Physico-chemical properties of erythrocytes in patients with fatty liver disease

The aim of the study was to study the associations of viscoelastic, electrical parameters of red blood cells and levels of fatty acids in their membranes and blood serum in patients with fatty liver disease.

Materials and methods. 84 men (average age 48.4±13.9 years) with fatty liver disease (FLD) of various origins (alcoholic, non-alcoholic, mixedalcohol + metabolic) with fibrosis grade 0–1 (FibroScan® 502 (Echosens, France) were examined. According to the test data from NashTest, AshTest as part of FibroMax, and ActiTest as part of FibroTest (BioPredictive, France), minimal necroinflammatory activity was found in 44 patients, and pronounced activity in 40 patients. The study of the electrical, viscoelastic parameters of erythrocytes was carried out using dielectrophoresis in an inhomogeneous alternating electric field using an electro-optical cell detection system. The content of fatty acids (FA) in the membranes of erythrocytes and blood serum was determined using gas chromatography/mass spectrometry (Agilent 7000B (USA).

Results. When analyzing the associations between the electrical, viscoelastic parameters of red blood cells and the levels of fatty acids in their membrane and blood serum, it was found that the indicators of red blood cells reflecting their ability to deform (amplitude of deformation at a frequency of 106Hz, degree of deformation at a frequency of 0.5×106 Hz), the state of the membranes (capacity), the amount of surface charge (dipole moment, velocity movement of cells to the electrodes), resistance of red blood cells (polarizability at different frequencies of the electric field, relative polarizability) directly correlated with the levels of omega‑3 PUFA in erythrocyte membranes, blood serum, both their total content and concentrations of individual FAs (eicosapentaenoic, docosapentaenoic, docosahexaenoic), the level of monounsaturated oleic FA, as well as two saturated FAs (pentadecanoic, margarine). On the contrary, there are associations with the predominant part of saturated FAs, both individual (especially highly reliable – palmitic, stearic), and the total content of monounsaturated, unsaturated, including polyunsaturated, ratios of omega‑6/ omega‑3 PUFA, SFA/UFA, SFA/PUFA turned out to be the opposite. The summarized rigidity of cells with high binding strength and statistical significance was associated with the erythrocyte content of palmitic acid (C 16:0) (r=0.674, p<0.000001), stearic acid (C 18:0) (r=0.443, p<0.000006), linoleic acid (C 18:2n‑6) (r=0.345, p=0.0006), total saturated fat content (r=0.640, p<0.000001), omega‑6/omega‑3 PUFA ratios (r=0.605, p<0.000001), SFA/PUFA (r=0.428, p=0.000015). The closest correlations for the level of membrane electrical conductivity were with erythrocyte levels of palmitic (r=0.551, p<0.000001), linoleic (r=0.470, p=0.000042), total contents of saturated (r=0.484, p<0.000001), unsaturated (r=0.468, p<0.000001), monounsaturated (r=0.504, p<0.000001), omega‑6 PUFA (r=0.414, p=0.00003), as well as with the omega‑6/omega‑3 PUFA ratio (r=0.568, p<0.000001). For the erythrocyte aggregation index, associations of high strength and statistical significance were found in relation to erythrocyte concentrations of palmitic acid (C 16:0) (r=0.691, p<0.000001), stearic acid (C 18:0) (r=0.486, p<0.000001), total SFA content (r=0.672, p<0.000001), ratios of omega‑6/omega‑3 PUFA (r=0.611, p<0.000001), SFA/UFA (r=0.475, p<0.000001), SFA/PUFA (r=0.478, p<0.000001). The indices of erythrocyte destruction were directly related to the total content of unsaturated FA in both erythrocyte membranes and blood serum, the erythrocyte level of digomo-γ-linolenic acid (r=0.205, p=0.046) and inversely to the content of C 15:0 (r= –0.224, p=0.028), C 17:0 (r= –0.151, p=0.039) and oleic acid C 18:1; c9 (r= –0.206, p=0.045).

Conclusion. The revealed associations indicate the dependence of the structural and functional parameters of red blood cells on the levels and ratios of fatty acids in their membranes and blood serum. Deterioration of the rheological properties of blood in patients with fatty liver disease is a factor associated with an increase in hypoxia in liver tissue with the potential for fibrogenesis and progression. The ability to modulate the electrical, viscoelastic parameters of red blood cells by exposure to fatty acids, including dietary ones, defines new additional treatment targets for patients with fatty liver disease.

1. Maev IV, Andreev DN, Kucheryavy Yu A. Metabolically associated fatty liver disease – a disease of the 21st century. Consilium Medicum. 2022; 24 (5): 325–332. (In Russ.). DOI: 10.26442/20751753.2022.5.201532

2. Sharma B, John S. Nonalcoholic Steatohepatitis (NASH). 2023 Apr 7. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan. PMID: 29262166.

3. Parthasarathy G, Revelo X, Malhi H. Pathogenesis of Nonalcoholic Steatohepatitis: An Overview. Hepatol Commun. 2020 Jan 14; 4 (4): 478–492. DOI: 10.1002/hep4.1479

4. Patrono D, De Stefano N, Vissio E. et al. How to Preserve Steatotic Liver Grafts for Transplantation. J Clin Med. 2023 Jun 12; 12 (12): 3982. DOI: 10.3390/jcm12123982

5. Ramalho FS, Fernandez-Monteiro I, Rosello-Catafau J, Peralta C. Hepatic microcirculatory failure. Acta Cir Bras. 2006;21 Suppl 1:48–53. DOI: 10.1590/s0102–86502006000700012

6. Brun JF, Varlet-Marie E, Myzia J. et al. Metabolic Influences Modulating Erythrocyte Deformability and Eryptosis. Metabolites. 2021 Dec 21; 12 (1): 4. DOI: 10.3390/metabo12010004

7. Farrell GC, Teoh NC, McCuskey RS. Hepatic microcirculation in fatty liver disease. Anat Rec (Hoboken). 2008 Jun; 291 (6): 684–92. DOI: 10.1002/ar.20715

8. Kim CY, Johnson H, Peltier S. et al. Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage. Front Physiol. 2022 Apr 26; 13: 868578. DOI: 10.3389/fphys.2022.868578

9. Rinella ME, Lazarus JV, Ratziu V. et al. NAFLD Nomenclature consensus group. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology. 2023 Dec 1; 78 (6): 1966–1986. DOI: 10.1097/HEP.0000000000000520

10. Yuan T, Cong Y, Meng J. et al. Arachidonic acid causes hidden blood loss-like red blood cell damage through oxidative stress reactions. J Surg Res. 2017 May 1; 211: 14–20. DOI: 10.1016/j.jss.2016.11.060

11. Kruchinina MV, Parulikova MV, Belkovets AV, Gromov AA. Possibilities of using electrical and viscoelastic parameters of erythrocytes for the diagnosis of steatohepatitis in patients with fatty liver disease. RMJ. Medical Review. 2023; 7 (5): 249–257. (In Russ.). DOI: 10.32364/2587-6821-2023-7-5-2

12. Kruchinina MV, Belkovets AV, Parulikova MV, Gromov AA. Fatty liver disease of mixed genesis: features of electrical and viscoelastic parameters of erythrocytes. Modern science: current problems of theory and practice. Series: Natural and technical sciences. 2023; 9: 185–196. (In Russ.). DOI: 10.37882/2223-2982.2023.09.13

13. Kruchinina MV, Osipenko MF, Parulikova MV, Gromov AA. Electrical and viscoelastic parameters of erythrocytes in diagnostic models for differentiating fatty liver disease of mixed genesis from non-alcoholic and alcoholic fatty liver disease. Russian Journal of Gastroenterology, Hepatology, Proctology. 2024; 34 (3): 38–52. (In Russ.). DOI:10.22416/1382-4376-2024-1215-3218

14. Kruchinina MV, Osipenko MF, Parulikova MV. et al. Diagnostic models for differentiating fatty liver disease of alcoholic and non-alcoholic genesis. Medical Council. 2024; 18 (15): 82–95. (In Russ.). DOI: 10.21518/ms2024-397

15. Kruchinina MV, Parulikova MV, Pershina NE, Kruchinina EV. Fatty acids of erythrocyte membranes as biomarkers of non-alcoholic fatty liver disease in men. Atherosclerosis. 2022; 18 (4): 362–380. (In Russ.). DOI: 10. 52727/2078-256Х-2022-18-4-362-380

16. Ivashkin VT, Maevskaya MV, Zharkova MS. et al. Clinical Practice Guidelines of the Russian Scientific Liver Society, Russian Gastroenterological Association, Russian Association of Endocrinologists, Russian Association of Gerontologists and Geriatricians and National Society for Preventive Cardiology on Diagnosis and Treatment of Non-Alcoholic Liver Disease. Russian Journal of Gastroenterology, Hepatology, Coloproctology. 2022; 32 (4): 104–140. (In Russ.). DOI: 10.22416/1382-4376-2022-32-4-104-140

17. Alcoholic liver disease (ALD) in adults. Clinical guidelines. 2021-2022-2023 (10.01.2022) / Approved by the Ministry of Health of the Russian Federation URL: http://disuria.ru/_ld/12/1219_kr21K70MZ.pdf (date accessed: 12.05.2024). Text: electronic. (In Russ.).

18. Recommendations of the VNOK experts on the diagnosis and treatment of metabolic syndrome (second revision). Cardiovascular therapy and prevention. 2009: 6 (2). (In Russ.).

19. Munteanu M, Tiniakos D, Anstee Q. et al. FLIP Consortium and the FibroFrance Group. Diagnostic performance of FibroTest, SteatoTest and ActiTest in patients with NAFLD using the SAF score as histological reference. Aliment Pharmacol Ther. 2016 Oct; 44 (8): 877–89. DOI: 10.1111/apt.13770

20. Chen Z, Ma Y, Cai J. et al. Serum biomarkers for liver fibrosis. Clin Chim Acta. 2022 Dec 1; 537: 16–25. DOI: 10.1016/j.cca.2022.09.022

21. Ferraioli G, Monteiro LBS. Ultrasound-based techniques for the diagnosis of liver steatosis. World J. Gastroenterol. 2019; 25: 6053–6062. DOI: 10.3748/wjg.v25.i40.6053

22. Generalov KV, Kruchinina MV, Safatov AS. et al. Dielectrophoresis in medicine. Novosibirsk: Offset-TM LLC, 2020. (In Russ.).

23. Kruchinina MV, Kruchinin VN, Prudnikova Ya I. et al. Study of the level of fatty acids in erythrocyte membranes and serum of patients with colorectal cancer in Novosibirsk. Advances in Molecular Oncology. 2018; 5 (2): 50–61. (In Russ.). DOI: 10.17650/2313-805X-2018-5-2-50-61

24. Kruchinina MV, Parulikova MV, Kurilovich SA. et al. Potential of the erythrocyte dielectrophoresis method in differentiating fatty liver disease of alcoholic and non-alcoholic genesis in men. Atherosclerosis. 2020; 16 (4): 27–42. (In Russ.). DOI: 10.15372/ATER 20200404

25. Kruchinina MV, Parulikova MV, Belkovets AV. et al. Features of the fatty acid profile of erythrocyte membranes in patients with fatty liver disease of alcoholic genesis. Medical Council. 2023; 17 (18): 146–158. (In Russ.). DOI: 10.21518/ms2023-391

26. Kruchinina MV, Belkovets AV, Parulikova MV, Gromov AA. Atherogenic features of the fatty acid profile of erythrocyte membranes in patients with fatty liver disease of mixed genesis. Atherosclerosis. 2023; 19 (4): 350–368. (In Russ.). DOI: 10.52727/2078-256X-2023-19-2-350-368

27. Mitra S, De A, Chowdhury A. Epidemiology of non-alcoholic and alcoholic fatty liver diseases. Transl Gastroenterol Hepatol. 2020 Apr 5; 5: 16. DOI: 10.21037/tgh.2019.09.08

28. Buzzetti E, Pinzani M, Tsochatzis EA. The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism. 2016 Aug; 65 (8): 1038–48. DOI: 10.1016/j.metabol.2015.12.012

29. Long MT, Gandhi S, Loomba R. Advances in non-invasive biomarkers for the diagnosis and monitoring of non-alcoholic fatty liver disease. Metabolism. 2020 Oct; 111S: 154259. DOI: 10.1016/j.metabol.2020.154259

30. Dushianthan A, Cusack R, Koster G. et al. Insight into erythrocyte phospholipid molecular flux in healthy humans and in patients with acute respiratory distress syndrome. PLoS One. 2019 Aug 27; 14 (8): e0221595. DOI: 10.1371/journal.pone.0221595

31. Anderson HL, Brodsky IE, Mangalmurti NS. The Evolving Erythrocyte: Red Blood Cells as Modulators of Innate Immunity. J Immunol. 2018 Sep 1; 201 (5): 1343–1351. DOI: 10.4049/jimmunol.1800565

32. Papadopoulos C, Tentes I, Anagnostopoulos K. Red Blood Cell Dysfunction in Non-Alcoholic Fatty Liver Disease: Marker and Mediator of Molecular Mechanisms. Maedica (Bucur). 2020 Dec; 15 (4): 513–516. DOI: 10.26574/maedica.2020.15.4.513

33. Papadopoulos C, Tentes I, Anagnostopoulos K. Erythrocytes Contribute to the Immunometabolic Cross-Talk. Immunometabolism. 3 (2): e210015, April 2021. DOI: 10.20900/immunometab20210015

34. Sun K, Zhang Y, D’Alessandro A. et al. Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia. Nat. Commun. 2016; 7: 12086. DOI: 10.1038/ncomms12086

35. Sun K, D’Alessandro A, Ahmed MH. et al. Structural and Functional Insight of Sphingosine 1-Phosphate-Mediated Pathogenic Metabolic Reprogramming in Sickle Cell Disease. Sci. Rep. 2017; 7: 15281. DOI: 10.1038/s41598-017-13667-8

36. Tutino V, De Nunzio V, Donghia R. et al. Significant Increase in Oxidative Stress Indices in Erythrocyte Membranes of Obese Patients with Metabolically-Associated Fatty Liver Disease. J Pers Med. 2024 Mar 18; 14 (3): 315. DOI: 10.3390/jpm14030315

37. Benson TW, Weintraub N, Kim HW. et al. A single high-fat meal provokes pathological erythrocyte remodeling and increases myeloperoxidase levels: Implications for acute coronary syndrome. Lab. Investig. 2018; 98: 1300–1310. DOI: 10.1038/s41374-018-0038-3

38. Ferreri C, Sansone A, Ferreri R. et al. Fatty Acids and Membrane Lipidomics in Oncology: A Cross-Road of Nutritional, Signaling and Metabolic Pathways. Metabolites. 2020; 10: 345. DOI: 10.3390/metabo10090345

39. Abbott SK, Else PL, Atkins TA, Hulbert AJ. Fatty acid composition of membrane bilayers: Importance of diet polyunsaturated fat balance. Biochim. Biophys. Acta. 2012; 1818: 1309–1317. DOI: 10.1016/j.bbamem.2012.01.011

40. Ferreri C, Masi A, Sansone A. et al. Fatty Acids in Membranes as Homeostatic, Metabolic and Nutritional Biomarkers: Recent Advancements in Analytics and Diagnostics. Diagnostics. 2016; 7: 1. DOI: 10.3390/diagnostics7010001

41. Monserrat-Mesquida M, Quetglas-Llabres M, Capo X. et al. Metabolic Syndrome is Associated with Oxidative Stress and Proinflammatory State. Antioxidants. 2020; 9: 236. DOI: 10.3390/antiox9030236

42. Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Curr. Biol. 2014; 24: R 453–R 462. DOI: 10.1016/j.cub.2014.03.034

43. Olusi S. O. Obesity is an independent risk factor for plasma lipid peroxidation and depletion of erythrocyte cytoprotectic enzymes in humans. Int. J. Obes. Relat. Metab. Disord. 2002; 26: 1159–1164. DOI: 10.1038/sj.ijo.0802066

44. Cazzola R, Rondanelli M, Russo-Volpe S. et al. Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes. J. Lipid. Res. 2004; 45: 1846–1851. DOI: 10.1194/jlr.M300509-JLR 200

45. Wupperfeld D, Fricker G, Bois De Fer B. et al. Essential phospholipids decrease apoptosis and increase membrane transport in human hepatocyte cell lines. Lipids Health Dis. 2022 Sep 24; 21 (1): 91. DOI: 10.1186/s12944-022-01698-8

46. Jang B, Han JW, Sung PS. et al. Hemorheological Alteration in Patients Clinically Diagnosed with Chronic Liver Diseases. J Korean Med Sci. 2016 Dec; 31 (12): 1943–1948. DOI: 10.3346/jkms.2016.31.12.1943

47. Lockwood SY, Erkal JL, Spence DM. Endothelium-derived nitric oxide production is increased by ATP released from red blood cells incubated with hydroxyurea. Nitric Oxide. 2014; 38: 1–7. DOI: 10.1016/j.niox.2014.02.003

48. Chen J, Chen J, Fu H. et al. Hypoxia exacerbates nonalcoholic fatty liver disease via the HIF‑2α/PPARα pathway. Am J Physiol Endocrinol Metab. 2019 Oct 1; 317 (4): E 710–E 722. DOI: 10.1152/ajpendo.00052.2019

49. Saraswathi V, Kumar N, Gopal T. et al. Lauric Acid versus Palmitic Acid: Effects on Adipose Tissue Inflammation, Insulin Resistance, and Non-Alcoholic Fatty Liver Disease in Obesity. Biology (Basel). 2020 Oct 22; 9 (11): 346. DOI: 10.3390/biology9110346

50. Yoo W, Gjuka D, Stevenson HL. et al. Fatty acids in non-alcoholic steatohepatitis: Focus on pentadecanoic acid. PLoS One. 2017 Dec 15; 12 (12): e0189965. DOI: 10.1371/journal.pone.0189965

51. Jenkins B, West JA, Koulman A. A review of odd-chain fatty acid metabolism and the role of pentadecanoic Acid (c15:0) and heptadecanoic Acid (c17:0) in health and disease. Molecules. 2015 Jan 30; 20 (2): 2425–44. DOI: 10.3390/molecules20022425

52. Khaw KT, Friesen MD, Riboli E, et al. Plasma phospholipid fatty acid concentration and incident coronary heart disease in men and women: the EPIC-Norfolk prospective study. PLoS Med. 2012; 9 (7): e1001255. DOI: 10.1371/journal.pmed.1001255

53. Forouhi NG, Koulman A, Sharp SJ. et al. Differences in the prospective association between individual plasma phospholipid saturated fatty acids and incident type 2 diabetes: the EPIC–InterAct case-cohort study. Lancet Diabetes Endocrinol. 2014 Oct; 2 (10): 810–8. DOI: 10.1016/S2213-8587 (14) 70146-9

54. Yuan T, Fan WB, Cong Y. et al. Linoleic acid induces red blood cells and hemoglobin damage via oxidative mechanism. Int J Clin Exp Pathol. 2015 May 1; 8 (5): 5044–52.

55. Kim CY, Larsen HJ, Spitalnik SL. et al. Low-Dose Dietary Fish Oil Improves RBC Deformability without Improving Post-Transfusion Recovery in Mice. Nutrients. 2023; 15: 4456. DOI: 10.3390/nu15204456

56. Banerjee A, Dey T, Majumder R. et al. Oleic acid prevents erythrocyte death by preserving haemoglobin and erythrocyte membrane proteins. Free Radic Biol Med. 2023 Jun; 202: 17–33. DOI: 10.1016/j.freeradbiomed.2023.03.019

57. Spengler MI, Svetaz MJ, Leroux MB. et al. Lipid peroxidation affects red blood cells membrane properties in patients with systemic lupus erythematosus. Clin Hemorheol Microcirc. 2014; 58 (4): 489–95. DOI: 10.3233/CH-131716

58. Liu ZL, Li H, Qiang Y. et al. Computational modeling of biomechanics and biorheology of heated red blood cells. Biophys J. 2021 Nov 2; 120 (21): 4663–4671. DOI: 10.1016/j.bpj.2021.09.038

59. Marrone A, Ballantyne J. Changes in dry state hemoglobin over time do not increase the potential for oxidative DNA damage in dried blood. PLoS One. 2009; 4: e5110. DOI: 10.1371/journal.pone.0005110

60. Pimenova T, Pereira CP, Gehrig P. et al. Quantitative mass spectrometry defines an oxidative hotspot in hemoglobin that is specifically protected by haptoglobin. J Proteome Res. 2010; 9: 4061–4070. DOI: 10.1021/pr100252e

61. Niki E. Lipid peroxidation: physiological levels and dual biological effects. Free Radic Biol Med. 2009 Sep 1; 47 (5): 469–84. DOI: 10.1016/j.freeradbiomed.2009.05.032

62. Wilson MT, Reeder BJ. The peroxidatic activities of Myoglobin and Hemoglobin, their pathological consequences and possible medical interventions. Mol Aspects Med. 2022 Apr; 84: 101045. DOI: 10.1016/j.mam.2021.101045

63. Chistyakov DV, Gavrish GE, Goriainov SV. et al. Oxylipin Profiles as Functional Characteristics of Acute Inflammatory Responses in Astrocytes Pre-Treated with IL-4, IL-10, or LPS. Int J Mol Sci. 2020 Mar 5; 21 (5): 1780. DOI: 10.3390/ijms21051780

64. Gutiérrez S, Svahn SL, Johansson ME. Effects of Omega-3 Fatty Acids on Immune Cells. Int J Mol Sci. 2019 Oct 11; 20 (20): 5028. DOI: 10.3390/ijms20205028

65. Bentzen PJ, Lang F. Effect of anandamide on erythrocyte survival. Cell. Physiol. Biochem. 2007; 20: 1033–1042. DOI: 10.1159/000110714

66. Takić M, Ranković S, Girek Z, et al. Current Insights into the Effects of Dietary α-Linolenic Acid Focusing on Alterations of Polyunsaturated Fatty Acid Profiles in Metabolic Syndrome. Int J Mol Sci. 2024 Apr 30; 25 (9): 4909. DOI: 10.3390/ijms25094909

67. Immenschuh S, Baumgart-Vogt E, Mueller S. Heme oxygenase-1 and iron in liver inflammation: a complex alliance. Curr Drug Targets. 2010 Dec; 11 (12): 1541–50. DOI: 10.2174/1389450111009011541

68. Rother RP, Bell L, Hillmen P, Gladwin MT. The clinical sequelae of intravascular hemolysis and extracellular plasma hemoglobin: a novel mechanism of human disease. JAMA. 2005 Apr 6; 293 (13): 1653–62. DOI: 10.1001/jama.293.13.1653

69. Camus SM, De Moraes JA, Bonnin P. et al. Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease. Blood. 2015 Jun 11; 125 (24): 3805–14. DOI: 10.1182/blood-2014-07-589283