1311
Glucagon enhances hepatic glycolysis and gluconeoAlthough in-vitro experiments have an effect on adipose tissue 28,29 a role of indicated in ketogenesis in man has not been estabglucagon The present data showing a close lished as yet. correlation between plasma-glucagon and blood ketone-body concentrations are highly suggestive.
genesis 25,27
It would be of great interest to see whether the of hyperglyczemia and hyperketonaemia could be delayed by the use of an inhibitor of glucagon secretion such as somatostatin. 11,1112 It is also possible that inhibition of glucagon secretion could be a useful adjunct to insulin therapy in the treatment of severe hyperglycaemia and ketoacidosis, especially in those cases associated with infection. However, in the full clinical syndrome many other factors are involved, such as increased catecholamine secretion, hypovolxmia, hypotension, and hypothermia, so that a cautious approach is necessary. We thank Mrs S. Warren, Mrs H. Darre, Miss B. Jacobsen, Mrs K. Just, and Mrs E. Bang for technical assistance; Prof. Rud Keiding, Department of Clinical Chemistry, University School of Aarhus, for his help; and Prof. K. Lundbaek for encouragement and guidance. This study was supported by the Wellcome Trust, the British Council, and Statens Laegevidenskabelige Forskningsrad, Denmark. Requests for reprints should be addressed to K. G. M. M. A.
USE OF FACTOR-VII-RICH PROTHROMBIN COMPLEX CONCENTRATE IN LIVER DISEASE I. W. DYMOCK THOMSON Departments of Medicine and Hœmatology, University Hospital of South Manchester
G. GREEN
L. POLLER
JEAN M.
onset
Addendum Similar findings with regard to glucagon have now been reported by J. E. Gerich and others (7. clin. Endocr. Metab. 1975, 40, 526) during 20 hours of insulin deprivation. They have also shown inhibition of the rise in hydroxybutyrate by somatostatin - (New Engl..,. Med. 1975,292, 985). REFERENCES
23.
Assan, R., Hautecouverture, G., Guillemant, S., Dauchy, F., Protin, P., Derot, M. Path. Biol. 1969, 17, 1095. Christensen, N. J. Scand. J. clin. Lab. Invest. 1971, 27, 227. Jacobs, H. S., Nabarro, J. D. N. Br. med. J. 1969, ii, 595. Cryer, P. E., Daughaday, W. H. Diabetes, 1970, 19, 519. Alberti, K. G., Hockaday, T. D. R. Diabetologia, 1973, 9, 13. Rocha, D. M., Santeusanio, F., Faloona, G. R., Unger, R. H. New Engl. J. Med. 1973, 288, 700. Beisel, W. R., Rappaport, M. I. ibid. 1969, 280, 541. Ørskov, H., Thomsen, H. G., Yde, H. Nature, 1968, 219, 193. Heding, L. Diabetologia, 1971, 7, 10. Christensen, N. J. Clin. Sci. 1973, 45, 163. Hohorst, H. J., Kreutz, F. H., Bücher, T. Biochem. Z. 1959, 332, 18. Bücher, T., Czok, R., Lamprecht, W., Latzko, E. in Methods of Enzymatic Analysis (edited by H.-U. Bergmeyer); p. 324. New York, 1963. Williamson, D. H., Mellanby, J., Krebs, H. A. Biochem. J. 1962, 82, 90. Eggstein, M., Kreutz, F. H. Klin. Wschr. 1966, 44, 262. Williamson, D. H., Lopes-Vieira, O., Walker, B. Biochem. J. 1967, 104, 497. Ho, R. J., Meng, H. C. Analyt. Biochem. 1969, 31, 426. Christensen, N. J. J. clin. Lab. Invest. 1967, 19, 379. Unger, R. H., Orci, L. Lancet, 1975, i, 14. Alford, F. P., Bloom, S. R., Nabarro, J. D. N., Hall, R., Besser, G. M., Cay, D. H., Kaston, A. J., Schally, A. V. ibid. 1974, ii, 974. Christensen, S. E., Hansen, Aa. P., Iversen, J., Lundbæk, K., Ørskov, H., Seyer-Hansen, K. Scand. J. clin. Lab. Invest. 1974, 34, 321. Madison, L. L., Seyffert, W. A., Jr., Unger, R. H., Barker, B. Metabolism, 1968, 17, 301. Luyckx, A. S., Lefebvre, P. J. Proc. Soc. exp. Biol. Med. 1969, 133, 524. Edwards, J. C., Taylor, K. W. Biochim. biophys. Acta, 1970, 215,
24.
Unger,
1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
13. 14.
15. 16. 17. 18. 19. 20.
21. 22.
310. R. H., Eisentraut, A. M., McCall, M. S., Madison, L. L. J. clin. Invest. 1962, 42, 682. 25. Unger, R. H. Diabetes, 1971, 20, 834. 26. Christensen, N. J. ibid. 1974, 23, 1. 27. Exton, J. H., Jefferson, L. S., Butcher, R. W., Park, C. R. Am. J. Med. 1966, 40, 709.
A prothrombin complex concentrate rich in factor VII has been used in the management of the clotting defect in thirteen patients with liver disease. Adequate correction of coagulation was achieved immediately after the infusion in all cases. Within 4 hours there was some deterioration and by 24 hours the results approximated to pre-infusion values. Liver biopsies were performed without hæmorrhagic complication in the immediate post-infusion period. There was no evidence of induced intravascular coagulation. Since other prothrombin complex concentrates have proved disappointing, both in their failure to correct the clotting defect and in their production of disseminated intravascular coagulation, this factor-VII-rich concentrate may be the treatment of choice in patients with liver disease who require temporary correction of their
Summary
coagulation defect. Introduction THE coagulation defect in hepatic disease is complex, but impairment of synthesis of clotting factors by the liver is a major component.1-3 In many instances of severe hepatic dysfunction the parenteral administration of vitamin Kl fails to induce a rise of clotting factors 11, VII, ix, and x: a bleeding diathesis and defective coagulation response ensue which preclude surgical procedures. In thirteen with or liver we tried acute chronic disease patients to overcome this clinical problem by improving blood coagulation with the parenteral administration of a prothrombin complex concentrate of factors 11, VII, ix, and x produced by the Oxford Haemophilia Centre. This procedure was adopted with a view to liver biopsy which would otherwise have been contraindicated. Because of the possible danger of thrombotic sequelae resulting from the infusion of clotting-factor concentrates, laboratory tests for disseminated intravascular coagulation were done. Patients and Methods Patients All patients admitted under our clinical care with suspected liver disease are screened for a bleeding disorder with the following tests: prothrombin-time, using British Comparative Thromboplastin; cephalin (partial thrombo-
28. 29. 30. 31. 32.
Gries, F. A., Berger, M., Herberg, L., Preiss, H., Hesse-Wortmann, C., Jahnke, K., Liebermeister, H. Med. Ernähr. 1969, 10, 99. Björntorp, B., Karlsson, M., Hovden, A. Acta med. scand. 1969, 185, 89. Iversen, J. Scand. J. clin. Lab. Invest. 1974, 33, 125. Koerker, D. J., Ruch, W., Chideckel, E., Palmer, J., Goodner, C. J., Ensinck, J., Gale, C. C. Science, 1974, 184, 482. Christensen, S. E., Hansen, Aa. P., Iversen, J., Lundbæk, K., Seyer-Hansen, K., Ørskov, H. Unpublished.
1312
plastin) time; factor-v assay; factor-VII assay; fibrinogen estimation ; thrombin/fibrinogen time; fibrin(ogen) degradation products, by staphylococcal clumping technique or by haemagglutination-inhibition assay, using human red blood-cells 1; euglobulin lysis-time; full bloodcount and platelet-count. If the prothrombin-time is abnormal, vitamin Ki is given in a dose of 10 mg. intramuscularly for 3 days, after which time the coagulation tests are repeated. In patients whose prothrombin-time was still 3 seconds or more longer than the control 72 hours after the administration of vitamin Kl, the concentrate was infused before liver
biopsy. Of the thirteen patients investigated seven had cirrhosis, chronic active hepatitis, two drug-induced hepatitis, and two collagen disorders associated with hepatocellular
two
dysfunction.
e
Concentrate Infusion The prothrombin complex concentrate was dissolved in 100 ml. of sterile water and infused over a period of 2030 minutes. The coagulation studies were repeated immediately after this infusion. Provided the prothrombintime was shortened to within 3 seconds of the normal range, liver biopsy was done without delay. One patient who did not respond adequately was given a second infusion. Post-infusion coagulation studies were also done at 4 hours and 24 tours. Blood was cross-matched as a precaution, and blood-pressure, pulse, and temperature were checked every 15 minutes during the 60-minute period from the start of the infusion.
Results
Clinical In all patients liver biopsy was performed without clinical evidence of abnormal bleeding. Neither overt thromboembolism nor adverse reactions due to the administration of the concentrate were observed. Adequate tissue for diagnostic histology was obtained in twelve of the thirteen patients.
Laboratory Prothrombin-time.-In every instance the pro-
Fig. 2—Effect of infusion on factor-VII levels.. was shortened to 15 seconds or less (normal range 11-13). In one patient the prothrombin-time was only shortened to 15 seconds, but this represented a 5-second improvement. The mean prothrombintime immediately after infusion was 13 seconds. 4 hours after the infusion the prothrombin-times had lengthened, but were still significantly shorter than the pre-infusion values. At 24 hours the prothrombintimes approximated to their pre-treatment values. The three patients with the longest prothrombin-times initially showed the poorest response to the administration of concentrate and the most incomplete and
time
transient corrections. Factor VII (fig. 2).-Pre-infusion f actor-vn levels ranged from 25% to 70% (mean 44%). Immediately post-infusion all patients had achieved factor-vn
longed prothrombin-time improved (fig. 1). Before the infusion prothrombin-times ranged from 15 to 20 seconds (mean 17): afterwards the prothrombin-
Fig. I-Effect of infusion
on
prothrombin-time.
Fig. 3-Cephalin (partial thromboplastin) times before various times after infusion.
and at
1313
limit of the normal range. But neither these nor any of the other patients showed a rise after the administration of concentrate.
Discussion There were two main aims in this study-first to assess the efficacy of a factor-VII-rich prothrombin concentrate in restoring the coagulation defect in liver disease, and second to monitor any tendency of this concentrate to induce intravascular clotting. Use of the Oxford factor-vu-rich prothrombin concentrate permitted diagnostic liver biopsy without obvious bleeding. In patients with a coagulation defect resulting from impaired liver function, the levels of factors u, VII, ix, and x sufficient to meet the haemostatic challenge of liver biopsy are not well established. Most workers feel that the procedure is unsafe when the prothrombin-time is 3 seconds longer than the normal contral4 Our patients had
Fig. 4-Thrombin-fibrinogen times before and
at
various
times after infusion.
activities of 100 % or greater, with a mean activity of 135 %. By 4 hours post-infusion the mean factorvii activity had fallen to 73 %, and by 24 hours it had almost returned to pre-infusion levels with a mean of 49 %. Patients with the lowest factor-vu activities initially had reverted to similar low levels by 24 hours post-infusion. Cephalin (partial thromboplastin) time.-In six patients prolonged cephalin-times were recorded pre-infusion (fig. 3), but there was a significant shortening at 4 hours post-infusion, the mean value falling from 60 to 51-5 seconds (normal 38-45 seconds). By 24 hours post-infusion the cephalin-times had returned to pre-treatment levels. The remaining seven patients who had relatively normal cephalin-times initially showed no appreciable shortening after infusion. Factor-V assay.-Factor-v levels were reduced preinfusion in twelve of the thirteen patients. Immediately post-infusion there was a significant rise (see Discussion). By 24 hours levels had returned to their initial values. Total fibrinogen (clot-opacity technique).-There was no significant change after the infusion. Thrombin-fibrinogen time (fig. 4).-All patients had prolonged thrombin-fibrinogen times initially which persisted after the infusion. In the immediate post-infusion sample there was, paradoxically, a further lengthening of the thrombin-fibrinogen time, presumably due to small amounts of heparin in the concentrate. By 4 hours this effect was no longer ’
demonstrable. Platelet-count.-In all patients the platelet-count remained
unchanged throughout the study. Euglobulin lysis-time.-No changes were observed
post-infusion.
Fibrin(ogen) five patients
breakdown
F.D.P.
were
products (F.D.P.).-In initially above the upper
initial prothrombin-times ranging from 15 to 20 seconds (normal 11-13). In this study we have stressed the importance of measuring factor vil. We have demonstrated the considerable contribution that deficiency of factor vii makes to the production of the coagulation defect of liver disease.5 Since factor VII also has the shortest half-life of the prothrombin complex clotting factorss it is the major component determining the duration of the coagulation response to this concentrate. A potential danger of clotting-factor concentrates is the induction of intravascular clotting?-9 Patients with liver disease may have pre-existing intravascular coagulation or impairment of clearance of activated clotting factors potentiating the risks of disseminated intravascular coagulation. It is reassuring, therefore, that our laboratory and clinical data provide no evidence for the induction of intravascular coagulation by this concentrate. The relevant laboratory tests,
including
titres, thrombin-fibrinogen times, fibrinogen levels, factor-v assays, and platelet-counts, showed no changes indicative of intravascular coagulation. Nor was there clinical or histological evidence F.D.P.
of thromboembolism.
experience patients with recently established usually have accelerated cephalintimes. The cephalin-time results from this study did not show any such change. In seven patients in whom the test was virtually normal initially, there was no evidence of acceleration post-infusion. Any slight activation of intrinsic " clotting due to infusion of clotting-factor concentrate might have been masked in the immediate post-infusion blood-samples by the small amount of heparin contained in the infusion, but by 4 hours any heparin should have been cleared. Some anomalous results need explanation. Immediately post-infusion the thrombin-fibrinogen time was slightly prolonged in some patients, an effect which we also attribute to the heparin content of the concentrate. Furthermore, the concentrate contains no appreciable amounts of factor v, yet the concentration In
our
venous
thrombosis
"
seemed to increase. This may have been an artefact due to the difficulty in preparing an artificial factor-v substrate. We have, in fact, shown that this substrate, prepared by the conventional method, was
1314
slightly deficient in factor 11 which may account for a spurious acceleration, post-infusion. Prothrombin complex concentrates have been used in a variety of congenital and acquired coagulation defects.8,10,1l Current preparations, with a high potency of factor ix, contain little or no factor vn. Besides being ineffective in restoring the coagulation defect in liver patients, their use has been associated with a high risk of disseminated intravascular coagulation.7,8 From our experience, therefore, the Oxford factor-VIIrich concentrate seems to be the preparation of choice in patients with liver disease requiring temporary correction of their coagulation defect. We thank Dr E. Bidwell of the Oxford Haemophilia Centre for supplies of prothrombin complex concentrate; Prof. J. M. Evanson for provision of clinical facilities; and S. R. Armitage and T. A. Morris for technical assistance. The work was supported by a Medical Research Council programme grant
to L. P.
’
Requests for reprints should be addressed Withington Hospital, Manchester M20 8LR.
to
L.
P.,
REFERENCES 1. 2.
3.
4. 5. 6. 7. 8. 9. 10.
Smith, H. P., Warner, E. D., Brinkhous, K. M. J. exp. Med. 1937, 66, 801. Rapaport, S. I., Ames, S. B., Mikkelsen, S., Goodman, J. R. New Engl. J. Med. 1960, 263, 278. Clark, R., Borirakchanyavat, V., Gazzard, B. G., Rake, M. O., Shilken, K. B., Flute, P. T., Williams, R. Gastroenterology, 1973, 65, 778. Sherlock, S. Diseases of the Liver and Biliary System. Oxford, 1969. Dymock, I. W., Tucker, J. S., Woolf, I. L., Poller, L., Thomson, J. M. Br. J. Hœmat. 1975, 29, 381. Loeliger, E. A., van der Esch, B., Matten, M. J. Thromb. Diath. hœmorrh. 1964, 10, 267. Kasper, C. K. New Engl. J. Med. 1973, 289, 160. Gazzard, B. G., Lewis, M. L., Ash, G., Rizza, C. R., Bidwell, E., Williams, R. Gut, 1974, 15, 993. Blatt, P. M., Lundblad, R. L., Kingdon, H. S., McLean, G., Roberts, H. R. Ann. intern. Med. 1974, 81, 766. Tullis, J. L., Mehin, M., Jurigian, P. New Engl. J. Med. 1965, 273,
667. 11. Bruning, P. F.,
Loeliger, E. A. Br. J. Hœmat. 1971, 21, 377.
LACK OF CAUSAL ASSOCIATION BETWEEN COXSACKIE B4 VIRUS INFECTION AND DIABETES P. H. BENNETT M. MILLER J. E. MAYNARD K. R. BERQUIST Epidemiology and Field Studies Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases, and Phoenix Laboratories Division, Bureau of Epidemiology, Center for Disease Control, Phoenix, Arizona, and Case Western Reserve University, Cleveland, Ohio, U.S.A.
S. E. DIPPE
An epidemic of Coxsackie B4 virus infection in an isolated group of islands in the Bering Sea in 1967 provided an opportunity to test the suggestion that infection with this virus might be associated with an increased incidence of diabetes. In 1973 islanders were tested by glucosetolerance tests and their two-hour plasma glucose levels were analysed in the light of serological evidence of CB4 infection five years earlier. There was no evidence of any increased prevalence of diabetes in those who had been infected in 1967.
Sum ary
introduction COXSACKIE B4 (CB4) virus prevalence has been shown to correlate with the seasonal variation in incidence of juvenile-onset diabetes,l and a higher prevalence of neutralising antibody titres to CB4, inversely related to the duration of symptoms, has been described in newly diagnosed juvenile diabetics more frequently than expected.2 Thus, CB4 or an antigenetically related virus may be implicated in the aetiology of diabetes mellitus. Experimental work supports this view. Intraperitoneal CB4 infection in mice can lead to degranulation and disruption of the pancreatic islets3 After inoculation with CB4 virus 6 of 14 CD1mice aged eight to nine weeks had hyperglycaemia in twelve to twenty-one days with degranulation of the beta cells and mononuclear-cell infiltration of some pancreatic islets.4 Another picornavirus, the M variant of encephalomyocarditis virus, has also induced diabetes in mice.5 This virus produces lesions exclusively in the pancreatic islets: some animals develop chronic hyperglycaemia and hypoinsulinasmia, and a few become ketotic.6 The severity and frequency of the diabetes syndrome, however, vary with the strain of mouse, suggesting that genetic factors may affect to diabetes. The occurrence of lymphocytic infiltration of the islets in human juvenile diabetics,’,’ and occasionally in late-onset diabetes,9 points to an autoimmune and/ or infectious process. This finding and the higher frequency of CB4 titres in juvenile diabetics lend credence to the hypothesis of an infectious aetiology in man, and indicate the need for a critical prospective evaluation of the possible role of CB4 and other viral infections in the pathogenesis of human diabetes. In 1967 there was a well-documented, extensive epidemic of CB4 infection in the isolated Pribilof Islands. Five years later the incidence of diabetes in the CB4 infected and non-infected persons was determined to ascertain whether or not those infected had had a greater risk of developing diabetes.
susceptibility
Methods The Pribilof Islands are in the Bering Sea north of the Aleutian Chain, some 700 miles (1100 km.) southwest of Anchorage, Alaska. Only two of four islands are inhabited-St. Paul has about 350 people and St. George has some 100 inhabitants. There is little migration to the islands, but there is considerable movement between them. The only transportation to the mainland is a weekly air service. The resident Aleuts are descendants of those brought to the islands from the Aleutian chain by the Russians in the 19th century to harvest the fur seal in its breeding area. This remains the only significant industry. All receive free medical care from a resident U.S. Public Health Service physician.
Epidemic 10 An epidemic illness, characterised mainly by headache, fever, myalgia, and sore throat, started in August, 1967, in St. Paul and spread explosively through the population. Throat cultures from 13 ill patients were inoculated into primary rhesus-monkey kidney, WI-38 human embryonic fibroblasts, and HEp2 cell cultures. 1/100 dilutions of viral isolates were passed again for preparation of a virus pool and mixed with 1/10 or 1/20 dilutions of Coxsackie antisera A9, Bl, B2, B3, B4, and B5.
After incubation