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213
Neuroendocrinology Letters ISSN 0172–780X
Copyright © 2002 Neuroendocrinology Letters
The effect of pyridoxine administration on melatonin
secretion in normal men
Rafael Luboshitzky
1
, U. Ophir
2
, Rachel Nave
3
, Rachel Epstein
3
,
Zila Shen-Orr
2
& Paula Herer
3
1. Endocrine Institute, Haemek Medical Center, Afula, Israel.
2. Endocrine Laboratory, Rambam Medical center, Haifa, Israel.
3. Sleep research Laboratory, Technion, Israel Institute of Technology, Haifa, Israel.
Correspondence to: Professor R. Luboshitzky, M.D.
Endocrine Institute,
Haemek Medical Center,
Afula, 18101,
ISRAEL
FA X
: +972 46495553;
E-MAIL: luboshitzky_ r@ clalit.org.il
Submitted: April 28, 2002
Accepted: May 1, 2002
Key words: melatonin; core body temperature; sleep; pyridoxine
Neuroendocrinology Letters 2002; 23:213–217 pii: NEL230302A02 Copyright © Neuroendocrinology Letters 2002
Abstract
OBJECTIVES
: To determine pineal response to pyridoxine in normal men.
MATERIAL AND METHODS: Twelve healthy men were given orally pyridoxine
(100 mg) or placebo at 1700h. Serum melatonin levels were determined
every 30 minutes with simultaneous measurement of core body temperature
between 1700h to 0300h. Polysomnographic sleep recordings were performed
between 1800h to 2000h.
RESULTS: Serum melatonin levels after both placebo and pyridoxine showed
a nocturnal rise occurring at 22:10±1:22h and 22:24±1:09h, respectively.
The melatonin onset, peak, mean and area under the curve (AUC) values
after pyridoxine (3.2±1.6 pg/ml, 47.2±22.6 pg/ml, 31.5±11.0 pg/ml and
173.5±138.4 pg/ml x min, respectively) were similar to the values after pla-
cebo administration (4.7±1.6 pg/ml, 53.9±26.0 pg/ml, 37.2±2.8 pg/ml and
205.3±137.8 pg/ml x min, respectively). CBT revealed a signifi cant nocturnal
decline but without signifi cant difference between pyridoxine and placebo.
Sleep amount and architecture were similar after the two treatments.
CONCLUSIONS: In adult man, the oral administration of 100 mg-pyridoxine
during the evening hours has no effect on melatonin secretion nor does it alter
CBT or sleep quality.
ORIGINAL ARTICLE
214
Introduction
Melatonin, the main hormone produced by the pi-
neal gland, displays a circadian rhythm peaking at
night [1]. Pinealocytes uses tryptophan as substrate
for melatonin synthesis, and melatonin levels change
as a function of tryptophan availability [2]. Pyridox-
ine is converted to its active coenzyme form, pyridoxal
phosphate (PLP). More than 60 PLP-dependent en-
zymes are known, including enzymes that participate
in decarboxylation reactions such as the decarboxyl-
ation of DOPA to dopamine and 5-hydroxytryptophan
to serotonin [3–4]. The activity of pyridoxine as a co-
enzyme in the tryptophan metabolism was described
in the kinurenine and methoxyindole pathways [5].
Pyridoxine acts as a coenzyme of 5-hydroxytryptophan
decarboxylase. The enzyme carboxylates 5-hydroxy-
tryptophan to serotonin, the immediate precursor of
melatonin [5]. The effect of pyridoxine on aromatic
amino acid decarboxylase activity supports a regula-
tory role of pyridoxine on the synthesis of neurotrans-
mitters [6–7]. Melatonin was shown to increase brain
pyridoxal phosphokinase activity, inhibition of gluta-
minergic neurotransmission, resulting in inhibitory
effects on central nervous system activity [8]. The
participation of endogenous melatonin in the normal
sleep-wake cycle regulation has been inferred from the
temporal relationships between melatonin cycle and
the 24-hour cycle in sleep propensity, and particularly
between the nocturnal melatonin onset and the noc-
turnal sleep gate [9–11]. The typical 24-hour sleep pro-
pensity pattern reveals a midafternoon sleepiness peak
followed by a forbidden zone for sleep, which is char-
acterized by very low sleep propensity in the early eve-
ning hours and then followed by the nocturnal sleep
gate. This term refers to a steep rise in sleepiness oc-
curring in the late evening hours [12]. Exogenous mel-
atonin given prior to an early evening nap, during the
forbidden zone for sleep at 1800–2000h,signifi cantly
shortened sleep latency and increased total sleep time.
These data suggested that timed administered melato-
nin can modify sleep propensity [13].
Exogenous melatonin administration has been
shown to lower core body temperature (CBT) by
0.2–0.4°C [14–17]. Acute exposure to bright light at
night elevated the nocturnal CBT and inhibits melato-
nin secretion [18]. This change in CBT was reversed by
a constant infusion of melatonin [19]. The time taken
to reach the maximum drop in CBT following melato-
nin administration was about 3 hours [17].
We hypothesized that pyridoxine may participate
in the nocturnal melatonin secretion and therefor can
modify other circadian rhythms as sleep and tempera-
ture when administered in the late afternoon hours. To
examine this hypothesis, we determined serum mela-
tonin levels, CBT and sleep quality in healthy young
adult men given a single oral dose of pyridoxine or pla-
cebo in the evening hours.
Material and Methods
Subjects and protocol
The study was approved by the institutional review
board (Helsinki committee) and all participants gave
their informed consent before the start of the study.
Twelve healthy males (aged: 22–26 years) participated
in the study. Participants were receiving no medica-
tions and during the study they were instructed to re-
frain from smoking, coffee and alcohol, and to have
7–8 hours of sleep per night, one week prior to the
study. The study comprised of two sessions, two weeks
apart.
On each experimental night, an IV catheter was in-
serted in an antecubital vein, kept patent by a slow
infusion of 0.9% NACL. Blood samples (2ml) were
collected every 30 minutes from 1700h to 0300h for
the determination of serum melatonin levels. Rectal
thermistor was inserted to record core body temper-
ature (CBT) between 1700h to 0300h. Subjects were
awake between 1700–1800h and between 2000–0300h,
with lights on (50 Lux at eye level) and assumed the
upright posture. Between 1800-2000h subjects were
lying in bed attempting to fall asleep. Conventional
sleep recordings were obtained to verify sleep quality.
Melatonin measurements
Blood was centrifuged, immediately separated and
stored at –20°C until assayed. Serum melatonin levels
were determined by radioimmunoassay (Buhlman
Lab., Albschvill, Switzerland). The assay sensitivity
was 0.3 pg/ml. The intra-assay coeffi cients of variation
(CV) were 4.9% and 5.8% for low (0.9–2.6 pg/ml), and
high (9.0–23.0 pg/ml), respectively. The interassay CVs
were 7.8% and 6.7%, respectively.
Core body temperature
CBT was monitored by rectal thermometer at one-
minute intervals between 1700–0300h, using the Min-
imitter series 2000 YSI, Yellow-Spring, USA.
Analysis of sleep stages
Electrodes were attached for the following electro-
physiological recordings: two electroencephalograms
(EEG levels C3-A2, C4-A1), two electrooculograms and
one electromyogram of the mentalis. Sleep stages were
scored in 30 seconds epochs according to conventional
criteria [20]. The following parameters were deter-
mined: total recording time (TRT), sleep latency (time
from lights off until 3 consecutive minutes of stage 2),
actual sleep time (AST=TRT– sleep latency+waking
periods), rapid eye movement (REM) latency (time
from beginning of sleep to the fi rst REM episode), sleep
effi ciency (AST/TRT) and percentages of sleep stage
2,3/4, and REM.
Medications
Subjects were given an oral dose of 100 mg- pyridox-
ine (vitamin B6, Pyridoxine HCL, Tarima, Maabarot,
Israel) or a look-alike placebo (starch) at 1700h, in a
double blind Latin square design.
Rafael Luboshitzky, U. Ophir, Rachel Nave, Rachel Epstein, Zila Shen-Orr & Paula Herer
215
Neuroendocrinology Letters ISSN 0172–780X Copyright © 2002 Neuroendocrinology Letters
Pyridoxine and melatonin secretion
Statistical analysis
The onset of the nocturnal melatonin rise was de-
fi ned as the time at which the fi rst of three consecutive
samples exceeded the mean levels of the day-time val-
ues (1700–2000h) by more than 1 SD. The integrated
melatonin values were determined as the area under
the curve (AUC) from the time of melatonin onset to
0300h. Independent t test were used to test the differ-
ences in serum melatonin levels, CBT and polysom-
nographic data between pyridoxine and placebo treat-
ments. Data are expressed as mean ± SD.
Results
In this study, we determined serum melatonin lev-
els, core body temperature and sleep quality in 12 men,
aged 23.4±2.6 years, after a single oral dose of 100 mg
pyridoxine or placebo given at 17:00h. Serum melato-
nin onset levels after pyridoxine (3.2 ±1.6 pg/ml) were
lower than the values after placebo (4.7±1.6 pg/ml),
occurring at 22:24±1:09h and 22:10±1:22h, respec-
tively (Table 1). Likewise, peak levels after pyridoxine
(47.2±22.6 pg/ml) were lower than the values after
placebo (53.9±26.0 pg/ml). The integrated amount of
melatonin secreted (area under the curve between
1700–0300h) after pyridoxine (173.5±138.4 pg/ml x h)
was lower than the value after placebo (205.3±137.8
pg/ml x h). The melatonin values after pyridoxine were
statistically not signifi cantly different from the values
after placebo. Core body temperature (CBT) curves
after both treatments were statistically not signifi -
cantly different. The peak CBT values after pyri doxine
and placebo were 37°C±0.27 and 36.9±0.26, respec-
tively; occurring at 02:30h ±0:45 and 02:30h ±0:50,
respectively (Figure 1). Quality of sleep was assessed
by actual sleep time and by sleep effi ciency. Sleep effi -
ciency was similar after both treatments and was rela-
tively low (66–69%). This is explained by the fact that
subjects had disturbed sleep, probably due to frequent
blood sampling not performed from a separate room
(Table 2).
Table 1. Nocturnal serum melatonin levels after an oral dose of
pyridoxine (100 mg) or placebo given at 1700h in normal men
(Data are mean ± SD)
Melatonin data Pyridoxine Placebo P value
Onset time (clock h) 22:24 ± 1:09 22:10 ± 1:22 NS
Onset level (pg/ml) 3.2 ± 1.6 4.7 ± 1.6 0.09
Peak level (pg/ml) 47.2 ± 22.6 53.9 ± 26.0 NS
Mean nocturnal rise
31.5 ± 11.0 37.2 ± 12.8 NS
(onset to 0300h (pg/ml)
AUC (onset to 0300h)
173.5 ± 138.4 205.3 ± 137.8 0.15
(pg/ml x h)
NS: Not signifi cant; AUC: Area under the curve
Table 2. Sleep recording data after pyridoxine or placebo adminis-
tration in normal men (Data are mean ± SD)
Sleep parameters Pyridoxine Placebo P value
Total recording time (h:min) 1:50 ± 0:03 1:44 ± 0:07 0.08
Actual sleep time (h: min) 1:23 ± 0:11 1:19 ± 0:16 NS
Sleep effi ciency (%) 69.4 ± 9.7 66.0 ± 12.5 NS
Sleep latency (minutes) 10 ± 3 15 ± 8 0.09
REM latency (minutes) 37 ± 30 49 ± 8 NS
Stage 1 (%) 4.5 ± 1.7 3.5 ± 2.9 NS
Stage 2 (%) 53.6 ± 9.4 46.3 ± 7.5 NS
Stage ¾ (%) 8,9 ± 7.7 16.7 ± 14.4 NS
REM (%) 12.4 ± 7.7 9.3 ± 10.7 NS
NS: not signifi cant
Figure 1. Serum melatonin levels and core body temperature values after pyridoxine and
placebo administration in normal men.
216
Rafael Luboshitzky, U. Ophir, Rachel Nave, Rachel Epstein, Zila Shen-Orr & Paula Herer
Discussion
In the present study we determined the effect of a
single oral dose of 100 mg pyridoxine given in the af-
ternoon on serum melatonin levels, core body tempera-
ture and sleep pattern in normal adult men. We used a
calculated dose related to body weight (1.5 mg/kg body
weight), which is much higher than the daily basal re-
quirement of pyridoxine, taking in account the intes-
tinal absorption and liver metabolism [21]. The three
circadian rhythms examined revealed similar results
after pyridoxine and placebo. Animal studies revealed
that in pyridoxine defi cient rats, melatonin synthesis
was reduced and the addition of pyridoxine restored
the levels of pineal melatonin to values observed in
control animals’ [22]. Also, the administration of mela-
tonin increased brain pyridoxal phosphokinase activity
[23]. A mild hypothermia was observed in female rats
during pyridoxine treatment. The hypothermic effect
appeared by day 3 of treatment. The reduction in core
body temperature was greater early in the day [24].
It is not clear whether the hypothermic effect of pyri-
doxine was mediated through the release of melatonin,
as melatonin levels were not determined in this study.
Positive immunohistochemical staining for pyridoxine-
5-phosphate oxidase was demonstrated in mammalian
hypothalamic paraventricular nucleus [25]. Also, pos-
itive staining to brain pyridoxal kinase was demon-
strated in human brain [26].
The effect of pyridoxine on dreaming was investi-
gated in adult men treated with 100 mg pyridoxine or
placebo prior to bedtime for 4 consecutive days. The
data suggested that pyridoxine may act by increasing
cortical arousal during periods of rapid eye movement
(REM) sleep. The authors suggested that this action of
pyridoxine might result from the conversion of trypto-
phan to serotonin [27].
Pharmacological doses of pyridoxine signifi cantly
decreased pituitary hormone levels [3]. Others failed to
observe any effect of acute or chronic pyridoxine treat-
ment on anterior pituitary hormones in amenorrheic
women [28]. In young children and infants single IV
dose of pyridoxine given at 21:00h, but not when given
at 09:00h, signifi cantly increased serum melatonin lev-
els three hours later. In this study, only two blood sam-
ples were obtained from each subject for the determi-
nation of melatonin levels [29]. These fi ndings are in
contrast with our results and may be due to differences
in the populations studied, the route and dose of pyri-
doxine administered and frequency of blood sampling.
Rat brain serotonin levels were increased after tryp-
tophan load. Coadministration of pyridoxine, signif-
icantly increased serotonin levels in the hypothala-
mus when tryptophan intake was in excess, but not
with a diet of basal tryptophan requirement [30]. Oral
L-5-hydroxytryptophan administered to normal chil-
dren in the late evening hours signifi cantly increased
serum melatonin levels [29].
In our study, oral administration of pyridoxine may
be associated with retarded absorption and hepatic me-
tabolism of pyridoxine, which may alter the available
dose of the vitamin in the circulation [31]. Consider-
ing that our subjects were in good health and probably
were not consuming tryptophan or pyridoxine in ex-
cess, which may account for the lack of effect of pyri-
doxine on melatonin levels in the current study.
Our data suggest that acute oral administration of
100 mg pyridoxine given in the afternoon to normal
adult men has no immediate effect on serum melatonin
levels, core body temperature or sleep quality.
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