Theoretical Chemistry Accounts

, Volume 130, Issue 4–6, pp 1211–1220 | Cite as

A Rheumatoid arthritis study using Raman spectroscopy

  • C. S. Carvalho
  • A. A. Martin
  • A. M. E. Santo
  • L. E. C. Andrade
  • M. M. Pinheiro
  • M. A. G. Cardoso
  • L. Raniero
Regular Article


Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints and can lead to a progressive destruction of articular cartilage and bone. In this study, the specificity and sensitivity of the RA diagnostic methods based on the receiver-operating characteristic curves for monitoring C-reactive protein (CRP) and rheumatoid factor (RF) were compared with the Raman spectroscopic diagnostic method developed in this work. Sera from 24 patients with rheumatoid arthritis and from 16 healthy individuals were analyzed to assess the biochemical composition and presence of inflammatory activity by the aforementioned methods. By comparing with the clinical results for specificity and sensitivity from the RF and CRP tests, we show that the overall results from the newly developed Raman method were significantly better, with a specificity of 96%, a sensitivity of 88%, and correctly identifying 92% of the RA and healthy individuals, while the RF test gave a specificity of 100% and a sensitivity of 54%, and the CRP test gave a specificity of 87% and a sensitivity of 58%, respectively.


Confocal Raman Rheumatoid arthritis Serum 



Rheumatoid arthritis


C-reactive protein


Rheumatoid factor


American College of Rheumatology


Acute-phase proteins


National Committee for Clinical Laboratory and Approved Standards


Charge-coupled device


Calcium fluoride


Principal components analysis


Principal component


Receiver-operating characteristic


Kolmogorov and Smirnov


Total cholesterol


Correlation coefficient


Total triglycerides


Low-density lipoprotein


Linear discriminant analysis


Universidade Federal de São Paulo


Vibrational biomedical spectroscopy laboratory


Universidade do Vale do Paraíba


Institute for Research and Development



We are grateful to patients help donating the samples and also for the great contribution and scientific discussion of the referees.


  1. 1.
    Jazayeri JA, Carrol GJ, Vernallis AB (2010) Interleukin-6 subfamily cytokines and rheumatoid arthritis: role of antagonists. Int Immunopharmacol 10:1–8CrossRefGoogle Scholar
  2. 2.
    Feldmann M, Brennan FM, Maini RN (1996) Role of cytokines in rheumatoid arthritis. Ann Rev Immunol 14:397–440CrossRefGoogle Scholar
  3. 3.
    Carteron NL (2000) Cytokines in rheumatoid arthritis: trials and tribulations. Mol Med Today 6:315–323CrossRefGoogle Scholar
  4. 4.
    Walsmith J, Roubenoff R (2002) Cachexia in rheumatoid arthritis. Int J Cardiol 85:89–99CrossRefGoogle Scholar
  5. 5.
    Van Boekel M, Vossenaar ER, Van Den Hoogen FHJ, Van Venrooij WJ (2002) Autoantibody systems in rheumatoid arthritis: Specificity, sensitivity and diagnostic value. Arthritis Res 4:87–93CrossRefGoogle Scholar
  6. 6.
    Khurana R, Berney SM (2005) Clinical aspects of rheumatoid arthritis. Pathophysiology 12:153–165CrossRefGoogle Scholar
  7. 7.
    Chang J, Kavanaugh A (2005) Novel therapies for rheumatoid arthritis. Pathophysiol 12:217–225CrossRefGoogle Scholar
  8. 8.
    Cheng YJ, Hootman JM, Murphy LB, Langmaid GA, Helmick CG (2010) Prevalence of doctor-diagnosed arthritis and arthritis-attributable activity limitation—United States, 2007–2009. Morbidity and mortality weekly report 59:1261–1265.
  9. 9.
    Arnett FC, Edworthy SM, Bloch DA, Mcshane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG (1988) The American rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–324CrossRefGoogle Scholar
  10. 10.
    Aletaha D, Neogi T, Silman AJ, Funovits J, Felson DT, Bingham CO, Birnbaum NS, Burmester GR, Bykerk VP, Cohen MD, Combe B, Costenbader KH, Dougados M, Emery P, Ferraccioli G, Hazes JMW, Hobbs K, Huizinga TWJ, Kavanaugh A, Kay J, Kvien TK, Laing T, Mease P, Ménard HA, Moreland LW, Naden RL, Pincus T, Smolen JS, Stanislawska-Biernat E, Symmons D, Tak PP, Upchurch KS, Vencovsky J, Wolfe F, Hawker G (2010) 2010 Rheumatoid arthritis classification criteria. Arthritis Rheum 62:2569–2581CrossRefGoogle Scholar
  11. 11.
    Vossenaar ER, Van Venrooij WJ (2004) Anti-CCP antibodies, a highly specific marker for (early) rheumatoid arthritis. Clin Applied Immunol Rev 4:239–262CrossRefGoogle Scholar
  12. 12.
    Sutton B, Corper A, Bonagura V, Taussig M (2000) The structure and origin of rheumatoid factors. Immunol Today 4:177–183CrossRefGoogle Scholar
  13. 13.
    Chaiamnuay S, Louis Bridges SJ (2005) The role of B cells and autoantibodies in rheumatoid arthritis. Pathophysiology 12:203–216CrossRefGoogle Scholar
  14. 14.
    Mewar D, Wilson AG (2006) Autoantibodies in rheumatoid arthritis: a review. Biomed Pharmacother 60:648–655CrossRefGoogle Scholar
  15. 15.
    Nijenhuis S, Zendman AJW, Vossenaar ER, Pruijin Ger JM, vanVenrooij W (2004) Autoantibodies to citrullinated proteins in rheumatoid arthritis: clinical performance and biochemical aspects of an RA-specific marker. Clin Chim Acta 350:17–34CrossRefGoogle Scholar
  16. 16.
    Ottaviani S, Mariani F, Ferrarotti I, Luisetti M (2011) C reactive protein and alpha 1-antitrypsin: relationship between levels and gene variants. Transl Res doi:10.1016/j.trsl.2010.12.014.
  17. 17.
    Sjowall C, Wettero J (2007) Pathogenic implications for autoantibodies against C-reactive protein and other acute phase proteins. Clin Chim Acta 378:13–23CrossRefGoogle Scholar
  18. 18.
    Levinson SS (2005) Brief review and critical examination of the use of hs-CRP for cardiac risk assessment with the conclusion that it is premature to use this test. Clin Chim Acta 356:1–8CrossRefGoogle Scholar
  19. 19.
    Heikkila K, Silander K, Salomaa V, Jousilahti P, Koskinen S, Pukkala E, Perola M (2011) C-reactive protein-associated genetic variants and cancer risk: findings from FINRISK 1992, FINRISK 1997 and Health 2000 studies. Eur J Cancer 47:404–412CrossRefGoogle Scholar
  20. 20.
    Marnell L, Mold C, Du Clos TW (2005) C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol 117:104–111CrossRefGoogle Scholar
  21. 21.
    Cassim B, Mody G, Bhoola KD (2002) Kallikrein cascade and cytokines in inflamed joints. Pharmacol Ther 94:1–34CrossRefGoogle Scholar
  22. 22.
    van Riel PLCM, Fransen J (2007) Established rheumatoid arthritis: clinical assessments. Best Pract Res Clin Rheumatol 21:807–825CrossRefGoogle Scholar
  23. 23.
    Moreno M, Raniero L, Arisawa EAL, Santo AME, Santos EAP, Bitar RA, Martin AA (2010) Raman spectroscopy study of breast disease. Theor Chem Acc 125:329–334CrossRefGoogle Scholar
  24. 24.
    Esmonde-White KA, Mandair GS, Raaii F, Jacobson JA, Miller BS, Urquhart AG, Roessler BJ, Morris MD (2009) Raman spectroscopy of synovial fluid as a tool for diagnosing osteoarthritis. J Biomed Opt 14:034013CrossRefGoogle Scholar
  25. 25.
    Hanlon EB, Manoharan R, Koo TW, Shafer KE, Motz JT, Fitzmaurice M, Kramer JR, Itzkan I, Dasari RR, Feld MS (2000) Prospects for in vivo Raman spectroscopy. Phys Med Biol 45:R1–R59CrossRefGoogle Scholar
  26. 26.
    CLSI (2003) Clinical and laboratory standards institute quality manual, 3rd edn. CLSI, Wayne Google Scholar
  27. 27.
    Lieber CA, Mahadevan-Jansen A (2003) Automated method for subtraction of fluorescence from biological Raman spectra. Appl Spectrosc 57:320–340CrossRefGoogle Scholar
  28. 28.
    Triola MF (2009) Elementary statistics plus MyStatLab student access kit, 11th edn. Addison-Wesley, Rio de Janeiro, ISBN-13:978-0321570895Google Scholar
  29. 29.
    Van Venrooij WJ, Van Beers JJBC, Pruijn GJM (2008) Anti-CCP antibody, a marker for the early detection of rheumatoid arthritis. The year in immunology. Ann N Y Acad Sci 1143:268–285CrossRefGoogle Scholar
  30. 30.
    Heidari B, Heidari P, Tayebi ME (2007) The value of changes in CRP and ESR for predicting treatment response in rheumatoid arthritis. APLAR J Rheumatol 10:23–28CrossRefGoogle Scholar
  31. 31.
    Lin F, Chen L, Liang R, Zhang Z, Wang J, Cai M, Li Y (2011) Pilot-scale production of low molecular weight peptides from corn wet milling byproducts and the antihypertensive effects in vivo and in vitro. Food Chem 124:801–807CrossRefGoogle Scholar
  32. 32.
    Perret-Guillaume C, Genet C, Herrmann FR, Benetos A, Hurst SA, Vischer UM (2011) Attitudes and approaches to decision making about antihypertensive treatment in elderly patients. Am Med Dir Assoc 12:121–128CrossRefGoogle Scholar
  33. 33.
    Fawcett T (2006) An introduction to ROC analysis. Pattern Recognit Lett 27:861–874CrossRefGoogle Scholar
  34. 34.
    Bizarro N, Mazzanti G, Tonutti E, Villalta D, Tozzoli R (2001) Diagnostic accuracy of the anti-citrulline antibody assay for rheumatoid arthritis. Clin Chem 47:1089–1093Google Scholar
  35. 35.
    Hoffman IEA, Peene I, Pottel H, Union A, Hulstaert F, Meheus L, Lebeer K, De Clercq L, Schatteman L, Poriau S, Mielants H, Veys EM, De Keyser F (2005) Diagnostic performance and predictive value of rheumatoid factor, anti-citrullinated peptide antibodies, and the HLA shared epitope for diagnosis of rheumatoid arthritis. Clin Chem 51:261–263CrossRefGoogle Scholar
  36. 36.
    Vander Cruyssen B, Peene I, Cantaert T, Hoffman IEA, De Rycke L, Veys EM, De Keyser F (2005) Anti-citrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis: specificity and relation with rheumatoid factor. Autoimmun Rev 4:468–474CrossRefGoogle Scholar
  37. 37.
    Pepys MB, Hirschfield GM (2003) C-reactive protein: a critical update. J Clin Investig 111:1805–1812Google Scholar
  38. 38.
    Rogowski O, Vered Y, Shapira I, Hirsh M, Zakut V, Berliner S (2005) Introducing the wide range C-reactive protein (wr-CRP) into clinical use for the detection of microinflamation. Clin Chim Acta 358:151–158CrossRefGoogle Scholar
  39. 39.
    Meyer KC, Decker C, Baughman R (2010) Toxicity and monitoring of immunosuppressive therapy used in systemic autoimmune diseases. Clin Chest Med 31:565–588CrossRefGoogle Scholar
  40. 40.
    Stamp L, Roberts R, Kennedy M, Barclay M, O’Donnell J, Chapman P (2006) The use of low dose methotrexate in rheumatoid arthritis-are we entering a new era of therapeutic drug monitoring and pharmacogenomics. Biomed Pharmacother 60:678–687CrossRefGoogle Scholar
  41. 41.
    Alarcón GS (2000) Methotrexate use in rheumatoid arthritis. A clinician’s perspective. Immunopharmacol 47:259–271CrossRefGoogle Scholar
  42. 42.
    Borchers AT, Keen CL, Cheema GS, Gershwin ME (2004) The use of methotrexate in rheumatoid arthritis. Semin Arthritis Rheumatol 34:465–483CrossRefGoogle Scholar
  43. 43.
    Kremer JM (1999) Methotrexate and leflunomide: biochemical basis for combination therapy in the treatment of rheumatoid arthritis. Semin Arthritis Rheumatol 29:14–26CrossRefGoogle Scholar
  44. 44.
    Park S-H, Choe J-Y, Kim S-K (2010) Assessment of liver fibrosis by transient elastography in rheumatoid arthritis patients treated with methotrexate. Joint Bone Spine 77:588–592CrossRefGoogle Scholar
  45. 45.
    Park Y-B, Choi HK, Kim M-Y, Lee W-K, Song J, Kim D-K, Lee S-K (2002) Effects of antirheumatic therapy on serum lipid levels in patients with rheumatoid arthritis: a prospective study. Am J Med 113:188–193CrossRefGoogle Scholar
  46. 46.
    Kermani TA, Luthra HS (2008) Folate supplementation and methotrexate in treatment of rheumatoid arthritis. Indian J Rheumatol 3:77–79CrossRefGoogle Scholar
  47. 47.
    Espinosa-Mansilla A, La Peña AM, Cañada-Cañada F, Gómez DG (2005) Determinations of xuoroquinolones and nonsteroidal anti-inflammatory drugs in urine by extractive spectrophotometry and photoinduced spectrofluorimetry using multivariate calibration. Anal Biochem 347:275–286CrossRefGoogle Scholar
  48. 48.
    Levy M, Zylber-Katz E, Rosenkranz B (1995) Clinical pharmacokinetics of dipyrone and its metabolites. Clin Pharmacokinet 28:216–234CrossRefGoogle Scholar
  49. 49.
    Papadakis JA, Ganotakis ES, Jagroop IA, Mikhailidis DP, Winder AF (1999) Effect of hypertension and its treatment on lipid, lipoprotein(a), fibrinogen, and bilirubin levels in patients referred for dyslipidemia. Am J Hypertens 12:673–681CrossRefGoogle Scholar
  50. 50.
    Krone W, Nagele H, Hamburg MD (1988) Effects of antihypertensives on plasma lipids and lipoprotein metabolism. Am Heart J 116:1729–1734CrossRefGoogle Scholar
  51. 51.
    Hadda V, Handa R, Aggarwal P, Lakshmy R, Kumar U, Pandey RM (2007) Disease activity and lipids in rheumatoid arthritis: a prospective study. Indian J Rheumatol 4:137–140CrossRefGoogle Scholar
  52. 52.
    Steiner G, Urowitz MB (2009) Lipid profiles in patients with rheumatoid arthritis: mechanisms and the impact of treatment. Semin Arthritis Rheumatol 38:372–381CrossRefGoogle Scholar
  53. 53.
    Seven A, Guzel S, Aslan M, Hamuryudan V (2008) Lipid, protein, DNA oxidation status in rheumatoid arthritis. Clin Biochem 14:538–543CrossRefGoogle Scholar
  54. 54.
    Pajevic S, Basser PJ (2003) Parametric and non-parametric statistical analysis of DT-MRI data. J Magn Reson 161:1–14CrossRefGoogle Scholar
  55. 55.
    Qi D, Berger AJ (2007) Chemical concentration measurement in blood serum and urine samples using liquid-core optical fiber Raman spectroscopy. Appl Opt 46:1726–1734CrossRefGoogle Scholar
  56. 56.
    Berger AJ, Koo T-W, Itzkan I, Horowitz G, Feld MS (1999) Multicomponent blood analysis by near-infrared Raman spectroscopy. Appl Opt 38:2916–2926CrossRefGoogle Scholar
  57. 57.
    Dàvila E, Parés D, Howell N (2007) Studies on plasma protein interactions in heat-induced gels by differential scanning calorimetry and FT-Raman spectroscopy. Food Hydrocoll 21:1144–1152CrossRefGoogle Scholar
  58. 58.
    Virkler K, Lednev IK (2010) Raman spectroscopic signature of blood and its potential application to forensic body fluid identification. Anal Bioanal Chem 396:525–534CrossRefGoogle Scholar
  59. 59.
    Rohleder D, Kocherscheidt G, Gerber K, Kiefer W, Kohler W, Mocks J, Petrich W (2005) Comparison of mid-infrared and Raman spectroscopy in the quantitative analysis of serum. J Biomed Opt 10:031108. doi:10.1117/1.1911847 Google Scholar
  60. 60.
    Herrero AM, Cambero MI, Ordóñez JA, La Hoz L, Carmona P (2009) Plasma powder as cold-set binding agent for meat system: rheological and Raman spectroscopy study. Food Chem 113:493–499CrossRefGoogle Scholar
  61. 61.
    Fleury F, Ianoul A, Berjot M, Feofanov A, Alix AJP, Nabiev I (1997) Camptothecin-binding site in human serum albumin and protein transformations induced by drug binding. FEBS Lett 411:215–220CrossRefGoogle Scholar
  62. 62.
    Xie Y, Zhang D, Jarori GK, Davisson VJ, Ben-Amotz D (2004) The Raman detection of peptide tyrosine phosphorylation. Anal Biochem 332:116–121CrossRefGoogle Scholar
  63. 63.
    Tristano AG (2009) Tyrosine kinases as targets in rheumatoid arthritis. Int Immunopharmacol 9:1–9CrossRefGoogle Scholar
  64. 64.
    Schrocksnadel K, Wirleitner B, Winkler C, Fuchs D (2006) Monitoring tryptophan metabolism in chronic immune activation. Clin Chim Acta 364:82–90CrossRefGoogle Scholar
  65. 65.
    McArthur JN, Dawkins PD, Smith MJH, Hamilton EBD (1971) Mode of action of antirheumatic drugs. Br Med J 2:667–679CrossRefGoogle Scholar
  66. 66.
    Rodenburg RJT, Van de Hoogen FHJ, Van de Putte LBA, Van Venrooij WJ (1998) Peripheral blood monocytes of rheumatoid arthritis patients do not express elevated TNF-α amd IL-8 mRNA levels. A comparison of monocyte isolation procedures. J Immunol Meth 221:169–175CrossRefGoogle Scholar
  67. 67.
    Badolato R, Oppenheim JJ (1996) Role of cytokines, acute-phase proteins, and chemokines in the progression of rheumatoid arthritis. Semin Arthritis Rheum 26:526–538CrossRefGoogle Scholar
  68. 68.
    Petibois C, Melin AM, Perromat A, Cazorla G, Déléris G (2000) Glucose and lactate concentration determination on single microsamples by Fourier-transform infrared spectroscopy. J Lab Clin Med 135:210–215CrossRefGoogle Scholar
  69. 69.
    Petibois C, Cazorla G, Cassaigne A, Déléris G (2001) Plasma protein contents determined by Fourier-transform infrared spectrometry. Clin Chem 47:730–738Google Scholar
  70. 70.
    Déléris G, Petibois C (2003) Applications of FT-IR spectrometry to plasma contents analysis and monitoring. Vib Spectrosc 32:129–136CrossRefGoogle Scholar
  71. 71.
    Barnaby OS, Cerny RL, Clarke W, Hage DS (2011) Comparison of modification sites formed on human serum albumin at various stages of glycation. Clin Chim Acta 412:277–285CrossRefGoogle Scholar
  72. 72.
    Rondeau P, Bourdon E (2011) The glycation of albumin: structural and functional impacts. Biochim doi:10.1016/j.biochi.2010.12.003.
  73. 73.
    Pezolet M, Pigeon-Gosselin M, Coulombe L (1976) Laser Raman investigation of the conformation of human immunoglobulin G. Biochim Biophys Acta 453:502–512Google Scholar
  74. 74.
    Sane SU, Cramer SM, Przybycien TM (1999) A holistic approach to protein secondary structure characterization using amide I band Raman spectroscopy. Anal Bioch 269:255–272CrossRefGoogle Scholar
  75. 75.
    Fraile MV, Blanco-Melgar M, Munõz M, Rodriguez GL, Gallego-Nicasio JA, Carmona P (2003) Structure and interactions of albumin-lipid systems as studied by infrared spectroscopy. J Mol Struct 651–653:231–236CrossRefGoogle Scholar
  76. 76.
    Sadler PJ, Tucker A, Viles JH (1994) Involvement of a lysibe residue in the N-terminal Ni2+ and Cu2+ binding site of serum albumins. Comparison with Co2+, Cd2+ and A13+. Eur J Biochem 220:193–200CrossRefGoogle Scholar
  77. 77.
    Dotzlaw H, Schulz M, Eggert M, Neeck G (2004) A pattern of protein expression in peripheral blood mononuclear cells distinguishes rheumatoid arthritis patients from healthy individuals arthritis patients from healthy individuals. Biochim Biophys Acta 1696:121–129Google Scholar
  78. 78.
    Jalkanen KJ, Suhai S (1996) N-acetyl-l-alanine N′-methylamide: a density functional analysis of the vibrational absorption and vibrational circular dichroism spectra. Chem Phys 208:81–116CrossRefGoogle Scholar
  79. 79.
    Diem M, Griffiths PR, Chalmers JM (eds) (2008) Vibrational spectroscopy for medical diagnosis. Culinary and hospitality industry publications services (CHIPS), WeimarGoogle Scholar
  80. 80.
    Romeo MJ, Dukor RK, Diem M (2008) Introduction to spectral imaging, and applications to diagnosis of lymph nodes. In: Diem M, Griffiths PR, Chalmers JM (eds) Vibrational spectroscopy for medical diagnostics, Wiley-VCH Verlag GmbH, WeinheimGoogle Scholar
  81. 81.
    Lasch P, Beekes M, Schmitt J, Naumann D (2007) Detection of preclinical scrapie from serum by infrared spectroscopy and chemometrics. Anal Bioanal Chem 387:1791–1800CrossRefGoogle Scholar
  82. 82.
    Huh SY, Chung AJ, Erickson D (2009) Surface enhanced Raman spectroscopy and its application to molecular and cellular analysis. Microfluid Nanofluid 6:285–297CrossRefGoogle Scholar
  83. 83.
    Sikirzhytski V, Virkler K, Lednev IK (2010) Discriminant analysis of Raman spectra for body fluid identification for forensic purposes. Sensors 10:2869–2884CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • C. S. Carvalho
    • 1
  • A. A. Martin
    • 1
  • A. M. E. Santo
    • 1
    • 2
  • L. E. C. Andrade
    • 3
  • M. M. Pinheiro
    • 3
  • M. A. G. Cardoso
    • 1
  • L. Raniero
    • 1
  1. 1.Laboratory of Biomedical Vibrational SpectroscopyUniversidade do Vale do ParaíbaSão José dos CamposBrazil
  2. 2.Departamento de Ciências Exatas e da TerraUniversidade Federal de São PauloDiademaBrazil
  3. 3.Departamento de Medicina, Divisão de ReumatologiaUniversidade Federal de São PauloSão PauloBrazil

Personalised recommendations