Genetic variability in melatonin concentrations in ewes originates in its synthesis, not in its catabolism

Luis A. Zarazaga1, Benoît Malpaux1, Daniel Guillaume1, Loys Bodin2, and Philippe Chemineau1

1 Neuroendocrinologie Sexuelle, Institut National de la Recherche Agronomique Physiologie de la Reproduction, 37380 Nouzilly; and 2 Institut National de la Recherche Agronomique, Station d'Amélioration Gènetique des Animaux, 31326 Castanet Tolosan, France

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated whether the genetic difference in plasma melatonin concentration in ewes was due to differences in the synthesis pathway from the pineal gland or in the catabolism of the hormone. Two groups of ewes [9 low (L) and 10 high (H)] were selected according to the breeding value of their mean nighttime plasma melatonin concentrations estimated at winter and summer solstices. In response to an identical dose of melatonin administered intravenously at 9:00 AM, no differences between groups were observed for any of the kinetic parameters: clearance rate, steady-state volume of distribution, terminal half-lives, and mean residence times. In the second experiment, two series of frequent blood samples were performed, one in the middle of the dark phase with samples taken every 5 min, and the other over 24 h with hourly samples. Highly significant differences between groups in nocturnal melatonin production rate were observed (L: 25.7 ± 2.8 vs. H: 63.1 ± 8.9 µg · kg-1 · h-1, P < 0.01). Thus the genetic differences in plasma melatonin concentrations in ewes originate in the synthesis pathway of the melatonin from the pineal gland rather than from differences in the catabolism of the hormone.

pineal gland; pharmacokinetics; clearance; half-life

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IN ALL MAMMALIAN SPECIES, melatonin is secreted into the blood circulation by the pineal gland with a nycthemeral rhythm characterized by high concentrations at night and low or undetectable concentrations during the day (1). The nocturnal plasma melatonin concentration results from an equilibrium that reflects not only the synthesis and liberation of melatonin from the pineal gland into the general circulation but also the catabolism of the hormone. The majority of the current data suggests that melatonin diffuses from the gland into the circulation immediately after synthesis, with no discernible storage or release mechanisms (1). Pharmacokinetic parameters, which allow estimation of the catabolism of the hormone, have been shown to be dose independent in sheep (6), humans (12), and rats (9). The major route of catabolism in rodents (11), humans (10a), and ewes (6) is hepatic hydroxylation into 6-hydroxymelatonin followed by sulfate or glucuronide conjugation; the conjugated forms are then excreted in the urine.

It is well documented that nocturnal plasma melatonin concentrations vary greatly among individual ewes (1, 13, 14) but are highly repeatable within a particular ewe (4), and it was recently shown that its variations are under strong genetic control in sheep (22). However, the physiological mechanism that produces this genetic variation is not known. Thus the objective of the present study was to discriminate between two possible physiological mechanisms that might explain the genetic differences between animals in plasma melatonin concentrations: changes in the catabolic and/or synthesis pathways. The purposes of these investigations were to examine the kinetic degradation parameters of melatonin in animals differing in their melatonin blood levels and to measure the plasma melatonin concentrations during frequent samples performed in the middle of the dark phase and in a complete 24-h cycle, and then, on the basis of these measurements and the individual pharmacokinetic parameters calculated earlier, to assess the endogenous production rate of melatonin in the two groups of animals.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

General Procedures

Animals and photoperiodic treatments. The whole Ile-de-France flock from which the experimental ewes were obtained is a large flock of about 2,500 animals, which is divided into six different families. At regular intervals sires are purchased from various private external flocks and are introduced to prevent inbreeding and maintain genetic connections with the French national scheme of genetic improvement of the Ile-de-France breed.

This experiment was carried out with 19 adult ewes (3.05 ± 0.23 yr; 69.1 ± 1.6 kg) selected from 399 ewes that had been used in a previous experiment in which the breeding value (7) regarding the endogenous melatonin plasma concentration was determined at the June and December solstices (22). The experimental ewes were not selected on the basis of their own melatonin levels but from their breeding value regarding melatonin levels, which takes into account their own adjusted melatonin levels and those of the values observed in related animals (see explanations in Ref. 22). Groups of 9 (Low group) and 10 (High group) ewes were chosen on the basis of extreme difference in their breeding value for mean nocturnal plasma melatonin concentrations (low melatonin concentrations, L group: 157.2 ± 5.8 pg/ml; and high melatonin concentrations, H group: 747.0 ± 15.9 pg/ml). The 9 ewes of the L group were born from 9 different dams and from 4 different sires and belonged to 3 different families. The 10 ewes of the H group were born from 9 different dams and from 6 different sires and belonged to 4 families. Dams and sires were different between the L and the H groups.

The experiment began in January, when, at the latitude of France, day length was 8 h and 39 min (sunrise 7:41 AM, sunset 4:20 PM, local time). Ewes were maintained under natural photoperiod and were fed daily with hay, straw, and corn. They had free access to water and mineral licks.

Hormonal analysis. Plasma melatonin concentrations were assayed in duplicate aliquots of 100 µl of blood plasma by radioimmunoassay by use of the technique of Fraser et al. (8) with an antibody raised by Tillet et al. (20). The limit of detection of the assay was 4 pg/ml. The inter- and intra-assay coefficients of variation, estimated from plasma pools every 100 unknown samples, were 4.1 and 10.6%, respectively.

Experiment 1: Catabolism of Exogenous Melatonin

Melatonin administration and blood sampling. Melatonin was administered during the day (at 9:00 AM) by intravenous administration in a single bolus. Crystalline melatonin (10 mg; purity >98%; Sigma Chemical, St. Louis, MO) was dissolved in 5 ml of absolute ethanol. Two milliliters of this solution were diluted in 78 ml of saline solution to obtain a final concentration of 50 µg/ml.

This melatonin solution was then administered (9:00 AM = time 0) into the right jugular vein through a catheter (Intraflon, Vigon, Paris, France). The dose administered was individually adjusted to ewe body weight by 3 µg/kg body wt0.75. Blood samples were collected from the left jugular vein in heparinized tubes (sodium heparinate) through a sterilized Teflon catheter. Blood samples (~3 ml each) were collected at times -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 min and then every 5 min for a period of 4 h 20 min. Plasma was immediately separated by centrifugation and stored at -20°C until radioimmunoassay of melatonin.

Pharmacokinetic analysis. The pharmacokinetic analysis aims to estimate as precisely as possible different parameters that describe the catabolism of the hormone. These parameters were estimated for each ewe and then were used for the statistical comparison between the two groups (experiment 1) and for the individual estimation of instantaneous melatonin production rate from the measurement of plasma concentrations of endogenous secretion (experiment 2).

Plasma melatonin concentrations obtained after intravenous melatonin administration were fitted to a biexponential equation, which corresponds to a two-compartment open model with melatonin administration and elimination from a central compartment. The volume of the central compartment, the plasma melatonin clearance (CL), the area under the plasma melatonin curve (AUC), the mean residence time (MRT), the melatonin steady-state volume of distribution (Vss), and the terminal half-life (t1/2) were estimated for each ewe by use of successive equations as described in Toutain and Oukessou (21) and Guillaume et al. (10).

Experiment 2: Plasma Melatonin Concentrations and Endogenous Melatonin Production Rate During Frequent Samplings

In this experiment, the same 19 ewes were used. Two series of jugular blood samples (~3 ml each) were carried out: the first series (1-2 wk after experiment 1) was an intensive period of 3 h in the middle of the dark period (11:00 PM-2:00 AM) with samples obtained every 5 min. In the second series (8-9 wk after experiment 1), blood samples were collected hourly for 24 h. In both cases, samples were obtained by jugular venipuncture. During the hours of darkness, samples were collected under dim red light (<1 lux at 20 cm) with care taken to avoid any direct illumination of the eyes.

The production rate of melatonin during the night, as during the day, was calculated by a classical formula used by Guillaume et al. (10). The AUC was calculated by a regression model. For each animal, a melatonin elevation was defined as the interval between the first and last value preceding and following the dark period that exceeded the baselines by more than 3 standard deviations of those respective baselines (14). The baselines were defined as the mean of the daytime samples during the day before and the day after the dark period.

Statistical Analysis

Samples that were below the limit of detection of the assay were arbitrarily given a limit of detection (4 pg/ml of plasma) for the statistical analysis. Statistical analyses of data were performed using SUPERANOVA (Abacus Concepts, Berkeley, CA) except for daytime values of experiment 2 of the two groups, which were analyzed by a Mann-Whitney nonparametric test (Statview; Abacus Concepts, Berkeley, CA), because six ewes had values that fell below the limit of detection of the assay. Unless otherwise stated, data are presented as means ± SE.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Catabolism of Exogenous Melatonin

The semilogarithmic plots of melatonin concentrations before and after a bolus melatonin administration (3 µg/kg body wt0.75) for both groups are shown in Fig. 1. The intravenous injection resulted in a peak in plasma melatonin concentrations in the first sample after melatonin injection (1 min). About 3 h after intravenous injection, plasma melatonin concentrations were similar to those observed before melatonin injection. Significant differences between groups in melatonin concentrations were observed for the three samples before melatonin injection (L: 5.6 ± 0.1; H: 12.8 ± 0.9 pg/ml, P < 0.05). Melatonin concentration in the first sample 1 min after injection was significantly higher in ewes of the H group (L: 2,980.5 ± 198.5; H: 3,728.3 ± 214.5 pg/ml, P < 0.05). No differences were observed afterward, during the 156 min after injection, until melatonin concentration became similar to concentrations before injection. In the last samples of the series, higher melatonin concentrations were observed in the H group (P < 0.05).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Semilogarithmic plots of plasma melatonin concentrations (means ± SE) in groups of Ile-de-France ewes (Low, n = 9, and High, n = 10) after administration of an iv bolus of melatonin (3 µg/kg body wt0.75). Dashed line, limit of detection of assay.

The mean kinetic parameters for both groups are given in Table 1. No differences between groups were observed for major kinetic parameters: CL (L: 1.99 ± 0.14; H: 1.99 ± 0.13 l · kg-1 · h-1), the Vss (L: 0.67 ± 0.06; H: 0.61 ± 0.05 l/kg), t1/2 values (L: 18.62 ± 2.44; H: 16.94 ± 1.88 min), and MRT (L: 20.52 ± 1.26; H: 19.19 ± 2.06 min).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Pharmacokinetic parameters of plasma melatonin concentrations after administration of an iv bolus of melatonin (3 µg/kg body wt0.75) in 2 groups of Ile-de-France ewes chosen as extreme for their breeding value on mean nighttime plasma melatonin concentrations

Plasma Melatonin Concentrations and Endogenous Melatonin Production Rate During Intensive Samplings

The patterns of mean plasma melatonin concentrations for the first intensive bleeding period of melatonin determination are presented in Fig. 2. Individual animals did not show any evidence of regular pulsatility (data not shown). Both groups presented typical nighttime melatonin concentrations, with significantly higher melatonin concentrations in the H group compared with the L group (L: 219.9 ± 21.8; H: 526.7 ± 65.8 pg/ml, P < 0.01).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   Mean nocturnal plasma melatonin concentrations (means ± SE) in groups of Low (n = 9) and High (n = 10) Ile-de-France ewes sampled during 3 h of the night (11:00 PM-2:00 AM) every 5 min. All samples were collected in the dark, under dim red light. Limit of detection of assay was 4 pg/ml of plasma.

Figure 3 shows the profile of melatonin concentration in the second intensive bleeding period. All ewes presented a clear day/night rhythm in their plasma melatonin concentrations, with high melatonin concentrations during the night and low melatonin concentrations during the day. Highly significant differences were observed between groups, with highest melatonin concentrations in the H group during the night and during the day (nocturnal period, L: 177.6 ± 15.5; H: 499.2 ± 53.4 pg/ml, ANOVA P < 0.01; diurnal period, L: 4.6 ± 0.3; H: 9.4 ± 0.7 pg/ml, Mann-Whitney P < 0.00021).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 3.   Patterns of mean plasma melatonin concentrations (means ± SE) in groups of Low (n = 9) and High (n = 10) Ile-de-France ewes sampled every hour during a 24-h period. Dark period, hatched bar, when samples were collected under dim red light. Graphic inset (right) has an expanded melatonin scale for samples taken between 10:00 AM and 6:00 PM. Dashed line, limit of detection of assay.

In the first bleeding period, melatonin production rate was higher in the H group (L: 25.70 ± 2.81; H: 63.09 ± 8.87 µg · kg-1 · h-1, P < 0.01). Similarly, in the second bleeding period, production rate from the H group was significantly higher (L: 23.75 ± 2.70; H: 72.14 ± 10.45 µg · kg-1 · h-1 during the night and L: 1.02 ± 0.15; H: 1.58 ± 0.14 µg · kg-1 · h-1 during the day, P < 0.01).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study indicates that the physiological origin of the genetic difference between the two extreme groups of ewes regarding their nocturnal plasma concentration of melatonin comes from differences in the synthesis pathway of melatonin from the pineal gland rather than from the catabolism of the hormone. Indeed, in experiment 1, no differences between groups were observed regarding melatonin pharmacokinetics, and therefore the catabolism of this hormone (variations in the hepatic blood flow or the enzymatic biotransformations in the liver) is not the origin of the difference in the nocturnal plasma melatonin concentrations between our two extreme groups of ewes. In contrast, when the kinetic parameters determined for each animal in this experiment are used, the results of experiment 2 clearly indicate that the differences in melatonin production rate are the cause of variations in melatonin blood levels.

Plasma melatonin clearance did not show any difference between groups of ewes with extreme melatonin concentrations. It is generally assumed that temporal variations in drug catabolism (i.e., clearance) are related to variations in hepatic blood flow (HBF) or enzymatic biotransformations in the liver, and many studies conducted in healthy subjects have given proof of such assumptions (3). Clearance is a basic pharmacokinetic parameter that expresses the body's capacity to metabolize a drug. Because melatonin is catabolized in the liver by hydroxylation at position 6 followed by conjugation mainly (70%) with sulfate in rodents (11), humans (10a), and after intravenous administration in sheep (6), the HBF is the major determinant of melatonin clearance, and we can assume that there are no differences between groups on HBF. It should also be noted that plasma half-life was not different between our two groups of ewes. Melatonin kinetics has been reported to be dose independent in different species, including sheep (6, 16), humans (12), rats (9), and pony mares (10). The existence of a significant difference between the two groups of ewes regarding the first value after injection is difficult to explain. We can suggest two possibilities. The first possible explanation is technical; even though we were extremely careful when timing the injections, a slight difference in sampling times relative to injection times between the two groups of ewes may have occurred and may have generated a slight delay after injection in one of the two groups. Because the peak is extremely brief, a significant difference in the concentrations may have occurred. The second explanation may be a difference in the volume of the compartments in which melatonin was diluted after injection. However, the absence of a difference between the two groups in the pharmacokinetic parameters does not support this second hypothesis.

Melatonin production rate, which was calculated for each ewe by use of the individual pharmacokinetic parameters found in the study of catabolism, was widely different between the two groups of ewes during the nocturnal period but also during the diurnal period. The existence of a difference between groups in the diurnal plasma concentration of melatonin was an unexpected result of this experiment, but despite the fact that the concentration detected in the H group was low, it was clearly distinguishable from the limit of detection of the assay and was higher than that of the L group. The question of whether this diurnal melatonin in the blood is from pineal origin is questionable. It could be indicative of the general activity of the pineal gland, more active in the H than in the L group, but this melatonin could also come from other sources (e.g., retina or digestive tract; Ref. 15). However, at least during the night, these differences in melatonin production rate indicate that the difference in plasma melatonin concentrations between the two groups of ewes is the synthesis pathway from the pineal gland into the blood. Differences in the development of the pineal gland (i.e., pineal size) may be responsible for the observed difference in the capacity to liberate melatonin into the blood. Pineal weight differed widely among adult ewes of the same breed (19). If so, some individuals could have high nocturnal plasma melatonin concentrations simply because they have larger pineal glands. Thus the genetic difference observed here between groups may be exerted during the development of the gland during intrauterine or early life.

The difference could arise more centrally, perhaps in the circadian system of the animals. It was recently demonstrated that lesion of the sheep suprachiasmatic nuclei (SCN) resulted in alterations of the circadian pattern of melatonin secretion. In such rams maintained under dim red light, melatonin rhythm of secretion was desynchronized among animals, but melatonin amplitude was also decreased after the surgical lesions of the SCN (18). Also, mathematical modeling suggests that the output of the circadian pacemaker can contribute to the large variability of melatonin rhythm (2).

Differences in the capacity of the gland to synthesize melatonin may also be responsible for the difference between groups. In sheep (5), as in other mammalian species such as hamsters (17), N-acetyltransferase (NAT) activity is a limiting factor of melatonin synthesis. Thus the ewes in the present experiment may differ in the ability of their NAT to synthesize melatonin, which may provide a first biochemical basis for the observed difference in the variability in melatonin blood levels in various mammalian species.

The determinations of the genetic effect on pineal weight, on the capacity to release melatonin, on enzymatic activities, and on the existence of different variants of various enzymes (such as tryptophan 5-hydroxylase, NAT, and hydroxyindol-O-methyltransferase) will be the future directions to develop to further identify the origin of the genetic difference between animals.

    ACKNOWLEDGEMENTS

The authors thank Agnès Daveau and Françoise Maurice-Mandon for advice in technical procedures, the breeders of the Institut National de la Recherche Agronomique Research Center of Nouzilly for supply and care of experimental animals, and Dr. D. Skinner for help in preparation of this manuscript.

    FOOTNOTES

L. A. Zarazaga was supported by a postdoctoral grant from Diputación General de Aragón and Institut National de la Recherche Agronomique-Direction Scientifique des Productions Animales.

Address for reprint requests: P. Chemineau, Neuroendocrinologie Sexuelle, INRA Physiologie de la Reproduction, 37380 Nouzilly, France (E-mail: Philippe.Chemineau{at}tours.inra.fr).

Received 27 August 1997; accepted in final form 3 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Arendt, J. Melatonin and the Mammalian Pineal Gland, edited by J. Arendt. London: Chapman & Hall, 1995, p. 331.

2.   Brown, E. N., Y. Choe, T. L. Shanahan, and C. A. Czeisler. A mathematical model of diurnal variations in human plasma melatonin levels. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E506-E516, 1997[Abstract/Free Full Text].

3.   Bruguerolle, B. Chronopharmacology. In: Biologic Rhythms in Clinical and Laboratory Medicine, edited by Y. Haus, and E. Haus. Paris: Springer Verlag, 1992, p. 114-137.

4.   Chemineau, P., I. Beltrán de Heredia, A. Daveau, and L. Bodin. High repeatability of the amplitude and duration of the nycthemeral rhythm of the plasma melatonin concentrations in the Ile-de-France ewes. J. Pineal Res. 21: 1-6, 1996[Medline].

5.   Coon, S. L., P. H. Roseboom, R. Baler, J. L. Weller, M. A. A. Namboodiri, E. V. Koonin, and D. C. Klein. Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis. Science 70: 1681-1683, 1995.

6.   English, J., C. J. Bojkowski, A. L. Poulton, A. M. Symons, and J. Arendt. Metabolism and pharmacokinetics of melatonin in the ewe. J. Pineal Res. 4: 351-358, 1987[Medline].

7.   Falconer, D. S. Introduction to Quantitative Genetics. London: Longman Group Ltd., 1960, chapt. 7, p. 365.

8.   Fraser, S., P. Cowen, M. Franklin, C. Franey, and J. Arendt. Direct radioimmunoassay for melatonin in plasma. Clin. Chem. 20: 396-397, 1983[Free Full Text].

9.   Gibbs, F. P., and J. Uriend. The half life of melatonin elimination from rat plasma. Endocrinology 109: 1796-1798, 1981[Abstract].

10.   Guillaume, D., N. Rio, and P. L. Toutain. Kinetic studies and production rate of melatonin in pony mares. Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R1236-R1241, 1995[Abstract/Free Full Text].

10a.   Jones, R. L., P. L. McGeer, and A. C. Greiner. Metabolism of exogenous melatonin in schizophrenic and non-schizophrenic volunteers. Clin. Chim. Acta 26: 281-285, 1969[Medline].

11.   Kopin, I. J., C. M. B. Pare, J. Axelrod, and H. Weissbach. The fate of melatonin in animals. J. Biol. Chem. 236: 3072-3075, 1961[Medline].

12.   Mallo, C. R., G. Zaidan, G. Galy, E. Vermeulen, J. Brun, G. Chazot, and B. Claustrat. Pharmacokinetics of melatonin in man after intravenous infusion and bolus injection. Eur. J. Clin. Pharmacol. 38: 297-301, 1990[Medline].

13.   Malpaux, B., S. M. Moenter, N. L. Wayne, C. J. I. Woodfill, and F. J. Karsch. Reproductive refractoriness of the ewe to inhibitory photoperiod is not caused by alteration of the circadian secretion of melatonin. Neuroendocrinology 48: 264-270, 1988[Medline].

14.   Malpaux, B., J. Robinson, M. B. Brown, and F. J. Karsch. Reproductive refractoriness of the ewe to inductive photoperiod is not caused by inappropriate secretion of melatonin. Biol. Reprod. 36: 1333-1341, 1987[Abstract].

15.   Pang, S. F., P. P. N. Lee, Y. S. Chang, and E. Ayre. Melatonin secretion and its rhythms in bilogical fluids. In: Melatonin: Biosynthesis, Physiological Effects, and Clinical Applications, edited by H. S. Yu, and R. J. Reiter. Boca Raton, FL: CRC, 1993, p. 129-153.

16.   Rollag, M. D., R. J. Morgan, and G. D. Niswender. Route of melatonin secretion in sheep. Endocrinology 102: 1-8, 1978[Medline].

17.   Rollag, M. D., E. S. Panke, W. Trakulrungsi, C. Trakulrungsi, and R. J. Reiter. Quantification of daily melatonin synthesis in the hamster pineal gland. Endocrinology 106: 231-236, 1980[Medline].

18.   Tessonneaud, A., A. Locatelli, M. Caldani, and M. C. Viguier-Martinez. Bilateral lesions of the suprachiasmatic nuclei alter the diurnal rhythm of melatonin secretion in sheep. J. Neuroendocrinol. 7: 145-152, 1995[Medline].

19.   Thimonier, J., and P. Mauléon. Variations saisonnières du comportement d'oestrus et des activités ovarienne et hypophysaire chez les ovins. Ann. Biol. Anim. Biochim. Biophys. 9: 233-250, 1969.

20.   Tillet, Y., J. P. Ravault, C. Selve, G. Evin, B. Castro, and M. P. Dubois. Conditions d'utilisation d'anticorps spécifiques pour la visualisatin immunohistochimique de la sérotonine et de la mélatonine dans la glande pinéale deu mouton. CR Acad. Sci. Paris 303: 77-82, 1986.

21.   Toutain, P. L., and M. Oukessou. Pharmacocinétique: éléments de méthodologie. Rec. Méd. Vét. Spéc. Anti-Infectieux 166: 195-203, 1990.

22.   Zarazaga, L. A., B. Malpaux, L. Bodin, and P. Chemineau. The large variability in melatonin blood levels in ewes is under strong genetic influence. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E607-E610, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 274(6):E1086-E1090
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society