ClinicalEndocrinology (1977) 6,291-298.

NORMAL PLASMA CALCITONIN: CIRCADIAN VARIATION AND RESPONSE TO STIMULI CARMEL J . HILLYARD, T . J . C. COOKE,* R . C. COOMBES,f IMOGEN M. A. EVANS A N D I. MACJNTYRE

Endocrine Unit and “Department of Surgery, Royal Postgraduate Medical School, London (Received 1.5 September 1976; revised 2 November 19 76; accepted 6 November 1976) SUMMARY

A simple, reproducible extraction method has been developed, which is capable of measuring calcitonin in normal individuals. Normal calcitonin levels show a circadian variation, with a peak around midday and respond to known stimuli for calcitonin release. Physiological studies on the role of calcitonin in normal subjects have been severely limited by insensitive assay methods and by the very low levels of circulating calcitonin. Although some workers (Tashjian et al., 1970) had previously reported the presence of circulating immunoreactive calcitonin in normal adults, such findings were not confirmed, and it is now generally agreed (Potts et al., 1972) that calcitonin levels are less than 0.1 pg/l in plasma or serum from normal adults. Until recently, therefore, it was only possible to measure calcitonin in disease states, such as medullary thyroid carcinoma (Clark et al., 1969; Tashjian et al., 1970; Deftos et al., 1971; Sizemore etal., 1973; Samaan et al., 1973; Milhaud et al., 1974) or other cancers (Silva et al., 1974; Coombes et al., 1974). We have now developed an extraction method which is capable of measuring calcitonin levels in most normal subjects, and in this paper we report the variation in plasma calcitonin throughout the day and the response to stimuli. PATIENTS AND METHODS

Sampling. 30 ml fasting blood was taken from patients with hypercalcaemia and Paget’s disease or from normal volunteers and placed in an ice-cold heparinized tube which was then centrifuged in a refrigerated centrifuge and the plasma immediately separated and frozen. Extraction method. 100 mg Spherosil XOA 400 (JJ’s (Chromatography) Ltd) beads and 2 ml 1 M HCl were added to 10 ml plasma. The container was then capped and shaken for 30 min, centrifuged and the supernatant removed by aspiration and discarded. The beads were then washed first with 2 ml 1 M H a , then with 2 ml distilled water. Calcitonin was eluted by shaking the beads in 2 ml 70% acetone in water for 30 min. The eluate was separated from the beads using a ‘Sinta’ funnel (Gallenkamp) under vacuum and mixed with 5 ml distilled water, frozen and lyophilized. Plasma extracts were then dissolved in 0.1 ml 0.1 M HCOOH and 0.4 ml 0.05 M phosphate buffer. Standard amounts of synthetic human calci-

t Present address: Royal Marsden Hospital, Sutton, Surrey. Correspondence: Professor I. MacIntyre, Royal Postgraduate Medical School, Ducane Road, London W12 OHS. 29 1

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Carmel J. Hillyard e t al.

tonin (MRC ref 70/50) were added to 10 ml aliquots of calcitonin-free plasma (obtained from athyroid patients and subjected to prior extraction with Spherosil or charcoal) and taken through the extraction procedure to provide a standard curve. Calcitonin assay. The radioimmunoassay for calcitonin was similar to that previously reported (Coombes et al., 1974) except that the samples were double-diluted in phosphate buffer containing 0.3% human serum albumin (Lister Institute) to prevent calcitonin adhering to the plastic tubes, as no unextracted plasma was present in the system. Circadian variation. Blood was taken from two volunteers throughout the worlung day and a further two volunteers gave blood samples from 08.00 until 24.00 hours. Normal range. Blood was taken from twenty-one fasting volunteers at 09.00 hours and a further sample was taken between 12.00 and 13.00 hours, after most subjects had eaten a meal. Calcium infisions. Calcium was given as calcium gluconate in isotonic saline, 5 mg kg-’ h-’ over 3 h. Blood samples were collected before and every 30 min during the infusion. Whisky test. Blood was taken from five volunteers before and at 5 and 15 min after administration of 50 ml whisky (Johnny Walker) orally (Dymling et al., 1976). In a second experiment, blood was taken from four volunteers before and 10 min after ingestion of 50 ml whisky. Reproducibility experiments. Ten aliquots of the same plasma sample were extracted and assayed in one assay. One aliquot of this was included in each subsequent assay.

L I I I I 3.125 6.25 12.5 25 50 Volume of extract ( P I ) I

I

8

16

I I 1 1 63 125 250 32 Colcitonin ( p g / t u b e )

‘ 0 I 100

I I 500 1000

Fig. 1. Comparison of displacement curves for unextracted ( 0 ) and extracted thetic calcitonin. Also shown are the curves for extracted blank (athyroid) (0)

plasma.

(A)

(m)

human synand normal

Normal plasma calcitonin

29 3

Degradation of plasma calcitonin. Blood from one normal subject was divided into four aliquots. One aliquot was centrifuged and frozen immediately and the others were left at room temperature for 30 min, 90 min and overnight before the plasma was frozen. RESULTS

Calcitonin assay (Fig. 1). The extracted standards gave similar curves t o the unextracted calcitonin with a recovery of between 75%and 85%. Circadian variation (Fig. 2). Circulating calcitonin levels rose during the morning t o a peak after lunch in all four subjects. Lunch

Milk

Lunch

100 -

q

-

90

-

80

-

70

m C

-

60

-

.{-

50

-

o

40 -

.-

c

0

5

5

30 -

Fig. 2. Calcitonin levels throughout the day in four normal subjects: - - - -,male; --, female. The evening meal in the two subjects studied until midnight commenced around 22.30 hours.

Normal range (Fig. 3 , Table 1). In twelve subjects, fasting plasma calcitonin levels were undetectable (

Normal plasma calcitonin: circadian variation and response to stimuli.

ClinicalEndocrinology (1977) 6,291-298. NORMAL PLASMA CALCITONIN: CIRCADIAN VARIATION AND RESPONSE TO STIMULI CARMEL J . HILLYARD, T . J . C. COOKE,*...
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