- Email: [email protected]
EFFECT OF CORTICOSTEROIDS ON SERUM GROWTH HORMONE
376
anxiety. Also, if termination is decided upon, the earlier the better, because of the increasing risk of operation. Patients sometimes play one doctor off against another, and try
to get one doctor to comment on what another doctor has said. For this reason, as few doctors as possible should be concerned in the matter. Lastly, if operation is decided upon, it should always be performed in a large public hospital, and never in a private
nursing-home. Summary
and Conclusion
The eventual outcome of maternal rubella in pregnancy fraught with anxiety for parents and doctors alike. Accurate widespead dissemination of the facts will help to allay unnecessary anxiety and allow a rational appraisal of the risks. When presented with the true situation, most parents will not press for termination of the pregnancy. Termination may be justifiable on account of the high risk of embryopathy (around 60%) if maternal rubella occurs during the second to fourth weeks after the first day of the last menstrual period. There is no legal justification for this, but prosecution of the doctor is unlikely. The legal reason for therapeutic abortion is that the anxiety resultant from the knowledge that the pregnancy might result in a damaged baby is such as to seriously impair the mental health of the mother. Now that the rubella virus can be grown, there is hope that one day it may be possible to prevent altogether what one writer has called " this miserable business ".
is
I should like to thank the gynaecologists and others who have sent me cases and given me advice, and Dr. A. Dugdale for reading and commenting on this paper. REFERENCES
Hill, A. B., Doll, R., Galloway, T. McL., Hughes, J. P. W. (1958) Brit. J. prev. soc. Med. 12, 1. Liggins, G. C., Phillips, L. I. (1963) Brit. med. J. i, 711. Lundström, R. (1962) Acta pœdiat., Stockh. 51, suppl. 133. Michaels, R. M., Mellin, G. W. (1960) Pœdiatrics, 26, 200. Pitt, D. B. (1961) Med. J. Aust. i, 881. Plotkin, S. A., Dudgeon, J. A., Ramsay, A. M. (1963) Brit. med. J. ii, 1296. Robinson, K., Williams, G. L., Morris, N., Ackner, B. (1962) Proc. R. Soc. Med. 55, 373. Siegel, M., Greenberg, M. (1960) New Engl. J. Med. 262, 389. Young, S. E. J., Ramsay, A. M. (1963) Brit. med. J. ii, 1295.
EFFECT OF CORTICOSTEROIDS ON SERUM GROWTH HORMONE M. HARTOG B.M. Oxon., M.R.C.P. SENIOR MEDICAL
REGISTRAR,
HAMMERSMITH
HOSPITAL, LONDON, W.12
M. A. GAAFAR M.D. Cairo HONORARY RESEARCH ASSISTANT
RUSSELL FRASER N.Z., F.R.C.P.
M.D.
PROFESSOR OF CLINICAL ENDOCRINOLOGY
From the
Department of Medicine, Postgraduate Medical School of London, W.12
CORTICOSTE :OIDS have long been known to interfere with normal growth, for their administration causes dwarfism both in animals (Evans et al. 1943) and in man (Blodgett et al. 1956); but the mechanism whereby corticosteroids have this effect is uncertain. They cause considerable protein wasting by their stimulation of gluconeogenesis, and corticotrophin has been shown to antagonise the actior of growth hormone on the epiphyseal cartilage of hypophysectomised rats (Becks et al. 1944). While the effect of corticosteroids on growth may be partly due to these and other peripheral effects, they might also suppress the secretion of growth hormone by the pituitary. For this reason, we have measured the serum
human growth hormone (H.G.H.) levels both in patients with Cushing’s syndrome and in patients who have been treated with corticosteroids, and compared them with those of normal subjects. In order to assess the potential secretory capacity of the pituitary, these measurements have been made before and during an insulin-tolerance test, since hypoglycaemia has been shown to cause a rise of serum H.G.H. (Roth et al. 1963). Methods Serum H.G.H. Assay The method of assay depends upon the changes produced in the amount of l3lI-labelled H.G.H. which will bind to an antibody to H.G.H. prepared in a rabbit when varying amounts of unlabelled H.G.H., whether in standard solutions or in the unknown sera, are added to the system. H.G.H. is labelled with radioiodine (l31J) by the technique of Hunter and Greenwood (1962), and specific activities of 150-200 {iC per &mgr;g. are obtained. The amount of labelled H.G.H. bound by the antiserum is measured by a double precipitation technique similar to that described by Utiger et al. (1962). After the incubates containing labelled H.G.H., varying amounts of standard or unknown serum, and anti-H.G.H. have stood at 4°C for 72 hours, an anti-rabbit y-globulin is added which will precipitate rabbit y-globulin, and so any growth hormone bound to it. The incubates stand again at 4°C for 24 hours and are then centrifuged, the precipitates are washed, the tubes are centrifuged again, and the precipitate left in the tubes is finally counted for its radioactivity in a well-type scintillation counter, or in an automatic Packard liquid-scintillation counter. Assays are set two levels of sensitivity, one capable of recognising between 0-1 and 0-2 !Lmg. which will measure concentrations in serum of 1.0-2.0 &mgr;mg. per ml. serum, and another which will measure concentrations of 4-0-6-0 !Lmg. per ml. serum (Hartog et al. 1964).
up at
Insulin-tolerance Tests These have all been done on patients lying quietly in bed in the ward after an overnight fast. For the previous three days the patients were taking either a full normal ward diet or a 300 g. carbohydrate diet. Soluble insulin was given intravenously, and 10 ml. blood specimens were taken for both blood-sugar and serum-H.G.H. determinations before the insulin injection and usually at 30, 60, 90, and 120 minutes afterwards. The dose of insulin used in the normal subjects was 0-1 units per kg. body-weight except for one who was given 0-2 units per kg. The patients with Cushing’s syndrome and those receiving corticosteroids were given 0-3 units per kg. except for three who were given 0-2 units per kg. In order to assess the extent of hypoglycaemia produced, a normal response to a standard insulin-tolerance test (0-1 units per kg.) may be defined as a fall in the 30 or 60-minute blood-sugar value to less than 60% of the fasting level. One of the normal subjects failed to show a fall of blood-sugar, but we are unable to account for this. This criterion has been used to assess the adequacy of the drop of blood-sugar in our patients who were given larger doses of insulin. Hypoglycsmic symptoms were experienced by almost all the patients, but none were severe enough to necessitate giving glucose and stopping the test. TABLE I-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN
SEVEN NORMAL
SUBJECTS
377 TABLE II-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN THREE PATIENTS WITH
CUSHING’S
SYNDROME AND SEVEN
PATIENTS RECEIVING CORTICOSTEROIDS
*
Or
equivalent dosage of another corticosteroid.
Patients Studied The normal subjects were volunteers from the nursing and other staff, and patients convalescing from non-endocrinological illnesses. The three patients with Cushing’s syndrome were all obvious cases severely affected by the disease. Two had previously had pituitary implants in which a subablative dose of radioactive material had been implanted in the pituitary (Joplin et al. 1961). In neither patient was there any evidence of hypopituitarism either clinically or from investigations. Neither patient had responded to pituitary implantation, and they were both later treated by bilateral subtotal adrenalectomy; in one the insulin-tolerance test was repeated one month after this operation. The third patient with Cushing’s syndrome had an inoperable adrenal carcinoma, and died three weeks after the insulin-tolerance test; at autopsy there were no metastatic deposits in the pituitary. Seven patients were tested whilst receiving corticosteroids for various conditions. One had agranulocytosis, one scleroderma, one haemolytic anaemia, and three rheumatoid arthritis; two of these three patients were girls with Still’s disease who were severely dwarfed. 12-hour
Fasting
Levels
Results H.G.H. of (Tables I and
II)
No abnormality emerged here, since the assay is not sensitive enough to measure many normal values, and does not, therefore, reveal subnormal values. H.G.H. Levels
during Insulin-tolerance
Tests
Normals (table 1).-All the subjects showed a rise in the levels of serum H.G.H. The 60-minute sample showed the greatest response, with a median peak value of 36 tmg. per ml. (range 18-69). We have attempted to obtain an indication of the " total response " by calculating the mean value for the 30, 60, and 90-minute samples; this was 26 mg. per ml. (range 15-42) for the six subjects who had estimations of serum H.G.H. done at these times. The one subject given twice the standard dose of insulin showed a rise of serum H.G.H. values which was similar to that in the others. Patients with Cushing’s syndrome or receiving corticosteroids (table II).-To try to ensure that the hypoglycaemic stress in these patients was at least equivalent to
that given to the normal subjects, they were given thrice or sometimes twice the standard insulin dose; they all showed a " normal " fall of blood-sugar as defined in the Methods section, and all of them except patients S2 and S7 experienced some hypoglycsemic symptoms. Table 11 shows that, during the insulin-tolerance test, no serum levels above 6 flmg. per ml. were encountered in the three patients with Cushing’s syndrome. The mean value of the 30, 60, and 90-minute samples in these patients was 2-8 flmg. per ml., which is lower than the corresponding normal value (p < 0’001). One patient was tested again one month after bilateral subtotal adrenalectomy, the results of which are shown in table ill. It will be seen that the serum H.G.H. now rose to 14 flmg. per ml. in the 60-minute sample, with a mean of the 30, 60, and 90-minute samples of 8 flmg. per ml. The patients receiving corticosteroids have been arbitrarily grouped into those on high doses and those on medium doses, according to the dose and duration of the corticosteroid therapy. In two of the patients who were receiving heavy doses of corticosteroids there was no discernible rise in the serum H.G.H. levels during the insulin-tolerance test. In the other five patients there was a rise to a median peak level of 26 mg. per ml. (range 20-42), the peak levels occurring in either the 30 or the 60-minute sample. Three of these five patients had samples measured at 30, 60, and 90 minutes, and the mean H.G.H. level for these times was 17 {jLmg. per ml. (range 11-21), which may not be different from the corresponding Two of the normal value of 26 tmg. per ml. (P>0-1). patients had also been tested before they began corticosteroid therapy (table ill). In both the response in H.G.H. was possibly less after the period of therapy, though it still fell within the lower normal range. Discussion
This study was begun in order to try to establish whether corticosteroids interfere with the production of growth hormone. Although our assay is not sensitive enough to reveal subnormal values of fasting serum H.G.H.,
TABLE III-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN ONE PATIENT BEFORE AND AFTER SUBTOTAL AND TWO PATIENTS BEFORE AND AFTER BEGINNING CORTICOSTEROID THERAPY
ADRENALECTOMY,
378 the normal rise of serum H.G.H. in response to hypoglycaemia. The normal subjects had a median peak value of 36 mg. per ml. (range 18-69), and the mean value for the 30, 60, and 90-minute samples was 26 fLmg. per ml. (range 15-42)-results which are comparable with the other published figures. Roth et al. (1963) did standard insulin-tolerance tests (0-1units per kg. body-weight) on six normal subjects. The peak level in one of them was 10 img. per ml., all the rest had peak levels of 35 fLmg. per ml. or above. Hunter and Greenwood (1964) report insulin and tolbutamide tests in three subjects with nonendocrinological illnesses. Their peak serum H.G.H. levels were 31-0, 23-9 and 23-0 fLmg. per ml.; the mean value for the serum H.G.H. levels measured at 30, 60, and 90 minutes was 14-9 jjunag. per ml. The highest value encountered during insulin-tolerance tests in the three patients with Cushing’s syndrome was only 6 mg. per ml. One patient who was retested one month after his Cushing’s syndrome had been treated by bilateral subtotal adrenalectomy showed a nearly normal rise. Two of the patients receiving corticosteroid showed no discernible rise of serum H.G.H. during insulintolerance tests, whilst the other five showed responses which were in the lower normal range. Patients with Cushing’s syndrome are known to be insulin-resistant (Fraser et al. 1941), and we have, therefore, given them and the patients receiving corticosteroids two or three times the standard insulin dosage. The fall of blood-sugar produced was at least as great as that in the normals, and therefore the failure of some of these patients to produce a normal rise of the serum H.G.H. is unlikely to depend on inadequate hypoglycsemia. One of the patients (S2), however, who showed no rise of the serum H.G.H., experienced no hypoglycaemic symptoms, and it might be argued that more profound hypoglyceeinia, sufficient to produce symptoms, would have evoked such a rise. These results, therefore, suggest that corticosteroids can interfere with the normal production of growth hormone in response to hypoglycaemia. The dosage of corticosteroid seems to be important. The patients who showed clearly defective responses had been exposed to high corticosteroid levels. The majority of the patients receiving corticosteroids who showed rises of serum H.G.H. that were within the normal. range had been treated for shorter periods or with smaller doses. Perhaps, therefore, it is prolonged and high-dosage corticosteroid treatment that suppresses the normal response of the pituitary. One patient, however, who had been on high doses for several months, still showed a response. It is clearly necessary to study more patients in order to define the dose and duration of corticosteroid therapy required to produce this effect, and to see how consistently the suppression occurs. it
can measure
Summary Serum levels of growth hormone were measured during insulin-tolerance tests in seven normal subjects, three patients with Cushing’s syndrome, and seven patients receiving corticosteroid therapy. In the normal subjects the median peak level attained during the test was 36 mg. per ml. (range 18-69), and the mean of the values at 30, 60, and 90 minutes was 26 fLmg. per ml. (range 15-42). The three patients with Cushing’s syndrome and two of those receiving corticosteroids scarcely showed any rise, while the other five showed responses in the lower normal range. One of the patients with Cushing’s syndrome showed some response when retested after subtotal
adrenalectomy.
We should like to thank the Medical Research Council for a grant for technical assistance; the clinical endocrinology committee of the Medical Research Council and the Department of Biochemistry, University of Cambridge, for supplies of human growth hormone; Dr. A. J. Fulthorpe, of Burroughs Wellcome Laboratories, Beckenham, Kent, for supplies of the horse anti-rabbit y-globulin; and the Department of Chemical Pathology, Hammersmith Hospital, for the blood-sugar estimations. REFERENCES
Becks, H., Simpson, M. E., Marx, W., Li, C. H., Evans, H. M. (1944) Endocrinology, 34, 311. Blodgett, F. M., Burgin, L., Iezzoni, D., Gribetz, D., Talbot, N. D. (1956) New Engl. J. Med. 254, 636. Evans, H. M., Li, C. H., Simpson, M. E. (1943) Endocrinology, 33, 237. Fraser, R., Albright, F., Smith, P. H. (1941) J. clin. Endocrin. 1, 297. Hartog, M., Meisser, B., Fraser, R. (1964) Unpublished. Hunter, W. M., Greenwood, F. C. (1962) Nature, Lond. 194, 495. (1964) Brit. med. J. i, 804. Joplin, G. F., Fraser, R., Steiner, R., Laws, J., Jones, E. (1961) Lancet, ii, -
-
1277.
Roth, J., Glick, S. M., Yalow, R. S., Berson, S. A. (1963) Science, 140, 987. Utiger, R. D., Parker, M. L., Daughaday, W. H. (1962) J. clin. Invest. 41, 254.
RESISTANCE TO HUMAN GROWTH HORMONE CAUSED BY SPECIFIC ANTIBODIES
ACQUIRED
A. PRADER
H. WAGNER
M.D. Zürich
M.D. Zürich
JUDITH SZÉKY
RUTH ILLIG
M.D.
M.D. Berne DEPARTMENT, UNIVERSITY OF ZÜRICH, SWITZERLAND
Budapest
OF THE PÆDIATRIC
J. L. TOUBER
D. MAINGAY
M.D. Amsterdam
M.D. Leiden
OF THE CENTRAL LABORATORY OF THE NETHERLANDS RED CROSS BLOOD-TRANSFUSION
SERVICE, AMSTERDAM
WE have treated nine children, aged 2 to 16 years, with hypopituitary dwarfism with human growth hormone (H.G.H.) in a uniform dosage schedule over periods ranging 36 months. Six of these children showed congrowth response. In the three others there was no growth effect at all. This was surprising because all nine children had shown a marked increase in nitrogen (N) retention in response to H.G.H. during a preliminary 5-day test period, and because none of the three growthresistant patients had been given cortisone, which might have inhibited growth. In an effort to explore the growth resistance to H.G.H. we searched for H.G.H. antibodies; in fact we found that such antibodies appeared in high titres during the first few months of treatment in the three growth-resistant patients, while no such antibodies or antibodies in low titres only could be identified in the other
from 9
to
siderable
six
patients. Methods
Growth-hormone
Preparation
1.5 U.S.P. units per mg.), prepared by the method of Raben (1957), was used for the short-term metabolic studies and for the long-term treatment of the nine children. The dry powder was dissolved in 0’N HC1 and water. The final pH of the solution was about 3. Despite great care in preparing the solution, it was not always clear. Some bottles showed slight turbidity, but this did not seem to influence the metabolic and the growth effect. The hxmagglutination assays were performed with H.G.H. (Raben), with H.G.H. (Organon) prepared by the method of Wilhelmi (1961) (1-8 U.S.P. units per mg.), and with a modified bovine growth hormone obtained from Laboratoire Choay H.G.H.
(about
(Paris). H.G.H. used for the radioimmuno-assays was prepared by the method of Raben, radioiodinated by the technique of Hunter and Greenwood (1962), and purified by gel filtration on Sephadex G ’ 200 (Touber and Maingay 1963). Passive Haemagglutination and Haemagglutination Inhibition Sheep erythrocytes treated with tannic acid and coated with H.G.H. are specifically agglutinated by H.G.H. anti-serum (Read ’
anxiety. Also, if termination is decided upon, the earlier the better, because of the increasing risk of operation. Patients sometimes play one doctor off against another, and try
to get one doctor to comment on what another doctor has said. For this reason, as few doctors as possible should be concerned in the matter. Lastly, if operation is decided upon, it should always be performed in a large public hospital, and never in a private
nursing-home. Summary
and Conclusion
The eventual outcome of maternal rubella in pregnancy fraught with anxiety for parents and doctors alike. Accurate widespead dissemination of the facts will help to allay unnecessary anxiety and allow a rational appraisal of the risks. When presented with the true situation, most parents will not press for termination of the pregnancy. Termination may be justifiable on account of the high risk of embryopathy (around 60%) if maternal rubella occurs during the second to fourth weeks after the first day of the last menstrual period. There is no legal justification for this, but prosecution of the doctor is unlikely. The legal reason for therapeutic abortion is that the anxiety resultant from the knowledge that the pregnancy might result in a damaged baby is such as to seriously impair the mental health of the mother. Now that the rubella virus can be grown, there is hope that one day it may be possible to prevent altogether what one writer has called " this miserable business ".
is
I should like to thank the gynaecologists and others who have sent me cases and given me advice, and Dr. A. Dugdale for reading and commenting on this paper. REFERENCES
Hill, A. B., Doll, R., Galloway, T. McL., Hughes, J. P. W. (1958) Brit. J. prev. soc. Med. 12, 1. Liggins, G. C., Phillips, L. I. (1963) Brit. med. J. i, 711. Lundström, R. (1962) Acta pœdiat., Stockh. 51, suppl. 133. Michaels, R. M., Mellin, G. W. (1960) Pœdiatrics, 26, 200. Pitt, D. B. (1961) Med. J. Aust. i, 881. Plotkin, S. A., Dudgeon, J. A., Ramsay, A. M. (1963) Brit. med. J. ii, 1296. Robinson, K., Williams, G. L., Morris, N., Ackner, B. (1962) Proc. R. Soc. Med. 55, 373. Siegel, M., Greenberg, M. (1960) New Engl. J. Med. 262, 389. Young, S. E. J., Ramsay, A. M. (1963) Brit. med. J. ii, 1295.
EFFECT OF CORTICOSTEROIDS ON SERUM GROWTH HORMONE M. HARTOG B.M. Oxon., M.R.C.P. SENIOR MEDICAL
REGISTRAR,
HAMMERSMITH
HOSPITAL, LONDON, W.12
M. A. GAAFAR M.D. Cairo HONORARY RESEARCH ASSISTANT
RUSSELL FRASER N.Z., F.R.C.P.
M.D.
PROFESSOR OF CLINICAL ENDOCRINOLOGY
From the
Department of Medicine, Postgraduate Medical School of London, W.12
CORTICOSTE :OIDS have long been known to interfere with normal growth, for their administration causes dwarfism both in animals (Evans et al. 1943) and in man (Blodgett et al. 1956); but the mechanism whereby corticosteroids have this effect is uncertain. They cause considerable protein wasting by their stimulation of gluconeogenesis, and corticotrophin has been shown to antagonise the actior of growth hormone on the epiphyseal cartilage of hypophysectomised rats (Becks et al. 1944). While the effect of corticosteroids on growth may be partly due to these and other peripheral effects, they might also suppress the secretion of growth hormone by the pituitary. For this reason, we have measured the serum
human growth hormone (H.G.H.) levels both in patients with Cushing’s syndrome and in patients who have been treated with corticosteroids, and compared them with those of normal subjects. In order to assess the potential secretory capacity of the pituitary, these measurements have been made before and during an insulin-tolerance test, since hypoglycaemia has been shown to cause a rise of serum H.G.H. (Roth et al. 1963). Methods Serum H.G.H. Assay The method of assay depends upon the changes produced in the amount of l3lI-labelled H.G.H. which will bind to an antibody to H.G.H. prepared in a rabbit when varying amounts of unlabelled H.G.H., whether in standard solutions or in the unknown sera, are added to the system. H.G.H. is labelled with radioiodine (l31J) by the technique of Hunter and Greenwood (1962), and specific activities of 150-200 {iC per &mgr;g. are obtained. The amount of labelled H.G.H. bound by the antiserum is measured by a double precipitation technique similar to that described by Utiger et al. (1962). After the incubates containing labelled H.G.H., varying amounts of standard or unknown serum, and anti-H.G.H. have stood at 4°C for 72 hours, an anti-rabbit y-globulin is added which will precipitate rabbit y-globulin, and so any growth hormone bound to it. The incubates stand again at 4°C for 24 hours and are then centrifuged, the precipitates are washed, the tubes are centrifuged again, and the precipitate left in the tubes is finally counted for its radioactivity in a well-type scintillation counter, or in an automatic Packard liquid-scintillation counter. Assays are set two levels of sensitivity, one capable of recognising between 0-1 and 0-2 !Lmg. which will measure concentrations in serum of 1.0-2.0 &mgr;mg. per ml. serum, and another which will measure concentrations of 4-0-6-0 !Lmg. per ml. serum (Hartog et al. 1964).
up at
Insulin-tolerance Tests These have all been done on patients lying quietly in bed in the ward after an overnight fast. For the previous three days the patients were taking either a full normal ward diet or a 300 g. carbohydrate diet. Soluble insulin was given intravenously, and 10 ml. blood specimens were taken for both blood-sugar and serum-H.G.H. determinations before the insulin injection and usually at 30, 60, 90, and 120 minutes afterwards. The dose of insulin used in the normal subjects was 0-1 units per kg. body-weight except for one who was given 0-2 units per kg. The patients with Cushing’s syndrome and those receiving corticosteroids were given 0-3 units per kg. except for three who were given 0-2 units per kg. In order to assess the extent of hypoglycaemia produced, a normal response to a standard insulin-tolerance test (0-1 units per kg.) may be defined as a fall in the 30 or 60-minute blood-sugar value to less than 60% of the fasting level. One of the normal subjects failed to show a fall of blood-sugar, but we are unable to account for this. This criterion has been used to assess the adequacy of the drop of blood-sugar in our patients who were given larger doses of insulin. Hypoglycsmic symptoms were experienced by almost all the patients, but none were severe enough to necessitate giving glucose and stopping the test. TABLE I-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN
SEVEN NORMAL
SUBJECTS
377 TABLE II-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN THREE PATIENTS WITH
CUSHING’S
SYNDROME AND SEVEN
PATIENTS RECEIVING CORTICOSTEROIDS
*
Or
equivalent dosage of another corticosteroid.
Patients Studied The normal subjects were volunteers from the nursing and other staff, and patients convalescing from non-endocrinological illnesses. The three patients with Cushing’s syndrome were all obvious cases severely affected by the disease. Two had previously had pituitary implants in which a subablative dose of radioactive material had been implanted in the pituitary (Joplin et al. 1961). In neither patient was there any evidence of hypopituitarism either clinically or from investigations. Neither patient had responded to pituitary implantation, and they were both later treated by bilateral subtotal adrenalectomy; in one the insulin-tolerance test was repeated one month after this operation. The third patient with Cushing’s syndrome had an inoperable adrenal carcinoma, and died three weeks after the insulin-tolerance test; at autopsy there were no metastatic deposits in the pituitary. Seven patients were tested whilst receiving corticosteroids for various conditions. One had agranulocytosis, one scleroderma, one haemolytic anaemia, and three rheumatoid arthritis; two of these three patients were girls with Still’s disease who were severely dwarfed. 12-hour
Fasting
Levels
Results H.G.H. of (Tables I and
II)
No abnormality emerged here, since the assay is not sensitive enough to measure many normal values, and does not, therefore, reveal subnormal values. H.G.H. Levels
during Insulin-tolerance
Tests
Normals (table 1).-All the subjects showed a rise in the levels of serum H.G.H. The 60-minute sample showed the greatest response, with a median peak value of 36 tmg. per ml. (range 18-69). We have attempted to obtain an indication of the " total response " by calculating the mean value for the 30, 60, and 90-minute samples; this was 26 mg. per ml. (range 15-42) for the six subjects who had estimations of serum H.G.H. done at these times. The one subject given twice the standard dose of insulin showed a rise of serum H.G.H. values which was similar to that in the others. Patients with Cushing’s syndrome or receiving corticosteroids (table II).-To try to ensure that the hypoglycaemic stress in these patients was at least equivalent to
that given to the normal subjects, they were given thrice or sometimes twice the standard insulin dose; they all showed a " normal " fall of blood-sugar as defined in the Methods section, and all of them except patients S2 and S7 experienced some hypoglycsemic symptoms. Table 11 shows that, during the insulin-tolerance test, no serum levels above 6 flmg. per ml. were encountered in the three patients with Cushing’s syndrome. The mean value of the 30, 60, and 90-minute samples in these patients was 2-8 flmg. per ml., which is lower than the corresponding normal value (p < 0’001). One patient was tested again one month after bilateral subtotal adrenalectomy, the results of which are shown in table ill. It will be seen that the serum H.G.H. now rose to 14 flmg. per ml. in the 60-minute sample, with a mean of the 30, 60, and 90-minute samples of 8 flmg. per ml. The patients receiving corticosteroids have been arbitrarily grouped into those on high doses and those on medium doses, according to the dose and duration of the corticosteroid therapy. In two of the patients who were receiving heavy doses of corticosteroids there was no discernible rise in the serum H.G.H. levels during the insulin-tolerance test. In the other five patients there was a rise to a median peak level of 26 mg. per ml. (range 20-42), the peak levels occurring in either the 30 or the 60-minute sample. Three of these five patients had samples measured at 30, 60, and 90 minutes, and the mean H.G.H. level for these times was 17 {jLmg. per ml. (range 11-21), which may not be different from the corresponding Two of the normal value of 26 tmg. per ml. (P>0-1). patients had also been tested before they began corticosteroid therapy (table ill). In both the response in H.G.H. was possibly less after the period of therapy, though it still fell within the lower normal range. Discussion
This study was begun in order to try to establish whether corticosteroids interfere with the production of growth hormone. Although our assay is not sensitive enough to reveal subnormal values of fasting serum H.G.H.,
TABLE III-SERUM H.G.H. LEVELS DURING INSULIN-TOLERANCE TESTS IN ONE PATIENT BEFORE AND AFTER SUBTOTAL AND TWO PATIENTS BEFORE AND AFTER BEGINNING CORTICOSTEROID THERAPY
ADRENALECTOMY,
378 the normal rise of serum H.G.H. in response to hypoglycaemia. The normal subjects had a median peak value of 36 mg. per ml. (range 18-69), and the mean value for the 30, 60, and 90-minute samples was 26 fLmg. per ml. (range 15-42)-results which are comparable with the other published figures. Roth et al. (1963) did standard insulin-tolerance tests (0-1units per kg. body-weight) on six normal subjects. The peak level in one of them was 10 img. per ml., all the rest had peak levels of 35 fLmg. per ml. or above. Hunter and Greenwood (1964) report insulin and tolbutamide tests in three subjects with nonendocrinological illnesses. Their peak serum H.G.H. levels were 31-0, 23-9 and 23-0 fLmg. per ml.; the mean value for the serum H.G.H. levels measured at 30, 60, and 90 minutes was 14-9 jjunag. per ml. The highest value encountered during insulin-tolerance tests in the three patients with Cushing’s syndrome was only 6 mg. per ml. One patient who was retested one month after his Cushing’s syndrome had been treated by bilateral subtotal adrenalectomy showed a nearly normal rise. Two of the patients receiving corticosteroid showed no discernible rise of serum H.G.H. during insulintolerance tests, whilst the other five showed responses which were in the lower normal range. Patients with Cushing’s syndrome are known to be insulin-resistant (Fraser et al. 1941), and we have, therefore, given them and the patients receiving corticosteroids two or three times the standard insulin dosage. The fall of blood-sugar produced was at least as great as that in the normals, and therefore the failure of some of these patients to produce a normal rise of the serum H.G.H. is unlikely to depend on inadequate hypoglycsemia. One of the patients (S2), however, who showed no rise of the serum H.G.H., experienced no hypoglycaemic symptoms, and it might be argued that more profound hypoglyceeinia, sufficient to produce symptoms, would have evoked such a rise. These results, therefore, suggest that corticosteroids can interfere with the normal production of growth hormone in response to hypoglycaemia. The dosage of corticosteroid seems to be important. The patients who showed clearly defective responses had been exposed to high corticosteroid levels. The majority of the patients receiving corticosteroids who showed rises of serum H.G.H. that were within the normal. range had been treated for shorter periods or with smaller doses. Perhaps, therefore, it is prolonged and high-dosage corticosteroid treatment that suppresses the normal response of the pituitary. One patient, however, who had been on high doses for several months, still showed a response. It is clearly necessary to study more patients in order to define the dose and duration of corticosteroid therapy required to produce this effect, and to see how consistently the suppression occurs. it
can measure
Summary Serum levels of growth hormone were measured during insulin-tolerance tests in seven normal subjects, three patients with Cushing’s syndrome, and seven patients receiving corticosteroid therapy. In the normal subjects the median peak level attained during the test was 36 mg. per ml. (range 18-69), and the mean of the values at 30, 60, and 90 minutes was 26 fLmg. per ml. (range 15-42). The three patients with Cushing’s syndrome and two of those receiving corticosteroids scarcely showed any rise, while the other five showed responses in the lower normal range. One of the patients with Cushing’s syndrome showed some response when retested after subtotal
adrenalectomy.
We should like to thank the Medical Research Council for a grant for technical assistance; the clinical endocrinology committee of the Medical Research Council and the Department of Biochemistry, University of Cambridge, for supplies of human growth hormone; Dr. A. J. Fulthorpe, of Burroughs Wellcome Laboratories, Beckenham, Kent, for supplies of the horse anti-rabbit y-globulin; and the Department of Chemical Pathology, Hammersmith Hospital, for the blood-sugar estimations. REFERENCES
Becks, H., Simpson, M. E., Marx, W., Li, C. H., Evans, H. M. (1944) Endocrinology, 34, 311. Blodgett, F. M., Burgin, L., Iezzoni, D., Gribetz, D., Talbot, N. D. (1956) New Engl. J. Med. 254, 636. Evans, H. M., Li, C. H., Simpson, M. E. (1943) Endocrinology, 33, 237. Fraser, R., Albright, F., Smith, P. H. (1941) J. clin. Endocrin. 1, 297. Hartog, M., Meisser, B., Fraser, R. (1964) Unpublished. Hunter, W. M., Greenwood, F. C. (1962) Nature, Lond. 194, 495. (1964) Brit. med. J. i, 804. Joplin, G. F., Fraser, R., Steiner, R., Laws, J., Jones, E. (1961) Lancet, ii, -
-
1277.
Roth, J., Glick, S. M., Yalow, R. S., Berson, S. A. (1963) Science, 140, 987. Utiger, R. D., Parker, M. L., Daughaday, W. H. (1962) J. clin. Invest. 41, 254.
RESISTANCE TO HUMAN GROWTH HORMONE CAUSED BY SPECIFIC ANTIBODIES
ACQUIRED
A. PRADER
H. WAGNER
M.D. Zürich
M.D. Zürich
JUDITH SZÉKY
RUTH ILLIG
M.D.
M.D. Berne DEPARTMENT, UNIVERSITY OF ZÜRICH, SWITZERLAND
Budapest
OF THE PÆDIATRIC
J. L. TOUBER
D. MAINGAY
M.D. Amsterdam
M.D. Leiden
OF THE CENTRAL LABORATORY OF THE NETHERLANDS RED CROSS BLOOD-TRANSFUSION
SERVICE, AMSTERDAM
WE have treated nine children, aged 2 to 16 years, with hypopituitary dwarfism with human growth hormone (H.G.H.) in a uniform dosage schedule over periods ranging 36 months. Six of these children showed congrowth response. In the three others there was no growth effect at all. This was surprising because all nine children had shown a marked increase in nitrogen (N) retention in response to H.G.H. during a preliminary 5-day test period, and because none of the three growthresistant patients had been given cortisone, which might have inhibited growth. In an effort to explore the growth resistance to H.G.H. we searched for H.G.H. antibodies; in fact we found that such antibodies appeared in high titres during the first few months of treatment in the three growth-resistant patients, while no such antibodies or antibodies in low titres only could be identified in the other
from 9
to
siderable
six
patients. Methods
Growth-hormone
Preparation
1.5 U.S.P. units per mg.), prepared by the method of Raben (1957), was used for the short-term metabolic studies and for the long-term treatment of the nine children. The dry powder was dissolved in 0’N HC1 and water. The final pH of the solution was about 3. Despite great care in preparing the solution, it was not always clear. Some bottles showed slight turbidity, but this did not seem to influence the metabolic and the growth effect. The hxmagglutination assays were performed with H.G.H. (Raben), with H.G.H. (Organon) prepared by the method of Wilhelmi (1961) (1-8 U.S.P. units per mg.), and with a modified bovine growth hormone obtained from Laboratoire Choay H.G.H.
(about
(Paris). H.G.H. used for the radioimmuno-assays was prepared by the method of Raben, radioiodinated by the technique of Hunter and Greenwood (1962), and purified by gel filtration on Sephadex G ’ 200 (Touber and Maingay 1963). Passive Haemagglutination and Haemagglutination Inhibition Sheep erythrocytes treated with tannic acid and coated with H.G.H. are specifically agglutinated by H.G.H. anti-serum (Read ’