Genetic variability in melatonin concentrations in ewes
originates in its synthesis, not in its catabolism
LUIS A. ZARAZAGA,1 BENOÎT MALPAUX,1 DANIEL GUILLAUME,1
LOYS BODIN,2 AND PHILIPPE CHEMINEAU1
1Neuroendocrinologie Sexuelle, Institut National de la Recherche Agronomique
Physiologie de la Reproduction, 37380 Nouzilly; and 2Institut National de la Recherche
Agronomique, Station d’Amélioration Gènetique des Animaux, 31326 Castanet Tolosan, France
pineal gland; pharmacokinetics; clearance; half-life
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 6hydroxymelatonin 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
E1086
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
General Procedures
Animals and photoperiodic treatments. The whole Ile-deFrance 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 6
0.23 yr; 69.1 6 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 6 5.8 pg/ml; and high melatonin concentrations,
H group: 747.0 6 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
0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.4 on June 18, 2017
Zarazaga, Luis A., Benoı̂t Malpaux, Daniel Guillaume, Loys Bodin, and Philippe Chemineau. Genetic
variability in melatonin concentrations in ewes originates in
its synthesis, not in its catabolism. Am. J. Physiol. 274
(Endocrinol. Metab. 37): E1086–E1090, 1998.—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 6 2.8 vs. H: 63.1 6 8.9 µg · kg21 · h21, 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.
MELATONIN SYNTHESIS AND CATABOLISM IN THE EWE
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
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 6 SE.
RESULTS
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 6 0.1; H: 12.8 6 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 6 198.5; H: 3,728.3 6
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).
The mean kinetic parameters for both groups are
given in Table 1. No differences between groups were
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
Fig. 1. Semilogarithmic plots of plasma melatonin concentrations
(means 6 SE) in groups of Ile-de-France ewes (Low, n 5 9, and High,
n 5 10) after administration of an iv bolus of melatonin (3 µg/kg body
wt0.75 ). Dashed line, limit of detection of assay.
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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 5
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 22, 21, 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 220°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 (t½ ) were estimated for each
ewe by use of successive equations as described in Toutain
and Oukessou (21) and Guillaume et al. (10).
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E1088
MELATONIN SYNTHESIS AND CATABOLISM IN THE EWE
Table 1. Pharmacokinetic parameters of plasma
melatonin concentrations after administration of an iv
bolus of melatonin (3 mg/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
Clearance, l · kg21 · h21
VC , l/kg
Vss , l/kg
Terminal half-life, min
High Group
(n 5 10)
1.99 6 0.14
0.87 6 0.12
0.67 6 0.06
18.62 6 2.44
(13.48–34.0)
20.52 6 1.26
32.62 6 2.05
1.99 6 0.13
0.78 6 0.13
0.61 6 0.05
16.94 6 1.88
(10.16–28.12)
19.19 6 2.06
32.35 6 2.23
NS
NS
NS
NS
NS
NS
Values are means 6 SE; those in parentheses are ranges. VC ,
volume of central compartment; Vss , noncompartmental steady-state
volume of distribution; MRT, mean residence time; AUC, area under
plasma curve; NS, nonsignificant.
observed for major kinetic parameters: CL (L: 1.99 6
0.14; H: 1.99 6 0.13 l · kg21 · h21 ), the Vss (L: 0.67 6 0.06;
H: 0.61 6 0.05 l/kg), t½ values (L: 18.62 6 2.44; H:
16.94 6 1.88 min), and MRT (L: 20.52 6 1.26; H:
19.19 6 2.06 min).
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 6 21.8; H: 526.7 6 65.8
pg/ml, P , 0.01).
Fig. 2. Mean nocturnal plasma melatonin concentrations (means 6
SE) in groups of Low (n 5 9) and High (n 5 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.
DISCUSSION
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.
Fig. 3. Patterns of mean plasma melatonin concentrations (means 6
SE) in groups of Low (n 5 9) and High (n 5 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.
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MRT, min
AUC, mg · min · ml21
Low Group
(n 5 9)
Statistical
Difference
Between
Groups
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 6 15.5; H: 499.2 6
53.4 pg/ml, ANOVA P , 0.01; diurnal period, L: 4.6 6
0.3; H: 9.4 6 0.7 pg/ml, Mann-Whitney P , 0.00021).
In the first bleeding period, melatonin production
rate was higher in the H group (L: 25.70 6 2.81; H:
63.09 6 8.87 µg · kg21 · h21, P , 0.01). Similarly, in the
second bleeding period, production rate from the H
group was significantly higher (L: 23.75 6 2.70; H:
72.14 6 10.45 µg · kg21 · h21 during the night and
L: 1.02 6 0.15; H: 1.58 6 0.14 µg · kg21 · h21 during the
day, P , 0.01).
MELATONIN SYNTHESIS AND CATABOLISM IN THE EWE
nin 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.
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.
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: [email protected]).
Received 27 August 1997; accepted in final form 3 March 1998.
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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
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MELATONIN SYNTHESIS AND CATABOLISM IN THE EWE
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