Life Sciences, Vol. Printed in the USA
51, pp.
143-149
Pergamon
Press
LACK OF DIURNAL RHYTHM IN PLASMA ATRIAL NATRIURETIC PEPTIDE M Follenlus, G Brandenberger and J Sam1 Laboratolre de Physmlogie et de Psychologm Envlronnementales CNRS/INRS, 21 rue Becquerel, 67087 Strasbourg Cedex, France (Received
an final
form May 6, 1992)
Summary The occurrence of a circadian rhythm in the concentratmn of circulating atrial natnuretlc peptide (ANP) IS not clearly estabhshed To investigate dmrnal changes, plasma levels of ANP were measured at 10-mm intervals for 24 hours in six normal volunteers The subjects were studied once during a normal sleep-wake cycle and once during a cycle with a shifted sleep period They received continuous enteral nutrition from 8 hours preceding the experiment until the end of the experiment, throughout this time the subjects remained in a supine position The mean ANP levels did not differ slgmficantly between the sleep periods and the periods spent awake in either of the protocols, which provides evidence of a lack of a sleep-related influence of ANP A slgmflcant llneanty of the mean ANP profile was observed, smoothing out the transient and randomly occurnng fluctuations In individual ANP concentration These results lead to the conclusion that ANP secretion is neither under the control of endogenous circadian rhythmicity nor is it affected by sleep-regulatory mechanisms Circadian and ultradlan rhythmmlty and the alternance of wakefulness and sleep are known to interact m modulating the overall dmrnal vananon of pltmtary and pituitary-dependent hormonal secretion (1) Whether these factors influence also the 24-hour profile of plasma atrial natriuretm pepnde (ANP) is not clear The existence of a circadian rhythm in the plasma concentration of the ANP is still matter of controversy A dmrnal vananon m plasma ANP has been described in some studies of young healthy volunteers (2-8) and in hypertensives (7), whereas it has been found to be age-dependent m hospitalized elderly patients (9) No or at least no major circadian variations have been described by other authors (10-12) The time of ANP peak values differed greatly between the studms The different results reported may well reflect different experimental condmons (posture, activity, diet and blood sampling) All these studms have been performed during a normal sleep-wake cycle and to our knowledge no information is available concerning the influence of sleep itself on the circulating ANP level The aim of this investigation was to determine, more precisely, the presence or absence of an intrinsic circadian rhythm in plasma ANP concentration under standardized condmons Supine subjects were studied during a normal sleep-wake cycle under continuous enteral nutrition with 10-rain blood sampling for ANP measurements The relationship with sleep was investigated under the basal conditions and also when the sleep period was shifted to dissocxate eventual interactions between sleep and the circadian rhythm Copyraght
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All raghts reserved.
144
L a c k of D i u r n a l R h y t h m in A N P
Vol.
51, No.
2, 1992
Methods
Subjects and procedure Six male volunteers, aged 20-26 years participated in this study They all had normal routines of work, meals and sleep, had no evidence of any disease and were not taking any me&cation All subjects gave their informed written consent and the study was approved by the Strasbourg Hospital Ethics Committee For the experiments subjects remained supine, even for carrying out bodily functions, from 1500 h on day 1 until 2300 h on day 2 The studies were conducted m a sound-proof airconditioned sleep chamber Before the subjects entered the chamber a catheter was inserted Into an antecubltal vein and was kept patent with a heparln containing sahne solution To avoid the influence of repeated meal and fired retake continuous enteral nutrition was exclusively administered This began 8 hours before blood sampling (SONDALIS, ISO, Sopharga, Puteaux, France 50% carbohydrate, 35% fat, 15% protean, 1 Cal.ml-1 and 90 ml.h-a) The subJeCts underwent two randomized studies with a one to two month interval between them Each subject was studied once during a normal 24 h sleep-wake cycle with nocturnal sleep from 2300 to 700 h and once during a 24 h cycle with an 8 hour shift of the sleep period (from 700 to 1500 h) Uninterrupted polygraphic sleep recordings were performed and sleep stages scored according to established criteria (13) When awake, the subjects read, hstened to music and watched television
Blood sampling and plasma sample measurements Blood was removed continuously using a peristaltic pump It was collected an an adjoining room at 10 mxn lnter'~als from 2300 h (day 1) to 2300 h (day 2) and sampled an tubes containing aprotman (500 KIU.ml-1) and EDTA (1 mg.ml-1) Samples were immediately centrifuged at 4°C and the plasma stored at - 2 5 ° C The maximum volume of blood removed averaged 200 ml for each 24 h cycle An equivalent volume of heparmlzed saline solution (500 IU heparln.ml-1 of 0 9 g NaCI.100 ml-1) was infused at the rate of blood removal After extraction on Sep-Pak C - 1 8 cartridges (Waters Associates) plasma ANP was measured by radlolmmunoassay using kits from Amersham (Buckinghamshire, England) with an adaptation for small plasma samples The detection limit was 12 pg.ml-1 The lntra-assay coefficient of ,~arlatlon (CV) for the duphcates assayed was 8% above 50 pg.ml-1 and 12% for levels less than 50 pg.ml-1 All samples from a given night were analysed m the same assay
Data analysis To determine the overall trend of the 24 h ANP profiles polynomial regression analysis was performed for 1, 2 and 3 degrees Mean levels for the sleep period and the period spent awake were compared by paired t-tests and a binomial test was used for assessing group significance Transient increases In ANP levels were identified by taking into account the CV in the rele,,ant concentration range An increase was considered as slgmflcant when the concentration Increased by 2 CV Results The 24 h ANP profile for one representative subject, studied during a normal sleep-wake cycle, is galen an Fig 1 ANP fluctuated shghtly throughout the experimental period and 3 to 7
Vol.
51,
No.
2,
Lack of Diurnal Rhythm
1992
in ANP
145
slgmflcant oscdlatlons of the ANP concentration were found among subjects These were transient increases superimposed on a basal level appearing randomly throughout the 24 hours Their relative increment ranged from 33 to 111% Slmdar transient mcreases were found when the sleep period was shifted The number varied from 2 to 5 among the subjects and their relatwe increment ranged from 30 to 112% m this case
REM 1 2 3 4
z
51, pp.
143-149
Pergamon
Press
LACK OF DIURNAL RHYTHM IN PLASMA ATRIAL NATRIURETIC PEPTIDE M Follenlus, G Brandenberger and J Sam1 Laboratolre de Physmlogie et de Psychologm Envlronnementales CNRS/INRS, 21 rue Becquerel, 67087 Strasbourg Cedex, France (Received
an final
form May 6, 1992)
Summary The occurrence of a circadian rhythm in the concentratmn of circulating atrial natnuretlc peptide (ANP) IS not clearly estabhshed To investigate dmrnal changes, plasma levels of ANP were measured at 10-mm intervals for 24 hours in six normal volunteers The subjects were studied once during a normal sleep-wake cycle and once during a cycle with a shifted sleep period They received continuous enteral nutrition from 8 hours preceding the experiment until the end of the experiment, throughout this time the subjects remained in a supine position The mean ANP levels did not differ slgmficantly between the sleep periods and the periods spent awake in either of the protocols, which provides evidence of a lack of a sleep-related influence of ANP A slgmflcant llneanty of the mean ANP profile was observed, smoothing out the transient and randomly occurnng fluctuations In individual ANP concentration These results lead to the conclusion that ANP secretion is neither under the control of endogenous circadian rhythmicity nor is it affected by sleep-regulatory mechanisms Circadian and ultradlan rhythmmlty and the alternance of wakefulness and sleep are known to interact m modulating the overall dmrnal vananon of pltmtary and pituitary-dependent hormonal secretion (1) Whether these factors influence also the 24-hour profile of plasma atrial natriuretm pepnde (ANP) is not clear The existence of a circadian rhythm in the plasma concentration of the ANP is still matter of controversy A dmrnal vananon m plasma ANP has been described in some studies of young healthy volunteers (2-8) and in hypertensives (7), whereas it has been found to be age-dependent m hospitalized elderly patients (9) No or at least no major circadian variations have been described by other authors (10-12) The time of ANP peak values differed greatly between the studms The different results reported may well reflect different experimental condmons (posture, activity, diet and blood sampling) All these studms have been performed during a normal sleep-wake cycle and to our knowledge no information is available concerning the influence of sleep itself on the circulating ANP level The aim of this investigation was to determine, more precisely, the presence or absence of an intrinsic circadian rhythm in plasma ANP concentration under standardized condmons Supine subjects were studied during a normal sleep-wake cycle under continuous enteral nutrition with 10-rain blood sampling for ANP measurements The relationship with sleep was investigated under the basal conditions and also when the sleep period was shifted to dissocxate eventual interactions between sleep and the circadian rhythm Copyraght
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All raghts reserved.
144
L a c k of D i u r n a l R h y t h m in A N P
Vol.
51, No.
2, 1992
Methods
Subjects and procedure Six male volunteers, aged 20-26 years participated in this study They all had normal routines of work, meals and sleep, had no evidence of any disease and were not taking any me&cation All subjects gave their informed written consent and the study was approved by the Strasbourg Hospital Ethics Committee For the experiments subjects remained supine, even for carrying out bodily functions, from 1500 h on day 1 until 2300 h on day 2 The studies were conducted m a sound-proof airconditioned sleep chamber Before the subjects entered the chamber a catheter was inserted Into an antecubltal vein and was kept patent with a heparln containing sahne solution To avoid the influence of repeated meal and fired retake continuous enteral nutrition was exclusively administered This began 8 hours before blood sampling (SONDALIS, ISO, Sopharga, Puteaux, France 50% carbohydrate, 35% fat, 15% protean, 1 Cal.ml-1 and 90 ml.h-a) The subJeCts underwent two randomized studies with a one to two month interval between them Each subject was studied once during a normal 24 h sleep-wake cycle with nocturnal sleep from 2300 to 700 h and once during a 24 h cycle with an 8 hour shift of the sleep period (from 700 to 1500 h) Uninterrupted polygraphic sleep recordings were performed and sleep stages scored according to established criteria (13) When awake, the subjects read, hstened to music and watched television
Blood sampling and plasma sample measurements Blood was removed continuously using a peristaltic pump It was collected an an adjoining room at 10 mxn lnter'~als from 2300 h (day 1) to 2300 h (day 2) and sampled an tubes containing aprotman (500 KIU.ml-1) and EDTA (1 mg.ml-1) Samples were immediately centrifuged at 4°C and the plasma stored at - 2 5 ° C The maximum volume of blood removed averaged 200 ml for each 24 h cycle An equivalent volume of heparmlzed saline solution (500 IU heparln.ml-1 of 0 9 g NaCI.100 ml-1) was infused at the rate of blood removal After extraction on Sep-Pak C - 1 8 cartridges (Waters Associates) plasma ANP was measured by radlolmmunoassay using kits from Amersham (Buckinghamshire, England) with an adaptation for small plasma samples The detection limit was 12 pg.ml-1 The lntra-assay coefficient of ,~arlatlon (CV) for the duphcates assayed was 8% above 50 pg.ml-1 and 12% for levels less than 50 pg.ml-1 All samples from a given night were analysed m the same assay
Data analysis To determine the overall trend of the 24 h ANP profiles polynomial regression analysis was performed for 1, 2 and 3 degrees Mean levels for the sleep period and the period spent awake were compared by paired t-tests and a binomial test was used for assessing group significance Transient increases In ANP levels were identified by taking into account the CV in the rele,,ant concentration range An increase was considered as slgmflcant when the concentration Increased by 2 CV Results The 24 h ANP profile for one representative subject, studied during a normal sleep-wake cycle, is galen an Fig 1 ANP fluctuated shghtly throughout the experimental period and 3 to 7
Vol.
51,
No.
2,
Lack of Diurnal Rhythm
1992
in ANP
145
slgmflcant oscdlatlons of the ANP concentration were found among subjects These were transient increases superimposed on a basal level appearing randomly throughout the 24 hours Their relative increment ranged from 33 to 111% Slmdar transient mcreases were found when the sleep period was shifted The number varied from 2 to 5 among the subjects and their relatwe increment ranged from 30 to 112% m this case
REM 1 2 3 4
z
