Departments of Neurology and Clinical Chemistry, University Hospital, Uppsala, Sweden

THE PLASMA PROLACTIN LEVELS IN MAN DURING PROLONGATION OF DARKNESS IN THE MORNING

By P. O. Osterman and L. Wide

ABSTRACT

plasma prolactin levels were studied in 4 healthy subjects with a sleep-waking cycle and a constant activity schedule before and after experimental alteration of the dark-light cycle. The subjects slept in the dark from 10.30 p. m. to 6.30 a. m. During a control period of 10 days the subjects stayed in rooms which were well lighted after 6.30 a. m. During the following 10 days darkness was prolonged by 4 hours until 10.30 a. m. Blood samples were obtained at 10.30 p. m., and each hour from 2.30 to 11.30 a. m. on days 9 and 10 of each period of investigation. In all the subjects an increase of the plasma prolactin level occurred during sleep. At about the time of waking the concentration fell rapidly. There was no apparent shift in the waking-related decrease in the prolactin level during the experimental period with extended darkness. Present data do not indicate that the dark-light transition in the morning is essential for the waking-related decrease in the prolactin level. The

constant

Studies on young adults with a "normal" sleep-waking cycle have shown that the 24-h secretory pattern of prolactin is characterized by episodic secretion and increased plasma levels during sleep (Sassin et al. 1972; Parker et al. 1973). From the published data it is not possible to determine whether the sleep-related increase in plasma prolactin is dependent on sleep, or if other phenomena, such as darkness, which are normally associated with sleep, cause the increased secretion. Therefore, further studies, with shifts of the sleepwaking cycle and the light-dark patterns, are required to clarify this issue. In the present investigation the plasma prolactin levels were studied in 4 healthy subjects with a constant sleep-waking cycle and a constant activity schedule before and after prolonging of darkness by 4 h in the morning.

MATERIALS AND METHODS Two 20-year-old males (HEA and LA), one 20-year-old nulliparous female (EW) and her father (BW), aged 49, were studied. All were healthy. The female had normal menstrual cycles. Blood samples were obtained from her on days 8 and 18-19 respec¬ tively of the menstrual cycle. The subjects slept in the dark from 10.30 p. m. to 6.30 a. m. They were not allowed to sleep between 6.30 a. m. and 10.30 a. m. During a control period of 10 days the subjects stayed in rooms which were well lighted after 6.30 a. m. During the following 10 days darkness was prolonged by 4 h until 10.30 a. m. (experimental period with extended darkness). The same schedule of activity was adopted for the waking hours during the control period and the experimental period. The schedule included physical exercise on a bicycle ergometer. From 6 p. m. the subjects fasted until breakfast, which was served each morning at 8 a. m. Blood sampling took place on days 9 and 10 of each period of investigation. During the control period the plasma prolactin concentration was determined in subjects BW and EW only in blood samples taken on day 10. A flexible venous cannula was inserted into an antecubital vein. The cannula was connected to a 20 cm long plastic tube which was attached to a 3-way stopcock. The cannula and tube were filled with heparinized, isotonic saline between samples. Ten ml of blood was removed and run into heparinized tubes at 10.30 p.m., and each hour from 2.30 a.m. to 11.30 a.m. The blood sampling usually did not wake the subjects. Heavily filtered, shielded torch-light was used when blood samples were obtained in the dark. During the waking hours in the dark the subjects wore a black bandage over their eyes and kept them closed during the blood sampling procedure. Plasma was immediately separated in a refrigerated centrifuge and stored at -20°C until assayed. Further details about the experimental design are given in a recent report, which deals with the level of plasma 11-hydroxycorticosteroids in the same experiment

(Osterman 1974). Prolactin in plasma was measured radioimmunologically by the use of 125I-labelled human prolactin and rabbit anti-human prolactin antibodies coupled to CNBr-activated ultrafine Sephadex particles (Wide et al. 1967; Wide 1969). The prolactin and antiprolactin preparations were supplied by National Institute of Arthritis, Metabolism and Digestive Diseases, National Institute of Health, Bethesda, and the ultrafine Sephadex particles by Pharmacia AB, Uppsala. The sensitivity of the assay was about 1 ng/ml. The prolactin assay showed no cross-reaction with either follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone or growth hormone in physiological concentrations. The plasma samples were assayed in duplicate in 100 µ\ aliquots, and for each person all samples were run in the same assay. Statistical methods In each study the mean prolactin level was calculated as the mean of the eleven samples taken between 10.30 p.m. and 11.30 a.m. (Table 1). In the graphs (Figs. 1 and 2) the plasma prolactin concentrations were plotted as deviations from the mean level, in order to facilitate the observations of any differences in phase between the

different curves. In each subject the mean values were calculated for the two consecutive studies, in the control period, and in the experimental period with extended darkness. Thus, one combined curve for each subject and each adaptation period was determined.

The

mean

prolactin

Table 1. levels (ng/ml) calculated as the mean of eleven between 10.30 p.m. and 11.30 a.m.

Control

Subject Day BW EW HEA LA

4.4 14.8

9

taken

Experimental period

period Day

samples

with extended darkness 10

10.7 15.7 5.3 15.5

Day 7.2

8.4 4.5 13.3

9

Day

10

5.3 9.8 4.3 11.0

For subjects BW and EW the values of the single study in the control period were used. The average values for the four combined curves of each period of investigation were calculated for each sampling time (Table 2) and the average values for the control period were compared with those of the experimental period with extended darkness by Student's i-test for paired observations. The mean values for the two consecutive studies in each period of investigation were used in order to reduce the bias caused by the lack of one control study in subjects BW and EW. This also seemed justified, as in each subject there was only a small difference in the mean prolactin levels for the two consecutive studies in each

adaptation period (Table 1).

RESULTS

subjects an elevation of the plasma prolactin concentration occurred during sleep (Fig. 1). At about the time of waking the concentration fell rapidly. The time, (7 a. m.) corresponding to the points of intersection between the descending prolactin curves and the mean levels, was the same for both the control period and the period with extended darkness (Fig. 2). Thus, there was no apparent shift in the waking-related decrease in the prolactin level during the experimental period with extended darkness. In each subject there was only a small difference, in the mean prolactin levels for the two consecutive studies in each period of investigation. In no case did this difference exceed 2.3 ng/ml (Table 1). The mean levels for the same adaptation period, however, varied considerably between the subjects. The average prolactin level, calculated as the mean of the four combined curves of each period of investigation (Table 2) was significantly higher (P

The plasma prolactin levels in man during prolongation of darkness in the morning.

The plasma prolactin levels were studied in 4 healthy subjects with a constant sleep-waking cycle and a constant activity schedule before and after ex...
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