553
patients in the acute stage of myocardial infarction-in
56%of a type likely to have serious consequences. Of the
many types of arrhythmias observed, some, such as
bundle-branch and atrio-ventricular block, merely reflected
the severe degree of myocardial damage. In others, such
as ventricular tachycardia or ventricular fibrillation, death
was quite likely to result from cardiac arrest even if the
underlying myocardial lesion was trivial. In a third group,
including atrial fibrillation or flutter, a poor prognosis
probably resulted from the reduction in coronary blood-
flow brought about by the arrhythmia, which aggravated
the underlying myocardial lesion. There seems no reason
to suppose that propranolol would be any less effective in
controlling or inhibiting these arrhythmias when they
follow infarction than in other situations, and it should
thereby considerably improve the prognosis. The truth of
this possibility cannot readily be established other than by
continuous E.c.G. monitoring of treated and control
groups of patients which is beyond the resources of this
hospital. Nevertheless, it is hoped that such a study will
be undertaken elsewhere, for the matter is of some
practical importance. If propranolol is found to be
effective in suppressing arrhythmias such as ventricular
tachycardia and ventricular fibrillation which may cause
sudden death from cardiac arrest, the application of such
treatment is not limited to the acute phase of infarction.
Similar mechanisms may be responsible for sudden and
unexpected death during the chronic and possibly
asymptomatic phase of the disease.
Side-effects from the administration of propranolol in
the doses used appear to be infrequent and trivial. None
was encountered in the present study. The most important
theoretical disadvantage of propranolol is the risk of
precipitating heart-failure by reducing the sympathetic
drive, and it is this consideration which has apparently
been responsible for the reluctance to employ -adrenergic
blocking agents in conditions where failure may supervene.
In fact, only 2 of the 45 patients treated with propranolol
developed heart-failure, compared with 4 of the 46 patients
not so treated. Higher dosage than that used in the
present study might add to the risk of inducing heart-
failure, but with a dose of 60 mg. a day the risk appears
to be small, and, in view of the possible benefits, seems
acceptable.
Summary
Propranolol, 10-20 mg. eight-hourly, was given to
45 patients with acute myocardial infarction. The
mortality was found to be only 16%compared with 35%
in a comparable control group of 46 patients. Propranolol
appears to be capable of significantly reducing the
mortality from acute myocardial infarction. It may act by
reducing the demand for oxygen of the myocardium while
increasing its efficiency, and also by inhibiting dangerous
arrhythmias. In the dosage used propranolol appears to
be without serious side-effects in myocardial infarction.
I wish
to thank my colleague Dr. H. P. Goldman for allowing me
to study patients under his care, my resident and nursing staff for
their assistance, Dr. R. H. E. Grant for the supply of Inderal
(propranolol), and Mr. C. Clarke for the statistical analysis.
REFERENCES
Alleyne, G.A.O., Dickinson, C. J., Dornhorst, A. C., Fulton, R. M., Green,
K. G., Hill, I. D., Hurst, P., Laurence, D. R., Pilkington, T., Prichard.
B. N. C., Robinson, B., Rosenheim, M. L. (1963) Br. med. J. ii, 1226.
Apthorp, G. H., Chamberlain, D. A., Hayward, G. W. (1964) Br. Heart J.
26, 218.
Chamberlain, D. A., Howard, J. (1964) ibid. p. 213.
Dornhorst, A. C., Robinson, B. F. (1962) Lancet, ii, 314.
Hamer, J., Grandjean, T., Melendez, L., Sowton, G. E. (1964) Br. med. J.
ii, 720.
DIAGNOSIS OF THE ADRENOGENITAL
SYNDROME BEFORE BIRTH
T. N. A. JEFFCOATE
M.D.Lpool, F.R.C.S.E., F.R.C.O.G.
J. R. H. FLIEGNER SHONA H. RUSSELL
M.B. Melb., M.R.C.O.G. M.B. Lpool, M.R.C.O.G.
OF THE DEPARTMENT OF OBSTETRICS AND GYNÆCOLOGY
J. C. DAVIS A. P. WADE
M.D. Lpool, M.C.Path. B.Sc., Ph.D. Lond., A.R.I.C.
OF THE DEPARTMENT OF ENDOCRINE PATHOLOGY
UNIVERSITY OF LIVERPOOL
THE need recognise the adrenogenital syndrome in
to
a baby possible requires little emphasis: if the
as soon as
diagnosis is missed at the time of birth, a female child
may be registered male; and in a baby of either sex the
as
neonatal period can be complicated by serious (and
sometimes fatal) electrolyte disturbances. Though the
sex-chromatin pattern and the chromosome content of the
nuclei of epithelial cells and leucocytes are helpful when
an anomaly in the development of the external genitalia
is suspected, the diagnosis rests mainly on demonstrating
a raised level of pregnanetriol and often also of
17-ketosteroids in the urine.
Since from at least the twenty-eighth week of pregnancy
onwards foetal urine contributes significantly to the liquor
amnii, it occurred to us that hormone assays carried out
on the liquor obtained by amniocentesis might disclose
before labour that a foetus was affected. All preparations
for its management could then be made before delivery.
This idea came to mind when there happened to be under
our care at the same time two pregnant women, each of
whom had given birth previously to children with the
adrenogenital syndrome. In view of the known strong
familial tendency, there was a fear that the pregnancies
would end in the birth of affected children. Accordingly,
amniocentesis before labour was carried out in both
women and also in three control pregnant women. The
liquor was examined for its content of pregnanetriol and
of 17-ketosteroids according to the methods described in
the appendix. The findings are shown in the accompanying
table.
Case-reports
Case 1
A woman, aged 30 years, had had six previous pregnancies.
These had resulted in three living normal male children, one
abortion, and two babies who had died in the neonatal period:
one of these, a normally developed male, died from pre-
maturity, the mothers pregnancy having been complicated by
abruptio placentx; the second, the outcome of the fifth
pregnancy and delivered normally at term, was registered as
a male but, having died from electrolyte imbalance when
11 days old, was seen at necropsy to be a virilised female with
typicalfeatures of the adrenogenital syndrome.
When this patient conceived again (last menstrual period
Sept. 7, 1964) the possibility of her carrying another affected
child was entertained. She was therefore admitted to hospital
at the 39th week of what had been an uneventful pregnancy,
and 150 ml. of liquor amnii was obtained by abdominal
amniocentesis on June 14, 1965. The pregnanetriol content
compared with that in case 2 was high. Labour did not ensue
DR. SNOW: REFERENCES—continued
Julian, D. G., Valentine, P. A., Miller, G. G. (1964) Am. J. Med. 37, 915.
Kadas, T. (1964) Br. med. J. i, 1384.
Keelan, P. J. R. B. (1965) ibid. i, 897.
Lucchesi, B. R. (1965) J. Pharmacol. 148, 94.
Rowlands, D. J., Howitt, G., Markman, P. (1965 Br. med. J. i, 891.
Sloman, J. G., Robinson, J. S., McLean, K. H. (1965) ibid. p. 895.
Sodi-Pallares, D. Unpublished.
Stock, J. P. P., Dale, N. (1963) Br. med. J. ii, 1230.
554
HORMONE LEVELS IN LIQUOR AMNII
when expected, and, with post-maturity as the indication, it was
induced by artificial rupture of the fore-waters on June 28
(2 weeks after amniocentesis). After an e1SY labour lasting
3 hours a female child weighing8 lb. (3630 g.) was delivered.
The phallus was enlarged, and, behind it, the labia were fused
to give an appearance suggestive of hypospadias in the male.
The diagnosis of adrenogenital syndrome was later fully
confirmed by chromosomal and biochemical studies.
Case 2
A woman, aged 38 years, had had two previous children, one
born in 1952 and the second in 1962. Both were girls with the
adrenogenital syndrome and had been fully investigated and
treated by one of us (T. N. A. J.). The older girl died from
infiuenzal bronchopneumonia at the age of 5/2 years, the
younger is still under supervision.
When this patient conceived for a third time (last menstrual
period Sept. 28, 1964) she was especially anxious about the
outcome. After a normal pregnancy she was admitted to
hospital at the 39th week, and 71 ml. of liquor amnii was
removed by abdominal amniocentesis on June 28, 1965. The
pregnanetriol content (see table) was much lower than in
case 1, and the baby, delivered on July 5, proved to be a normal
female weighing 8 lb. 7 oz. (3830 g.).
Case 3
A woman, aged 18 years, primigravid and with mild pre-
eclampsia, underwent abdominal amniocentesis when labour
was one day overdue. 150 ml. of liquor was obtained; the
pregnanetriol content was relatively low (see table). 2 days after
amniocentesis this patient delivered a normal female child
weighing 7 lb. 1 oz. (3210 g.).
Cases 4 and 5
Two women, aged 26 and 19 years, had labour induced
surgically at or nelr term, one on account of mild hypertension
and the other on the ground of maturity. Liquor was in each
case collected through the Drew Smythe catheter used for the
induction. Both patients produced normal male babies of
good size.
Discussion
The table shows that the liquor from case 1, who
subsequently gave birth to an infant with adrenal hyper-
plasia, gave a much greater concentration of both preg-
nanetriol and of 17-ketosteroids than the liquor from the
women carrying unaffected foetuses. For the reasons given
in the appendix, the identification of the substance
measured as pregnanetriol is reasonably certain. The
steroid values are expressed as ug. per litre, because the
total volume of liquor in the uterus was not estimated.
The liquor was examined late in pregnancy, between
the 35th and 40th weeks. If; as the evidence suggests
(Jeffcoate and Scott 1959), the foetal kidneys excrete freely
from at least the 20th week of pregnancy and the fcetal
adrenal gland is functioning by the 12th to 14th week,
differences in the steroid levels in the liquor in the
adrenogenital syndrome seem likely to be detectable long
before term. From the practical standpoint, however, the
diagnosis need only be made in the last 2 or 3 weeks of
pregnancy to give the obstetrician and paediatrician
adequate warning of the impending birth of a child
affected by the syndrome.
The value of observations such as are described here
might be increased if the assays were combined with an
assessment of the total volume of liquor at the time of
amniocentesis. Nevertheless, since all the women were at
about the same stage of pregnancy, were carrying babies
of average size, and gave no clinical evidence of either
polyhydramnios or oligohydramnios, it seems reasonable
to conclude that the high level of pregnanetriol and
17-ketosteroids in the liquor of case 1 is related to the
adrenal hyperplasia in the fcetus. In support of this
conclusion we made one further possibly significant
observation in a case of anencephaly-a condition in which
the fcetal adrenals are usually hypoplastic.
Case 6
A woman, aged 19 years and primigravid, was discovered to
be carrying an anencephalic foetus when difficulty in deter-
mining the nature of the presenting part prompted an X-ray
examination. There was no clinical evidence of polyhydramnios.
Labour was induced by amniotomy at the 39th week of
pregnancy, and 300 ml. of liquor amnii was collected through
a Drew Smythe catheter. The baby, a female weighing
4 lb. 13 oz. (2180 g.), when born two days later, had an incom-
plete degree of anencephaly and did not die until a few days
later. At necropsy the adrenals were present, but they were
smaller than normal, together weighing only 7 g.
The levels of steroids in case 6, which are remarkably
lower than in any of the cases in which the foetus was
normal, match with the associated fcetal adrenal hypo-
plasia, and go to confirm that hormone assays of the liquor
amnii do in fact reflect adrenal cortical activity. And it
still seems likely that the hormone-excretion products
reach the liquor by way of foMai urine.
Confirmation of our findings is clearly necessary before
their significance can be fully assessed. But the adreno-
genital syndrome is relatively uncommon, and our
experience in one case is reported in the hope that others
who may have to deal with a pregnant woman who has
previously had an affected child might test the method.
With this in mind, full details of the biochemical methods
employed are appended.
Appendix
ESTIMATION OF PREGNANETRIOL IN LIQUOR AMNII
Extraction of steroids from liquor amnii has hitherto been
confined to unconjugated compounds (Baird and Bush 1960,
Lambert and Pennington 1965). In the present study, pre-
liminary experiments indicated that the pregnanetriol was
probably present as a glucuronoside; the isolation procedure
therefore included enzyme hydrolysis. Purification on alumina
followed the method of Leon and Bulbrook (1960); the
pregnanetriol was estimated as a 17-ketosteroid after periodate
oxidation (Metcalf 1963).
1. The liquor was poured into 5 vols. of ethanol and the
mixture was refrigerated overnight. The flocculent precipitate
was removed by centrifuging, washed with % little ethanol, and
the combined solutions evaporated to dryness. The removal
of this precipitate facilitated subsequent extraction.
2. The residue was dissolved in methanol (70% v/v, 50 ml.)
and the solution was extracted with light petroleum 40-60
(2 x 50 ml.). The aqueous methanol solution was evaporated
to dryness.
3. The residue was dissolved in water (50 ml.) and extracted
with chloroform (2 x 50 ml.) (unconjugated steroids).
4. The aqueous solution was adjusted to pH 3-5 with acetic
acid and a few drops of hydrochloric acid. Conjugated steroids

were extracted by the ammonium sulphate-ether/ethanol
method (Edwards et al. 1953).
5. The conjugated residue was dissolved in 0-5 M acetate
buffer, pH 4-0 (10 ml.) containing monobasic potassium
phosphate (50 mg.) and limpet &bgr;-glucuronidase (25,000 u).
The mixture was incubated overnight at 37°C and extracted
with ether-ethyl acetate 1:1mixture (3 x 20 ml.). The combined
extracts were washed with N sodium hydroxide (2 x 10 ml.)
and water (4 x 10 ml.), dried and evaporated to dryness.
6. The residue was chromatographed on alumina as described
by Leon and Bulbrook (1960), except that the pregnanediol
fraction and the pregnanetriol pre-wash (both 3% ethanol//
benzene eluants) were combined.
7. The pregnanetriol content of the appropriate fraction
from the alumina column was determined by oxidising one half
of the residue with sodium periodate (Metcalf 1963), followed
by a micro-Zimmermann reaction on half the ketosteroid
fraction. Using aetiocholanolone as standard, the result is
expressed as pregnanetriol by multiplying by 1-15.
The conjugate extraction stage can perhaps be omitted, and
the enzyme hydrolysis carried out immediately after extracting
unconjugated compounds. It was included in these experiments
to aid in eliminating enzyme inhibitors, if present.
In the sodium periodate oxidation procedure, the whole of
the chloroform extract was evaporated to dryness. The
Zimmermann reaction was carried out by dissolving the residue
in ethanol (0-05 ml.), and adding m-dinitrobenzene solution
(0-05 ml. 1% w/v in ethanol) and tetramethylammonium
hydroxide (25% 0-05 ml.). After mixing thoroughly, and
incubating for 1 hour at 25°C, 30% aqueous ethanol (0-5 ml.)
was added and the mixture extracted with ether (0-75 ml.)
(James 1961). The ether was pipetted into a 0-5 ml. cuvette
(1 cm. light path) which was then covered. The test and
standard (aEtiochoIanolone, 2 g.) were read against a blank at
440, 515, and 590 m and Allens correction applied (Allen
1950).
This method of estimating pregnanetriol, instead of the more
usual sulphuric acid reaction, was decided upon because of its
greater specificity. The amount of pregnanetriol was expected
to be small, and, if contaminated by much non-steroid material,
reaction with sulphuric acid might give an ambiguous result.
IDENTIFICATION OF THE MATERIAL ESTIMATED
AS PREGNANETRIOL
The small amount of material precluded many possible tests,
but on the appropriate fraction prepared from each sample of
liquor the following tests were carried out.
1. Half the pregnanetriol fraction, together with pure
pregnanetriol, was chromatographed on a thin layer of silica gel
in the system chloroform/methanol 12-3:1. After drying, the
plate was sprayed with 1 % periodic acid in 75 % 0-4 N ethanolic
sulphuric acid (Bush 1955), and heated for 10 minutes at 60°C.
It was further sprayed with Zimmermann reagents (3 N
ethanolic potassium hydroxide and 2% ethanolic m-dinitro-
benzene). A pink spot was given by both standard and
unknown. Each was the same distance from the origin. This
showed that the unknown ran parallel with pregnanetriol, and
that it could be oxidised to a 17-ketone.
2. Half the material from the sodium periodate oxidation
was chromatographed on a thin layer of silica gel in the system
ethyl acetate/cyclohexane 45:55, together with androsterone,
Eetiocholanolone, and 11-hydroxyaetiocholanolone. Treat-
ment of the plate with Zimmermann reagents revealed the
unknown as having the same Rf value as mtiocholanolone, which
is the 17-ketosteroid formed by oxidising pregnanetriol with
periodate.
This evidence strongly suggests pregnanetriol was present
in the fraction eluted from alumina.
ESTIMATION OF 17-KETOSTEROIDS
17-ketosteroids, if present, would be eluted from the
alumina column in the first fraction (0-8% ethanol/benzene).
This was tested by means of the micro-Zimmermann reaction.
A positive reaction was obtained in all cases. Thin-layer
555
chromatography on silica gel showed from one to three spots
in different samples. One spot had the same Rf value as
androsterone. No further attempts at identification were made.
REFERENCES
Allen, W. M. (1950) J. clin. Endocr. Metab. 10, 71.
Baird, C. W., Bush, I. E. (1960) Acta endocr., Copenh. 34, 97.
Bush, I. E. (1955) Biochem. 59, xiv.
Edwards, R. W. H., Kellie, A. E., Wade, A. P. (1953) Mem. Soc. Endocr.
2, 53.
James, V. H. T. (1961) J. Endocr. 22, 195.
Jeffcoate, T. N. A., Scott, J. S. (1959) Can. med. Ass. J. 80, 77.
Lambert, M., Pennington, G. W. (1965) J. Endocr. 32, 287.
Leon, Y. A., Bulbrook, R. D. (1960) ibid. 20, 236.
Metcalf, M. G. (1963) ibid. 26, 415.
PLASMA-GROWTH-HORMONE LEVELS
IN UNTREATED ACROMEGALY
AND AFTER RADIOACTIVE IMPLANTS
INTO THE PITUITARY
F. C. GREENWOOD
M.Sc., Ph.D. Lond.
OF THE DIVISION OF CHEMISTRY AND BIOCHEMISTRY, IMPERIAL CANCER
RESEARCH FUND, LINCOLNS INN FIELDS, LONDON, W.C.2
HELEN
J. STEWART
M.B. Glasg.
BRITISH EMPIRE CANCER CAMPAIGN ASSISTANT,
SURGICAL UNIT, ROYAL INFIRMARY, CARDIFF
A. P. M. FORREST
B.Sc., M.D., Ch.M. St. And., F.R.C.S., F.R.C.S.E., F.R.C.S.G.
PROFESSOR OF SURGERY, WELSH NATIONAL SCHOOL OF MEDICINE
R. G. WOOD
M.Sc. Manc., F.Inst.P., A.M.I.E.E.
CHIEF PHYSICIST,
SOUTH WALES AND MONMOUTHSHIRE RADIOTHERAPY SERVICE, CARDIFF
ACROMEGALY is well recognised as an endocrine disease
in which the secretion of pituitary growth-hormone may
be excessive. There has been varied success with pro-
cedures designed to reduce the overactivity of the
pituitary gland-irradiation with X-rays (Johnsen 1952),
surgical hypophysectomy (Hamberger et al. 1959, Ray and
Horwith 1964), (x-particle irradiation (Lawrence et al.
1962), and implantation of radioactive isotopes (Joplin
et al. 1961, Molinatti et al. 1962).
The development of reliable methods for measuring the
concentration of growth hormone in plasma (Glick et al.
1963, Hunter and Greenwood 1964a) has provided a
direct method of assessing pituitary activity in this
syndrome. These groups of workers have confirmed that
acromegalic patients, unselected for any degree of clinical
activity, have abnormally high plasma-levels of growth
hormone. Concentrations after overnight fasting were
also high and have been shown to be notably lowered after
treatment by a-particle irradiation (Linfoot and Green-
wood 1965). Unlike healthy people, in whom a rise in
blood-sugar completely suppresses the secretion of growth
hormone (Roth et al. 1963, Hunter and Greenwood
1964b), acromegalics have persistently high levels even
when given oral glucose (Roth et al. 1963, Linfoot and
Greenwood 1965).
We have studied the effect of the implantation of
radioisotopes into the pituitary of acromegalic patients on
the plasma-levels of growth hormone.
Clinical Material and Methods
Estimations of growth hormones were carried out in 15
patients with acromegaly, diagnosed on clinical and radiological
grounds and treated by radioactive implantation of the pituitary
(tables i and II).
In 8 of these patients estimations were carried out before and