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Current Status of Assay Methods of Melatonin
Current Status of Assay Methods of Melatonin J. Arendt Division of Clinical Biochemistry, Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH, UK
ABSTRACT Current available methodology for the assay of melatonin is briefly reviewed. Areas of controversy, due probably to technical problems, are identified, together with observations confirmed by reports from different laboratories. Some technical improvements are suggested. KEYWORDS Melatonin assay. TEXT In a rapidly moving field such as the study of pineal indoleamines, it is likely that an assessment of the "current status" of assay methodology will be out of date long before it appear in print. Nevertheless trends can be recognised and areas of controversy identified. Wurtman and Axelrod!s (1965) formulation of the pineal gland as a neuroendocrine transducer, and the consequent interest in pineal products as possible humoral effectors of photic signals, led to intensified efforts to measure the methoxyindoleamines in tissues and body fluids. Circulating melatonin in particular was, and is, considered to be largely, if not exclusively (Lewy and coworkers, 1980; Arendt, Forbes, Brown and Marston, 1980) of pineal origin. While several reports demonstrate its presence and synthesis in the retina and the Harderian gland (Cardinali and Wurtman, 1972; Pang and coworkers, 1976), it is possible that from these sites of origin it does not reach the general circulation. From 1970 to 1975, it was possible to measure melatonin using the tadpole bioassay of Ralph and Lynch (1970). This assay, whilst considered to be specific, was particularly tedious and necessitated a complicated tadpole breeding programme. In addition, the sensitivity was insufficient for determinations in sequential blood samples : of great importance in the study of a rhythmic variable. During the years 1975-1978 an expolsion of publications concerning the measurement of melatonin occurred, the vast majority using radioimmunoassay (RIA) followed by gas-chromatography-mass-spectrometry (GC-MS) and gas-chromatography (GC). A review of this methodology has appeared (Arendt, 1978). From 1978 until the time
3
J. Arendt
4
of writing there has been little basic change in methodological approach - as assays have been refined, reported values have generally speaking decreased, and areas of controversy have appeared which may or may not be of methodological origin. Clearly an assay has to be useful within the confines of the system it is proposed to investigate, and the potential user must choose a method according to his or her own requirements. For example clinical screening for pathological disturbance should be rapid and preferably cheap. Where gross differences in measured values are diagnostic,then some degree of noise and assay variation is tolerable. Very few literature reports of gross clinical melatonin variations exist, and these are unconfirmed (Barber, Smith and Hughes, 1978; Silman and coworkers, 1979). Where subtle differences are important, for example phase-changes in circadian rhythm, then a high degree of assay precision and accuracy is required. In the case of one application of melatonin assay - assessment of central nervous system rhythmic function - a precise measurement of pineal $-receptor stimulation is required, and interference by other methoxy-indoles, whose synthetic control is differently mediated, is not tolerable. In the case of urinary melatonin assay, it is likely that a system recognising either 6-hydroxymelatonin (Lewy and coworkers, 1980) or both 6-hydroxymelatonin and melatonin, may well provide more information on pineal output than measurement of melatonin separately (Lynch and coworkers, 1975), in view of the small percentage of melatonin thought to be excreted unchanged (Kopin and coworkers, 1961). Criteria for melatonin RIA acceptability (which, with slight changes in terminology, can also be applied to other assay systems) are generally considered to be as follows: 1)
negligible cross-reactivity with analogues, at the concentrations likely to be present in a given sample;
2)
parallelism of serial dilutions of sample with the standard curve;
3)
good recovery of added radioactive and non-radioactive melatonin;
4)
low assay blank (preferably below the limit of detection of the standard curve;
5)
acceptable intra- and inter-assay variation (melatonin assays tend to have a rather large assay variation and account should be taken of this, when present, in the interpretation of results);
6)
demonstration of the chromatographic identity of immunoreactivity with melatonin (although quite large discrepancies are sometimes found between chromatographically validated assays of similar samples);
7)
comparison with as near an absolutely specific method as possible.
Recent reports of low to undetectable melatonin levels following short-term pinealectomy (Lewy and coworkers, 1980; Arendt and coworkers, 1980), or suppression of pineal indoleamine synthesis (Arendt and coworkers, 1980) may provide a more physiologically meaningful assessment of assay performance. Most published RIAfs fulfil the criteria 1) - 6) (for references see Arendt, 1978). In view of the prevailing opinion that GC-MS provides the most specific available technique, several authors have made successful, if limited GC-MS/RIA comparisons,
Current Status of Assay Methods of Melatonin however this is clearly a difficult undertaking for the majority of laboratories. It is to be hoped that inter-laboratory comparative studies will include at least one laboratory performing GC-MS assay so that the maximum number of workers may compare their own results with this highly specific but expensive technique. Clearly, with RIA, every sample cannot be validated : with a good GC-MS system every sample is validated. A compromise solution, both from the point of view of assay specificity, capital investment, running expenses and throughput, may be found in high-performance liquid chromatography (HPLC) separation of melatonin from extracted material, followed by RIA or electrochemical detection (Goldman, Hamm and Erickson, 1980). Whether the sensitivity required for plasma determinations will be achieved by electrochemical detection remains to be seen. Comparison of results from different assay systems in order to pinpoint the ! best f method for a given situation is clearly difficult. In general, for a small molecule obtainable in a pure state, such as melatonin, provided that recovery of added compound from the (so far inevitable) extraction procedures is good, then the lower the reported values, the more specific the assay is likely to be. Amongst the published RIA methods, that of Rollag and Niswender (1976) using iodinated melatonin analogue as tracer has the greatest sensitivity, although not necessarily the lowest values. Unless every sample is validated, it is more correct to refer to measured immunoreactive material as melatonin-like. It cannot be emphasised too strongly that the establishment of physiological melatonin variations in an adequately large population is an essential prerequisite to pathological studies. However, the complete absence of 24-hour variations is, according to the vast majority of authors an infrequent observation in normal mammals, and would appear to be good evidence of disturbed pineal and/or central nervous rhythmic function. Indeed the areas of least controversy in the measurement of melatonin include its almost uniform darkphase rise in all species studied to date (Lynch and coworkers, 1975; Kennaway and coworkers, 1976; Rollag and Niswender, 1976; Arendt and coworkers, 1977; Wilkinson and coworkers, 1977; Per low and coworkers, 1980; Vivien-Roe* Is and coworkers, 1979; Owens, G e m and Ralph, 1980). In addition, several authors have shown a lack of immediate response of human circulating melatonin to artificial light in the dark-phase (Jimerson and coworkers, 1977; Arendt, 1978; Lynch and coworkers, 1978) compared to other species (Rollag and Niswender, 1976; Illnerova and Vanacek, 1979; Perlow and coworkers, 1980) and disturbed plasma melatonin variations in psychiatric disease (Wirz-Justice and Arendt, 1979; Lewy and coworkers, 1979; Wetterberg and coworkers, 1979; Mendlewicz and coworkers, 1979). Most other observations are not yet repeated in competing laboratories. Good general agreement is found amongst different laboratories with respect to pineal and plasma melatonin levels in rats and sheep measured by RIA. Recent results in adult human values show very large (up to 50-fold) variations from the lowest reported daytime levels (Lewy and Markey, 1978 - GC-MS) to the highest (Mendlewicz and coworkers, 1979 - RIA), probably due in part to differences in assay specificity. In addition reports of high "spikes" of circulating melatonin in man during day and night (Weitzman and coworkers, 1978; Mullen and coworkers, 1980), are unconfirmed. In the authors laboratory daytime melatonin in normal volunteers sampled at hourly intervals, is almost invariably low to undetectable. It is as yet impossible to revue urinary melatonin assays due to their paucity (Lynch and coworkers, 1975; Akerstedt and coworkers, 1980). A major obstacle has been the apparent lack of specificity of direct RIA for urinary melatonin (Arendt, 1978). We have successfully modified RIA (Arendt and coworkers, 1977) to the determination of melatonin in neonatal urine, with demonstrable chromatographic specificity (Marston and coworkers, 1980). In the course of these investigations it became evident that melatonin is unstable over periods of more
5
6
J . Arendt than three months in frozen urine. Preliminary observations in neonatal urine, assayed rapidly after collection, indicate that infants develop a 24-hour melatonin rhythm at different r a t e s , even under similar environmental conditions.
Every melatonin a s s a y i s t ' s ideal must be an automated system, not requiring extraction, of sub-picogram s e n s i t i v i t y , absolute s p e c i f i c i t y , throughput measured in hundreds per day, readily available, and cheap to run. I t does not exist yet. For the moment, future prospects for melatonin assay include greater automation of GC-MS assay, HPLC coupled with sensitive detection methods, and direct plasma RIA without extraction using sufficiently sensitive and specific antibodies. ACKNOWLEDGEMENT
Some of t h e work r e p o r t e d h e r e was f i n a n c i a l l y supported by t h e Medical Research Council and t h e A g r i c u l t u r a l Research Council. REFERENCES A k e r s t e d t , T . , Frbberg, J . E . , F r i b e r g , Y. and Wetterberg, L. (1979) Melatonin e x c r e t i o n , body temperature and s u b j e c t i v e a r o u s a l during 64 hours of s l e e p d e p r i v a t i o n . Psychoneuroendocrinol., 4_y 219-225. Arendt, J . , Wetterberg, L . , Heyden, T . , Sizonenko, P.C. and Paunier, L. (1977). Radioimmunoassay of melatonin : human serum and c e r e b r o s p i n a l f l u i d . Hormone R e s . , 8^, 65-75. Arendt, J . (1978). Melatonin assays i n body f l u i d s . J . Neural Transm. Suppl. 13_, 265-278. Arendt, J . , Forbes, J . M . , Brown, W.B. and Marston, A. (1980). Effect of p i n e a l ectomy on immunoassayable melatonin i n sheep. J . E n d o c r i n o l . , 85, 1-2P. Arendt, J . , Ho, A.K., Laud, C , Marston, A., Nohria, V., Smith, J . A . and Symons, A.M. (1980) D i f f e r e n t i a l e f f e c t of b e n s e r a z i d e (Ro4-4602) on t h e c o n c e n t r a t i o n s of indoleamines i n r a t p i n e a l and hypothalamus. B r i t . J . Pharmac. In p r e s s . Axelrod, J . (1974) The Pineal Gland : A Neurochemical Transducer. Science, 184, 1341-1348. Barber, S.G., Smith, J . A . and Hughes, R.C. (1978) Melatonin as a tumour marker i n a p a t i e n t with p i n e a l tumour. B r i t . Med. J . , i i , 328. C a r d i n a l i , D.P. and Wurtman, R . J . (1972). Hydroxyindole-o-methyltransferase in r a t p i n e a l , r e t i n a and Harderian gland. E n d o c r i n o l . , 9 1 , 247-252 Goldman, M.E., Hamm, H. and Erickson, C.K. (1980). Determination of melatonin by high-performance l i q u i d chromatography with e l e c t r o c h e m i c a l d e t e c t i o n . J . Chromatog., 190, 217-220 I l l n e r o v a , H . , Backstrttm, M., Saaf, J . , Wetterberg, L. and Vangbo, B. (1978). Melatonin i n r a t p i n e a l gland and serum; r a p i d p a r a l l e l d e c l i n e a f t e r l i g h t exposure a t n i g h t . Neurosci. L e t t e r s , 9^ 189-193 Jimerson, D.C., Lynch, H . J . , P o s t , R.M., Wurtman, R . J . and Bunney, W.E. (1977) Urinary melatonin rhythms during s l e e p d e p r i v a t i o n i n depressed p a t i e n t s and normals. Life S c i . , 20, 1501-1508. Kennaway, D . J . , F r i t h , E . G . , P h i l l i p o i i , G., Matthews, C. and Seamark, R.F. (1977) A s p e c i f i c radioimmunoassay f o r melatonin i n b i o l o g i c a l t i s s u e s and f l u i d s and i t s v a l i d a t i o n by gas-chromatography-mass-spectrometry. Endocrinology, 1 0 1 , 119-123. Kopin, I . J . , Pare, C.M.B., Axelrod, J . and Weissbach, H. (1961). The f a t e of melatonin i n animals. J . B i o l . Chem., 236, 3072-3075.
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Lewy, A.J. and Markey, S.P. (1978) Analysis of melatonin in human plasma by gas-chromatography n e g a t i v e chemical i o n i z a t i o n mass s p e c t r o m e t r y . S c i e n c e , 2 0 1 , 741-743. Lewy, A . J . , Wehr, T.A., Gold, P.W. and Goodwin, F.K. (1979) Plasma melatonin in manic-depressive i l l n e s s . In E. Usdin, I . J . Kopin and J . Barchas ( E d s . ) , Catecholamines : Basic and C l i n i c a l F r o n t i e r s , Pergamon P r e s s , New Y o r k , p l l 7 3 . Lewy, A . J . , Tetsuo, M., Markey, S . P . , Goodwin, F.K. and Kopin, I . J . (1980) Pinealectomy a b o l i s h e s plasma melatonin in t h e r a t . J . C l i n . Endocrinol. Metab., 50^, 204-205. Lynch, H . J . , Wurtman, R . J . , Moskowitz, M.A., A r d i e r , M.C. and Ho., M.H. (1975) Daily rhythm in human u r i n a r y m e l a t o n i n . S c i e n c e , 187, 169-171. Lynch, H . J . , Jimerson, D.C., Ozaki, Y., P o s t , R.M., Bunney, W.E. J r . , and Wurtman, R.J. (1978) Entrainment of rhythmic melatonin s e c r e t i o n i n man t o a 12-hour phase s h i f t in t h e l i g h t / d a r k c y c l e . Life S c i . , 2 3 , 1557-1563. Marston, A., Arendt, J . , Campbell, C , Armstrong-Esther, C., Hawkins, L. and Goodison, S. (1980). Assay of melatonin in n e o n a t a l u r i n e ( A b s t r . ) 4th European Neuroscience Meeting. Mendlewicz, J . , Linkowski, P . , Branchey, L., Weinberg, U., Weitzman, E.D. and Branchey, M. (1979). Abnormal 24-hour p a t t e r n of melatonin s e c r e t i o n in d e p r e s s i o n . Lancet, i i , 1362. Mullen, P . E . , Hooper, J . , Leone, R., L i n s e l l , C., McKeon, P . , Silman, R. and Smith, I . (1980). The 24-hour p a t t e r n of s e c r e t i o n of melatonin and 5-methoxytryptophol in man. ( A b s t r . ) XI I n t e r n a t i o n a l Congress of the I n t e r n a t i o n a l S o c i e t y of Psychoneuroendocrinoftgy. Owens, D.W., Gern, W.A. and Ralph, C.L. (1980). Melatonin in the blood and c e r e b r o s p i n a l f l u i d of the green sea t u r t l e (Chelonia mydas) Gen.and Comp. E n d o c r i n o l . , 40, 180-187. Pang, S . F . , Brown, G.M., Grota, L.J. and Rodman, R.L. (1976).Radioimmunoassay of melatonin i n p i n e a l g l a n d s , h a r d e r i a n g l a n d s , r e t i n a s and s e r a of r a t s or chickens. Fed.Proc.Fed.Am.Socs. exp. B i o l . , 3 5 . , 6 9 1 . Perlow, M . J . , Reppert, S.M., Tamarkin, L . , Wyatt, R.J. and K l e i n , D.C. (1980). Photic r e g u l a t i o n of the melatonin rhythm : monkey and man are n o t the same. Brain R e s . , 182, 211-216. Ralph, C.L. and Lynch, H.J. (1970) A q u a n t i t a t i v e melatonin b i o a s s a y . Gen. Comp. E n d o c r i n o l . , 15, 334-338. Rollag, M.D. and Niswender, G.D. (1976) Radioimmunoassay of serum c o n c e n t r a t i o n s of melatonin i n sheep exposed to d i f f e r e n t l i g h t i n g regimes. Endocrinology, 9 8 , 482-489. Silman, R . E . , Leone, R.M., Hooper, R . J . L . and Preece, M.A. (1979) Melatonin, the p i n e a l gland and human p u b e r t y . N a t u r e , 282, 301-303. Tetsuo, M., Markey, S.P. and Kopin, I . J . (1980) Measurement of 6-hydroxymelatonin i n human u r i n e and i t s d i u r n a l v a r i a t i o n s . Life S c i . In p r e s s . Vivien-Roels, B . , Arendt, J . and Bradtke, J . (1979). Circadian and c i r c a n n u a l f l u c t u a t i o n s of p i n e a l indoleamines ( s e r o t o n i n and melatonin) in Testudo hermanni Gmelin ( R e p t i l a C h e l o n i a ) . Gen. Comp. E n d o c r i n o l . , 37, 197-210. Weitzman, E . D . , Weinberg, U., D ' E l e t t o , R., Lynch, H . , Wurtman, R . J . , C z e i s l e r , Ch., E r l i c h , S. (1978). S t u d i e s of t h e 24-hour rhythm of melatonin i n man. J . Neural. Trans. Suppl. 13, 325-329. Wetterberg, L . , B e c k - F r i i s , J . , Aperia, B. and P e t t e r s o n , U. (1979) Melatonin/ c o r t i s o l r a t i o in d e p r e s s i o n . Lancet, i i , 1361. Wilkinson, M., Arendt, J . , Bradtke, J . and Z i e g l e r , D. de. (1977) Determination of dark-induced e l e v a t i o n of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y with simultaneous radioimmunoassay of melatonin i n p i n e a l , serum and p i t u i t a r y of the male r a t . J . E n d o c r i n o l . 72, 243-244. W i r z - J u s t i c e , A. and Arendt, J . (1979) D i u r n a l , mentrual cycle and s e a s o n a l indole rhythms i n man and t h e i r m o d i f i c a t i o n in a f f e c t i v e d i s o r d e r s . In J . O b i o l s , C. B a l l u s , E. Gonzalez Monclus and J . Pujol (Eds.) Biological P s y c h i a t r y Today. E l s e v i e r / N o r t h Holland P r e s s , pp.294-302. CSP - B
ABSTRACT Current available methodology for the assay of melatonin is briefly reviewed. Areas of controversy, due probably to technical problems, are identified, together with observations confirmed by reports from different laboratories. Some technical improvements are suggested. KEYWORDS Melatonin assay. TEXT In a rapidly moving field such as the study of pineal indoleamines, it is likely that an assessment of the "current status" of assay methodology will be out of date long before it appear in print. Nevertheless trends can be recognised and areas of controversy identified. Wurtman and Axelrod!s (1965) formulation of the pineal gland as a neuroendocrine transducer, and the consequent interest in pineal products as possible humoral effectors of photic signals, led to intensified efforts to measure the methoxyindoleamines in tissues and body fluids. Circulating melatonin in particular was, and is, considered to be largely, if not exclusively (Lewy and coworkers, 1980; Arendt, Forbes, Brown and Marston, 1980) of pineal origin. While several reports demonstrate its presence and synthesis in the retina and the Harderian gland (Cardinali and Wurtman, 1972; Pang and coworkers, 1976), it is possible that from these sites of origin it does not reach the general circulation. From 1970 to 1975, it was possible to measure melatonin using the tadpole bioassay of Ralph and Lynch (1970). This assay, whilst considered to be specific, was particularly tedious and necessitated a complicated tadpole breeding programme. In addition, the sensitivity was insufficient for determinations in sequential blood samples : of great importance in the study of a rhythmic variable. During the years 1975-1978 an expolsion of publications concerning the measurement of melatonin occurred, the vast majority using radioimmunoassay (RIA) followed by gas-chromatography-mass-spectrometry (GC-MS) and gas-chromatography (GC). A review of this methodology has appeared (Arendt, 1978). From 1978 until the time
3
J. Arendt
4
of writing there has been little basic change in methodological approach - as assays have been refined, reported values have generally speaking decreased, and areas of controversy have appeared which may or may not be of methodological origin. Clearly an assay has to be useful within the confines of the system it is proposed to investigate, and the potential user must choose a method according to his or her own requirements. For example clinical screening for pathological disturbance should be rapid and preferably cheap. Where gross differences in measured values are diagnostic,then some degree of noise and assay variation is tolerable. Very few literature reports of gross clinical melatonin variations exist, and these are unconfirmed (Barber, Smith and Hughes, 1978; Silman and coworkers, 1979). Where subtle differences are important, for example phase-changes in circadian rhythm, then a high degree of assay precision and accuracy is required. In the case of one application of melatonin assay - assessment of central nervous system rhythmic function - a precise measurement of pineal $-receptor stimulation is required, and interference by other methoxy-indoles, whose synthetic control is differently mediated, is not tolerable. In the case of urinary melatonin assay, it is likely that a system recognising either 6-hydroxymelatonin (Lewy and coworkers, 1980) or both 6-hydroxymelatonin and melatonin, may well provide more information on pineal output than measurement of melatonin separately (Lynch and coworkers, 1975), in view of the small percentage of melatonin thought to be excreted unchanged (Kopin and coworkers, 1961). Criteria for melatonin RIA acceptability (which, with slight changes in terminology, can also be applied to other assay systems) are generally considered to be as follows: 1)
negligible cross-reactivity with analogues, at the concentrations likely to be present in a given sample;
2)
parallelism of serial dilutions of sample with the standard curve;
3)
good recovery of added radioactive and non-radioactive melatonin;
4)
low assay blank (preferably below the limit of detection of the standard curve;
5)
acceptable intra- and inter-assay variation (melatonin assays tend to have a rather large assay variation and account should be taken of this, when present, in the interpretation of results);
6)
demonstration of the chromatographic identity of immunoreactivity with melatonin (although quite large discrepancies are sometimes found between chromatographically validated assays of similar samples);
7)
comparison with as near an absolutely specific method as possible.
Recent reports of low to undetectable melatonin levels following short-term pinealectomy (Lewy and coworkers, 1980; Arendt and coworkers, 1980), or suppression of pineal indoleamine synthesis (Arendt and coworkers, 1980) may provide a more physiologically meaningful assessment of assay performance. Most published RIAfs fulfil the criteria 1) - 6) (for references see Arendt, 1978). In view of the prevailing opinion that GC-MS provides the most specific available technique, several authors have made successful, if limited GC-MS/RIA comparisons,
Current Status of Assay Methods of Melatonin however this is clearly a difficult undertaking for the majority of laboratories. It is to be hoped that inter-laboratory comparative studies will include at least one laboratory performing GC-MS assay so that the maximum number of workers may compare their own results with this highly specific but expensive technique. Clearly, with RIA, every sample cannot be validated : with a good GC-MS system every sample is validated. A compromise solution, both from the point of view of assay specificity, capital investment, running expenses and throughput, may be found in high-performance liquid chromatography (HPLC) separation of melatonin from extracted material, followed by RIA or electrochemical detection (Goldman, Hamm and Erickson, 1980). Whether the sensitivity required for plasma determinations will be achieved by electrochemical detection remains to be seen. Comparison of results from different assay systems in order to pinpoint the ! best f method for a given situation is clearly difficult. In general, for a small molecule obtainable in a pure state, such as melatonin, provided that recovery of added compound from the (so far inevitable) extraction procedures is good, then the lower the reported values, the more specific the assay is likely to be. Amongst the published RIA methods, that of Rollag and Niswender (1976) using iodinated melatonin analogue as tracer has the greatest sensitivity, although not necessarily the lowest values. Unless every sample is validated, it is more correct to refer to measured immunoreactive material as melatonin-like. It cannot be emphasised too strongly that the establishment of physiological melatonin variations in an adequately large population is an essential prerequisite to pathological studies. However, the complete absence of 24-hour variations is, according to the vast majority of authors an infrequent observation in normal mammals, and would appear to be good evidence of disturbed pineal and/or central nervous rhythmic function. Indeed the areas of least controversy in the measurement of melatonin include its almost uniform darkphase rise in all species studied to date (Lynch and coworkers, 1975; Kennaway and coworkers, 1976; Rollag and Niswender, 1976; Arendt and coworkers, 1977; Wilkinson and coworkers, 1977; Per low and coworkers, 1980; Vivien-Roe* Is and coworkers, 1979; Owens, G e m and Ralph, 1980). In addition, several authors have shown a lack of immediate response of human circulating melatonin to artificial light in the dark-phase (Jimerson and coworkers, 1977; Arendt, 1978; Lynch and coworkers, 1978) compared to other species (Rollag and Niswender, 1976; Illnerova and Vanacek, 1979; Perlow and coworkers, 1980) and disturbed plasma melatonin variations in psychiatric disease (Wirz-Justice and Arendt, 1979; Lewy and coworkers, 1979; Wetterberg and coworkers, 1979; Mendlewicz and coworkers, 1979). Most other observations are not yet repeated in competing laboratories. Good general agreement is found amongst different laboratories with respect to pineal and plasma melatonin levels in rats and sheep measured by RIA. Recent results in adult human values show very large (up to 50-fold) variations from the lowest reported daytime levels (Lewy and Markey, 1978 - GC-MS) to the highest (Mendlewicz and coworkers, 1979 - RIA), probably due in part to differences in assay specificity. In addition reports of high "spikes" of circulating melatonin in man during day and night (Weitzman and coworkers, 1978; Mullen and coworkers, 1980), are unconfirmed. In the authors laboratory daytime melatonin in normal volunteers sampled at hourly intervals, is almost invariably low to undetectable. It is as yet impossible to revue urinary melatonin assays due to their paucity (Lynch and coworkers, 1975; Akerstedt and coworkers, 1980). A major obstacle has been the apparent lack of specificity of direct RIA for urinary melatonin (Arendt, 1978). We have successfully modified RIA (Arendt and coworkers, 1977) to the determination of melatonin in neonatal urine, with demonstrable chromatographic specificity (Marston and coworkers, 1980). In the course of these investigations it became evident that melatonin is unstable over periods of more
5
6
J . Arendt than three months in frozen urine. Preliminary observations in neonatal urine, assayed rapidly after collection, indicate that infants develop a 24-hour melatonin rhythm at different r a t e s , even under similar environmental conditions.
Every melatonin a s s a y i s t ' s ideal must be an automated system, not requiring extraction, of sub-picogram s e n s i t i v i t y , absolute s p e c i f i c i t y , throughput measured in hundreds per day, readily available, and cheap to run. I t does not exist yet. For the moment, future prospects for melatonin assay include greater automation of GC-MS assay, HPLC coupled with sensitive detection methods, and direct plasma RIA without extraction using sufficiently sensitive and specific antibodies. ACKNOWLEDGEMENT
Some of t h e work r e p o r t e d h e r e was f i n a n c i a l l y supported by t h e Medical Research Council and t h e A g r i c u l t u r a l Research Council. REFERENCES A k e r s t e d t , T . , Frbberg, J . E . , F r i b e r g , Y. and Wetterberg, L. (1979) Melatonin e x c r e t i o n , body temperature and s u b j e c t i v e a r o u s a l during 64 hours of s l e e p d e p r i v a t i o n . Psychoneuroendocrinol., 4_y 219-225. Arendt, J . , Wetterberg, L . , Heyden, T . , Sizonenko, P.C. and Paunier, L. (1977). Radioimmunoassay of melatonin : human serum and c e r e b r o s p i n a l f l u i d . Hormone R e s . , 8^, 65-75. Arendt, J . (1978). Melatonin assays i n body f l u i d s . J . Neural Transm. Suppl. 13_, 265-278. Arendt, J . , Forbes, J . M . , Brown, W.B. and Marston, A. (1980). Effect of p i n e a l ectomy on immunoassayable melatonin i n sheep. J . E n d o c r i n o l . , 85, 1-2P. Arendt, J . , Ho, A.K., Laud, C , Marston, A., Nohria, V., Smith, J . A . and Symons, A.M. (1980) D i f f e r e n t i a l e f f e c t of b e n s e r a z i d e (Ro4-4602) on t h e c o n c e n t r a t i o n s of indoleamines i n r a t p i n e a l and hypothalamus. B r i t . J . Pharmac. In p r e s s . Axelrod, J . (1974) The Pineal Gland : A Neurochemical Transducer. Science, 184, 1341-1348. Barber, S.G., Smith, J . A . and Hughes, R.C. (1978) Melatonin as a tumour marker i n a p a t i e n t with p i n e a l tumour. B r i t . Med. J . , i i , 328. C a r d i n a l i , D.P. and Wurtman, R . J . (1972). Hydroxyindole-o-methyltransferase in r a t p i n e a l , r e t i n a and Harderian gland. E n d o c r i n o l . , 9 1 , 247-252 Goldman, M.E., Hamm, H. and Erickson, C.K. (1980). Determination of melatonin by high-performance l i q u i d chromatography with e l e c t r o c h e m i c a l d e t e c t i o n . J . Chromatog., 190, 217-220 I l l n e r o v a , H . , Backstrttm, M., Saaf, J . , Wetterberg, L. and Vangbo, B. (1978). Melatonin i n r a t p i n e a l gland and serum; r a p i d p a r a l l e l d e c l i n e a f t e r l i g h t exposure a t n i g h t . Neurosci. L e t t e r s , 9^ 189-193 Jimerson, D.C., Lynch, H . J . , P o s t , R.M., Wurtman, R . J . and Bunney, W.E. (1977) Urinary melatonin rhythms during s l e e p d e p r i v a t i o n i n depressed p a t i e n t s and normals. Life S c i . , 20, 1501-1508. Kennaway, D . J . , F r i t h , E . G . , P h i l l i p o i i , G., Matthews, C. and Seamark, R.F. (1977) A s p e c i f i c radioimmunoassay f o r melatonin i n b i o l o g i c a l t i s s u e s and f l u i d s and i t s v a l i d a t i o n by gas-chromatography-mass-spectrometry. Endocrinology, 1 0 1 , 119-123. Kopin, I . J . , Pare, C.M.B., Axelrod, J . and Weissbach, H. (1961). The f a t e of melatonin i n animals. J . B i o l . Chem., 236, 3072-3075.
Current Status of Assay Methods of Melatonin
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Lewy, A.J. and Markey, S.P. (1978) Analysis of melatonin in human plasma by gas-chromatography n e g a t i v e chemical i o n i z a t i o n mass s p e c t r o m e t r y . S c i e n c e , 2 0 1 , 741-743. Lewy, A . J . , Wehr, T.A., Gold, P.W. and Goodwin, F.K. (1979) Plasma melatonin in manic-depressive i l l n e s s . In E. Usdin, I . J . Kopin and J . Barchas ( E d s . ) , Catecholamines : Basic and C l i n i c a l F r o n t i e r s , Pergamon P r e s s , New Y o r k , p l l 7 3 . Lewy, A . J . , Tetsuo, M., Markey, S . P . , Goodwin, F.K. and Kopin, I . J . (1980) Pinealectomy a b o l i s h e s plasma melatonin in t h e r a t . J . C l i n . Endocrinol. Metab., 50^, 204-205. Lynch, H . J . , Wurtman, R . J . , Moskowitz, M.A., A r d i e r , M.C. and Ho., M.H. (1975) Daily rhythm in human u r i n a r y m e l a t o n i n . S c i e n c e , 187, 169-171. Lynch, H . J . , Jimerson, D.C., Ozaki, Y., P o s t , R.M., Bunney, W.E. J r . , and Wurtman, R.J. (1978) Entrainment of rhythmic melatonin s e c r e t i o n i n man t o a 12-hour phase s h i f t in t h e l i g h t / d a r k c y c l e . Life S c i . , 2 3 , 1557-1563. Marston, A., Arendt, J . , Campbell, C , Armstrong-Esther, C., Hawkins, L. and Goodison, S. (1980). Assay of melatonin in n e o n a t a l u r i n e ( A b s t r . ) 4th European Neuroscience Meeting. Mendlewicz, J . , Linkowski, P . , Branchey, L., Weinberg, U., Weitzman, E.D. and Branchey, M. (1979). Abnormal 24-hour p a t t e r n of melatonin s e c r e t i o n in d e p r e s s i o n . Lancet, i i , 1362. Mullen, P . E . , Hooper, J . , Leone, R., L i n s e l l , C., McKeon, P . , Silman, R. and Smith, I . (1980). The 24-hour p a t t e r n of s e c r e t i o n of melatonin and 5-methoxytryptophol in man. ( A b s t r . ) XI I n t e r n a t i o n a l Congress of the I n t e r n a t i o n a l S o c i e t y of Psychoneuroendocrinoftgy. Owens, D.W., Gern, W.A. and Ralph, C.L. (1980). Melatonin in the blood and c e r e b r o s p i n a l f l u i d of the green sea t u r t l e (Chelonia mydas) Gen.and Comp. E n d o c r i n o l . , 40, 180-187. Pang, S . F . , Brown, G.M., Grota, L.J. and Rodman, R.L. (1976).Radioimmunoassay of melatonin i n p i n e a l g l a n d s , h a r d e r i a n g l a n d s , r e t i n a s and s e r a of r a t s or chickens. Fed.Proc.Fed.Am.Socs. exp. B i o l . , 3 5 . , 6 9 1 . Perlow, M . J . , Reppert, S.M., Tamarkin, L . , Wyatt, R.J. and K l e i n , D.C. (1980). Photic r e g u l a t i o n of the melatonin rhythm : monkey and man are n o t the same. Brain R e s . , 182, 211-216. Ralph, C.L. and Lynch, H.J. (1970) A q u a n t i t a t i v e melatonin b i o a s s a y . Gen. Comp. E n d o c r i n o l . , 15, 334-338. Rollag, M.D. and Niswender, G.D. (1976) Radioimmunoassay of serum c o n c e n t r a t i o n s of melatonin i n sheep exposed to d i f f e r e n t l i g h t i n g regimes. Endocrinology, 9 8 , 482-489. Silman, R . E . , Leone, R.M., Hooper, R . J . L . and Preece, M.A. (1979) Melatonin, the p i n e a l gland and human p u b e r t y . N a t u r e , 282, 301-303. Tetsuo, M., Markey, S.P. and Kopin, I . J . (1980) Measurement of 6-hydroxymelatonin i n human u r i n e and i t s d i u r n a l v a r i a t i o n s . Life S c i . In p r e s s . Vivien-Roels, B . , Arendt, J . and Bradtke, J . (1979). Circadian and c i r c a n n u a l f l u c t u a t i o n s of p i n e a l indoleamines ( s e r o t o n i n and melatonin) in Testudo hermanni Gmelin ( R e p t i l a C h e l o n i a ) . Gen. Comp. E n d o c r i n o l . , 37, 197-210. Weitzman, E . D . , Weinberg, U., D ' E l e t t o , R., Lynch, H . , Wurtman, R . J . , C z e i s l e r , Ch., E r l i c h , S. (1978). S t u d i e s of t h e 24-hour rhythm of melatonin i n man. J . Neural. Trans. Suppl. 13, 325-329. Wetterberg, L . , B e c k - F r i i s , J . , Aperia, B. and P e t t e r s o n , U. (1979) Melatonin/ c o r t i s o l r a t i o in d e p r e s s i o n . Lancet, i i , 1361. Wilkinson, M., Arendt, J . , Bradtke, J . and Z i e g l e r , D. de. (1977) Determination of dark-induced e l e v a t i o n of p i n e a l N - a c e t y l t r a n s f e r a s e a c t i v i t y with simultaneous radioimmunoassay of melatonin i n p i n e a l , serum and p i t u i t a r y of the male r a t . J . E n d o c r i n o l . 72, 243-244. W i r z - J u s t i c e , A. and Arendt, J . (1979) D i u r n a l , mentrual cycle and s e a s o n a l indole rhythms i n man and t h e i r m o d i f i c a t i o n in a f f e c t i v e d i s o r d e r s . In J . O b i o l s , C. B a l l u s , E. Gonzalez Monclus and J . Pujol (Eds.) Biological P s y c h i a t r y Today. E l s e v i e r / N o r t h Holland P r e s s , pp.294-302. CSP - B