Serum melatonin levels are associated with ...

Journal of Critical Care xxx (2015) xxx–xxx

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Serum melatonin levels are associated with mortality in severe septic patients☆,☆☆ Leonardo Lorente a,⁎, María M. Martín b, Pedro Abreu-González c, Thais de la Cruz d, José Ferreres e, Jordi Solé-Violán f, Lorenzo Labarta g, César Díaz h, Alejandro Jiménez i, Juan M. Borreguero-León j a

Intensive Care Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna, 38320, Santa Cruz de Tenerife, Spain Intensive Care Unit, Hospital Universitario Nuestra Señora Candelaria, Crta Rosario s/n. Santa Cruz Tenerife, 38010, Spain Deparment of Phisiology, Faculty of Medicine, University of the La Laguna, Ofra, s/n. La Laguna, 38320, Santa Cruz de Tenerife, Spain d Faculty of Pharmacy, University of the La Laguna, Avda. Astrofísico Francisco Sánchez s/n, La Laguna, Tenerife, 38071, Spain e Intensive Care Unit, Hospital Clínico Universitario de Valencia, Avda. Blasco Ibáñez n°17-19, Valencia, 46004, Spain f Intensive Care Unit, Hospital Universitario Dr. Negrín, Barranco de la Ballena s/n. Las Palmas de Gran Canaria, 35010, Spain g Intensive Care Unit, Hospital San Jorge de Huesca, Avenida Martínez de Velasco n°36, Huesca, 22004, Spain h Intensive Care Unit, Hospital Insular, Plaza Dr. Pasteur s/n. Las Palmas de Gran Canaria, 35016, Spain i Research Unit, Hospital Universitario de Canarias, Ofra, s/n. La Laguna, 38320, Santa Cruz de Tenerife, Spain j Laboratory Department, Hospital Universitario de Canarias, La Laguna, Santa Cruz de Tenerife, Spain b c

a r t i c l e

i n f o

Keywords: Melatonin Sepsis Mortality Outcome

a b s t r a c t Objective: Melatonin in septic patients has been scarcely explored and only in studies of small sample size (maximum 20 patients). Thus, the objective of this study was to determine whether serum melatonin levels are associated with severity, oxidant and inflammatory state, and mortality in a large series of septic patients. Methods: A prospective, observational, multicenter study was performed in 6 Spanish intensive care units with 201 severe septic patients. Serum levels of melatonin were measured at moment of severe sepsis diagnosis. The end point was 30-day mortality. Results: Non-surviving patients (n = 71) showed higher serum melatonin levels (P b .001) than survivors (n = 130). Multiple logistic regression analysis showed that serum melatonin levels were associated with 30-day mortality (odds ratio, 1.022; 95% confidence interval, 1.001-1.043; P = .04), controlling for serum tumor necrosis factor–α levels, serum interleukin 6 levels and age. Serum melatonin levels were positively associated with serum levels of malondialdehyde as biomarker of oxidative stress, interleukin-6 and lactate, and with SOFA score. Conclusions: The novel finding of our study was that serum melatonin levels are associated with mortality in septic patients. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Melatonin is a lipophilic amino acid derived from tryptophan that is synthesized by the pineal gland with a circadian rhythm with high values during the night time and low values during the day time. Additionally, melatonin is synthesized in other organs, such as the retina, gastrointestinal tract, thymus, bone marrow, and in lymphocytes, but without circadian rhythm. Melatonin has a very short half-life and is

☆ Funding: This study was supported by a grant from Instituto de Salud Carlos III (FIS-PI14-00220) (Madrid, Spain) and co-financed with Fondo Europeo de Desarrollo Regional (FEDER). ☆☆ Conflicts of interest: None. ⁎ Corresponding author. E-mail addresses: [email protected] (L. Lorente), [email protected] (M.M. Martín), [email protected] (P. Abreu-González), [email protected] (T. de la Cruz), [email protected] (J. Ferreres), [email protected] (J. Solé-Violán), [email protected] (L. Labarta), [email protected] (C. Díaz), [email protected] (A. Jiménez), [email protected] (J.M. Borreguero-León).

predominantly metabolized to 6-sulfatoxymelatonin in the liver and excreted in urine [1]. Melatonin is involved in the regulation of sleep [2] and has antiinflammatory, antioxidant, and antiapoptotic effects, as well as preserving mitochondrial function [3–14]. Melatonin has anti-inflammatory effects that reduce proinflammatory cytokines, such as interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)–α; and also increase antiinflammatory cytokines, such as IL-10. The antioxidant effects of melatonin make it a potent scavenger of reactive oxygen species (ROS) and cause up-regulation of several antioxidant enzymes (such as glutathione peroxidase, glutathione reductase, etc.). Apoptotic cell death occurs through different pathways and the intrinsic or mitochondrial pathway can be activated by ROS; thus, the antioxidant effects of melatonin also reduce the apoptosis. Besides, the antioxidant effects of melatonin also protect the respiratory enzyme complex and mitochondrial deoxyribonucleic acid DNA by ROS. In addition, it has been found in septic rats that the administration of melatonin has been found to increase the levels of different antioxidant compounds, reduce malondialdehyde (MDA) levels as biomarker of lipid peroxidation, improve mitochondrial

http://dx.doi.org/10.1016/j.jcrc.2015.03.023 0883-9441/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Lorente L, et al, Serum melatonin levels are associated with mortality in severe septic patients, J Crit Care (2015), http:// dx.doi.org/10.1016/j.jcrc.2015.03.023

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L. Lorente et al. / Journal of Critical Care xxx (2015) xxx–xxx

function, decrease TNF-α and interleukin-6 levels, decrease nitric oxide, reduce biochemical markers of organ dysfunction, and increase survival rates [15–23]. However, melatonin in septic patients has been scarcely explored and only in studies of small sample size (maximum 20 patients) [24–30]. In one study of 14 septic patients was found a negative correlation between Acute Physiology and Chronic Health Evaluation II (APACHE II) scores and serum melatonin levels [28]. On the other hand, in one study with 20 septic pediatric patients were found higher serum melatonin concentrations in septic patients than in controls, and in non-survivors than in survivor septic patients [27], although the sample size was too small to demonstrate that serum melatonin levels are associated with mortality. Thus, the findings of the previous small studies appear contradictory. It could be possible that nonsurvivor septic patients showed low serum melatonin levels and this may contribute to death, or that non-survivor septic patients showed high serum melatonin levels to compensate the response to sepsis, although those high levels were insufficient to compensate the unfavourable situation leading to death. Thus, the objective of this study was to determine whether serum melatonin levels are associated with severity, oxidant and inflammatory state, and mortality in a large series of septic patients. 2. Methods 2.1. Design and subjects A prospective, observational, multicenter study was carried out in 6 intensive care units (ICUs) between 2008 and 2009. The institutional ethical review boards of the 6 Spanish hospitals involved approved this study: Hospital Universitario de Canarias (La Laguna. Santa Cruz de Tenerife), Hospital Universitario Nuestra Señora de Candelaria (Santa Cruz de Tenerife), Hospital Universitario Dr Negrín (Las Palmas de Gran Canaria), Hospital Clínico Universitario de Valencia (Valencia), Hospital San Jorge (Huesca), and Hospital Insular (Las Palmas de Gran Canaria). Written informed consent from the patients or from their family members was obtained. A total of 201 patients with severe sepsis were included. Severe sepsis was defined according to the International Sepsis Definitions Conference criteria [31]. Patients aged less than 18 years; pregnant and breastfeeding women; and patients with human immunodeficiency virus (HIV); white blood cell count b1000/μL; solid or hematological tumor; or immunosuppressive, steroid, or radiation therapy were excluded. We used use an incidental sample, and this patient cohort was the same that we used previously for other objectives [32]. We recorded the following categorical variables for each patient: sex, diabetes mellitus, chronic renal failure defined as glomerular filtration rate b60 mL/min per 1.73 m 2 , chronic obstructive pulmonary disease (COPD), site of infection, microorganism responsible, bloodstream infection, and empirical antimicrobial treatment. The following continuous variables for each patient were recorded: age, pressure of arterial oxygen/fraction of inspired oxygen (PaO2/FIO2) ratio, serum creatinine, serum bilirubin, leukocytes, serum lactate, platelets, international normalized ratio (INR), activated partial thromboplastin time (aPTT), APACHE II score [33], and Sepsis-related Organ Failure Assessment (SOFA) score [34]. 2.2. Endpoint The endpoint of the study was 30-day mortality. 2.3. Blood samples Blood samples from 201 patients were collected at the time of the diagnosis of severe sepsis. Serum was allowed to clot for 10 minutes at

room temperature, then centrifuged at 1000g for 15 minutes, and the supernatant was immediately stored in aliquot at −80°C to the end of the recruitment process. We measured serum levels of MDA to assess oxidative status [35], with TNF-α and interleukin (IL)-6 as pro-inflammatory cytokines. All determinations were performed by laboratory technicians blinded to all clinical data. 2.4. Serum melatonin analysis Melatonin assay was carried out in the Physiology Department of the Faculty of Medicine, University of La Laguna (Tenerife, Spain). Serum melatonin was determined by ELISA methods, using a commercial kit purchased from Immuno Biological Laboratories (IBL Hamburg GmbH, Hamburg, Germany). The detection limit of this assay was 0.13 pg/mL; the intra- and inter-assay coefficients of variation were 6.4% and 11.1%, respectively. 2.5. Serum MDA analysis MDA assay was carried out in the Physiology Department of the Faculty of Medicine, University of La Laguna (Tenerife, Spain). We used a thiobarbituric acid-reactive substance assay to evaluate plasma lipid peroxidation, according to Kikugawa et al [36]. Briefly, plasma (200 μL) was mixed with trichloroacetic acid (1.5 mL, pH 3.5), sodium dodecyl sulphate (200 μL, 8.1%), and thiobarbituric acid (2.5 mL, 0.8%). 50 μL of butylated hydroxytoluene (0.8%) was added to the assay mixture to prevent auto-oxidation of the sample. The mixture was kept at 5°C for exactly 1 hour and was heated only afterwards at 100°C for 1 hour. After n-butanol extraction and centrifugation, each sample in duplicate was placed in a 96-well plate and read at 535 nm using a spectrophotometer reader (Benchmark Plus, Bio-Rad, Hercules, CA, USA). The detection limit of this assay was 0.079 nmol/mL; the intra- and interassay CV were 1.82% and 4.01%, respectively. 2.6. Serum TNF-α analysis Assays for TNF-α were performed at the Laboratory Department, Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain). TNF-α levels were measured in serum by solid-phase chemiluminescent immunometric assays (Immulite, Siemens Healthcare Diagnostics Products, Llanberis, United Kingdom). The intra- and interassay CV were 3.6% and 6.5%, respectively; and the detection limit for the assay was 1.7 pg/mL. 2.7. Serum IL-6 analysis Assays for IL-6 were performed at the Laboratory Department, Hospital Universitario de Canarias (La Laguna, Santa Cruz de Tenerife, Spain). IL-6 levels were measured in serum by solid-phase, enzymelabeled, chemiluminescent sequential immunometric assay (Immulite 1000, Siemens Healthcare Diagnostics Products, Llanberis, Gwynedd, United Kingdom). The intra- and inter-assay CV were 6.2% and 7.5%, respectively; and the detection limit for the assay was 2.0 pg/mL. 2.8. Statistical methods Continuous variables are reported as medians and interquartile ranges, and the comparisons between groups were carried out using Mann-Whitney U test. Categorical variables are reported as frequencies and percentages, and the comparisons between groups were carried out with χ2 test. We plotted a receiver operating characteristic (ROC) curve using survival at 30 days as classification variable, and serum melatonin levels as prognostic variable. Analysis of survival at 30 days with Kaplan-Meier method curve and comparisons by log-rank test were carried out using serum melatonin levels higher than 19.5 pg/mL and lower



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Table 1 Comparison between survivor and non-survivor patients in baseline characteristics Survivors (n = 130) Gender male Diabetes mellitus Chronic renal failure Chronic obstructive pulmonary disease Ischemic heart disease Site of infection ·Respiratory ·Abdominal ·Neurological ·Urinary ·Skin ·Endocarditis Microorganism responsibles ·Unknown ·Gram-positive ·Gram-negative ·Fungii ·Anaerobe Bloodstream infection Empiric antimicrobial treatment adequate ·Unknown due to negative cultures ·Adequate ·Unknown due to antigenuria diagnosis ·Inadequate Betalactamic plus aminoglycoside (%) aminoglycoside, n (%) Betalactamic plus quinolone Hydrocortisone Age, y PaO2/FIO2 ratio Creatinine (mg/dL) Bilirubin (mg/dL) Leukocytes (cells/L) Lactate (mmol/L) Platelets (cells/L) INR aPTT (s) SOFA score APACHE II score Melatonin (pg/mL) Malondialdehyde (nmol/mL) TNF-α (pg/mL) Interleukin-6 (pg/mL) ICU length of stay Mechanical ventilation duration

Non-survivors (n = 71)

87 (66.9) 35 (26.9) 6 (4.6) 13 (10.0) 13 (10.0)

47 (66.2) 24 (33.8) 5 (7.0) 10 (14.1) 5 (7.0)

74 (56.9) 35 (26.9) 3 (2.3) 7 (5.4) 5 (3.8) 6 (4.6)

42 (59.2) 20 (28.2) 0 3 (4.2) 3 (4.2) 3 (4.2)

70 (53.8) 28 (21.5) 32 (24.6) 2 (2.3) 1(0.8) 19 (14.6)

42 (59.2) 15 (21.1) 14 (19.7) 3 (4.2) 1 (1.4) 9 (12.7)

71 (54.6) 51 (39.2) 3 (2.3) 6 (4.6) 24 (18.5) 70 (53.8) 67 (51.9) 56 (47-68) 170 (115-260) 1.20 (0.80-2.00) 0.90 (0.41-1.70) 15.2 (10.1-20.3) 2.00 (1.10-3.60) 191 (130-273) 1.28 (1.10-1.54) 32 (28-42) 9 (7-11) 19 (14-22) 6.86 (2.73-17.35) 2.78 (2.05-4.05) 31.8 (20.0-50.3) 104 (39.9-529.0) 16 (8-32) 14 (7-29)

42 (59.2) 25 (35.2) 2 (2.8) 2 (2.8) 14 (19.7) 38 (53.5) 50 (70.4) 65 (56-74) 180 (101-264) 1.70 (1.05-2.90) 1.20 (0.50-2.59) 15.3 (8.6-22.4) 3.80 (1.45-6.50) 124 (76-223) 1.45 (1.21-1.94) 38 (29-46) 12 (9-15) 23 (20-28) 15.18 (4.35-34.89) 3.94 (2.77-8.11) 39.9 (18.2-76.8) 634 (57.1-1000.0) 5 (2-12) 5 (2-12)

P .99 .33 .52 .49 .61 .82

.55 .99 .49 .67 .99 .83 .94

.85 .99 .02 .001 .54 .006 .24 .83 b.001 .001 b.001 .006 b.001 b.001 b.001 b.001 .21 .002 b.001 b.001

Data are shown as number and percentage or as median and interquartile 25-75; PaO2/FIO2 = pressure of arterial oxygen/fraction inspired oxygen.

than 15.6 pg/mL as the independent variable, and survival at 30 days as the dependent variable. Multiple logistic regression analysis was carried out to test the independent contribution of serum melatonin levels on the prediction of 30-day mortality, controlling for TNF-α levels, serum IL-6 levels and age. Odds ratio and 95% confidence intervals (CI) were calculated as measures of the clinical impact of the predictor variables. We used Spearman’s rank correlation coefficient to determine the association between continuous variables. A P value of less than 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 17.0 (SPSS Inc, Chicago, IL) and NCSS 2000 (Kaysville, UT). 3. Results Table 1 shows the comparison of baseline characteristics between survivors (n = 130) and non-survivors (n = 71) septic patients. No Table 2 Multiple logistic regression analyses to predict mortality at 30 days

Serum melatonin levels (pg/mL) Serum Interleukin-6 levels (pg/mL) Serum TNF-α levels (pg/mL) Age (years)

Odds ratio

95% CI

P

1.022 1.001 1.002 1.019

1.001-1.043 1.000-1.001 0.998-1.005 0.989-1.050

.04 .04 .39 .21

differences were observed regarding sex, diabetes mellitus, chronic renal failure, COPD, ischemic heart disease, site of infection, microorganism responsible, bloodstream infection, antimicrobial treatment, PaO2/ FIO2 ratio, bilirubin, leukocytes, and TNF-α. However, we found that non-survivors showed higher age, creatinine, lactate, INR, aPTT, SOFA and APACHE II scores, higher serum levels of melatonin (P b .001), MDA (P b .001) and IL-6 (P = .002), and lower platelet count than survivors (Table 2). Receiver operating characteristic (ROC) analysis showed that the area under the curve of serum melatonin levels to predict 30-day survival was 0.66 (95% CI, 0.583-0.730; P b .001) (Fig. 1). We have not found significant differences in the comparison of areas of ROC curves of serum melatonin levels with SOFA score (P = .35), APACHE II score (P = .16), lactate (P = .78) and age (P = .72) to discriminate 30-day mortality. Kaplan-Meier survival analysis showed that the risk of death in septic patients with serum melatonin levels higher than 19.5 pg/mL was higher than in patients with serum melatonin levels lower than 15.6 pg/mL (hazard ratio, 2.3; 95% CI, 1.34-3.93; P b .001) (Fig. 2). We plotted serum melatonin levels of survivor and non-survivor septic patients at 30 days (Fig. 3). Multiple logistic regression analysis showed that serum melatonin levels were associated with 30-day mortality (odds ratio, 1.022; 95%


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Fig. 3. Dot-plot of serum melatonin levels in survivors and non-survivors at 30 days.

Fig. 1. Receiver operation characteristic analysis using serum melatonin levels, serum lactate levels and SOFA score as predictors of mortality at 30 days.

CI, 1.001-1.043; P = .04), controlling for serum TNF-α levels, serum IL-6 levels and age (Table 2). Serum melatonin levels were positively associated with serum levels of MDA, IL-6 and lactate, and with SOFA score (Table 3).

4. Discussion To our knowledge, this study includes the largest series providing data on circulating melatonin levels in septic patients. And the most relevant and novel findings of our study were that serum melatonin levels are associated with severity, oxidant state, inflammatory state, and mortality in septic patients. We found higher serum melatonin levels in non-surviving than surviving septic patients, in consonance with the findings of a previous study of 20 critically ill pediatric patients with sepsis [27]. However, the sample size in that study was too small to demonstrate that serum melatonin levels are independently associated with survival. The larger sample size of our study allowed regression analysis which showed, for the first time, the independent contribution of melatonin levels for the prediction of 30-day mortality.

Another interesting finding of our study was a positive association between serum melatonin levels and several indicators of severity in sepsis, including serum lactate levels, SOFA score and APACHE II score. This contrasts with the results of a previous study of 14 septic patients where a negative correlation was found between APACHE II scores and serum melatonin levels [28]. The difference in sample size could contribute to the discrepancy of findings between the 2 studies. Another interesting new finding of our study was a positive association between serum melatonin levels and circulating MDA and IL-6 levels in septic patients. Taken together, these results indicate that serum melatonin levels may be of great pathophysiological significance in septic patients. In summary, we found higher serum melatonin, MDA and IL-6 levels in non-surviving than surviving septic patients, and a positive association of serum melatonin levels with SOFA score, and circulating levels of lactate, IL-6, and MDA levels. These findings could mean that in nonsurvivors with increased inflammatory and oxidative state, the increase in serum melatonin was insufficient to compensate for this unfavourable situation. We found that non-survivor patients received more frequently hydrocortisone that survivor patients, which reflects the severity of nonsurvivor patients. The strengths of our study are the large sample size and that it was a multicenter study. However, our study has certain limitations. First,

Table 3 Correlation of serum melatonin levels with serum levels of MDA, TNF-α, interleukin-6 and lactic acid, SOFA score, APACHE II score, age, ICU length of stay, and mechanical ventilation duration Melatonin levels MDA (nmol/mL) TNF-α (pg/mL) Interleukin-6 (pg/mL) Lactate (mmol/L) SOFA score (punctuation) APACHE II score (punctuation) Age (y) ICU length of stay (d) Mechanical ventilation duration (d) Fig. 2. Survival curves at 30 days using serum melatonin higher than 19.5 pg/mL and lower than 15.6 pg/mL.

ρ = 0.23 P = .001 ρ = 0.13 P = .06 ρ = 0.17 P = .02 ρ = 0.24 P = .001 ρ = 0.23 P = .001 ρ = 0.14 P = .047 ρ = 0.11 P = .14 ρ = −0.21 P = .003 ρ = −0.19 P = .006

ρ = Spearman rank correlation coefficient.



measuring other cytokines and compounds of oxidant/antioxidant states would be desirable to better evaluate their inter-relationships. Second, respect to the blood samples timing, we have not recorded exactly the moment when were performed and serum melatonin levels were not determined during follow-up. Third, apoptotic and mitochondrial function were not tested. Four, the area of ROC is not high and there is a great overlap of values between dead and alive patients at 30 days; thus, the sole use of serum melatonin levels to predict 30-day survival in septic patients should be taken with caution. Finally, blood samples were not obtained at the same time of day for all patients; and they were obtained at the time of severe sepsis diagnosis. It is well known that melatonin secretion presents a circadian rhythm with higher values during the dark than the light period. However, we would argue that this disadvantage is of minor importance in patients in ICU cubicles. We have measured light intensity in our ICUs and found that it oscillates between 2.8 lux (light period, corresponding to greater activity in the ICU) and 0.2 lux (dark period, corresponding to rest and less activity). Thus the light/dark ratio of light intensity in the ICU is approximately 14/1. By contrast, most humans are under environmental conditions and light intensity varies 0.1 lux (dark period) and 1000 lux (light period); thus, under normal conditions, the light/dark ratio of light intensity is 10000/1. We think that the findings of our study will not change the daily clinical practice; however, serum melatonin levels could help in the mortality prediction of some severe septic patients. In addition, we believe that the more important point of our study are that we report, for the first time, an association between serum melatonin levels and mortality in septic patients, and that our study represents the larger series reporting circulating melatonin levels in septic patients. Thus, we think that the results of our study could open the interest for research about other pathway in sepsis. 5. Conclusions The novel findings of our study was that serum melatonin levels are associated with mortality in septic patients. Key messages Serum melatonin levels are associated with mortality in septic patients. Serum melatonin levels are associated with oxidant state in septic patients. Serum melatonin levels are associated with inflammatory state in septic patients. Abbreviations

MDA SOFA APACHE aPTT CI COPD ICU INR PaO2/FIO2

malondialdehyde Sepsis-related Organ Failure Assessment Acute Physiology and Chronic Health Evaluation activated partial thromboplastin time confidence intervals chronic obstructive pulmonary disease Intensive Care Unit international normalized ratio pressure of arterial oxygen/fraction inspired oxygen

Statement of authorship LLo conceived, designed and coordinated the study, participated in acquisition and interpretation of data, and drafted the manuscript.

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MMM, PAG, TDLC, JF, JSV, LLa, CD, JMBL participated in acquisition of data. AJ participated in the interpretation of data. All authors revised the manuscript critically for important intellectual content and made the final approval of the version to be published. Acknowledgments This study was supported by a grant from Instituto de Salud Carlos III (FIS-PI-14-00220) (Madrid, Spain) and co-financed by Fondo Europeo de Desarrollo Regional (FEDER). References [1] Cagnacci A. Melatonin in relation to physiology in adult humans. J Pineal Res 1996; 21:200–13. [2] Dawson D, Encel N. Melatonin and sleep in humans. J Pineal Res 1993;15:1–12. [3] Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res 2011;51:1–16. [4] Mauriz JL, Collado PS, Veneroso C, Reiter RJ, Gonzalez-Gallego J. A review of the molecular aspects of melatonin's anti-inflammatory actions: recent insights and news perspectives. J Pineal Res 2013;54:1–14. 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