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ELECTROPHORESIS OF HUMAN PYRUVATE KINASE
920 PROPRANOLOL AND HYPOGLYCÆMIA SiR,-Kotler et al.1 noted that propranolol precipitated hypoglycxmia in an insulin-dependent diabetic but did not change his total insulin requirement. Sussman et al. suggested that propranolol " can be a potent stimulator of insulin secretion, and the hypoglycasmia observed ... may have been due to raised circulating-insulin levels ". Since, whichever view is true, propranolol can induce hypoglycxmia, we wondered whether this effect might be of value in the management of the labile diabetic. Our objective was to flatten the unpredictable hyperglycxmic peaks, and, if possible, to reduce the variable insulin requirement. The risks involved3 and the uncertainties of the proposed treatment were fully explained to and consent obtained from 4 outpatient labile diabetics before adding oral propranolol, in varying dosage and timing, to their daily treatment schedules. We give here a brief summary of the results. Case 1.-A 42-year-old man with diabetes for 27 years had developed progressive bilateral retinitis in the past 8 years and increasingly disabling coronary insufficiency in the past year. A 2000 calorie diet with 40 units isophane (NPH) insulin and supplementary crystalline insulin had failed to achieve good control of his persistent hyperglycxmia. Propranolol, 40 mg., t.i.d., was started. On the afternoon of the 2nd day the patient had a severe hypoglycaemic reaction after the noontime urine had been aglycosuric in contrast to the usually heavy glycosuria. Reduction of the daily isophane-insulin dosage to 35 then 25 units and the propranolol to 40 mg. b.d. failed to prevent the daily hypoglycxmic reactions. The patient refused to cooperate after the 8th day. Incidentally the exertional angina was not improved by the treatment. Case 2.-This 47-year-old woman with diabetes for 22 years, poorly controlled with an 1800 calorie diet and 23 units isophane plus 7 units crystalline insulin, was started on a single daily 40 mg. dose of propranolol at noon, when the glycosuria was most intense. She had a severe hypoglycaemic reaction 6 hours later, and again at the same time next day. The propranolol was reduced to 20 mg. daily, without further reactions but with unaltered glycosuria, during the next week. The daily dosage was again increased to 40 mg. at 8 A.M. and 10 mg. at bedtime, then continued at this level for the next 8 weeks. No reduction followed in the persistent glycosuria or the morning blood-sugar levels. Case 3.-A 34-year-old woman with diabetes for 19 years was poorly controlled with a 1500 calorie diet and 50 units isophane insulin plus 15-30 units supplementary crystalline insulin. Glycosuria was almost constant and attacks of hypoglycaEmia increased when more rigid control was attempted. In the past 8 years there was progressive bilateral retinitis and persistent heavy proteinuria. With the addition of 20 mg. propranolol at 10 A.M. and 6 P.M. daily the glycosuria decreased No further to a 2-plus reaction in all 4 daily specimens. hypoglycxmia reactions occurred while continuing with a single dose of 50 units isophane insulin daily. This improved status has persisted during the past 6 months but the heavy proteinuria has continued and fresh retinal haemorrhages have recurred at intervals. This seeming improvement in control may be due in part to a coincidental change in the diabetic state attributable to Kimmelstiel-Wilson nephropathy. Case 4.-This 47-year-old woman with diabetes for 25 years, poorly controlled with an 1800 calorie diet and 50 units isophane insulin plus supplementary crystalline insulin, had frequent hypoglycaemic reactions, which were especially disturbing because of a previous attack of myocardial infarction with recurrent anginal pain. The morning dose of isophane insulin was reduced to 25 units; then 20 mg. of propranolol was given at 10 A.M., 4 P.M. and 9 P.M. daily. In the next 9 days the fasting blood-sugar levels remained high, glycosuria was un1. Kotler, M. W., Berman, L., Rubenstein, A. H. Lancet, 1966, ii, 1389. 2. Sussman, K. E., Stjernholm, M. R., Vaughan, G. D. ibid. 1967, i, 626. 3. Abramson, E. A., Arky, R. A., Woeber, K. A. ibid. 1966, ii, 1386. Bewsher, P. D. ibid. 1967, i, 104. Mackintosh, T. F. ibid. Reveno, W. S., Rosenbaum, H. Harper Hosp. Bull. 1967, 25, 251. Lancet,
1967, i, 939.
altered, and the exertional anginal pain persisted. No hypoglycsemic reactions occurred even after gradual increase in insulin
dosage
to
40 units
isophane insulin daily.
In summary, it may be assumed that in the labile diabetic, p-adrenergic blockade, by opposing catecholamine action, might enhance hypoglycaEmia in sufficient degree to overcome the hyperglycaemia resulting from the multiple factors at play in this disease. This assumption would necessarily ascribe a dominant role to the catecholamines and a weak compensatory It is probable, or defensive reaction to these multiple factors. however, when propranolol is effective that there is already a trend toward hypoglycaemia and that the hypoglycaemia it induces is the result of -blockade interfering with the attainment of normoglycsemia. Propranolol does not induce hypoglycasmia in normal individuals, though it does so after p-adrenergic blockade, as Dr. Divitiis and his colleagues (April 6, p. 749) have shown from their response to the tolbutamide-load test. Accordingly its use would have no practical value in the management of the brittle diabetic. Department of Medicine, Harper Hospital and
Wayne University College of Medicine, Detroit, Michigan 48201.
WILLIAM S. REVENO HERBERT ROSENBAUM.
ELECTROPHORESIS OF HUMAN PYRUVATE KINASE letter SIR,ŇThe (March 9, p. 529) by Dr. Blume and colconfirms leagues my prior report1 of two electrophoretic components in human red cells. The symposiumat which my starch-gel electrophoretic studies were reported was cited in the letter but with reference only to a presentation which did not involve pyruvate kinase. Details of my methods of electrophoresis and staining have been described.’1 The staining method is based on the conversion of pyruvate to lactate by exogenous lactic-acid-dehydrogenase with ultraviolet visualisation of pyruvate kinase by zones of reduced-nicotinamideadenosine-dinucleatide extinction. Electrophoresis of more than 300 haemolysates revealed two red-cell components (fast and slow). Extracts of liver also proved to have two similar components, while leucocytes and other tissues were found to have only single (slow) components. The patterns observed on 1. Townes, P. L. in International Symposium on Hereditary Disorders of Erythrocyte Metabolism, Duarte, California, February, 1967; p. 259 (in the press).
Starch-gel electrophoresis of red-cell lysate (R.B.C.), and extracts of leucocytes (W.B.C.), liver, and muscle. (Photographed in ultraviolet light.)
921
starch-gel electrophoresis at pH 8-6 (triethanolamine buffer) are shown in the accompanying figure. A variety of buffers were examined, with the finding that only limited separation of the components occurred at pH values near neutrality. The least separation (approximately 1 cm.) observed by Dr. Blume and colleagues at pH 6-2 is in keeping with my earlier experience, but the discrimination is less satisfactory than that afforded by the greater separation (4-5 cm.) at the higher pH. The suggestion that the two red"
Antral mucosal extracts had no obvious effect in 3 fasting subiects, as follows:
on
absorption
cell components are " isozymes is questionable in view of my studies1 which suggest that the slow component is a molecular aggregate of the fast component. Division of Genetics, University of Rochester School of Medicine, Rochester, New York, U.S.A.
PHILIP L. TOWNES.
A similar effect had been found earlier in 3 anxmic gastrectomised subjects with a whole-hog-stomach extract (’ Pepsac
given orally.2 NUTRITION AT GREAT HEIGHTS SIR,-Dr. Bender (April 6, p. 759) doubts the results of my studies on South American highland Indians who " behave disobediently towards at least one of the laws of nutrition". My observations show that these " laws " do not have general validity, and that during years or generations of living under extreme nutritional conditions man may adapt his metabolism to the circumstances. Unfortunately, it is impossible to make such long laboratory experiments in man, and there is therefore reason to accept and utilise the experimental conditions which Nature herself has created for man, even if the results differ from the traditional " laws ". Hospital de Ninos, Salta, Argentine.
Support for this work was provided by the research grants mittee of the United Manchester Hospitals, Manchester 13. Department of Internal Medicine, University of Texas, Southwestern Medical School, Dallas, Texas 75235.
com-
L. A. TURNBERG.
ARNE HOEYGAARD.
GASTRIC FACTOR IN IRON ABSORPTION SiR,—Ihave obtained some data in human experiments which tend to support the conclusion of Dr. Murray and Miss Stein (March 23, p. 614) that there is a factor in gastric juice which may enhance iron absorption. Tests were performed in normal volunteers, aged 23-28 years. Iron absorption was measured using [5 9Fe]-ferriccitrate, specific activity 3-15 mC per mg. (Radiochemical Centre, Amersham). 5[1. C 59Fe was dissolved in 100 ml. of water with 100 mg. ascorbic acid. This solution was sipped during the ingestion of a standard meal containing about 5 mg. of iron.2 In other tests, on subjects fasted for 12 hours, 5 {C 59Fe was dissolved in 50 ml. water with 100 mg. ascorbic acid, and 5 mg. inorganic iron as ferrous sulphate. Absorption was estimated from measurements of radioactivity excreted in the stools for 7-10 days. 2 weeks were allowed to elapse between tests on the same subject. Gastric juice produced in response to histamine stimulation was obtained from patients with iron-
deficiency anaemia, immediately cooled, and adjusted to pH 7 with 0-1N sodium hydroxide. After filtration through gauze to remove large particles, the juice was concentrated 8-10 times by suspension inCarbowax 6000 ’ in dialysis sacs at 4°C.3 Specimens of human stomach were obtained at operation from patients subjected to partial gastrectomy for duodenal ulcer. Extracts were made of the mucosa of the body of the stomach and of the antrum in isotonic saline in the cold and the residue removed by filtration. 5 ml. of the extract of mucosa or of the gastric-juice concentrate was given with the iron solution to assess their effect on absorption. Gastric-juice concentrate enhanced the absorption of iron given with food in 3 normal subiects, as follows:
Extracts of the gastric mucosa from the body of the stomach enhanced the absorption of iron in 5 fasting normal subjects, as follows: 2. 3.
The present tests were performed with neutralised solutions, and any effect due to acid, which might be raised in objection to the findings of Dr. Murray and Miss Stein, was therefore eliminated. The results tend to confirm their interpretation that there is an iron-absorption-enhancing factor in the gastric juice of anaemic subjects and also to suggest that this factor might arise in the mucosa of the body of the stomach but not in the antrum.
Turnberg, L. A. Q. Jl Med. 1966, 35, 107. Ishimori, A., Glass, G. B. J. Clin. Chem. 1961, 7,
457.
SERUM-&agr;-FŒTOPROTEIN AND PRIMARY HEPATIC CANCER of embryo-specific x-foetoprotein patients with primary carcinoma of the liver has been reported by Tatarinov4 in four cases. A project on the diagnosis and treatment of primary cancer of the
SiR,—The (A.F.P.) in the
presence
sera
of
liver in Bantu mine-workers has been in progress in the Witwatersrand area, South Africa, for the past three years; in this period sera have been collected at monthly intervals from suspected patients referred to a central mine hospital. Many studies have been performed and have partly been reported.5 The sera were tested for the presence of A.F.P. by a standard Ouchterlony immunodiffusion technique4 using an antiserum prepared by injecting rabbits with serum from a fivemonth-old stillborn fcetus and absorbing the rabbit antiserum with pooled normal human serum. 6 Immunoelectrophoresis confirmed the presence of a single antigenic component in the reacting sera tested. Sera from 194 cases, a total of 405 samples, were available for study: 132 of the 194 were proved to have a primary cancer of the liver by biopsy (19 cases) or by post-mortem examination (113 cases); only 1 patient, who was lost to follow-up, had a negative biopsy but was considered to be an obvious case of primary cancer of the liver (see table). The remainder were Tatarinov, Y. S. Fedn Proc. Fedn Am. Socs exp. Biol. (translation supplement), 1966, 25, T344. 5. South African Primary Liver Cancer Research Group. S. Afr. med. J. 1967, 41, 309. 6. Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826.
4.
ANALYSIS OF NO. OF CASES OF PRIMARY CANCER OF LIVER TESTED
1967, i, 939.
altered, and the exertional anginal pain persisted. No hypoglycsemic reactions occurred even after gradual increase in insulin
dosage
to
40 units
isophane insulin daily.
In summary, it may be assumed that in the labile diabetic, p-adrenergic blockade, by opposing catecholamine action, might enhance hypoglycaEmia in sufficient degree to overcome the hyperglycaemia resulting from the multiple factors at play in this disease. This assumption would necessarily ascribe a dominant role to the catecholamines and a weak compensatory It is probable, or defensive reaction to these multiple factors. however, when propranolol is effective that there is already a trend toward hypoglycaemia and that the hypoglycaemia it induces is the result of -blockade interfering with the attainment of normoglycsemia. Propranolol does not induce hypoglycasmia in normal individuals, though it does so after p-adrenergic blockade, as Dr. Divitiis and his colleagues (April 6, p. 749) have shown from their response to the tolbutamide-load test. Accordingly its use would have no practical value in the management of the brittle diabetic. Department of Medicine, Harper Hospital and
Wayne University College of Medicine, Detroit, Michigan 48201.
WILLIAM S. REVENO HERBERT ROSENBAUM.
ELECTROPHORESIS OF HUMAN PYRUVATE KINASE letter SIR,ŇThe (March 9, p. 529) by Dr. Blume and colconfirms leagues my prior report1 of two electrophoretic components in human red cells. The symposiumat which my starch-gel electrophoretic studies were reported was cited in the letter but with reference only to a presentation which did not involve pyruvate kinase. Details of my methods of electrophoresis and staining have been described.’1 The staining method is based on the conversion of pyruvate to lactate by exogenous lactic-acid-dehydrogenase with ultraviolet visualisation of pyruvate kinase by zones of reduced-nicotinamideadenosine-dinucleatide extinction. Electrophoresis of more than 300 haemolysates revealed two red-cell components (fast and slow). Extracts of liver also proved to have two similar components, while leucocytes and other tissues were found to have only single (slow) components. The patterns observed on 1. Townes, P. L. in International Symposium on Hereditary Disorders of Erythrocyte Metabolism, Duarte, California, February, 1967; p. 259 (in the press).
Starch-gel electrophoresis of red-cell lysate (R.B.C.), and extracts of leucocytes (W.B.C.), liver, and muscle. (Photographed in ultraviolet light.)
921
starch-gel electrophoresis at pH 8-6 (triethanolamine buffer) are shown in the accompanying figure. A variety of buffers were examined, with the finding that only limited separation of the components occurred at pH values near neutrality. The least separation (approximately 1 cm.) observed by Dr. Blume and colleagues at pH 6-2 is in keeping with my earlier experience, but the discrimination is less satisfactory than that afforded by the greater separation (4-5 cm.) at the higher pH. The suggestion that the two red"
Antral mucosal extracts had no obvious effect in 3 fasting subiects, as follows:
on
absorption
cell components are " isozymes is questionable in view of my studies1 which suggest that the slow component is a molecular aggregate of the fast component. Division of Genetics, University of Rochester School of Medicine, Rochester, New York, U.S.A.
PHILIP L. TOWNES.
A similar effect had been found earlier in 3 anxmic gastrectomised subjects with a whole-hog-stomach extract (’ Pepsac
given orally.2 NUTRITION AT GREAT HEIGHTS SIR,-Dr. Bender (April 6, p. 759) doubts the results of my studies on South American highland Indians who " behave disobediently towards at least one of the laws of nutrition". My observations show that these " laws " do not have general validity, and that during years or generations of living under extreme nutritional conditions man may adapt his metabolism to the circumstances. Unfortunately, it is impossible to make such long laboratory experiments in man, and there is therefore reason to accept and utilise the experimental conditions which Nature herself has created for man, even if the results differ from the traditional " laws ". Hospital de Ninos, Salta, Argentine.
Support for this work was provided by the research grants mittee of the United Manchester Hospitals, Manchester 13. Department of Internal Medicine, University of Texas, Southwestern Medical School, Dallas, Texas 75235.
com-
L. A. TURNBERG.
ARNE HOEYGAARD.
GASTRIC FACTOR IN IRON ABSORPTION SiR,—Ihave obtained some data in human experiments which tend to support the conclusion of Dr. Murray and Miss Stein (March 23, p. 614) that there is a factor in gastric juice which may enhance iron absorption. Tests were performed in normal volunteers, aged 23-28 years. Iron absorption was measured using [5 9Fe]-ferriccitrate, specific activity 3-15 mC per mg. (Radiochemical Centre, Amersham). 5[1. C 59Fe was dissolved in 100 ml. of water with 100 mg. ascorbic acid. This solution was sipped during the ingestion of a standard meal containing about 5 mg. of iron.2 In other tests, on subjects fasted for 12 hours, 5 {C 59Fe was dissolved in 50 ml. water with 100 mg. ascorbic acid, and 5 mg. inorganic iron as ferrous sulphate. Absorption was estimated from measurements of radioactivity excreted in the stools for 7-10 days. 2 weeks were allowed to elapse between tests on the same subject. Gastric juice produced in response to histamine stimulation was obtained from patients with iron-
deficiency anaemia, immediately cooled, and adjusted to pH 7 with 0-1N sodium hydroxide. After filtration through gauze to remove large particles, the juice was concentrated 8-10 times by suspension inCarbowax 6000 ’ in dialysis sacs at 4°C.3 Specimens of human stomach were obtained at operation from patients subjected to partial gastrectomy for duodenal ulcer. Extracts were made of the mucosa of the body of the stomach and of the antrum in isotonic saline in the cold and the residue removed by filtration. 5 ml. of the extract of mucosa or of the gastric-juice concentrate was given with the iron solution to assess their effect on absorption. Gastric-juice concentrate enhanced the absorption of iron given with food in 3 normal subiects, as follows:
Extracts of the gastric mucosa from the body of the stomach enhanced the absorption of iron in 5 fasting normal subjects, as follows: 2. 3.
The present tests were performed with neutralised solutions, and any effect due to acid, which might be raised in objection to the findings of Dr. Murray and Miss Stein, was therefore eliminated. The results tend to confirm their interpretation that there is an iron-absorption-enhancing factor in the gastric juice of anaemic subjects and also to suggest that this factor might arise in the mucosa of the body of the stomach but not in the antrum.
Turnberg, L. A. Q. Jl Med. 1966, 35, 107. Ishimori, A., Glass, G. B. J. Clin. Chem. 1961, 7,
457.
SERUM-&agr;-FŒTOPROTEIN AND PRIMARY HEPATIC CANCER of embryo-specific x-foetoprotein patients with primary carcinoma of the liver has been reported by Tatarinov4 in four cases. A project on the diagnosis and treatment of primary cancer of the
SiR,—The (A.F.P.) in the
presence
sera
of
liver in Bantu mine-workers has been in progress in the Witwatersrand area, South Africa, for the past three years; in this period sera have been collected at monthly intervals from suspected patients referred to a central mine hospital. Many studies have been performed and have partly been reported.5 The sera were tested for the presence of A.F.P. by a standard Ouchterlony immunodiffusion technique4 using an antiserum prepared by injecting rabbits with serum from a fivemonth-old stillborn fcetus and absorbing the rabbit antiserum with pooled normal human serum. 6 Immunoelectrophoresis confirmed the presence of a single antigenic component in the reacting sera tested. Sera from 194 cases, a total of 405 samples, were available for study: 132 of the 194 were proved to have a primary cancer of the liver by biopsy (19 cases) or by post-mortem examination (113 cases); only 1 patient, who was lost to follow-up, had a negative biopsy but was considered to be an obvious case of primary cancer of the liver (see table). The remainder were Tatarinov, Y. S. Fedn Proc. Fedn Am. Socs exp. Biol. (translation supplement), 1966, 25, T344. 5. South African Primary Liver Cancer Research Group. S. Afr. med. J. 1967, 41, 309. 6. Gitlin, D., Boesman, M. J. clin. Invest. 1966, 45, 1826.
4.
ANALYSIS OF NO. OF CASES OF PRIMARY CANCER OF LIVER TESTED