Impact of L-Citrulline on Lipid Profile and Inflammatory Markers in Mice with Diet-Induced Atherosclerosis

ORIGINAL ARTICLE
Zainab Abdlkadhim Aboshnin

 
For citation: Aboshnin ZA. Impact of L-Citrulline on Lipid Profile and Inflammatory Markers in Mice with Diet-Induced Atherosclerosis. International Journal of Biomedicine. 2025;15(1):205-209. doi:10.21103/Article15(1)_OA27
 
Originally published March 5, 2025

Abstract: 

Background: Lipid profile is the primary indicator of atherosclerosis disorder. Several strategies and therapeutic agents are targeted to reduce the risk of atherosclerosis by manipulating the lipid profile and inflammatory mediators. This study aimed to investigate the effect of citrulline on atherosclerosis and inflammation.
Methods and Results: Eighteen adult male mice were assigned randomly for the following groups (six mice for each group): the control group, which received a standard chow diet; the induced group, which received a chow diet rich with 5% cholesterol; the L-citrulline-treated group, which received a chow diet rich with cholesterol, 5% together with 100 mg/kg of citrulline. The study lasted for ten weeks; the mice monitoring continued during and after completing the study course. After ten weeks of experiments, the blood sample was collected from the animals and centrifuged at 1500rpm for 15 minutes to obtain the serum. The ELISA technique was used to measure the level of TNF-α and ET-1. TC, HDL-C, LDL-C, VLDL-C, and TG were evaluated using the colorimetric enzymatic method and the Friedewald equation.
The lipid profile components and inflammatory markers were similar at the baseline in the study groups. After ten weeks, the group of mice that received a cholesterol-rich diet showed a significant increase in all lipid profile components, excluding HDL-C, and the level of the inflammatory markers (ET-1 and TNF-α) compared with control mice. In the citrulline-treated group, the blood levels of TC, VLDL-C, LDL-C, and TNF-α were lowered insignificantly compared to the cholesterol-rich diet group. At the same time, in the citrulline-treated group, the levels of TG and ET-1 decreased significantly, and the HDL-C level increased compared to the cholesterol-rich diet group.
Conclusion: A 10-week intake of L-citrulline against the background of a cholesterol-rich diet reduces the blood TG level and proinflammatory marker (ET-1) in experimental mice. L-citrulline use may have clinical benefits in the treatment of atherosclerosis in humans.

Keywords: 
lipid profile • inflammatory markers • L-citrulline
References: 
  1. Abd-Ulhussein HK, Rizij FA. Evaluation of the effect of dapagliflozin on atherosclerosis progression by interfering with inflammatory and oxidative stress pathways in rabbits. Med J Babylon. 2019;16:20-24.
  2. Björkegren JLM, Lusis AJ. Atherosclerosis: Recent developments. Cell. 2022 May 12;185(10):1630-1645. doi: 10.1016/j.cell.2022.04.004. Epub 2022 May 2. PMID: 35504280; PMCID: PMC9119695.
  3. Mahmood KA, Ewadh MJ, Al-Saad SF. Assessment of cystatin C and CCL14 as predictive and diagnostic biomarkers for contrast-induced nephropathy. Regul Mech Biosyst. 2024;15(3):610–612.
  4. Ueng KC, Chiang CE, Chao TH, Wu YW, Lee WL, Li YH, Ting KH, Su CH, Lin HJ, Su TC, Liu TJ, Lin TH, Hsu PC, Wang YC, Chen ZC, Jen HL, Lin PL, Ko FY, Yen HW, Chen WJ, Hou CJ. 2023 Guidelines of the Taiwan Society of Cardiology on the Diagnosis and Management of Chronic Coronary Syndrome. Acta Cardiol Sin. 2023 Jan;39(1):4-96. doi: 10.6515/ACS.202301_39(1).20221103A. Erratum in: Acta Cardiol Sin. 2023 Nov;39(6):946. PMID: 36685161; PMCID: PMC9829849.
  5. Abe A, Harada-Abe M, Ueda M, Katano T, Nakajima M, Muraga K, Suda S, Nishiyama Y, Okubo S, Mishina M, Katsura K, Katayama Y. Aortic arch atherosclerosis in ischaemic stroke of unknown origin affects prognosis. Cerebrovasc Dis Extra. 2014 May 9;4(2):92-101. doi: 10.1159/000362434. PMID: 24926306; PMCID: PMC4035681.
  6. De J, Mukhopadhyay A, Saha PK. Study of serum lipid profile in pregnancy induced hypertension. Indian J Clin Biochem. 2006 Sep;21(2):165-8. doi: 10.1007/BF02912935. PMID: 23105637; PMCID: PMC3453987.
  7. Mora S, Rifai N, Buring JE, Ridker PM. Comparison of LDL cholesterol concentrations by Friedewald calculation and direct measurement in relation to cardiovascular events in 27,331 women. Clin Chem. 2009 May;55(5):888-94. doi: 10.1373/clinchem.2008.117929. PMID: 19395440; PMCID: PMC2692888.
  8. Urschel K, Cicha I. TNF-α in the cardiovascular system: from physiology to therapy. International Journal of Interferon, Cytokine and Mediator Research. 2015 Jul 9:9-25.
  9. Park HY, Kim SW, Seo J, Jung YP, Kim H, Kim AJ, Kim S, Lim K. Dietary Arginine and Citrulline Supplements for Cardiovascular Health and Athletic Performance: A Narrative Review. Nutrients. 2023 Mar 3;15(5):1268. doi: 10.3390/nu15051268. PMID: 36904267; PMCID: PMC10005484.
  10. Khalaf D, Krüger M, Wehland M, Infanger M, Grimm D. The Effects of Oral l-Arginine and l-Citrulline Supplementation on Blood Pressure. Nutrients. 2019 Jul 22;11(7):1679. doi: 10.3390/nu11071679. PMID: 31336573; PMCID: PMC6683098.
  11. Cylwik D, Mogielnicki A, Buczko W. L-arginine and cardiovascular system. Pharmacol Rep. 2005 Jan-Feb;57(1):14-22. PMID: 15849373.
  12. Park HY, Kim SW, Seo J, Jung YP, Kim H, Kim AJ, Kim S, Lim K. Dietary Arginine and Citrulline Supplements for Cardiovascular Health and Athletic Performance: A Narrative Review. Nutrients. 2023 Mar 3;15(5):1268. doi: 10.3390/nu15051268. PMID: 36904267; PMCID: PMC10005484.
  13. Momiyama Y, Adachi H, Fairweather D, Ishizaka N, Saita E. Inflammation, Atherosclerosis and Coronary Artery Disease. Clin Med Insights Cardiol. 2016 Aug 28;8(Suppl 3):67-70. doi: 10.4137/CMC.S39423. PMID: 27594791; PMCID: PMC5003124.
  14. van Diepen JA, Berbée JF, Havekes LM, Rensen PC. Interactions between inflammation and lipid metabolism: relevance for efficacy of anti-inflammatory drugs in the treatment of atherosclerosis. Atherosclerosis. 2013 Jun;228(2):306-15. doi: 10.1016/j.atherosclerosis.2013.02.028. Epub 2013 Mar 1. PMID: 23518178.
  15. Khatana C, Saini NK, Chakrabarti S, Saini V, Sharma A, Saini RV, Saini AK. Mechanistic Insights into the Oxidized Low-Density Lipoprotein-Induced Atherosclerosis. Oxid Med Cell Longev. 2020 Sep 15;2020:5245308. doi: 10.1155/2020/5245308. PMID: 33014272; PMCID: PMC7512065.
  16. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002 Mar 5;105(9):1135-43. doi: 10.1161/hc0902.104353. PMID: 11877368.
  17. Ross R. The pathogenesis of atherosclerosis--an update. N Engl J Med. 1986 Feb 20;314(8):488-500. doi: 10.1056/NEJM198602203140806. PMID: 3511384.
  18. Winterbourn CC. Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol. 2008 May;4(5):278-86. doi: 10.1038/nchembio.85. PMID: 18421291.
  19. Wang HH, Garruti G, Liu M, Portincasa P, Wang DQ. Cholesterol and Lipoprotein Metabolism and Atherosclerosis: Recent Advances In reverse Cholesterol Transport. Ann Hepatol. 2017 Nov;16(Suppl. 1: s3-105.):s27-s42. doi: 10.5604/01.3001.0010.5495. PMID: 29080338.
  20. Zhang H, Temel RE, Martel C. Cholesterol and lipoprotein metabolism: Early Career Committee contribution. Arterioscler Thromb Vasc Biol. 2014 Sep;34(9):1791-4. doi: 10.1161/ATVBAHA.114.304267. PMID: 25142876; PMCID: PMC4142559.
  21. Rajendran P, Rengarajan T, Thangavel J, Nishigaki Y, Sakthisekaran D, Sethi G, Nishigaki I. The vascular endothelium and human diseases. Int J Biol Sci. 2013 Nov 9;9(10):1057-69. doi: 10.7150/ijbs.7502. PMID: 24250251; PMCID: PMC3831119.
  22. Kattoor AJ, Pothineni NVK, Palagiri D, Mehta JL. Oxidative Stress in Atherosclerosis. Curr Atheroscler Rep. 2017 Sep 18;19(11):42. doi: 10.1007/s11883-017-0678-6. PMID: 28921056.
  23. Morita M, Hayashi T, Ochiai M, Maeda M, Yamaguchi T, Ina K, Kuzuya M. Oral supplementation with a combination of L-citrulline and L-arginine rapidly increases plasma L-arginine concentration and enhances NO bioavailability. Biochem Biophys Res Commun. 2014 Nov 7;454(1):53-7. doi: 10.1016/j.bbrc.2014.10.029. Epub 2014 Oct 14. PMID: 25445598.
  24. Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev. 2006 Fall-Winter;24(3-4):275-90. doi: 10.1111/j.1527-3466.2006.00275.x. PMID: 17214603.
  25. Ignarro LJ, Napoli C. Novel features of nitric oxide, endothelial nitric oxide synthase, and atherosclerosis. Curr Atheroscler Rep. 2004 Jul;6(4):281-7. doi: 10.1007/s11883-004-0059-9. PMID: 15191702.
  26. Mori M, Gotoh T. Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Commun. 2000 Sep 7;275(3):715-9. doi: 10.1006/bbrc.2000.3169. PMID: 10973788.
  27. Moncada S, Bolaños JP. Nitric oxide, cell bioenergetics and neurodegeneration. J Neurochem. 2006 Jun;97(6):1676-89. doi: 10.1111/j.1471-4159.2006.03988.x. PMID: 16805776.
  28. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4651-5. doi: 10.1073/pnas.88.11.4651. PMID: 1675786; PMCID: PMC51723.
  29. Ignarro LJ, Buga GM, Wei LH, Bauer PM, Wu G, del Soldato P. Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation. Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):4202-8. doi: 10.1073/pnas.071054698. Epub 2001 Mar 20. PMID: 11259671; PMCID: PMC31203.
  30. Napoli C, Ackah E, De Nigris F, Del Soldato P, D'Armiento FP, Crimi E, Condorelli M, Sessa WC. Chronic treatment with nitric oxide-releasing aspirin reduces plasma low-density lipoprotein oxidation and oxidative stress, arterial oxidation-specific epitopes, and atherogenesis in hypercholesterolemic mice. Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12467-70. doi: 10.1073/pnas.192244499. Epub 2002 Sep 3. PMID: 12209007; PMCID: PMC129468.
  31. Aji W, Ravalli S, Szabolcs M, Jiang XC, Sciacca RR, Michler RE, Cannon PJ. L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice. Circulation. 1997 Jan 21;95(2):430-7. doi: 10.1161/01.cir.95.2.430. PMID: 9008461.
  32. El-Kirsh AA, Abd El-Wahab HM, Abd-Ellah Sayed HF. The effect of L-arginine or L-citrulline supplementation on biochemical parameters and the vascular aortic wall in high-fat and high-cholesterol-fed rats. Cell Biochem Funct. 2011 Jul;29(5):414-28. doi: 10.1002/cbf.1766. Epub 2011 Jun 3. PMID: 21638297.
  33. Fu WJ, Haynes TE, Kohli R, Hu J, Shi W, Spencer TE, Carroll RJ, Meininger CJ, Wu G. Dietary L-arginine supplementation reduces fat mass in Zucker diabetic fatty rats. J Nutr. 2005 Apr;135(4):714-21. doi: 10.1093/jn/135.4.714. PMID: 15795423.
  34. Siasos G, Tousoulis D, Vlachopoulos C, Antoniades C, Stefanadi E, Ioakeimidis N, Andreou I, Zisimos K, Papavassiliou AG, Stefanadis C. Short-term treatment with L-arginine prevents the smoking-induced impairment of endothelial function and vascular elastic properties in young individuals. Int J Cardiol. 2008 Jun 6;126(3):394-9. doi: 10.1016/j.ijcard.2007.04.057. Epub 2007 Jun 22. PMID: 17588688.
  35. Lin CC, Tsai WC, Chen JY, Li YH, Lin LJ, Chen JH. Supplements of L-arginine attenuate the effects of high-fat meal on endothelial function and oxidative stress. Int J Cardiol. 2008 Jul 21;127(3):337-41. doi: 10.1016/j.ijcard.2007.06.013. Epub 2007 Jul 30. PMID: 17659795.
  36. Bode-Böger SM, Muke J, Surdacki A, Brabant G, Böger RH, Frölich JC. Oral L-arginine improves endothelial function in healthy individuals older than 70 years. Vasc Med. 2003 May;8(2):77-81. doi: 10.1191/1358863x03vm474oa. PMID: 14518608.
  37. Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, Spickler W, Schulze F, Böger RH. Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: impact on nitric oxide metabolism. Br J Clin Pharmacol. 2008 Jan;65(1):51-9. doi: 10.1111/j.1365-2125.2007.02990.x. Epub 2007 Jul 27. PMID: 17662090; PMCID: PMC2291275.
  38. Ochiai M, Hayashi T, Morita M, Ina K, Maeda M, Watanabe F, Morishita K. Short-term effects of L-citrulline supplementation on arterial stiffness in middle-aged men. Int J Cardiol. 2012 Mar 8;155(2):257-61. doi: 10.1016/j.ijcard.2010.10.004. Epub 2010 Nov 9. PMID: 21067832.
  39. Morita M, Sakurada M, Watanabe F, Yamasaki T, Doi H, Ezaki H, Morishita K, Miyakex T. Effects of Oral L-Citrulline Supplementation on Lipoprotein Oxidation and Endothelial Dysfunction in Humans with Vasospastic Angina. Immunol Endocr Metab Agents Med Chem. 2013 Sep;13(3):214-220. doi: 10.2174/18715222113139990008. PMID: 26005507; PMCID: PMC4435567.
  40. Asgary MR, Mirghazanfari SM, Hazrati E, Hadi V, Mehri Ardestani M, Bani Yaghoobi F, Hadi S. The Effect of L-Citrulline Supplementation on Outcomes of Critically Ill Patients under Mechanical Ventilation; a Double-Blind Randomized Controlled Trial. Arch Acad Emerg Med. 2023 Jan 1;11(1):e11. doi: 10.22037/aaem.v11i1.1774. PMID: 36620736; PMCID: PMC9807954.
  41. Joffin N, Jaubert AM, Durant S, Barouki R, Forest C, Noirez P. Citrulline counteracts overweight- and aging-related effects on adiponectin and leptin gene expression in rat white adipose tissue. Biochim Open. 2015 Jun 5;1:1-5. doi: 10.1016/j.biopen.2015.05.001. PMID: 29632824; PMCID: PMC5889428.
  42. Barkhidarian B, Seyedhamzeh S, Hashemy SI, Nematy M, Rahbari A, Ranjbar R, Safarian M. Effects of arginine and citrulline supplementation on inflammatory markers in critically ill patients. J Nutr Sci Diet. 2016;2 doi: 10.1177/0148607101025004182. 
  43. Breuillard C, Bonhomme S, Couderc R, Cynober L, De Bandt JP. In vitro anti-inflammatory effects of citrulline on peritoneal macrophages in Zucker diabetic fatty rats. Br J Nutr. 2015 Jan 14;113(1):120-4. doi: 10.1017/S0007114514002086. Epub 2014 Nov 13. PMID: 25391524.
  44. Barkhidarian B, Hashemi SI, Nematy M, Rahbar A, Ranjbar R, Safarian M. Effects of arginine and citrulline supplementation on inflammatory markers in critically ill patients. Journal of Nutritional Sciences and Dietetics. 2016.
  45. Miczke A, Suliburska J, Pupek-Musialik D, Ostrowska L, Jabłecka A, Krejpcio Z, Skrypnik D, Bogdański P. Effect of L-arginine supplementation on insulin resistance and serum adiponectin concentration in rats with fat diet. Int J Clin Exp Med. 2015 Jul 15;8(7):10358-66. PMID: 26379826; PMCID: PMC4565209.
  46. Bode-Böger SM, Scalera F, Ignarro LJ. The L-arginine paradox: Importance of the L-arginine/asymmetrical dimethylarginine ratio. Pharmacol Ther. 2007 Jun;114(3):295-306. doi: 10.1016/j.pharmthera.2007.03.002. Epub 2007 Apr 1. PMID: 17482266.

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Received January 16, 2025.
Accepted February 25, 2025.
©2025 International Medical Research and Development Corporation.