THRESHOLD ADRENOCORTICAL SENSITIVITY IN MAN AND ITS POSSIBLE APPLICATION TO CORTICOTROPHIN BIOASSAY

697 J. H. Darrell, Prof. N. R. Grist, Dr. J. R. Hobbs, Dr. D. A. McSwiggan, Dr. E. G. J. Olsen, and Dr. R. G. Sommerville for their immunological and pathological examinations. Requests for reprints should be addressed to A. K. Dr.

REFERENCES

Andrews, P. S., Marmion, B. P. (1959) Br. med. J. ii, 983. Baylon, H., Bloch, F., Giroud, P., Coumel, H. (1952) Bull. Mém. Soc. méd. Hôp., Paris 68, 67. Beck, M. D., Bell, J. A., Shaw, E. W., Huebner, R. J. (1949) Publ. Hlth Rep., Wash. 64, 41. Burnet, F. M., Freeman, M. (1937) Med. J. Aust. ii, 299. Clinicopathological conference, Postgraduate Medical School of London (1963) Br. med. J. i, 1143. Derrick, E. H. (1937) Med. J. Aust. ii, 281. Evans, A. D. (1963) Practitioner, 191, 605. Powell, D. E. B., Burrell, C. D. (1959) Lancet, i, 864. Ferguson, I. C., Craik, J. E., Grist, N. R. (1962) J. clin. Path. 15, 235. Grist, N. R. (1967) Personal communication. Ross, C. A. C., Sommerville, R. G. (1967) Lancet, i, 727. Gsell, O. (1962) Schweiz. med. Wschr. 92, 1219. Hillis, B. R. (1967) Personal communication. Hobbs, J. R., Sommerville, R. G., McSwiggan, D. A. (1967) Lancet, i, 1109. Huebner, R. J., Jellison, W. L., Beck, M. D. (1949) Ann. intern. Med. 30, 495. Lepeschkin, E. (1952) Am. J. med. Sci. 224, 318. Ludwig, H. (1956) Schweiz. med. Wschr. 86, 490. Marmion, B. P. (1962) J. Hyg. Epidem. Microbiol. Immun. 6, 79. McCoy, J. H., Stoker, M. G. P., Malloch, R. A., Moore, B. (1953) Lancet, i, 503. — Higgins, F. E., Bridges, J. B., Edwards, A. T. (1960) Br. med. J. ii, 1264. McIver, M. (1962) Med. J. Aust. ii, 379. Mitchell, R., Grist, N. R., Bazaz, G., Kenmuir, A. C. F. (1966) J. Path. Bact. 91, 317. Nicolau, S. (1963) Bull. Soc. Path. exot. 56, 690. Ormsbee, R. A., Parker, H., Pickens, E. G. (1955) J. infect. Dis. 96, 162. Parker, R. T., Menon, P. G., Merideth, A. M., Snyder, M. J., Woodward, T. E. (1954) J. Immun. 73, 383. Powell, O. (1960) Aust. Ann. Med. 9, 214. Price, W. H., Emerson, H., Nagel, H., Blumberg, R., Talmadge, S. (1958) Am. J. Hyg. 67, 154. Robson, A. O., Shimmin, C. D. G: L. (1959) Br. med. J. ii, 980. Smadel, J. E., Ley, H. L., Diercks, F. H., Cameron, J. A. P. (1952) Am. J. Hyg. 56, 294. Smith, W. G., Evans, A. D. (1960) Lancet, ii, 846. Stephan, E., Saliba, E. (1963) Archs Mal. Cœur, 56, 1161. Stoker, M. G. P., Fiset, P. (1956) Can. J. Microbiol. 2, 310. Tapie, J., Delaude, A., Cassagneau, J., Le Tallec, Fermond (1960) Bull. Mém. Soc. méd. Hôp., Paris, 76, 65. Zinsser, H. (1934) Am. J. Hyg. 20, 513. —

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corticosteroid levels, which resulted from its prior administration, made small changes in circulating corticosteroid levels more apparent. In dexamethasonesuppressed subjects the mean minimal effective dose of synacthen necessary to produce an adrenal response was less than 30 ng. Based on these studies a simple procedure has been devised which may prove of value for assaying corticotrophin in man and also for assessing whether altered adrenocortical sensitivity is a feature of any clinical disorder. Introduction THE determination of plasma-steroid concentrations before and after the administration of corticotrophin constitutes the basis of tests of adrenocortical function. Usually, pharmacological quantities of corticotrophin are administered, and the tests consequently assess the maximum secretory capacity of the adrenal cortex. Investigation of adrenocortical sensitivity requires a different approach, and involves quantitative administration of physiological amounts of corticotrophin. For this purpose ’Synacthen ’ (a synthetic polypeptide identical in structure with the N-terminal twenty-four aminoacids of corticotrophin) is a very suitable material because solutions can be prepared in accurate and known concentrations by

weight. We have determined the adrenocortical response to single intravenous injections of nanogram amounts of synacthen in an attempt to devise a test of adrenocortical sensitivity. The procedure evolved may also prove of value as a means of assaying corticotrophin and its synthetic analogues for clinical use. Volunteers and Methods Thirty volunteers were investigated; they had no clinical or

biochemical evidence of endocrine, hepatic, or renal dysfunction. Tests were started between 8 and 10 A.M. using an indwelling needle inserted into a forearm vein. Doublingamounts of synacthen from 31.25 to 250 ng. were injected intravenously at 30-minute or 60-minute intervals, and bloodsamples were taken immediately before, and at exactly timed THRESHOLD ADRENOCORTICAL intervals after, each injection. Finally, each volunteer received an injection of 250 -g. of peptide to assess their maximum SENSITIVITY IN MAN AND ITS POSSIBLE adrenocortical response, the zero and 30-minute values being APPLICATION TO CORTICOTROPHIN compared with those reported previously (Wood, Frankland, BIOASSAY James, and Landon 1965). Peptides may adsorb onto glass and other surfaces: studies J. LANDON with ["8’I]-labelled synacthen and others using a radioimmunoM.D. Lond. assay capable of determining picogram amounts of this peptide SENIOR LECTURER IN CHEMICAL PATHOLOGY, showed that such losses could be considerable. Thus in AND TO THE MEDICAL UNIT experiments in which the 250 -g. contents of ampoules of V. H. T. JAMES MARGARET J. WHARTON synacthen were diluted in isotonic saline solution to concentraPh.D. Lond. M.B. Lond., M.R.C.P., D.C.H. tions suitable for the present study, losses onto the two glass PROFESSOR OF CHEMICAL REGISTRAR syringes and the litre bottle of saline solution used for this ENDOCRINOLOGY TO THE MEDICAL UNIT purpose ranged from 36 to 70%. Adsorption losses, which were ST. MARY’S HOSPITAL MEDICAL SCHOOL, LONDON W.2 not prevented by the addition of human serum-albumin at a final concentration of 5 mg. per ml., were not significaht if the M. FRIEDMAN peptide was dissolved in acidified saline solution of pH 2.0 M.D. W’srand, M.R.C.P.E. (Landon et al. 1967). A 0-02% solution of hydrochloric acid in SENIOR PÆIATRIC REGISTRAR, UNIVERSITY COLLEGE HOSPITAL, 0-15M sodium chloride proved suitable and caused no disLONDON W.C.1 comfort when injected intravenously in the 0.25 to 2.0 ml. amounts used. Plasma - 11 - hydroxycorticosteroids were Summary determined after Plasma-11-hydroxycorticosteroid (11-OHc.s.) values were single intravenous injec- determined a semiautomated modification (James et al. 1967) by tions of nanogram amounts of ’ Synacthen ’ (identical in of the fluorimetric procedure of Mattingly (1962). structure to the twenty-four N-terminal aminoacids of Results corticotrophin). Adsorption losses onto glass were prevented by administering the peptide in acidified saline Plasma-11-0Hc.s. After Synacthen without Dexasolution. Dexamethasone, in doses sufficient to suppress methasone Suppression In twelve controls, given single intravenous injections of endogenous corticotrophin secretion, did not impair adrenocortical function significantly. The low baseline saline solution only, the mean plasma-11-OHc.s. cono3

698 PLASMA-11-OHC.S. RESPONSES

TO SINGLE INTRAVENOUS

centration fell progressively from 17 to 11 g. per 100 ml. during the 6-hour period within which all subsequent tests were completed. Five of six subjects given synacthen had an increment of 2 .g. per 100 ml. or more, after 62-5 ng. and the other after 125 ng. (see table). The response was transient, peak values being found 15 minutes after the injection of nanogram amounts of synacthen in more than 85% of instances. After the injection of 250 {jLg. plasma-11-OHc.s. levels continued to rise throughout the 30-minute test period.

INJECTIONS

OF SYNACTHEN

of synacthen in more than 90% of instances. Based on this observation a simpler investigational procedure was devised. Doubling-doses of the peptide were injected at hourly intervals into a further ten dexamethasone-pretreated subjects and only basal and 15-minute bloodsamples were taken, except after the 250 (J.g. injection when an additional sample was taken after 30 minutes.

Effect of Dexamethasone on Basal’ Plasma-ll-0Hc.s. and Response to Synacthen The mean (-4- S.D.) basal plasma-11-OHc.s. concentration between 8 and 10 A.M. in twenty-six controls who had received 2 mg. of dexamethasone orally at 10 P.M. the previous evening and again 2 hours before venepuncture was 3-4 (± 1.39) g. per 100 ml. (range 2-7 g. per 100 ml.). This is significantly lower (p < 0-001) than the value of 1W(± 1-9) .g. per 100 ml. found in one hundred and five subjects who had not received dexamethasone. These values are, in part, the result of spurious fluorescence since in one subject the sum of his plasma hydrocortisone and corticosterone concentrations (0.62 and 0.04 .g. per 100 ml., respectively), as determined by a double-isotope procedure (Fraser and James 1967), was less than a third the total corticosteroid level (3 g. per 100 ml.) determined by the semiautomated fluorimetric technique. Two subjects (nos. 1 and 5) were retested with synacthen while receiving an intravenous infusion of dexamethasone at a rate of 1 mg. per hour. The infusion, which was started 2 hours before the first injection and continued throughout the test, did not impair the response (fig. 1). The table includes the results obtained in ten subjects pretreated with dexamethasone (2 mg. orally night and morning) in whom multiple blood-samples were taken after each injection of synacthen. A considerable variability of response is apparent which may relate to the individual’s body-weight. Thus the largest plasma-11OHc.s. increments at each dose level were recorded in the two lightest volunteers-nos. 5 and 9 who weighed 99 lb. (45 kg.) and 93 lb. (42 kg.), respectively. One volunteer (no. 1) with a relatively poor response weighed 256 lb. (116 kg.). Peak plasma-11-OHc.s. levels were found 15 minutes after the injection of nanogram amounts

Fig. 1-Plasma-11-OHc.s. after single intravenous infections of synacthen to two controls while receiving, and while not receiving, constant intravenous infusion of dexamethasone at 1 mg. per hour.

a

a rate

of

699

Six other dexamethasone-suppressed subjects served as controls and received hourly injections of saline solution only. In this group, plasma-11-OHc.s. concentrations remained at, or below, 3 !log. per 100 ml. (fig. 2). The plasma-11-OHc.s. increment, above the basal. value, found 15 minutes after the injection of each dose of synacthen in the individual subjects is shown in fig. 2. The administration of 31.25 ng. resulted in a significant rise both as compared with the basal level and with the mean value recorded in subjects given saline solution (P< 0-001). There was no significant difference between the 15-minute increments after 250 ng. and 250 !log. of synacthen. However, whereas with 250 ng. plasmacorticosteroid values fall after 15 minutes, with the 250 !log. dose the mean value continued to rise from 22-4 (iL 5-6) tig. per 100 ml. at 15 minutes to 27-2 (± 5-4) !log. per 100 ml. at 30 minutes. A linear log-dose/response relationship seems to exist over the range 31-25 to 250 ng. By continuing this line (fig. 2) until it reaches zero the mean minimal effective dose of synacthen in this group of twenty control subjects can be established. This is

approximately

20 ng.

Discussion The human adrenal is very sensitive to the steroidogenic effects of corticotrophin, and the intravenous injection of 31-25 ng. of synacthen usually induced a plasma-11-OHc.s. rise. This is only about 10-4 of the amount used in one test of adrenocortical function (Wood, Frankland, James, and Landon 1965) which emphasises that such procedures make use of pharmacological quantities of corticotrophin. This does not reduce their value in assessing the reserve capacity of the gland but means that they do not determine its sensitivity. Natural corticotrophin preparations would be unsuitable for determining sensitivity because of difficulties in administering them on a quantitative basis. Synacthen, however, is ideal since its dosage can be determined accurately by weight. Care must be taken to avoid losses of corticotrophin by adsorption on to glass or other surfaces, which may be considerable (Stouffer and Lipscomb 1963). Fortunately, in the case of synacthen, this can be prevented by dissolving it in acid solutions (James, Landon et al. 1967, Landon et al. 1967). In initial studies synacthen was administered to resting controls, but difficulties arose in interpreting the findings. The considerable variation between individual’s basal corticosteroid levels may have influenced the response; it was not certain whether endogenous corticotrophin secretion might contribute; the tests were all performed in the morning at a time when circulating corticosteroid concentrations are falling as a consequence of their circadian rhythm, and thus failure to demonstrate this fall after the administration of an amount of synacthen insufficient to induce an actual rise might be indicative of a steroidogenic effect. Studies were, therefore, undertaken in which the participants received pretreatment with dexamethasone in amounts which totally suppress endogenous corticotrophin secretion (James and Landon 1964), in an attempt to overcome these difficulties. We found that dexamethasone did not impair the response to synacthen and that its prior administration proved of value in lowering basal corticosteroid levels, thereby making more apparent any small adrenocortical response. The suppression of endogenous corticotrophin secretion, by surgical or chemical means, is a recognised prerequisite of all animal bioassays for corticotrophin, and would seem essential for this type of study in man. A simple procedure based on determining plasma-11-OHc.s. values immediately before and again 15 minutes after the injection of nanogram amounts of synacthen would seem to offer a suitable means of assessing adrenal sensitivity. A similar procedure may also have value in the assay, for clinical use, of corticotrophin or its synthetic analogues. Available assays, such as the determination of adrenal ascorbic-acid depletion in the hypophysectomised rat, are far from ideal. We have found a significant discrepancy between the activity of synacthen and

synthetic porcine adrenocorticotrophic as determined by the Sayers’ assay (Sayers et al. 1948) and that based on

hormone

minimal-effective-dose Fig. 2-Mean (± S.D.) of plasma-11-OHc.s. increments 15 minutes after the administration of single intravenous injections of saline solution (X), or synacthen (0) to controls subjects.

studies

in

man

(unpublished observations). However, before the present procedure can be advanced for assay purposes many addi-

700

tional studies

are required. Thus it will be essential determine whether the earlier injections significantly influence the later responses; whether more reproducible responses can be obtained between individuals if the peptide is administered in amounts related to body-weight or body-surface area; whether an individual’s response varies from day to day; whether a longer interval should be left between each injection; and whether similar or

to

better results are obtained using a more specific method for determining plasma-corticosteroid values. Requests for reprints should be addressed to J. L. REFERENCES H. T., Landon, J. (1964) in The Structure and Function of Corticosteroids (edited by J. R. Pasqualini and M. F. Jayle); p. 55. London. Fraser, R. (1967) Mem. Soc. Endocr. 17, 141. Townsend, J., Fraser, R. (1967) J. Endocr. 37, xxviii. Landon, J., Livanou, D., Greenwood, F. C. (1967) Biochem. J. (in the press) Mattingly, D. (1962) J. clin. Path. 15, 374. Sayers, M. A., Sayers, G., Woodbury, L. A. (1948) Endocrinology, 42, 379. Stouffer, J. E., Lipscomb, H. S. (1963) ibid. 72, 91. Wood, J. B., Frankland, A. W., James, V. H. T., Landon, J. (1965) Lancet, i, 243.

James, V. —

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EFFECT OF DEAFFERENTATION ON HUMAN PHYSIOLOGICAL TREMOR C. D. MARSDEN M.B., M.Sc. Lond., M.R.C.P. LECTURER IN ST.

THOMAS’S SCHOOL,

Cantab., M.R.C.P.

REGISTRAR IN

MEDICINE,

HOSPITAL MEDICAL LONDON

J. C. MEADOWS M.B. ST.

NEUROLOGY, THOMAS’S HOSPITAL, LONDON S.E. 1

S.E.1

R. S. WATSON City, Grad.Inst.P.

G. W. LANGE Ph.D. Lond.

B.Sc.

OF THE AUTONOMICS

DIVISION, NATIONAL PHYSICAL LABORATORY, TEDDINGTON, MIDDLESEX

Physiological finger tremor has been studied in a patient whose right arm had previously been totally deafferented surgically. A welldefined 9.5 c.p.s. tremor peak was present in both the deafferented arm and the normal limb, indicating that physiological tremor arises independently of sensory feedback and that the tremor peak is not solely the result of inherent delay in operation of the stretch-reflex mechanism, as has been suggested. Peripheral &bgr;-adrenergic stimulation also increased tremor of the deafferented limb, indicating that adrenaline can augment physiological tremor independently of an action on muscle spindles. Summary

Introduction

WHENEVER

needle,

we

hold a full glass or thread a that our hands shake. The mech-

attempt

we are aware

to

anisms underlying physiological tremor are uncertain, and its relation to pathological tremors such as that of Parkinson’s disease is unknown. Records of the movement of the outstretched fingertip reveal a complex waveform (fig. 1). Frequency analysis shows this to include oscillations at all frequencies up to as high as 50 cycles

per second (c.p.s.), with a well-defined peak between 7 and 12 c.p.s. (fig. 2) (Halliday and Redfearn 1956). In a task where the oscillation of tremor is near-isotonic, as in holding the hand outstretched, this peak may account for as much as 40% of the total fluctuation of velocity between 1 and 20 c.p.s. in maintaining the posture. The 7-12 c.p.s. tremor peak has attracted much attention, for it may represent oscillation in a particular neuronal pathway concerned with the regulation of muscle contraction. Some workers hold that the tremor peak around 9 c.p.s. is the result of inherent delay in operation of the muscle length servo-loop or stretch-reflex mechanism (Halliday and Redfearn 1956, Hammond et al. 1956, Lippold et al. 1957). Thus fluctuations in muscle length evoke muscle-spindle discharge which reflexly elicits muscle contraction in an attempt to restore the extrafusal

fibre to its previous length. However, a time-delay must exist between the initial change in muscle length and the reflexly evoked compensatory contraction. This time-lag comprises delay due to the response of the spindles, conduction in afferent and efferent nerve-fibres, synaptic and neuromuscular transmission, and muscle contraction. The sum total of these delays has been calculated to be approximately 110 milliseconds, thus generating a potential 9 c.p.s. oscillation in muscle contraction. The key role of the muscle spindle in this theory is apparent. Tremor commonly increases under conditions of emotional, mental, and physical stress. Release of catecholamines may be responsible, for adrenaline increases the amplitude of physiological tremor by stimulation of peripheral (3-adrenergic receptors (Marsden et al. 1967). The site of action of adrenaline in this response has yet to be defined; one possibility is that adrenaline has a direct effect upon the muscle spindle (Paintal 1959, Calma and Kidd 1962). We have examined the role of the muscle spindle in physiological tremor in a patient with a totally deafferented arm.

Case-report A 51-year-old woman underwent a right upper lobectomy for a peripheral alveolar-cell carcinoma of the lung in 1966. Increasingly severe pain in the right arm, associated with numbness and some weakness of the hand, led to her admission to the Maida Vale Hospital in July, 1967, for consideration of palliative surgery. Examination then revealed a right Horner’s syndrome and neurological abnormalities in the right arm, consisting of weakness of the small muscles of the hand and finger flexors, diminished sensation, and loss of reflexes in the distribution of the c7, 8 and Tl roots. A right cervical hemi-

laminectomy

was

performed

on

July

14

(Mr. Lindsay Symon)

which revealed extradural tumour, and infiltration of the c8 anterior and posterior roots extending into the cervical cord. All posterior roots from c5 to T2 inclusive and the c8 anterior root were divided surgically. The patient consented to the tests 2 weeks later, after an uneventful postoperative recovery. Examination then revealed total loss of all forms of sensation up to but not including the shoulder on the right, absent reflexes in the right arm, considerable weakness of grip and finger flexion, with less marked weakness of finger extension. In addition there was impaired appreciation of pinprick in the left leg; the subsequent course indicated that this was due to early cervical-cord compression.

Methods

Fig. 1-3-second records

of tremor of both hands.

Tremor was detected in both hands by accelerometers (Ether BLA 2, weight 2-25 g.) strapped over the dorsal surfaces of the terminal interphalangeal joints of the index fingers. In all experiments, tremor was recorded with the patient’s wrists and fingers actively extended and the arm fully supported to the wrist. Owing to the patient’s motor deficit, it