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Oral administration and distribution of melatonin in human serum, saliva and urine
Life Sciences, Vol. 37, pp. 489-495 Printed in the U.S.A.
ORAL
ADMINISTRATION
AND
Pergamon Press
D I S T R I B U T I O N OF M E L A T O N I N SALIVA AND URINE
Olli V a k k u r i I, J u h a n i L e p p ~ l u o t o 2 and A n t t i Departments
IN H U M A N
SERUM,
Kauppila 3
of
P h y s i o l o g y I and O b s t e t r i c s and G y n a e c o l o g y 3 U n i v e r s i t y of O u l u K a j a a n i n t i e 52 A, 90220 Oulu, F i n l a n ~ L a b o r a t o r i e s for N e u r o e n d o c r i n o l o g y = The S a l k I n s t i t u t e for B i o l o g i c a l S t u d i e s La Jolla, CA, U.S.A. (Received in final form May 29, 1985) Summary
In o r d e r to e v a l u a t e f o r f u t u r e p h y s i o l o g i c a l a n d pharmacological studies the extent to w h i c h orally administered melatonin is f o u n d in h u m a n serum and s a l i v a and e x c r e t e d into u r i n e w e m e a s u r e d serum, s a l i v a and urine concentrations of m e l a t o n i n by r a d i o i m m u n o assay after oral administration of 100 m g m e l a t o n i n . Elevated melatonin concentrations were observed with p e a k v a l u e s of 435 n m o l / l in s e r u m a n d 241 n m o l / l in s a l i v a at 60 min. Elimination was monophasic following first-order kinetics. The half-lives for serum and s a l i v a m e l a t o n i n w e r e 41 and 38 min, r e s p e c t i v e l y . The results suggest that melatonin is p a s s i v e l y secreted into s a l i v a w h i c h r e f l e c t s c l o s e l y the c h a n g e s in s e r u m melatonin. S a l i v a s a m p l i n g is thus u s e f u l in s t u d i e s on p e r i p h e r a l m e l a t o n i n b o t h in p h y s i o l o g i c a l a n d e x p e r i m e n t a l conditions. U r i n a r y e x c r e t i o n of m e l a t o n i n was 0.01 % of t h e a m o u n t of m e l a t o n i n ingested. In h i g h performance liquid chromatography urine extracts were f o u n d to c o n t a i n a l s o a m i n o r u n k n o w n i m m u n o r e a c t i v e c o m p o n e n t w h i c h we s u g g e s t to be s o m e u n k n o w n m e t a b o l i t e of m e l a t o n i n o The f u n c t i o n s of m e l a t o n i n , a p i n e a l h o r m o n e , are still o b s c u r e in m a n . S y n t h e t i c m e l a t o n i n h a s b e e n u s e d in p h a r m a c o l o g i c a l d o s e s in m a n y s t u d i e s to c l a r i f y its e f f e c t s in t h e h u m a n body. L a r g e p h a r m a c o l o g i c a l d o s e s h a v e a l s o b e e n u s e d for m a n y c l i n i c a l p u r p o s e s ( I ) . W h e n e v a l u a t i n g r e s p o n s e s of m e l a t o n i n in d i f f e r e n t t r i a l s it is i m p o r t a n t to k n o w t h e c o n c e n t r a t i o n s which follow oral a d m i n i s t r a t i o n of m e l a t o n i n . H o w e v e r , d a t a on the fate of an oral d o s e of m e l a t o n i n are scanty. So far m o s t s t u d i e s c o n c e r n i n g the p h a r m a c o k i n e t i c s and m e t a b o l i s m of ~ e l a t o n ~ have been carried out in the rat and the m o u s e u t i l i z i n g J H - or " ~ C - m e l a t o n i n (2-8). In h u m a n s o n l y W e t t e r b e r g et al. (9) in one s u b j e c t and W a l d h a u s e r et al. (I0) have m e a s u r e d s e r u m c o n c e n t r a t i o n s of m e l a t o n i n a f t e r its o r a l a d m i n i s t r a t i o n . In t h e p r e s e n t s t u d y w e h a v e m e a s u r e d s i m u l t a n e o u s l y serum, s a l i v a and u r i n e c o n c e n t r a t i o n s of m e l a t o n i n
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pemgamon Press Ltd.
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a f t e r its oral a d m i n i s t r a t i o n to h e a l t h y s u b j e c t s using a s p e c i f i c and s e n s i t i v e r a d i o i m m u n o a s s a y (RIA) d e v e l o p e d in our l a b o r a t o r y (]I). The m e a s u r e m e n t of s a l i v a c o n c e n t r a t i o n s w a s i n c l u d e d in the study in o r d e r to c l a r i f y the s o u r c e of saliva m e l a t o n i n o b s e r v e d r e c e n t l y in m e a s u r a b l e c o n c e n t r a t i o n s ( 1 2 ) . B a s e d on u r i n e d e t e r m i n a t i o n s the d e g r a d a t i o n of m e l a t o n i n was a l s o calculated.
Methods Five h e a l t h y m a l e v o l u n t e e r s , aged 25-48 years, p a r t i c i p a t e d in this study giving their informed consents. Their body weights r a n g e d f r o m 59 to 78 kg. In t h e m o r n i n g at 0 9 1 5 h ( t w o h o u r s a f t e r a l i g h t b r e a k f a s t ) t h e s u b j e c t s i n g e s t e d a d o s e of 100 m g s y n t h e t i c m e l a t o n i n (Sigma C h e m i c a l Co., St. Louis, MO, U.S.A.) in a single g e l a t i n c a p s u l e w i t h 100 ml of water. Blood s a m p l e s (810 ml) w e r e w i t h d r a w n by a n t e c u b i t a l v e n i p u n c t u r e 0, 15, 30, 60, 120, 240 a n d 360 m i n a f t e r the i n t a k e of m e l a t o n i n into glass tubes and they were allowed to c l o t o v e r n i g h t . Sera were s e p a r a t e d by c e n t r i f u g a t i o n ( 1 , 4 0 0 x g for 10 m i n at +4°C) a n d s t o r e d at -20°C until a s s a y e d for m e l a t o n i n . Saliva samples were t a k e n 0, 30, 60, 120 a n d 240 m i n a f t e r t h e i n t a k e by s h e d d i n g 3-5 ml m i x e d s a l i v a into p o l y e t h y l e n e tubes w h i c h were centrifuged (18,000xg for 10 m i n at +4°C). The s u p e r n a t a n t s were s t o r e d at -20°C before assays. Urine was collected 2 and 6 h after oral a d m i n i s t r a t i o n of m e l a t o n i n and the v o l u m e s w e r e r e c o r d e d for the c a l c u l a t i o n of renal clearance. M e l a t o n i n was e x t r a c t e d and a s s a y e d from the r e f e r e n c e s a m p l e s of s e r u m and s a l i v a as d e s c r i b e d p r e v i o u s l y (11,12). Other serum and saliva samples (0.2 m l ) were extracted with I m l of c h l o r o f o r m , u r i n e s a m p l e s (0.5 ml) w i t h 2 ml of c h l o r o f o r m . After l i g h t v o r t e x i n g (30 s) a n d c e n t r i f u g a t i o n ( 1 , 4 0 0 x g f o r 20 m i n at +4°C) the a q u e o u s p h a s e was d i s c a r d e d and the c h l o r o f o r m phase was w a s h e d w i t h 0.4 m l ( u r i n e s a m p l e s w i t h I ml) of d i s t i l l e d w a t e r . After centrifugation ( 1 , 4 0 0 x g f o r 10 m i n at +4°C) t h e a q u e o u s phase was d i s c a r d e d and the c h l o r o f o r m p h a s e w a s e v a p o r a t e d to d r y n e s s u n d e r vacuum. The r e s i d u e was d i s s o l v e d in 0.5-1.0 ml of RIA b u f f e r and d i l u t e d for m e l a t o n i n R I A (11) if necessary. B e c a u s e o u r R I A w a s n o t c o m p l e t e l y s p e c i f i c f o r m e l a t o n i n in urine determinations (11) the i m m u n o r e a c t i v i t y in the u r i n e extracts was further studied by h i g h - p e r f o r m a n c e liquid chromatography (HPLC). A pooled urine sample (50 pl u r i n e c o l l e c t e d f r o m all s u b j e c t s t w o h o u r s a f t e r a d m i n i s t r a t i o n ) was e x t r a c t e d w i t h c h l o r o f o r m (I ml) and the c h l o r o f o r m p h a s e w a s h e d with distilled water (0.5 m l ) . After evaporation of t h e c h l o r o f o r m p h a s e the r e s i d u e w a s d i s s o l v e d in 0.01 % t r i f l u o r o acetic acid (E. Merck, Darmstadt, Germany). A sample c o r r e s p o n d i n g to 2 ~i o r i g i n a l u r i n e was s u b j e c t e d to HPLC (Varian Liquid Chromatograph, M o d e l 5020, V a r i a n A s s o c i a t e s , Inc., P a l o Alto, California, U.S.A.). A C18 column (MicroPak MCH-10, V a r i a n ) a n d a p r o p a n o l g r a d i e n t e l u t i o n f r o m 20 % to 74 % w a s u s e d in r e v e r s e p h a s e t e c h n i q u e . Synthetic melatonin and 6-hydroxym e l a t o n i n ( S i g m a C h e m i c a l Co., St. L o u i s , MO, U.S.A.) w e r e r u n in s i m i l a r HPLC conditions.
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Results Oral administration of 100 m g m e l a t o n i n resulted in e l e v a t e d melatonin concentrations in h u m a n s e r u m a n d s a l i v a (Fig. I). The maximum concentrations b o t h in s e r u m (435 n m o l / l ) a n d s a l i v a (241 nmol/l) were reached a t 60 m i n . Thereafter the elimination of melatonin in s e r u m a n d s a l i v a w a s m o n o p h a s i c in a s e m i l o g plot. The half-lives calculated f r o m t h e p l o t w e r e 40.8 ± 3.5 m i n f o r serum melatonin a n d 37.8 ± 7.6 m i n ( m e a n f SD, n = 5 ) f o r s a l i v a melatonin. The concentrations in the s e r u m w e r e at all t i m e s 1.84.7 t i m e s higher t h a n in t h e s a l i v a (the m e a n ratio of s e r u m melatonin to s a l i v a melatonin was 2.7). Six hours after the i n t a k e the m e a n s e r u m m e l a t o n i n c o n c e n t r a t i o n was 8 nmol/l. Urin a r y e x c r e t i o n of m e l a t o n i n w a s a b o u t 17.3 ± 6.3 n m o l / h d u r i n g the first two hours and approximately 6.5 ± 3.4 n m o l / h ( m e a n ± SD, n=5) d u r i n g the s u b s e q u e n t f o u r hours. It c a n be c a l c u l a t e d that a b o u t 0.01 % of i n g e s t e d melatonin was excreted into urine un-
1.0
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I
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2
3
4
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FIG.
oral administration
I.
Serum (o------o) a n d s a l i v a (o o) m e l a t o n i n male subjects after oral administration of 100 point represents the m e a n ± SE (n=5).
levels in h e a l t h y mg melatonin. Each
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changed during the first six hours. Urinary m e l a t o n i n w a s 1.1 + 0.6 m l / m i n ( m e a n -+ SD, n=5).
clearance
of
Serum and saliva concentrations of m e l a t o n i n exhibited large individual variations, especially at 30 m i n a n d 60 m i n . The o v e r a l l m e a n c o e f f i c i e n t s of v a r i a t i o n w e r e 88 a n d 85 % for s e r u m and saliva melatonin, respectively. In a d d i t i o n the p r o f i l e s of s e r u m and s a l i v a m e l a t o n i n v a r i e d so t h a t in t h r e e s u b j e c t s a p e a k value was clearly observed at 60 m i n , w h e r e a s in t h e o t h e r t w o subjects the concentration values were still high at 120 min reducing thereafter. No s i d e - e f f e c t s w e r e n o t e d by the s u b j e c t s . Fig. 2 s h o w s H P L C of a s a m p l e of u r i n e e x t r a c t s . The main immunoreactivity co-eluted with synthetic melatonin, but a contaminating immunoreactivity was detectable (20 % of t o t a l immunoreactivity). This eluted before melatonin, but later than 6-hydroxymelatonin.
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20 min 2.
H P L C of a u r i n e e x t r a c t c o r r e s p o n d i n g to 2 Z1 o r i g i n a l u r i n e . Column: MicroPak MCH-10, 300 x 4 m m (10 ~ m ) ; s o l v e n t A: 0.01 % t r i f l u o r o a c e t i c acid; s o l v e n t B: 2 - p r o p a n o l ; g r a d i e n t f r o m 20 % B to 74 % B in A d u r i n g 21 m i n ; f l o w - r a t e 1.0 m l / m i n . 6 - O H - M T a n d M T = e l u t i o n p o s i t i o n s of 6 - h y d r o x y m e l a t o n i n and melatonin.
Discussion In t h i s s t u d y w e f o u n d that orally administered m e l a t o n i n in d o s e s of 1.4 m g / k g e m e r g e d in h u m a n s e r u m a n d s a l i v a a l m o s t p a r a l lelly. The mean peak serum and saliva melatonin levels were o b s e r v e d at 60 m i n a f t e r the intake. T h e r e a f t e r the e l i m i n a t i o n of s e r u m a n d s a l i v a melatonin followed first-order kinetics. S i m i l a r m e l a t o n i n k i n e t i c s has b e e n o b s e r v e d by R e p p e r t and K l e i n
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(7), G i b b s a n d V r i e n d (8) in t h e r a t a n d b y R e p p e r t e~t al. (13) in the r h e s u s m o n k e y a f t e r s y s t e m i c a d m i n i s t r a t i o n of 5 H - m e l a t o n i n . In a p r e l i m i n a r y s t u d y on o n e m a l e s u b j e c t (9) a s i m i l a r p r o f i l e w a s o b t a i n e d f o r s e r u m m e l a t o n i n a f t e r its o r a l a d m i n i s t r a t i o n w i t h t h e e x c e p t i o n of t h e p e a k c o n c e n t r a t i o n at 30 min. In t h e l a t t e r s t u d y m e l a t o n i n w a s i n g e s t e d in a w a t e r s o l u t i o n , w h i c h m i g h t e x p l a i n t h e e a r l i e r p e a k v a l u e in s e r u m m e l a t o n i n . In a r e c e n t s t u d y W a l d h a u s e r et al. (10) found that a f t e r oral a d m i n i s tration melatonin l e v e l s in h u m a n s e r u m r e m a i n e d e l e v a t e d f o r a b o u t 1.5 h, as w a s a l s o f o u n d in t w o s u b j e c t s in t h e p r e s e n t study. T h o s e a u t h o r s o b s e r v e d g r e a t d i f f e r e n c e s in a b s o r p t i o n of i n g e s t e d m e l a t o n i n b e t w e e n subjects, w h i c h is in a c c o r d a n c e w i t h our observations of 1 0 - 2 0 - f o l d v a r i a t i o n s in s e r u m a n d s a l i v a melatonin. Thus, variable gastrointestinal absorption of m e l a t o n i n has to be t a k e n into a c c o u n t w h e n s y n t h e t i c m e l a t o n i n is a d m i n i s t e r e d to h u m a n s orally. In a g r e e m e n t w i t h t h e f i n d i n g s of W e t t e r b e r g et al. (9) a n d W a l d h a u s e r et al. (10) w e o b s e r v e d t h a t t h e p e a k s e r u m m e l a t o n i n c o n c e n t r a t i o n s w e r e 3 0 0 - 3 7 0 0 times, and six h o u r s a f t e r the i n t a k e still 10-30 t i m e s , h i g h e r than p h y s i o l o g i c a l s e r u m c o n c e n t r a t i o n s at n i g h t (11). T h e r e f o r e it can be c o n c l u d e d that in m o s t s t u d i e s t h e d o s e of m e l a t o n i n u s e d to e v o k e p h y s i o l o g i c a l r e s p o n s e s h a s b e e n far too large if n o r m a l e n d o g e n o u s m e l a t o n i n c o n c e n t r a t i o n s at n i g h t are used as a r e f e r e n c e level. F r o m the p r e s e n t r e s u l t s it c a n be e s t i m a t e d that a melatonin d o s e of 1.0-1.5 ~ g / k g is e n o u g h to r a i s e b l o o d m e l a t o n i n to h i g h n o c t u r n a l c o n c e n t r a t i o n s . In s h e e p a n d g o a t s o r a l d o s e s of 2 m g in t h e d a y t i m e h a v e b e e n found s u i t a b l e to r a i s e p l a s m a m e l a t o n i n c o n c e n t r a t i o n s w i t h i n the n o r m a l n i g h t - t i m e r a n g e (14). In t h e p r e s e n t s t u d y m e l a t o n i n w a s f o u n d in h u m a n s a l i v a in c o n c e n t r a t i o n s w h i c h t h r o u g h o u t the e x p e r i m e n t c o r r e l a t e d to 2156 % of s e r u m c o n c e n t r a t i o n s . A s i m i l a r r a t i o w a s r e c e n t l y found also between serum and saliva concentrations of e n d o g e n o u s m e l a t o n i n at n i g h t (12). Taking into account also the parallel p r o f i l e s of s e r u m a n d s a l i v a m e l a t o n i n it c a n be c o n c l u d e d t h a t m e l a t o n i n , l i k e s t e r o i d h o r m o n e s (15), diffuses passively from blood into saliva. Hence, simple, stress-free and noninvasive s a l i v a s a m p l i n g is of c l i n i c a l v a l u e in m o n i t o r i n g c i r c u l a t i n g melatonin. In e a r l i e r s t u d i e s the h a l f - l i f e of m e l a t o n i n has b e e n r e p o r t e d to be b e t w e e n 2 a n d 44 m i n . T h e p r e s e n t h a l f - l i f e for blood m e l a t o n i n in h u m a n s w a s 41 m i n as m e a s u r e d by o u r R I A (11) a f t e r chloroform e x t r a c t i o n . T h i s v a l u e is of t h e s a m e m a g n i t u d e as t h o s e r e p o r t e d by W e t t e r b e r g et al. (9), W a l d h a u s e r et al. (10) after oral administration and Iguchi et al. (16) a f t e r iv injection, but far g r e a t e r t h a n those r e p o r t e d for b l o o d m e l a t o n i n in the rat (17) or total body 3 H - m e l a t o n i n in the m o u s e (3) a f t e r systemic administration. R o l l a g et al. (18) h a v e o b t a i n e d a halfl i f e of 3 - 1 0 m i n a f t e r iv i n j e c t i o n of m e l a t o n i n in t h e s h e e p , b u t Kennaway & Seamark (14) f o u n d a h a l f - l i f e of 30 m i n a f t e r subcutaneous injection. In the s h e e p s e r u m m e l a t o n i n r e m a i n e d elevated for several hours after oral administration, which was p r o b a b l y due to the m i x i n g of m e l a t o n i n w i t h the c o n t e n t s of the c o m m o d i o u s r u m e n and its s u b s e q u e n t s l o w r e l e a s e (14).
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T h e w i d e v a r i e t y in the r e s u l t s c o n c e r n i n g t h e h a l f - l i f e of m e l a t o n i n m a y be p a r t l y due to d i f f e r e n t routes of a d m i n i s t r a t i o n , different m e t h o d s u s e d for the d e t e r m i n a t i o n of m e l a t o n i n or s p e c i e s - s p e c i f i c d i f f e r e n c e s , but m a i n l y to the c a l c u l a t i o n of the h a l f - l i f e as d i s c u s s e d by G i b b s a n d V r i e n d (8). All half-lives reported are not comparable with each other because short halflives have been calculated d u r i n g the d i s t r i b u t i o n phase and longer h a l f - l i v e s d u r i n g the e l i m i n a t i o n phase. K o p i n et al. (3) have reported two half-lives: the first (2 m i n ) d u r i n g the d i s t r i b u t i o n phase and the segond (35 min) d u r i n g the e l i m i n a t i o n p h a s e a f t e r iv i n j e c t i o n of ] H - m e l a t o n i n in t h e m o u s e . A l t h o u g h the s e c o n d h a l f - l i f e d e p i c t s b e t t e r h a l v i n g of m e l a t o n i n the first v a l u e has b e e n w i d e l y u s e d to c h a r a c t e r i z e p h a r m a c o k i n e t i c s of melatonin. Melatonin is metabolized chiefly in the liver by 6h y d r o x y l a t i o n (2-5, 19) and a c c o r d i n g to a r e c e n t o b s e r v a t i o n (20) by O - d e m e t h y l a t i o n , b u t a l s o in t h e b r a i n b y k y n u r e n a m i n e f o r m a t i o n (21). P a r d r i d g e and M i e t u s (22) have c a l c u l a t e d in the rat that m e l a t o n i n is u n i d i r e c t i o n a l l y c l e a r e d 92-97 % on a single pass t h r o u g h o u t the liver by a n o n s a t u r a b l e m e c h a n i s m . In a c c o r d ance with these findings we found that exogenous melatonin is almost completely metabolized. D u r i n g t h e f i r s t six h o u r s o n l y 0.01 % of i n g e s t e d m e l a t o n i n w a s f o u n d u n c h a n g e d in t h e u r i n e , w h i c h is o v e r I0 t i m e s l e s s t h a n in the s t u d y of W e t t e r b e r g et al. (9). The clearance values of 1 m l / m i n refer to p a s s i v e r e a b s o r p t i o n of m e l a t o n i n in renal tubules. As in o u r p r e v i o u s u r i n e d e t e r m i n a t i o n s (11) we o b s e r v e d a l s o in the p r e s e n t determinations an u n k n o w n immunoreactivity, a l t h o u g h the u r i n e s a m p l e used in HPLC was 500 t i m e s s m a l l e r than in the p r e v i o u s s t u d y . T a k i n g i n t o a c c o u n t a l s o the e l u t i o n p o s i t i o n of this u n k n o w n i m m u n o r e a c t i v i t y b e t w e e n m e l a t o n i n and 6h y d r o x y m e l a t o n i n , it can be c o n c l u d e d that it is s o m e m e t a b o l i t e of m e l a t o n i n . The c h a r a c t e r i z a t i o n of this u n k n o w n m e t a b o l i t e is in p r o g r e s s in our laboratory. D e s p i t e the high d o s e of m e l a t o n i n the s u b j e c t s in the p r e s e n t study did not e x p e r i e n c e d r o w s i n e s s or other s u b j e c t i v e f e e l i n g s r e p o r t e d in s o m e p r e v i o u s s t u d i e s (23-26). In t h i s r e s p e c t t h e a d m i n i s t r a t i o n t i m e is p r o b a b l y the m o s t c r i t i c a l factor, b e c a u s e t h e r e is a d i u r n a l v a r i a t i o n in m e l a t o n i n b i n d i n g s i t e s . In the m o r n i n g t h e r e is i n s e n s i t i v i t y d u e to m e l a t o n i n r e c e p t o r d o w n regulation (27). Thus, melatonin may have significance in the i n d u c t i o n of n o c t u r n a l sleep.
Acknowledgements This
study was
s u p p o r t e d by the E m i l
A a l t o n e n Foundation.
References I. A.B. L E R N E R a n d J.J. N O R D L U N D , J. N e u r a l T r a n s m . , 3 3 9 - 3 4 7 (1978). 2. I.J. K O P I N , C.M.B. PARE, J° A X E L R O D a n d H. W E I S S B A C H , Biophys. Acta 4 0 377-378 (1960).
Suppl.
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3. I.J. K O P I N , C.M.B. P A R E , J. A X E L R O D a n d H. W E I S S B A C H , J. B i o l . C h e m . 236 3 0 7 2 - 3 0 7 5 (1961). 4. S. K V E D E R a n d W.M. M C I S A A C , J. B i o l . C h e m . 2 3 6 321 4 - 3 2 2 0 (1961). 5. R.G. T A B O R S K Y , P. D E L V I G S a n d I.H. P A G E , J. M e d . C h e m . 8 8 5 5 8 5 8 (1965). 6. D.P. C A R D I N A L I , M.T. H Y Y P P A a n d R.J. W U R T M A N , Neuroendoc r i n o l o g y 1 2 3 0 - 4 0 (1973). 7. S.M. R E P P E R T a n d D.C. KLEIN, E n d o c r i n o l o g y 102 5 8 2 - 5 8 8 (1978). 8. F.P. G I B B S and J. V R I E N D , E n d o c r i n o l o g y 109 1 7 9 6 - 1 7 9 8 (I 981 ). °. L. W E T T E R B E R G , O. E R I K S S O N , Y. F R I B E R G a n d B. V A N G B O , Clin. Chim. A c t a 86 1 6 9 - 1 7 7 (1978). I0. F. W A L D H A U S E R , M. W A L D H A U S E R , H.R. L I E B E R M A N , M.-H. D E N G , H.J. L Y N C H and R.J. W U R T M A N , N e u r o e n d o c r i n o l o g y 3 9 3 0 7 - 3 1 3 (1984). 11 . O. VAKKURI, J. LEPPALUOTO and O. VUOLTEENAHO, Acta Endocrinol. ( C o p e n h . ) 106 1 5 2 - 1 5 7 (1984). 12. O. V A K K U R I , A c t a P h y s i o l . S c a n d . ( 1 9 8 5 ) , in p r e s s . 1 3. S . M . R E P P E R T , M.J. P E R L O W , L. T A M A R K I N and D.C. K L E I N , Endocrinology 104 295-301 (1979). 1 4. D.J. K E N N A W A Y a n d R.F. S E A M A R K , A u s t . J. B i o l . Sci. 33 3 4 9 353 (1980). 15. R.F. V I N I N G a n d R.A. M C G I N L E Y , Front. oral Physiol. 5 21-32 (1984). 16 H. I G U C H I , K.-I. K A T O a n d H. I B A Y A S H I , J. C l i n . E n d o c r i n o l . M e t a b . 54 1 0 2 5 - 1 0 2 7 (1982). 17 Y. O Z A K I , H.J. L Y N C H a n d R.J. W U R T M A N , Endocrinology 98 14181 4 2 4 (1976). 18 M.D. R O L L A G , R.J. M O R G A N a n d G.D. N I S W E N D E R , Endocrinology 102 I-8 (1978). 19 R.L. J O N E S , P.L. M C G E E R a n d A.C. G R E I N E R , C l i n . C h i m . A c t a 26 2 8 1 - 2 8 5 (1969). 20 R.M. L E O N E a n d R.E. S I L M A N , Endocrinology 11 4 1825-1832 (1984). 21 F. H I R A T A , O. H A Y A I S H I , T. T O K U Y A M A a n d S. S E N O H , J. B i o l . Chem. 249 1 3 1 1 - 1 3 1 3 (1974). 22. W.M. P A R D R I D G E a n d L.J. M I E T U S , J. N e u r o c h e m . 34 1 761 -I 7 6 3 (1980). 23. F. A N T O N - T A Y , J.L. D I A Z a n d A. F E R N A N D E Z - G U A R D I O L A , L i f e Sci. I0 841 -850 (I 971 ). 24. J.J. N O R D L U N D a n d A.B. L E R N E R , J. C l i n . E n d o c r i n o l . M e t a b . 45 7 6 8 - 7 7 4 (I 977). 25. H. C R A M E R , J. R U D O L P H , U. C O N S B R U C H a n d K. K E N D E L , Adv. Biochem. Psychopharmacol. 11 187-191 (I 974). 26. L. V O L L R A T H , P. S E M M a n d G. G A M M E L , A d v . B i o s c i . 29 3 2 7 - 3 2 9 (1981). 27. M.I. V A C A S a n d D.P. C A R D I N A L I , Neurosci. Lett. 15 2 5 9 - 2 6 3 (1979).
ORAL
ADMINISTRATION
AND
Pergamon Press
D I S T R I B U T I O N OF M E L A T O N I N SALIVA AND URINE
Olli V a k k u r i I, J u h a n i L e p p ~ l u o t o 2 and A n t t i Departments
IN H U M A N
SERUM,
Kauppila 3
of
P h y s i o l o g y I and O b s t e t r i c s and G y n a e c o l o g y 3 U n i v e r s i t y of O u l u K a j a a n i n t i e 52 A, 90220 Oulu, F i n l a n ~ L a b o r a t o r i e s for N e u r o e n d o c r i n o l o g y = The S a l k I n s t i t u t e for B i o l o g i c a l S t u d i e s La Jolla, CA, U.S.A. (Received in final form May 29, 1985) Summary
In o r d e r to e v a l u a t e f o r f u t u r e p h y s i o l o g i c a l a n d pharmacological studies the extent to w h i c h orally administered melatonin is f o u n d in h u m a n serum and s a l i v a and e x c r e t e d into u r i n e w e m e a s u r e d serum, s a l i v a and urine concentrations of m e l a t o n i n by r a d i o i m m u n o assay after oral administration of 100 m g m e l a t o n i n . Elevated melatonin concentrations were observed with p e a k v a l u e s of 435 n m o l / l in s e r u m a n d 241 n m o l / l in s a l i v a at 60 min. Elimination was monophasic following first-order kinetics. The half-lives for serum and s a l i v a m e l a t o n i n w e r e 41 and 38 min, r e s p e c t i v e l y . The results suggest that melatonin is p a s s i v e l y secreted into s a l i v a w h i c h r e f l e c t s c l o s e l y the c h a n g e s in s e r u m melatonin. S a l i v a s a m p l i n g is thus u s e f u l in s t u d i e s on p e r i p h e r a l m e l a t o n i n b o t h in p h y s i o l o g i c a l a n d e x p e r i m e n t a l conditions. U r i n a r y e x c r e t i o n of m e l a t o n i n was 0.01 % of t h e a m o u n t of m e l a t o n i n ingested. In h i g h performance liquid chromatography urine extracts were f o u n d to c o n t a i n a l s o a m i n o r u n k n o w n i m m u n o r e a c t i v e c o m p o n e n t w h i c h we s u g g e s t to be s o m e u n k n o w n m e t a b o l i t e of m e l a t o n i n o The f u n c t i o n s of m e l a t o n i n , a p i n e a l h o r m o n e , are still o b s c u r e in m a n . S y n t h e t i c m e l a t o n i n h a s b e e n u s e d in p h a r m a c o l o g i c a l d o s e s in m a n y s t u d i e s to c l a r i f y its e f f e c t s in t h e h u m a n body. L a r g e p h a r m a c o l o g i c a l d o s e s h a v e a l s o b e e n u s e d for m a n y c l i n i c a l p u r p o s e s ( I ) . W h e n e v a l u a t i n g r e s p o n s e s of m e l a t o n i n in d i f f e r e n t t r i a l s it is i m p o r t a n t to k n o w t h e c o n c e n t r a t i o n s which follow oral a d m i n i s t r a t i o n of m e l a t o n i n . H o w e v e r , d a t a on the fate of an oral d o s e of m e l a t o n i n are scanty. So far m o s t s t u d i e s c o n c e r n i n g the p h a r m a c o k i n e t i c s and m e t a b o l i s m of ~ e l a t o n ~ have been carried out in the rat and the m o u s e u t i l i z i n g J H - or " ~ C - m e l a t o n i n (2-8). In h u m a n s o n l y W e t t e r b e r g et al. (9) in one s u b j e c t and W a l d h a u s e r et al. (I0) have m e a s u r e d s e r u m c o n c e n t r a t i o n s of m e l a t o n i n a f t e r its o r a l a d m i n i s t r a t i o n . In t h e p r e s e n t s t u d y w e h a v e m e a s u r e d s i m u l t a n e o u s l y serum, s a l i v a and u r i n e c o n c e n t r a t i o n s of m e l a t o n i n
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pemgamon Press Ltd.
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a f t e r its oral a d m i n i s t r a t i o n to h e a l t h y s u b j e c t s using a s p e c i f i c and s e n s i t i v e r a d i o i m m u n o a s s a y (RIA) d e v e l o p e d in our l a b o r a t o r y (]I). The m e a s u r e m e n t of s a l i v a c o n c e n t r a t i o n s w a s i n c l u d e d in the study in o r d e r to c l a r i f y the s o u r c e of saliva m e l a t o n i n o b s e r v e d r e c e n t l y in m e a s u r a b l e c o n c e n t r a t i o n s ( 1 2 ) . B a s e d on u r i n e d e t e r m i n a t i o n s the d e g r a d a t i o n of m e l a t o n i n was a l s o calculated.
Methods Five h e a l t h y m a l e v o l u n t e e r s , aged 25-48 years, p a r t i c i p a t e d in this study giving their informed consents. Their body weights r a n g e d f r o m 59 to 78 kg. In t h e m o r n i n g at 0 9 1 5 h ( t w o h o u r s a f t e r a l i g h t b r e a k f a s t ) t h e s u b j e c t s i n g e s t e d a d o s e of 100 m g s y n t h e t i c m e l a t o n i n (Sigma C h e m i c a l Co., St. Louis, MO, U.S.A.) in a single g e l a t i n c a p s u l e w i t h 100 ml of water. Blood s a m p l e s (810 ml) w e r e w i t h d r a w n by a n t e c u b i t a l v e n i p u n c t u r e 0, 15, 30, 60, 120, 240 a n d 360 m i n a f t e r the i n t a k e of m e l a t o n i n into glass tubes and they were allowed to c l o t o v e r n i g h t . Sera were s e p a r a t e d by c e n t r i f u g a t i o n ( 1 , 4 0 0 x g for 10 m i n at +4°C) a n d s t o r e d at -20°C until a s s a y e d for m e l a t o n i n . Saliva samples were t a k e n 0, 30, 60, 120 a n d 240 m i n a f t e r t h e i n t a k e by s h e d d i n g 3-5 ml m i x e d s a l i v a into p o l y e t h y l e n e tubes w h i c h were centrifuged (18,000xg for 10 m i n at +4°C). The s u p e r n a t a n t s were s t o r e d at -20°C before assays. Urine was collected 2 and 6 h after oral a d m i n i s t r a t i o n of m e l a t o n i n and the v o l u m e s w e r e r e c o r d e d for the c a l c u l a t i o n of renal clearance. M e l a t o n i n was e x t r a c t e d and a s s a y e d from the r e f e r e n c e s a m p l e s of s e r u m and s a l i v a as d e s c r i b e d p r e v i o u s l y (11,12). Other serum and saliva samples (0.2 m l ) were extracted with I m l of c h l o r o f o r m , u r i n e s a m p l e s (0.5 ml) w i t h 2 ml of c h l o r o f o r m . After l i g h t v o r t e x i n g (30 s) a n d c e n t r i f u g a t i o n ( 1 , 4 0 0 x g f o r 20 m i n at +4°C) the a q u e o u s p h a s e was d i s c a r d e d and the c h l o r o f o r m phase was w a s h e d w i t h 0.4 m l ( u r i n e s a m p l e s w i t h I ml) of d i s t i l l e d w a t e r . After centrifugation ( 1 , 4 0 0 x g f o r 10 m i n at +4°C) t h e a q u e o u s phase was d i s c a r d e d and the c h l o r o f o r m p h a s e w a s e v a p o r a t e d to d r y n e s s u n d e r vacuum. The r e s i d u e was d i s s o l v e d in 0.5-1.0 ml of RIA b u f f e r and d i l u t e d for m e l a t o n i n R I A (11) if necessary. B e c a u s e o u r R I A w a s n o t c o m p l e t e l y s p e c i f i c f o r m e l a t o n i n in urine determinations (11) the i m m u n o r e a c t i v i t y in the u r i n e extracts was further studied by h i g h - p e r f o r m a n c e liquid chromatography (HPLC). A pooled urine sample (50 pl u r i n e c o l l e c t e d f r o m all s u b j e c t s t w o h o u r s a f t e r a d m i n i s t r a t i o n ) was e x t r a c t e d w i t h c h l o r o f o r m (I ml) and the c h l o r o f o r m p h a s e w a s h e d with distilled water (0.5 m l ) . After evaporation of t h e c h l o r o f o r m p h a s e the r e s i d u e w a s d i s s o l v e d in 0.01 % t r i f l u o r o acetic acid (E. Merck, Darmstadt, Germany). A sample c o r r e s p o n d i n g to 2 ~i o r i g i n a l u r i n e was s u b j e c t e d to HPLC (Varian Liquid Chromatograph, M o d e l 5020, V a r i a n A s s o c i a t e s , Inc., P a l o Alto, California, U.S.A.). A C18 column (MicroPak MCH-10, V a r i a n ) a n d a p r o p a n o l g r a d i e n t e l u t i o n f r o m 20 % to 74 % w a s u s e d in r e v e r s e p h a s e t e c h n i q u e . Synthetic melatonin and 6-hydroxym e l a t o n i n ( S i g m a C h e m i c a l Co., St. L o u i s , MO, U.S.A.) w e r e r u n in s i m i l a r HPLC conditions.
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Results Oral administration of 100 m g m e l a t o n i n resulted in e l e v a t e d melatonin concentrations in h u m a n s e r u m a n d s a l i v a (Fig. I). The maximum concentrations b o t h in s e r u m (435 n m o l / l ) a n d s a l i v a (241 nmol/l) were reached a t 60 m i n . Thereafter the elimination of melatonin in s e r u m a n d s a l i v a w a s m o n o p h a s i c in a s e m i l o g plot. The half-lives calculated f r o m t h e p l o t w e r e 40.8 ± 3.5 m i n f o r serum melatonin a n d 37.8 ± 7.6 m i n ( m e a n f SD, n = 5 ) f o r s a l i v a melatonin. The concentrations in the s e r u m w e r e at all t i m e s 1.84.7 t i m e s higher t h a n in t h e s a l i v a (the m e a n ratio of s e r u m melatonin to s a l i v a melatonin was 2.7). Six hours after the i n t a k e the m e a n s e r u m m e l a t o n i n c o n c e n t r a t i o n was 8 nmol/l. Urin a r y e x c r e t i o n of m e l a t o n i n w a s a b o u t 17.3 ± 6.3 n m o l / h d u r i n g the first two hours and approximately 6.5 ± 3.4 n m o l / h ( m e a n ± SD, n=5) d u r i n g the s u b s e q u e n t f o u r hours. It c a n be c a l c u l a t e d that a b o u t 0.01 % of i n g e s t e d melatonin was excreted into urine un-
1.0
<
"5 E 0.1 ¢.. ¢-
o
I
I
I
I
I
I
I
1
2
3
4
5
6
h
Time
after
FIG.
oral administration
I.
Serum (o------o) a n d s a l i v a (o o) m e l a t o n i n male subjects after oral administration of 100 point represents the m e a n ± SE (n=5).
levels in h e a l t h y mg melatonin. Each
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Melatonin After Oral Administration
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changed during the first six hours. Urinary m e l a t o n i n w a s 1.1 + 0.6 m l / m i n ( m e a n -+ SD, n=5).
clearance
of
Serum and saliva concentrations of m e l a t o n i n exhibited large individual variations, especially at 30 m i n a n d 60 m i n . The o v e r a l l m e a n c o e f f i c i e n t s of v a r i a t i o n w e r e 88 a n d 85 % for s e r u m and saliva melatonin, respectively. In a d d i t i o n the p r o f i l e s of s e r u m and s a l i v a m e l a t o n i n v a r i e d so t h a t in t h r e e s u b j e c t s a p e a k value was clearly observed at 60 m i n , w h e r e a s in t h e o t h e r t w o subjects the concentration values were still high at 120 min reducing thereafter. No s i d e - e f f e c t s w e r e n o t e d by the s u b j e c t s . Fig. 2 s h o w s H P L C of a s a m p l e of u r i n e e x t r a c t s . The main immunoreactivity co-eluted with synthetic melatonin, but a contaminating immunoreactivity was detectable (20 % of t o t a l immunoreactivity). This eluted before melatonin, but later than 6-hydroxymelatonin.
-100
MT 200 O
E 6-OH-MT
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10 FIG.
20 min 2.
H P L C of a u r i n e e x t r a c t c o r r e s p o n d i n g to 2 Z1 o r i g i n a l u r i n e . Column: MicroPak MCH-10, 300 x 4 m m (10 ~ m ) ; s o l v e n t A: 0.01 % t r i f l u o r o a c e t i c acid; s o l v e n t B: 2 - p r o p a n o l ; g r a d i e n t f r o m 20 % B to 74 % B in A d u r i n g 21 m i n ; f l o w - r a t e 1.0 m l / m i n . 6 - O H - M T a n d M T = e l u t i o n p o s i t i o n s of 6 - h y d r o x y m e l a t o n i n and melatonin.
Discussion In t h i s s t u d y w e f o u n d that orally administered m e l a t o n i n in d o s e s of 1.4 m g / k g e m e r g e d in h u m a n s e r u m a n d s a l i v a a l m o s t p a r a l lelly. The mean peak serum and saliva melatonin levels were o b s e r v e d at 60 m i n a f t e r the intake. T h e r e a f t e r the e l i m i n a t i o n of s e r u m a n d s a l i v a melatonin followed first-order kinetics. S i m i l a r m e l a t o n i n k i n e t i c s has b e e n o b s e r v e d by R e p p e r t and K l e i n
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(7), G i b b s a n d V r i e n d (8) in t h e r a t a n d b y R e p p e r t e~t al. (13) in the r h e s u s m o n k e y a f t e r s y s t e m i c a d m i n i s t r a t i o n of 5 H - m e l a t o n i n . In a p r e l i m i n a r y s t u d y on o n e m a l e s u b j e c t (9) a s i m i l a r p r o f i l e w a s o b t a i n e d f o r s e r u m m e l a t o n i n a f t e r its o r a l a d m i n i s t r a t i o n w i t h t h e e x c e p t i o n of t h e p e a k c o n c e n t r a t i o n at 30 min. In t h e l a t t e r s t u d y m e l a t o n i n w a s i n g e s t e d in a w a t e r s o l u t i o n , w h i c h m i g h t e x p l a i n t h e e a r l i e r p e a k v a l u e in s e r u m m e l a t o n i n . In a r e c e n t s t u d y W a l d h a u s e r et al. (10) found that a f t e r oral a d m i n i s tration melatonin l e v e l s in h u m a n s e r u m r e m a i n e d e l e v a t e d f o r a b o u t 1.5 h, as w a s a l s o f o u n d in t w o s u b j e c t s in t h e p r e s e n t study. T h o s e a u t h o r s o b s e r v e d g r e a t d i f f e r e n c e s in a b s o r p t i o n of i n g e s t e d m e l a t o n i n b e t w e e n subjects, w h i c h is in a c c o r d a n c e w i t h our observations of 1 0 - 2 0 - f o l d v a r i a t i o n s in s e r u m a n d s a l i v a melatonin. Thus, variable gastrointestinal absorption of m e l a t o n i n has to be t a k e n into a c c o u n t w h e n s y n t h e t i c m e l a t o n i n is a d m i n i s t e r e d to h u m a n s orally. In a g r e e m e n t w i t h t h e f i n d i n g s of W e t t e r b e r g et al. (9) a n d W a l d h a u s e r et al. (10) w e o b s e r v e d t h a t t h e p e a k s e r u m m e l a t o n i n c o n c e n t r a t i o n s w e r e 3 0 0 - 3 7 0 0 times, and six h o u r s a f t e r the i n t a k e still 10-30 t i m e s , h i g h e r than p h y s i o l o g i c a l s e r u m c o n c e n t r a t i o n s at n i g h t (11). T h e r e f o r e it can be c o n c l u d e d that in m o s t s t u d i e s t h e d o s e of m e l a t o n i n u s e d to e v o k e p h y s i o l o g i c a l r e s p o n s e s h a s b e e n far too large if n o r m a l e n d o g e n o u s m e l a t o n i n c o n c e n t r a t i o n s at n i g h t are used as a r e f e r e n c e level. F r o m the p r e s e n t r e s u l t s it c a n be e s t i m a t e d that a melatonin d o s e of 1.0-1.5 ~ g / k g is e n o u g h to r a i s e b l o o d m e l a t o n i n to h i g h n o c t u r n a l c o n c e n t r a t i o n s . In s h e e p a n d g o a t s o r a l d o s e s of 2 m g in t h e d a y t i m e h a v e b e e n found s u i t a b l e to r a i s e p l a s m a m e l a t o n i n c o n c e n t r a t i o n s w i t h i n the n o r m a l n i g h t - t i m e r a n g e (14). In t h e p r e s e n t s t u d y m e l a t o n i n w a s f o u n d in h u m a n s a l i v a in c o n c e n t r a t i o n s w h i c h t h r o u g h o u t the e x p e r i m e n t c o r r e l a t e d to 2156 % of s e r u m c o n c e n t r a t i o n s . A s i m i l a r r a t i o w a s r e c e n t l y found also between serum and saliva concentrations of e n d o g e n o u s m e l a t o n i n at n i g h t (12). Taking into account also the parallel p r o f i l e s of s e r u m a n d s a l i v a m e l a t o n i n it c a n be c o n c l u d e d t h a t m e l a t o n i n , l i k e s t e r o i d h o r m o n e s (15), diffuses passively from blood into saliva. Hence, simple, stress-free and noninvasive s a l i v a s a m p l i n g is of c l i n i c a l v a l u e in m o n i t o r i n g c i r c u l a t i n g melatonin. In e a r l i e r s t u d i e s the h a l f - l i f e of m e l a t o n i n has b e e n r e p o r t e d to be b e t w e e n 2 a n d 44 m i n . T h e p r e s e n t h a l f - l i f e for blood m e l a t o n i n in h u m a n s w a s 41 m i n as m e a s u r e d by o u r R I A (11) a f t e r chloroform e x t r a c t i o n . T h i s v a l u e is of t h e s a m e m a g n i t u d e as t h o s e r e p o r t e d by W e t t e r b e r g et al. (9), W a l d h a u s e r et al. (10) after oral administration and Iguchi et al. (16) a f t e r iv injection, but far g r e a t e r t h a n those r e p o r t e d for b l o o d m e l a t o n i n in the rat (17) or total body 3 H - m e l a t o n i n in the m o u s e (3) a f t e r systemic administration. R o l l a g et al. (18) h a v e o b t a i n e d a halfl i f e of 3 - 1 0 m i n a f t e r iv i n j e c t i o n of m e l a t o n i n in t h e s h e e p , b u t Kennaway & Seamark (14) f o u n d a h a l f - l i f e of 30 m i n a f t e r subcutaneous injection. In the s h e e p s e r u m m e l a t o n i n r e m a i n e d elevated for several hours after oral administration, which was p r o b a b l y due to the m i x i n g of m e l a t o n i n w i t h the c o n t e n t s of the c o m m o d i o u s r u m e n and its s u b s e q u e n t s l o w r e l e a s e (14).
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T h e w i d e v a r i e t y in the r e s u l t s c o n c e r n i n g t h e h a l f - l i f e of m e l a t o n i n m a y be p a r t l y due to d i f f e r e n t routes of a d m i n i s t r a t i o n , different m e t h o d s u s e d for the d e t e r m i n a t i o n of m e l a t o n i n or s p e c i e s - s p e c i f i c d i f f e r e n c e s , but m a i n l y to the c a l c u l a t i o n of the h a l f - l i f e as d i s c u s s e d by G i b b s a n d V r i e n d (8). All half-lives reported are not comparable with each other because short halflives have been calculated d u r i n g the d i s t r i b u t i o n phase and longer h a l f - l i v e s d u r i n g the e l i m i n a t i o n phase. K o p i n et al. (3) have reported two half-lives: the first (2 m i n ) d u r i n g the d i s t r i b u t i o n phase and the segond (35 min) d u r i n g the e l i m i n a t i o n p h a s e a f t e r iv i n j e c t i o n of ] H - m e l a t o n i n in t h e m o u s e . A l t h o u g h the s e c o n d h a l f - l i f e d e p i c t s b e t t e r h a l v i n g of m e l a t o n i n the first v a l u e has b e e n w i d e l y u s e d to c h a r a c t e r i z e p h a r m a c o k i n e t i c s of melatonin. Melatonin is metabolized chiefly in the liver by 6h y d r o x y l a t i o n (2-5, 19) and a c c o r d i n g to a r e c e n t o b s e r v a t i o n (20) by O - d e m e t h y l a t i o n , b u t a l s o in t h e b r a i n b y k y n u r e n a m i n e f o r m a t i o n (21). P a r d r i d g e and M i e t u s (22) have c a l c u l a t e d in the rat that m e l a t o n i n is u n i d i r e c t i o n a l l y c l e a r e d 92-97 % on a single pass t h r o u g h o u t the liver by a n o n s a t u r a b l e m e c h a n i s m . In a c c o r d ance with these findings we found that exogenous melatonin is almost completely metabolized. D u r i n g t h e f i r s t six h o u r s o n l y 0.01 % of i n g e s t e d m e l a t o n i n w a s f o u n d u n c h a n g e d in t h e u r i n e , w h i c h is o v e r I0 t i m e s l e s s t h a n in the s t u d y of W e t t e r b e r g et al. (9). The clearance values of 1 m l / m i n refer to p a s s i v e r e a b s o r p t i o n of m e l a t o n i n in renal tubules. As in o u r p r e v i o u s u r i n e d e t e r m i n a t i o n s (11) we o b s e r v e d a l s o in the p r e s e n t determinations an u n k n o w n immunoreactivity, a l t h o u g h the u r i n e s a m p l e used in HPLC was 500 t i m e s s m a l l e r than in the p r e v i o u s s t u d y . T a k i n g i n t o a c c o u n t a l s o the e l u t i o n p o s i t i o n of this u n k n o w n i m m u n o r e a c t i v i t y b e t w e e n m e l a t o n i n and 6h y d r o x y m e l a t o n i n , it can be c o n c l u d e d that it is s o m e m e t a b o l i t e of m e l a t o n i n . The c h a r a c t e r i z a t i o n of this u n k n o w n m e t a b o l i t e is in p r o g r e s s in our laboratory. D e s p i t e the high d o s e of m e l a t o n i n the s u b j e c t s in the p r e s e n t study did not e x p e r i e n c e d r o w s i n e s s or other s u b j e c t i v e f e e l i n g s r e p o r t e d in s o m e p r e v i o u s s t u d i e s (23-26). In t h i s r e s p e c t t h e a d m i n i s t r a t i o n t i m e is p r o b a b l y the m o s t c r i t i c a l factor, b e c a u s e t h e r e is a d i u r n a l v a r i a t i o n in m e l a t o n i n b i n d i n g s i t e s . In the m o r n i n g t h e r e is i n s e n s i t i v i t y d u e to m e l a t o n i n r e c e p t o r d o w n regulation (27). Thus, melatonin may have significance in the i n d u c t i o n of n o c t u r n a l sleep.
Acknowledgements This
study was
s u p p o r t e d by the E m i l
A a l t o n e n Foundation.
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