RAPID SCREENING TEST FOR ADRENAL CORTICAL FUNCTION

1046 shock.

Injection of 500 ml. of a 3% saline solution resulted in

RAPID SCREENING TEST FOR ADRENAL CORTICAL FUNCTION

18% reduction of the output of urine measured in 30minute periods before and after the beginning of infusion,

an

Injection of 15 milli-units of vasopressin (’ Pitressin’), however, caused a 90% antidiuresis in the 30-minute periods. Treatment consisted of prednisolone 5 mg. t.i.d., desiccated thyroid gr. 2 daily, depot-testosterone cyclopentylpropionate 100 mg. intramuscularly once, and depot-restradiol cyclopentylpropionate 3 mg. intramuscularly once. The patient gradually became more alert, her appetite was restored, and she gained weight. Dihydrochlorothiazide 25 mg. b.i.d. had little effect on urinary volume, but vasopressin 10 units b.i.d. controlled urinary output to within the normal range. After 3 weeks of treatment the patient was discharged in a fairly satisfactory condition. At home she continued to take prednisolone and thyroid, but vasopressin, depot-testosterone, and depot-restradiol were stopped. In May, 1963-i.e., a year after onset of the disease-the polydipsia gradually disappeared, and the urinary volume fell which lasted 20 minutes.

to

normal. At the

same

time

menses

recommenced, but

were

,irregular and scanty. The patient said that before this improvement she drank large quantities of Korean rice wine (about 8% alcohol) instead of drinking water. Soon afterwards she stopped taking all medication of her own accord. In February, 1964, she returned in poor physical condition and we admitted her for the second time. She was again pale and weak. Depot fat was normal, and the skin was slightly pale, dry, and cold. The eyebrows were still scanty in the outer third. Axillary hair was still absent. Pubic hair seemed to be slightly increased in amount. Blood-pressure 110/70 mm. Hg. Daily urine output ranged between 1500 and 2200 ml., specific gravity 1-015-1-010; red blood-cells 4,300,000 per c.mm., haemoglobin 11 g. per 100 ml., white cells 7500 per c.mm. (eosinophils 25%, total eosinophil count 1500 per c.mm.). Fasting blood-sugar 100 mg. per 100 ml. 1311 uptake 11 % in 24 hours. The patient left hospital without permission before further tests, such as the Thorn test and the nicotine test, could be done, and

never

returned.

Discussion

Our patient had hypopituitarism and also symptoms of diabetes insipidus, both apparently related to the

postpartum haemorrhage. According to Sheehan and Whitehead (1963) and Whitehead (1963) the posterior lobe of the pituitary and the nucleus

supraopticus and paraventricularis of the hypothalamus become atrophic to some degree in most cases of postpartum pituitary necrosis. Nevertheless, clinical manifestations of diabetes insipidus in this syndrome are very unusual. This is partly explained by the wide distribution of the cells which produce antidiuretic hormone. In our patient, the hypothalamo/posterior-pituitary system and gonadotrophin-producing function of the anterior lobe were apparently at least partly restored 12 months after the onset of the disease. Recovery from diabetes insipidus has been shown to take place occasionally in these circumstances (Sheehan and Whitehead 1963).

Summary In a patient with Sheehan’s syndrome associated with diabetes insipidus polyuria and polydipsia began immediately after a complicated delivery and disappeared completely after 12 months. At the same time irregular and scanty menstrual periods reappeared. REFERENCES

Dingman, J. F., Thorn, G. W. (1958) in Principles of Internal Medicine (edited by T. R. Harrison); p. 553. New York. Góth, A. (1963) Brit. med. J. i, 240. Sheehan, H. L., Whitehead, R. (1963) J. Path. Bact. 85, 145. Whitehead, R. (1963) ibid. 86, 55.

D. MATTINGLY M.B. Lond., M.R.C.P. DIRECTOR, POSTGRADUATE MEDICAL INSTITUTE,

UNIVERSITY OF EXETER

P. M. DENNIS N.Z., M.R.A.C.P.

M.B.

RESEARCH ASSISTANT

J.

C. L. COPE Oxon., F.R.C.P.

PEARSON

B.A. Cantab.

D.M.

RESEARCH ASSISTANT POSTGRADUATE MEDICAL

PHYSICIAN

SCHOOL, HAMMERSMITH, LONDON, W.12

Two biochemical principles have lately been introduced which have greatly helped to bring precision to the clinical investigation of adrenal cortical function in man. These are: (a) the use of isotopic methods to determine the actual secretion-rate of cortisol (Cope and Black 1958); and (b) the development of fluorescence methods for quick measurements of plasma-corticosteroid levels (Silber et al. 1958, De Moor et al. 1960b, Stewart et al. 1961, and Mattingly 1962), which give an indirect index of the status at the time of the adrenal cortex and are easy to apply in the clinic and laboratory. The isotopic method, on the other hand, gives a reliable measure of the mean adrenal cortical activity over 24 or 48 hours, but the technical skill and time needed in the analyses inevitably limit its use to better-equipped laboratories with experience in handling both isotopes and steroids. We describe here a method which combines the simplicity of the plasma method with some of the clinically more valuable information obtained by the isotopic secretion-rate method. The facility and speed with which the fluorescent corticosteroid technique can be applied to plasma has led some workers to apply the same technique to urine samples. These attempts may have been encouraged by the knowledge that urine ordinarily contains small amounts of free cortisol, which increase rapidly in states of increased adrenal activity (Cope and Black 1959). Since the particular reaction in the fluorescent test is a response to 11-hydroxylated corticosteroids, the fluorogenic substances measured are for convenience referred to as

11-hydroxycorticosteroids (11-o.H.c.s.).

De Moor et al. (1960a) investigated the use of this fluorescent method in urine and found relatively low values in states and high values in hyperadrenal disorders. They concluded that when applied to urine the fluorescent method offered a useful " screening" test for adrenal cortical function. But, although this group indicated the type of changes to be expected in adrenal disorders and suggested the potential clinical value of the method, their evidence of the reliability of the technique was based on the agreement of the results with clinical impression and diagnosis. Plainly, caution should be shown in accepting this method for quantitative estimates, partly because of its suspected lack of specificity in urine, but also because the data of De Moor et al. (1960a) themselves showed that measurements of the urinary excretion of the fluorogenic substances were only slightly below the normal in two cases of hypopituitarism and were reduced to a mean of only half the normal in three patients with Addison’s disease. Accordingly, to define more precisely the clinical reliability of such a simple method we compared the results of the urinary fluorescence method with actual estimates of the cortisol secretion-rate obtained simultaneously by isotopic means in a series of fifty-four patients with a wide

hypoadrenal

1047

variety of disorders. A much higher degree of correlation between the two was found than was expected. The correlation was, in fact, appreciably closer than is seen when the urinary excretion of 17-oxogenic steroids is compared with the cortisol secretion-rate. Since the fluorescence test takes only 11/2 hours, and yet furnishes comparable information to that given by estimation of the 17-ketogenic steroid which takes several hours to complete, it seems to have great advantages.

The coefficient of variation over the normal range of 80-330 g. is 8%, which is similar to the results obtained with plasma.

Accuracy When cortisol standards ranging from 0-1 to 5 fLg. were taken through the procedure a linear relation was found between the intensity of fluorescence and the concentration of steroid. Cortisol recovery was determined on nine occasions by adding 1 0 fLg. of cortisol to one of a pair of duplicate samples of urine. The recovery ranged from 93 to 101 %, with a mean of 972°,i°.

Method those described by Mattingly (1962) for The reagents for urine. 2 ml. samples of a fresh modified plasma slightly thoroughly mixed 24-hour collection of urine are extracted by Normal Values Urinary 11-o.H.c.s. excretion was measured in fortyshaking with 15 ml. of pure methylene chloride in a 20 ml. two convalescent hospital patients whose ages ranged from glass-stoppered tube. The upper aqueous layer is sucked off 20 to 70 years. Patients with endocrine, hepatic, or renal and replaced by 2-0 ml. of 1-0 N sodium hydroxide. After shaking for about 20 seconds the tube is stood for about 10 disorders were excluded. None of the patients was acutely minutes to allow complete separation of the two layers and the ill at the time of the collections and none was being exposed soda layer is removed by suction. In later estimations the wash to recognisable stressful procedures. Urinary excretion of was with 2 ml. of 0’ 1 N sodium hydroxide, followed by a wash 11-o.H.c.s. ranged from 78 to 372 g. per 24 hours when with 2-5 ml. of water, and this produced comparable results. as cortisol equivalents. expressed 10 ml. of the washed methylene chloride extract is then added The mean value for the twenty-one adult males was to 5 ml. of a reagent composed of 7 vols. of concentrated sul229 and for fLg. twenty-one adult females was 178 {ig. phuric acid and 3 vols. of pure ethanol. The mixture is then shaken vigorously for 20 seconds and allowed to separate into per day. two layers. The now supernatant methylene chloride is sucked Values Encountered in Disease off, and the lower acid layer is transferred to a glass cuvette Range of The lower limits of the estimation were determined by for fluorimetry. the 11-o.H.c.s. excretion in severe hypoadrenalism. Three The instrument used was a direct-reading fluorimeter (Model with untreated Addison’s disease gave values of of Electronic but other fluoriInstruments Ltd.) patients 27A, designs 19 g., 24 (ig. and 56 g. per day. One patient with clinimeter have proved suitable. A mercury-vapour lamp provides the exciting light, and the primary filter (’ Chance ’ OB 10 cally severe hypopituitarism had an excretion of 21 jjt.g., blue) has a peak transmission at 430 m[J.. Emitted light is and two healthy persons who were under dexamethasone passed through two secondary filters (’ Chance ’ OGR 1 and suppression gave values of zero and 70 g. respectively. OY 3) which give a peak transmission at 540 mil, whilst The mean value for the six hypoadrenal subjects was thus’ excluding light below 510 m[J.. The yellow filter must be nearest 32 pg. per day. By contrast, the highest value so far to the photomultiplier cell. encountered was in a patient with Cushing’s syndrome A reagent blank of 2 ml. distilled water and a standard of whose 2 (ig. cortisol in 2 ml. water are both carried through the prohyperplastic adrenals were being stimulated with cedure and are used to calibrate the fluorimeter. corticotrophin. Her excretion reached 21,400 4g. per day. If the sensitivity control of this instrument is set at range 2 Correlation with Cortisol Secretion-rate to give a reading of 100 with the 2 .g. cortisol standard, then A really useful test should do more than merely confirm the reading for the unknown on the same range represents clinical impression, but should be comparable a general cortisol equivalents of 11-o.H.C.S. in g. per 100 ml. urine. with a more reliable standard of reference. At present the Since the fluorescence increases slowly after mixing, it must be cortisol secretion-rate, determined by means of isotopes, read after a fixed interval, and 13 minutes has been found convenient. With practice three samples can be assayed in duplioffers the most reliable and accurate estimate of the procate at this interval. With urines giving a fluorescence in excess duction of cortisol in the human adrenal cortex. The of the standard a diluted sample of urine should be used. urinary 11-o.H.c.s. excretion in a series of fifty-four Turbid urines should be centrifuged before assay, and only patients was compared with the cortisol secretion-rate fresh or deep-frozen samples should be used, because the nonestimated simultaneously by means of either14C-labelled specific fluorescence has been found to increase appreciably in or tritium-labelled cortisol. Three groups of cases were urine stored for more than 24 hours, even when kept at +4°C. completely excluded from these comparisons: (a) pregnant The purity of reagents and the scrupulous cleanliness of women; (b) patients receiving metyrapone; and (c) patients for 11-o.H.c.s. in are needed glassware determining plasma equally essential in the urine estimation. Full details have been in whom adrenal cortical activity was rapidly changing. are

’

given by Mattingly (1962). Precision precision

Results

The precision of the method was assessed by the criteria suggested by Brown et al. (1957). Standard deviations for different urine concentrations were calculated from the differences between duplicate estimations (Snedecor 1952). 2-0 ml. samples of urine were used. The results were as follows: STANDARD DEVIATION OF DUPLICATE DETERMINATIONS

patients were included. Pregnant women were excluded because of the possible risk of interference from oestrogens and also because of the undesirability of submitting such women to isotopic radiation. Patients on metyrapone are likely to undergo rapid changes not only in adrenal activity but also in the quality of this activity; hence the isotopic secretion-rate technique may give an erroneous result. The degree of correlation between the 11-o.H.c.s. All other

excretion in the urine and .the cortisol secretion-rate is shown in fig. 1 in which, for convenience, both urinary 11-o.H.c.s. and cortisol secretion-rate are plotted on a logarithmic scale. The cortisol secretion-rates ranged from 3 to 480 mg. daily, and the urinary 11-o.H.c.s. excretion from 50 to

1048

costeroid in the urines of the hypoadrenal patients already mentioned was probably negligible. Hence the amount of non-specific substance present as contaminant is likely to vary from nil to 70 g. per day, the latter representing about 30% of the mean normal figure. Close correlation of the urinary 11-o.H.c.s. values with adrenal activity is therefore unlikely to be found in the lowest ranges of

activity.

Fig. 1-Relation between cortisol secretion-rate and urinary excretion of 11-hydroxycorticosteroids. Both plotted on log scale.

The correlation coefficient between their logarithms is 0-95, and this is significant at the 1% level. This correlation may be compared with that observed between the cortisol secretion-rate and the urinary 17oxogenic steroid excretion in a comparable series of patients (fig. 2). In this series, which also excludes those with cortisol secretion-rates below 3 mg. daily, the correlation coefficient between their logarithms is found to be only 0-80, but this is also significant at the 1% level. The standard error of the estimate of cortisol secretionrate determined from the urinary 11-o.H.c.s. excretion is 0-14 when both are expressed by their logarithms. This is equivalent to a standard error range from +38 to - 28 % of the cortisol secretion-rate. The corresponding result for deriving the cortisol secretion-rate from the urinary excretion of 17-oxogenic steroids is 0-23 for the logarithms, equivalent to a standard error range of +71 to - 42 % Nature of the Fluorogenic Steroids Attempts to determine the nature of the substances estimated in this test have been only partly successful. Non-specific and possibly non-steroidal substances undoubtedly account for a small and probably variable part of the fluorescence measured. The amount of free corti-

21,400

g.

daily.

&mid ot;

2-Relation between cortisol secretion-rate and urinary tion of 17-oxogenic steroids. Both plotted on log scale.

Fig.

excre-

The three main steroid components active in yielding this type of fluorescence are cortisol, 20-hydroxycortisol, and corticosterone. When the urine extracts are run on suitable paper chromatographic systems, 60-80% of the recovered fluorogenic substance usually runs in the zones corresponding to cortisol and to 20-hydroxycortisol in varying proportions. The proportion of the total 11o.H.c.s. which can be accounted for by the free cortisol present varies from 20 to 80% of the total in different urines. Less than 20% of the fluorescence can usually be recovered from the zone corresponding to corticosterone. That a significant part of the urinary fluorogenic steroid was in fact 20-hydroxycortisol was shown by the run of the steroid in a number of samples in three different chromatographic systems at a rate indistinguishable from that of authentic 20-hydroxycortisol. In this respect our results differ from those of De Moor et al. (1962) who estimated that 54-7% of the total fluorescence was due to cortisol and 22-8°,o to corticosterone, but were unable to detect the presence of 20"hydroxycortisol. Discussion The attraction of a urinary fluorescence method is essentially its speed and relative simplicity. The justification of the test for clinical screening purposes lies in the observed degree of correlation between the test and a relatively reliable standard of reference-the isotopic estimation of the cortisol secretion-rate. The relatively close agreement between this secretion-rate and the urinary 11-o.H.c.s. determined in this simple manner was unexpected. One of the urinary components estimated is free cortisol, and our previous attempts to correlate urinary cortisol excretion with the cortisol secretion-rate had been discouraging (Cope and Black 1959). But cortisol is only one of the urinary steroids measured by the test and it is not always the main one. The proportion of the total 11-o.H.c.s. which cortisol represents is very variable, and the total urinary fluorogenic steroid excretion apparently correlates more closely with the cortisol secretionrate than the excretion of free cortisol itself. Although it is convenient to refer to the substances estimated as urinary 11-hydroxycorticosteroids, there is little doubt that a variable quantity of contaminating nonspecific substances are included in the estimation. Hence cessation of adrenal cortical action does not produce zero values in the test, and correlation between the 11-o.H.c.s. excretion and the true cortisol secretion-rate must therefore be poor at low levels of activity. But this disadvantage is found in all urinary steroid analyses which do not include the automatic internal corrections provided by the use of isotopes. Because of this relatively high blank value we excluded from our statistical analyses all cases with a cortisol secretion-rate below 3 mg. daily, though these are shown in figs. 1 and 2. Like the 17-oxogenic steroid estimation, the 11-o.H.c.s. is likely to prove of greater clinical value in states of normal or raised adrenal cortical activity than in states of low activity. A value of 220 g. or less gives a 95% probability of the cortisol secretion being within or below the normal

1049 range, and a value of over 1500 {jLg. gives a 95 % probability of a cortisol secretion above the upper limit of normal. For a value of 500 tg. there is a 66% probability of secretion being above the normal range. Because the magnitude of the 11-o.H.c.s. excretion is determined in part by the amount of free cortisol in the urine, which rises sharply with increasing cortisol secretion, the 11-o.H.c.s. excretion may be expected to rise more rapidly than the 17-oxogenic steroid excretion, which is mainly determined by the quantity of cortisol metabolites being produced. Figs. 1 and 2 show that a tenfold rise in cortisol secretion-rate will result in a twenty-seven times rise in mean 11-o.H.c.s. excretion, but a rise in mean excretion of 17-oxogenic steroids only seventeen and a half times. Thus, the 11-o.H.c.s. determination may be expected to prove more sensitive in revealing enhanced adrenal cortical activity in the same way as the urinary cortisol excretion (Cope and Black 1959). The test should be

especially valuable, therefore, for screening suspected cases of Cushing’s syndrome or for observing the response to corticotrophin stimulation. Summary A simple and rapid method of measuring the daily excretion of urinary 11-hydroxycorticosteroids demonstrated that their excretion correlated more closely with the cortisol secretion-rate determined by isotopes, than did the excretion of 17-oxogenic steroids. Since six estimations of 11-hydroxycorticosteroids can be completed within one and a half hours, the test has great advantages over the assay of 17-oxogenic steroids. Our thanks are due to the Medical Research Council for a grant; and to the Wellcome Trust and the Wellington Medical Research Foundation for grants to Dr. P. M. Dennis. REFERENCES

Brown, J. B., Bulbrook, R. D., Greenwood, F. C. (1957) J. Endocrin. 16, 41. Cope, C. L., Black, E. G. (1958) Clin. Sci. 17, 147. (1959) Brit. med. J. ii, 1117. De Moor, P., Raskin, M., Steeno, O. (1960a) Ann. endocr., Paris, 21, 477. Steeno, O., Raskin, M., Hendrikx, A. (1960b) Acta endocr., Copenhagen, 33, 297. Osinski, P., Deckx, R., Steeno, O. (1962) Clin. chim. Acta, 7, 475. Mattingly, D. (1962) J. clin. Path. 15. 374. Silber, R. H., Busch, R. D., Oslapas, R. (1958) Clin. Chem. 4, 278. Snedecor, G. W. (1952) Biometrics, 8, 85. Stewart, C. P., Albert-Recht, F., Osman, L. M. (1961) Clin. chim. Acta, 6, —

—

—

—

These eventful years covered the rejection, after a dour struggle before a Select Committee of the House of Lords, of the Bill for the recognition and registration of osteopaths, and the B.M.A.’s own committees of inquiry into nutrition and into the treatment of fractures, and a policy statement on a General Medical Service for the Nation. They also saw the reorganisation of medicine

Clinical Diabetes Mellitus Editors: MAx ELLENBERG, M.D. ; assistant attending physician, Mount Sinai Hospital, New York, and HAROLD RIFKIN, M.D., clinical professor of medicine, Albert Einstein College of Medicine, New York. New York and London: McGraw-Hill. 1964. Pp. 448. 104s. 6d.

Reviews of Books Lord HILL

OF

LUTON. Heineman: London. 1964.

Pp. 260.

35s.

Charles Hill has played many parts, and his recollections provide material for a readable autobiography which follows his fortunes from an impoverished childhood in Islington to his life peerage and chairmanship of the Independent Television

Authority. He was only two years old when his father died and his mother had to return to ill-paid work to support the family. He went to a London elementary school and then with a highly competitive scholarship to grammar school, Cambridge, and the London Hospital, from which he qualified in 1927, and to which (after locums in general practice) he returned as houseofficer. After he left the London he went to a temporary post at a mental hospital in Nottingham, where he read for his D.P.H. This prepared him for an excursion into public health, and he became the deputy medical officer of health for Oxford. In 1930 he saw an advertisement for an assistant secretary to the British Medical Association. His application was successful and he remained with the B.M.A. for 18 years as assistant secretary, deputy secretary, and for 6 years as secretary.

the demands of war, the Medical

made a most successful broadcast for the Conservative party. In the short Parliament of 1950-51 he was a back-bench Opposition member. When the Conservatives came to power in 1951 he became parliamentary secretary to the Ministry of Food. In Eden’s ministry he was Postmaster General, and under Macmillan Chancellor of the Duchy of Lancaster with a seat in the cabinet and responsibility for the coordination of the Government information services. In 1962, when he was Minister for Housing, Local Government, and Welsh Affairs, he lost ministerial office in the Cabinet reshuffle. The following year he was made a life peer and was appointed chairman of the Independent Television Authority. This interesting and unpredictable saga Lord Hill himself finds difficult to believe. He writes with wonder, almost of awe, of his own progress. Setting out with no such goals as those he achieved, he feels that most that has happened to him has happened by chance. But when opportunity knocked he was never heedless, timorous, or ill prepared. As he says himself, " given the predetermined end, we could argue that even the most thoughtless actions, the most trivial incidents, have their place in an intricate pattern. Certainly I can see now that there were times when I acted as I did because of motives and desires that I grasped only very much later ".

696.

Both Sides of the Hill

to meet

Planning Commission, the Beveridge report, and professional and parliamentary arguments before the National Health Service Act. In all these Charles Hill played his part, and he remembers the struggles with relish and shrewdly sketches the protagonists. In 1945, while still the secretary of the B.M.A., he had stood unsuccessfully as an independent candidate for a university seat. He had also, by weekly broadcasts in a fruity, easily recognised voice, built a national reputation as the Radio Doctor. These simple, easily understood, and persuasive broadcasts weighed with the Conservative party when they persuaded him in 1947 to become (with the consent of the council of -the B.M.A.) the Liberal and Conservative candidate for Luton. In the General Election of 1950 Hill won this seat from Labour, and

THIS book contains thirty-four chapters which originally appeared seriatim in the New York State Journal of Medicine, and thus each is a self-sufficient contribution. A feature of the synthesis is the wide range, from basic consideration of the metabolism of carbohydrate, protein, and fat, to the role of zinc and trace metals, dental and oral aspects, and the employability of the diabetic patient. The chapter by Dr. R. Levine and Dr. M. Goldstein on the action of insulin deserves special praise as a competent and thoughtful summary of a difficult subject. The reader will be interested by the many approaches and disciplines bearing on diabetes, and he cannot fail to increase his understanding of the syndrome; for no one man could describe so wide an area in this way. The book can be recommended as a stimulating survey of diabetes mellitus.

Clinical

Psychiatry for

Practitioners and Students

2nd ed. IAN SKOTTOWE, M.D., F.R.C.P., D.P.M., psychiatrist, Warneford Hospital, Oxford. London:J. & A. Churchill. 1964. Pp. 306. 42s. 6d.

BASIC textbooks of psychiatry are blooming, and there is now wide choice for the medical student or the general practitioner. Dr. Skottowe’s second edition (the first was in 1953) has the merits of sound clinical basis and high quality of writing. On a