Clinical Endocrinology ( 1 979) 11,533-539.
THE CIRCADIAN VARIATION OF IMMUNOREACTIVE LIPOTROPHIN AND ITS RELATIONSHIP TO ACTH AND GROWTH HORMONE IN MAN P. E. M U L L E N , W. J . J E F F C O A T E , C . LINSELL, R. H O W A R D A N D LESLEY H . R E E S The Institute of Psychiatry, De Crespigny Park, London, SE.5 8.4F and the Departments of Endocrinology and Chemical Pathology, St. Bartholomew’s Hospital. London, EClA 7BE (Rereitled 29 September 1978: revised 9 Junuur-v 1979: accepted 25 Mqv 1979)
SUMMARY
There is a clear circadian rhythm of plasma immunoreactive LPH in man with the trough occurring between 22.00 h and 03.00 h and the peak between 07.00 h and 08.00 h, immediately after waking. At all times circulating LPH and ACTH levels follow each other closely. However, no correlation was observed between LPH levels and either plasma G H or the stage of sleep. Beta-lipotrophin (8-LPH) was chemically isolated from pituitaries in 1964 (Li, 1964). The fat mobilizing properties from which it derived its name were rapidly shown to be a non-specific effect, probably of little physiological significance. In 1974 studies in man revealed that the immunoreactivity in human plasma samples and pituitary tissue, previously attributed to P-melanocyte stimulating hormone (/?-MSH),was in fact largely due to the presence of human P-LPH (Ph-LPH) (Bloomfield et al., 1974; Lowry & Scott, 1975). The further discovery of the opioid peptides with their structural relationship to 8-LPH (Hughes etal., 1975; Li & Chung, 1976; Ling et al., 1976) has raised the possibility that P-LPH is a precursor molecule for the body’s ‘natural analgesic’ (Jacquet & Marks, 1976; Guillemin, 1976; British Medical Journal, 1978). ACTH and P-LPH are reported to be present in the same cells of the pituitary (Moriarty, 1973; Phifer et al., 1974; Pelletier et al., 1977)and the two appear to be released together following acute stress (Krieger et al., 1977, Jeffcoate et al., 1978). Studies on immunoreactive human P-MSH (‘Ph-MSH’) also suggested a correlation with ACTH in human plasma under a variety of conditions (Abe et al., 1969). Tanaka and his colleagues recently reported the concentrations of ‘Ph-MSH’ in three normal subjects and showed a diurnal variation with highest values at about the time of waking and lowest values shortly after retiring to bed (Tanaka et al., 1978). The present work wasdesigned to study circadian variation in plasma LPH levels under Correspondence: Dr. P. E. Mullen, Institute of Psychiatry, De Crespigny Park, London SE5 A F 0300-0664/79/1 100-0533$02.00
0 1979 Blackwell Scientific Publications 533
S 34
P . E. Mullen el al.
resting, non-stressed conditions using an N-terminal LPH radioimmunoassay which shows cross-reaction with y-LPH but no cross-reaction with ‘Ph-MSH’ (Jeffcoate et a/., 1978). METHODS Suhjccrs The subjects were all normal volunteers with no history of significant physical or psychiatric disease and who were not receiving any medication. There were five males and one female ranging in age from 21 years to 33 years. They were admitted to a metabolic ward equipped with a sleep laboratory for the study. The first 24 h was spent acclimatizing to the ward and sleep laboratory. The subjects were fed at 07.00, 12.00, 15.00 and 18.00 h with no restriction on intake but no additional snacks. Alcohol was forbidden and no caffeine containing beverages were permitted after 19.00 h. During the day the subjects were encouraged to maintain a normal degree of physical activity. N o sleeping was permitted during the day. The study commenced on the second day with an indwelling intravenous catheter being introduced at either 09.00 h or 21 .OO h . The patency of the catheter was maintained by a slow infusion of heparin saline delivered by a syringe strapped to the arm during the day and via a drip at night. The volume of the infusion over the 24 h varied between 300 and 500 ml. Blood sampling at night was accomplished from the control room via a 7 foot low-dead-space extension tube, thus leaving the subject undisturbed. The subjects entered the sleep laboratory at approximately 22.30 h and lights were extinguished at 23.00 h and turned on again at 07.00 h. One subject who was sleep deprived had intensive sampling performed between 05.00 and 07.15 h for comparison with the sleeping subjects.
Sample handling Between twenty-four and thirty samples were taken during the 24 h, constituting a total blood loss of between 200 and 240 ml. The sampling schedule was varied to some extent in response to changes in sleep stage with the objective of obtaining samples during each phase of sleep. On each occasion 8 ml blood was taken into a heparinized plastic tube. The blood was centrifuged immediately at 2500 rpm for 10 min at room temperature, and the plasma separated and frozen in contact with dry ice. Samples were kept frozen at - 20‘C. until required for assay. Staging of sleep The electroencephalogram (EEG), electromyogram (EMC) and electro-oculogram (EOG) were recorded throughout the night. The resulting sleep records were scored visually according to the criteria of Rechtschaffen & Kales (1968). Three independent raters scored the records and, on an epoch by epoch comparison, they had interrater reliabilities between 85% and 93% with a correlation for all three raters given by Kappa of 0.82 with an error of 0.01 (Cohen, 1960). Radio immunoassay LPH was measured using an extracted, homologous radioimmunoassay using purified human fl-LPH (Dr. P. J . Lowry) as standard and tracer. The antiserum employed is directed towards the amino-terminal and shows cross-reaction with yh-LPH, but none
Circadian Liar ia t ion of lip0 rropk in
535
with ‘Ph-MSH’, purified human ACTH, a-MSH, methionine enkephalin or purified &endorphin or any other pituitary peptide or glycoprotein hormone. Full assay details have been published elsewhere (Jeffcoate et al., 1978). The ACTH radioimmunoassay employs an amino-terminal antiserum which has no cross-reaction with p-LPH related peptides and has been published in detail elsewhere (Rees et a l . , 1971). Growth hormone (GH) was assayed by conventional homologous double-antibody radioimmunoassay, the antiserum having been raised in a rabbit against purified human G H . RESULTS Secretory pattern of N-terminal LPH irnniunoreactiait?) The six subjects all showed their highest values of the day between 07.00 h and 08.00 h, levels fluctuating throughout the day (Fig. 1 ) with an apparent circadian periodicity. To establish whether the observed fluctuations reflected a true circadian variation a two-way analysis of variance between individuals and between hourly time periods was undertaken. Between individuals the F value was 30.8 with degrees of freedom of 5 and 1 15, which is significant (P
THE CIRCADIAN VARIATION OF IMMUNOREACTIVE LIPOTROPHIN AND ITS RELATIONSHIP TO ACTH AND GROWTH HORMONE IN MAN P. E. M U L L E N , W. J . J E F F C O A T E , C . LINSELL, R. H O W A R D A N D LESLEY H . R E E S The Institute of Psychiatry, De Crespigny Park, London, SE.5 8.4F and the Departments of Endocrinology and Chemical Pathology, St. Bartholomew’s Hospital. London, EClA 7BE (Rereitled 29 September 1978: revised 9 Junuur-v 1979: accepted 25 Mqv 1979)
SUMMARY
There is a clear circadian rhythm of plasma immunoreactive LPH in man with the trough occurring between 22.00 h and 03.00 h and the peak between 07.00 h and 08.00 h, immediately after waking. At all times circulating LPH and ACTH levels follow each other closely. However, no correlation was observed between LPH levels and either plasma G H or the stage of sleep. Beta-lipotrophin (8-LPH) was chemically isolated from pituitaries in 1964 (Li, 1964). The fat mobilizing properties from which it derived its name were rapidly shown to be a non-specific effect, probably of little physiological significance. In 1974 studies in man revealed that the immunoreactivity in human plasma samples and pituitary tissue, previously attributed to P-melanocyte stimulating hormone (/?-MSH),was in fact largely due to the presence of human P-LPH (Ph-LPH) (Bloomfield et al., 1974; Lowry & Scott, 1975). The further discovery of the opioid peptides with their structural relationship to 8-LPH (Hughes etal., 1975; Li & Chung, 1976; Ling et al., 1976) has raised the possibility that P-LPH is a precursor molecule for the body’s ‘natural analgesic’ (Jacquet & Marks, 1976; Guillemin, 1976; British Medical Journal, 1978). ACTH and P-LPH are reported to be present in the same cells of the pituitary (Moriarty, 1973; Phifer et al., 1974; Pelletier et al., 1977)and the two appear to be released together following acute stress (Krieger et al., 1977, Jeffcoate et al., 1978). Studies on immunoreactive human P-MSH (‘Ph-MSH’) also suggested a correlation with ACTH in human plasma under a variety of conditions (Abe et al., 1969). Tanaka and his colleagues recently reported the concentrations of ‘Ph-MSH’ in three normal subjects and showed a diurnal variation with highest values at about the time of waking and lowest values shortly after retiring to bed (Tanaka et al., 1978). The present work wasdesigned to study circadian variation in plasma LPH levels under Correspondence: Dr. P. E. Mullen, Institute of Psychiatry, De Crespigny Park, London SE5 A F 0300-0664/79/1 100-0533$02.00
0 1979 Blackwell Scientific Publications 533
S 34
P . E. Mullen el al.
resting, non-stressed conditions using an N-terminal LPH radioimmunoassay which shows cross-reaction with y-LPH but no cross-reaction with ‘Ph-MSH’ (Jeffcoate et a/., 1978). METHODS Suhjccrs The subjects were all normal volunteers with no history of significant physical or psychiatric disease and who were not receiving any medication. There were five males and one female ranging in age from 21 years to 33 years. They were admitted to a metabolic ward equipped with a sleep laboratory for the study. The first 24 h was spent acclimatizing to the ward and sleep laboratory. The subjects were fed at 07.00, 12.00, 15.00 and 18.00 h with no restriction on intake but no additional snacks. Alcohol was forbidden and no caffeine containing beverages were permitted after 19.00 h. During the day the subjects were encouraged to maintain a normal degree of physical activity. N o sleeping was permitted during the day. The study commenced on the second day with an indwelling intravenous catheter being introduced at either 09.00 h or 21 .OO h . The patency of the catheter was maintained by a slow infusion of heparin saline delivered by a syringe strapped to the arm during the day and via a drip at night. The volume of the infusion over the 24 h varied between 300 and 500 ml. Blood sampling at night was accomplished from the control room via a 7 foot low-dead-space extension tube, thus leaving the subject undisturbed. The subjects entered the sleep laboratory at approximately 22.30 h and lights were extinguished at 23.00 h and turned on again at 07.00 h. One subject who was sleep deprived had intensive sampling performed between 05.00 and 07.15 h for comparison with the sleeping subjects.
Sample handling Between twenty-four and thirty samples were taken during the 24 h, constituting a total blood loss of between 200 and 240 ml. The sampling schedule was varied to some extent in response to changes in sleep stage with the objective of obtaining samples during each phase of sleep. On each occasion 8 ml blood was taken into a heparinized plastic tube. The blood was centrifuged immediately at 2500 rpm for 10 min at room temperature, and the plasma separated and frozen in contact with dry ice. Samples were kept frozen at - 20‘C. until required for assay. Staging of sleep The electroencephalogram (EEG), electromyogram (EMC) and electro-oculogram (EOG) were recorded throughout the night. The resulting sleep records were scored visually according to the criteria of Rechtschaffen & Kales (1968). Three independent raters scored the records and, on an epoch by epoch comparison, they had interrater reliabilities between 85% and 93% with a correlation for all three raters given by Kappa of 0.82 with an error of 0.01 (Cohen, 1960). Radio immunoassay LPH was measured using an extracted, homologous radioimmunoassay using purified human fl-LPH (Dr. P. J . Lowry) as standard and tracer. The antiserum employed is directed towards the amino-terminal and shows cross-reaction with yh-LPH, but none
Circadian Liar ia t ion of lip0 rropk in
535
with ‘Ph-MSH’, purified human ACTH, a-MSH, methionine enkephalin or purified &endorphin or any other pituitary peptide or glycoprotein hormone. Full assay details have been published elsewhere (Jeffcoate et al., 1978). The ACTH radioimmunoassay employs an amino-terminal antiserum which has no cross-reaction with p-LPH related peptides and has been published in detail elsewhere (Rees et a l . , 1971). Growth hormone (GH) was assayed by conventional homologous double-antibody radioimmunoassay, the antiserum having been raised in a rabbit against purified human G H . RESULTS Secretory pattern of N-terminal LPH irnniunoreactiait?) The six subjects all showed their highest values of the day between 07.00 h and 08.00 h, levels fluctuating throughout the day (Fig. 1 ) with an apparent circadian periodicity. To establish whether the observed fluctuations reflected a true circadian variation a two-way analysis of variance between individuals and between hourly time periods was undertaken. Between individuals the F value was 30.8 with degrees of freedom of 5 and 1 15, which is significant (P
