0013-7227/79/1042-0295$02.

00
Endocrinology Vol. 104, No. 2
Copyright © 1979 by The Endocrine Society Printed in U.S.A.

A Diurnal Melatonin Rhythm in Primate Cerebrospinal

Fluid*
STEVEN M. REPPERT, MARK J. PERLOW, LAWRENCE TAMARKIN, AND DAVID C. KLEIN
Section on Neuroendocrinology, Laboratory of Development Neurobiology, National Institute of Child
Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014; and the
Laboratory of Clinical Psychopharmacology, National Institute of Mental Health, St. Elizabeth's
Hospital, Washington D.C. 20032

ABSTRACT. Melatonin was measured in cerebrospinal fluid
(CSF) withdrawn continuously from partially restrained rhesus
monkeys. There was a daily rhythm in CSF melatonin with peak
night values 2- to >15-fold higher than day values. The increase
occurred shortly after lights were turned off, and the decrease
occurred soon after lights were turned on. There was substantial
variation in the magnitude of the rhythm among animals. How-
ever, there was little day to day variation in the rhythm of
individual animals studied for 3 or 6 consecutive days. Although
the concentration of melatonin in CSF was lower than that in
plasma, the changes in CSF melatonin concentrations seemed to
reflect large daily changes in plasma melatonin concentrations.
(Endocrinology 104: 295, 1979)

A S is apparently true of all warm-blooded animals

. (1-6), there is a daily melatonin rhythm in blood
and urine of primates (7-12). Our knowledge of the
[12-h light, 12-h dark (LD 12:12)] with the lights on from
0600-1800 h EST. Each chair was in a separate sound-atten-
uated chamber.
factors involved in the regulation of this rhythm in pri-
mates is limited in part, because a useful experimental Sample collection
model has not been available. In an attempt to overcome
this limitation, we have investigated the possibility of CSF was continuously collected from an indwelling catheter
using cerebrospinal fluid (CSF) continuously removed (13) which was inserted as follows. Animals were anesthetized
with ketamine HC1; a styleted 18-gauge spinal needle was then
from the partially restrained rhesus monkey to monitor
percutaneously inserted between lumbar vertebrae (L3-4) into
circulating melatonin. The results of our efforts presented
the subarachnoid space. A polyethylene catheter was inserted
in this report indicate this is an excellent model to use through the needle hub and advanced cephalad in the subarach-
for chronic studies of the regulation of melatonin. noid space so that the tip terminated in the high cervical to low
cisternal region. CSF was continuously withdrawn (~1 ml/h)
Materials and Methods from the awake animal by a peristaltic pump and was auto-
matically collected into polypropylene tubes as either 90- or
Animals 120-min fractions. CSF was collected for 3 days before the start
Adult male rhesus monkeys (5.5-6.5 kg) were purchased from of an experiment. The CSF remained at room temperature
the NIH primate colony and adapted to chronic restraint in (20-22 C) for about 100 min before refrigeration (4 C); the 100-
primate chairs for at least 3 weeks before the start of an min lag period was calculated from the dead space of the tubing
experiment. Purina monkey chow was placed on feeding trays and the rate of withdrawal. The refrigerated samples were
between 0900-1200 h each morning; water was available ad removed every 24 h and stored at -70 C until analysis. It was
libitum. Lighting (15 watt cool-white fluorescent light; West- determined by thin layer chromatography (TLC) (14) that [3H]
inghouse Electric, Pittsburg, PA) was automatically controlled melatonin was stable in CSF for at least 24 h at 20 and 4 C.
Blood samples were collected from an indwelling venous
catheter percutaneously inserted into a saphenous vein. The
Received August 7, 1978. catheter was advanced so that the tip terminated in the vena
Address all correspondence and requests for reprints to: Dr. Steven cava. Blood samples (2 ml) were withdrawn at intermittent
M. Reppert, Section on Neuroendocrinology, Laboratory of Develop- intervals into heparinized syringes, transferred to polypropylene
mental Neurobiology, National Institute of Child Health and Human tubes, and stored at 4 C for 24 h. The blood was then centri-
Development, National Institutes of Health, Building 6, Room 128,
Bethesda, Maryland 20014. fuged; the plasma was stored at -70 C until analysis. It was
* A portion of these studies was presented at the 60th Annual determined by TLC (14) that [3H]melatonin incubated in
Meeting of The Endocrine Society, Miami, FL, June 1978. plasma was stable for at least 24 h at 4 C.
295

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296 REPPERT ET AL. Endo • 1979
Vol 104 • No 2

Melatonin RIA 60 -
_l I i I 1

CSF and plasma melatonin were measured by the RIA of CSF

Rollag and Niswender, using their antiserum 1055 (2). Samples Y = 0.92X + 15.5
_ r = 0.998
(400 /xl) of CSF and plasma were assayed in duplicate. Mela- 50 -
tonin in each sample was extracted into 5 vol glass-distilled
chloroform (Burdick and Jackson Laboratories Inc., Muskegon, CD

MI), which was then sequentially washed with 1 vol of 0.1 M S 40 -

sodium bicarbonate, pH 10.0, and 1 vol water. A sample (1.5 Q
LLJ /
ml) of the washed chloroform was taken to dryness under >-
/ I
I I 1 I I I I

140
vacuum in the dark (45 C). The residue from CSF samples was
dissolved in 500 /xl PBS-gel (0.01 M phosphate buffer with 0.9%
30 -
/
4
y Plasma
120 _ Y = 0.81X + 18.2
r = 0.996
^
/ /
sodium chloride and 0.1% gelatin, pH 7.0) and then assayed for 100 - -

melatonin, as previously described (2). The extraction efficiency 20 -

y
A 80 -
/
-
for CSF samples was 92-95%, as determined in several assays.
The residue from plasma samples was dissolved in 300 ju.1 / 60 A -

40 -
phosphate-buffered saline (PBS) gel, which was further ex- 10-
tracted with 2 ml glass-distilled petroleum ether (Burdick and 20 -
Jackson Laboratories Inc., Muskegon, MI); a 200-jul sample of 0 _ _
i i i I I I I

the extracted buffer was then assayed for melatonin. The ex- 0 - 0 20 40 60 80 100 120 140 _
1 i i 1 1 I 1
traction efficiency for plasma samples using two extraction
steps was about 80%. A final antibody dilution of 1:256,000 was 0 10 20 30 40 50 60
used for the assay of CSF and plasma. The limits of sensitivity MELATONIN ADDED (pg/ml)
varied among assays from 0.5-1.0 pg/tube. RIA data were FIG. 1. Quantitative recovery of melatonin added to CSF and plasma.
analyzed using a computer program based on a log-logit linear- The values represent the mean (±SD) of six (CSF) and eight (plasma)
ization of the standard curve (15). All samples for an individual determinations.
animal were determined in a single assay. The data are pre-
sented as the mean of duplicate determinations. The values 35% of each other for day CSF samples (3-9 pg/ml) and 10% of
were not corrected for extraction efficiency. each other for night CSF samples (10-40 pg/ml). The values
This method was found to be valid and reliable for measuring for the pooled plasma sample were within 30% of each other.
melatonin in monkey CSF and plasma as follows.
Precision. A 100-ml pool of monkey CSF and plasma was
Quantitative recovery of melatonin. Authentic melatonin (Re- prepared. In every assay, two 400-jtd samples of each pool were
gis Chemical Co., Chicago, IL; 4-61 pg for CSF and 4-125 pg inserted between every 20 samples and assayed in the same
for plasma) was added to 1-ml aliquots from a pool of day-night manner as the unknowns. The intra- and interassay coefficients
CSF and a pool of day plasma samples. Melatonin was quanti- of variation (±SD) for the CSF pool containing 11.5 ± 2.3 pg/ml
tatively recovered from CSF and plasma (Fig. 1). The slopes of (n = 67) of immunoreactivity were 10% and 20%, respectively;
the regression line, unconnected for extraction efficiency, were for the plasma pool containing 15.4 ± 3.3 pg/ml (n = 26) of
0.92 (CSF) and 0.81 (plasma). The values of the y intercept immunoreactivity, the coefficients were 15% and 21%, respec-
which represented the endogenous melatonin concentration tively.
was 16 pg (CSF) and 18 pg (plasma). The correlation coefficients
of the relationship between the amount measured and the pH]Melatonin1 studies
amount added were 0.998 (CSF) and 0.996 (plasma).
[2-aminoethyl-2-3H]iV-Acetyl-5-methoxytryptamine ([3H]-
Parallel inhibition curves for the melatonin standard and melatonin; SA, 35 Ci/mmol; New England Nuclear Corp., Bos-
extracted samples. Pooled CSF and plasma samples (100 ml ton, MA) was >99% radiochemically pure, as judged by one-
each) were extraced with 5 vol chloroform. The chloroform was dimensional TLC (see below). The [3H]melatonin stock solution
taken to dryness, dissolved in 1 ml PBS-gel, and serially diluted (28 /XM; ethanol-water, 96:4) was diluted with sterile saline for
in the same manner as the melatonin standard. The serially injections (1:100) and infusions (1:32).
diluted plasma extracts were extracted with petroleum ether The amount of [3H]melatonin in a sample was determined
before analysis, as described above. The resultant loge-logit by extracting the [3H]melatonin in 150 or 200 \i\ sample with 5
slopes of the inhibition curves for the melatonin standard vol water-saturated chloroform. The aqueous layer was re-
(-0.81), the extracted CSF sample (-0.83), and the extracted moved and saved for further analysis of [3H]melatonin metab-
plasma sample (-0.83) were not significantly different (P > olites2 (see below). The chloroform was washed sequentially
0.05).
1
Quantitative validation of pooled CSF and plasma samples The term [3H]melatonin refers to the radiolabeled compound be-
by gas chromatography-mass spectrometry (GCMS). Pooled fore injection and to the radioactive compound that was found to be
chloroform extractable and to comigrate with authentic melatonin by
samples of CSF obtained at night and during the day for three one-dimensional TLC.
animals and a pooled day plasma sample were analyzed by RIA 2
The term [3H]melatonin metabolites refers to all nonchloroform-
and GCMS (16). The results of the two methods were within extractable radiolabeled compounds recovered from plasma and CSF.

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 PRIMATE CSF MELATONIN 297

with 1 vol 0.1 M sodium bicarbonate, pH 10.0, and water. After showed a slow nocturnal increase, with peak values oc-
the last wash, 0.5 mol chloroform was dried in a scintillation curring 8 h after the lights were turned off. Peak night
vial and the radioactivity was determined as described (14). values ranged from 12-40 pg/ml (50-172 pM) among the
This procedure extracts 90-94% Qf added [3H]melatonin from six animals. The magnitude of the melatonin rhythm,
monkey plasma and CSF. low day to high night values, was 2- to > 15-fold. A
Essentially all (88-94%) of the radioactivity in pools of chlo- decrease in CSF melatonin occurred at about the same
roform extracts of CSF and plasma for each animal studied
comigrated with authentic melatonin in one-dimensional TLC time the lights were turned on in the morning.
(chloroform-methanol-acetic acid, 90:10:1, silica gel) (14). The The daily pattern of melatonin in CSF was studied for
reported values were not corrected for this loss. 3-6 days (Figs. 3 and 4). In contrast to the variation
The [3H]melatonin metabolites were estimated by measuring among individual animals, the daily pattern of melatonin
the water-soluble radioactivity remaining after chloroform ex- in CSF was remarkably similar from day to day for an
traction. One half of the volume of extracted sample was mixed individual.
with 10 vol solubilizer (N.C.S; Amersham/Searle Corp., Arling-
ton Height, IL) counting fluor was added, and the samples were Comparison of CSF and plasma melatonin
counted.
The amounts of melatonin in CSF and plasma obtained
Results at the same time from two monkeys were determined
(Fig. 5). The 24-h pattern of melatonin in both the plasma
CSF melatonin and CSF was similar. In both animals, the day CSF levels
CSF melatonin obtained from six monkeys during 24- were about one fourth of the corresponding plasma levels,
h sampling periods was measured (Fig. 2). The daytime but night CSF levels were similar to the night plasma
values ranged from <2 to 8 pg/ml(<9 to 34 pM). levels.
In five of the six animals examined, a rapid increase in
CSF melatonin occurred within the first 2-h period after [*H]Melatonin transfer to CSF from plasma
the lights were turned off; peak night time levels occurred After an iv injection to three animals, [3H]melatonin
6-8 h into darkness. The remaining monkey (animal 375) disappeared from the circulation in a rapid and multi-
phasic manner (Fig. 6), similar to that observed in other

30-

—j
30-
— i — —._ _ — . }
706

201-
rJl nI

r1
: it
10h

V
LIGHTING
SCHEDULE 1200 2400 1200 1200 2400 1200

CLOCK TIME (hrs)

FIG. 2, The diurnal pattern of CSF melatonin for six animals. CSF was LIGHTING
SCHEDULE 0600 1800 0600 1800 0600 1800 0600 1800
collected as 120-min fractions for four animals (animals 669, 508, 842,
CLOCK TIME (hrs)
and 706) and as 90-min fractions for two animals (animals 375 and 707).
The lower limit of detection for each assay was 2 pg/ml. The times of FIG. 3. The diurnal pattern of CSF melatonin for individual animals
the year the samples were obtained are as follows: animals 669, 508, studied for 3 consecutive days. CSF was collected as 120-min fractions.
and 842, Nov.; animal 375, May; animal 706, Dec; animal 707, June. The lower limit of detection was 2 pg/ml.

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298 REPPERT ET AL. Endo • 1979
Vol 104 • No 2

707 the termination of the infusion were similar to those seen

40 in the [3H]melatonin injection studies.

Discussion
c 30
The results of this study clearly indicate that a diurnal
melatonin rhythm is present in blood and CSF of the
§ adult male rhesus monkey. In addition, the melatonin
UJ rhythm is closely coordinated with environmental light-
8 io ing under the lighting regime studied (LD 12:12); high
melatonin values (>10 pg/ml; >43 pM) are seen only
during the dark period of a 24-h day. The presence of a
LIGHTING 24-h rhythm in the concentration of melatonin in CSF
SCHEDULE
0600 1800 0600 1800 0600 1800 0600 1800 0600 1800 0600 1800 raises the possibility that the CSF may be an important
CLOCK TIME (hrs) route of communication between the pineal gland and
FIG. 4. The diurnal pattern of CSF melatonin for an animal studied the rest of the brain.
for 6 consecutive days. CSF was collected as 90-min fractions. The The [3H]melatonin studies indicate that melatonin
lower limit of detection was 2 pg/ml.

mammals (17-20). The peak concentrations of [3H]mel-

atonin measured at 3 min after the injection were 120
pg/ml (517 pM) for animals 842 and 669, and 54 pg/ml
(233 pM) for animal 615; these levels seem to be within
the physiological range of the nocturnal levels reported
for primates (6, 7, 9-12). After the initial rapid phase, the
disappearance of [3H]melatonin from the circulation was
linear (ti/2 = 30 min) from 15-150 min after the injection.
[3H]Melatonin was rapidly transferred into the CSF and
was first detectable within 10 min after the systemic
injection. The CSF levels rapidly increased, with peak
values occurring 30-40 min after the injection. These
were about 10-20% of the peak plasma levels measured
at 2 min; [3H]mleatonin disappeared from CSF in a
monophasic manner, with a ti/2 of about 40 min during
the period studied.
[3H]Melatonin metabolites rapidly appeared in plasma
after the systemic injection of [3H]melatonin (Fig. 7); the
[3H]melatonin metabolites increased throughout the
sampling period; 2 h after the injection, they represented
over 90% of the total plasma radioactivity. The appear-
ance of [3H]melatonin metabolites in CSF was slower
and less pronounced; 2 h after the injection, they repre-
sented about 63% of total CSF radioactivity.
Plasma and CSF were studied during and after a 2-h
iv infusion of [3H]melatonin (Fig. 8). [3H]Melatonin rap-
idly crossed from blood into CSF; at relative steady state
levels, occurring at about 90 min into the infusion, the
concentration of [3H]melatonin in CSF was about 30-40%
of that in the plasma. The steady state plasma (130-150 LIGHTING
pg/ml; 560-646 pM) [3H]melatonin levels established by SCHEDULE 0600 1800 0600 1800
the infusion seem to be in the physiological range for the CLOCK TIME (hrs)
night time levels reported for primates (6, 7, 9-12); the FIG. 5. The diurnal pattern of melatonin in plasma and CSF. Plasma
corresponding steady state CSF [3H]melatonin levels and CSF were collected over the same 24-h time period for each animal.
were 40-56 pg/ml (172-241 pM). The rates of disappear- The CSF was collected as 120-min fractions. The lower limit of detec-
ance of [3H]melatonin from plasma and CSF seen after tion was 2 pg/ml.

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 PRIMATE CSF MELATONIN 299

•——• Plasma
[3H] MELATONIN -0 CSF
50 i i i i i i i i i i i i i i i i i i i 11111 1 1 1 1 1 1 1 1

| INFUSION | R49

i
<

1
1
1 1 1 1 1
10-

5- 7 ^A

1
-* oxf° -

0
/ \
1- /
0.1 - /
60 120 180 0 60 120 180 0 60
- / -
TIME AFTER SYSTEMIC INJECTION (min) 0.5 - o —
3 - / -
FIG. 6. [ H]Melatonin in blood and CSF. Two animals (nos. 842, 669) - / _
were given an iv bolus injection of 90 /xCi (0.6 jug) [3H]melatonin and /
- 0
animal 614 was given 70 /xCi (0.5 jug). CSF was collected as 10-min
fractions, and the data are plotted at the midpoint of each collection cc
II II
period. Plasma was obtained at the times indicated. 3 0.1 71 t t 1 f i t 1 f i n 1 1 1 1 1 r^ II II ^ I 1 n 11 ii

o 706 :
< 50h1 INFUSION |

10 -

5
- ?
- $
V\ -

1
1- 1
'- 1
- / -
0.5
~6
-

60
TIME AFTER INTERJECTION (min)
FIG. 7. [;'H]Melatonin metabolites in plasma and CSF after an iv bolus
injection of [3H]melatonin. These data were collected during the ex-
periment described in Fig. 6. Values for [3H]melatonin metabolites are
given as a percentage of the total radioactivity present in plasma or
CSF at the times indicated. Each value is the mean (±SE) of three
animals.
rapidly crosses into CSF and equilibrates to about 40%
of plasma levels. The differences between CSF and
plasma melatonin may be explained by a difference in
protein binding. About 70% of the melatonin in plasma
1 1 1 1 1 1 1 1 1 1 1 1 1
0.1 0 120 180 60 240 300
TIME (min)
FIG. 8. ['H]Melatonin in blood and CSF during and after an iv
[3H]melatonin infusion. ['H]Melatonin was infused at a constant rate
of 7 /iCi/min (47 ng/min) for 132 (animal 706) and 140 min (animal
842). CSF was collected as 10-min fractions, and the data are plotted at
the midpoint of each collection period. Plasma was obtained at the
times indicated.
120 is bound to albumin; however, none is bound to proteins
in CSF (24). Accordingly, only 30% of plasma melatonin
would be available as free melatonin to cross into the
CSF. Consistent with this explanation are our findings
that CSF melatonin is lower than plasma levels in both
the [3H]melatonin infusion studies and in studies looking
at endogenous plasma and CSF melatonin levels. A CSF-
plasma melatonin relationship similar to that seen in the
rhesus monkey is also seen in adult humans (11, 20) and
in sheep (21), where CSF melatonin is lower than the
corresponding plasma melatonin levels.
Our finding that CSF melatonin is lower than or equal

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300 REPPERT ET AL. Kndo 1979
Vol 104 , No 2

to plasma melatonin is in contrast to what has been tected. A relatively smaller amount of metabolites ap-
reported in the calf (4) and children undergoing therapy peared in the CSF and their appearance was delayed.
for leukemia (22), where CSF melatonin is higher than The identity of the metabolites appearing in CSF in our
that seen in plasma. The reason for the higher values in study is unknown. However, circulating melatonin is
calves is now known but may represent either technical known to be primarily metabolized to the conjugates of
or physiological differences. The calf CSF was obtained 6-hydroxymelatonin, which occurs in the liver (17, 18).
from the lateral ventricle and the monkey samples were These polar compounds probably will not readily cross
obtained from the cisternal subarachnoid space. Thus, into the CSF. This could explain the delayed appearance
the difference in CSF melatonin might reflect differential of metabolites in CSF and the lower concentration of
local concentrations within the ventricular system. How- these metabolites relative to [3H]melatonin in CSF as
ever, this does not seem to be the case in humans, where compared with plasma. Other possibilities are that me-
the concentrations of CSF melatonin obtained from the tabolites in CSF reflect only metabolism of melatonin by
ventricles and lumbar subarachnoid space are essentially the brain (26) or a combination of peripheral and brain
the same; both are lower than corresponding plasma metabolites.
melatonin levels (20). Higher CSF melatonin values The subcellular mechanism involved in generating the
might also occur if there was a high concentration of a melatonin rhythm in the rhesus may be the same as that
melatonin-binding protein in calf CSF. Another possible described in the rodent, in that an increase in melatonin
explanation involves the different modes of sample col- release is due to an increase in melatonin production.
lection. In our primate studies, CSF was withdrawn con- Some evidence in support of this is the report that pineal
tinuously at a rate which is about one half the calculated serotonin in the rhesus, as is true of the rodent, decreases
production rate (25), and CSF samples were used for at night, presumably because there is an increased en-
analysis only after a 3-day period of continuous CSF zymatic conversion of serotonin to melatonin (23). It will
withdrawal, allowing for adaption to sampling. In the calf be interesting to determine whether the entire biochem-
study, large volumes of CSF were acutely withdrawn ical and neural regulatory scheme which functions in the
from the lateral ventricles. This could have resulted in rhesus to generate a diurnal rhythm in melatonin is
acute pressure changes in the ventricles at each sampling similar to that described in the rodent (27).
interval and could result in leakage of interstitial fluid Based on our findings, the chaired monkey seems to
from around the pineal gland into the CSF and falsely be an excellent subject for longitudinal studies of the
elevated CSF melatonin levels. This would not occur in factors regulating melatonin production. It will be im-
our experiment because ventricular pressure would re- portant to determine the validity of the chaired monkey
main constant with continuous slow withdrawal of CSF as a model of human pineal physiology.
(1 ml/h).
Acknowledgments
The observation in leukemic children of higher CSF
melatonin levels is distinctly different from that found in We would like to thank Drs. M. D. Rollag and G. W. Niswender for
their generous supply of the antibody to melatonin (1055), Drs. A. Lewy
normal adults (11) as well as in adults with intracranial and S. Markey (NIMH) for assaying melatonin by GCMS, and Mr. A.
disease (20). These high CSF levels in the child study Anderson and Ms. Delores Braun for technical assistance.
could result from the effects of leukemia or leukemic
therapy (cytotoxic drugs and central nervous system References
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The other variable to consider, as in the calf study, is Persistent rhythms of pineal and serum melatonin in cockerels in
continuous darkness, J Endocrinol 63:319, 1974.
age. 2. Rollag, M., and G. Niswender, Radioimmunoassay of serum con-
Our studies indicate the daily rhythms in plasma and centrations of melatonin in sheep exposed to different lighting
CSF melatonin are similar. This is not surprising in view regimens, Endocrinology 98: 482, 1976.
3. Ozaki, Y., H. J. Lynch, and R. J. Wurtman, Melatonin in rat pineal,
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CSF and plasma. It is not clear whether melatonin is Melatonin: daily cycle in plasma and cerebrospinal fluid of calves,
Science 195: 686,1977.
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 PRIMATE CSF MELATONIN
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