BACKGROUND: Vibrational and chiroptical spectroscopy of blood plasma is an advanced experimental diagnostic approach that allows the identification of disease-specific molecular patterns. This study aimed to test its potential to distinguish between different stages of metabolic dysfunction-associated steatotic liver disease (MASLD) including liver fibrosis. METHODS: We analyzed blood plasma samples from 29 patients with metabolic dysfunction-associated steatohepatitis (MASH) and 24 MASLD patients with simple steatosis using Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy and electronic circular dichroism (ECD) spectroscopy to distinguish: (i) MASH; (ii) MASH with moderate to advanced fibrosis; and (iii) overall fibrosis. RESULTS: FTIR spectroscopy distinguished MASH from simple steatosis with a sensitivity of 73% and a specificity of 92%, with an area under the receiver operating characteristic curve (AUROC) of 0.92, p < 0.001. In addition, FTIR spectroscopy identified MASH with moderate to advanced fibrosis (F2-3) from MASH/steatosis with no to mild fibrosis (F0-1) with a sensitivity of 87% and specificity of 78% (AUROC 0.91, p < 0.001). Furthermore, regardless of the MASH/steatosis diagnosis, fibrosis (F1-3) was distinguished from F0 by the combination of data from the three spectroscopic methods with a sensitivity of 79% and a specificity of 88% (AUROC 0.91, p < 0.001). Both FTIR and Raman spectroscopy alone discriminated all target groups with an AUROC > 0.82. CONCLUSIONS: This pilot study revealed the potential of blood plasma spectroscopy to identify and differentiate patients with various stages of MASLD.

4th Department of Internal Medicine 1st Faculty of Medicine and General University Hospital Prague Charles University Prague 12808 Czech Republic

Department of Analytical Chemistry University of Chemistry and Technology Prague Prague 6 166 28 Czech Republic

Department of Pathology 1st Faculty of Medicine and General University Hospital Prague Charles University Prague 12800 Czech Republic

Institute of Medical Biochemistry and Laboratory Diagnostics 1st Faculty of Medicine General University Hospital Prague Charles University Prague 12108 Czech Republic

Institute of Photonics and Electronics Czech Academy of Sciences Prague 18200 Czech Republic

Regional Hospital Liberec Liberec 460 01 Czech Republic

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Rinella ME, Lazarus JV, Ratziu V, Francque SM, Sanyal AJ, Kanwal F, et al. A multisociety Delphi consensus statement on new fatty liver disease nomenclature. Hepatology. 2023;78(6):1966–86. PubMed PMC

Targher G, Byrne CD, Tilg H. MASLD: a systemic metabolic disorder with cardiovascular and malignant complications. Gut. 2024;73(4):691–702. PubMed

Estes C, Anstee QM, Arias-Loste MT, Bantel H, Bellentani S, Caballeria J, et al. Modeling NAFLD disease burden in china, france, germany, italy, japan, spain, united kingdom, and united States for the period 2016–2030. J Hepatol. 2018;69(4):896–904. PubMed

Parthasarathy G, Revelo X, Malhi H. Pathogenesis of nonalcoholic steatohepatitis: an overview. Hepatol Commun. 2020;4(4):478–92. PubMed PMC

Asrani SK, Devarbhavi H, Eaton J, Kamath PS. Burden of liver diseases in the world. J Hepatol. 2019;70(1):151–71. PubMed

Llovet JM, Willoughby CE, Singal AG, Greten TF, Heikenwalder M, El-Serag HB, et al. Nonalcoholic steatohepatitis-related hepatocellular carcinoma: pathogenesis and treatment. Nat Rev Gastroenterol Hepatol. 2023;20(8):487–503. PubMed PMC

Sanyal AJ, Van Natta ML, Clark J, Neuschwander-Tetri BA, Diehl A, Dasarathy S, et al. Prospective study of outcomes in adults with nonalcoholic fatty liver disease. N Engl J Med. 2021;385(17):1559–69. PubMed PMC

Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med. 2001;344(7):495–500. PubMed

Tamaki N, Ajmera V, Loomba R. Non-invasive methods for imaging hepatic steatosis and their clinical importance in NAFLD. Nat Rev Endocrinol. 2022;18(1):55–66. PubMed PMC

Davison BA, Harrison SA, Cotter G, Alkhouri N, Sanyal A, Edwards C, et al. Suboptimal reliability of liver biopsy evaluation has implications for randomized clinical trials. J Hepatol. 2020;73(6):1322–32. PubMed

Noureddin M, Truong E, Gornbein JA, Saouaf R, Guindi M, Todo T, et al. MRI-based (MAST) score accurately identifies patients with NASH and significant fibrosis. J Hepatol. 2022;76(4):781–7. PubMed

Mozes FE, Lee JA, Vali Y, Selvaraj EA, Jayaswal ANA, Boursier J, et al. Diagnostic accuracy of non-invasive tests to screen for at-risk MASH-An individual participant data meta-analysis. Liver Int. 2024;44(8):1872–85. PubMed

Zhou B, Xiao JF, Tuli L, Ressom HW. LC-MS-based metabolomics. Mol Biosyst. 2012;8(2):470–81. PubMed PMC

Yang CR, Lin WJ, Shen PC, Liao PY, Dai YC, Hung YC, et al. Phenotypic and metabolomic characteristics of mouse models of metabolic associated steatohepatitis. Biomark Res. 2024;12(1):6. PubMed PMC

Vrtelka O, Kralova K, Fouskova M, Habartova L, Hribek P, Urbanek P, et al. Vibrational and chiroptical analysis of blood plasma for hepatocellular carcinoma diagnostics. Analyst. 2023;148(12):2793–800. PubMed

Ten-Doménech I, Rienda I, Pérez-Rojas J, Pareja E, Moreno-Torres M, Castell JV, et al. Progress and challenges of mid-infrared spectroscopy for liver characterization focusing on steatosis, fibrosis and cancer. Appl Spectrosc Rev. 2024;59(4):578–99.

Le Corvec M, Charpentier F, Kachenoura A, Bensaid S, Henno S, Bardou-Jacquet E, et al. Fast and non-invasive medical diagnostic using mid infrared sensor: the AMNIFIR project. Irbm. 2016;37(2):116–23.

Bensaid S, Kachenoura A, Costet N, De Ledinghen V, Vergniol J, Lainé F, et al. editors. Early diagnosis of NAFLD-NASH transition using mid infrared spectroscopy. 2016 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC); 2016: IEEE. PubMed

Anty R, Morvan M, Le Corvec M, Canivet CM, Patouraux S, Gugenheim J, et al. The mid-infrared spectroscopy: A novel non-invasive diagnostic tool for NASH diagnosis in severe obesity. JHEP Rep. 2019;1(5):361–8. PubMed PMC

Anty R, Le Corvec M, Iannelli A, Patouraux S, Saint-Paul M-C, Schneck A-S, et al. Mid-infrared spectroscopy has a high sensitivity and specificity for point-of-care diagnosis of non-alcoholic steato-hepatitis. J Hepatol. 2016;64(2):S177–S.

Aitekenov S, Sultangaziyev A, Abdirova P, Yussupova L, Gaipov A, Utegulov Z, et al. Raman, infrared and Brillouin spectroscopies of biofluids for medical diagnostics and for detection of biomarkers. Crit Rev Anal Chem. 2023;53(7):1561–90. PubMed

Synytsya A, Judexova M, Hruby T, Tatarkovic M, Miskovicova M, Petruzelka L, et al. Analysis of human blood plasma and Hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA). Anal Bioanal Chem. 2013;405(16):5441–53. PubMed

Tatarkovic M, Fisar Z, Raboch J, Jirak R, Setnicka V. Can chiroptical spectroscopy be used for the analysis of blood plasma? Chirality. 2012;24(11):951–5. PubMed

Keiderling TA. Structure of condensed phase peptides: insights from vibrational circular dichroism and Raman optical activity techniques. Chem Rev. 2020;120(7):3381–419. PubMed

Tatarkovic M, Synytsya A, Stovickova L, Bunganic B, Miskovicova M, Petruzelka L, et al. The minimizing of fluorescence background in Raman optical activity and Raman spectra of human blood plasma. Anal Bioanal Chem. 2015;407(5):1335–42. PubMed

Dvorak K, Stritesky J, Petrtyl J, Vitek L, Sroubkova R, Lenicek M, et al. Use of non-invasive parameters of non-alcoholic steatohepatitis and liver fibrosis in daily practice–an exploratory case-control study. PLoS ONE. 2014;9(10):e111551. PubMed PMC

Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig L, et al. STARD 2015: an updated list of essential items for reporting diagnostic accuracy studies. Radiology. 2015;277(3):826–32. PubMed

Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology. 2005;41(6):1313–21. PubMed

Brunt EM. Nonalcoholic steatohepatitis. Semin Liver Dis. 2004;24(1):3–20. PubMed

Hribek P, Vrtelka O, Kralova K, Klasova J, Fouskova M, Habartova L, et al. Efficacy of blood plasma spectroscopy for early liver cancer diagnostics in obese patients. Ann Hepatol. 2024;29(5):101519. PubMed

Severcan F, Bozkurt O, Gurbanov R, Gorgulu G. FT-IR spectroscopy in diagnosis of diabetes in rat animal model. J Biophotonics. 2010;3(8–9):621–31. PubMed

Tsouka S, Kumar P, Seubnooch P, Freiburghaus K, St-Pierre M, Dufour JF, et al. Transcriptomics-driven metabolic pathway analysis reveals similar alterations in lipid metabolism in mouse MASH model and human. Commun Med (Lond). 2024;4(1):39. PubMed PMC

Lambert JE, Ramos-Roman MA, Browning JD, Parks EJ. Increased de Novo lipogenesis is a distinct characteristic of individuals with nonalcoholic fatty liver disease. Gastroenterology. 2014;146(3):726–35. PubMed PMC

Min HK, Kapoor A, Fuchs M, Mirshahi F, Zhou H, Maher J, et al. Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metab. 2012;15(5):665–74. PubMed PMC

Musso G, Gambino R, Cassader M. Cholesterol metabolism and the pathogenesis of non-alcoholic steatohepatitis. Prog Lipid Res. 2013;52(1):175–91. PubMed

Liu J, Chen Y, Qian J, Cui R, Demirkan A, Zheng Y. Novel genetic insights into the roles of amino acids in metabolic dysfunction-associated steatotic liver disease. medRxiv [Internet]. 2024 Apr 12]:[2024.04. 09.24305556 p.]. Available from: 10.1101/2024.04.09.24305556

Masarone M, Troisi J, Aglitti A, Torre P, Colucci A, Dallio M, et al. Untargeted metabolomics as a diagnostic tool in NAFLD: discrimination of steatosis, steatohepatitis and cirrhosis. Metabolomics. 2021;17:1–13. PubMed

Gao F, Lu DC, Zheng TL, Geng S, Sha JC, Huang OY, et al. Fully connected neural network-based serum surface-enhanced Raman spectroscopy accurately identifies non-alcoholic steatohepatitis. Hepatol Int. 2023;17(2):339–49. PubMed PMC

Gurian E, Giraudi P, Rosso N, Tiribelli C, Bonazza D, Zanconati F, et al. Differentiation between stages of non-alcoholic fatty liver diseases using surface-enhanced Raman spectroscopy. Anal Chim Acta. 2020;1110:190–8. PubMed

Crocombe RA. Portable spectroscopy. Appl Spectrosc. 2018;72(12):1701–51. PubMed

Sala A, Cameron JM, Jenkins CA, Barr H, Christie L, Conn JJ, et al. Liquid biopsy for pancreatic cancer detection using infrared spectroscopy. Cancers. 2022;14(13):3048. PubMed PMC

Schie IW, Rüger J, Mondol AS, Ramoji A, Neugebauer U, Krafft C, et al. High-throughput screening Raman spectroscopy platform for label-free cellomics. Anal Chem. 2018;90(3):2023–30. PubMed

Cameron JM, Sala A, Antoniou G, Brennan PM, Butler HJ, Conn JJ, et al. A spectroscopic liquid biopsy for the earlier detection of multiple cancer types. Br J Cancer. 2023;129(10):1658–66. PubMed PMC

Vrtělka O, Králová K, Fousková M, Setnička V. Comprehensive assessment of the role of spectral data pre-processing in spectroscopy-based liquid biopsy. Spectrochim Acta Part A Mol Biomol Spectrosc. 2025:126261. PubMed

Vališ J, Fousková M, Janstová D, Habartová L, Petrtýl J, Petruželka L, et al. Automated classification pipeline for real-time in vivo examination of colorectal tissue using Raman spectroscopy. Spectrochim Acta Part A Mol Biomol Spectrosc. 2024;313:124152. PubMed

Newsome PN, Sasso M, Deeks JJ, Paredes A, Boursier J, Chan WK, et al. FibroScan-AST (FAST) score for the non-invasive identification of patients with non-alcoholic steatohepatitis with significant activity and fibrosis: a prospective derivation and global validation study. Lancet Gastroenterol Hepatol. 2020;5(4):362–73. PubMed PMC