- Email: [email protected]
ADRENOCORTICAL STEROIDS AND THE CEREBROSPINAL FLUID
1280 If patients of blood-group A have an increased susceptibility prostatic carcinoma, they should present clinically at an earlier age than those of other blood-groups. The mean age of diagnosis of patients with prostatic carcinoma of blood-group A was 69-0 years (standard error = ::1::0.73 years), and for bloodgroup 0 it was 72-1years (standard error 0-93 years). This difference is highly significant (p < 0-001).
to
Therefore, people of blood-group A seem to have an susceptibility to carcinoma of the prostate and they present clinically at an earlier age. Our findings should be regarded as preliminary observations in view of the small sample size. increased
Our thanks are due to Prof. V. W. Dix and Mr. G. C. Tresidder for allowing us to study their patients, and for their advice and encouragement. London
Hospital Medical College, London, E.1.
J. B. BOURKE J. P. GRIFFIN.
SPIRONOLACTONE AND GYNÆCOMASTIA SIR,-We were very interested to read the letter by Dr. W. G. Smith (Oct. 27). We had just seen a 24-yearold white male with chronic glomerulonephritis who
developed gynsecomastia after having been placed on chlorothiazide (’Diuril’) and spironolactone (’Aldactone’). There was no apparent other reason for his gynxcomastia. We were unable to find the association in the literature until we saw Dr. Smith’s letter. RONALD A. RESTIFO Department of Medicine, Walson Army Hospital, T. ALBERT FARMER. Fort Dix, New Jersey. NON-ESTERIFIED FATTY ACIDS AND FATTY LIVER IN HEREDITARY FRUCTOSE INTOLERANCE
SIR,-Patients with hereditary fructose intolerance (H.F.I.) develop marked hypoglycaemia and gross hepatomegaly when allowed free access to fructose-containing foods. The hepatomegaly seems to be caused mainly by accumulation of lipids.23 The liver enlarges rapidly after fructose administration and diminishes on a fructose-free diet in a few days.3 In one of the patients studied here hyperlipxmia was also found.3 Determination of plasma non-esterified fatty acid (N.E.F.A.) level4 in two of our patients with H.F.I. before and after an oral fructose load (0-40 g. per kg.) gave the following results:
concentration,67 and rapidly esterifies them
to form triglycerides and phospholipids.8 With an intact liver cell the hepatic accumulation of lipids is followed by an increased release of lipoprotein-bound lipids from the liver into the circulation, and consequently by hyperlipsemia. A similar mechanism may also be responsible for the fatty liver and hyperlipxmia in the von Gierke type of glycogenosis, where increased plasma N.E.F.A. levels have lately been demonstrated.9 Hereditary fructose intolerance offers a unique opportunity to observe the metabolic effects of hypoglycasmia not induced by insulin. First Department of Medicine and Children’s Hospital, University of Helsinki, Helsinki, Finland.
AMBULANT TREATMENT OF SPINAL TUBERCULOSIS SIR,-In your annotation of Oct. 27 you say there is little doubt that the combination of chemotherapy and anterior spinal fusion as advocated by Hodgson and Stock reduces both the length of stay in hospital and the risk of deformity. With the ambulant treatment, few of the patients need hospital inpatient treatment; and as for the risk of deformity no advocate of the radical surgical
approach, to our knowledge, has published a series giving degree of collapse before and after treatment. Without this, discussion on the risk of deformity remains speculative. Since the results of simple ambulant treatment compare favourably with other methods, it may be the best treatment for the majority of patients with spinal tuberculosis and, as with pulmonary tuberculosis, surgery will be reserved for selected cases not responding to the medical regimen.
’the
patients developed the characteristic hypoglycamic during the test. In healthy children a similar fructose load caused only insignificant changes in the plasma N.E.F.A. Both
level. The huge elevation of plasma N.E.F.A. after fructose administration indicates a mobilisation of fat stores, and is apparently a consequence of the hypoglycsemia. One or both of the following mechanisms may produce this phenomenon: an increased lipolysis of adipose-tissue glycerides due to a hypoglycasmia-induced release of catecholamines, or a decreased rate of re-esterification of fatty acids in adipose tissue due to a lack of available glucose (and a-glycerophosphate). The demonstration of this plasma N.E.F.A. response makes it evident that the fructose-induced hypoglycamlia of H.F.I. is not caused by increased secretion of insulin, as has been suggested.5 The high plasma N.E.F.A. level probably explains the development of fatty liver in untreated H.F.I. It is known that the liver extracts plasma non-esterified fatty acids in proportion to their 1. 2. 3.
4. 5.
Froesch, E. R., Prader, A., Wolf, H. P., Labhart, A. Helv. paediat. Acta, 1959, 14, 99. Jeune, M., Planson, E., Cotte, J., Bonnefoy, S., Nivelon, J.-L., Skosowsky, J. Pédiatrie, 1961, 14, 605. Perheentupa, J., Pitkanen, E., Nikkilä, E. A., Somersalo, O., Hakosalo, J. Ann. pœdiat. Fenn. 1962, 8, 221. Trout, D. L., Estes, E. H., Jr., Friedberg, S. J. J. Lipid Res. 1960, 1, 199. Dormandy, T. L., Porter, R. J. Lancet, 1961, i, 1189.
P. G. KONSTAM A. BLESOVSKY.
St. Bartholomew’s Hospital, E.C.1. London, E.C.!.
to our correspondents for giving an reference to incorrect their paper, which appeared in the British Journal of Surgery (1962, 50, 26).-ED. L.
:1 <:1 < :I < We apologise
ADRENOCORTICAL STEROIDS AND THE CEREBROSPINAL FLUID
SIR,-Since writing have come across further interest.
response
E. A. NIKKILÄ J. PERHEENTUPA.
you under this title (Aug. 25) I early work which may stimulate
to
some
Dixon and Halliburton 10-13 showed that the choroid plexuses obtained from Dr. Mott contained cholesterin-like substance, which, injected intravenously, produced a striking increase in cerebrospinal fluid production, without, except in disease states of the plexus, passing through into the fluid itself. Could this cholesterin-like substance have been the steroid in question? Dixon and Halliburton suggested that it might be a product of brain metabolism rather than of adrenocortical origin, and this might still be so. They considered the cerebrospinalfluid circulation to be a route, additional to the arteriovenous one common to all tissues, whereby carbon dioxide is removed from the central nervous system, the brain’s predominantly
carbohydrate metabolism being particularly susceptible
to
carbon-dioxide accumulation or oxygen lack. The extracellular space in which the cerebrospinal fluid circulates then becomes a sea in which the central nervous system has gills rather than kidneys, and the humoral mechanism controlling this might be more likely to have its origin in the specialised 6. 7. 8. 9. 10. 11. 12. 13.
Fine, M. B., Williams, R. H. Amer. J. Physiol. 1960, 199, 403. Spitzer, J. J., McElroy, W. T., Jr. ibid. p. 876. Stein, Y., Shapiro, B. ibid. 1959, 196, 1238. Howell, R. R., Ashton, D. M., Wyngaarden, J. B. Pediatrics, 1962, 29, 553, Dixon, W. E., Halliburton, W.D. J. Physiol. 1910, 40, xxx. Dixon, W. E., Halliburton, W. D. ibid. 1913-14, 47, 215. Dixon, W. E., Halliburton, W. D. ibid. 1914, 48, 128, 317. Dixon, W. E., Halliburton, W. D. ibid. 1915-16, 50, 198.
1281
metabolic conditions for which it system of tissues in general.
caters
rather than share the
That there may be some degree of integration, however, is suggested by the embryological development of structures around the third ventricle. Having concluded that the cerebrospinal fluid was formed by secretion rather than dialysis, Dixon and Halliburton refer to the choroid gland ", and the third ventricular part of this structure develops from the thalam-encephalic (diencephalic) roof plate. The pituitary gland, and indirectly therefore the adrenal cortex, is controlled from the thalamencephalic floor. The roof plate also gives rise to the pineal gland, which in reptiles secretes directly into the third ventricle, giving this part of the choroid gland " a particular significance perhaps, especially if the pineal body should prove in the end to play a role in the humoral mechanism "
"
!
in question.
J. P. CRAWFORD. VIRILISATION AND MALFORMATION OF A FEMALE INFANT SIR,-Wilkins et aLl drew attention to the masculinisation of female infants whose mothers had received androgens or progestogens during pregnancy. Their findings have since been confirmed by others. Your correspondent Dr. Dubowitz (Aug. 25) describes another case of striking masculinisation of a female infant whose mother, a primipara, had received a small quantity of ethisterone early in pregnancy. For many yearsAmenorone ’has been used extensively as a pregnancy test, since it causes withdrawal bleeding in nonpregnant cases and no bleeding in pregnancy; so far, no congenital abnormalities following its use have been reported. I have myself employed it for many years in secondary amenorrhoea, some patients later becoming pregnant. No developmental abnormalities ever came to my notice. In the series reviewed by Wilkins2 and Grumbach et al.,3 the degree of masculinisation depended upon the time of gestation when hormone administration was started. If the hormone was given from the 4th-8th week onwards, most cases showed phallic enlargement and a urogenital sinus. If treatment was started later in embryonic life, phallic enlargement was the only abnormality. The urogenital sinus was not a true one but was an ante-chamber of the vagina formed by labial fusion. In this chamber the urethral orifice was normal in relation to the
vaginal opening. More marked abnormalities of female infants were observed where the mother, without hormone treatment, had shown signs ofvirilisation. Wilkins et al.1 described such a case of a mother in a cushingoid state with deepening of the voice, gross obesity, and facial hirsutism so marked that the husband told the doctor he was afraid his wife was turning into a man. The resulting infant was a female pseudohermaphrodite thought to be a boy with hypospadias until the true sex was discovered 5 years later. There are other cases in which the cause of virilisation in a female child is obscure, there being no evidence either of hormones administered to the mother or of androgens being excessively produced by her during pregnancy. Perloff et awl.4 described such a case of a twin pregnancy, both females, one normal, the other presenting as a non-adrenal pseudohermaphrodite with a urethral orifice opening at the tip of the phallus and almost complete Jabioscrotal fusion. Many such instances have been described. Suffice it to say that non-adrenal pseudohermaphroditism can arise in the absence of androgenic influences coming from the mother or from medicaments supplied. The case described by Dr. Dubowitz presents abnormalities more marked than any of those described by Wilkins and Grumbach et. al. Not merely is there an enlarged clitoris and 1. 2. 3. 4.
Wilkins, L., Jones, H. W., Holman,
G. H., Stempfel, R. S. J. clin. Endocr. 1958, 18, 559. Wilkins, L. J. Amer. med. Ass. 1960, 172, 1028 Grumbach, M. M., Ducharme, J., Moloshok, R. E. J. clin. Endocr. 1959,
18, 1369. Perloff, W. H., Conger,
K.
B., Levy, L. M. ibid. 1953, 13, 783.
labioscrotal fusion but also
an
absent
anus
and
a
rectovesico-
vaginal communication suggestive of a cloaca, although obviously not one in its true embryonic sense. Moreover, the Miillerian ducts have failed to fuse, giving rise to a double uterus and a double vagina. The amount of hormone given was very small and the time of administration rather later than in the previously mentioned series. The available evidence, therefore, seems to make it most unlikely that administered ethisterone was the factor responsible for the virilisation. Dr. Dubowitz mentions the absence of chimaerism in the blood of the male twin. He must have had in mind the possibility of a " freemartin " effect. Freemartins were first described by Lillie,5 who discovered a placental communication in bovine dizygotic twins which produced pseudohermaphroditism in the female twin through the androgenic activity of the male twin. Human freemartins have never been described, though intrauterine placental vascular communication has been inferred because of the presence of blood chimaeras. Woodruff et al.1,s have described 5 such cases in which two different bloodgroups were found in the same twin, and drumsticks were noticed in the leucocytes of the peripheral blood. Dr. Dubowitz does not mention an examination of leucocytes for drumsticks in his male twin. Such an examination may provide evidence of a human freemartin. The urinary 17-ketosteroid excretion of the male twin as well as his bone age may give information about androgenic activity in this twin. Dr. Dubowitz does not mention the mother’s condition during her pregnancy. She may have shown signs of excessive androgen output which may have been responsible for the infant’s abnormalities. She might have exhibited signs of virilisation-e.g., acne, or android hirsutism. Even a few hairs on the face, or breasts, or abdomen would suggest an increased androgen output. These hairs remain permanently and would be noticeable even now. The twin pregnancy occurred only after 6 years of married life, and this may well have some bearing on the mother’s endocrine state. Dr. Dubowitz’s case may belong to the same category as Perloff’s pseudohermaphrodite twin which was probably caused by a faulty determinant in the developing embryo. Endocrine Clinic, Charing Cross Hospital, London, W.C.2.
J. ADLER.
A MICROMETHOD FOR CHROMOSOME ANALYSIS ON PERIPHERAL BLOOD-CULTURES
SIR,-Chromosome studies in the newborn are often difficult because of the small volumes of blood that are obtainable. On the other hand, the white-cell count is higher in the first years (and especially the first days) of life than in adults. In this laboratory we have used the following micromethod, which is based on the principles of Moorhead et al. It has yielded good results with as little as 0-1 ml. of capillary blood, even in adults. Aspirate 0-2 ml. of capillary blood into a fine pipette, which has been coated inside with heparin solution by sucking it up and then blowing it out. Transfer the heparinised blood to a small test-tube (1 cm. x 8 cm.) containing 1 ml. of AB-positive serum. Shake gently. Place the tube at an angle of 45 °C at room-temperature for 15-45 minutes. When most of the red cells have settled down, aspirate the supernatant with a syringe, into which 2 ml. of medium 199 (Glaxo) has previously been drawn. Mix in the syringe by shaking gently. Inject aliquots of 1 ml. into each of three small test-tubes. Add one drop of phytohemagglutinin (Wellcome) and incubate for 3 days. Add one drop of a 0-04% solution of demecolcine (’ Colcemid ’) and incubate for a further 4 hours. Then spin down the contents of the tubes, add 1 ml. of an 0-95% solution of sodium citrate, and leave the tubes for 15 minutes at 37°C. Spin and fix in a mixture of methanol and glacial Lillie, F. R. Science, 1916, 43, 611. Woodruff, M. F. A., Fox, M., Buckton, K. A., Jacobs, P. A. Lancet, 1962, i, 192. 7. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., Hungerford, D. A. Exp. Cell Res. 1960, 20, 613.
5. 6.
-
to
Therefore, people of blood-group A seem to have an susceptibility to carcinoma of the prostate and they present clinically at an earlier age. Our findings should be regarded as preliminary observations in view of the small sample size. increased
Our thanks are due to Prof. V. W. Dix and Mr. G. C. Tresidder for allowing us to study their patients, and for their advice and encouragement. London
Hospital Medical College, London, E.1.
J. B. BOURKE J. P. GRIFFIN.
SPIRONOLACTONE AND GYNÆCOMASTIA SIR,-We were very interested to read the letter by Dr. W. G. Smith (Oct. 27). We had just seen a 24-yearold white male with chronic glomerulonephritis who
developed gynsecomastia after having been placed on chlorothiazide (’Diuril’) and spironolactone (’Aldactone’). There was no apparent other reason for his gynxcomastia. We were unable to find the association in the literature until we saw Dr. Smith’s letter. RONALD A. RESTIFO Department of Medicine, Walson Army Hospital, T. ALBERT FARMER. Fort Dix, New Jersey. NON-ESTERIFIED FATTY ACIDS AND FATTY LIVER IN HEREDITARY FRUCTOSE INTOLERANCE
SIR,-Patients with hereditary fructose intolerance (H.F.I.) develop marked hypoglycaemia and gross hepatomegaly when allowed free access to fructose-containing foods. The hepatomegaly seems to be caused mainly by accumulation of lipids.23 The liver enlarges rapidly after fructose administration and diminishes on a fructose-free diet in a few days.3 In one of the patients studied here hyperlipxmia was also found.3 Determination of plasma non-esterified fatty acid (N.E.F.A.) level4 in two of our patients with H.F.I. before and after an oral fructose load (0-40 g. per kg.) gave the following results:
concentration,67 and rapidly esterifies them
to form triglycerides and phospholipids.8 With an intact liver cell the hepatic accumulation of lipids is followed by an increased release of lipoprotein-bound lipids from the liver into the circulation, and consequently by hyperlipsemia. A similar mechanism may also be responsible for the fatty liver and hyperlipxmia in the von Gierke type of glycogenosis, where increased plasma N.E.F.A. levels have lately been demonstrated.9 Hereditary fructose intolerance offers a unique opportunity to observe the metabolic effects of hypoglycasmia not induced by insulin. First Department of Medicine and Children’s Hospital, University of Helsinki, Helsinki, Finland.
AMBULANT TREATMENT OF SPINAL TUBERCULOSIS SIR,-In your annotation of Oct. 27 you say there is little doubt that the combination of chemotherapy and anterior spinal fusion as advocated by Hodgson and Stock reduces both the length of stay in hospital and the risk of deformity. With the ambulant treatment, few of the patients need hospital inpatient treatment; and as for the risk of deformity no advocate of the radical surgical
approach, to our knowledge, has published a series giving degree of collapse before and after treatment. Without this, discussion on the risk of deformity remains speculative. Since the results of simple ambulant treatment compare favourably with other methods, it may be the best treatment for the majority of patients with spinal tuberculosis and, as with pulmonary tuberculosis, surgery will be reserved for selected cases not responding to the medical regimen.
’the
patients developed the characteristic hypoglycamic during the test. In healthy children a similar fructose load caused only insignificant changes in the plasma N.E.F.A. Both
level. The huge elevation of plasma N.E.F.A. after fructose administration indicates a mobilisation of fat stores, and is apparently a consequence of the hypoglycsemia. One or both of the following mechanisms may produce this phenomenon: an increased lipolysis of adipose-tissue glycerides due to a hypoglycasmia-induced release of catecholamines, or a decreased rate of re-esterification of fatty acids in adipose tissue due to a lack of available glucose (and a-glycerophosphate). The demonstration of this plasma N.E.F.A. response makes it evident that the fructose-induced hypoglycamlia of H.F.I. is not caused by increased secretion of insulin, as has been suggested.5 The high plasma N.E.F.A. level probably explains the development of fatty liver in untreated H.F.I. It is known that the liver extracts plasma non-esterified fatty acids in proportion to their 1. 2. 3.
4. 5.
Froesch, E. R., Prader, A., Wolf, H. P., Labhart, A. Helv. paediat. Acta, 1959, 14, 99. Jeune, M., Planson, E., Cotte, J., Bonnefoy, S., Nivelon, J.-L., Skosowsky, J. Pédiatrie, 1961, 14, 605. Perheentupa, J., Pitkanen, E., Nikkilä, E. A., Somersalo, O., Hakosalo, J. Ann. pœdiat. Fenn. 1962, 8, 221. Trout, D. L., Estes, E. H., Jr., Friedberg, S. J. J. Lipid Res. 1960, 1, 199. Dormandy, T. L., Porter, R. J. Lancet, 1961, i, 1189.
P. G. KONSTAM A. BLESOVSKY.
St. Bartholomew’s Hospital, E.C.1. London, E.C.!.
to our correspondents for giving an reference to incorrect their paper, which appeared in the British Journal of Surgery (1962, 50, 26).-ED. L.
:1 <:1 < :I < We apologise
ADRENOCORTICAL STEROIDS AND THE CEREBROSPINAL FLUID
SIR,-Since writing have come across further interest.
response
E. A. NIKKILÄ J. PERHEENTUPA.
you under this title (Aug. 25) I early work which may stimulate
to
some
Dixon and Halliburton 10-13 showed that the choroid plexuses obtained from Dr. Mott contained cholesterin-like substance, which, injected intravenously, produced a striking increase in cerebrospinal fluid production, without, except in disease states of the plexus, passing through into the fluid itself. Could this cholesterin-like substance have been the steroid in question? Dixon and Halliburton suggested that it might be a product of brain metabolism rather than of adrenocortical origin, and this might still be so. They considered the cerebrospinalfluid circulation to be a route, additional to the arteriovenous one common to all tissues, whereby carbon dioxide is removed from the central nervous system, the brain’s predominantly
carbohydrate metabolism being particularly susceptible
to
carbon-dioxide accumulation or oxygen lack. The extracellular space in which the cerebrospinal fluid circulates then becomes a sea in which the central nervous system has gills rather than kidneys, and the humoral mechanism controlling this might be more likely to have its origin in the specialised 6. 7. 8. 9. 10. 11. 12. 13.
Fine, M. B., Williams, R. H. Amer. J. Physiol. 1960, 199, 403. Spitzer, J. J., McElroy, W. T., Jr. ibid. p. 876. Stein, Y., Shapiro, B. ibid. 1959, 196, 1238. Howell, R. R., Ashton, D. M., Wyngaarden, J. B. Pediatrics, 1962, 29, 553, Dixon, W. E., Halliburton, W.D. J. Physiol. 1910, 40, xxx. Dixon, W. E., Halliburton, W. D. ibid. 1913-14, 47, 215. Dixon, W. E., Halliburton, W. D. ibid. 1914, 48, 128, 317. Dixon, W. E., Halliburton, W. D. ibid. 1915-16, 50, 198.
1281
metabolic conditions for which it system of tissues in general.
caters
rather than share the
That there may be some degree of integration, however, is suggested by the embryological development of structures around the third ventricle. Having concluded that the cerebrospinal fluid was formed by secretion rather than dialysis, Dixon and Halliburton refer to the choroid gland ", and the third ventricular part of this structure develops from the thalam-encephalic (diencephalic) roof plate. The pituitary gland, and indirectly therefore the adrenal cortex, is controlled from the thalamencephalic floor. The roof plate also gives rise to the pineal gland, which in reptiles secretes directly into the third ventricle, giving this part of the choroid gland " a particular significance perhaps, especially if the pineal body should prove in the end to play a role in the humoral mechanism "
"
!
in question.
J. P. CRAWFORD. VIRILISATION AND MALFORMATION OF A FEMALE INFANT SIR,-Wilkins et aLl drew attention to the masculinisation of female infants whose mothers had received androgens or progestogens during pregnancy. Their findings have since been confirmed by others. Your correspondent Dr. Dubowitz (Aug. 25) describes another case of striking masculinisation of a female infant whose mother, a primipara, had received a small quantity of ethisterone early in pregnancy. For many yearsAmenorone ’has been used extensively as a pregnancy test, since it causes withdrawal bleeding in nonpregnant cases and no bleeding in pregnancy; so far, no congenital abnormalities following its use have been reported. I have myself employed it for many years in secondary amenorrhoea, some patients later becoming pregnant. No developmental abnormalities ever came to my notice. In the series reviewed by Wilkins2 and Grumbach et al.,3 the degree of masculinisation depended upon the time of gestation when hormone administration was started. If the hormone was given from the 4th-8th week onwards, most cases showed phallic enlargement and a urogenital sinus. If treatment was started later in embryonic life, phallic enlargement was the only abnormality. The urogenital sinus was not a true one but was an ante-chamber of the vagina formed by labial fusion. In this chamber the urethral orifice was normal in relation to the
vaginal opening. More marked abnormalities of female infants were observed where the mother, without hormone treatment, had shown signs ofvirilisation. Wilkins et al.1 described such a case of a mother in a cushingoid state with deepening of the voice, gross obesity, and facial hirsutism so marked that the husband told the doctor he was afraid his wife was turning into a man. The resulting infant was a female pseudohermaphrodite thought to be a boy with hypospadias until the true sex was discovered 5 years later. There are other cases in which the cause of virilisation in a female child is obscure, there being no evidence either of hormones administered to the mother or of androgens being excessively produced by her during pregnancy. Perloff et awl.4 described such a case of a twin pregnancy, both females, one normal, the other presenting as a non-adrenal pseudohermaphrodite with a urethral orifice opening at the tip of the phallus and almost complete Jabioscrotal fusion. Many such instances have been described. Suffice it to say that non-adrenal pseudohermaphroditism can arise in the absence of androgenic influences coming from the mother or from medicaments supplied. The case described by Dr. Dubowitz presents abnormalities more marked than any of those described by Wilkins and Grumbach et. al. Not merely is there an enlarged clitoris and 1. 2. 3. 4.
Wilkins, L., Jones, H. W., Holman,
G. H., Stempfel, R. S. J. clin. Endocr. 1958, 18, 559. Wilkins, L. J. Amer. med. Ass. 1960, 172, 1028 Grumbach, M. M., Ducharme, J., Moloshok, R. E. J. clin. Endocr. 1959,
18, 1369. Perloff, W. H., Conger,
K.
B., Levy, L. M. ibid. 1953, 13, 783.
labioscrotal fusion but also
an
absent
anus
and
a
rectovesico-
vaginal communication suggestive of a cloaca, although obviously not one in its true embryonic sense. Moreover, the Miillerian ducts have failed to fuse, giving rise to a double uterus and a double vagina. The amount of hormone given was very small and the time of administration rather later than in the previously mentioned series. The available evidence, therefore, seems to make it most unlikely that administered ethisterone was the factor responsible for the virilisation. Dr. Dubowitz mentions the absence of chimaerism in the blood of the male twin. He must have had in mind the possibility of a " freemartin " effect. Freemartins were first described by Lillie,5 who discovered a placental communication in bovine dizygotic twins which produced pseudohermaphroditism in the female twin through the androgenic activity of the male twin. Human freemartins have never been described, though intrauterine placental vascular communication has been inferred because of the presence of blood chimaeras. Woodruff et al.1,s have described 5 such cases in which two different bloodgroups were found in the same twin, and drumsticks were noticed in the leucocytes of the peripheral blood. Dr. Dubowitz does not mention an examination of leucocytes for drumsticks in his male twin. Such an examination may provide evidence of a human freemartin. The urinary 17-ketosteroid excretion of the male twin as well as his bone age may give information about androgenic activity in this twin. Dr. Dubowitz does not mention the mother’s condition during her pregnancy. She may have shown signs of excessive androgen output which may have been responsible for the infant’s abnormalities. She might have exhibited signs of virilisation-e.g., acne, or android hirsutism. Even a few hairs on the face, or breasts, or abdomen would suggest an increased androgen output. These hairs remain permanently and would be noticeable even now. The twin pregnancy occurred only after 6 years of married life, and this may well have some bearing on the mother’s endocrine state. Dr. Dubowitz’s case may belong to the same category as Perloff’s pseudohermaphrodite twin which was probably caused by a faulty determinant in the developing embryo. Endocrine Clinic, Charing Cross Hospital, London, W.C.2.
J. ADLER.
A MICROMETHOD FOR CHROMOSOME ANALYSIS ON PERIPHERAL BLOOD-CULTURES
SIR,-Chromosome studies in the newborn are often difficult because of the small volumes of blood that are obtainable. On the other hand, the white-cell count is higher in the first years (and especially the first days) of life than in adults. In this laboratory we have used the following micromethod, which is based on the principles of Moorhead et al. It has yielded good results with as little as 0-1 ml. of capillary blood, even in adults. Aspirate 0-2 ml. of capillary blood into a fine pipette, which has been coated inside with heparin solution by sucking it up and then blowing it out. Transfer the heparinised blood to a small test-tube (1 cm. x 8 cm.) containing 1 ml. of AB-positive serum. Shake gently. Place the tube at an angle of 45 °C at room-temperature for 15-45 minutes. When most of the red cells have settled down, aspirate the supernatant with a syringe, into which 2 ml. of medium 199 (Glaxo) has previously been drawn. Mix in the syringe by shaking gently. Inject aliquots of 1 ml. into each of three small test-tubes. Add one drop of phytohemagglutinin (Wellcome) and incubate for 3 days. Add one drop of a 0-04% solution of demecolcine (’ Colcemid ’) and incubate for a further 4 hours. Then spin down the contents of the tubes, add 1 ml. of an 0-95% solution of sodium citrate, and leave the tubes for 15 minutes at 37°C. Spin and fix in a mixture of methanol and glacial Lillie, F. R. Science, 1916, 43, 611. Woodruff, M. F. A., Fox, M., Buckton, K. A., Jacobs, P. A. Lancet, 1962, i, 192. 7. Moorhead, P. S., Nowell, P. C., Mellman, W. J., Battips, D. M., Hungerford, D. A. Exp. Cell Res. 1960, 20, 613.
5. 6.
-