Daily Patterns of Secretion of Neurohypophysial Hormones in Man: Effect of Age Mary L

Experimental Physiology (1998), 83, 409-418 Printed in Great Britain

DAILY PATTERNS OF SECRETION OF NEUROHYPOPHYSIAL HORMONES IN MAN: EFFECT OF AGE MARY L. FORSLING*, H. MONTGOMERYt, D. HALPINtt, R. J. WINDLE AND D. F. TREACHERt Departments of Obstetrics and Gynaecology and t Medicine, United Medical and Dental Schools, St Thomas's Campus, Lambeth Palace Road, London SE] 7EH, UK (MANUSCRIPT RECEIVED 8 DECEMBER 1997, ACCEPTED 14 JANUARY 1998)

SUMMARY The neurohypophysial hormone vasopressin contributes to control of urine output and, while urine flow shows a clear daily rhythm, there has been debate as to whether this is true of neurohypophysial hormones. A study was performed on fifteen adult males, with a mean age of 25 years, over a 24 h period, nine blood samples being taken at regular intervals for the determination of neurohypophysial hormones and indices of fluid balance. Samples were taken via an indwelling cannula so that sleep was undisturbed. A daily variation in the plasma concentrations of oxytocin and vasopressin was demonstrated with concentrations reaching a nadir in the late afternoon. Concentrations of both hormones peaked at 02.00 h. Vasopressin concentrations were inversely correlated with packed cell volume, indicating that the altered hormone release was affecting fluid retention. Consistent with this was the observation that the relationship of plasma osmolality to vasopressin depended on the time of day. To determine the effect of ageing, a similar study was performed on nine healthy elderly subjects with a mean age of 70 years. The nocturnal peak of vasopressin was markedly attenuated, while oxytocin release was similar to that in the younger group. These observations confirm the existence of a daily rhythm in the plasma concentrations of neurohypophysial hormones and indicate that the amplitude of the vasopressin change decreases with age.

INTRODUCTION Changes in circulating vasopressin concentrations have obvious implications for fluid balance, but recent evidence suggests that oxytocin may also play a role in sodium balance in some species (Verbalis, Mangione & Stricker, 1991; Windle & Forsling, 1991). To understand pathological changes in fluid balance, as for example in nocturia, it is necessary to establish normal daily variations in circulating concentrations of vasopressin and possibly oxytocin and their relationship to changes in indices of fluid balance. Many of the circadian changes are predictive in nature serving to switch on a function of importance for anticipated demands, so that if vasopressin does contribute to the nocturnal fall in urine flow in man, as suggested by the experiments of Papper & Rosenbaum (1952), one would expect to see an increase in plasma vasopressin concentrations, unrelated to plasma osmolality, in the early part of the night.

t Present address: Department of Respiratory Medicine, Royal Devon and Exeter Hospital, Exeter EX2 3DN, UK. * Corresponding author: [email protected] 410 M. L. FORSLING AND OTHERS Daily rhythms have been shown in the levels of neurohypophysial hormones in the hypothalamus (Noto, Hashomoto, Doi, Nakajima & Kato, 1983), pituitary (Dyball, Forsling, Patterson & Peysner, 1988) and plasma of the rat (Mohring, Kohrs, Mohring, Petri, Homsy & Haack, 1978; Greeley, Morris, Eldridge & Kizer, 1982). The plasma concentrations of the hormones increase during the hours of daylight, falling again during the hours of darkness. A daily rhythm in the secretion of vasopressin has not been demonstrated in all species studied, and in the rat its demonstration depends on a number of factors, including the timing of the samples (Wells, Windle, Peysner & Forsling, 1993); there may also be a genetic component. A daily rhythm of vasopressin secretion with a nocturnal rise has been observed in humans by some authors (George et al. 1975; Linkola, Ylikahri, Fyhrquist & Wallenius, 1978) and has provided the rationale for investigation and treatment of enuresis (Rittig, Knudsen, Norgaard, Pedersen & Djurhus, 1989). Others have questioned whether neurohypophysial hormone secretion in man shows a daily rhythm (Katz, Smith, Lock & Loeffel, 1979; Richards et al. 1987; Forsling, 1993), while Drumer et al. (1987) noted a daily rhythm in plasma vasopressin, but they studied recumbent subjects and posture could affect the results, altering patterns seen in normally active subjects. There is a similar debate concerning plasma oxytocin concentrations in humans. Landgraf, Hacker & Buhl (1982) described a fall in plasma concentrations during the day, although Amico Tenicel, Johnstone & Robinson, 1983) were unable to confirm these observations. Data obtained from clinical studies and from studies in the rat clearly show that the ageing process is accompanied by alterations in the ability to regulate water excretion and there is also a change in the circadian pattern of urine production. The increased susceptibility of the aged animal or human to significant derangement of water balance could result from a number of factors including altered thirst sensation (Phillips, Johnston & Gray, 1993), altered synthesis and release of vasopressin and altered renal concentrating ability (Pavo et al. 1995). There is no loss of vasopressin-producing cells in the hypothalamic magnocellular nuclei in man (Van der Woude et al. 1995) and Helderman, Vestal, Rowe, Tobin, Andres & Robertson (1978) found no change in plasma vasopressin concentrations with ageing, although Frolkis, Golovchenkop, Medved & Frolkis (1982) report a progressive increase. The response to hypertonic saline (Raskind et al. 1995) or water loading appears to be unaffected by age (Crowe, Forsling, Rolls, Phillips, Ledingham & Smith, 1987), while the response to ortho- stasis is attenuated (Rowe, Minaker, Sparrow & Robertson, 1982). Ageing affects the circadian pattern of many functions, but no observations have been performed on this aspect of the neurohypophysial system. In order to determine whether neurohypophysial hormones show a daily rhythm under normal conditions, a study has now been performed on the daily pattern of neurohypophysial hormone release in a young group of men carrying out their normal daily activities, the changes being related to indices of fluid balance. The pattern of neurohypophysial hormone secretion was compared with that in a fully active, healthy group of elderly men.

METHODS Subjects Observations were performed on fifteen healthy male subjects in the age range 22-40 years (mean 25 years) and nine healthy male subjects in the age range 60-75 (mean 70 years). All subjects were normotensive with normal renal function, with no significant past or current medical history and on no regular medication. Each was studied over a single 24 h period. The subjects spent the 36 h prior to the DAILY RHYTHMS OF NEUROHYPOPHYSIAL HORMONES 411 study following their normal daily routine at the St Thomas' Hospital and during both this period and during the study the subjects abstained from alcohol, tobacco and vigorous exercise. The meal times were standardized, and the subjects kept a diary of their activities and fluid and food intakes and were weighed. At 16.30 h a forearm vein was cannulated and after about 30 min of recumbent rest 12 ml of blood were withdrawn through the cannula. Over the following 24 h, eight further blood samples were taken (seven in the elderly subjects), each after 30 min recumbency, for the determination of packed cell volume, plasma osmolality, sodium, potassium, vasopressin and oxytocin. All subjects went to bed at 23.30 h when the lights were switched off and rose at 08.00 h the following day when lighting was restored. The investigation was approved by the local Ethical Committee and all subjects gave their written informed consent. Analyses Plasma osmolality was determined by the method of freezing point depression (Digmatic osmometer model 3D; Advanced Instruments Inc., Needham Heights, MA, USA). Sodium concentration was measured using a flame photometer (410C; Corning, Halstead, Essex, UK). Packed cell volume was determined in duplicate using heparinized microhaematocrit tubes (Hawksley & Sons, Lancing, Sussex, UK). Plasma vasopressin concentration was determined by a radioimmunoassay after prior extraction (Forsling, 1985) with SepPak C18 cartridges (Water Associates Inc, Millford, MA, USA) using the First International Standard for vasopressin (77/50 1). The lower limit of detection was 0. 12 + 0-02 pmol F'. The intra-assay coefficient of variation was 7-7 % and the interassay coefficient of variation was 11.9 % for 2-5 pmol. Oxytocin was assayed against the Fourth International Standard (76/575) as described by Windle & Forsling (1993), with an intra-assay coefficient of variation of 4.1 % and an interassay coefficient of variation of 9-0 % for 2.0 pmol 1'. Statistical analysis Results are presented as the mean + S.E.M. and values were compared using repeated measures analysis of variance. Areas under the curve were calculated using the trapezium rule as described by Altman (1991) and values in the young and elderly compared using Student's unpaired t test. Regression analysis was performed to determine the correlation between parameters. A value of P < 0 05 was taken as significant.

RESULTS Fluid balance All subjects showed a similar pattern of fluid intake. In the younger group, osmolality exhibited a significant rise of 10 mosmol kg-' during the afternoon (P < 0.01) (Fig. 1). Plasma sodium concentrations peaked at the same time, although the increase was not statistically significant. Packed cell volume showed a progressive rise (P < 0-04) from 02.00 h reaching a peak at 13.00 h. The subjects were in overall fluid balance during the study, as judged by the body weight at the beginning and end of the study. The values for packed cell volume and plasma sodium in the elderly group were not significantly different from those in the younger group. In the elderly group, packed cell volume showed the same trend over the 24 h period as that in the younger group, with values increasing from 39. 1 + 2-2 % at 02.00 h to 43-3 ± 2-0 % at 12.00 h, but the change was not statistically significant; plasma sodium concentrations also showed no significant change. Plasma concentrations ofneurohypophysial hormones Plasma concentrations of the posterior pituitary hormones in the young group showed a clear pattern over the 24 h period studied, as shown in Figs 2 and 3. Analysis of variance showed an effect of time on both oxytocin (P <0.01) and vasopressin (P <0.05). Mean plasma vasopressin concentrations reached a peak of 3*1 + 1.0 pmol I-' at 02.00 h with a second peak of 2.2 ± 0*6 pmol 1F at 06.00 h. All subjects showed an increase between 24.00 412 M. L. FORSLING AND OTHERS

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Fig. 1. Plasma osmolality and packed cell volume over 24 h in a group of 15 young subjects. Values are given as means + S.E.M. and 0.200 h of approximately 40 % from the mean value. In addition, ten of the subjects showed a second peak before rising. Vasopressin concentrations fell progressively during the day to a nadir of 0-8 + 0.2 pmol 1F between 16.00 and 20.00 h. Analysis of variance showed no effect of time in the elderly and the area under the curve of 24-3 + 6-1 pmol h 1F1 in the young group was significantly greater than that of 12-8 + 1-65 pmol h 1F in the elderly group (P<0-05). DAILY RHYTHMS OF NEUROHYPOPHYSIAL HORMONES 413

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Fig. 2. Plasma vasopressin concentrations over 24 h in a group of 15 young subjects (A) and 9 elderly subjects (B). Values are given as means + S.E.M.

In the young group, oxytocin concentrations were lowest during the afternoon and evening, the nadir being 1.91 + 0.31 pmol 1-'. After midnight, concentrations rose steeply to a mean maximum 4-63 + 1-34 pmol F' at 02.00 h (Fig. 2). In contrast to the pattern of vasopressin secretion, two peaks were seen in only five subjects. Analysis of variance showed an effect of time. The profile of hormone release in the elderly group was the same and the concentrations reached were not significantly different in the two groups, neither was there any significant difference in the areas under the curve. 414 M. L. FORSLING AND OTHERS

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Fig. 3. Plasma oxytocin concentrations over 24 h in a group of 15 young subjects (A) and 9 elderly subjects (B). Values are given as means + S.E.M.

For both oxytocin and vasopressin there was variation in the magnitude and the timing of the increases, but the morning decrease in concentrations of both hormones occurred before the subjects rose or drank. If overall values for 24 h were taken, no significant correlation was seen between neurohypophysial hormone concentrations and osmolality or sodium concentrations, but a significant correlation was seen at individual time points apart from 14.00 and 06.00 h. However, vasopressin was inversely correlated to packed cell volume over the 24 h period (P < 0.05). DAILY RHYTHMS OF NEUROHYPOPHYSIAL HORMONES 415

DISCUSSION The results showed a clear pattern of vasopressin release over the 24 h in studies in the young group of subjects studied while in fluid balance and undergoing their normal activities. Plasma concentrations tended towards a maximum at midnight and fell to a minimum in the late afternoon. There was, however, some variation between individuals, with differences both in the magnitude and the timing of the peak concentration. Landgraf et al. (1982) also noted such variation between individuals, and Rubin, Poland, Ravesun, Gonin & Tower (1975) indicated that there was marked variation in the same individual over successive days and nights. Such variation in the pattern of release could account for the failure of some observers to detect a significant change in circulating concentrations of the hormone. A similar pattern of release was seen with oxytocin, suggesting that a similar mechanism may control the patterns of release of both neurohypophysial hormones. One of the main factors influencing vasopressin release is the osmolality of the plasma, and, in man, a linear relationship between plasma osmolality has been demonstrated by many authors (Baylis & Robertson, 1980). However, in the present studies, while a correlation was seen at individual time points, there was no overall correlation between plasma osmolality and vasopressin concentrations over 24 h. The neurohypophysial hormone concentrations fell before the subject awoke and took in fluid, and circulating concentrations of vasopressin also fell at a time when plasma osmolality was rising, suggesting that the altered vasopressin secretion was being reflected in changes in water retention rather than being driven by changes in plasma osmolality, an observation consistent with the inverse correlation of vasopressin and packed cell volume. A similar observation has been made in the rat; at individual time points vasopressin concentrations did show a direct correlation with plasma osmolality, but this was not true when all data for the 24 h cycle were taken as a whole (Windle, Forsling & Guzek, 1992). Uhlich, Weber, Groschel-Stewart & Ruschlau (1975) also reported diurnal variations in plasma concentrations independent of plasma osmolality. Furthermore, studies in the rat showed that the slope of the osmoregulatory line varies with the time of day (Windle & Forsling, 1995), suggesting that the pattern of vasopressin release is not purely governed by the level of hydration and the relationship to osmolality is dynamic and subject to several influences. The present measurements were performed by design on normally active subjects and not in an artificially constrained environment, so that the normal patterns of vasopressin release could be identified. Thus changes in posture could influence vasopressin release. However, samples were taken after 30 min recumbency and Richards et al. (1987) found that a rhythm was still present in fully recumbent subjects. Our protocol was similar to that of Rittig et al. (1989), who also found a nocturnal increase in plasma vasopressin concentrations. However, they only took two samples during the hours of darkness, so that no clear patterns or correlations could be discerned. Most biological functions are not static, but rhythmic, showing clear changes over 24 h. Such rhythms might be considered as a dynamic extension of the principle of homeostasis, serving to switch on or augment functions of immediate value and to switch off functions no longer required. In the case of vasopressin, the plasma concentrations rise around midnight before the effects of overnight dehydration would be seen. The ensuing enhanced water reabsorption by the kidney should prevent or reduce any increase in plasma osmolality or fall in plasma volume, which was the case. The highest concentrations observed during the night were not as great in the elderly group as in the younger subjects and not significantly higher than those found during waking hours. 416 M. L. FORSLING AND OTHERS

Age-related changes in daily rhythms of other endocrine sytems have been documented in a variety of species including man (Czeisler, Chiasera & Duffy, 1991) and show an age-related damping of the changes. The physiological mechanisms underlying the age-related changes in the circadian system are unknown, but studies in the rat suggest that they are related to a decrease in the ability of the hypothalamus to secrete catecholamines (Turek, Penev, Zhang, Van Reeth, Takahashi & Zee, 1995). The reduced vasopressin secretion seen during the night in elderly subjects would appear not to reflect their possibly lower level of physical activity, as exercise does not influence the diurnal pattern of release in the rat (Forsling, 1993). Nocturia is a frequent symptom in the elderly male, usually attributed to prostate pathology. However, 30 % of patients investigated by urologists do not have pathology to account for their nocturia. It has been suggested that certain of these patients may have obstructive sleep apnoea syndrome, where nocturnal polyuria is a frequently reported symptom (Whyte, Allen, Jeffery, Gould & Douglas, 1989). Both in this group and in those who have neither prostatic problems nor obstructive sleep apnoea, it may be that attenuation of the circadian rhythm of vasopressin, demonstrated in this study, could be responsible for the nocturia in otherwise healthy elderly males. The mechanism underlying this daily rhythm in the human is unknown, but studies in the rat suggest pineal involvement. Pinealectomy suppresses the rhythm of neurohypophysial hormone secretion in the rat (Forsling, Stoughton, Zhou, Kelestimur & Demaine, 1993), while administration of melatonin inhibits release of oxytocin and vasopressin from the rat hypothalamus (Yasin, Costa, Besser, Hucks, Grossman & Forsling, 1993) in vitro and in vivo (Bojanowska & Forsling, 1997). Since melatonin is released during the hours of darkness, this observation would be consistent with the observed fall in neurohypophysial hormone concentrations in the rat during this period. Melatonin release could also contribute to the altered responsiveness to an osmotic challenge over 24 h, since pinealectomy affects not only osmotically stimulated hormone release but also neuronal activation as indicated by Fos production (Windle, Luckman, Stoughton & Forsling, 1996). Thus, as for body temperature (Cagnacci, Elliot & Yen, 1992) and plasma cortisol, the daily rhythms of neurohypophysial hormones could depend on the underlying pattern of melatonin secretion influencing, inter alia, the activity of the suprachiasmatic nucleus. It has yet to be established if similar mechanisms play a role in the human, although if this were the case one would expect melatonin to have a stimulatory effect on vasopressin release. In the context of the observations in the elderly, it is well known that there is a decline in the amplitude of the melatonin rhythm with age (Iguchi, Kato &Ibayashi, 1982).

Financial support from The Wellcome Trust and The Special Trustees for St Thomas' Hospital is gratefully acknowledged. The authors would also like to acknowledge the assistance of Debbie Orbell.

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