Docosahexaenoic Acid Modulates Oxidative Stress and Monoamines Levels in Brain of Streptozotocin-Induced Diabetic Rats.
Keywords:
Diabetes- Docosahexaenoic acid- Oxidative stress-Neurotransmitters- Insulin.
Abstract
As diabetes mellitus (DM) is growing in many countries, a lower prevalence of DM type2 and other glucose metabolism disorders in populations consuming larger amounts of n-3 polyunsaturated fatty acids, existing mainly in fish was observed. Docosahexaenoic acid (DHA), is required for central nervous system continuous maintenance for brain functioning and as an important signaling molecule. The aim is to highlight the role of DHA in controlling glycemic measures and modulating the oxidant/ antioxidant status and neurotransmitters in rats’ brains of diabetic rats. Diabetes was induced with a single s.c. injection of Streptozotocin (STZ) (6.0 mg/0.5 ml/100 g body weight). Experimental male Wister rats (n=40) were randomly divided into four groups of control, DHA group STZ-diabetic, and STZ + DHA. All rats were decapitated after 30 days to evaluate glucose and insulin levels, brain oxidative stress and monoamines level were also estimated. DHA administration significantly improved fasting blood glucose and insulin levels compared to the DHA+STZ group and decreased 8-hydroxy-2′-deoxyguanosine level in their urine. In addition, DHA treatment to STZ treated rats showed decreased the elevated malondialdehyde content and advanced oxidation protein product and significantly increased glutathione content in the brain of DHA+STZ-treated rats, and decreased rat brain elevated monoamines level. In conclusion; DHA modulated the elevated oxidative stress and neurotransmitters levels also, acetylcholinesterase activity in diabetic rat brain via enhancing insulin level in their sera.
References
(1) Pardeshi R, Bolshette N, Gadhave K et al. Docosahexaenoic Acid Increases the Potency of Soluble Epoxide Hydrolase Inhibitor in Alleviating Streptozotocin-Induced Alzheimer’s Disease-Like Complications of Diabetes. Front Pharmacol 2019; 10: 1-13. https://doi.org/10.3389/fphar.2019.00288
(2) Sarbolouki S, Javanbakht MH, Derakhshanian H et al. Eicosapentaenoic acid improves insulin sensitivity and blood sugar in overweight type 2 diabetic mellitus patients: A double-blind randomised clinical trial. Sing Med J 2013; 54:387-90.
https://doi: 10.11622/smedj.2013139.
(3) Mozaffarian D, Wu JH. (n-3) fatty acids and cardiovascular health: are effects of EPA and DHA shared or complementary?. J Nutr 2012; 142: 614S–625S. https://doi: 10.3945/jn.111.149633
(4) Grosso G, Micek A, Marventano S et al. Dietary n-3 PUFA, fish consumption and depression: A systematic review and meta-analysis of observational studies. J Affect Disord 2016; 205: 269–281. https://doi: 10.1016/ j.jad. 2016.08.011. Epub 2016 Aug 16.
(5) Smink W, Gerrits WJ, Gloaguen M et al. Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyun-saturated fatty acids in liver and brain of growing pigs. Animal 2012; 6: 262–270. https://doi.org/10.1017/S1751731111001479
(6) Bazan G, Eady N, Khoutorova L et al. Novel aspirin-triggered neuroprotectin D1 attenuates cerebral ischemic injury after experimental stroke. Exp Neurol 2012; 236:122–130. https://DOI: 10.1016/j.expneurol.2012.04.007
(7) Uchiyama S, Yamaguchi M. Alteration in serum and bone component findings induced in streptozotocin-diabetic rats is restored by zinc acexamate. Int J Mol Med 2003; 12:949–954.
https://doi.org/10.3892/ijmm.12.6.949
(8) Mannaa F, Ahmed HH, Estefan SF et al. Saccharomyces cerevisiae intervention for relieving flutamide-induced hepatotoxicity in male rats. Die Pharm Int J Pharm Sci 2005; 60:689–695
(9) Hussein J, Refaat E, Morsy S et al. Green tea attenuates experimental hepatitis in context of oxidative stress. J Appl Pharm Sci 2013; 3:124. https://DOI: 10.7324/JAPS.2013.31222
(10) Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 1969; 6:24–27.
(11) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1982; 95:351–358.
(12) Beutler E. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61:882–888.
(13) Hussein J, Abo Elmatty D, Medhat D et al. Flaxseed oil attenuates experimental liver hepatitis. Der Pharm Lett 2016; 8:142–150.
(14) Larsen K. Creatinine assay by a reaction-kinetic principle. Clin Chim Acta 1972; 41:209–217
(15) Hussein J, El-Khayat Z, Abdel Latif Y et al. Evaluation of brain monoamines in diabetic rats treated with quercetin. J Chem Pharm Res 2014; 6: 384–390
(16) Ellman GL, Courtney KD, Andres V Jr et al. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; .7:88–95
(17) El-Yamany NA, Mahmoud SM, Abdel Moneim AE. The Ameliorative Effect of Panax Ginseng Extract on Norepinephrine Dopamine and Serotonin Levels in Different Brain Regions of Alloxan-Induced Diabetic Rats. Egy J Zoo 2004; 43:347-366.
(18) Mohamed MI, Sharkawy MA, AbdelRahman T et al. Effect of Panax Ginseng on The Activity of Cholinesterase in Different Tissues of Experimentally-Induced Diabetes in Rats. J Egypt Soc Toxicol 2007; 37: 95-106.
(19) Hussein J, El-Naggar M, Badawy E. et al. Homocysteine and Asymmetrical Dimethylarginine in Diabetic Rats Treated with Docosahexaenoic Acid–Loaded Zinc Oxide Nanoparticles. Appl Biochem Biotechnol 2020; E-published Jan. 10.1007/s12010-020-03230-z https://doi.org/10.1007/s12010-020-03230-z.
(20) Hussein J, Attia MF, ElBana M et al. Solid state synthesis of docosahexaenoic acid-loaded zinc oxide nanoparticles as a potential antidiabetic agent in rats. Int J Bio Macromol. 2019; 140:1305–1314. https://DOI: 10.1016/j.ijbiomac.2019.08.201
(21) Oh DY, Talukdar S, Bae EJ et al. GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell 2020; 142: 687–98. https://doi: 10.1016/j.cell.2010.07.041
(22) Casañas-Sánchez V, Pérez JA, Fabelo N et al. Addition of docosahexaenoic acid, but not arachidonic acid, activates glutathione and thioredoxin antioxidant systems in murine hippocampal HT22 cells: Potential implications in neuroprotection. J Neurochem 2014; 131: 470–483. https://DOI: 10.1111/jnc.12833
(23) Jeong YK, Kim H. A Mini-Review on the Effect of Docosahexaenoic Acid (DHA) on Cerulein-Induced and Hypertriglyceridemic Acute Pancreatitis Y A Mini-Review on the Effect of Docosahexaenoic Acid (DHA) on Cerulein-Induced and Hypertriglyceridemic Acute Pancreatitis. Int J Mol Sci 2017; 18:2239. https://DOI: 10.3390/ijms18112239
(24) Li ZG, Zhang W, Grunberger G et al. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 2002; 946:221–231. https://DOI: 10.1016/s0006-8993(02)02887-1
(25) Alvarez-Nölting R, Arnal E, Barcia JM et al. Protection by DHA of early hippocampal changes in diabetes: possible role of CREB and NF-κB. Neurochem Res 2012; 37:105–115. https://DOI: 10.1007/s11064-011-0588-x
(26) Merad-Boudia M, Nicole A, Santiard-Baron D et al. Mitochondrial impairment as an early event in the process of apoptosis induced by glutathione depletion in neu¬ronal cells: relevance to Parkinson's disease. Biochem Pharmacol 1998; 56:645–655.
(27) Casanas-Sanchez V, Perez JA, Fabelo N, Quinto-Alemany D, Díaz M L () Docosahexaenoic (DHA) modulates phospholipid hydroperoxide glutathione peroxidase (Gpx4) gene expression to ensure self-protection from oxidative damage in hippocampal cells. Front Physiol 2015; 6:203. https://doi.org/10.3389/fphys.2015.00203
(28) Arnal E, Miranda M, Barcia J et al. Lutein and docosahexaenoic acid prevent cortex lipid peroxidation in streptozotocin-induced diabetic rat cerebral cortex. Neuroscience 2010; 166 :271–278. https://DOI: 10.1016/j.neuroscience.2009.12.028.
(29) Sakai C, Ishida M, Ohba H et al.. Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA ; damage in vascular endothelial cells. PLoS ONE 2017; 12: e0187934.
(30) Haag M. Essential fatty acids and the brain, Can. J Psychiatry 2003; 48:195–203. https://doi.org/10.1177/070674370304800308
(31) Schwarzenberg SJ, Georgieff MK. Committee on Nutrition Advocacy for improving nutrition in the first 1000 days to support childhood development and adult health Pediatrics. 2018; 141. https://DOI: 10.1542/peds.2017-3716
(32) Hashimoto M, Hossain S, Al Mamun A et al. . Docosahexaenoic acid: one molecule diverse functions. Cri Rev Biotech 2017; 37: 579-597.
https://doi: 10.1080/07388551.2016.1207153. Epub 2016 Jul 17.
(33) Mun JG, Legette LC, Ikonte CJ et al. Choline and DHA in Maternal and Infant Nutrition: Synergistic Implications in Brain and Eye Health. Nutrients 2019; 11: 1125. https://DOI: 10.3390/nu11051125
(34) Pifferi F, Roux F, Langelier B et al. (n-3) polyunsaturated fatty acid deficiency reduces the expression of both isoforms of the brain glucose transporter GLUT1 in rats. J Nutr 2005; 135: 2241-6. https://DOI: 10.1093/jn/135.9.2241
(35) Champeil-Potokar G, Chaumontet C, Guesnetp L et al. Docosahexaenoic acid (22: 6n-3) enrichment of membrane phosphor- lipids increases gap junction coupling capacity in cultured astrocytes. Eur J Neurosci 2006; 24: 3084-3090. https://doi: 10.1111/j.1460-9568.2006.05185.x.
(36) Jones CR, Arai T, Rapoport SI. Evidence for the involvement of docosahexaenoic acid in cholinergic stimulated signal transduction at the synapse. Neurochem Res. 1997; 22:663–670. https://DOI: 10.1023/a:1027341707837
(37) Rapoport SI, Ramadan E, Basselin M. Imaging plasma docosahexaenoic acid (DHA) incorporation into the brain in vivo,as a biomarker of brain DHA: Metabolism and neurotransmission. OCL 2011; 18: 246-250. https://DOI: 10.1051/ocl.2011.0396
(38) Kim HY, Spector AA, Xiong ZM. A synaptogenic amide N-docosahexaenoyl- ethanolamide promotes hippocampal development. Prostaglandins Lipid Med 2011; 96:114–120. https://doi: 10.1016/j.prostaglandins.2011.07.002
(39) Zimmer L, Hembert S, Durand G et al. Chronic n-3 polyunsaturated fatty acid diet-deficiency acts on dopamine metabolism in the rat frontal cortex: a microdialysis study. Neurosci Lett 1998; d240:177–181. https://DOI: 10.1016/s0304-3940(97)00938-5
(40) Mathieu G, Denis S, Langelier B et al. DHA enhances the noradrenaline release by SH-SY5Y cells. Neurochem Int 2010; 56:94–100.
(41) Onuki Y, Morishita M, Chiba Y et al. Docosahexaenoic acid and eicosapentae- noic acid induce changes in the physical properties of a lipid bilayer model membrane. Chem Pharm Bull 2006; 54:68–71. https://DOI: 10.1248/cpb.54.68
(42) Hashimoto M, Hossain S, Shimada T, Shido O. Docosahexaenoic acid-induced protective effect against impaired learning in amyloid beta-infused rats is associated with increased synaptosomal membrane fluidity. Clin Exp Pharmacol Physiol 2006; 33: 934–939.
(43) Bandarra NM, Lopes PA, Martins SV et al. Docosahexaenoic acid at the sn-2 position of structured triacylglycerols improved n-3 polyunsaturated fatty acid assimilation in tissues of hamsters. Nutr Res 2016; 36:452–463. https://DOI: 10.1016/j.nutres.2015.12.015 PMID: 27101763
(44) Marcheselli VL, Hong S, Lukiw WJ et al. Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. J Biol Chem 2003; 278: 43807–43817. https://DOI: 10.1074/jbc.M305841200
(45) Zhao Y, Calon F, Julien C et al. Doco-sahexaenoic acid-derived neuro-protectin D1 induces neuronal survival via secretase and PPARc-mediated mechanisms in Alzheimer’s disease models. PLoS One 2011; 6: e15816. DOI: 10.1371/journal.pone.0015816
(2) Sarbolouki S, Javanbakht MH, Derakhshanian H et al. Eicosapentaenoic acid improves insulin sensitivity and blood sugar in overweight type 2 diabetic mellitus patients: A double-blind randomised clinical trial. Sing Med J 2013; 54:387-90.
https://doi: 10.11622/smedj.2013139.
(3) Mozaffarian D, Wu JH. (n-3) fatty acids and cardiovascular health: are effects of EPA and DHA shared or complementary?. J Nutr 2012; 142: 614S–625S. https://doi: 10.3945/jn.111.149633
(4) Grosso G, Micek A, Marventano S et al. Dietary n-3 PUFA, fish consumption and depression: A systematic review and meta-analysis of observational studies. J Affect Disord 2016; 205: 269–281. https://doi: 10.1016/ j.jad. 2016.08.011. Epub 2016 Aug 16.
(5) Smink W, Gerrits WJ, Gloaguen M et al. Linoleic and α-linolenic acid as precursor and inhibitor for the synthesis of long-chain polyun-saturated fatty acids in liver and brain of growing pigs. Animal 2012; 6: 262–270. https://doi.org/10.1017/S1751731111001479
(6) Bazan G, Eady N, Khoutorova L et al. Novel aspirin-triggered neuroprotectin D1 attenuates cerebral ischemic injury after experimental stroke. Exp Neurol 2012; 236:122–130. https://DOI: 10.1016/j.expneurol.2012.04.007
(7) Uchiyama S, Yamaguchi M. Alteration in serum and bone component findings induced in streptozotocin-diabetic rats is restored by zinc acexamate. Int J Mol Med 2003; 12:949–954.
https://doi.org/10.3892/ijmm.12.6.949
(8) Mannaa F, Ahmed HH, Estefan SF et al. Saccharomyces cerevisiae intervention for relieving flutamide-induced hepatotoxicity in male rats. Die Pharm Int J Pharm Sci 2005; 60:689–695
(9) Hussein J, Refaat E, Morsy S et al. Green tea attenuates experimental hepatitis in context of oxidative stress. J Appl Pharm Sci 2013; 3:124. https://DOI: 10.7324/JAPS.2013.31222
(10) Trinder P. Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 1969; 6:24–27.
(11) Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1982; 95:351–358.
(12) Beutler E. Improved method for the determination of blood glutathione. J Lab Clin Med 1963; 61:882–888.
(13) Hussein J, Abo Elmatty D, Medhat D et al. Flaxseed oil attenuates experimental liver hepatitis. Der Pharm Lett 2016; 8:142–150.
(14) Larsen K. Creatinine assay by a reaction-kinetic principle. Clin Chim Acta 1972; 41:209–217
(15) Hussein J, El-Khayat Z, Abdel Latif Y et al. Evaluation of brain monoamines in diabetic rats treated with quercetin. J Chem Pharm Res 2014; 6: 384–390
(16) Ellman GL, Courtney KD, Andres V Jr et al. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; .7:88–95
(17) El-Yamany NA, Mahmoud SM, Abdel Moneim AE. The Ameliorative Effect of Panax Ginseng Extract on Norepinephrine Dopamine and Serotonin Levels in Different Brain Regions of Alloxan-Induced Diabetic Rats. Egy J Zoo 2004; 43:347-366.
(18) Mohamed MI, Sharkawy MA, AbdelRahman T et al. Effect of Panax Ginseng on The Activity of Cholinesterase in Different Tissues of Experimentally-Induced Diabetes in Rats. J Egypt Soc Toxicol 2007; 37: 95-106.
(19) Hussein J, El-Naggar M, Badawy E. et al. Homocysteine and Asymmetrical Dimethylarginine in Diabetic Rats Treated with Docosahexaenoic Acid–Loaded Zinc Oxide Nanoparticles. Appl Biochem Biotechnol 2020; E-published Jan. 10.1007/s12010-020-03230-z https://doi.org/10.1007/s12010-020-03230-z.
(20) Hussein J, Attia MF, ElBana M et al. Solid state synthesis of docosahexaenoic acid-loaded zinc oxide nanoparticles as a potential antidiabetic agent in rats. Int J Bio Macromol. 2019; 140:1305–1314. https://DOI: 10.1016/j.ijbiomac.2019.08.201
(21) Oh DY, Talukdar S, Bae EJ et al. GPR120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects. Cell 2020; 142: 687–98. https://doi: 10.1016/j.cell.2010.07.041
(22) Casañas-Sánchez V, Pérez JA, Fabelo N et al. Addition of docosahexaenoic acid, but not arachidonic acid, activates glutathione and thioredoxin antioxidant systems in murine hippocampal HT22 cells: Potential implications in neuroprotection. J Neurochem 2014; 131: 470–483. https://DOI: 10.1111/jnc.12833
(23) Jeong YK, Kim H. A Mini-Review on the Effect of Docosahexaenoic Acid (DHA) on Cerulein-Induced and Hypertriglyceridemic Acute Pancreatitis Y A Mini-Review on the Effect of Docosahexaenoic Acid (DHA) on Cerulein-Induced and Hypertriglyceridemic Acute Pancreatitis. Int J Mol Sci 2017; 18:2239. https://DOI: 10.3390/ijms18112239
(24) Li ZG, Zhang W, Grunberger G et al. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 2002; 946:221–231. https://DOI: 10.1016/s0006-8993(02)02887-1
(25) Alvarez-Nölting R, Arnal E, Barcia JM et al. Protection by DHA of early hippocampal changes in diabetes: possible role of CREB and NF-κB. Neurochem Res 2012; 37:105–115. https://DOI: 10.1007/s11064-011-0588-x
(26) Merad-Boudia M, Nicole A, Santiard-Baron D et al. Mitochondrial impairment as an early event in the process of apoptosis induced by glutathione depletion in neu¬ronal cells: relevance to Parkinson's disease. Biochem Pharmacol 1998; 56:645–655.
(27) Casanas-Sanchez V, Perez JA, Fabelo N, Quinto-Alemany D, Díaz M L () Docosahexaenoic (DHA) modulates phospholipid hydroperoxide glutathione peroxidase (Gpx4) gene expression to ensure self-protection from oxidative damage in hippocampal cells. Front Physiol 2015; 6:203. https://doi.org/10.3389/fphys.2015.00203
(28) Arnal E, Miranda M, Barcia J et al. Lutein and docosahexaenoic acid prevent cortex lipid peroxidation in streptozotocin-induced diabetic rat cerebral cortex. Neuroscience 2010; 166 :271–278. https://DOI: 10.1016/j.neuroscience.2009.12.028.
(29) Sakai C, Ishida M, Ohba H et al.. Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA ; damage in vascular endothelial cells. PLoS ONE 2017; 12: e0187934.
(30) Haag M. Essential fatty acids and the brain, Can. J Psychiatry 2003; 48:195–203. https://doi.org/10.1177/070674370304800308
(31) Schwarzenberg SJ, Georgieff MK. Committee on Nutrition Advocacy for improving nutrition in the first 1000 days to support childhood development and adult health Pediatrics. 2018; 141. https://DOI: 10.1542/peds.2017-3716
(32) Hashimoto M, Hossain S, Al Mamun A et al. . Docosahexaenoic acid: one molecule diverse functions. Cri Rev Biotech 2017; 37: 579-597.
https://doi: 10.1080/07388551.2016.1207153. Epub 2016 Jul 17.
(33) Mun JG, Legette LC, Ikonte CJ et al. Choline and DHA in Maternal and Infant Nutrition: Synergistic Implications in Brain and Eye Health. Nutrients 2019; 11: 1125. https://DOI: 10.3390/nu11051125
(34) Pifferi F, Roux F, Langelier B et al. (n-3) polyunsaturated fatty acid deficiency reduces the expression of both isoforms of the brain glucose transporter GLUT1 in rats. J Nutr 2005; 135: 2241-6. https://DOI: 10.1093/jn/135.9.2241
(35) Champeil-Potokar G, Chaumontet C, Guesnetp L et al. Docosahexaenoic acid (22: 6n-3) enrichment of membrane phosphor- lipids increases gap junction coupling capacity in cultured astrocytes. Eur J Neurosci 2006; 24: 3084-3090. https://doi: 10.1111/j.1460-9568.2006.05185.x.
(36) Jones CR, Arai T, Rapoport SI. Evidence for the involvement of docosahexaenoic acid in cholinergic stimulated signal transduction at the synapse. Neurochem Res. 1997; 22:663–670. https://DOI: 10.1023/a:1027341707837
(37) Rapoport SI, Ramadan E, Basselin M. Imaging plasma docosahexaenoic acid (DHA) incorporation into the brain in vivo,as a biomarker of brain DHA: Metabolism and neurotransmission. OCL 2011; 18: 246-250. https://DOI: 10.1051/ocl.2011.0396
(38) Kim HY, Spector AA, Xiong ZM. A synaptogenic amide N-docosahexaenoyl- ethanolamide promotes hippocampal development. Prostaglandins Lipid Med 2011; 96:114–120. https://doi: 10.1016/j.prostaglandins.2011.07.002
(39) Zimmer L, Hembert S, Durand G et al. Chronic n-3 polyunsaturated fatty acid diet-deficiency acts on dopamine metabolism in the rat frontal cortex: a microdialysis study. Neurosci Lett 1998; d240:177–181. https://DOI: 10.1016/s0304-3940(97)00938-5
(40) Mathieu G, Denis S, Langelier B et al. DHA enhances the noradrenaline release by SH-SY5Y cells. Neurochem Int 2010; 56:94–100.
(41) Onuki Y, Morishita M, Chiba Y et al. Docosahexaenoic acid and eicosapentae- noic acid induce changes in the physical properties of a lipid bilayer model membrane. Chem Pharm Bull 2006; 54:68–71. https://DOI: 10.1248/cpb.54.68
(42) Hashimoto M, Hossain S, Shimada T, Shido O. Docosahexaenoic acid-induced protective effect against impaired learning in amyloid beta-infused rats is associated with increased synaptosomal membrane fluidity. Clin Exp Pharmacol Physiol 2006; 33: 934–939.
(43) Bandarra NM, Lopes PA, Martins SV et al. Docosahexaenoic acid at the sn-2 position of structured triacylglycerols improved n-3 polyunsaturated fatty acid assimilation in tissues of hamsters. Nutr Res 2016; 36:452–463. https://DOI: 10.1016/j.nutres.2015.12.015 PMID: 27101763
(44) Marcheselli VL, Hong S, Lukiw WJ et al. Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. J Biol Chem 2003; 278: 43807–43817. https://DOI: 10.1074/jbc.M305841200
(45) Zhao Y, Calon F, Julien C et al. Doco-sahexaenoic acid-derived neuro-protectin D1 induces neuronal survival via secretase and PPARc-mediated mechanisms in Alzheimer’s disease models. PLoS One 2011; 6: e15816. DOI: 10.1371/journal.pone.0015816
Published
2025/12/21
Section
Originalni rad / Original article