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Robert Antoni Olek, Joanna Jolanta Samulak, Angelika Katarzyna Sawicka, Dace Hartmane, Solveiga Grinberga, Osvalds Pugovics, Wieslawa Lysiak-Szydlowska, "Increased Trimethylamine N-Oxide Is Not Associated with Oxidative Stress Markers in Healthy Aged Women", Oxidative Medicine and Cellular Longevity, vol. 2019, Article ID 6247169, 6 pages, 2019. https://doi.org/10.1155/2019/6247169
Increased Trimethylamine N-Oxide Is Not Associated with Oxidative Stress Markers in Healthy Aged Women
Increased plasma trimethylamine N-oxide (TMAO) levels have been associated with cardiovascular diseases (CVD). L-carnitine induces TMAO elevation in human blood, and thus, it has been suggested as developing atherosclerosis. The aim of this study was to determine the relation between selected markers of oxidative stress and plasma TMAO concentration induced by L-carnitine supplementation for 24 weeks in healthy aged women. Twenty aged women were supplemented during 24 weeks with either 1500 mg L-carnitine-L-tartrate () or isonitrogenous placebo () per day. Fasting blood samples were taken from antecubital vein. L-carnitine supplementation induced an increase in TMAO, but not in γ-butyrobetaine (GBB). Moreover, there were no significant changes in serum ox-LDL, myeloperoxidase, protein carbonyls, homocysteine, and uric acid concentrations due to supplementation. Significant reduction in white blood cell counts has been observed following 24-week supplementation, but not attributable to L-carnitine. Our results in healthy aged women indicated no relation between TMAO and any determined marker of oxidative stress over the period of 24 weeks. At the same time, plasma GBB levels were not affected by L-carnitine supplementation. Further clinical studies of plasma GBB level as a prognostic marker are needed.
Important role in initiation and progression of multiple cardiovascular diseases (CVD) such as atherosclerosis, hypertension, and coronary heart disease plays endothelial dysfunction . Endothelial dysfunction has been strongly associated to reactive oxygen species (ROS) production and dysregulation of oxidant-antioxidant balance . The well-defined mediator of endothelial dysfunction is oxidized LDL (ox-LDL), which leads to the formation of “foam cells” [3, 4]. The main oxidant responsible for LDL oxidation is hypochlorous acid, produced by the myeloperoxidase (MPO) [5–7]. Epidemiological studies have shown that higher serum MPO is recognized as both a risk factor for the development of coronary artery disease  and can be predictive of future cardiac events and outcome [9–11].
In recent years, the role of microbiome in the pathophysiology of CVD has gained significant interest. Intestinal microbiota metabolism and CVD have been linked through trimethylamine N-oxide (TMAO) . Increased plasma TMAO levels have been associated with increased risk for major adverse cardiovascular events defined as death, myocardial infarction, or stroke [13–16]. TMAO may be produced by the intestinal microbiota from L-carnitine, via the microbiota-dependent intermediate metabolite γ-butyrobetaine (GBB) [17, 18]. Since dietary L-carnitine induces TMAO elevation in human blood [19–21], it has been suggested that L-carnitine increases atherosclerosis . On the contrary, L-carnitine treatment has been demonstrated to attenuate the development of endothelial dysfunction in spontaneously hypertensive rats [22, 23] and many studies presented L-carnitine as an antioxidant effectively scavenging ROS in various in vitro and in vivo models .
The aim of the current study was to determine the association between selected markers of oxidative stress and plasma TMAO concentration induced by L-carnitine supplementation for 24 weeks in healthy aged women.
2. Materials and Methods
The participants of the study were originally recruited to another study aimed at evaluating the effect of L-carnitine supplementation on skeletal muscle function  (individuals with CVD; liver and kidney diseases; gastrointestinal disorders, including stomach ulcers and erosions; neuromuscular disease; diabetes; and other severe chronic diseases were excluded during the recruitment process). The Independent Bioethics Commission for Research at Medical University of Gdansk has approved the study protocol (NKBBN/354-304/2015). Before starting the experimental procedure, all subjects gave their written informed consent. Twenty women in the age ranged from 65 to 70 years were supplemented during 24 weeks with either 1500 mg L-carnitine-L-tartrate () or isonitrogenous placebo () per day. The participants were examined 3 times throughout the period of the study, prior to the study, after 12 and 24 weeks of supplementation.
2.2. Blood Sampling
Blood samples were taken from antecubital vein. White blood cell (WBC) count and differential leukocyte count were determined using an automated hematology analyzer (Sysmex XT 2000, Global Medical Instrumentation, Inc.) in the whole blood. Plasma and serum were obtained by centrifugation at 2000 g at 4°C for 10 min. Aliquots were stored at -80°C for later analyses.
2.3. Biochemical Determination
Plasma TMAO and GBB were determined by the UPLC/MS/MS method as described previously . Serum ox-LDL and MPO concentrations were determined by the enzyme immunoassay method using commercially available kits (ox-LDL—Immunodiagnostik AG, Bensheim, Germany; MPO—Abnova Corp., Taipei, Taiwan); protein carbonyls (PC), spectrophotometrically using Protein Carbonyl Colorimetric Assay Kit (Cayman Chemical, Michigan, USA); homocysteine (Hcy), by immunochemiluminescence method using Immulite 2000 XPi (Siemens Healthcare Diagnostics Inc.); and uric acid (UA), using standard automatic analyzer Cobas6000 (Roche Diagnostics, Mannheim, Germany).
2.4. Statistical Analyses
All calculations were performed using software Statistica 13.1 (Dell Inc., Tulsa, OK, USA). The analysis of variance (ANOVA) for repeated measurements was performed to examine the interaction between the treatment and time. In case the ANOVA yielded a significant effect, a Tukey-Kramer test was used for post hoc comparisons. A probability level was considered statistically significant. All data are expressed as (standard error).
L-carnitine supplementation induced a tenfold increase in TMAO, observed in the midpoint of the study and maintained elevated until the end of the supplementation period (Figure 1(a)). At the same time points, plasma GBB in the supplemented group were not different () compared to placebo (Figure 1(b)).
The data of all determined oxidative stress biomarkers are summarized in Table 1. There were no significant changes in PC, ox-LDL, MPO, UA, and Hcy serum concentrations due to 24-week supplementation.
PC: protein carbonyls; ox-LDL: oxidized low-density lipoprotein; MPO: myeloperoxidase; UA: uric acid; Hcy: homocysteine.
Significant reduction in WBC, mostly in lymphocyte and monocyte counts, has been observed following 24-week supplementation, but not attributable to L-carnitine (Table 2). Despite significant decrease in lymphocyte counts, the mean values of the neutrophil-to-lymphocyte ratio (NLR) remained at the level ≤1.8 (Table 2).
main time effect. Leuko: leukocytes; Neutro: neutrophils; Lympho: lymphocytes; NLR: neutrophil-to-lymphocyte ratio (NLR); Mono: monocytes.
Similar to previously reported studies [19–21], L-carnitine supplementation induced increased plasma TMAO in humans. TMAO elevation was not related to any determined markers of oxidative stress nor WBC counts in aged women.
Since TMAO may directly act as an oxidant , animal treatment by TMAO in drinking water induces ROS generation [28, 29]. Moreover, inhibition of TMAO production in pathophysiological condition attenuates oxidative stress [30–32]. Thus, elevated circulating TMAO has been presented as a contributing factor in endothelial , cardiac , and renal  dysfunctions in animal models. Although we observed a 10-fold increase in plasma TMAO concentration of L-carnitine supplemented group, we could not measure TMAO-evoked changes in the oxidant/antioxidant status using serum PC or ox-LDL in the human subjects. Similarly, Fukami and colleagues  indicated that 6-month oral L-carnitine supplementation in hemodialysis patients significantly increased plasma TMAO, but markers of oxidative stress (malondialdehyde) and vascular injury (vascular cell adhesion molecule and intercellular adhesion molecule) decreased.
Despite a number of studies showing a positive correlation between elevated plasma TMAO concentration and an increased risk for major adverse cardiovascular events [13–16], higher plasma TMAO may be merely a marker of other cardiovascular risk factors, such as disturbed gut-blood barrier , high salt intake , or low glomerular filtration rates (GFR) . GFR of the subjects participating in the current study were within the normal range , and four months after cessation of L-carnitine treatment, TMAO reached a level comparable to the values observed before supplementation started [36, 37]. Furthermore, recent study showed that chronic, low-dose TMAO may be beneficial for the circulatory system .
GBB, intermediate in gut microbial metabolism of L-carnitine, has also been considered as a proatherogenic factor [18, 39–41]. Increase in plasma GBB concentration was shown to be related to the development of atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice [18, 40]. Moreover, higher circulating GBB has been presented in carotid atherosclerosis patients  and has been associated with increase in total atheroma volume after cardiac transplantation . On the contrary, L-carnitine supplementation did not induce elevation in plasma GBB neither in our subjects nor in healthy pregnant women . GBB is produced during L-carnitine biodegradation by Enterobacteriaceae such as Escherichia coli  and excreted primarily in feces . Therefore, it seems plausible that disturbed gut-blood barrier may increase penetration of L-carnitine metabolites into the bloodstream , suggesting gut-blood barrier permeability as a diagnostic marker in CVD .
The pathogenesis of human CVD is linked to various ROS sources . Some of the markers, associated with oxidative stress, have been proposed as clinical prognostic indicators for patients with CVD [9, 11, 46–51]. MPO promotes oxidative modification of proteins and lipids in CVD via various mechanisms . According to EPIC-Norfolk Prospective Population Study , MPO level is associated with the risk of CVD in apparently healthy individuals. In the Aging and Longevity Study in the Sirente geographic area, the lowest all-cause mortality risk was observed in the group with plasma μg/L . Similarly, the level of circulating UA has been considered as a predictor of all-cause mortality in CVD patients . Despite its important antioxidant effect, the mechanisms whereby UA promotes atherosclerosis are probably via UA-derived free radicals’ generation . As a substrate for MPO, UA is oxidized to the urate radical and then urate hydroperoxide , suggesting that UA may affect the progression of endothelial dysfunction . Indeed, the risk for mortality increases markedly at [~450 μmol/L] . Epidemiological studies have also investigated the relationship between CVD and Hcy levels in the blood [50, 51, 55]. Hcy impairs endothelial function by producing hydrogen peroxide  and superoxide anion  and can enhance LDL oxidation . Still none of these markers have changed in the supplementation period, despite changes in the TMAO level, and stayed within the normal ranges.
L-carnitine has a protective effect on the oxidative-induced decrease in low-molecular-weight thiols and lipid peroxidation in plasma , and L-carnitine supplementation reduces ox-LDL in patients with diabetes . However, neither ox-LDL nor PC concentrations were affected by L-carnitine supplementation in the present study. This may be due to the lack of oxidative stress, since ox-LDL [61, 62] and PC  values were similar to previously reported in healthy control subjects at corresponding ages.
Significant reduction in WBC, mostly in lymphocyte and monocyte counts, has been observed following 24-week supplementation, but not attributable to L-carnitine. Since the study protocol started in winter and finished in summer, it seems plausible that seasonal variations may be responsible for the variations in complete blood count [64, 65]. At the same time, NLR, an index of systemic inflammation associated with subclinical atherosclerosis , maintained at the level of ≤1.8, comparable to the control subjects . predicts with 80% probability of the carotid plaques, and gives 97% probability . Moreover, other inflammatory markers, i.e., vascular cell adhesion molecule, intercellular adhesion molecule, L-selectin, P-selectin, C-reactive protein, tumor necrosis factor α, and interleukin-6, were not affected by the L-carnitine supplementation .
Our results in healthy aged women indicated no relation between TMAO and any determined marker of oxidative stress over the period of 24 weeks. At the same time, plasma GBB levels were not affected by L-carnitine supplementation. Further clinical studies of plasma GBB level as a prognostic marker are needed.
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
We have not received any financial support or other benefits from commercial sources for the work reported in this manuscript. None of the authors have financial interests that could create a potential conflict of interest or the appearance of a conflict of interest with regard to this work.
The authors would like to thank a group of subjects participating in the study. This study was supported by the National Science Centre in Poland (2014/15/B/NZ7/00893).
- G. Amodio, O. Moltedo, R. Faraonio, and P. Remondelli, “Targeting the endoplasmic reticulum unfolded protein response to counteract the oxidative stress-induced endothelial dysfunction,” Oxidative Medicine and Cellular Longevity, vol. 2018, Article ID 4946289, 13 pages, 2018.
- R. Stocker and J. F. Keaney Jr., “Role of oxidative modifications in atherosclerosis,” Physiological Reviews, vol. 84, no. 4, pp. 1381–1478, 2004.
- J. A. Berliner and J. W. Heinecke, “The role of oxidized lipoproteins in atherogenesis,” Free Radical Biology & Medicine, vol. 20, no. 5, pp. 707–727, 1996.
- T. Kita, N. Kume, M. Minami et al., “Role of oxidized LDL in atherosclerosis,” Annals of the New York Academy of Sciences, vol. 947, no. 1, pp. 199–206, 2001.
- E. A. Podrez, H. M. Abu-Soud, and S. L. Hazen, “Myeloperoxidase-generated oxidants and atherosclerosis,” Free Radical Biology & Medicine, vol. 28, no. 12, pp. 1717–1725, 2000.
- E. Malle, G. Marsche, J. Arnhold, and M. J. Davies, “Modification of low-density lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid,” Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, vol. 1761, no. 4, pp. 392–415, 2006.
- A. I. Abdo, B. S. Rayner, D. M. van Reyk, and C. L. Hawkins, “Low-density lipoprotein modified by myeloperoxidase oxidants induces endothelial dysfunction,” Redox Biology, vol. 13, pp. 623–632, 2017.
- N. Teng, G. J. Maghzal, J. Talib, I. Rashid, A. K. Lau, and R. Stocker, “The roles of myeloperoxidase in coronary artery disease and its potential implication in plaque rupture,” Redox Report, vol. 22, no. 2, pp. 51–73, 2017.
- S. Baldus, C. Heeschen, T. Meinertz et al., “Myeloperoxidase serum levels predict risk in patients with acute coronary syndromes,” Circulation, vol. 108, no. 12, pp. 1440–1445, 2003.
- J. A. Vita, M. L. Brennan, N. Gokce et al., “Serum myeloperoxidase levels independently predict endothelial dysfunction in humans,” Circulation, vol. 110, no. 9, pp. 1134–1139, 2004.
- M. C. Meuwese, E. S. G. Stroes, S. L. Hazen et al., “Serum myeloperoxidase levels are associated with the future risk of coronary artery disease in apparently healthy individuals: the EPIC-Norfolk prospective population study,” Journal of the American College of Cardiology, vol. 50, no. 2, pp. 159–165, 2007.
- R. A. Koeth, Z. Wang, B. S. Levison et al., “Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis,” Nature Medicine, vol. 19, no. 5, pp. 576–585, 2013.
- E. G. Gruppen, E. Garcia, M. A. Connelly et al., “TMAO is associated with mortality: impact of modestly impaired renal function,” Scientific Reports, vol. 7, no. 1, article 13781, 2017.
- T. Suzuki, L. M. Heaney, S. S. Bhandari, D. J. L. Jones, and L. L. Ng, “Trimethylamine N-oxide and prognosis in acute heart failure,” Heart, vol. 102, no. 11, pp. 841–848, 2016.
- W. H. W. Tang, Z. Wang, D. J. Kennedy et al., “Gut microbiota-dependent trimethylamine N-oxide (TMAO) pathway contributes to both development of renal insufficiency and mortality risk in chronic kidney disease,” Circulation Research, vol. 116, no. 3, pp. 448–455, 2015.
- W. H. W. Tang, Z. Wang, B. S. Levison et al., “Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk,” The New England Journal of Medicine, vol. 368, no. 17, pp. 1575–1584, 2013.
- C. J. Rebouche, D. L. Mack, and P. F. Edmonson, “L-carnitine dissimilation in the gastrointestinal tract of the rat,” Biochemistry, vol. 23, no. 26, pp. 6422–6426, 2002.
- R. A. Koeth, B. S. Levison, M. K. Culley et al., “γ-butyrobetaine is a proatherogenic intermediate in gut microbial metabolism of L-carnitine to TMAO,” Cell Metabolism, vol. 20, no. 5, pp. 799–812, 2014.
- K. Fukami, S. Yamagishi, K. Sakai et al., “Oral L-carnitine supplementation increases trimethylamine-N-oxide but reduces markers of vascular injury in hemodialysis patients,” Journal of Cardiovascular Pharmacology, vol. 65, no. 3, pp. 289–295, 2015.
- M. J. Miller, B. L. Bostwick, A. D. Kennedy et al., “Chronic oral L-carnitine supplementation drives marked plasma TMAO elevations in patients with organic acidemias despite dietary meat restrictions,” JIMD Reports, vol. 30, pp. 39–44, 2016.
- H. D. Vallance, A. Koochin, J. Branov et al., “Marked elevation in plasma trimethylamine-N-oxide (TMAO) in patients with mitochondrial disorders treated with oral l-carnitine,” Molecular Genetics and Metabolism Reports, vol. 15, pp. 130–133, 2018.
- R. Bueno, M. Alvarez de Sotomayor, C. Perez-Guerrero, L. Gomez-Amores, C. M. Vazquez, and M. D. Herrera, “L-carnitine and propionyl-L-carnitine improve endothelial dysfunction in spontaneously hypertensive rats: different participation of NO and COX-products,” Life Sciences, vol. 77, no. 17, pp. 2082–2097, 2005.
- M. A. de Sotomayor, C. Mingorance, R. Rodriguez-Rodriguez, E. Marhuenda, and M. D. Herrera, “l-carnitine and its propionate: improvement of endothelial function in SHR through superoxide dismutase-dependent mechanisms,” Free Radical Research, vol. 41, no. 8, pp. 884–891, 2009.
- M. Mohammadi, A. Hajhossein Talasaz, and M. Alidoosti, “Preventive effect of l-carnitine and its derivatives on endothelial dysfunction and platelet aggregation,” Clinical Nutrition ESPEN, vol. 15, pp. 1–10, 2016.
- A. Sawicka, D. Hartmane, P. Lipinska, E. Wojtowicz, W. Lysiak-Szydlowska, and R. Olek, “l-carnitine supplementation in older women. A pilot study on aging skeletal muscle mass and function,” Nutrients, vol. 10, no. 2, p. 255, 2018.
- S. Grinberga, M. Dambrova, G. Latkovskis et al., “Determination of trimethylamine-N-oxide in combination with L-carnitine and γ-butyrobetaine in human plasma by UPLC/MS/MS,” Biomedical Chromatography, vol. 29, no. 11, pp. 1670–1674, 2015.
- B. Brzezinski and G. Zundel, “Formation of disulphide bonds in the reaction of SH group-containing amino acids with trimethylamine N-oxide. A regulatory mechanism in proteins,” FEBS Letters, vol. 333, no. 3, pp. 331–333, 1993.
- Y. Hu, Y. Zhao, L. Yuan, and X. Yang, “Protective effects of tartary buckwheat flavonoids on high TMAO diet-induced vascular dysfunction and liver injury in mice,” Food & Function, vol. 6, no. 10, pp. 3359–3372, 2015.
- Y. Ke, D. Li, M. Zhao et al., “Gut flora-dependent metabolite trimethylamine-N-oxide accelerates endothelial cell senescence and vascular aging through oxidative stress,” Free Radical Biology & Medicine, vol. 116, pp. 88–100, 2018.
- T. Li, C. Gua, B. Wu, and Y. Chen, “Increased circulating trimethylamine N-oxide contributes to endothelial dysfunction in a rat model of chronic kidney disease,” Biochemical and Biophysical Research Communications, vol. 495, no. 2, pp. 2071–2077, 2018.
- G. Sun, Z. Yin, N. Liu et al., “Gut microbial metabolite TMAO contributes to renal dysfunction in a mouse model of diet-induced obesity,” Biochemical and Biophysical Research Communications, vol. 493, no. 2, pp. 964–970, 2017.
- T. Li, Y. Chen, C. Gua, and X. Li, “Elevated circulating trimethylamine N-oxide levels contribute to endothelial dysfunction in aged rats through vascular inflammation and oxidative stress,” Frontiers in Physiology, vol. 8, p. 350, 2017.
- K. Chen, X. Zheng, M. Feng, D. Li, and H. Zhang, “Gut microbiota-dependent metabolite trimethylamine N-oxide contributes to cardiac dysfunction in western diet-induced obese mice,” Frontiers in Physiology, vol. 8, p. 139, 2017.
- K. Jaworska, T. Huc, E. Samborowska et al., “Hypertension in rats is associated with an increased permeability of the colon to TMA, a gut bacteria metabolite,” PLoS One, vol. 12, no. 12, article e0189310, 2017.
- K. Bielinska, M. Radkowski, M. Grochowska et al., “High salt intake increases plasma trimethylamine N-oxide (TMAO) concentration and produces gut dysbiosis in rats,” Nutrition, vol. 54, pp. 33–39, 2018.
- J. J. Samulak, A. K. Sawicka, D. Hartmane et al., “L-carnitine supplementation increases trimethylamine-N-oxide but not markers of atherosclerosis in healthy aged women,” Annals of Nutrition & Metabolism, vol. 74, no. 1, pp. 11–17, 2019.
- J. J. Samulak, A. K. Sawicka, E. Samborowska, and R. A. Olek, “Plasma trimethylamine-N-oxide following cessation of L-carnitine supplementation in healthy aged women,” Nutrients, vol. 11, no. 6, p. 1322, 2019.
- T. Huc, A. Drapala, M. Gawrys et al., “Chronic, low-dose TMAO treatment reduces diastolic dysfunction and heart fibrosis in hypertensive rats,” American Journal of Physiology. Heart and Circulatory Physiology, vol. 315, no. 6, pp. H1805–H1820, 2018.
- K. Skagen, M. Trøseid, T. Ueland et al., “The carnitine-butyrobetaine-trimethylamine-N-oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis,” Atherosclerosis, vol. 247, pp. 64–69, 2016.
- Y. Zhao, N. Yang, J. Gao et al., “The effect of different L-carnitine administration routes on the development of atherosclerosis in ApoE knockout mice,” Molecular Nutrition & Food Research, vol. 62, no. 5, 2018.
- M. Trøseid, C. C. K. Mayerhofer, K. Broch et al., “The carnitine-butyrobetaine-TMAO pathway after cardiac transplant: impact on cardiac allograft vasculopathy and acute rejection,” The Journal of Heart and Lung Transplantation, 2019.
- U. Keller, C. van der Wal, G. Seliger, C. Scheler, F. Röpke, and K. Eder, “Carnitine status of pregnant women: effect of carnitine supplementation and correlation between iron status and plasma carnitine concentration,” European Journal of Clinical Nutrition, vol. 63, no. 9, pp. 1098–1105, 2009.
- H. P. Kleber, “Bacterial carnitine metabolism,” FEMS Microbiology Letters, vol. 147, no. 1, pp. 1–9, 1997.
- C. J. Rebouche, “Quantitative estimation of absorption and degradation of a carnitine supplement by human adults,” Metabolism, vol. 40, no. 12, pp. 1305–1310, 1991.
- M. Ufnal and K. Pham, “The gut-blood barrier permeability - a new marker in cardiovascular and metabolic diseases?” Medical Hypotheses, vol. 98, pp. 35–37, 2017.
- S. Giovannini, G. onder, C. Leeuwenburgh et al., “Myeloperoxidase levels and mortality in frail community-living elderly individuals,” The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, vol. 65A, no. 4, pp. 369–376, 2010.
- A. G. Ioachimescu, D. M. Brennan, B. M. Hoar, S. L. Hazen, and B. J. Hoogwerf, “Serum uric acid is an independent predictor of all-cause mortality in patients at high risk of cardiovascular disease: a preventive cardiology information system (PreCIS) database cohort study,” Arthritis and Rheumatism, vol. 58, no. 2, pp. 623–630, 2008.
- G. Ndrepepa, S. Braun, H. U. Haase et al., “Prognostic value of uric acid in patients with acute coronary syndromes,” The American Journal of Cardiology, vol. 109, no. 9, pp. 1260–1265, 2012.
- L. Tamariz, A. Harzand, A. Palacio, S. Verma, J. Jones, and J. Hare, “Uric acid as a predictor of all-cause mortality in heart failure: a meta-analysis,” Congestive Heart Failure, vol. 17, no. 1, pp. 25–30, 2011.
- L. J. Fortin and J. Genest Jr., “Measurement of homocyst(e)ine in the prediction of arteriosclerosis,” Clinical Biochemistry, vol. 28, no. 2, pp. 155–162, 1995.
- O. Nygård, S. E. Vollset, H. Refsum et al., “Total plasma homocysteine and cardiovascular risk profile: The Hordaland homocysteine study,” JAMA, vol. 274, no. 19, pp. 1526–1533, 1995.
- T. Neogi, J. George, S. Rekhraj, A. D. Struthers, H. Choi, and R. A. Terkeltaub, “Are either or both hyperuricemia and xanthine oxidase directly toxic to the vasculature? A critical appraisal,” Arthritis and Rheumatism, vol. 64, no. 2, pp. 327–338, 2012.
- F. C. Meotti, G. N. L. Jameson, R. Turner et al., “Urate as a physiological substrate for myeloperoxidase: implications for hyperuricemia and inflammation,” The Journal of Biological Chemistry, vol. 286, no. 15, pp. 12901–12911, 2011.
- R. P. Silva, L. A. C. Carvalho, E. S. Patricio et al., “Identification of urate hydroperoxide in neutrophils: a novel pro-oxidant generated in inflammatory conditions,” Free Radical Biology & Medicine, vol. 126, pp. 177–186, 2018.
- J. Geisel, B. Hennen, U. Hübner, J. P. Knapp, and W. Herrmann, “The impact of hyperhomocysteinemia as a cardiovascular risk factor in the prediction of coronary heart disease,” Clinical Chemistry and Laboratory Medicine, vol. 41, no. 11, pp. 1513–1517, 2003.
- G. Starkebaum and J. M. Harlan, “Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine,” The Journal of Clinical Investigation, vol. 77, no. 4, pp. 1370–1376, 1986.
- D. Lang, M. B. Kredan, S. J. Moat et al., “Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 20, no. 2, pp. 422–427, 2000.
- B. Pfanzagl, F. Tribl, E. Koller, and T. Möslinger, “Homocysteine strongly enhances metal-catalyzed LDL oxidation in the presence of cystine and cysteine,” Atherosclerosis, vol. 168, no. 1, pp. 39–48, 2003.
- J. Kolodziejczyk, J. Saluk-Juszczak, and B. Wachowicz, “L-carnitine protects plasma components against oxidative alterations,” Nutrition, vol. 27, no. 6, pp. 693–699, 2011.
- M. Malaguarnera, M. Vacante, T. Avitabile, M. Malaguarnera, L. Cammalleri, and M. Motta, “L-carnitine supplementation reduces oxidized LDL cholesterol in patients with diabetes,” The American Journal of Clinical Nutrition, vol. 89, no. 1, pp. 71–76, 2009.
- N. Koubaa, A. Nakbi, S. Hammami et al., “Association of the C677T MTHFR polymorphism with homocysteine, ox-LDL levels, and thiolactonase activities in the severity of coronary syndrome,” Clinical and Applied Thrombosis/Hemostasis, vol. 16, no. 5, pp. 515–521, 2010.
- G. Basati, M. Pourfarzam, A. Movahedian, S. Z. Samsamshariat, and N. Sarrafzadegan, “Reduced plasma adiponectin levels relative to oxidized low density lipoprotein and nitric oxide in coronary artery disease patients,” Clinics, vol. 66, no. 7, pp. 1129–1135, 2011.
- U. Cakatay, R. Kayali, and H. Uzun, “Relation of plasma protein oxidation parameters and paraoxonase activity in the ageing population,” Clinical and Experimental Medicine, vol. 8, no. 1, pp. 51–57, 2008.
- M. Maes, W. Stevens, S. Scharpe et al., “Seasonal variation in peripheral blood leukocyte subsets and in serum interleukin-6, and soluble interleukin-2 and-6 receptor concentrations in normal volunteers,” Experientia, vol. 50, no. 9, pp. 821–829, 1994.
- B. Liu and E. Taioli, “Seasonal variations of complete blood count and inflammatory biomarkers in the US population - analysis of NHANES data,” PLoS One, vol. 10, no. 11, article e0142382, 2015.
- H. Acet, F. Ertaş, M. A. Akıl et al., “New inflammatory predictors for non-valvular atrial fibrillation: echocardiographic epicardial fat thickness and neutrophil to lymphocyte ratio,” The International Journal of Cardiovascular Imaging, vol. 30, no. 1, pp. 81–89, 2014.
- S. Demirkol, S. Balta, M. Unlu et al., “Neutrophils/lymphocytes ratio in patients with cardiac syndrome X and its association with carotid intima-media thickness,” Clinical and Applied Thrombosis/Hemostasis, vol. 20, no. 3, pp. 250–255, 2014.
- T. Corriere, S. di Marca, E. Cataudella et al., “Neutrophil-to-lymphocyte ratio is a strong predictor of atherosclerotic carotid plaques in older adults,” Nutrition, Metabolism, and Cardiovascular Diseases, vol. 28, no. 1, pp. 23–27, 2018.
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