A Specific Radioimmunoassay for Melatonin in Biological Tissue and Fluids and its Validation by Gas Chromatography-Mass Spectrometry D. J. KENNAWAY,1 R. G. FRITH, 2 G. PHILLIPOU, 2 C. D. MATTHEWS,1 AND R. F. SEAMARK1 1
Department of Obstetrics and Gynaecology, The University of Adelaide, 5000, and 2The Queen Elizabeth Hospital, Woodville, South Australia 5011 ABSTRACT. A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon the extraction procedure and column (Lipidex 5000) chromatography. In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography-mass spectrometry using [2H3]melatonin as an
I
NVESTIGATIONS on the endocrine function of the pineal gland have been severely hampered by the lack of a reliable, practical assay for melatonin, a presumptive major pineal hormone. That melatonin occurs in plasma has been indicated by sensitive bioassays (1) and its presence confirmed more recently by mass spectrometric methods (2,3), but these specialized procedures are generally unsuited to routine assay of large numbers of samples. Recently, several practical radioimmunoassay procedures for melatonin in plasma (4-6) or pineal tissues (7) have been reported and studies on melatonin content during various environmental and endocrinological studies initiated (4-7). This report concerns the development of a radioimmunoassay based on an antibody raised to an easily prepared compound of N-acetylserotonin and bovine serum albumin (BSA) which is suited to determinaReceived September 2, 1976. DJ.K. was supported by the Sir John Gellibrand Memorial Scholarship.
South
Australia
internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22-76 pg/ml), donkey (24-128 pg/ml), cow (20-320 pg/ml), camel (29-221 pg/ml) and a scincid lizard (20500 pg/ml) established. A nocturnal rise in plasma melatonin content occurred in all species. Melatonin was found in the plasma of ewes 2-12 weeks after pinealectomy, but the nocturnal rise was abolished. The results establish a nyctohemeral variation in plasma melatonin in a wide variety of species, and indicate that sources of melatonin other than the pineal may assume precedence following pinalectomy. (Endocrinology 101: 119, 1977)
tions of melatonin in both tissues and body fluids on a routine basis. In addition to the usual criterion of reliability, the method is validated by the technique of selected ion monitoring (8,9). Its application to determination of the plasma melatonin content of a variety of species kept under diurnal lighting conditions is reported. Materials and Methods Animals Sheep, mainly Merino breed, were obtained from the flocks of the Mortlock Research Centre, Mintaro, South Australia. For intensive bleeding regimens, animals were housed individually in air-conditioned pens illuminated between 0700 and 2100 h. A period of at least two weeks was allowed for the animals to acclimatize before bleeding commenced. Blood was continuously sampled at the rate of 12 ml/h from the left jugular vein using a vinyl cannula, id 0.86 mm, od 1.2 mm (Dural Plastics, Dural, N.S.W. Australia) placed inside a second larger vinyl cannula, id 2 mm, od 3.0 mm. Heparinized saline, 250 U/ml, was continuously
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infused via the outside lumen at 6 ml/h during collections. Samples were collected over 30 min intervals. Studies with a dye (Indocyanine Green, Hynson, Westcott and Dunning, Inc., Baltimore, Maryland) added to the heparinized saline, showed that the samples were contaminated with less than 2% of the heparinized saline when collected by this procedure. Pinealectomy was carried out according to the procedure of Roche et al. (10) as modified by Obst (unpublished). The cattle, Brahman and Jersey breed, were obtained from Struan Research Centre, and the camel, donkey and pigs, from the Waite Agricultural Research Institute, South Australia, and were kept on open pastures. Blood samples were collected by venipuncture. The rats (Hooded Wistar strain) and lizards were housed at constant temperature under a diurnal (13L:11D) lighting regimen. Blood samples were collected by heart puncture. Reagents All solvents were the highest purity grades available and were redistilled prior to use. Dry 1,2-dimethoxyethane was obtained by distillation from sodium-wire. Deuterated methyl iodide and acetonitrile (both > 99% 2H3) were purchased from R/M Isotopes Co., Maynard, Maine. Tritiated melatonin (specific activity 26 Ci/mmol) was purchased from New England Nuclear Corporation, Boston, Mass., and used without further purification. The indole standards, except N-formyl-Lkynurenine (Calbiochem, San Diego, Ca.) were obtained from Sigma, St. Louis, Mo. O and Nacetyl indoles, unavailable commercially, were prepared using acetic anhydride/pyridine (11), and their purity confirmed by thin layer chromatography (tic). Preparation of acetylserotonin
4-N,N-dimethylaminomethyl)-N-
Paraformaldehyde (4.2 mg, 0.14 mmol) and dimethylamine (16/u.l, 0.14 mmol, 40% aqueous) in ethanol (5 ml) were stirred at room temperature for 15 min. N-acetyl-serotonin (30 mg, 0.14 mmol) was then added and the whole mixture stirred overnight at room temperature under N2. The solvent was then removed in vacuo and the residue chromatographed on Sorbsil. Elution with methanol:chloroform (1:19) gave the major component (17 mg), shown to be pure by tic and identified by proton magnetic resonance (PMR) spectroscopy (Varian T-60 instru-
Endo • 1977 Vol 101 • No 1
ment, samples dissolved in deuterated acetonitrile, with tetramethylsilane as internal standard) as 4-(N,N-dimethylaminomethyl)-N-acetylserotonin. Preparation of [2H3]melatonin N-acetylserotonin (50 mg, 0.23 mmol) and sodium hydroxide (18 mg, 0.45 mmol) in 1,2dimethoxyethane (5 ml) were stirred at room temperature for 5 min in the dark. Deuterated methyl iodide (100 mg, 0.74 mmol) was then added and the reaction mixture stirred for a further 4 h at which time the excess base was neutralized (5% dilute hydrochloric acid) and the solvent removed on a rotary evaporator. The remaining residue was preabsorbed and chromatographed on neutral alumina grade II (5 g), care again being taken to exclude sunlight. The column was developed stepwise with the increasing amounts of chloroform in benzene (1-50%). It was then eluted with chloroform to give [2H3]melatonin (43 mg, 81%), which was recrystallized from benzene to yield pale yellow crystals nip 115.5-117 C (lit. (12) 116118 C); tic and gas chromatography (GC) showed the product to be homogeneous; its isotopic composition as determined by mass spectrometry was 99.1% 2H3. Preparation of antibody The antigen was formed by condensing Nacetylserotonin with the Mannich adduct of BSA and formaldehyde as described by Grota and Brown (13). The molar ratio of hapten to carrier protein, as determined by spectrometry, was 50:1. Four rabbits were injected sc at 3 dorsal sites with an emulsion of 7 mg of the lyophilized antigen, 2 ml 0.05M phosphate buffer, pH 7.9, and 2 ml complete Freund's adjuvant. Booster injections of 1-2 mg were given monthly. Blood from an ear vein was collected 9-10 days after these injections into lithium heparin coated tubes and centrifuged at 4000 rpm. Plasma was divided into 1 ml aliquots and stored frozen until used. Antibody was detectable after the second booster injection in one rabbit (R8) and after subsequent booster injections in the other rabbits. For use, 1 ml of plasma containing a suitable concentration of antibody was vortexed with 4 ml of a 0.4% solution of Rivanol (2-ethoxy-6,9diaminoacridine lactate, K & K Laboratories, Plainview, New York) in distilled water and allowed to stand for 10 min at room temperature (14). Then 300 mg activated charcoal was added,
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MELATONIN RADIOIMMUNOASSAY the suspension was vortexed, and allowed to stand at room temperature for 10 min. Five ml distilled water was added and the tubes were centrifuged at 4000 rpm for 10 min at 4 C. The antibody was stable at this dilution for several months at 4 C. Assay of melatonin by radioimmunoassay Extraction procedure. Plasma (2-4 ml) was mixed with an equal volume of 0.5M borate buffer, pH 10.0, and extracted for 30 min with 4-5 volumes of chloroform using a gentle rocking motion to avoid emulsion formation. A Paton extractor (Paton Industries Pty. Ltd., Beaumont, South Australia) was adapted for this purpose. Tissues (5-100 mg) were homogenized in 1 ml 0.5N NaOH, then 2 ml 0.5M borate buffer, pH 10.0, was added and the mixture extracted with chloroform. Following extraction, the samples were centrifuged to fully resolve the phases, the aqueous layer was then aspirated, and the chloroform evaporated at 37 C under N2.
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Known amounts of melatonin (30-1000 pg) were taken through the assay procedure as reference standards. Assay of melatonin by selected ion monitoring Extraction and isolation procedure. Individual pineal glands were homogenized in a solution of NaOH (1 ml, 0.5N) and borate buffer (2.0 ml, 0.5N, pH 10), followed by extraction with chloroform (10 ml). The chloroform extract was then evaporated to dryness and the residue chromatographed on Lipidex 5000 as described above. An aliquot was taken for RIA and 40 ng deuterated melatonin added to the remainder and the solvent removed. The pentafluoro-propionyl (PFP) ester was then formed (15) and the product subjected to selected ion monitoring.
Selected ion monitoring. GC-MS was carried out with an AEI MS-30 mass spectrometer, interfaced to a Pye GC (column, 1.0 m x 2 mm id; 1% OV-225 on 100/120 mesh support; temp., 230 C; helium flow, 25 ml/min) via a single stage glass Chromatographij. The residue was taken up and jet separator (S.G.E., Melbourne, Australia). transferred to columns (3.2 x 175 mm) of Lipidex Quantitation was achieved by selective ion moni5000 (Packard Instrument Co., Downers Grove toring (SIM) of the molecular ions of the correPFP derivatives of melatonin and 111.) in 0.5 ml of the solvent mixture CHC1 3 : sponding 2 [ H ]melatonin (M/z 360 and 363, respectively; 3 light petroleum (1:1). The first 0.5 ml eluate was per ion, 200 m sec; resolution, 1000; dwell time discarded and a further 4 ml solvent was added ionizing current, 300 /u,A). and the eluate discarded. A further 5 ml solvent was added, and the eluate containing the melaResults tonin collected into tubes (12 x 100 mm) and the solvent evaporated at 37 C under a stream of nitrogen. After use, the columns were washed Characterization of the antigen with 10 ml chloroform:methanol (2:1) and stored The mechanism of antigen formation inin solvent. Columns were repeatedly used in this volved condensation between N-acetylway for up to 4 months without renewal. serotonin and the Mannich adduct of BSA Assay procedure. Assay buffer (0.5 ml, 0.1M and formaldehyde (16). Although antigens phosphate, pH 7.4, 0.15% gelatin, 0.1% NaN3, have been previously prepared by this route, 0.9% NaCl) was added to the extracts, followed characterization has only been based on by 100 jitl antibody stock (diluted 1:500 prior cross-reactivity studies (13,17). Accordingly, we conducted a detailed into use with assay buffer) and 100 /JL\ of [3H] melatonin solution (10,000 cpm) to a final volume vestigation on the "Mannich" reaction of of 800 fi\. The tube contents were gently mixed, N-acetylserotonin, using a model system in incubated at 37 C for 30 min, and then allowed which dimethylamine replaced BSA as the to stand overnight at 4 C. Ice-cold saturated source of the secondary amine moiety. With (NH4)2SO4 (800 fx\) solution was then added, the a variety of solvent systems, including that tubes vortexed for 20 sec, and then centrifuged used in the antigen preparation, only one at 4000 rpm at 4 C for 15 min. The supernatant was carefully decanted into scintillation vials, 5 major product was formed as assessed by tic. ml toluene-based scintillator added and the vials This material was isolated by column briefly vortexed. The two phase system was chromatography and in pure form showed a allowed to equilibrate for 2 - 3 h, and the un- marked propensity to undergo a Reverse bound radioactivity determined. Mannich reaction (18).
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KENNAWAY ET AL. * •» • »
Melatonin Pineal Mid-dark Plasma Mid-light Plasma
iz o
o o
30
100
300
1000
3000
10000
pg MLT or u\ plasma or IXJytryptophol 5-M»rho»yindot»oc«tic acid S-Mttrtoiytryptophan 5-Hydro»y-N-oc«tyl»«rotonin 5-Hydroaytryptomin« 3-Hydro»ytrypfophol 3-HydronyindoUacOk acid 3-Hydroxytryptophan N-Ac*tyltryptam«n« Tryptomin. Tryptophol lndol*a»tic acid Tryptophon
Circadian rhythm and effect of pinealectomy
Kynur«nin« N-Formyl Kynurmin* 3-M«rhyltryptamin« 5-M«thyl-N-oc*ty1tryptafnn* O-Acttyl- 5 -ffttthoaytryptophol O-Ac*tyl tryptophol
14 chloroform ; pttroUum spirit (| : |)
19 tthyl occtot*
:
p*trol«um spirit 60-80 (1:1)
f lution Volum* (ml)
FIG. 2. Elution pattern of various indoles on a column of Lipidex 5000 (3.2 x 175 mm) and their crossreactivity with the antibody to the compound of Nacetyl-serotonin and the Mannich adduct of BSA and formaldehyde. Details of chromatography and conditions of assay are given in text. Solvent mixtures used for elution are indicated at the bottom of the figure. Cross-reactivity, determined as the amount of compound required to achieve 50% displacement of [3H]melatonin, is indicated as follows: • , 1 pmol; f2 4 nmol; D, > 80 nmol.
Figure 6 (a,b) shows the results of more intensive studies on the circadian rhythm in 5 intact and 3 pinealectomized ewes. Blood samples were obtained over 30 min intervals for 24 h. Melatonin is present in all samples analyzed. In the undisturbed intact sheep, a marked rise in plasma melatonin was evident within 30 min of the start of the dark period. The levels then remained high until the lights were switched on. Interestingly, this rise in plasma melatonin was abolished by pinealectomy. It is of interest also that ewe 245, which was pinealectomized 14 days prior to sampling, had much lower levels of circulating melatonin than that found in the two ewes (288, 22) which
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124
M/Z360 VWKV'*
/I
1 2
Endo • 1977 Vol 101 • No 1
KENNAWAY ET AL.
3 4
min
5
M/z363
2
3
min
(b) FIG. 4. Selected ion current profile of (a) extract from sheep pineal tissue (b) water blank. Upper channel, (M/z 360) monitoring endogenous melatonin; lower channel, (M/z 363) monitoring internal standard.
were pinealectomized 3 months previously and in which the plasma levels remained at dark period values. Melatonin in human plasma Melatonin was determined at 4 h intervals, during a period of 24 h in the plasma of 2 male and 3 female patients hospitalized for non-endocrine disorders during autumn (mid-March-early April) with the results shown in Fig. 6c. A nocturnal rise in plasma melatonin content was evident and day time samples were all below 20 pg/ml. To confirm the low day time values, plasma samples were obtained in late summer (February) at 1100 h from 5 male and 3 female members of the laboratory staff aged 18-32. The melatonin concentrations determined were all less than 14 pg/ml. Melatonin in cattle plasma The occurrence of melatonin in cattle plasma was investigated in three month old
heifers, two of the Brahman and one of the Jersey breed. Blood samples were obtained at hourly intervals through a 24 h period in mid-summer (December). The results are presented in Fig. 6d. Melatonin levels were low during day time but a marked rise occurred with the onset of night (2000 h) and the rise was sustained throughout the period of darkness. Melatonin in other species Blood was also collected from rats, a scincid lizard (Tiliqua rugosa), chickens, donkeys, a pig and a camel at mid-light and mid-dark. Table 1 shows the results obtained. Plasma levels of melatonin were higher at mid-dark that at mid-light in all species examined. Discussion The RIA described employs an antibody which reacts with a variety of N-acetylated indoles and for absolute specificity is dependent upon the extraction procedure and column chromatography. While these additional steps place some constraint on the number of samples which may be conveniently assayed, they result in a fraction for assay which is extensively purified and largely freed of competing substances. It also has the advantage that the antiserum used to quantitate melatonin may, 200
ff
y - 0 957x-16 r - 0 986
150
« (0
o
I 100 E 50
0
50
100
Selected Ion Monitoring
150
200
ng /gland
FIG. 5. Relationship of results of determinations of melatonin content of individual sheep pineal gland by the melatonin RIA and SIM procedures.
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MELATONIN RADIOIMMUNOASSAY
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The good agreement gives added confidence in the RIA, as of the various procedures available for the analysis of indoles 160 and related compounds, those based on se120 lected ion monitoring procedures are un80 doubtedly the most specific and reliable 40 (8,9). This technique has now been successfully applied to the detection of melatonin in a variety of biological extracts (2,3,15), 300 and the PFP ester employed in the present study established as the derivative of choice ^200 (15). Other derivatives such as the Ni trimethylsilyl and N,N-diethyl compounds uMOO _a have proven less satisfactory, for although z they have intense ions (M/z 245, 232 and z HUMAN 201, 188, respectively) they are prone to greater adsorption losses when chromatographed in picogram amounts (Phillipou, G., unpublished observations). The advantages of a stable-isotope labelled compound as the internal standard in SIM have been adequately discussed 300 elsewhere (8,9,21). For the preparation of deuterated melatonin, N-acetylserotonin 200 was chosen as the precursor since its 5100 hydroxyl group (phenolic) may be methylated by standard procedures utilizing diazomethane or methyl iodide (11). However, 1400h 1800h 2200h 0200h 0600h 10OOh CLOCK T I M E with diazomethane only low specific incorporation of deuterium may be achieved FIG. 6. Plasma melatonin determinations in sheep, human and cattle plasma, (a). Mean melatonin con- (22) and, accordingly, the procedure using tent (mean ± SEM) in plasma continuously sampled deuterated methyl iodide was developed as over 30 min intervals, throughout a 24 h period in 5 detailed in Materials and Methods. The intact ewes, (b) The melatonin content of 3 individual method is generally applicable to the labelpinealectomized ewes sampled similarly; (c and d) Individual data for single determinations made at ling of other 5-methoxyindoles. The content intervals throughout a period of 24 h of 5 humans and of melatonin determined for the sheep 3 cattle. Dark bars indicate the dark period. pineal is comparable with that found in pineal tissue of other species (23). The present data confirm that melatonin with only slight modification of the extracoccurs in plasma of man (3,4,24,25), sheep tion and chromatography procedures, be (6), rat (26) and chicken (2,27) and estabused to assay N-acetylserotonin and certain other indoles in the same sample. TABLE 1. Plasma melatonin concentrations in single The specificity of the assay for both pineal samples obtained at mid-light and mid-dark in a variety tissue and plasma samples was checked by of animal species* the use of GC-MS. Excellent correlation Mid-light Species Mid-dark Sex was found in determinations of both the Rattus norvegicus (rat) 9 6.3 ± 3 (5) 75 ± 27 (8) melatonin content of sheep pineal tissue, a Tiliqua rugosa 9,6 35 ±5.9(10) 240 ± 4 3 (10) tissue rich in content of other indoles in- Gallus gallus (chicken) 9 50 (2) 200 (3) 9 24.5 (2) 108 (2) cluding N-acetylserotonin, 5-methoxy- Equus asinus (donkey) Sus scrofa (pig) 6 22 (1) 76 (1) tryptophol and 5-hydroxytryptophol (20), Camelus dromedarius (camel) 29 (1) 221 (1) likely to react in RIA procedures for mela* Values are means (pg/ml) ± SEM, number of animals is shown in tonin, and of a sample of sheep plasma. parentheses. SHEEP
(a)
200
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lishes its presence in the plasma of the establishes its presence in the plasma of the pig, donkey, cow, camel and a lizard even at mid-light. The data have also shown that for all species examined the plasma levels were higher at mid-dark than at mid-light. The data on the content and nocturnal rise in sheep plasma are very similar to that recently obtained by Rollag and Niswender (6) using an RIA procedure employing a more specific antibody but no chromatography step. The sudden rise in plasma melatonin with the onset of darkness, the sustained elevation during the dark period and the immediate fall with the start of the lights on period in the undisturbed intact ewes indicate that the 24 h organization of melatonin secretion is strongly correlated with the light-dark changes. Of particular interest is that the nocturnal rise in plasma melatonin in the sheep was found to be abolished by pinealectomy. However, it is important to note the melatonin was still present in the plasma of the 3 pinealectomized ewes studied. Two of these ewes, pinealectomized 3 months prior to investigation, maintained dark period plasma levels of melatonin, and the other ewe (245), pinealectomized 2 weeks prior to the study, maintained light period plasma levels of melatonin. To confirm that the ewes were successfully pinealectomized, one of the ewes (288) showing high melatonin levels, was sacrificed and the completeness of the removal of pineal tissue confirmed, by histological investigation. In this regard there is now considerable evidence to suggest that melatonin may be produced by tissues other than the pineal gland. The original argument for the pineal being the principal site of melatonin synthesis was based on data suggesting that hydroxyindole - O - methyl - transferase (HIOMT), a key enzyme in melatonin synthesis, was localized exclusively in the pineal (28). However, there are now several reports on the occurrence of HIOMT in other tissues, including blood cells (29), Harderian gland and retina (30). Recently, indirect but compelling data have been
Endo • 1977 Vol 101 . No 1
presented by Koslow (36) to indicate that melatonin may be synthesized in extrapineal sites. He showed, by SIM, that the melatonin content of the hypothalamus of rats was unaltered one month after pinealectomy. If the extra-pineal sites of melatonin synthesis can assume prominence following pinealectomy, as suggested by the present data, this may contribute toward an explanation for the many conflicting reports stemming from investigations of the endocrine function of the pineal gland (31). Previous investigations on the occurrence of melatonin in human plasma by Arendt et al. (4) indicated a mean plasma melatonin content in 14 subjects of 130 pg/ml during daylight and 183 pg/ml at night. However, while other investigators have confirmed by bioassay the occurrence of melatonin in samples obtained at night, they were unable to detect melatonin in either blood (24,25) or urine (32) during periods of light. Our results in human subjects indicate that melatonin may occur in plasma in daylight but at levels usually below 20 pg/ml. It is difficult to explain the discrepancy between this report and that of Arendt et al. (4). Possibly seasonal variations in daytime plasma levels may occur (25) or their antibody may have responded to plasma indoles not included in the cross-reaction studies, or other non-polar substances (33). The nocturnal rise in plasma melatonin is of interest in relation to other known circadian rhythms in the human. Major alterations in CNS activity and neuroendocrine functions related to light-dark regimes and different stages of sleep have been described (34). At least 4 hypothalamic-pituitary systems have temporal patterns of secretion which are closely linked to the 24 h sleep-wake activity in man, including ACTH, HGH, prolactin and TSH. LH released during early and late puberty also appears to be linked with a sleep-wake cycle, but the existence of a diurnal cycle in plasma FSH concentration is somewhat controversial. The 24 h pattern in plasma melatonin most resembles that described for prolactin which has a nocturnal rise which has been
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MELATONIN RADIOIMiMUNOASSAY shown to be enhanced by sleeping. However, Vaughan et al. (25), using a bioassay, have data which indicates that the nocturnal rise in plasma melatonin is, at least in the short term, independent of alterations to both light and sleep phases. Further definition of the relationship of melatonin release to sleep and other circadian rhythms in the human is required. As regards the occurrence of melatonin in the other species, a nocturnal rise in melatonin has also been found for rats (26) and chickens (27), the plasma concentrations determined by these investigators being very similar to that found in the present study. No previous information on the occurrence of melatonin in cattle, camel, donkey or pig blood has been reported. The finding of a nychtohemeral rise in plasma melatonin content ofTiliqua rugosa was of interest, in view of the known occurrence of a diurnal rhythm in the HIOMT activity of the pineal organ in many reptilian species (35). Acknowledgments We gratefully acknowledge the collaboration of Professor W. V. Macfarlane and Drs. W. Breed, B. Firth, and J. Obst in obtaining some plasma samples, and Ann Le Conui and K. Porter for excellent technical and surgical assistance. We also wish to acknowledge use of the facilities of the Organic Chemistry Department, The University of Adelaide, South Australia 5000.
References 1. Ralph, C. L., and H. J. Lynch, Gen Comp Endocrinol 15: 334, 1970. 2. Pelham, R. W., C. L. Ralph, and I. M. Campbell, Biochem Biophys Res Commun 46: 1236, 1972. 3. Smith, I., P. E. Mullen, R. E. Snedden, and B. W. Wilson, Nature (Lond) 260: 718, 1976. 4. Arendt, J., L. Paunier, and P. C. Sizonenko,./ Clin Endocrinol Metab 40: 347, 1975. 5. Levine, L., and L. J. Riceberg, Res Commun Chem Pathol Pharmacol 10: 693, 1975. 6. Rollag, M. D., andG. D. Niswender,Endocrinology 98: 482, 1976. 7. Wurzburger, R. J., K. Kawashima, R. L. Miller, and S. Spector, Life Sci 18: 867, 1976. 8. Lawson, A. M., and G. H. Draffan, Prog Medicinal Chem 12: 1, 1975. 9. Falkner, F. C , B. J. Sweetman, and J. T. Watson, Appl Spectros Rev 10: 5, 1975.
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10. Roche, J., F. J. Karsch, D. L. Foster, S. Takagi, and P. J. Dziuk, Biol Reprod 2: 251, 1970. 11. Fieser, L. F., and M. Fieser, Reagents for Organic Synthesis, vol. 1, J. Wiley, New York, 1967. 12. Merck Index, ed. 8, Merck Inc., Rathway, 1968, p. 650. 13. Grota, L. J., and G. M. Brown, Can J Biochem 52: 196, 1974. 14. Abraham, G. E., J Clin Endocrinol Metah 29: 866, 1969. 15. Koslow, S. H., and A. R. Green, Adv Biochem Psychopharmacol 7: 33, 1973. 16. Remers, W. A., In Houlihan, W. J. (ed.), The Chemistry of Heterocyclic Compounds, Indoles, part 1, Wiley-Interscience, New York, 1972, p. 95. 17. Ranadive, N. S., and A. H. Sehon, Can J Biochem 45: 1701, 1967. 18. Monti, S. A., and G. D. Castillo, Jr., y Org Chem 35: 3764, 1970. 19. Monti, S. A., and W. O. Johnson, Tetrahedron 26: 3685, 1970. 20. Ebels, I., and A. E. M. Horwitzbresser, J Neural Trans 38: 31, 1976. 21. Fentiman, A. F., Jr., and R. L. Foltz, J Labelled Comp Radiopharmacol 12: 69, 1976. 22. Hattox, S. E., and R. C. Murphy, Biomed Mass Spectrom 2: 272, 1975. 23. Quay, W. B., Pineal Chemistry in Cellular and Physiological Mechanisms, Charles C Thomas, Springfield, 111., 1974, p. 150. 24. Pelham, R. W., G. M. Vaughan, K. L. Sandcock, and M. K. Vaughan, J Clin Endocrinol Metab 37: 341, 1973. 25. Vaughan, G. M., R. W. Pelham, S. F. Pang, L. L. Loughlin, K. M. Wilson, K. L. Sandcock, M. K. Vaughan, S. H. Koslow, and R. J. Reiter, / Clin Endocrinol Metab 42: 752, 1976. 26. Pang, S. F., and C. I. Ralph, Gen Comp Endocrinol 27: 125, 1975. 27. Pelham, R. W., Endocrinology 96: 543, 1975. 28. Axelrod, J., and H. Weissbach,./ Biol Chem 236: 211, 1961. 29. Rosengarten, H., E. Meller, and A. J. Friedhoff, Res Comm Chem Pathol Pharmacol 4: 457, 1972. 30. Vlahakes, G. J., and R. J. Wurtman, Biochim Biophys Ada 261: 194, 1972. 31. Reiter, R. J., Ann Rev Physiol 35: 305, 1973. 32. Lynch, H. J., R. J. Wurtman, M. A. Moskowitz, M. C. Archer, and M. H. Ho, Science 187: 169, 1975. 33. Rosa, U., R. Malvano, and E. Rolled, In Crosignani, P. G., and V. H. T. James (eds.), Recent Progress in Productive Endocrinology, Academic Press, London, 1974, p. 127. 34. Weitzman, E. D., R. M. Boyar, S. Kapen, and L. Hellman, Recent ProgHorm Res 31: 399, 1975. 35. Quay, W. B., Life Sci 4: 983, 1965. 36. Koslow, S. H., Adv Biochem Psychopharmacol 11: 95, 1974.
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Department of Obstetrics and Gynaecology, The University of Adelaide, 5000, and 2The Queen Elizabeth Hospital, Woodville, South Australia 5011 ABSTRACT. A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon the extraction procedure and column (Lipidex 5000) chromatography. In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography-mass spectrometry using [2H3]melatonin as an
I
NVESTIGATIONS on the endocrine function of the pineal gland have been severely hampered by the lack of a reliable, practical assay for melatonin, a presumptive major pineal hormone. That melatonin occurs in plasma has been indicated by sensitive bioassays (1) and its presence confirmed more recently by mass spectrometric methods (2,3), but these specialized procedures are generally unsuited to routine assay of large numbers of samples. Recently, several practical radioimmunoassay procedures for melatonin in plasma (4-6) or pineal tissues (7) have been reported and studies on melatonin content during various environmental and endocrinological studies initiated (4-7). This report concerns the development of a radioimmunoassay based on an antibody raised to an easily prepared compound of N-acetylserotonin and bovine serum albumin (BSA) which is suited to determinaReceived September 2, 1976. DJ.K. was supported by the Sir John Gellibrand Memorial Scholarship.
South
Australia
internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22-76 pg/ml), donkey (24-128 pg/ml), cow (20-320 pg/ml), camel (29-221 pg/ml) and a scincid lizard (20500 pg/ml) established. A nocturnal rise in plasma melatonin content occurred in all species. Melatonin was found in the plasma of ewes 2-12 weeks after pinealectomy, but the nocturnal rise was abolished. The results establish a nyctohemeral variation in plasma melatonin in a wide variety of species, and indicate that sources of melatonin other than the pineal may assume precedence following pinalectomy. (Endocrinology 101: 119, 1977)
tions of melatonin in both tissues and body fluids on a routine basis. In addition to the usual criterion of reliability, the method is validated by the technique of selected ion monitoring (8,9). Its application to determination of the plasma melatonin content of a variety of species kept under diurnal lighting conditions is reported. Materials and Methods Animals Sheep, mainly Merino breed, were obtained from the flocks of the Mortlock Research Centre, Mintaro, South Australia. For intensive bleeding regimens, animals were housed individually in air-conditioned pens illuminated between 0700 and 2100 h. A period of at least two weeks was allowed for the animals to acclimatize before bleeding commenced. Blood was continuously sampled at the rate of 12 ml/h from the left jugular vein using a vinyl cannula, id 0.86 mm, od 1.2 mm (Dural Plastics, Dural, N.S.W. Australia) placed inside a second larger vinyl cannula, id 2 mm, od 3.0 mm. Heparinized saline, 250 U/ml, was continuously
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infused via the outside lumen at 6 ml/h during collections. Samples were collected over 30 min intervals. Studies with a dye (Indocyanine Green, Hynson, Westcott and Dunning, Inc., Baltimore, Maryland) added to the heparinized saline, showed that the samples were contaminated with less than 2% of the heparinized saline when collected by this procedure. Pinealectomy was carried out according to the procedure of Roche et al. (10) as modified by Obst (unpublished). The cattle, Brahman and Jersey breed, were obtained from Struan Research Centre, and the camel, donkey and pigs, from the Waite Agricultural Research Institute, South Australia, and were kept on open pastures. Blood samples were collected by venipuncture. The rats (Hooded Wistar strain) and lizards were housed at constant temperature under a diurnal (13L:11D) lighting regimen. Blood samples were collected by heart puncture. Reagents All solvents were the highest purity grades available and were redistilled prior to use. Dry 1,2-dimethoxyethane was obtained by distillation from sodium-wire. Deuterated methyl iodide and acetonitrile (both > 99% 2H3) were purchased from R/M Isotopes Co., Maynard, Maine. Tritiated melatonin (specific activity 26 Ci/mmol) was purchased from New England Nuclear Corporation, Boston, Mass., and used without further purification. The indole standards, except N-formyl-Lkynurenine (Calbiochem, San Diego, Ca.) were obtained from Sigma, St. Louis, Mo. O and Nacetyl indoles, unavailable commercially, were prepared using acetic anhydride/pyridine (11), and their purity confirmed by thin layer chromatography (tic). Preparation of acetylserotonin
4-N,N-dimethylaminomethyl)-N-
Paraformaldehyde (4.2 mg, 0.14 mmol) and dimethylamine (16/u.l, 0.14 mmol, 40% aqueous) in ethanol (5 ml) were stirred at room temperature for 15 min. N-acetyl-serotonin (30 mg, 0.14 mmol) was then added and the whole mixture stirred overnight at room temperature under N2. The solvent was then removed in vacuo and the residue chromatographed on Sorbsil. Elution with methanol:chloroform (1:19) gave the major component (17 mg), shown to be pure by tic and identified by proton magnetic resonance (PMR) spectroscopy (Varian T-60 instru-
Endo • 1977 Vol 101 • No 1
ment, samples dissolved in deuterated acetonitrile, with tetramethylsilane as internal standard) as 4-(N,N-dimethylaminomethyl)-N-acetylserotonin. Preparation of [2H3]melatonin N-acetylserotonin (50 mg, 0.23 mmol) and sodium hydroxide (18 mg, 0.45 mmol) in 1,2dimethoxyethane (5 ml) were stirred at room temperature for 5 min in the dark. Deuterated methyl iodide (100 mg, 0.74 mmol) was then added and the reaction mixture stirred for a further 4 h at which time the excess base was neutralized (5% dilute hydrochloric acid) and the solvent removed on a rotary evaporator. The remaining residue was preabsorbed and chromatographed on neutral alumina grade II (5 g), care again being taken to exclude sunlight. The column was developed stepwise with the increasing amounts of chloroform in benzene (1-50%). It was then eluted with chloroform to give [2H3]melatonin (43 mg, 81%), which was recrystallized from benzene to yield pale yellow crystals nip 115.5-117 C (lit. (12) 116118 C); tic and gas chromatography (GC) showed the product to be homogeneous; its isotopic composition as determined by mass spectrometry was 99.1% 2H3. Preparation of antibody The antigen was formed by condensing Nacetylserotonin with the Mannich adduct of BSA and formaldehyde as described by Grota and Brown (13). The molar ratio of hapten to carrier protein, as determined by spectrometry, was 50:1. Four rabbits were injected sc at 3 dorsal sites with an emulsion of 7 mg of the lyophilized antigen, 2 ml 0.05M phosphate buffer, pH 7.9, and 2 ml complete Freund's adjuvant. Booster injections of 1-2 mg were given monthly. Blood from an ear vein was collected 9-10 days after these injections into lithium heparin coated tubes and centrifuged at 4000 rpm. Plasma was divided into 1 ml aliquots and stored frozen until used. Antibody was detectable after the second booster injection in one rabbit (R8) and after subsequent booster injections in the other rabbits. For use, 1 ml of plasma containing a suitable concentration of antibody was vortexed with 4 ml of a 0.4% solution of Rivanol (2-ethoxy-6,9diaminoacridine lactate, K & K Laboratories, Plainview, New York) in distilled water and allowed to stand for 10 min at room temperature (14). Then 300 mg activated charcoal was added,
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MELATONIN RADIOIMMUNOASSAY the suspension was vortexed, and allowed to stand at room temperature for 10 min. Five ml distilled water was added and the tubes were centrifuged at 4000 rpm for 10 min at 4 C. The antibody was stable at this dilution for several months at 4 C. Assay of melatonin by radioimmunoassay Extraction procedure. Plasma (2-4 ml) was mixed with an equal volume of 0.5M borate buffer, pH 10.0, and extracted for 30 min with 4-5 volumes of chloroform using a gentle rocking motion to avoid emulsion formation. A Paton extractor (Paton Industries Pty. Ltd., Beaumont, South Australia) was adapted for this purpose. Tissues (5-100 mg) were homogenized in 1 ml 0.5N NaOH, then 2 ml 0.5M borate buffer, pH 10.0, was added and the mixture extracted with chloroform. Following extraction, the samples were centrifuged to fully resolve the phases, the aqueous layer was then aspirated, and the chloroform evaporated at 37 C under N2.
121
Known amounts of melatonin (30-1000 pg) were taken through the assay procedure as reference standards. Assay of melatonin by selected ion monitoring Extraction and isolation procedure. Individual pineal glands were homogenized in a solution of NaOH (1 ml, 0.5N) and borate buffer (2.0 ml, 0.5N, pH 10), followed by extraction with chloroform (10 ml). The chloroform extract was then evaporated to dryness and the residue chromatographed on Lipidex 5000 as described above. An aliquot was taken for RIA and 40 ng deuterated melatonin added to the remainder and the solvent removed. The pentafluoro-propionyl (PFP) ester was then formed (15) and the product subjected to selected ion monitoring.
Selected ion monitoring. GC-MS was carried out with an AEI MS-30 mass spectrometer, interfaced to a Pye GC (column, 1.0 m x 2 mm id; 1% OV-225 on 100/120 mesh support; temp., 230 C; helium flow, 25 ml/min) via a single stage glass Chromatographij. The residue was taken up and jet separator (S.G.E., Melbourne, Australia). transferred to columns (3.2 x 175 mm) of Lipidex Quantitation was achieved by selective ion moni5000 (Packard Instrument Co., Downers Grove toring (SIM) of the molecular ions of the correPFP derivatives of melatonin and 111.) in 0.5 ml of the solvent mixture CHC1 3 : sponding 2 [ H ]melatonin (M/z 360 and 363, respectively; 3 light petroleum (1:1). The first 0.5 ml eluate was per ion, 200 m sec; resolution, 1000; dwell time discarded and a further 4 ml solvent was added ionizing current, 300 /u,A). and the eluate discarded. A further 5 ml solvent was added, and the eluate containing the melaResults tonin collected into tubes (12 x 100 mm) and the solvent evaporated at 37 C under a stream of nitrogen. After use, the columns were washed Characterization of the antigen with 10 ml chloroform:methanol (2:1) and stored The mechanism of antigen formation inin solvent. Columns were repeatedly used in this volved condensation between N-acetylway for up to 4 months without renewal. serotonin and the Mannich adduct of BSA Assay procedure. Assay buffer (0.5 ml, 0.1M and formaldehyde (16). Although antigens phosphate, pH 7.4, 0.15% gelatin, 0.1% NaN3, have been previously prepared by this route, 0.9% NaCl) was added to the extracts, followed characterization has only been based on by 100 jitl antibody stock (diluted 1:500 prior cross-reactivity studies (13,17). Accordingly, we conducted a detailed into use with assay buffer) and 100 /JL\ of [3H] melatonin solution (10,000 cpm) to a final volume vestigation on the "Mannich" reaction of of 800 fi\. The tube contents were gently mixed, N-acetylserotonin, using a model system in incubated at 37 C for 30 min, and then allowed which dimethylamine replaced BSA as the to stand overnight at 4 C. Ice-cold saturated source of the secondary amine moiety. With (NH4)2SO4 (800 fx\) solution was then added, the a variety of solvent systems, including that tubes vortexed for 20 sec, and then centrifuged used in the antigen preparation, only one at 4000 rpm at 4 C for 15 min. The supernatant was carefully decanted into scintillation vials, 5 major product was formed as assessed by tic. ml toluene-based scintillator added and the vials This material was isolated by column briefly vortexed. The two phase system was chromatography and in pure form showed a allowed to equilibrate for 2 - 3 h, and the un- marked propensity to undergo a Reverse bound radioactivity determined. Mannich reaction (18).
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KENNAWAY ET AL. * •» • »
Melatonin Pineal Mid-dark Plasma Mid-light Plasma
iz o
o o
30
100
300
1000
3000
10000
pg MLT or u\ plasma or IXJytryptophol 5-M»rho»yindot»oc«tic acid S-Mttrtoiytryptophan 5-Hydro»y-N-oc«tyl»«rotonin 5-Hydroaytryptomin« 3-Hydro»ytrypfophol 3-HydronyindoUacOk acid 3-Hydroxytryptophan N-Ac*tyltryptam«n« Tryptomin. Tryptophol lndol*a»tic acid Tryptophon
Circadian rhythm and effect of pinealectomy
Kynur«nin« N-Formyl Kynurmin* 3-M«rhyltryptamin« 5-M«thyl-N-oc*ty1tryptafnn* O-Acttyl- 5 -ffttthoaytryptophol O-Ac*tyl tryptophol
14 chloroform ; pttroUum spirit (| : |)
19 tthyl occtot*
:
p*trol«um spirit 60-80 (1:1)
f lution Volum* (ml)
FIG. 2. Elution pattern of various indoles on a column of Lipidex 5000 (3.2 x 175 mm) and their crossreactivity with the antibody to the compound of Nacetyl-serotonin and the Mannich adduct of BSA and formaldehyde. Details of chromatography and conditions of assay are given in text. Solvent mixtures used for elution are indicated at the bottom of the figure. Cross-reactivity, determined as the amount of compound required to achieve 50% displacement of [3H]melatonin, is indicated as follows: • , 1 pmol; f2 4 nmol; D, > 80 nmol.
Figure 6 (a,b) shows the results of more intensive studies on the circadian rhythm in 5 intact and 3 pinealectomized ewes. Blood samples were obtained over 30 min intervals for 24 h. Melatonin is present in all samples analyzed. In the undisturbed intact sheep, a marked rise in plasma melatonin was evident within 30 min of the start of the dark period. The levels then remained high until the lights were switched on. Interestingly, this rise in plasma melatonin was abolished by pinealectomy. It is of interest also that ewe 245, which was pinealectomized 14 days prior to sampling, had much lower levels of circulating melatonin than that found in the two ewes (288, 22) which
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124
M/Z360 VWKV'*
/I
1 2
Endo • 1977 Vol 101 • No 1
KENNAWAY ET AL.
3 4
min
5
M/z363
2
3
min
(b) FIG. 4. Selected ion current profile of (a) extract from sheep pineal tissue (b) water blank. Upper channel, (M/z 360) monitoring endogenous melatonin; lower channel, (M/z 363) monitoring internal standard.
were pinealectomized 3 months previously and in which the plasma levels remained at dark period values. Melatonin in human plasma Melatonin was determined at 4 h intervals, during a period of 24 h in the plasma of 2 male and 3 female patients hospitalized for non-endocrine disorders during autumn (mid-March-early April) with the results shown in Fig. 6c. A nocturnal rise in plasma melatonin content was evident and day time samples were all below 20 pg/ml. To confirm the low day time values, plasma samples were obtained in late summer (February) at 1100 h from 5 male and 3 female members of the laboratory staff aged 18-32. The melatonin concentrations determined were all less than 14 pg/ml. Melatonin in cattle plasma The occurrence of melatonin in cattle plasma was investigated in three month old
heifers, two of the Brahman and one of the Jersey breed. Blood samples were obtained at hourly intervals through a 24 h period in mid-summer (December). The results are presented in Fig. 6d. Melatonin levels were low during day time but a marked rise occurred with the onset of night (2000 h) and the rise was sustained throughout the period of darkness. Melatonin in other species Blood was also collected from rats, a scincid lizard (Tiliqua rugosa), chickens, donkeys, a pig and a camel at mid-light and mid-dark. Table 1 shows the results obtained. Plasma levels of melatonin were higher at mid-dark that at mid-light in all species examined. Discussion The RIA described employs an antibody which reacts with a variety of N-acetylated indoles and for absolute specificity is dependent upon the extraction procedure and column chromatography. While these additional steps place some constraint on the number of samples which may be conveniently assayed, they result in a fraction for assay which is extensively purified and largely freed of competing substances. It also has the advantage that the antiserum used to quantitate melatonin may, 200
ff
y - 0 957x-16 r - 0 986
150
« (0
o
I 100 E 50
0
50
100
Selected Ion Monitoring
150
200
ng /gland
FIG. 5. Relationship of results of determinations of melatonin content of individual sheep pineal gland by the melatonin RIA and SIM procedures.
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MELATONIN RADIOIMMUNOASSAY
125
The good agreement gives added confidence in the RIA, as of the various procedures available for the analysis of indoles 160 and related compounds, those based on se120 lected ion monitoring procedures are un80 doubtedly the most specific and reliable 40 (8,9). This technique has now been successfully applied to the detection of melatonin in a variety of biological extracts (2,3,15), 300 and the PFP ester employed in the present study established as the derivative of choice ^200 (15). Other derivatives such as the Ni trimethylsilyl and N,N-diethyl compounds uMOO _a have proven less satisfactory, for although z they have intense ions (M/z 245, 232 and z HUMAN 201, 188, respectively) they are prone to greater adsorption losses when chromatographed in picogram amounts (Phillipou, G., unpublished observations). The advantages of a stable-isotope labelled compound as the internal standard in SIM have been adequately discussed 300 elsewhere (8,9,21). For the preparation of deuterated melatonin, N-acetylserotonin 200 was chosen as the precursor since its 5100 hydroxyl group (phenolic) may be methylated by standard procedures utilizing diazomethane or methyl iodide (11). However, 1400h 1800h 2200h 0200h 0600h 10OOh CLOCK T I M E with diazomethane only low specific incorporation of deuterium may be achieved FIG. 6. Plasma melatonin determinations in sheep, human and cattle plasma, (a). Mean melatonin con- (22) and, accordingly, the procedure using tent (mean ± SEM) in plasma continuously sampled deuterated methyl iodide was developed as over 30 min intervals, throughout a 24 h period in 5 detailed in Materials and Methods. The intact ewes, (b) The melatonin content of 3 individual method is generally applicable to the labelpinealectomized ewes sampled similarly; (c and d) Individual data for single determinations made at ling of other 5-methoxyindoles. The content intervals throughout a period of 24 h of 5 humans and of melatonin determined for the sheep 3 cattle. Dark bars indicate the dark period. pineal is comparable with that found in pineal tissue of other species (23). The present data confirm that melatonin with only slight modification of the extracoccurs in plasma of man (3,4,24,25), sheep tion and chromatography procedures, be (6), rat (26) and chicken (2,27) and estabused to assay N-acetylserotonin and certain other indoles in the same sample. TABLE 1. Plasma melatonin concentrations in single The specificity of the assay for both pineal samples obtained at mid-light and mid-dark in a variety tissue and plasma samples was checked by of animal species* the use of GC-MS. Excellent correlation Mid-light Species Mid-dark Sex was found in determinations of both the Rattus norvegicus (rat) 9 6.3 ± 3 (5) 75 ± 27 (8) melatonin content of sheep pineal tissue, a Tiliqua rugosa 9,6 35 ±5.9(10) 240 ± 4 3 (10) tissue rich in content of other indoles in- Gallus gallus (chicken) 9 50 (2) 200 (3) 9 24.5 (2) 108 (2) cluding N-acetylserotonin, 5-methoxy- Equus asinus (donkey) Sus scrofa (pig) 6 22 (1) 76 (1) tryptophol and 5-hydroxytryptophol (20), Camelus dromedarius (camel) 29 (1) 221 (1) likely to react in RIA procedures for mela* Values are means (pg/ml) ± SEM, number of animals is shown in tonin, and of a sample of sheep plasma. parentheses. SHEEP
(a)
200
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KENNAWAY ET AL.
lishes its presence in the plasma of the establishes its presence in the plasma of the pig, donkey, cow, camel and a lizard even at mid-light. The data have also shown that for all species examined the plasma levels were higher at mid-dark than at mid-light. The data on the content and nocturnal rise in sheep plasma are very similar to that recently obtained by Rollag and Niswender (6) using an RIA procedure employing a more specific antibody but no chromatography step. The sudden rise in plasma melatonin with the onset of darkness, the sustained elevation during the dark period and the immediate fall with the start of the lights on period in the undisturbed intact ewes indicate that the 24 h organization of melatonin secretion is strongly correlated with the light-dark changes. Of particular interest is that the nocturnal rise in plasma melatonin in the sheep was found to be abolished by pinealectomy. However, it is important to note the melatonin was still present in the plasma of the 3 pinealectomized ewes studied. Two of these ewes, pinealectomized 3 months prior to investigation, maintained dark period plasma levels of melatonin, and the other ewe (245), pinealectomized 2 weeks prior to the study, maintained light period plasma levels of melatonin. To confirm that the ewes were successfully pinealectomized, one of the ewes (288) showing high melatonin levels, was sacrificed and the completeness of the removal of pineal tissue confirmed, by histological investigation. In this regard there is now considerable evidence to suggest that melatonin may be produced by tissues other than the pineal gland. The original argument for the pineal being the principal site of melatonin synthesis was based on data suggesting that hydroxyindole - O - methyl - transferase (HIOMT), a key enzyme in melatonin synthesis, was localized exclusively in the pineal (28). However, there are now several reports on the occurrence of HIOMT in other tissues, including blood cells (29), Harderian gland and retina (30). Recently, indirect but compelling data have been
Endo • 1977 Vol 101 . No 1
presented by Koslow (36) to indicate that melatonin may be synthesized in extrapineal sites. He showed, by SIM, that the melatonin content of the hypothalamus of rats was unaltered one month after pinealectomy. If the extra-pineal sites of melatonin synthesis can assume prominence following pinealectomy, as suggested by the present data, this may contribute toward an explanation for the many conflicting reports stemming from investigations of the endocrine function of the pineal gland (31). Previous investigations on the occurrence of melatonin in human plasma by Arendt et al. (4) indicated a mean plasma melatonin content in 14 subjects of 130 pg/ml during daylight and 183 pg/ml at night. However, while other investigators have confirmed by bioassay the occurrence of melatonin in samples obtained at night, they were unable to detect melatonin in either blood (24,25) or urine (32) during periods of light. Our results in human subjects indicate that melatonin may occur in plasma in daylight but at levels usually below 20 pg/ml. It is difficult to explain the discrepancy between this report and that of Arendt et al. (4). Possibly seasonal variations in daytime plasma levels may occur (25) or their antibody may have responded to plasma indoles not included in the cross-reaction studies, or other non-polar substances (33). The nocturnal rise in plasma melatonin is of interest in relation to other known circadian rhythms in the human. Major alterations in CNS activity and neuroendocrine functions related to light-dark regimes and different stages of sleep have been described (34). At least 4 hypothalamic-pituitary systems have temporal patterns of secretion which are closely linked to the 24 h sleep-wake activity in man, including ACTH, HGH, prolactin and TSH. LH released during early and late puberty also appears to be linked with a sleep-wake cycle, but the existence of a diurnal cycle in plasma FSH concentration is somewhat controversial. The 24 h pattern in plasma melatonin most resembles that described for prolactin which has a nocturnal rise which has been
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MELATONIN RADIOIMiMUNOASSAY shown to be enhanced by sleeping. However, Vaughan et al. (25), using a bioassay, have data which indicates that the nocturnal rise in plasma melatonin is, at least in the short term, independent of alterations to both light and sleep phases. Further definition of the relationship of melatonin release to sleep and other circadian rhythms in the human is required. As regards the occurrence of melatonin in the other species, a nocturnal rise in melatonin has also been found for rats (26) and chickens (27), the plasma concentrations determined by these investigators being very similar to that found in the present study. No previous information on the occurrence of melatonin in cattle, camel, donkey or pig blood has been reported. The finding of a nychtohemeral rise in plasma melatonin content ofTiliqua rugosa was of interest, in view of the known occurrence of a diurnal rhythm in the HIOMT activity of the pineal organ in many reptilian species (35). Acknowledgments We gratefully acknowledge the collaboration of Professor W. V. Macfarlane and Drs. W. Breed, B. Firth, and J. Obst in obtaining some plasma samples, and Ann Le Conui and K. Porter for excellent technical and surgical assistance. We also wish to acknowledge use of the facilities of the Organic Chemistry Department, The University of Adelaide, South Australia 5000.
References 1. Ralph, C. L., and H. J. Lynch, Gen Comp Endocrinol 15: 334, 1970. 2. Pelham, R. W., C. L. Ralph, and I. M. Campbell, Biochem Biophys Res Commun 46: 1236, 1972. 3. Smith, I., P. E. Mullen, R. E. Snedden, and B. W. Wilson, Nature (Lond) 260: 718, 1976. 4. Arendt, J., L. Paunier, and P. C. Sizonenko,./ Clin Endocrinol Metab 40: 347, 1975. 5. Levine, L., and L. J. Riceberg, Res Commun Chem Pathol Pharmacol 10: 693, 1975. 6. Rollag, M. D., andG. D. Niswender,Endocrinology 98: 482, 1976. 7. Wurzburger, R. J., K. Kawashima, R. L. Miller, and S. Spector, Life Sci 18: 867, 1976. 8. Lawson, A. M., and G. H. Draffan, Prog Medicinal Chem 12: 1, 1975. 9. Falkner, F. C , B. J. Sweetman, and J. T. Watson, Appl Spectros Rev 10: 5, 1975.
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10. Roche, J., F. J. Karsch, D. L. Foster, S. Takagi, and P. J. Dziuk, Biol Reprod 2: 251, 1970. 11. Fieser, L. F., and M. Fieser, Reagents for Organic Synthesis, vol. 1, J. Wiley, New York, 1967. 12. Merck Index, ed. 8, Merck Inc., Rathway, 1968, p. 650. 13. Grota, L. J., and G. M. Brown, Can J Biochem 52: 196, 1974. 14. Abraham, G. E., J Clin Endocrinol Metah 29: 866, 1969. 15. Koslow, S. H., and A. R. Green, Adv Biochem Psychopharmacol 7: 33, 1973. 16. Remers, W. A., In Houlihan, W. J. (ed.), The Chemistry of Heterocyclic Compounds, Indoles, part 1, Wiley-Interscience, New York, 1972, p. 95. 17. Ranadive, N. S., and A. H. Sehon, Can J Biochem 45: 1701, 1967. 18. Monti, S. A., and G. D. Castillo, Jr., y Org Chem 35: 3764, 1970. 19. Monti, S. A., and W. O. Johnson, Tetrahedron 26: 3685, 1970. 20. Ebels, I., and A. E. M. Horwitzbresser, J Neural Trans 38: 31, 1976. 21. Fentiman, A. F., Jr., and R. L. Foltz, J Labelled Comp Radiopharmacol 12: 69, 1976. 22. Hattox, S. E., and R. C. Murphy, Biomed Mass Spectrom 2: 272, 1975. 23. Quay, W. B., Pineal Chemistry in Cellular and Physiological Mechanisms, Charles C Thomas, Springfield, 111., 1974, p. 150. 24. Pelham, R. W., G. M. Vaughan, K. L. Sandcock, and M. K. Vaughan, J Clin Endocrinol Metab 37: 341, 1973. 25. Vaughan, G. M., R. W. Pelham, S. F. Pang, L. L. Loughlin, K. M. Wilson, K. L. Sandcock, M. K. Vaughan, S. H. Koslow, and R. J. Reiter, / Clin Endocrinol Metab 42: 752, 1976. 26. Pang, S. F., and C. I. Ralph, Gen Comp Endocrinol 27: 125, 1975. 27. Pelham, R. W., Endocrinology 96: 543, 1975. 28. Axelrod, J., and H. Weissbach,./ Biol Chem 236: 211, 1961. 29. Rosengarten, H., E. Meller, and A. J. Friedhoff, Res Comm Chem Pathol Pharmacol 4: 457, 1972. 30. Vlahakes, G. J., and R. J. Wurtman, Biochim Biophys Ada 261: 194, 1972. 31. Reiter, R. J., Ann Rev Physiol 35: 305, 1973. 32. Lynch, H. J., R. J. Wurtman, M. A. Moskowitz, M. C. Archer, and M. H. Ho, Science 187: 169, 1975. 33. Rosa, U., R. Malvano, and E. Rolled, In Crosignani, P. G., and V. H. T. James (eds.), Recent Progress in Productive Endocrinology, Academic Press, London, 1974, p. 127. 34. Weitzman, E. D., R. M. Boyar, S. Kapen, and L. Hellman, Recent ProgHorm Res 31: 399, 1975. 35. Quay, W. B., Life Sci 4: 983, 1965. 36. Koslow, S. H., Adv Biochem Psychopharmacol 11: 95, 1974.
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