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Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke
G Model NSL-29658; No. of Pages 4
ARTICLE IN PRESS Neuroscience Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Plenary article
Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke Thomas Ritzenthaler a , Isabelle Lhommeau b , Samuel Douillard b , Tea Hee Cho a , Jocelyne Brun c , Thierry Patrice b , Norbert Nighoghossian a,d , Bruno Claustrat c,e,∗ a
Cerebrovascular Unit, Neurology Department, Pierre Wertheimer Hospital, 59 Boulevard Pinel, 69677 Bron, France Laser Department, Laënnec Hospital, 44093 Nantes, France c Hormone Laboratory, Hospices Civils de Lyon, 59 Boulevard Pinel, 69677 Bron, France d UMR-CNRS 5220, INSERM U1044, Université de Lyon, France e INSERM, U846, Department of Chronobiologie, 18 Avenue du Doyen Lépine, 69500 Bron, France b
a r t i c l e Keywords: Melatonin Oxidative stress Stroke, AFMK Antioxidant 6-Sulfatoxymelatonin
i n f o
a b s t r a c t Oxidative stress is a leading cause of neuronal damage in ischemic stroke. Melatonin may play a role in the antioxidant response. Melatonin and its metabolites may be involved in the modulation of oxidative stress in human acute stroke. No data are available in humans to establish this relationship. In this context, on the first and the fifth days post-stroke, we assessed serum total antioxidant capacity (TAC) and urine levels of melatonin, 6-sulfatoxymelatonin (aMT6S), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), the last compound being produced in the brain after reaction of melatonin with reactive oxygen species. Compared to controls’ values, TAC and levels of melatonin and aMT6S were reduced, without difference between the first and the fifth days post-stroke, whereas AFMK levels remained in the normal range at both time points. Melatonin catabolism might be speeded up in acute ischemic stroke in order to increase the antioxidant response. © 2013 Published by Elsevier Ireland Ltd.
1. Introduction Oxidative stress is an imbalance between free reactive oxygen species (ROS) and their neutralization by antioxidant defense systems. The ischemic cascade in stroke involves several mechanisms (excitotoxicity, inflammation, and mitochondrial dysfunction) and increased oxidative stress. In addition, defense against oxidative radicals is impaired [2]. Melatonin, an indolamine hormone mainly secreted by the pineal gland, can directly and indirectly detoxify free radicals [24] and contributes to the serum total antioxidant capacity (TAC) in humans [4]. Especially, a low affinity cytosolic binding site for the radiolabelled 2-[125I]iodomelatonin designated as MT3 was identified as the same protein as human oxidoreductase 2 (QR2 protein) [6]. QR2 is also known as N-ribosyldihydronicotinamide: quinone oxidoreductase 2 (EC1.10.99.2) and is a detoxifying and antioxidant enzyme. Further, this hormone shows protective effects against ischemic damage in animals [19]. Melatonin treatment improved the survival rate and neural functioning of the mice by reduction of stroke-induced free radical production [9]. Nocturnal urine levels of
∗ Corresponding author at: Hormone Laboratory, Hospices Civils de Lyon, 59 Boulevard Pinel, 69677 Bron, France. E-mail address: [email protected] (B. Claustrat).
melatonin and of 6-sulfatoxymelatonin (aMT6S), the main hepatic metabolite of melatonin, are both indexes of the hormone secretion [5,22]. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) is generated from melatonin via several pathways including enzymatic, pseudo-enzymatic and interaction with a variety of ROS [26]. It displays an in vitro antioxidant capacity and is the last melatoninrelated compound taking part in the process by which melatonin and its metabolites successively scavenge ROS, referred as the free radical scavenging cascade [27]. In order to counteract ROS overgeneration, melatonin catabolism might be increased after stroke injury and could lead to increased production of AFMK. In a previous study, we reported decreased melatonin and aMT6S excretion during the acute stage of ischemic stroke [25]. In this paper, we assessed kinetics of serum TAC and urine AFMK, melatonin and aMT6S levels in acute stroke. 2. Materials and methods 2.1. Patients and controls Consecutive patients with a first hemispheric ischemic stroke were prospectively included. Exclusion criteria were a past medical history of stroke, renal or hepatic failure, or acute hemorrhagic stroke. The neurological status of the patients was evaluated using the National Institute of Health Stroke Scale (NIHSS) at baseline
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.02.073
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
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(Day 0), on day 1 (D1) and day 5 (D5). Acute ischemic stroke was documented by multimodal brain MRI (DWI, gradient echo, FLAIR, and MR angiography). The study was approved by the Local Ethical Board and all patients gave written consent before enrolment. The control groups consisted of healthy volunteers with an age range of 60–70 years for the study of melatonin levels (n = 50), aMT6S levels (n = 81), and AFMK levels (n = 10) and an age range of 50–70 years for the TAC study (n = 49).
DCF fluorescence (excitation 488 nm, emission 525 nm) over time measured [21]. Values were corrected for hemolysis (measured at 413 nm) and baseline absorbance (at 650 nm), then the corrected value was divided by the DCF fluorescence AUC value for a serum pool from 75 healthy donors used as a reference. A ratio higher than 1 indicates a greater AUC fluorescence than the reference pool used and thus a lower capacity of the given serum to neutralize 1 O2 and SOS as a function of time, whereas a lower value indicated a higher capacity.
2.2. Melatonin-related compound assays 2.4. Statistical analysis Urine samples were collected from 8 pm to 8 am during the first and the fifth nights after stroke (N1 and N5). Light intensity during urine collection was maintained below 50 lx in order to limit inhibition of melatonin secretion. Melatonin, aMT6S, and AFMK levels were determined by radioimmunoassay as described previously [7,16,17]. AFMK assay included a previous extraction followed by partition chromatography on celite column, in order to improve specificity of radioimmunoassay. Results are expressed as ng/h, which integrates the volume and time span of urine collection.
As the data were not normally distributed (Shapiro test), the results are expressed as the median and interquartile range. Wilcoxon and Friedmann tests were used to compare medians. Statistical significance was set at p < 0.05. All tests were performed using R v.2.13.0. (R development core team, Vienna, Austria, http://www.r-project.org/). 3. Results
2.3. Oxidative stress assay
3.1. Patients
We measured the TAC in serum of blood sampled at 7 am on D1 and D5. A subgroup of patients was assessed on D0 before administration of recombinant human tissue plasminogen activator [r-tPA; 0.9 mg/kg intravenous, starting 0–3 h (average 2 h 40 min) after stroke]. The principle of the measurement is to analyze the speed of neutralization of secondary reactive oxygen species and/or peroxides (SOS) induced by photodynamically induced singlet oxygens (1 O2 ) (produced by addition of a final concentration of 5 g/ml of rose Bengal to 5% serum and irradiation at 514 nm at 20 J/cm2 ) by measuring the fluorescent product DCF generated from DCFH. Activated DCFH was added to each sample immediately after the end of light delivery and the area under the curve (AUC) for the change in
Between May 2009 and December 2010, 75 patients were enrolled in the study, of which 33 were excluded (19 because of a lack of a urine sample, 12 were discharged before D5, and 2 died before D5), leaving 42 who completed the protocol; these consisted of 15 women and 27 men (age range: 27.7–88.5 years; median age = 73.1 years). Vascular risk factors included hyperlipemia (n = 20), hypertension (n = 16), and diabetes mellitus (n = 6). NIHSS scores on D0, D1, and D5 were, respectively, 12.00 [7.25–17.00], 8.00 [3.00–16.75], and 6.00 [2.25–13.75]. Each patient was classified according to TOAST criteria [1]: stroke was related to a cardioembolic source in 20, large-artery atherosclerosis in 16, small-vessel occlusion in 1, or a rare cause of stroke in 2, or was
Fig. 1. Urinary excretion of melatonin (a), aMT6S (b), or AFMK (c) and the TAC (d) in controls and patients on day 1 and day 5. AUC ratio higher than 1 indicates a lower TAC.
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
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cryptogenic in 3. Thirty-seven patients received r-tPA treatment that was started, on average, 2 h 40 min after stroke onset. 3.2. Biological data Data are represented in Fig. 1. Compared to the controls, patients’ urine levels of melatonin and aMT6S were reduced during N1 (2.96 [1.45–4.92] vs. 5.60 [3.66–9.50], p < 0.001 for melatonin, 163.23 [71.0–296.81] vs. 257.90 [127.60–341.20], p < 0.01 for aMT6S), whereas AFMK excretion did not show any significant difference between the groups (1.43 [0.93–2.66] vs. 1.40 [1.00–1.83]). Further, levels of melatonin, aMT6S, and AFMK levels did not change significantly between N1 and N5 (2.58 [1.38–5.26], 162.38 [77.85–284.15] and 1.64 [0.90–2.86] respectively at N5). TAC was reduced in patients on D1 compared to controls (2.05 [1.52–2.45] vs. 0.85 [0.79–1.05], p < 0.001) and remained low on D5 (1.93 [1.46–2.37]). In addition, TAC was stable in the subgroup of patients (n = 18) who were assessed 3 times on D0 (1.79 [1.24–2.08]), D1 (2.21 [1.52–2.58]), and D5 (2.08 [1.70–2.50]) (p = 0.31). In addition, r-tPA treatment did not significantly modify the TAC, which was 1.72 [1.21–2.06] before, and 2.21 [1.44–2.50] after, administration (p = 0.29). 4. Discussion Our study is the first one reporting the association of decreased melatonin secretion and serum TAC. We measured urinary excretion of melatonin, aMT6S, and AFMK and the serum TAC. In agreement with previous reports [3,11,25], patients displayed decreased melatonin and aMT6S excretion during N1, compared to controls, and this difference was maintained on N5. Further, the present study included the simultaneous measurement of AFMK, a product of melatonin oxidation, for which data in humans are very scarce. We observed that AFMK levels remained in the normal range during the first five nights following acute ischemic stroke. These data suggest that the free radical scavenging cascade generated by melatonin was hampered at the AFMK level. The decrease in melatonin and aMT6S excretion might be related to a reduction in hormone secretion, since these parameters are close indexes of pineal melatonin production. In this case, since the urinary AFMK profile is superimposed on the melatonin one in healthy people [10], accordingly, a decrease in AFMK levels would have been expected, unless melatonin catabolism to AFMK pathway was increased, in order to counteract the additional oxidative stress damage. The reported in vitro increase in free radical production associated with increased AFMK levels and simultaneous consumption of intracellular melatonin may support this hypothesis [12]. Direct assessment of free radical pathways is not currently available in clinical practice, as the life-time of these molecules is very short [15] and they are produced locally. We measured the TAC, which reflects the capacity of all known and unknown antioxidants and their synergistic action [14]. In agreement with results obtained using other methods [13,20], we observed a decrease in the TAC, using a new competitive approach that explores all the oxidative pathways after 1 O2 production, a physiological excited form of oxygen produced by photoreactions [18,21]. Analysis of the deactivation of 1 O2 therefore provides a picture of global antioxidant status at a given moment. The DCFH-DCF system is routinely used to detect with reasonable accuracy ROS generated, regardless of the source. Our results showed increased SOS levels after 1 O2 delivery and support the idea of a reduction in antioxidant defenses, at least in the peripheral blood. A reduced TAC and melatonin excretion was seen on the first day of symptoms (D0 was less than 4.5 h after stroke onset) and
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persisted for at least five days. The question remains whether decreased melatonin and TAC levels appear concomitantly with, or prior to, stroke. A recent study of a population-based prospective cohort of women suggested that dietary TAC is inversely correlated with risk of total stroke in cerebrovascular disease-free women [23]. We suspect that melatonin catabolism is increased, due to the overproduction of free radicals during acute ischemic stroke. This hypothesis should be validated by an experimental approach in a model of ischemic stroke. The maintenance of AFMK levels in the normal range and the simultaneous decrease of TAC levels are in agreement with the reduction in melatonin bioavailability. Finally, given the decreased melatonin levels seen in acute ischemic stroke and the experimental results showing the potential therapeutic interest of this hormone [8], a melatonin supplementation to restore the antioxidant capacity may deserve clinical assessment. References [1] H.P. Adams, B.H. Bendixen, L.J. Kappelle, J. Biller, B.B. Love, D.L. Gordon, E.E. Marsh, Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment, Stroke 24 (1993) 35–41. [2] C.L. Allen, U. Bayraktutan, Oxidative stress and its role in the pathogenesis of ischaemic stroke, Int J Stroke 4 (2009) 461–470. [3] Y. Beloosesky, J. Grinblat, M. Laudon, B. Grosman B., J.Y. Streifler, N. Zisapel, Melatonin rhythms in stroke patients, Neurosci. Lett. 319 (2002) 103–106. ˜ C. Osuna, J.M. Guerrero, [4] S. Benot, R. Goberna, R.J. Reiter, S. Garcia-Maurino, Physiological levels of melatonin contribute to the antioxidant capacity of human serum, J. Pineal Res. 27 (1999) 59–64. [5] C.J. Bojkowski, J. Arendt, M.C. Shih, S.P. Markey, Melatonin secretion in humans assessed by measuring its metabolite, 6-sulfatoxymelatonin, Clin. Chem. 33 (1987) 1343–1348. [6] J.A. Boutin, Melatonin binding site MT3 is QR2. State of the art, J. Soc. Biol. 201 (2007) 97–103. [7] J. Brun, B. Claustrat, M. David, Urinary melatonin, LH, oestradiol, progesterone excretion during the menstrual cycle or in women taking oral contraceptives, Acta Endocrinol. 116 (1987) 145–149. [8] M. Cervantes, G. Moralí, G. Letechipía-Vallejo, Melatonin and ischemiareperfusion injury of the brain, J. Pineal Res. 45 (2008) 1–7. [9] C.M. Chern, J.F. Liao, Y.H. Wang, Y.C. Shen, Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice, Free Radic. Biol. Med. 52 (2012) 1634–1647. [10] B. Claustrat, Do time of day and genetics influence melatonin pharmacokinetics? in: XI Congress of the European Biological Rhythms Society, Strasbourg August 22–28, 2009, Oral Communication. [11] P. Fiorina, G. Lattuada, O. Ponari, C. Silvestrini, P. DallAglio, Impaired nocturnal melatonin excretion and changes of immunological status in ischemic stroke patients Lancet 347 (1996) 692–693. [12] T.W. Fischer, T.W. Sweatman, I. Semak, R.M. Sayre, J. Wortsman, A. Slominski, Constitutive and UV-induced metabolism of melatonin in keratinocytes and cell-free systems, FASEB J. 20 (2006) 1564–1566. [13] S.E. Gariballa, T.P. Hutchin, A.J. Sinclair, Antioxidant capacity after acute ischaemic stroke, Q. J. Med. 95 (2002) 685–690. [14] A. Ghiselli, M. Serafini, F. Natella, C. Scaccini, Total antioxidant capacity as a tool to assess redox status: critical view and experimental data, Free Radic. Biol. Med. 29 (2000) 1106–1114. [15] A. Gomes, E. Fernandes, L.J.F. Lima, Fluorescence probes used for detection of reactive oxygen species, J. Biochem. Biophys. Methods 65 (2005) 45–80. [16] C. Harthé, B. Claustrat, J. Brun, G. Chazot, Direct 6-sulfatoxymelatonin radioimmunoassay in plasma with use of an iodinated tracer, Clin. Chem. 37 (1991) 536–539. [17] C. Harthé, D. Claudy, H. Déchaud, B. Vivien-Roels, P. Pévet, B. Claustrat, Radioimmunoassay of N-acetyl-N-formyl-5-methoxykynuramine (AFMK): a melatonin oxidative metabolite, Life Sci. 73 (2003) 1587–1597. [18] L. Lhommeau, S. Douillard, E. Bigot, I. Benoit, M. Krempf, T. Patrice, Serum resistance to singlet oxygen in patients with diabetes mellitus in comparison to healthy donors, Metab. Clin. Exp. 60 (2011) 1340–1348. [19] H.W. Lin, E.J. Lee, Effects of melatonin in experimental stroke models in acute, sub-acute, and chronic stages, Neuropsychiatr. Dis. Treat. 5 (2009) 157–162. [20] L. Nanetti, F. Raffaelli, A. Vignini, C. Perozzi, M. Silvestrini, M. Bartolini, L. Provinciali, L. Mazzanti, Oxidative stress in ischaemic stroke, Eur. J. Clin. Invest. 41 (2011) 1318–2132. [21] D. Olivier, S. Douillard, I. Lhommeau, E. Bigot, T. Patrice, Secondary oxidants in human serum exposed to singlet oxygen: the influence of hemolysis, Photochem. Photobiol. Sci. 8 (2009) 1476–1486. [22] T. Pääkkönen, T.M. Mäkinen, J. Leppäluoto, O. Vakkuri, H. Rintamäki, L.A. Palinkas, J. Hassi, Urinary melatonin: a noninvasive method to follow human pineal function as studied in three experimental conditions, J. Pineal Res. 40 (2006) 110–115.
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
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[23] S. Rautiainen, S. Larsson, J. Virtamo, A. Wolk, Total antioxidant capacity of diet and risk of stroke: a population-based prospective cohort of women, Stroke 43 (2012) 335–340. [24] R.J. Reiter, D.X. Tan, L. Fuentes-Broto, Melatonin: a multitasking molecule, Prog. Brain Res. 181 (2010) 127–151. [25] N. Ritzenthaler T, J. Nighoghossian, A.M. Berthiller, T.H. Schott, L.D. Cho, J. Brun, B. Claustrat, Nocturnal urine melatonin and 6-sulphatoxymelatonin excretion at the acute stage of ischaemic stroke, J. Pineal Res. 46 (2009) 349–352.
[26] D.X. Tan, L.C. Manchester, S. Burkhardt, R.M. Sainz, J.C. Mayo, R. Kohen, E. Shohami, Y.S. Huo, R. Hardeland, R.J. Reiter, N1-acetyl-N2-formyl-5methoxykynuramine, a biogenic amine and melatonin metabolite, functions as a potent antioxidant, FASEB J. 15 (2001) 2294–2296. [27] D.X. Tan, L.C. Manchester, M.P. Terron, L.J. Flores, R.J. Reiter, One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? J. Pineal Res. 42 (2007) 28–42.
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
ARTICLE IN PRESS Neuroscience Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Plenary article
Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke Thomas Ritzenthaler a , Isabelle Lhommeau b , Samuel Douillard b , Tea Hee Cho a , Jocelyne Brun c , Thierry Patrice b , Norbert Nighoghossian a,d , Bruno Claustrat c,e,∗ a
Cerebrovascular Unit, Neurology Department, Pierre Wertheimer Hospital, 59 Boulevard Pinel, 69677 Bron, France Laser Department, Laënnec Hospital, 44093 Nantes, France c Hormone Laboratory, Hospices Civils de Lyon, 59 Boulevard Pinel, 69677 Bron, France d UMR-CNRS 5220, INSERM U1044, Université de Lyon, France e INSERM, U846, Department of Chronobiologie, 18 Avenue du Doyen Lépine, 69500 Bron, France b
a r t i c l e Keywords: Melatonin Oxidative stress Stroke, AFMK Antioxidant 6-Sulfatoxymelatonin
i n f o
a b s t r a c t Oxidative stress is a leading cause of neuronal damage in ischemic stroke. Melatonin may play a role in the antioxidant response. Melatonin and its metabolites may be involved in the modulation of oxidative stress in human acute stroke. No data are available in humans to establish this relationship. In this context, on the first and the fifth days post-stroke, we assessed serum total antioxidant capacity (TAC) and urine levels of melatonin, 6-sulfatoxymelatonin (aMT6S), and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), the last compound being produced in the brain after reaction of melatonin with reactive oxygen species. Compared to controls’ values, TAC and levels of melatonin and aMT6S were reduced, without difference between the first and the fifth days post-stroke, whereas AFMK levels remained in the normal range at both time points. Melatonin catabolism might be speeded up in acute ischemic stroke in order to increase the antioxidant response. © 2013 Published by Elsevier Ireland Ltd.
1. Introduction Oxidative stress is an imbalance between free reactive oxygen species (ROS) and their neutralization by antioxidant defense systems. The ischemic cascade in stroke involves several mechanisms (excitotoxicity, inflammation, and mitochondrial dysfunction) and increased oxidative stress. In addition, defense against oxidative radicals is impaired [2]. Melatonin, an indolamine hormone mainly secreted by the pineal gland, can directly and indirectly detoxify free radicals [24] and contributes to the serum total antioxidant capacity (TAC) in humans [4]. Especially, a low affinity cytosolic binding site for the radiolabelled 2-[125I]iodomelatonin designated as MT3 was identified as the same protein as human oxidoreductase 2 (QR2 protein) [6]. QR2 is also known as N-ribosyldihydronicotinamide: quinone oxidoreductase 2 (EC1.10.99.2) and is a detoxifying and antioxidant enzyme. Further, this hormone shows protective effects against ischemic damage in animals [19]. Melatonin treatment improved the survival rate and neural functioning of the mice by reduction of stroke-induced free radical production [9]. Nocturnal urine levels of
∗ Corresponding author at: Hormone Laboratory, Hospices Civils de Lyon, 59 Boulevard Pinel, 69677 Bron, France. E-mail address: [email protected] (B. Claustrat).
melatonin and of 6-sulfatoxymelatonin (aMT6S), the main hepatic metabolite of melatonin, are both indexes of the hormone secretion [5,22]. N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) is generated from melatonin via several pathways including enzymatic, pseudo-enzymatic and interaction with a variety of ROS [26]. It displays an in vitro antioxidant capacity and is the last melatoninrelated compound taking part in the process by which melatonin and its metabolites successively scavenge ROS, referred as the free radical scavenging cascade [27]. In order to counteract ROS overgeneration, melatonin catabolism might be increased after stroke injury and could lead to increased production of AFMK. In a previous study, we reported decreased melatonin and aMT6S excretion during the acute stage of ischemic stroke [25]. In this paper, we assessed kinetics of serum TAC and urine AFMK, melatonin and aMT6S levels in acute stroke. 2. Materials and methods 2.1. Patients and controls Consecutive patients with a first hemispheric ischemic stroke were prospectively included. Exclusion criteria were a past medical history of stroke, renal or hepatic failure, or acute hemorrhagic stroke. The neurological status of the patients was evaluated using the National Institute of Health Stroke Scale (NIHSS) at baseline
0304-3940/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.neulet.2013.02.073
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
G Model NSL-29658; No. of Pages 4
ARTICLE IN PRESS T. Ritzenthaler et al. / Neuroscience Letters xxx (2013) xxx–xxx
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(Day 0), on day 1 (D1) and day 5 (D5). Acute ischemic stroke was documented by multimodal brain MRI (DWI, gradient echo, FLAIR, and MR angiography). The study was approved by the Local Ethical Board and all patients gave written consent before enrolment. The control groups consisted of healthy volunteers with an age range of 60–70 years for the study of melatonin levels (n = 50), aMT6S levels (n = 81), and AFMK levels (n = 10) and an age range of 50–70 years for the TAC study (n = 49).
DCF fluorescence (excitation 488 nm, emission 525 nm) over time measured [21]. Values were corrected for hemolysis (measured at 413 nm) and baseline absorbance (at 650 nm), then the corrected value was divided by the DCF fluorescence AUC value for a serum pool from 75 healthy donors used as a reference. A ratio higher than 1 indicates a greater AUC fluorescence than the reference pool used and thus a lower capacity of the given serum to neutralize 1 O2 and SOS as a function of time, whereas a lower value indicated a higher capacity.
2.2. Melatonin-related compound assays 2.4. Statistical analysis Urine samples were collected from 8 pm to 8 am during the first and the fifth nights after stroke (N1 and N5). Light intensity during urine collection was maintained below 50 lx in order to limit inhibition of melatonin secretion. Melatonin, aMT6S, and AFMK levels were determined by radioimmunoassay as described previously [7,16,17]. AFMK assay included a previous extraction followed by partition chromatography on celite column, in order to improve specificity of radioimmunoassay. Results are expressed as ng/h, which integrates the volume and time span of urine collection.
As the data were not normally distributed (Shapiro test), the results are expressed as the median and interquartile range. Wilcoxon and Friedmann tests were used to compare medians. Statistical significance was set at p < 0.05. All tests were performed using R v.2.13.0. (R development core team, Vienna, Austria, http://www.r-project.org/). 3. Results
2.3. Oxidative stress assay
3.1. Patients
We measured the TAC in serum of blood sampled at 7 am on D1 and D5. A subgroup of patients was assessed on D0 before administration of recombinant human tissue plasminogen activator [r-tPA; 0.9 mg/kg intravenous, starting 0–3 h (average 2 h 40 min) after stroke]. The principle of the measurement is to analyze the speed of neutralization of secondary reactive oxygen species and/or peroxides (SOS) induced by photodynamically induced singlet oxygens (1 O2 ) (produced by addition of a final concentration of 5 g/ml of rose Bengal to 5% serum and irradiation at 514 nm at 20 J/cm2 ) by measuring the fluorescent product DCF generated from DCFH. Activated DCFH was added to each sample immediately after the end of light delivery and the area under the curve (AUC) for the change in
Between May 2009 and December 2010, 75 patients were enrolled in the study, of which 33 were excluded (19 because of a lack of a urine sample, 12 were discharged before D5, and 2 died before D5), leaving 42 who completed the protocol; these consisted of 15 women and 27 men (age range: 27.7–88.5 years; median age = 73.1 years). Vascular risk factors included hyperlipemia (n = 20), hypertension (n = 16), and diabetes mellitus (n = 6). NIHSS scores on D0, D1, and D5 were, respectively, 12.00 [7.25–17.00], 8.00 [3.00–16.75], and 6.00 [2.25–13.75]. Each patient was classified according to TOAST criteria [1]: stroke was related to a cardioembolic source in 20, large-artery atherosclerosis in 16, small-vessel occlusion in 1, or a rare cause of stroke in 2, or was
Fig. 1. Urinary excretion of melatonin (a), aMT6S (b), or AFMK (c) and the TAC (d) in controls and patients on day 1 and day 5. AUC ratio higher than 1 indicates a lower TAC.
Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073
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cryptogenic in 3. Thirty-seven patients received r-tPA treatment that was started, on average, 2 h 40 min after stroke onset. 3.2. Biological data Data are represented in Fig. 1. Compared to the controls, patients’ urine levels of melatonin and aMT6S were reduced during N1 (2.96 [1.45–4.92] vs. 5.60 [3.66–9.50], p < 0.001 for melatonin, 163.23 [71.0–296.81] vs. 257.90 [127.60–341.20], p < 0.01 for aMT6S), whereas AFMK excretion did not show any significant difference between the groups (1.43 [0.93–2.66] vs. 1.40 [1.00–1.83]). Further, levels of melatonin, aMT6S, and AFMK levels did not change significantly between N1 and N5 (2.58 [1.38–5.26], 162.38 [77.85–284.15] and 1.64 [0.90–2.86] respectively at N5). TAC was reduced in patients on D1 compared to controls (2.05 [1.52–2.45] vs. 0.85 [0.79–1.05], p < 0.001) and remained low on D5 (1.93 [1.46–2.37]). In addition, TAC was stable in the subgroup of patients (n = 18) who were assessed 3 times on D0 (1.79 [1.24–2.08]), D1 (2.21 [1.52–2.58]), and D5 (2.08 [1.70–2.50]) (p = 0.31). In addition, r-tPA treatment did not significantly modify the TAC, which was 1.72 [1.21–2.06] before, and 2.21 [1.44–2.50] after, administration (p = 0.29). 4. Discussion Our study is the first one reporting the association of decreased melatonin secretion and serum TAC. We measured urinary excretion of melatonin, aMT6S, and AFMK and the serum TAC. In agreement with previous reports [3,11,25], patients displayed decreased melatonin and aMT6S excretion during N1, compared to controls, and this difference was maintained on N5. Further, the present study included the simultaneous measurement of AFMK, a product of melatonin oxidation, for which data in humans are very scarce. We observed that AFMK levels remained in the normal range during the first five nights following acute ischemic stroke. These data suggest that the free radical scavenging cascade generated by melatonin was hampered at the AFMK level. The decrease in melatonin and aMT6S excretion might be related to a reduction in hormone secretion, since these parameters are close indexes of pineal melatonin production. In this case, since the urinary AFMK profile is superimposed on the melatonin one in healthy people [10], accordingly, a decrease in AFMK levels would have been expected, unless melatonin catabolism to AFMK pathway was increased, in order to counteract the additional oxidative stress damage. The reported in vitro increase in free radical production associated with increased AFMK levels and simultaneous consumption of intracellular melatonin may support this hypothesis [12]. Direct assessment of free radical pathways is not currently available in clinical practice, as the life-time of these molecules is very short [15] and they are produced locally. We measured the TAC, which reflects the capacity of all known and unknown antioxidants and their synergistic action [14]. In agreement with results obtained using other methods [13,20], we observed a decrease in the TAC, using a new competitive approach that explores all the oxidative pathways after 1 O2 production, a physiological excited form of oxygen produced by photoreactions [18,21]. Analysis of the deactivation of 1 O2 therefore provides a picture of global antioxidant status at a given moment. The DCFH-DCF system is routinely used to detect with reasonable accuracy ROS generated, regardless of the source. Our results showed increased SOS levels after 1 O2 delivery and support the idea of a reduction in antioxidant defenses, at least in the peripheral blood. A reduced TAC and melatonin excretion was seen on the first day of symptoms (D0 was less than 4.5 h after stroke onset) and
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persisted for at least five days. The question remains whether decreased melatonin and TAC levels appear concomitantly with, or prior to, stroke. A recent study of a population-based prospective cohort of women suggested that dietary TAC is inversely correlated with risk of total stroke in cerebrovascular disease-free women [23]. We suspect that melatonin catabolism is increased, due to the overproduction of free radicals during acute ischemic stroke. This hypothesis should be validated by an experimental approach in a model of ischemic stroke. The maintenance of AFMK levels in the normal range and the simultaneous decrease of TAC levels are in agreement with the reduction in melatonin bioavailability. Finally, given the decreased melatonin levels seen in acute ischemic stroke and the experimental results showing the potential therapeutic interest of this hormone [8], a melatonin supplementation to restore the antioxidant capacity may deserve clinical assessment. References [1] H.P. 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Please cite this article in press as: T. Ritzenthaler, et al., Dynamics of oxidative stress and urinary excretion of melatonin and its metabolites during acute ischemic stroke, Neurosci. Lett. (2013), http://dx.doi.org/10.1016/j.neulet.2013.02.073