Clinical Implications of Alloxan Diabetes

CLINICAL IMPLICATIONS OF ALLOXAN DIABETES C. CABELL BAILEY,

M.D. #

AND PHILIP LECOMPTE,

M.D.t

WHEN alloxan, a pure chemical substance related structurally to uric acid, is injected into rabbits,l rats,2 dogs,S monkeys,4 pigeons 5 or turtles,6 it destroys the islets of Langerhans in the pancreas and produces diabetes. Animals with diabetes so produced have clinical symptoms which are indistinguishable from those of human diabetes. Polydipsia, polyuria and polyphagia may be marked and there is often extreme weight loss if the diabetes is not controlled with insulin. One such diabetic rat weighing 120 gm. excreted 72 cc. of urine in twenty-four hours in contrast to the usual 3 to 5 cc. Such animals become listless and dehydrated. Rabbits and rats with severe alloxan diabetes, if untreated with insulin, may develop diabetic acidosis and coma and may present blood chemical changes similar to those found in humans with diabetic coma. Lowering of the carbon dioxide content of the blood,7 marked lipemia,8 hypercholesteremia,9 acetonemia1 and an. increase of the blood inorganic phosphates lo, have all been reported. In markedly lipemic rabbits lipemia retinalis is easily seen. Kendall and his coworkers 9 report that rabbits dying in diabetic coma have been found to have hypercholesteremia reaching 780 mg. per 100 cc. and hyperlipemia ranging to 18.5 gm. per 100 cc. Clinically, animals dying in diabetic coma often have deep respirations, simulating the Kussmaul breathing of the diabetic coma patient. Diet.-Alloxan diabetic rats placed on a high fat diet containing 90 per cent margarine and 10 per cent casein plus a salt mixture show a marked decrease and finally a disappearance of glycosuria.l 1 When the original high carbohydrate diet is given again, however, glycosuria returns. A sudden change from a high carbohydrate to a high fat diet produces ketonuria in varying degrees in the rat and similar ketonuria has been reported in human diabetics when such a change is made in the diet. It has further been shown by Mar-

From the George F. Baker Clinic, Boston, Elliott P. Joslin, Medical Director. o Research Fellow in Medicine, Harvard Medical School; Physician, New England Deaconess Hospital. t Instructor in Pathology, Harvard Medical School; Pathologist, Faulkner Hospital. 427

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tinez l2 in Houssay's laboratory that ·a high fat diet increases the sen.. sitiveness of young white rats to the diabetogenic action of alloxan; whereas, a higll protein diet makes the rat more resistant than normal to alloxan. . It is known that hyperthyroidism affects the blood sugar ,in humans and for this reason in the George F. Baker Clinic a diagnosis of di.abetes is not made in a person with hypertllyroidism un~ess tIle blood sugar reaches a level of 200 mg., in contrast to the usual diagnostic level of 170 mg. The effect of thyroid is likewise evident in the rat given alloxan, for a thyroidectomy previous to the injection of alloxan makes the rat mucI1 lTIOre resistant to its diabetogenic action. l3 In contrast, the feeding of thyroid extract to rats increases their sensitivity to the diabetogenic action of alloxan. Thiouracil has an even greater inhibitory effect than thyroidectomy.l4 Alloxan diabetic animals have been valuable in studying tIle vitamin requireInents in diabetes. Lowry and .Hegsted15 present evidence that rats ma~e diabetic with alloxan showed no increased tendency to develop signs of thiamine deficiency when placed upon a thiamine deficient diet as, compared with the normal rat. They further state that the action of thiamine is not impaired in the diabetic animal and from their experiments conclude that the requirement of this vitamin is no greater than that of normal animals. Cataracts.-The anin1al made diabetic with alloxan tends to develop diabetic cataracts, especially if the diabetes is severe and uncontrolled with insulin. s In rats definite cataracts can usually be detected as early as one or two months and .often become mature, frequently \vith complete. blindness, in two to: four months. A siInilar cataract may at times be seen in diabetic humans, especially among younger persons whose diabetes is severe and imperfectly controlled. In the diabetic animal attempts have been nlade to determine the cause of these lens changes. Some, claim that diets deficient in l·iboHavin or tryptopl1ane may produce cataracts in animals. It has recently been shown16 that the supplementary injection of ri.boHavin, pyridoxine, thiamine or tryptopllane does not prevent the develop,ment of cataracts in rats with severe· alloxan diabetes. Animals with mild diabetes or with diabetespartittlly controlled with insulin SllOW much less tendency to;,deYelop·.. cataracts and wl1en they do occur they tend to remain immature.

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PATHOLOGY OF ALLOXAN DIABETES COMPARED WITH HUMAN DIABETES

The outstanding pathological finding in anin1als given alloxan is the striking effect upon the islets of Langerhans in the pancreas. If the proper dose is used the damage to other organs is usually minimal and reversible. As early as five minutes after injection a slight reduction in the number of granules in the beta cells (which presumably form i~sulin) may be seen, and by the end of one hour the nuclei of the cenb·al beta cells have become shrunken and early cytoplasmic disintegration is visible. Within twenty-follf hours the central beta cells are completely destroyed and tile center of the islets contains only debris surrounded by a collar of intact alpha ceJls. The chronic lesion found after two montlls shows islets smaller than normal and composed alnlost ~ntirely of alpha cells. The production of diabetes by the injection of repeated small doses produces a somewhat different picture. Some of the islet cells may appear normal, others may show loss of granules and pyknosis of tlleir nuclei, associated occasionally with clear vacuoles in the cytoplasm, the so-called "hydropic change." Occasionally, D1itotic figures are seen in the beta cells. Mitotic figures in acinar cells and in the lining epithelillm of the small pancreatic ducts are also occasionally observed. Significant changes in other organs are essentially confined to necrosis of the epithelium of the renal tubules, especially in rats, fatty infiltration of the liver in dogs, and small foci of necrosis and occasional mitotic :figures in the adrenal glands. A comparison of tllese changes \vith the pathological alterations in human diabetes reveals, at first glance, but few points of similarity. Acute necrosis of the islet cells is exceedingly rare in human diabetes. Hydropic degeneration has been described but is infrequent, occurring in approximately 5 per cent. The most frequent lesions found in the human diabetic pancreas, according to Warren,17 are hyalinization of some of the islets in approximately 40 per cent and fibrosis in some islets in approximately 25 per cent. In ap·proximately 25 per cent no pancreatic lesion has been demonstrated. A few pancreases show hydropic degeneration, hypertrophi.ed islets or lymphocytic infiltration in sonle of the islets. Certainly the acute pathological changes found after alloxan do no simulate this picture, although one would not expect acute changes produced ,\'ith a chemical substance to resemble chronic changes seen after years of diabetes. The opportunity to examine a hlunan pancreas within a fe\v'days after the onset of diabetes is indeed rare. It should be elnphasized, perhaps, that the con-

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clition of the beta cells in the human pancreas and their number relative to the number of alpha cells have not been adequately investigated, due to the lack of useful staining methods for differentiating beta and alpha cells. Perhaps the further development of such methods will permit extension of the work of Gomori,18 who reported a marked reduction in the ratio of beta to alpha cells in a few human diabetic pancreases studied with special stains. If such findings can be confirmed, there may prove to be more similarity between the lesions of human and chronic alloxan diabetes than is now apparent. Some of the chronic degenerative changes frequently found in various organs in human diabetes, such as intercapillary glomerulosclerosis and severe arteriosclerosis, have not been reported in animals made diabetic by means of alloxan. Such lesions, however, may require a long period for their development and, if avimals with alloxan diabetes are kept alive for years, these changes may eventually be found. The Effect of Alloxan on Humans.-Alloxan has been given to humans with varying results. Brunschwig and his associates19 , 20 were the first to administer alloxan to humans, all of whom had evidence of metastatic carcinoma, and one had an islet cell carcinoma with clinical hyperinsulinism. In three patients little or no change in the blood sugar was observed after the patients received alloxan. A fourth patient experienced a chill, nausea and cyanosis lasting for several hours but developed no evidence of diabetes. A fifth patient who received the largest single injection of this group (600 mg. per kilogram) died six hours after the termination of the injection. Approximately three hours before death the blood sugar had fallen to 16 mg. per 100 cc., and there were clinical signs of an insulin reaction. These, however, disappeared with intravenous 50 per cent dextrose. At postmortem examination a microscopic study of the liver showed rather diffuse degenerative changes in the hepatic cells. The pancreas revealed questionable evidences of injury to a number of cells in some of the islets, although many islets were not a~ected. Of course, evidence of widespread carcinoma was found. The sixth patient who received alloxan had an islet cell carcinoma with metastasis and evidence of hyperinsulinism. He received several series of alloxan injections and after each there was a period of freedom from attacks for ten to twenty-one days. This patient finally died and on postmortem examination no evidence of damage to the normal islet cell tissue of the pancreas nor of the metastatic nodules in the liver could be found.

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The extreme danger accompanying the administration of alloxan to humans is illustrated by a case of islet cell carcinoma with metastases treated with alloxan by. Rynearson. 21 This patient received alloxan and died a short time later with pathological evidence of extensive liver . necrosis, presumably due to the toxic effect of alloxan. The blood sugar in this patient reached 428 mg. per 100 cc. before death. Conn22 presents evidence that alloxan may destroy the normal islets of Langerhans in the pancreas. His patient, suffering from hyperinsulinism, was treated with alloxan and although there was a marked change in the character of the glucose tolerance curve, hypoglycemic attacks continued. At exploration an islet cell tumor was removed from the pancreas, but histological study failed to reveal damage to the tumor cells. A biopsy of the pancreas nonetheless revealed definite damage to the islets of Langerhans. Following operation the patient was diabetic and has remained so for a period of three months' observation. Recently Talbot and Bailey 23 treated an 8 month old baby girl who had 'evidence of hyperinsulinism with alloxan.. This child's fasting blood sugars were constantly low, frequently below 30 mg. per 100 cc., a.nd hypoglycemic convulsions occurred almost daily. Laparotomy failed to reveal a pancreatic adenoma. Accordingly a series of injections of alloxan was instigated, beginning with 10 mg. per kilogram and gradually increasing to 100 mg. After seven injections evidence of hypoglycemia disappeared and the fasting blood sugar was normal. After an interval of three weeks a tendency to hypoglycemia returned and, accordingly a second series of eight alloxan injections were given. Following the latter all evidence of hypoglycemia disappeared and the child has remai~ed free from evidence of hyperinsulinism to the present time, eleven months later. Nevertheless, the use of alloxan in humans at present is to b·e condemned, for its administration is accompanied by extreme danger. ·Even in cases with hyperinsulinism the chances are great that an islet cell tumor can be discovered at operation and successfully removed with complete cure. Only in the extremely rare case of hyperinsulinism without islet cell tumor can any beneficial results be hoped for with alloxan and even then a subtotal pancreatectomy is usually the safer procedure. CONCLUSION

The injection of alloxan into animals produces necrosis of the islands of Langerhans and diabetes, with· clinical symptoms' indistinguishable from those found in human diabetes. ,Diabetes so pro-

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LE COl\1PTE

ducecl can be modified by diet or insulin. Diabetic co-mplications which may be seen in hUlnans, including diabetic acidosis 'aild':coma and diabetic cataracts, are evident also,in animals made diabetic with alloxan. A comparison is lnade of the pancreatic pathology of,alloxanand hu~an diabetes. Alloxan injected into humans may destroy the normal islets of Langerhans. Islet cell adenon1as, however, appear resistant to -its action. Furthermore, since alloxan may produce liver necrosis and fatal toxic reactions, its use in hUlllans is not advi.sed.

REFERENCES 1. Bailey, C. C. and Bailey, O. T.:The Production of ,Diabetes Mellitus in Rab.. bits with Alloxan. J.A.~/I.A., 122:1165, 1943. 2. Dunn, J. S. and McLetchie, N. G. B.: Experhnental Alloxan Diabetes in the Rat. Lancet, 245:384--387, 1943. 3. Goldner, M. G. and Gomori, G.: Alloxan Diabetes in the' Dog. Endocrinology, 88:297, 1943. 4. Bannerjee, S.: Alloxan Diabetes in Monkeys. Lancet, 247:658, 1944. 5. Goldner, M. G. and Gomori, G.: Effect of Alloxan on Carbohydrate and Uric Acid Metabolisnl of the Pigeon. Proc. Soc. Exper. BioI. & Med., 58: 31, 1945. 6. Gracia Ranlos, J.: A Contribution to Our Knowledge of the Phannacology of Alloxan. Revista de la Sociedad Mexicana de Historia Natural, 5:25, 1944. (Abst. -from BioI. Abst., 19:2641 [Feb.] 1945.) 7. Bailey, C. C. and Leech, R. S.: Unpublished data. 8. Bailey, C. C., Bailey, O. T. and Leech, R. S.: Alloxan Diabetes 'vith Diabetic Complications. New England J. ~fed., 280:535, 1944. 9. Kendall, F. E., Meyer, W., Lewis, L. and Victor, J.: Alloxan Diabetes in Rabbits.Proc. Soc. Exper. Biol. & ~Ied., 60:190-195, 1945. lOo Franks, M., Friedgood, C. E. and Kaplan, N. 0.: Metabolism in Diabetic Coma Produced by Alloxan. Proc. M. Fed. Clin. Research, 2:41-42, 1945. 11. Burn, J. H., Lewis, rr. H. C. and Kelsey, F. D.: The Di.etary Control of Alloxan Diabetes in Rats. Brit. M. J., Dec. 9, 1944, p. 752. 12. Martinez, C.: Accion del Aloxano y Dieta. Rev. Soc. argent. de bioI., 21: 332, 1945. 13. Martinez, C.: Thyroid and Sensitivity to Intravenous Alloxan. Rev. Soc. Argent. de biol., 21 :2.54-25'8, 1945. 14. 11artinez, C.: Accion del tiuracilo sobre la diabetes aloxanica y pancreatica de la rata. Rev. Soc. argent. de biol., 1946, en prensa. 15. Lowry, P. T. and Hegsted,D.M.: The Thiamine Requirement in Alloxan Diabetes. J. Lab. & elin. Med., 30:839--843, 1945. 16. Bailey,'C. C., Bailey, O. T. and Leech, R. S.: In press. 17. 'Warren, S.: The Pathology of Diabetes Mellitus, 2nd ed. Philadelphia, Lea & Febiger, 1938.

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18. GOlnori, G.: Observations \vith })ifferential Stains on IIuInan Islets of Lan.. gerhans. A.m. ]. Path., 17:395, 1941. 19. Brunschwig, A. and Allen, ]. G.: Specific Injurious Action of Alloxan upon Pancreatic Islets and Convoluted Tubules of the Kidney. Cancel' Research, 4: L!5, 1944. 20. Brunschwig, A., AlIen, J. G., O\\rens, F. ~I. and Thomton, 1". F.: Alloxan in the rfreatment of Insulin-Producing Cell (~arcinoma of !lancreas. J.A.M.A., 124:212, 1944. 21. Rynearson, E. 11.: To be published. 22. Conn, J. \V.: J. Lab. & CHn. Med. In press. 23. 1'albot, N. and Bailey, C. C.: 1'0 be published.