INGEMANSSON J. J. C. KÜHL S. LINDKAER JENSEN G. LUNDQVIST J. F. REHFELD T. W. SCHWARTZ Institute of Medical Biochemistry, University of Aarhus; Department of Clinical Chemistry, Bispebjerg Hospital,

Copenhagen; Department of Surgery C, Rigshospitalet, Copenhagen; Denmark; Department of Endocrinology and Immunochemistry, Ayerst Research Laboratories, Montreal, Canada; Department of Surgery, University of Lund; and Department of Clinical Chemistry, University of Uppsala, Sweden

Summary

case of a tumour producing somatostatin-like immunoreactivity and

The first

bioactivity is presented. The pancreatic tumour was composed of cells indistinguishable from islet D cells. Radioimmunoassay of blood-samples obtained by tumour-vein catheterisation revealed very high levels of somatostatin immunoreactivity. On gel chromatography found to contain at least 4 differimmunoreactive components, one of which eluted in the position of synthetic somatostatin. Extracts from the tumour were potent in inhibiting insulin and glucagon secretion from isolated perfused porcine pancreas. Clinical abnormalities included hypochlorhydria, steatorrhœa, and diabetic glucose tolerance. Conceivably some of these abnormalities may be related to somatostatin hypersecretion from the pancreatic tumour.

tumour extracts were

ent

report here the first recorded

growth-hormone release-inhibiting hormone, somatostatin, is a powerful inhibitor also of insulin, glucagon, and gastrin secretion. 1In accordance with its

of

a

pure somatosta-

Case-report A 55-year-old woman was first admitted to hospital in 1961 because of abdominal pains and atoxic goitre. From 1965 she had diarrhoea (4-6 stools daily), and her abdominal pains became more severe. Laboratory tests confirmed hypochlorhydria and steatorrhoea (76 g fat/24 h). No evidence for pernicious anarmia was obtained, and examination of the upper gastrointestinal tract and a xylose test were normal. In 1971 she had episodes of headache, tachycardia, and possibly flushing. Urinary 5-hydroxyindoleacetic acid and vanillomandelic acid values were, however, normal. During a cholecystectomy a tumour localised in the head of the pancreas and liver metastases were detected. Examination of biopsy specimens showed the tumour to be of endocrine type. Retrospective analyses of X-rays from 1969 showed that the tumour had already been present then. Gastric secretory studies revealed a basal acid output of 0-5meq/h and a pentagastrin-stimulated peak acid output of 1-55 meq/h. At operation pancreas, duodenum, and liver metastases were resected. Postoperatively, her condition deteriorated rapidly and she died. Necropsy revealed central liver necrosis without evidence of tumour residuals.

Methods

Specimens of tissue obtained at operation were either freezedried and vapour-fixed or fixed in Bouin’s fluid before embedding in paraffin. For electron microscopy specimens were fixed in 2.5% glutaraldehyde, postfixed in osmic acid and embedded in Epon 812. The remainder of the tumour was frozen to the temperature of liquid nitrogen and stored at -70"C. The tissue was stained for light-microscopical demonstration of islet A, B, and D cells and by immunocytochemical methods for insulin, glucagon, gastrin, cholecystokinin, vasoactive intestinal polypeptide, secretin, gastric inhibitory polypeptide, motilin, somatostatin, human pancreatic polypeptide, and substance P.6The site of antigen-antibody reaction was revealed either with immunofluorescence or with the PAP method of Sternberger.11 Controls were those recommended by Sternberger8 and included the application of antigen-inactivated antisera. Blood obtained by antecubital-vein puncture was assayed radioimmunochemically for insulin, glucagon, gastrin, and pancreatic polypeptide. In addition, blood obtained by selective tumour-vein catheterisation was assayed for somatostatin. Before radioimmunoassay, somatostatin was extracted from plasma according to the method ot Anmura et al." Somatostatin antibodies were raised in rabbits after conjugation of synthetic somatostatin to bovine serum albumin, and ’1-tyrosyl-somatostatin (Beckman, Geneva) was used as tracer. Tissue from the hepatic metastases was extracted by the method of Arimura et al.and applied to a Sephadex G 25 column and eluted with acetic acid 0.2 mol/1. 4 ml fractions were collected and assayed for somatostatin immunoreactivity. In addition, pooled fractions were assayed for their potency in inhibiting insulin, glucagon, and pancreatic polypeptide secretion from isolated perfused porcine pancreas. 10

Introduction THE

case

tumour.

Results The

cells stained strongly with the silverof Hellerstrom and Hellman and method impregnation showed an ultrastructural appearance characteristic of tumour

667 D cells (fig. 1). Almost all tumour cells displayed a strong granular cytoplasmic staining with the somatostatin antiserum (fig. 2). All controls were negative. Potent antisera against other hormones or hormone can-

didates failed to stain the tumour cells. The extraturnoral pancreatic parenchyma appeared normal except for a pronounced hyperplasia of extrainsular pancreatic creatic polypeptide cells. Radioimmunoassay showed the tumour to contain large amounts of somatostatin immunoreactivity, which upon gel chromatography could be separated in one minor and three major peaks, one of which eluted in a position closely corresponding to that of synthetic somatostatin (fig. 3). From the gel chromatography the fractions were collected into four pools and tested on isolated perfused porcine pancreas. With each fraction a powerful inhibition of insulin and glucagon secretion was obtained, whereas pancreatic-polypeptide

secretion was only slightly affected

(fig. 4).

Blood-samples obtained by selective catheterisation of pancreatic and hepatic veins contained high levels (up to 107 ng/ml somatostatin equivalents) of somatostatin immunoreactivity, whereas blood obtained in similar fashion from patients with other types of endocrine pancreatic

tumours

contained below 3

ng/ml

somatostatin

equivalents. Peripheral-blood samples showed insulin, pancreatic polypeptide, and gastrin concentrations in the low-to-normal range (insulin 3 .u/ml, pancreatic polypeptide 25 pg/ml, gastrin 127 pg/ml) and glucagon concentrations (10 pg/ml) significantly below the normaf range (38-150 pg/ml). Discussion

Immunochemical, biological, and cytochemical eviour patient had a pancre-

dence strongly indicates that atic somatostatin-producing

tumour.

She had

hypo-

EFFLUENT VOLUME

(ml)

Fig. 1-Electron micrograph

of ultrathin section from the pan-

creatic tumour.

The cells are filled with cytoplasmic granules indistinguishable from those of normal pancreatic D cells.

Fig. 2-Section

(x 6600.)

from the pancreatic tumour stained with tostatin antiserum (PAP technique).

All

tumour

’500.)

cells

display

soma-

intense somatostatin immunoreactivity.

Fig. 3--Gel chromatography on Sephadex G25 column (20mm x 600 mm) of extract from tumour tissue. One minor and three major somatostatin-iminunoreactive components are seen. Void volume (Vo) is defined by 1251-albumin and total volume (Vt) by 22Na.

Fig. 4-Effects of crude tumour extract (1) and four different pooled fractions (2-5) from the gel-chromatographic fractionation on insulin and glucagon secretion from isolated perfused porcine pancreas. The lower portion illustrates the levels of glucose in the perfusing medium. Both with crude extract and with the separate fractions a pronounced inhibition of insulin and glucagon secretion is obtained.

668

chlorhydria, steatorrhoea, and impaired glucose tolerance. Retrospective analysis of our tumour records has revealed another patient with a pancreatic D-cell tumour and achlorhydria, steatorrhoea, and diabetes mellitus. The symptoms may be explained by the inhibitory effects of somatostatin on gastric acid’ 1-13 and pancreatic-enzyme secretion.2 14 Somewhat surprisingly, however, serum-gastrin levels were within the normal range, although synthetic somatostatin has been shown to suppress gastrin secretion."’" The significantly lowered levels of glucagon in the present case may have been caused by the somatostatin hypersecretion. The impaired glucose tolerance observed in both patients is, however, difficult to explain. Measurements of insulin and glucagon responses during a glucose-tolerance test in somatostatinoma patients may help to explain this apparent association. At any rate, it seems that the combination of steatorrhoea, achlorhydria, and impaired glucose tolerance should alert clinicians to the possible presence of a somatostatinoma. The question whether the somatostatin-like compound found in peripheral tissues displays biological properties similar to the hypothalamic peptide is of great importance. Somatostatin immunoreactivity extracted from the D-cell tumour could be separated into at least four different components, two of which eluted in the position of the two components found in normal pancreas by Arimura and others.9 Biological testing showed that the extracted components, like synthetic somatosta.tin, were potent inhibitors of insulin and glucagon release. Thus, the somatostatin-immunoreactive material produced by the normal pancreatic D-cell is likely to be inhibitory to the release of insulin and glucagon. Interestingly, quite high somatostatin levels were found in the venous effluent from the tumour. Thus, neoplastic D cells, at least, seem to deliver their products to the bloodstream. It is intriguing that the hypersecretion of this strongly inhibitory peptide does not give more severe symptoms than noted in the present case. This work was supported by grants from Landsforeningen til Kraettens bekaempelse, the Danish Medical Research Council (no. 51-7174), the Swedish Medical Research Council (no. 4534), and the M. Stephens Foundation. The authors are greatly indebted to Prof. A. Maunsbach, Institute of Anatomy, University of Aarhus, for gener-

ously putting electron-microscopy facilities at our disposal. Requests for reprints should be addressed to L.-I. L., Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark.

REFERENCES 1. Lancet, 1975, i, 1323. 2. Konturek, S. J. Scand. J. Gastroent.

1976, 11, 1.

3. Hökfelt, T., Efendic, S., Hellerström, C., Johansson, O., Luft, R., Arimura, A. Acta endocr., Copenh. 1975, 200, suppl., 5. 4. Polak, J. M., Pearse, A. G. E., Grimelius, L., Bloom, S. R., Arimura, A. Lancet, 1975, i, 1220. 5. Dubois, M. P. Proc. natn. Acad. Sci. U.S.A. 1975, 72, 1340. 6. Larsson, L. -I., Grimelius, L., Håkanson, R., Rehfeld, J. F., Stadil, F., J., Angervall, L., Sundler, F. Am. J. Path. 1975, 79, 271. 7. Larsson, L. -I., Schwartz, T., Lundqvist, G., et al. ibid. 1976, 85, 675. 8. Sternberger, L. A. Immunocytochemistry. New Jersey, 1974. 9. Arimura, A., Sato, H., Dupont, A., Nishi, N., Schally, A. V. Science,

Hoist,

1975, 189, 1007. 10. Lindkaer Jensen, S., Kühl, C., Vagn Nielsen, O., Hoist, J. J. Scand. J. Gastroent. 1976, 37, suppl., 57. 11. Bloom, S. R., Mortimer, C. H., Thorner, M. O., et al. Lancet, 1974, ii, 1106. 12. Barros, D’sa, A. A. J., Bloom, S. R., Baron, J. H. ibid. 1975, i, 886. 13. Gomez-Pan, A., Reed, J. D., Albmus, M., et al. ibid. p. 888. 14. Creutzfeldt, W., Lankisch, P. G., Fölsch, U. R. Dt. med. Wschr. 1957, 100, 1135.

NEOCORTICAL CHOLINERGIC NEURONS IN ELDERLY PEOPLE P. WHITE

C. R. HILEY L. H. CARRASCO I. E. I. WILLIAMS D. M. BOWEN

M. J. GOODHARDT J. P. KEET

Miriam Marks Department of Neurochemistry, Institute of Neurology, National Hospital, Queen Square, London; Department of Pharmacology and Therapeutics, University of Liverpool; Geriatric Department, University College Hospital Medical School, St. Pancras Hospital, London; Department of Neuropathology, Runwell Hospital, Wickford, Essex; and Psycho-Geriatric Assessment Unit (Charles Andrews Clinic), Barncoose Hospital, Redruth, Cornwall

Choline acetyltransferase activity (presynaptic cholinergic system) and high affinity binding of cholinergic antagonists (postsynaptic cholinergic system) were measured in brain tissue removed after death from both mentally normal and demented old people. Muscarinic receptor binding sites in frontal cortex decreased with advancing years only in old people without appreciable morphological evidence of senile degeneration. Preliminary data for temporal lobe suggested that also in Pick’s disease the density of receptor binding sites is reduced. The markers are not significantly reduced in cases of mixed senile and vascu-

Summary

lar dementia. However, in non-vascular senile dementia of the Alzheimer type, there were indications that the presynaptic marker is selectively depleted. Therefore, centrally acting anticholinesterases might be beneficial, particularly in the early stages of the disease.

Introduction ALZHEIMER’S disease or senile dementia’ is a cause’ of distressing morbidity in elderly people. There is usually pronounced thinning of the cortical ribbon, which seems to reflect neuronal loss,3 4 - and senile degeneration (senile plaque formation and neurofibrillary degeneration) is intense. Altered presynaptic axon terminals and presynaptic axons associated with senile plaques have also been described.5 Choline acetyltransferase (C.A.T.) activity, a potential marker of the presynaptic cholinergic system, appears to be reduced in neocortex obtained post mortem5-7 and at biopsy.8 C.A.T. and acetylcholinesterase activities are possibly also reduced in extraneocortical limbic areas.6 y-aminobutyric-acid-containing neurons may be spared,68 suggesting that the cholinergic system is particularly vulnerable. In elderly people with dementia and appreciable cerebrovascular disease, senile degeneration and neocortical neuronal loss can be as intense as in Alzheimer’s disease.4 Brains of this type, classified post mortem as cases of mixed senile and vascular dementia,9 may occur in patients diagnosed by clinical criteria as having "multiinfarct" dementia. 10 The rare heredodegenerative condition, Pick’s disease, is another dementia of middle and later life and is characterised by loss of neurons mainly from frontal and temporal lobe. Markers of cholinergic neurons have not been systematically examined in either disorder. In another hereditary disease, Huntington’s chorea, dementia is a common finding. C.A.T. activity and the density of muscarinic receptor binding sites are reduced in the striatum. 11 12 Occasionally it may be dif-