Age-related changes in melatonin synthesis in rat extrapineal tissues

HAL Id: hal-00493098 https://hal.archives-ouvertes.fr/hal-00493098 Submitted on 18 Jun 2010 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Age-related changes in melatonin synthesis in rat extrapineal tissues M Sanchez-Hidalgo, C Alarcon de la Lastra, Mp Carrascosa-Salmoral, Mc Naranjo, A Gomez-Corvera, B Caballero, Jm Guerrero To cite this version: M Sanchez-Hidalgo, C Alarcon de la Lastra, Mp Carrascosa-Salmoral, Mc Naranjo, A Gomez-Corvera, et al.. Age-related changes in melatonin synthesis in rat extrapineal tissues. Experimental Gerontology, Elsevier, 2009, 44 (5), pp.328. �10.1016/j.exger.2009.02.002�. �hal-00493098� Accepted Manuscript Age-related changes in melatonin synthesis in rat extrapineal tissues M Sanchez-Hidalgo, C Alarcon de la Lastra, MP Carrascosa-Salmoral, MC Naranjo, A Gomez-Corvera, B Caballero, JM Guerrero PII: S0531-5565(09)00024-2 DOI: 10.1016/j.exger.2009.02.002 Reference: EXG 8579 To appear in: Experimental Gerontology Received Date: 20 October 2008 Revised Date: 19 December 2008 Accepted Date: 11 February 2009 Please cite this article as: Sanchez-Hidalgo, M., de la Lastra, C.A., Carrascosa-Salmoral, M., Naranjo, M., Gomez- Corvera, A., Caballero, B., Guerrero, J., Age-related changes in melatonin synthesis in rat extrapineal tissues, Experimental Gerontology (2009), doi: 10.1016/j.exger.2009.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 1 Age-related changes in melatonin synthesis in rat extrapineal tissues M Sanchez-Hidalgo 1,2, C Alarcon de la Lastra 2, MP Carrascosa-Salmoral 3, MC Naranjo 3, A Gomez-Corvera 3, B Caballero 4, JM Guerrero1, *. 1, Department of Clinical Biochemistry, Biomedicine Institute of Seville (IBiS)/CSIC, University of Seville, Virgen del Rocío University Hospital, Seville, Spain. 2, Department of Pharmacology, Faculty of Pharmacy, University of Seville, Seville, Spain, 3, Department of Medical Biochemistry and Molecular Biology, University of Seville School of Medicine, Seville, Spain 4, Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain. * Corresponding author: Dr. Juan M. Guerrero Department of Clinical Biochemistry Virgen del Rocío University Hospital Manuel Siurot Street, s/n 41013, Seville (Spain) Phone: +34 955013243 e-mail: [email protected], [email protected] Keywords: extrapineal melatonin, NAT, HIOMT, aging, neuroimmunomodulation ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Abstract In the search of new therapeutic targets improving the quality of life of elderly, melatonin, “the chemical expression of darkness”, seems to play a remarkable role in aging process possibly due to its antioxidant, immunoenhancer and antiaging properties. The present study was designed to elucidate effects of aging in melatonin extrapineal synthesis and investigate evident age-related alterations in the action mechanisms involved. The presence of the two key enzymes involved in melatonin synthesis, arylalkylamine- N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) was analyzed in thymus, spleen, liver, kidney and heart of 3- and 12 monthold rats using real time PCR as well as its functionality by enzymatic activity assays. In addition, extrapineal melatonin content was measured by a competitive enzyme immunoassay (ELISA). The results of this study reveal that all rat tissues studied including thymus, and for the first time, spleen, liver, kidney and heart have the necessary machinery to synthesize melatonin. Moreover, we report an age-related decline in rat extrapineal melatonin synthesis with a consequent HIOMT functionality decrease in spleen, liver and heart during physiological aging. On the contrary, NAT enzymatic activity maintains unchanged without evident alterations with advancing age. Moreover, diminished melatonin concentrations were measured in these tissues cited above during aging except in the thymus, where, surprisingly, melatonin content, NAT/HIOMT expression, and enzymatic functionality assays revealed no significant alterations with age. As a conclusion, we report evident age-related changes in melatonin synthesis in some rat peripheral organs. We suggest that thymus may develop compensatory mechanisms to counteract the loss of immune activity and consequently, the loss of this potent antioxidant, during physiological aging. ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 1. Introduction Melatonin (N-actetyl-5-methoxy-tryptamine), an indolamine derived from the amino-acid tryptophan via synthesis of serotonin, was first isolated by Lerner et al in 1958 (Lerner et al., 1958). Melatonin, is produced by the pineal gland and secreted into the blood in a circadian manner. Its pineal production is triggered by darkness in the retina and the pineal gland, conferring the diurnal rhythm of melatonin, while in the peripheral system it is continuously produced in the gastrointestinal tract, especially in the stomach, and is triggered by innate immune response in the immune competent cells. Melatonin participates in diverse physiological functions and has great functional versatility related to the regulation of circadian rhythms and seasonal behaviour (Reiter, 1991), sexual development (Roy and Belsham, 2001), retinal physiology (Lundmark et al., 2006), osteogenesis (Sanchez-Hidalgo et al., 2007), tumour inhibition, antioxidant, anti-aging (Anisimov et al., 2006) and immunoenhancer properties (Guerrero and Reiter, 2002). More recently, it has been also implicated in bone homeostasis, cardiovascular, neurological and gastrointestinal systems (Pandi- Perumal et al., 2006). The enzymatic machinery for melatonin biosynthesis in pinealocytes was first identified by Axelrod in 1974 (Axelrod, 1974). Its biosynthesis is mediated by the sequential action of AA-NAT and HIOMT. The primary neurotransmitter, noradrenalinee, is released during the night from the postganglionic symphatic fibers of the superior cervical ganglion that terminate in the pinealocytes where it couples to - adrenergic receptors. This leads to an activation of adenylate cyclase with a consequent rise in cAMP levels, promoting the novo protein synthesis and stimulating the ratelimiting enzyme in melatonin production, arylalkylamine-N-acetyltransferase (AA- ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 NAT) (Klein et al., 1981). As a result, AA-NAT N-acetylates serotonin to N- acetylserotonin (NAS) and, as long as NAS is generated, is quickly O-methylated by hydroxyindole-O-methyltransferase (HIOMT) to finally produce melatonin (Axelrod y Weissbach, 1960). The dramatic rise in AA-NAT (10- to 100-fold) leads to melatonin synthesis which is released during periods of darkness in a circadian manner (Reiter, 1991). In contrast, the nocturnal increase in pineal HIOMT activity is weak but significant. HIOMT gene expression is high during daytime but displays an additional two fold increase at night. Once synthesized and released into blood at night, melatonin is transferred through biological membranes and is found throughout the organism in all body tissues within a very short period. This presence does not necessarily indicate an endogenous synthesis because the high lipophilic nature permits its transfer. To date, extrapineal melatonin synthesis has been demonstrated only in retina, harderian gland, brain, gut, ovary, testes, inner ear and immune system including bone marrow, lymphocytes, macrophages and, more recently, in thymus (Kvetnoy, 1999; Bubenik, 2002; Carrillo-Vico et al., 2004; Slominski et al., 2005). In general, while pineal melatonin seems to act as a typical hormone reaching target cells through the bloodstream, extrapineal melatonin may play a key role as an intra-, auto- and paracrine signal molecule in those tissues where it is synthesized (Carrillo-Vico et al., 2004). The consequences of aging are also seen in melatonin production. The secretory dynamic of pineal melatonin attenuates gradually with age, resulting in a loss of nocturnal peak of melatonin with nearly undetectable plasma levels at 20-22 months of age in rodents (Reiter et al., 1980) and at 60-70 in humans (Touitou et al., 1985). Recent studies have suggested that age-related decline in melatonin concentration involves a loss of its antioxidant ability against damage produced by free radicals (Reiter et al., 2002). Thus, the loss of this potent antioxidant during aging may be consequential of ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 the onset of age-related diseases. In fact, some studies in rodents have suggested that such diminished melatonin secretion may accelerate aging (Reiter, 1995) whereas melatonin administration has an immune reconstituting effect in aging (Tian et al., 2003). As mentioned above, pineal and serum melatonin levels decrease with advancing age, however little is known about the physiological roles of melatonin in extrapineal tissues during aging. The relevance of pineal and extrapineal melatonin in maintaining immune surveillance and defence response and the implication of reducing melatonin production by aging could support the idea that age-related decrease in extrapineal melatonin production should be very important for the developing of age-related diseases. Furthermore, the present study was designed to obtain a better understanding about melatonin produced in situ in rat peripheral organs, including the immune system, with age. That is why we studied the presence of the two key enzymes involved in melatonin synthesis, NAT and HIOMT, in thymus, spleen, liver, kidney and heart of rats of 3- and 12 month-old using real time PCR as well as its functionality by enzymatic activity assays. In addition, the extrapineal melatonin content was measured by a competitive enzyme immunoassay (ELISA). 2. Material and methods 2.1. Animals and tissue preparations Experiments were performed on male Wistar rats (3- and 12 month-old) supplied by Production and Experimentation Animal Services from the University of Granada, Spain. Animals were placed singly in cages at a controlled room temperature 24-25C, humidity 70-75%, under controlled photoperiods of 12 h light: 12 h darkness (lights were turned off daily from 20:00 to 08:00h) and were maintained with a normal laboratory diet (Panlab, Barcelona, Spain) and water ad libitum. The experiments followed a protocol approved by the local animal Ethics Committee and the Local Government and all ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 experiments were in accordance to the recommendations of the European Union regarding animal experimentation (Directive of the European Counsel 86/609/EC). When required, 3- and 12 month-old rats were sacrificed by decapitation between 12:00 and 15:00 h, and the organs were carefully removed under sterile conditions, briefly rinsed in normal saline solution, frozen on solid CO2and stored at -80ºC until their use for RNA extraction, enzyme activities and melatonin determinations. 2.2. Total RNA extraction and reverse transcription Total RNA was extracted from samples by a modification of Chomczynski and Sacchi´s method using TriPure Isolation Reagent (Roche, Mannheim, Germany) as a denaturing solution and appropriate chloroform volume. After cell lysis and RNA extraction, RNA was precipitated with isopropanol, and the pellet was washed in 75% ethanol. The RNA samples were recovered by centrifugation at 14000 g for 5 min and then dried. Each RNA pellet was dissolved in 50 or 100 μL RNase-free water and the quantified spectrophotometrically at 260 nm. In an attempt to discard DNA contamination before cDNA synthesis, the RNA samples underwent a purification process. A determined volume of reaction buffer 10x (100 mM Tris pH 7.5, 25 mM MgCl2, 5 mM CaCl2) and DNAsa I (Roche, 2U/ 10 μg ARN) were added to RNA extracted volume and incubated at 37 ºC for 30 min. The enzymatic inactivation was at 65ºC for 10 min. The samples were centifuged at 2500g at 4ºC 5 min after phenolchloroform addition and RNA was precipitated with absolute ethanol, dissolved in 50 μL RNase-free water and the quantity and quality of RNA was assessed spectrophotometrically at 260 nm and 260/280 nm, respectively. Single-strand cDNA was then synthesized using the following method: 5 μg of RNA was denatured in 19 μL RNase-free water at 85ºC for 10 min, and then rapidly ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 chilled on ice. Then, 21 μL of a mixture formed by 1 X reverse transcription (RT) buffer, 20 mM dithiothreitol (DTT), 2´-deoxyribonucleoside-5´-triphosphates (0.5 mM of each), 40 U recombinant RNasin ribonuclease inhibitor, 0.5 μg oligo (dT)15 primer and 200 U Moloney murine leukemia virus reverse transcriptase (M-MLV RT) were added to give a final volume of 40 μL and incubated for 60 min at 42ºC (all reagents were purchased from Promega, Madison, WI, USA). The RT reaction was terminated by placing it on ice after deactivation at 95ºC for 5 min. 2.3. Real Time RT-PCR Real time quantitative PCR was performed by the MiniOpticon cycler system (BioRad) using the 2 X SensiMix DNA Kit (Quantace). The cDNA was amplified in a reaction containing 5 μL of RT product as template DNA (200 ng), 1 X Sensi Mix (reaction buffer, dNTPs, Taq DNA polymerase, 3 mM MgCl2), 1 X SYBR Green and 0.2 μM sense and antisense primers of the gene under study or 0.1 μM for the housekeeping gene (hprt), in a final volume of 25 μL. The sequences of primers are shown in Table 1. The PCR reaction started by a 10 min activation of hot-start DNA polymerase at 95ºC followed by 45 cycles of target cDNA amplification. The template was initially denatured at 95ºC 10 s followed by 45-cycle program with 20 s annealing at 57ºC (AA-NAT) or 45 s annealing at 58-60ºC (HIOMT) and 20 s elongation at 72ºC and acquisition of SYBR Green fluorescence. In addition, the ubiquitously expressed hprt mRNA was used to monitor the quality of RNAs and the efficiency of the RT and PCR processes. During PCR the intercalate agent is separated resulting in increased fluorescence. The MiniOpticon instrument reads each well every few seconds and measures the increase in fluorescence which is a direct consequence of the target amplification during PCR. The results are reported as threshold cycle (Ct) values, the ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 melatonin biosynthetic enzymes using the relative quantification method. Regarding the immune system, enzymatic machinery expression involved in melatonin synthesis has been demonstrated in lymphoid organs including thymus (Naranjo et al., 2007), bone marrow (Tan et al., 1999; Conti et al., 2000), peripheral blood mononuclear leucocytes (Pozo et al., 2004) and in human cultured lymphocytes (Carrillo-Vico et al., 2004). Our results confirmed that all extrapineal tissues studied express both necessary key enzymes for melatonin synthesis, as opposed to Stefulj et al., who showed that neither rat immune system (thymus and spleen) nor other peripheral organs (kidney, liver and heart) contain reliable amounts NAT and/or HIOMT (Stefulj et al., 2001). This discrepancy might be due to a different amplification method used (PCR conventional vs. real time PCR). AA-NAT enzyme seems to be present in all peripheral tissues studied from 3- and 12 month-old rats, except in kidney and spleen of 12 month-old rats where we clearly observed a trend to decrease with age. However, NAT enzymatic assay revealed that NAT enzyme continues to be functional in all cited tissues in spite of aging process. Likewise, HIOMT expression study revealed the presence of this enzyme in all of them as well as a significant increase in spleen and heart during aging. On the contrary, HIOMT expression levels diminished significantly in liver of 12 month-old rats. Unexpectedly, the diurnal rhythm of melatonin content did not follow the same pattern as the enzymes. This discrepancy in expression/activity agrees with the published literature (Bernard et al., 1993; Sakamoto and Ishida, 1998). Melatonin content measurement revealed a significant decrease in spleen, liver and heart from middle-aged rats whereas melatonin levels were significantly higher in thymus and kidney. Particularly, the increase observed in melatonin content in the kidney is according to the increase in NAT activity, suggesting that there is a direct ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 correlation between both parameters. Nevertheless, these results agree with those obtained in HIOMT enzymatic activity assay. When melatonin levels and HIOMT functionality decrease significantly in spleen, heart and liver during rat physiological aging process, NAT enzymatic activity remains unchanged (spleen and heart) or increases (liver) possibly due to a feedback mechanism by which the organism attempts to compensate the drop in this potent antioxidant. Particularly, the age-related decrease in melatonin levels observed in these tissues did not result in a corresponding reduction in NAT activity. These data suggest that HIOMT enzymatic activity determines the level of melatonin synthesis in the above mentioned tissues during aging. In accordance with Simonneaux and Ribelayga, any modification in HIOMT activity could modulate, consequently, the melatonin production phase (Simonneaux and Ribelayga, 2003). In this sense, HIOMT enzyme may be the first key enzyme involved in melatonin synthesis that suffers the consequences of aging. In agreement with previous results reported by Liu and Borjigin we suggest that HIOMT should be considered the ratelimiting enzyme in the melatonin synthesis during aging (Liu and Borjigin, 2005). With advancing age, the amount of free radicals increases considerably in cells, leading them to cell death. In this sense, although the melatonin physiological role in extrapineal tissues is not fully known, we should take into account its antioxidant and anti-aging properties (Reiter et al., 2002). It has been demonstrated that melatonin can prevent age-related diseases (Karasek, 2004) and some studies in rodents suggest that such diminished melatonin secretion may accelerate aging (Reiter, 1995). Likewise, an accumulation of oxidative products in many organs has been described after pinealectomy in aging rats (Reiter et al., 1999).Consequently, if melatonin levels contribute to the antioxidant capacity, such ability would be reduced in advanced age and could have severe effects. As a result, an age-related decline in melatonin levels ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 16 may contribute to an increase in the oxidative damage and the onset of age-related degenerative diseases due to an accumulation of free radicals. Endogenous melatonin may act as free radical scavenger protecting the cells against oxidative damage associated to the aging. Surprisingly, of the rat extrapineal tissues studied, we have to highlight the results obtained in thymus for thymic mRNA expression and enzyme activity studies as well as the melatonin content measured since these parameters remained unchanged during aging. This could be explained by taking into account the bidirectional communication circuit between pineal melatonin and the immune system (Skwarlo-Sonta et al., 2003; Markus et al., 2007). The pineal gland, via the rhythmic synthesis and release of melatonin, influences the development, differentiation and function of the immune system. This connection is supported by studies which have demonstrated a correlation between melatonin production and circadian and seasonal variations in the immune system, effects of surgical or functional pinealectomy on the immune system, as well as the presence of melatonin receptors in the immune system (Guerrero and Reiter, 2002). Our results suggest that a feedback circuit may exist between the thymus and the pineal which balances the immunologic function in the body. In this sense, when the pineal gland starts to degenerate, the thymus may develop compensatory mechanisms such as maintaining NAT enzymatic functionality to counteract the loss of immune activity. A bloodstream melatonin retention cannot completely be ruled out. Thus, we suggest that, despite action mechanisms involved in melatonin synthesis fail with advancing age, melatonin may be retained by the thymus from the bloodstream or intracellular space for transport to other organs where it exerts its effects or synthesized by the thymus, or both, counteracting the age-associated alterations. Our results agree with the high melatonin levels described in bone marrow (Tan et al., 1999), rat thymus ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17 (Naranjo et al., 2007) and those reported by Jimenez-Jorge et al. (2005). Pinealectomized rats have clearly increased thymic melatonin content evidencing an endogenous synthesis and suggesting that either pineal melatonin could be inhibiting thymic melatonin synthesis and when rat pinealecthesized melatonin is absent, the thymus could show a compensatory rise in melatonin production (Jimenez-Jorge et al., 2005). Taken together, our study reveals that all rat extapineal tissues studied including thymus, and for the first time, spleen, liver, kidney and heart have the necessary machinery to synthesize melatonin. Moreover, we report an age-related decline in rat extrapineal melatonin synthesis in some organs. When melatonin levels and HIOMT functionality decrease in some organs during rat physiological aging process, the thymus would try to counteract the loss of this potent antioxidant by maintaining NAT enzymatic activity unchanged via pineal gland axis. Nonetheless, further studies are required in order to obtain a better understanding about the role of melatonin in these extrapineal tissues under physiological conditions as well as the relevance of extrapineal melatonin synthesis, including the regulation on the immune system, with advancing age. Acknowledgements This work was supported by grants of the Andalucía (P06-CTS-1604) and Spanish (PI 060091; RD06/00130001) governments. MSH gratefully acknowledges support from a Postgraduate National Program of FPU fellowship and financial sponsorship from the Spanish MEC. ACCEPTED MANUSCRIPT 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18 References Anisimov, V.N., Popovich, I.G., Zabezhinski, M.A., Anisimov, S.V., Vesnushkin, G.M.,Vinogradova, I.A., 2006. Melatonin as antioxidant, geroprotector and anticarcinogen. Biochim Biophys Acta. 1757, 573-89. 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Age- and sex-associated modification of plasma melatonin concentrations in man. Relationship to pathology, malignant or not, and autopsy findings. Acta Endocrinol (Copenh). 108(1), 135-44. ACCEPTED MANUSCRIPT 100 150 200 250 12 month old 3 month old * HIOMT activity pmol Mel / mg prot * h) Figure 4 0 50 Thymus Spleen Liver Kidney Heart * * HIOMT activity ( pmol Mel / mg prot * h) ACCEPTED MANUSCRIPT 3 4 12 month old 3 month old *** 10 20 30 40 50 * Melatonin content (pg MEL / g protein) Figure 5 0 1 2 Thymus Spleen Liver Kidney Heart * * * * * Melatonin content (pg MEL /