A Novel Quantitative Method for the Detection of Lipofuscin, the Main By-Product of Cellular Senescence, in Fluids

  • Sophia V. Rizou
  • Konstantinos Evangelou
  • Vassilios Myrianthopoulos
  • Iordanis Mourouzis
  • Sophia Havaki
  • Aikaterini Athanasiou
  • Panagiotis V. S. Vasileiou
  • Aggelos Margetis
  • Athanassios Kotsinas
  • Nikolaos G. Kastrinakis
  • Petros Sfikakis
  • Paul Townsend
  • Emmanuel Mikros
  • Constantinos Pantos
  • Vassilis G. GorgoulisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1896)


Lipofuscin accumulation is a hallmark of senescence. This nondegradable material aggregates in the cytoplasm of stressed or damaged cells due to metabolic imbalance associated with aging and age-related diseases. Indications of a soluble state of lipofuscin have also been provided, rendering the perspective of monitoring such processes via lipofuscin quantification in liquids intriguing. Therefore, the development of an accurate and reliable method is of paramount importance. Currently available assays are characterized by inherent pitfalls which demote their credibility. We herein describe a simple, highly specific and sensitive protocol for measuring lipofuscin levels in any type of liquid. The current method represents an evolution of a previously described assay, developed for in vitro and in vivo senescent cell recognition that exploits a newly synthesized Sudan Black-B analog (GL13). Analysis of human clinical samples with the modified protocol provided strong evidence of its usefulness for the exposure and surveillance of age-related conditions.

Key words

Lipofuscin GL13 (SenTraGorSenescence Biological fluids Aging Age-related diseases 



We would like to thank Dr. Alexandros Papalampros and Dr. Dimitrios Papadopoulos for providing material for this investigation. This work was financially supported by the “SYNTRAIN” ITN Horizon 2020 Grant No 722729, the NKUA SARG grants 70/3/12128, 70/3/8916, 70/3/1135 and the Welfare Foundation for Social & Cultural Sciences (KIKPE) Greece.

Conflict of interest:

The authors wish to declare no conflict of interest.

Patent pending: UK Patent Application No GB1803531.1.


  1. 1.
    Georgakopoulou EA, Tsimaratou K, Evangelou K et al (2013) Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging (Albany NY) 5:37–50CrossRefGoogle Scholar
  2. 2.
    Evangelou K, Gorgoulis VG (2017) Sudan Black B, The specific histochemical stain for lipofuscin: a novel method to detect senescent cells. Methods Mol Biol 1534:111–119CrossRefGoogle Scholar
  3. 3.
    Bartkova J, Rezaei N, Liontos M et al (2006) Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444:633–637CrossRefGoogle Scholar
  4. 4.
    Gorgoulis VG, Halazonetis TD (2010) Oncogene-induced senescence: the bright and dark side of the response. Curr Opin Cell Biol 22:816–827CrossRefGoogle Scholar
  5. 5.
    Halazonetis TD, Gorgoulis VG, Bartek J (2008) An oncogene-induced DNA damage model for cancer development. Science 319:1352–1355CrossRefGoogle Scholar
  6. 6.
    Herbig U, Ferreira M, Condel L et al (2006) Cellular senescence in aging primates. Science 311:1257CrossRefGoogle Scholar
  7. 7.
    Lopez-Otin C, Blasco MA, Partridge L et al (2013) The hallmarks of aging. Cell 153:1194–1217CrossRefGoogle Scholar
  8. 8.
    Evangelou K, Lougiakis N, Rizou SV et al (2017) Robust, universal biomarker assay to detect senescent cells in biological specimens. Aging Cell 16:192–197CrossRefGoogle Scholar
  9. 9.
    Galanos P, Vougas K, Walter D et al (2016) Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol 18:777–789CrossRefGoogle Scholar
  10. 10.
    Komseli ES, Pateras IS, Krejsgaard T et al (2018) A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence. BMC Genomics 19:37CrossRefGoogle Scholar
  11. 11.
    Barbouti A, Evangelou K, Pateras IS et al (2018) In situ evidence of cellular senescence in Thymic Epithelial Cells (TECs) during human thymic involution. Mech Ageing Dev. pii:S0047-6374(17)30300-7Google Scholar
  12. 12.
    Ivy G, Kanai S, Ohta M et al (1988) Lipofuscin-like substances accumulate rapidly in brain, retina and internal organs with cysteine protease inhibition. Adv Exp Med Biol 266:31–45Google Scholar
  13. 13.
    Ivy G, Roopsingh R, Kanai S et al (1996) Leupeptin causes an accumulation of lipofuscin-like substances and other signs of aging in kidneys of young rats: further evidence for the protease inhibitor model of aging. Ann N Y Acad Sci 786:12–23CrossRefGoogle Scholar
  14. 14.
    Brunk UT, Terman A (2002) Lipofuscin: mechanisms of age-related accumulation and influence on cell function. Free Radic Biol Med 33:611–619CrossRefGoogle Scholar
  15. 15.
    Terman A, Gustafsson B, Brunk UT (2006) The lysosomal-mitochondrial axis theory of postmitotic aging and cell death. Chem Biol Interact 163:29–37CrossRefGoogle Scholar
  16. 16.
    Terman A, Kurz T, Navratil M et al (2010) Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging. Antioxid Redox Signal 12(4):503–535CrossRefGoogle Scholar
  17. 17.
    Höhn A, Grune T (2013) Lipofuscin: formation, effects and role of macroautophagy. Redox Biol 19(1):140–144CrossRefGoogle Scholar
  18. 18.
    Jung T, Höhn A, Grune T (2014) The proteasome and the degradation of oxidized proteins: partII protein oxidation and proteasomal degradation. Redox Biol 2:99–104CrossRefGoogle Scholar
  19. 19.
    König J, Ott C, Hugo M et al (2017) Mitochondrial contribution to lipofuscin formation. Redox Biol 11:673–681CrossRefGoogle Scholar
  20. 20.
    Korolchuk VI, Miwa S, Carroll B et al (2017) Mitochondria in cell senescence: is mitophagy the weakest link? EBioMedicine 21:7–13CrossRefGoogle Scholar
  21. 21.
    Gaspar J, Mathieu J, Alvarez PJJ (2016) A rapid platform to generate lipofuscin and screen therapeutic drugs for efficacy in lipofuscin removal. Mater Meth Technol 10:1–9 ISSN 1314-7269Google Scholar
  22. 22.
    Sheehy MR (2002) A flow-cytometric method for quantification of neurolipofuscin and comparison with existing histological and biochemical approaches. Arch Gerontol Geriatr 34:233–248CrossRefGoogle Scholar
  23. 23.
    Di Guardo G (2015) Lipofuscin, lipofuscin-like pigments and autofluorescence. Eur J Histochem 59:2485CrossRefGoogle Scholar
  24. 24.
    Seehafer SS, Pearce DA (2006) You say lipofuscin, we say ceroid: defining autofluorescent storage material. Neurobiol Aging 27:576–588CrossRefGoogle Scholar
  25. 25.
    Bluhm BA, Brey T (2001) Age determination in the Antarctic shrimp Notocrangon antarcticus (Crustacea: Decapoda), using the autofluorescent pigment lipofuscin. Mar Biol 138:247–257CrossRefGoogle Scholar
  26. 26.
    Bluhm BA, Brey T, Klages M (2001) The autofluorescent age pigment lipofuscin: key to age, growth and productivity of the Antarctic amphipod Waldeckia obesa (Chevreux, 1905). J Exp Mar Bio Ecol 258:215–235CrossRefGoogle Scholar
  27. 27.
    Cassidy KM (2008) Use of extractable lipofuscin as an age biomarker to determine age structure of ghost shrimp (Neotrypaea californiensis) populations in west coast estuaries. Dissertation. Oregon State UniversityGoogle Scholar
  28. 28.
    Harvey HR, Secor DH, Ju SJ (2008) The use of extractable lipofuscin for age determination of crustaceans: reply to Sheehy. Mar Ecol Prog Ser 353:307–311CrossRefGoogle Scholar
  29. 29.
    Puckett JB, Secor DΗ, Ju SJ (2008) Validation and application of lipofuscin-based age determination for Chesapeake Bay Blue Crabs Callinectes sapidus. Trans Am Fish Soc 137:1637–1649CrossRefGoogle Scholar
  30. 30.
    Sparrow JR, Nakanishi K, Parish CA (2000) The lipofuscin fluorophore A2E mediates blue light induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 41:1981–1989PubMedGoogle Scholar
  31. 31.
    Meredith GE, Totterdell S, Petroske E et al (2002) Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson’s disease. Brain Res 956:156–165CrossRefGoogle Scholar
  32. 32.
    Moreira PI, Siedlak SL, Wang X et al (2007) Increased autophagic degradation of mitochondria in Alzheimer disease. Autophagy 3:614–615CrossRefGoogle Scholar
  33. 33.
    Nozynski J, Zakliczynski M, Konecka-Mrowka D et al (2013) Advanced glycation end products and lipofuscin deposits share the same location in cardiocytes of the failing heart. Exp Gerontol 48:223–228CrossRefGoogle Scholar
  34. 34.
    Beregi E, Regius O (1983) Lipofuscin in lymphocytes and plasma cells in aging. Arch Gerontol Geriatr 2:229–235CrossRefGoogle Scholar
  35. 35.
    Skoumalová A, Mádlová P, Topinková E (2012) End products of lipid peroxidation in erythrocyte membranes in Alzheimer's disease. Cell Biochem Funct 30:205–210CrossRefGoogle Scholar
  36. 36.
    Feng FK, E LL, Kong XP et al (2015) Lipofuscin in saliva and plasma and its association with age in healthy adults. Aging Clin Exp Res 27:573–580CrossRefGoogle Scholar
  37. 37.
    Wu CX, Wei XB (2006) Influence of effective parts of Zingiber officinale on senium of rats resulting from high fat die. J Shandong Med Coll 3:010Google Scholar
  38. 38.
    Hegedus ZL, Frank HA, Steinman TI et al (1988) Elevated levels of plasma lipofuscins in patients with chronic renal failure. Arch Int Physiol Biochim 96:211–221PubMedGoogle Scholar
  39. 39.
    Tsuchida M, Miura T, Mizutani K et al (1985) Fluorescent substances in mouse and human sera as a parameter of in vivo lipid peroxidation. Biochim Biophys Acta 834:196–204CrossRefGoogle Scholar
  40. 40.
    Roumen RM, Hendriks T, de Man BM et al (1994) Serum lipofuscin as a prognostic indicator of adult respiratory distress syndrome and multiple organ failure. Br J Surg 81:1300–1305CrossRefGoogle Scholar
  41. 41.
    Sutherland WH, Williams MJ, de Jong SA (2007) Plasma protein lipofuscin-like fluorophores in men with coronary artery disease treated with statins. Arch Med Res 38:757–763CrossRefGoogle Scholar
  42. 42.
    Skoumalová A, Ivica J, Šantorová P et al (2011) The lipid peroxidation products as possible markers of Alzheimer's disease in blood. Exp Gerontol 46:38–42CrossRefGoogle Scholar
  43. 43.
    Kuznetsov A, Frorip A, Maiste A et al (2015) Visible auto-fluorescence in biological fluids as biomarker of pathological processes and new monitoring tool. J Innov Opt Health Sci 8(3):1541003–1541009CrossRefGoogle Scholar
  44. 44.
    Tomečková V (2016) Monitoring of heart ischemia in blood serum. Spectral Anal Rev 4:11–22CrossRefGoogle Scholar
  45. 45.
    Chmátalová Z, Vyhnálek M, Laczó J et al (2016) Analysis of lipophilic fluorescent products in blood of Alzheimer's disease patients. J Cell Mol Med 20:1367–1372CrossRefGoogle Scholar
  46. 46.
    Madhuri S, Vengadesan N, Aruna P et al (2003) Native fluorescence spectroscopy of blood plasma in the characterization of oral malignance. Photochem Photobiol 78:197–204CrossRefGoogle Scholar
  47. 47.
    Sheehy MR, Roberts BE (1991) An alternative explanation for anomalies in "soluble lipofuscin" fluorescence data from insects, crustaceans, and other aquatic species. Exp Gerontol 26:495–509CrossRefGoogle Scholar
  48. 48.
    Yin D (1996) Biochemical basis of lipofuscin, ceroid, and age pigment-like fluorophores. Free Radic Biol Med 21:871–888CrossRefGoogle Scholar
  49. 49.
    Mochizuki Y, Park MK, Mori T et al (1995) The difference in autofluorescence features of lipofuscin between brain and adrenal. Zool Sci 12:283–288CrossRefGoogle Scholar
  50. 50.
    Croce AC, Bottiroli G (2014) Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. Eur J Histochem 58:2461CrossRefGoogle Scholar
  51. 51.
    Rózanowska M, Pawlak A, Rózanowski B et al (2004) Age-related changes in the photoreactivity of retinal lipofuscin granules: role of chloroform-insoluble components. Invest Ophthalmol Vis Sci 45:1052–1060CrossRefGoogle Scholar
  52. 52.
    Otsuki J, Nagai Y, Matsuyama Y et al (2012) The influence of the redox state of follicular fluid albumin on the viability of aspirated human oocytes. Syst Biol Reprod Med 58:149–153CrossRefGoogle Scholar
  53. 53.
    Otsuki J, Nagai Y, Chiba K (2007) Lipofuscin bodies in human oocytes as an indicator of oocyte quality. J Assist Reprod Genet 24:263–270CrossRefGoogle Scholar
  54. 54.
    Siakotos AN, Watanabe I, Pennington K et al (1973) Procedures for the mass isolation of pure lipofuscins from normal human heart and liver. Biochem Med 7:25–38CrossRefGoogle Scholar
  55. 55.
    Chang J, Wang Y, Shao L et al (2016) Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat Med 22:78–83CrossRefGoogle Scholar
  56. 56.
    Sheehy MRJ (2008) Questioning the use of biochemical extraction to measure lipofuscin for age determination of crabs: comment on Ju et al. (1999, 2001). Mar Ecol Prog Ser 353:303–306CrossRefGoogle Scholar
  57. 57.
    Crowley CE, Gandy RL, Daly KL et al (2014) Problems associated with a lipofuscin extraction method used to age blue crabs Callinectes sapidus cultured in Florida, USA. Aquat Biol 21:85–92CrossRefGoogle Scholar
  58. 58.
    Manjunath S, Bola Sadashiva SR, Satyamoorthy K, et al (2014) Nature of autofluorescence in human serum albumin under its native, unfolding and digested forms. Proc SPIE 8935, advanced biomedical and clinical diagnostic systems. XII, 8935:893520Google Scholar
  59. 59.
    Wolfbeis SO, Leiner M (1985) Mapping of the total fluorescence of human blood serum as a new method for its characterization. Anal Chim Acta 167:203–215CrossRefGoogle Scholar
  60. 60.
    Hegedus ZL, Altschule MD, Frank HA et al (1985) Increase in plasma lipofuscin levels of stored blood. Crit Care Med 13:155–159CrossRefGoogle Scholar
  61. 61.
    Cequier-Sánchez E, Rodríguez C, Ravelo AG et al (2008) Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. J Agric Food Chem 56:4297–4303CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sophia V. Rizou
    • 1
  • Konstantinos Evangelou
    • 1
    • 2
  • Vassilios Myrianthopoulos
    • 3
    • 4
  • Iordanis Mourouzis
    • 5
  • Sophia Havaki
    • 1
  • Aikaterini Athanasiou
    • 6
  • Panagiotis V. S. Vasileiou
    • 1
  • Aggelos Margetis
    • 1
  • Athanassios Kotsinas
    • 1
  • Nikolaos G. Kastrinakis
    • 1
  • Petros Sfikakis
    • 7
  • Paul Townsend
    • 8
  • Emmanuel Mikros
    • 3
    • 4
  • Constantinos Pantos
    • 5
  • Vassilis G. Gorgoulis
    • 1
    • 8
    • 9
    • 10
    Email author
  1. 1.Molecular Carcinogenesis Group, Department of Histology and Embryology, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  2. 2.Department of Anatomy-Histology-Embryology, Medical SchoolUniversity of IoanninaIoanninaGreece
  3. 3.Division of Pharmaceutical Chemistry, School of PharmacyNational and Kapodistrian University of AthensAthensGreece
  4. 4.PharmaInformatics UnitAthena Research CenterAthensGreece
  5. 5.Department of Pharmacology, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  6. 6.Department of Obstetrics and GynecologyWeill Cornell MedicineNew YorkUSA
  7. 7.First Department of Propaedeutic Internal Medicine and Rheumatology Unit, Medical SchoolNational and Kapodistrian University of AthensAthensGreece
  8. 8.Faculty Institute for Cancer Sciences, Manchester Academic Health Sciences CentreUniversity of ManchesterManchesterUK
  9. 9.Biomedical Research FoundationAcademy of AthensAthensGreece
  10. 10.Center for New Biotechnologies and Precision MedicineMedical School, National and Kapodistrian University of AthensAthensGreece

Personalised recommendations