- Email: [email protected]
HYPOGLYCÆMIC EFFECT OF ADIPOSE-TISSUE EXTRACTS IN ADRENALECTOMISED RATS
602 RESULTS
Following the injection of the enzyme the behaviour of the clottable
purified coagulant radioactivity in the
essentially the same as that described elsefor the crude venom; the initial rapid fall (figs. 1-3) associated with the formation of disseminated microclots was followed by a rise in protein-bound non-clottable radioactivity (figs. 2 and 3) due to the appearance of labelled breakdown products arising from the dissolving microthrombi. Plasma-haemoglobin began to rise during the fall of labelled fibrinogen, and peak values coincided with the release of solubilised fibrin from the sites of deposition (figs. 2 and 3). The degree of haemolysis correlated with the rate of disappearance of fibrinogen; at slower rates the haemoglobinaemia was less (fig. 1). The injection of 59Fe-labelled red cells before or up to 15 minutes after the venom resulted in a simultaneous rise in the level of free haemoglobin and the amount of 59Fe radioactivity in the plasma (fig. 2). However, when the labelled red cells were injected 20 minutes or more after the venom there was little rise in radioactivity despite the plasma where
was
9
pronounced haemoglobinaemia (fig. 3). In rabbits which had been made anbrinogenaemic by the infusion of plasmin, injection of the coagulant enzyme caused no haemolysis. If clot lysis was prevented by pretreating the animals with soya-bean trypsin inhibitor and e-aminocaproic acid, haemolysis still occurred. Rapid infusion of the fibrinopeptides from 280 mg. of fibrinogen into a rabbit failed to produce haemolysis. The haemoglobinaemia can be observed only in vivo, as the purified coagulant enzyme of the venom when incubated with washed red cells
or
whole blood
causes
the venom-clots lyse more rapidly. The number and size of thrombi forming in blood-vessels will greatly depend, therefore, on the rate at which the intravascular fibrinogen pool is being converted into fibrin. Thus the interdependence between this rate and the haemoglobinaemia is just another expression of the relationship between the extent of red-cell destruction and the histological findings in the small vessels.3-5 The mechanism of haemolysis is still under investigation and will be the subject of a fuller report. Our findings support the previous evidence that there exists a close relationship between disseminated intravascular clotting and haemolysis, and that such states may exist in human disease. Although the morphological changes in the red cells were slight in these acute experiments, the presence of fragmented red cells have been reported in other experiments,3-5 and this change in human disease may provide a valuable clue to an underlying pathological process which may be successfully treated with heparin.12 The batchof A.R.V. used was a generous gift by Dr. G. W. Tunnah, Twyford Laboratories Ltd., London. Requests for reprints should be addressed to E. R. Biophysics Division, National Institute for Medical Research, Mill Hill, London N.W.7
M.R.C. Group for the Study of Hæmolytic Mechanisms, Royal Postgraduate Medical School, London W.12 *
neither
OF ADIPOSE-TISSUE EXTRACTS IN
DISCUSSION
9. Regoeczi, E., Gergely, J., McFarlane, A. S. J. clin. Invest. 1966, 45, 1202. 10. Pederson, H. J., Marr, J., Barboriak, J. J., Tebo, T. H., Johnson, S. A. Fedn Proc. Fedn Am. Socs exp. Biol. 1965, 24, 452. 11. Regoeczi, E. Z. Tropenmed. Parasit. 1966, 17, 144.
United States Public Health Service.
HYPOGLYCÆMIC EFFECT
haemolysis nor an increase in osmotic fragility. The onset of haemoglobinasmia during the course of defibrination9 suggests that haemolysis occurs during the trapping of red cells in microthrombi which accumulate temporarily in small blood-vessels. The rise in plasmahsemoglobin coincides with the release of solubilised fibrin from microthrombi (figs. 2 and 3). This suggests that hsemoglobinsemia may depend on the release of damaged red cells into the circulation. Somewhat against this hypothesis is the fact that haemoglobinaemia occurred when lysis of the microthrombi was prevented by pretreatment with soya-bean trypsin inhibitor and e-aminocaproic acid, at a time when no release of soluble fibrin or, presumably, of damaged red cells occurred. Nevertheless, it seems probable that red-cell damage is sustained in the initial stages of microthrombus formation, and that hxmoglobinxmia results from leakage of haemoglobin either from red cells held within the microthrombi, or from red cells damaged on passage through vessels undergoing thrombosis, yet failing to be trapped. This accords with the electron-micrographic studies of Pederson et al.,10 who observed lysis of red cells in newly formed clots in vivo, and the presence of fibrin on the surface of adjacent intact red cells. Our studies with 59Fe-labelled red cells confirm that red-cell damage was sustained in the initial stages of microthrombus formation. They do not, however, show exactly how the red-cell damage is brought about. The fibrin monomers produced by A.R.v. on the one hand and by thrombin on the other are not identical," and
Special research fellow,
E. REGOECZI Tuebingen M. L. RUBENBERG* M.D. Washington M. C. BRAIN D.M. Oxon., M.R.C.P. M.D.
ADRENALECTOMISED RATS Summary
Injection of partly purified preparations of extracts of adipose tissue (A.T.E.) from the
epididymis of rats into adrenalectomised rats reduced concentrations of blood-glucose significantly. The active principle in the A.T.E. preparations was heat-stable (100°C for 5 minutes) and ultrafiltrable through an ’ Amicon UM-2 ’ membrane, which allows the passage of substances with a molecular weight below 600. Absorption of the heated and ultrafiltered A.T.E. (pH 6·0) was at its height at 249 mµ and at its lowest at 231 mµ. In IN sodium hydroxide absorption was highest at 251 mµ and lowest at 235 mµ. The hypoglycæmic effect of A.T.E. in the adrenalectomised rats may be due, at least in part, to the activation and utilisation of their circulating inactive bound insulin by the tissues under the stimulation of A.T.E. INTRODUCTION
INSULIN activity in health is probably regulated by the balance between two forms of circulating insulin called free and bound.13 Free insulin is released by the pancreas under the stimulation of glucose, is biologically active, and has properties similar to those of crystalline insulin. Bound insulin, we suggested, represents an inactive metabolite of free insulin, and, in appropriate circumstances, the metabolism of free to bound insulin, and the activation of bound-insulin is catalysed by extra pancreatic tissues.13 In patients with the adult-onset type of diabetes the rate of 12.
Brain, M. C., Baker, L. R. I., McBride, J. A., Rubenberg, M. L. Unpublished. 13. Antoniades, H. N. On the Nature and Treatment of Diabetes; p. 194. Amsterdam, 1965.
603 HYPOGLYCaeMIC EFFECTS OF EXTRACTS OF RAT EPIDIDYMAL ADIPOSE TISSUE IN ADRENALECTOMISED RATS
transformation of free
to bound-insulin is increased, and the rate of utilisation of bound-insulin is decreased.13 Inactive bound-insulin accumulates in the blood of these patients, which in turn, can prevent the normal utilisation of active, free insulin by the tissues.14 The observation that extracts of adipose tissue (A.T.E.) can stimulate the activation of human or rat serum-boundinsulin in vitro in isolated muscle,15-17 prompted the idea that the factors in A.T.E. responsible for this effect could also stimulate the activation and utilisation of circulating bound-insulin in vivo, and thus increase the rate of glucose utilisation. This possibility was tested by injecting purified A.T.E. preparations into insulin-sensitive animals, such as adrenalectomised rats.
various intervals after the injection. Blood-glucose concentrations were measured with a’Technicon Auto-Analyzer’. Groups of three to four rats were injected with A.T.E. and an equal number of rats with control 0- 15M sodium chloride. RESULTS
Injection of partly purified A.T.E. into adrenalectomised produced a significant gradual decline in their bloodglucose concentrations as compared with injections of 0- 15M sodium chloride alone into control rats (see accompanying table). Similar results were obtained with heated A.T.E. and with heated-ultrafiltered A.T.E. (see table). rats
DISCUSSION
A.T.E. MATERIALS AND METHODS
Partly purified preparations of A.T.E. were obtained from pooled rat-epididymal tissue pads, homogenised in a ’Waring’ blender in cold distilled water. The homogenate was passed through a nylon cloth (100 mesh), centrifuged, the liquid phase collected, cooled at 2-5°C and brought to 60% ethanol concentration under continuous stirring. The mixture was centrifuged at -5°C for 30 minutes in an’International’ refrigerated centrifuge, and the precipitate was discarded. The *
supernatant fluid was diluted with cold distilled water and lyophilised. The dry powder was collected and stored in a desiccator at -15°C. About 200 mg. of dry powder was obtained from 1 kg. of pooled adipose-tissue pads. The partly purified A.T.E. was further purified by heating at 100°C solutions of these preparations in 0’15Af sodium chloride for 5 minutes. The inactive precipitate was discarded by centrifugation. This process produced a roughly threefold purification of the original preparation without affecting its activity. Ultrafiltration of heated A.T.E. preparations through ’Amicon UM-2’ filters (which permit the passage of substances with a molecular weight below 600) produced an additional twofold purification on a dry-weight basis. The filtrate retained the activity of the preparation. Absorption spectra of heated and ultrafiltered preparations of A.T.E. were measured in a ’Cary, model 14 ’ recording spectrophotometer. Density was measured with a 1 cm. cell, in 0- 15M sodium chloride, pH 6-0, and in 1N sodium hydroxide. 1 ml. of 0-15M sodium chloride (with or without A.T.E.) was injected into the jugular vein of each rat (’ Charles River Laboratories ’, male CD rats, weighing 100-115 g.). The rats were injected about a week after adrenalectomy. Food was removed from their cages 2 hours before injection, and the rats were narcotised lightly before the injection with 50% carbon dioxide and 50% oxygen. Blood-samples (0-1 ml.) were collected from the tails of the rats before injection and at 14. 15. 16. 17.
Antoniades, H. N. Lancet, 1965, ii, 159. Antoniades, H. N., Gundersen, K. Endocrinology, 1961, 68, Shaw, W. N., Shuey, E. W. Biochemistry, 1963, 2, 286. Antoniades, H. N. Diabetes, 1966, 15, 889.
36.
findings suggest that the active substance in the preparation is heat-stable, and has a molecular
These
weight of less than
600. The maximum absorption of the heated and ultrafiltered A.T.E. (pH 6-0) was at 249 m and the minimum was at 231 m{.L. In a solution of 1N sodium hydroxide the maximum was at 251 m and the minimum at 235 m. Whether the substance exhibiting these absorption spectra is the active principle in A.T.E. or whether it is an inactive contaminant is not known.
Partly purified extracts of rat kidney and heart (heated and heated-ultrafiltered preparations) were not effective in adrenalectomised rats, even when injected at concentrations equivalent to four times the A.T.E. concentration. Liver extracts also seem to be inactive in rats. Antoniades and Gundersen showed that extracts of kidney and muscle did not activate bound-insulin in isolated muscle in vitro. 15 The
in adrenalectomised due to an increased rate of utilisation of circulating bound-insulin by the tissues under the stimulation of A.T.E. This possibility is supported by the demonstration that A.T.E. can activate human or rat bound-insulin on isolated muscle.15-17 Preliminary studies indicated that the concentration of bound-insulin declined in the blood of adrenalectomised rats 3-4 hours after the injection of A.T.E. The concentration of bound-insulin was not affected in control rats injected with 0.15M sodium chloride. Other effects of A.T.E., such as pancreatic stimulation, are not excluded. Hypoglycaemia in adrenalectomised rats with A.T.E. was accompanied by an increase in the rate of incorporation of glucose carbon in their muscle (hemidiaphragm) glycogen and in the fat of their epididymal adipose tissue. These effects are similar to those produced by the injection of crystalline or bound insulin in these rats.I8 The concentration of bound-
hypoglycxmic effect of A.T.E.
rats may be
18.
Antoniades, H. N., Gershoff, S. N. Endocrinology, 1966, 78, 1079.
604 insulin in adrenalectomised rats is about 300-400 microunits per ml. of serum. Intact rats, with 400-500 microunits of bound-insulin per ml. of serum, 17 did not respond to injections of A.T.E. Intact rats, however, are less sensitive to insulin than adrenalectomised rats. The blood-glucose levels of intact rats were not affected by injections of 10 milliunits of crystalline insulin.18 19 On the other hand, injection of 5 milliunits of crystalline insulin produced hypoglycaemia in adrenalectomised rats.188 The effect of A.T.E. administration in newly discovered 19.
Rafaelsen, O. J., Lauris, V., Renold, A. E. Diabetes, 1965, 14, 19.
Reviews of Books Textbook of Obstetrics and Gynecology Editor: DAVID N. DANFORTH, PH.D., M.D., F.A.C.O.G., F.A.C.S.,
professor and chairman, department of obstetrics and gynecology, Northwestern University Medical School. London and New York: Harper & Row. 1966. Pp. 1146. E10. FORTY-THREE authors from more than 30 medical schools in the United States of America and Canada have contributed to this new textbook, which is designed to meet the needs of the student and of the young specialist in training. After an account of important milestones in the history of obstetrics and gynaecology the opening chapters deal with the basic sciences including anatomy, physiology, embryology, endocrinology, and genetics. As in other leading American texts these subjects are described in depth and are a strong feature of the book. There is a brief but helpful section on the principles of genetic inheritance and genetic counselling. The clinical teaching both in obstetrics and in gynaecology is largely acceptable, but in some matters (compared with practice in the United Kingdom) there is a different emphasis, and of course certain terms have one meaning in the United States and another here: for example, abortion is said to be termination of pregnancy before 20 weeks’ gestation (and not 28 weeks as in the United Kingdom). Three different definitions are given for perinatal mortality-a term which is clearly laid down and understood in this country. The high cost of hospital care in the United States is reflected in the advice that patients with suspected placenta prsevia may be allowed home (if they live near hospital) once the bleeding has stopped. This is risky, even though the women are seen regularly as outpatients. There are clear, well-illustrated descriptions of standard operative procedures, but more could have been said about the many important ways in which socioeconomic factors can influence reproduction and the diseases which affect the female genital tract. The book is well produced, with double columns to the page and the text suitably illustrated by large numbers of tables, diagrams, and photographs, all of high quality. Students for a higher diploma will appreciate especially the basic, scientific content of this work, and will recognise where advice or practice is at variance with accepted principles in this country.
Principles
of Resuscitation
STANLEY FELDMAN, B.SC., M.B., F.F.A. R.C.S., consultant anaathetist, Westminster Hospital, London; HAROLD ELLIS, D.M., 1967. F.R.C.S., professor of surgery, Westminster Hospital. Pp. 128. Oxford: &bgr;lackwell Scientific Publications. Philadelphia : F. A. Davis Company. 30s.;$6-00.
THIS small book presents in concise, readable, and straightforward fashion the principles and practice of resuscitation. Carriage of oxygen to the tissues and the causes of hypoxia are first considered, and then the various forms of artificial ventilation and the treatment of pneumothorax and airway obstruction. Tracheostomy and laryngotomy are clearly described. The major part of the book deals with cardiac arrest, the most urgent of all emergencies, emphasising that the mainstay of diagnosis is the disappearance of major pulses. Diagnosis, treatment
untreated diabetics of the maturity-onset type, who have a high concentration of circulating bound-insulin, is being
investigated. These studies were supported by Public Health Service grants AM-08381 and HE-06302 National Institutes of Health. Requests for reprints should be addressed to H. N. A., Protein Foundation Laboratories, 281 South Street, Jamaica Plain 02130, Massachusetts. Protein Foundation Laboratories, Jamaica Plain, and the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts, U.S.A.
HARRY N. ANTONIADES Ph.D. Athens.
(including the pros and cons of open and closed cardiac massage), the use of defibrillation and drugs, and aftercare are all clearly if briefly considered. The section on the ethics of resuscitation raises the difficult problem of successful resuscitation with severe mental and physical sequela:. The illustrations are clear and helpful. In the next edition the authors could with advantage add sections on the principles of bloodtransfusion for severe haemorrhage and the emergency diagnosis and treatment of pulmonary embolism. Young housemen and senior students will particularly benefit from this book, which fits snugly into the pocket of a white coat.
Thymic
Tumours
Their Association with Myasthenia Gravis and their Treatment
by Radiotherapy. J. F. HALE, M.A., D.M., M.R.C.P., physician, Luton and Dunstable Hospital; E. F. SCOWEN, M.D., D.sC., F.R.C.P., F.R.C.P.E., F.R.C.S., F.C.PATH., professor of medicine, University of London, and physician to St. Bartholomew’s Hospital. London: Lloyd-Luke. 1967. Pp. 93. 30s. of a series of 64 cases will be of all concerned in the treatment of thymic tumours or myasthenia gravis, or of both together. It can also be recommended to those who have a more academic interest in the elusive functions of the thymus gland. The study is predominantly clinical, and the practical problems of treatment Whilst it is disappointing to learn are critically examined. that surgical removal of the tumour does not confer any obvious benefit on the victim of myasthenia gravis, recognition of this fact will save a good deal of suffering and-as it would appearfutile labour. On the other hand, radiotherapy (discussed in part II) is regarded by the authors as an essential ingredient of treatment for the myasthenic patient with a thymic tumour. The fascinating problem of the relation of thymic tumours to myasthenia gravis is analysed (pp. 55-62). There seem to be no grounds for contending that myasthenia gravis promotes the development of a thymic tumour, or vice versa. In the light of recent research the authors favour the hypothesis that both these conditions are promoted by a common cause, and that this is most probably a disturbance of the body’s autoimmune process. This conclusion will, it may be hoped, provoke further research. A bibliography of 290 references is
THIS interest
scholarly analysis
to
appended. Introduction
to
Environmental
Physiology
Environmental Extremes and Mammalian Survival. G. EDGAR FOLK, JR., PH.D., professor, department of physiology, University of Iowa, Iowa City, Iowa. London: Henry Kimpton. 1966.
Pp.
308. 90s.
THIS book deals with the effects of heat, cold, high or low pressure, hibernation, light, biological rhythms, and temperature regulation. Primarily of interest to physiologists, it should be useful to those concerned with the relation between health and environment. The rapidly growing volume of work on biological rhythms is well described, as might be expected from the author’s notable contributions in this field. The style is often obscure, and indeed sections of the book read as if they were lecturer’s notes. Temperatures are sometimes expressed in Fahrenheit and at other times in Centigrade, and the references seem to be arbitrary. But, despite minor blemishes, this volume can be recommended to the critical student.
Following the injection of the enzyme the behaviour of the clottable
purified coagulant radioactivity in the
essentially the same as that described elsefor the crude venom; the initial rapid fall (figs. 1-3) associated with the formation of disseminated microclots was followed by a rise in protein-bound non-clottable radioactivity (figs. 2 and 3) due to the appearance of labelled breakdown products arising from the dissolving microthrombi. Plasma-haemoglobin began to rise during the fall of labelled fibrinogen, and peak values coincided with the release of solubilised fibrin from the sites of deposition (figs. 2 and 3). The degree of haemolysis correlated with the rate of disappearance of fibrinogen; at slower rates the haemoglobinaemia was less (fig. 1). The injection of 59Fe-labelled red cells before or up to 15 minutes after the venom resulted in a simultaneous rise in the level of free haemoglobin and the amount of 59Fe radioactivity in the plasma (fig. 2). However, when the labelled red cells were injected 20 minutes or more after the venom there was little rise in radioactivity despite the plasma where
was
9
pronounced haemoglobinaemia (fig. 3). In rabbits which had been made anbrinogenaemic by the infusion of plasmin, injection of the coagulant enzyme caused no haemolysis. If clot lysis was prevented by pretreating the animals with soya-bean trypsin inhibitor and e-aminocaproic acid, haemolysis still occurred. Rapid infusion of the fibrinopeptides from 280 mg. of fibrinogen into a rabbit failed to produce haemolysis. The haemoglobinaemia can be observed only in vivo, as the purified coagulant enzyme of the venom when incubated with washed red cells
or
whole blood
causes
the venom-clots lyse more rapidly. The number and size of thrombi forming in blood-vessels will greatly depend, therefore, on the rate at which the intravascular fibrinogen pool is being converted into fibrin. Thus the interdependence between this rate and the haemoglobinaemia is just another expression of the relationship between the extent of red-cell destruction and the histological findings in the small vessels.3-5 The mechanism of haemolysis is still under investigation and will be the subject of a fuller report. Our findings support the previous evidence that there exists a close relationship between disseminated intravascular clotting and haemolysis, and that such states may exist in human disease. Although the morphological changes in the red cells were slight in these acute experiments, the presence of fragmented red cells have been reported in other experiments,3-5 and this change in human disease may provide a valuable clue to an underlying pathological process which may be successfully treated with heparin.12 The batchof A.R.V. used was a generous gift by Dr. G. W. Tunnah, Twyford Laboratories Ltd., London. Requests for reprints should be addressed to E. R. Biophysics Division, National Institute for Medical Research, Mill Hill, London N.W.7
M.R.C. Group for the Study of Hæmolytic Mechanisms, Royal Postgraduate Medical School, London W.12 *
neither
OF ADIPOSE-TISSUE EXTRACTS IN
DISCUSSION
9. Regoeczi, E., Gergely, J., McFarlane, A. S. J. clin. Invest. 1966, 45, 1202. 10. Pederson, H. J., Marr, J., Barboriak, J. J., Tebo, T. H., Johnson, S. A. Fedn Proc. Fedn Am. Socs exp. Biol. 1965, 24, 452. 11. Regoeczi, E. Z. Tropenmed. Parasit. 1966, 17, 144.
United States Public Health Service.
HYPOGLYCÆMIC EFFECT
haemolysis nor an increase in osmotic fragility. The onset of haemoglobinasmia during the course of defibrination9 suggests that haemolysis occurs during the trapping of red cells in microthrombi which accumulate temporarily in small blood-vessels. The rise in plasmahsemoglobin coincides with the release of solubilised fibrin from microthrombi (figs. 2 and 3). This suggests that hsemoglobinsemia may depend on the release of damaged red cells into the circulation. Somewhat against this hypothesis is the fact that haemoglobinaemia occurred when lysis of the microthrombi was prevented by pretreatment with soya-bean trypsin inhibitor and e-aminocaproic acid, at a time when no release of soluble fibrin or, presumably, of damaged red cells occurred. Nevertheless, it seems probable that red-cell damage is sustained in the initial stages of microthrombus formation, and that hxmoglobinxmia results from leakage of haemoglobin either from red cells held within the microthrombi, or from red cells damaged on passage through vessels undergoing thrombosis, yet failing to be trapped. This accords with the electron-micrographic studies of Pederson et al.,10 who observed lysis of red cells in newly formed clots in vivo, and the presence of fibrin on the surface of adjacent intact red cells. Our studies with 59Fe-labelled red cells confirm that red-cell damage was sustained in the initial stages of microthrombus formation. They do not, however, show exactly how the red-cell damage is brought about. The fibrin monomers produced by A.R.v. on the one hand and by thrombin on the other are not identical," and
Special research fellow,
E. REGOECZI Tuebingen M. L. RUBENBERG* M.D. Washington M. C. BRAIN D.M. Oxon., M.R.C.P. M.D.
ADRENALECTOMISED RATS Summary
Injection of partly purified preparations of extracts of adipose tissue (A.T.E.) from the
epididymis of rats into adrenalectomised rats reduced concentrations of blood-glucose significantly. The active principle in the A.T.E. preparations was heat-stable (100°C for 5 minutes) and ultrafiltrable through an ’ Amicon UM-2 ’ membrane, which allows the passage of substances with a molecular weight below 600. Absorption of the heated and ultrafiltered A.T.E. (pH 6·0) was at its height at 249 mµ and at its lowest at 231 mµ. In IN sodium hydroxide absorption was highest at 251 mµ and lowest at 235 mµ. The hypoglycæmic effect of A.T.E. in the adrenalectomised rats may be due, at least in part, to the activation and utilisation of their circulating inactive bound insulin by the tissues under the stimulation of A.T.E. INTRODUCTION
INSULIN activity in health is probably regulated by the balance between two forms of circulating insulin called free and bound.13 Free insulin is released by the pancreas under the stimulation of glucose, is biologically active, and has properties similar to those of crystalline insulin. Bound insulin, we suggested, represents an inactive metabolite of free insulin, and, in appropriate circumstances, the metabolism of free to bound insulin, and the activation of bound-insulin is catalysed by extra pancreatic tissues.13 In patients with the adult-onset type of diabetes the rate of 12.
Brain, M. C., Baker, L. R. I., McBride, J. A., Rubenberg, M. L. Unpublished. 13. Antoniades, H. N. On the Nature and Treatment of Diabetes; p. 194. Amsterdam, 1965.
603 HYPOGLYCaeMIC EFFECTS OF EXTRACTS OF RAT EPIDIDYMAL ADIPOSE TISSUE IN ADRENALECTOMISED RATS
transformation of free
to bound-insulin is increased, and the rate of utilisation of bound-insulin is decreased.13 Inactive bound-insulin accumulates in the blood of these patients, which in turn, can prevent the normal utilisation of active, free insulin by the tissues.14 The observation that extracts of adipose tissue (A.T.E.) can stimulate the activation of human or rat serum-boundinsulin in vitro in isolated muscle,15-17 prompted the idea that the factors in A.T.E. responsible for this effect could also stimulate the activation and utilisation of circulating bound-insulin in vivo, and thus increase the rate of glucose utilisation. This possibility was tested by injecting purified A.T.E. preparations into insulin-sensitive animals, such as adrenalectomised rats.
various intervals after the injection. Blood-glucose concentrations were measured with a’Technicon Auto-Analyzer’. Groups of three to four rats were injected with A.T.E. and an equal number of rats with control 0- 15M sodium chloride. RESULTS
Injection of partly purified A.T.E. into adrenalectomised produced a significant gradual decline in their bloodglucose concentrations as compared with injections of 0- 15M sodium chloride alone into control rats (see accompanying table). Similar results were obtained with heated A.T.E. and with heated-ultrafiltered A.T.E. (see table). rats
DISCUSSION
A.T.E. MATERIALS AND METHODS
Partly purified preparations of A.T.E. were obtained from pooled rat-epididymal tissue pads, homogenised in a ’Waring’ blender in cold distilled water. The homogenate was passed through a nylon cloth (100 mesh), centrifuged, the liquid phase collected, cooled at 2-5°C and brought to 60% ethanol concentration under continuous stirring. The mixture was centrifuged at -5°C for 30 minutes in an’International’ refrigerated centrifuge, and the precipitate was discarded. The *
supernatant fluid was diluted with cold distilled water and lyophilised. The dry powder was collected and stored in a desiccator at -15°C. About 200 mg. of dry powder was obtained from 1 kg. of pooled adipose-tissue pads. The partly purified A.T.E. was further purified by heating at 100°C solutions of these preparations in 0’15Af sodium chloride for 5 minutes. The inactive precipitate was discarded by centrifugation. This process produced a roughly threefold purification of the original preparation without affecting its activity. Ultrafiltration of heated A.T.E. preparations through ’Amicon UM-2’ filters (which permit the passage of substances with a molecular weight below 600) produced an additional twofold purification on a dry-weight basis. The filtrate retained the activity of the preparation. Absorption spectra of heated and ultrafiltered preparations of A.T.E. were measured in a ’Cary, model 14 ’ recording spectrophotometer. Density was measured with a 1 cm. cell, in 0- 15M sodium chloride, pH 6-0, and in 1N sodium hydroxide. 1 ml. of 0-15M sodium chloride (with or without A.T.E.) was injected into the jugular vein of each rat (’ Charles River Laboratories ’, male CD rats, weighing 100-115 g.). The rats were injected about a week after adrenalectomy. Food was removed from their cages 2 hours before injection, and the rats were narcotised lightly before the injection with 50% carbon dioxide and 50% oxygen. Blood-samples (0-1 ml.) were collected from the tails of the rats before injection and at 14. 15. 16. 17.
Antoniades, H. N. Lancet, 1965, ii, 159. Antoniades, H. N., Gundersen, K. Endocrinology, 1961, 68, Shaw, W. N., Shuey, E. W. Biochemistry, 1963, 2, 286. Antoniades, H. N. Diabetes, 1966, 15, 889.
36.
findings suggest that the active substance in the preparation is heat-stable, and has a molecular
These
weight of less than
600. The maximum absorption of the heated and ultrafiltered A.T.E. (pH 6-0) was at 249 m and the minimum was at 231 m{.L. In a solution of 1N sodium hydroxide the maximum was at 251 m and the minimum at 235 m. Whether the substance exhibiting these absorption spectra is the active principle in A.T.E. or whether it is an inactive contaminant is not known.
Partly purified extracts of rat kidney and heart (heated and heated-ultrafiltered preparations) were not effective in adrenalectomised rats, even when injected at concentrations equivalent to four times the A.T.E. concentration. Liver extracts also seem to be inactive in rats. Antoniades and Gundersen showed that extracts of kidney and muscle did not activate bound-insulin in isolated muscle in vitro. 15 The
in adrenalectomised due to an increased rate of utilisation of circulating bound-insulin by the tissues under the stimulation of A.T.E. This possibility is supported by the demonstration that A.T.E. can activate human or rat bound-insulin on isolated muscle.15-17 Preliminary studies indicated that the concentration of bound-insulin declined in the blood of adrenalectomised rats 3-4 hours after the injection of A.T.E. The concentration of bound-insulin was not affected in control rats injected with 0.15M sodium chloride. Other effects of A.T.E., such as pancreatic stimulation, are not excluded. Hypoglycaemia in adrenalectomised rats with A.T.E. was accompanied by an increase in the rate of incorporation of glucose carbon in their muscle (hemidiaphragm) glycogen and in the fat of their epididymal adipose tissue. These effects are similar to those produced by the injection of crystalline or bound insulin in these rats.I8 The concentration of bound-
hypoglycxmic effect of A.T.E.
rats may be
18.
Antoniades, H. N., Gershoff, S. N. Endocrinology, 1966, 78, 1079.
604 insulin in adrenalectomised rats is about 300-400 microunits per ml. of serum. Intact rats, with 400-500 microunits of bound-insulin per ml. of serum, 17 did not respond to injections of A.T.E. Intact rats, however, are less sensitive to insulin than adrenalectomised rats. The blood-glucose levels of intact rats were not affected by injections of 10 milliunits of crystalline insulin.18 19 On the other hand, injection of 5 milliunits of crystalline insulin produced hypoglycaemia in adrenalectomised rats.188 The effect of A.T.E. administration in newly discovered 19.
Rafaelsen, O. J., Lauris, V., Renold, A. E. Diabetes, 1965, 14, 19.
Reviews of Books Textbook of Obstetrics and Gynecology Editor: DAVID N. DANFORTH, PH.D., M.D., F.A.C.O.G., F.A.C.S.,
professor and chairman, department of obstetrics and gynecology, Northwestern University Medical School. London and New York: Harper & Row. 1966. Pp. 1146. E10. FORTY-THREE authors from more than 30 medical schools in the United States of America and Canada have contributed to this new textbook, which is designed to meet the needs of the student and of the young specialist in training. After an account of important milestones in the history of obstetrics and gynaecology the opening chapters deal with the basic sciences including anatomy, physiology, embryology, endocrinology, and genetics. As in other leading American texts these subjects are described in depth and are a strong feature of the book. There is a brief but helpful section on the principles of genetic inheritance and genetic counselling. The clinical teaching both in obstetrics and in gynaecology is largely acceptable, but in some matters (compared with practice in the United Kingdom) there is a different emphasis, and of course certain terms have one meaning in the United States and another here: for example, abortion is said to be termination of pregnancy before 20 weeks’ gestation (and not 28 weeks as in the United Kingdom). Three different definitions are given for perinatal mortality-a term which is clearly laid down and understood in this country. The high cost of hospital care in the United States is reflected in the advice that patients with suspected placenta prsevia may be allowed home (if they live near hospital) once the bleeding has stopped. This is risky, even though the women are seen regularly as outpatients. There are clear, well-illustrated descriptions of standard operative procedures, but more could have been said about the many important ways in which socioeconomic factors can influence reproduction and the diseases which affect the female genital tract. The book is well produced, with double columns to the page and the text suitably illustrated by large numbers of tables, diagrams, and photographs, all of high quality. Students for a higher diploma will appreciate especially the basic, scientific content of this work, and will recognise where advice or practice is at variance with accepted principles in this country.
Principles
of Resuscitation
STANLEY FELDMAN, B.SC., M.B., F.F.A. R.C.S., consultant anaathetist, Westminster Hospital, London; HAROLD ELLIS, D.M., 1967. F.R.C.S., professor of surgery, Westminster Hospital. Pp. 128. Oxford: &bgr;lackwell Scientific Publications. Philadelphia : F. A. Davis Company. 30s.;$6-00.
THIS small book presents in concise, readable, and straightforward fashion the principles and practice of resuscitation. Carriage of oxygen to the tissues and the causes of hypoxia are first considered, and then the various forms of artificial ventilation and the treatment of pneumothorax and airway obstruction. Tracheostomy and laryngotomy are clearly described. The major part of the book deals with cardiac arrest, the most urgent of all emergencies, emphasising that the mainstay of diagnosis is the disappearance of major pulses. Diagnosis, treatment
untreated diabetics of the maturity-onset type, who have a high concentration of circulating bound-insulin, is being
investigated. These studies were supported by Public Health Service grants AM-08381 and HE-06302 National Institutes of Health. Requests for reprints should be addressed to H. N. A., Protein Foundation Laboratories, 281 South Street, Jamaica Plain 02130, Massachusetts. Protein Foundation Laboratories, Jamaica Plain, and the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts, U.S.A.
HARRY N. ANTONIADES Ph.D. Athens.
(including the pros and cons of open and closed cardiac massage), the use of defibrillation and drugs, and aftercare are all clearly if briefly considered. The section on the ethics of resuscitation raises the difficult problem of successful resuscitation with severe mental and physical sequela:. The illustrations are clear and helpful. In the next edition the authors could with advantage add sections on the principles of bloodtransfusion for severe haemorrhage and the emergency diagnosis and treatment of pulmonary embolism. Young housemen and senior students will particularly benefit from this book, which fits snugly into the pocket of a white coat.
Thymic
Tumours
Their Association with Myasthenia Gravis and their Treatment
by Radiotherapy. J. F. HALE, M.A., D.M., M.R.C.P., physician, Luton and Dunstable Hospital; E. F. SCOWEN, M.D., D.sC., F.R.C.P., F.R.C.P.E., F.R.C.S., F.C.PATH., professor of medicine, University of London, and physician to St. Bartholomew’s Hospital. London: Lloyd-Luke. 1967. Pp. 93. 30s. of a series of 64 cases will be of all concerned in the treatment of thymic tumours or myasthenia gravis, or of both together. It can also be recommended to those who have a more academic interest in the elusive functions of the thymus gland. The study is predominantly clinical, and the practical problems of treatment Whilst it is disappointing to learn are critically examined. that surgical removal of the tumour does not confer any obvious benefit on the victim of myasthenia gravis, recognition of this fact will save a good deal of suffering and-as it would appearfutile labour. On the other hand, radiotherapy (discussed in part II) is regarded by the authors as an essential ingredient of treatment for the myasthenic patient with a thymic tumour. The fascinating problem of the relation of thymic tumours to myasthenia gravis is analysed (pp. 55-62). There seem to be no grounds for contending that myasthenia gravis promotes the development of a thymic tumour, or vice versa. In the light of recent research the authors favour the hypothesis that both these conditions are promoted by a common cause, and that this is most probably a disturbance of the body’s autoimmune process. This conclusion will, it may be hoped, provoke further research. A bibliography of 290 references is
THIS interest
scholarly analysis
to
appended. Introduction
to
Environmental
Physiology
Environmental Extremes and Mammalian Survival. G. EDGAR FOLK, JR., PH.D., professor, department of physiology, University of Iowa, Iowa City, Iowa. London: Henry Kimpton. 1966.
Pp.
308. 90s.
THIS book deals with the effects of heat, cold, high or low pressure, hibernation, light, biological rhythms, and temperature regulation. Primarily of interest to physiologists, it should be useful to those concerned with the relation between health and environment. The rapidly growing volume of work on biological rhythms is well described, as might be expected from the author’s notable contributions in this field. The style is often obscure, and indeed sections of the book read as if they were lecturer’s notes. Temperatures are sometimes expressed in Fahrenheit and at other times in Centigrade, and the references seem to be arbitrary. But, despite minor blemishes, this volume can be recommended to the critical student.