Skip to main content
SpringerLink
Log in
Book cover

Advanced Protocols in Oxidative Stress I pp 259–276Cite as

Paraoxonases (PON1, PON2, PON3) Analyses In Vitro and In Vivo in Relation to Cardiovascular Diseases

  • Protocol

Part of the book series: Methods In Molecular Biology ((MIMB,volume 477))

Abstract

Mammalian paraoxonases (PON1, PON2, PON3) are a unique family of calcium-dependent hydrolases, with enzymatic activities toward a broad range of substrates (lactones, thiolactones, carbonates, esters, phosphotriesters). Although PONs physiological substrates were not yet identified, some studies suggest that they could be some lactones, or some specific oxidized phospholipids, or products of both enzymatic and nonenzymatic oxidation of arachidonic and docosahexaenoic acid, as well as N-acyl-homoserine lactones (which are quorum-sensing signals of pathogenic bacteria). Since no endogenous substrates for PONs activity determination are available yet, synthetic substrates such as paraoxon, phenyl acetate, and several lactones are used for PONs activity assays. All three members of the PON family (PON 1/2/3) were shown to protect from atherosclerosis development. Their anti-atherogenic biological activities were studied in vitro using serum or cell cultures, and also in vivo, using PON 1/2/3 knockout or transgenic mice, as well as humans – healthy volunteers and atherosclerotic patients (diabetics, hypercholesterolemics, and hypertensives).

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Aviram M, Rosenblat M (2004) Paraoxonases 1, 2 and 3, oxidative stress, and macrophage foam cell formation during atherosclerosis development. Free. Radic. Biol. Chem. 37:1304–1316.

    Article  CAS  Google Scholar 

  2. Fuhrman B, Volkova N, Aviram M (2005) Paraoxonase 1 (PON1) is present in postprandial chylomicrons. Atherosclerosis 180:55–61.

    Article  CAS  PubMed  Google Scholar 

  3. Khersonsky O, Tawfik DS (2005) Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 44:6371–6382.

    Article  CAS  PubMed  Google Scholar 

  4. Gaidukov L, Tawfik DS (2005) High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with apoA-I. Biochemistry 44:11843– 11854.

    Article  CAS  PubMed  Google Scholar 

  5. Harel M, Aharoni A, Gaidukov L, Brumshtein B, Khersonsky O, Meged R, Dvir H, Ravelli RBG, McCarthy A, Toker L, Silman I, Sussman JL, Tawfic DS (2004) Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. Nat. Struc. Mol. Biol. 11:412–419.

    Article  CAS  Google Scholar 

  6. Rozenberg O, Rosenblat M, Coleman R, Shih DM, Aviram M (2003) Paraoxonase (PON1)-deficiency is associated with increased macrophage oxidative stress: studies in PON1-knockout mice. Free. Radic. Biol. Med. 34:774–784.

    Article  CAS  PubMed  Google Scholar 

  7. Rozenberg O, Shih DM, Aviram M (2005) Paraoxonase 1 (PON1) attenuates macrophage oxidative status: studies in PON1 transfected cells and in PON1 transgenic mice. Atherosclerosis 181:9–18.

    Article  CAS  PubMed  Google Scholar 

  8. Aviram M, Rosenblat M, Bisgaier CL, Newton RS, Primo-Parmo SL, La Du BN (1998) Paraoxonase inhibits high-density lipoprotein oxidation and preserves its functions. A possible peroxidative role for paraoxonase. J. Clin. Invest. 101:1581–1590.

    Article  CAS  PubMed  Google Scholar 

  9. Aviram M, Hardak E, Vaya J, Mahmood S, Milo S, Hoffman A, Billicke S, Daraganov D, Rosenblat M (2000) Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation 101:2510–2517.

    CAS  PubMed  Google Scholar 

  10. Rozenberg O, Shih DM, Aviram M (2003) Human serum paraoxonase (PON1) decreases macrophage cholesterol biosynthesis: a possible role for its phodpholipase-A2 activity and lysophosphatidylcholine formation. Arterioscler. Thromb. Vasc. Biol. 23:461–467.

    Article  CAS  PubMed  Google Scholar 

  11. Rosenblat M, Vaya J, Shih DM, Aviram M (2005) Paraoxonase 1 (PON1) enhances HDL-mediated macrophage cholesterol efflux via the ABCA1 transporter in association with increased HDL binding to the cells: a possible role for lysophosphatidylcholine. Atherosclerosis 179:69–77.

    Article  CAS  PubMed  Google Scholar 

  12. Rosenblat M, Gaidukov L, Khersonsky O, Vaya J, Oren R, Tawfik DS (2006) The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux. J. Biol. Chem. 281:7657–7665.

    Article  CAS  PubMed  Google Scholar 

  13. Gaidukov L, Rosenblat M, Aviram M, Tawfik DS (2006) The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux. J. Lipid. Res. 47:2492–2502.

    Article  CAS  PubMed  Google Scholar 

  14. Rosenblat M, Draganov D, Watson CE, Bisgaier CL, La Du BN, Aviram M (2003) Mouse macrophage paraoxonase 2 (PON2) activity is increased whereas cellular PON3 activity is decreased under oxidative stress. Arterioscler. Thromb. Vasc. Biol. 23:468–474.

    Article  CAS  PubMed  Google Scholar 

  15. Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S,, Grijalva VR, Navab M, Fogelman AM, Reddy ST (2001) Paraoxonase-2 is an ubiquitously expressed protein with antioxidant properties, and is capable of preventing cell-mediated oxidative modification of low-density lipoprotein. J. Biol. Chem. 276:44444–44449.

    Article  CAS  PubMed  Google Scholar 

  16. Ng CL, Hama SY, Bourquard N, Navab M, Reddy ST (2006) Adenovirus mediated expression of human paraoxonase 2 protects against the development of atherosclerosis in apolipoprotein E-deficient mice. Mol. Genet. Metab. 89:368–373.

    Article  CAS  PubMed  Google Scholar 

  17. Ng CJ, Bourquard N, Grijalva V, Hama S, Shih SM, Navab M, Fogelman AM, Lusis AJ (2006) Paraoxonase-2-deficiency aggravates atherosclerosis in mice despite lower apolipoprotein-B-containing lipoproteins: anti-atherogenic role for paraoxonase 2. J. Biol. Chem. 281:29491–29500.

    Article  CAS  PubMed  Google Scholar 

  18. Horke S, Witte I, Wilgenbus P, Kruger M, Strand D, Forstermann U (2007) Paraoxonase-2 reduces oxidative stress in vascular cells and decreases endoplasmic reticulum stress-induced caspase activation. Circulation 115:2055–2064.

    Article  CAS  PubMed  Google Scholar 

  19. Shiner M, Fuhrman B, Aviram M (2006) A biphasic U-shape effect of cellular oxidative stress on the macrophage anti-oxidant paraoxonase 2 (PON2) enzymatic activity. Biochem. Biophys. Res. Commun. 349:1094–1099.

    Article  CAS  PubMed  Google Scholar 

  20. Shiner M, Fuhrman B, Aviram M (2004) Paraoxonase 2 (PON2) expression is upregulated via a reduced nicotinamide-adenine-dinucleotide-phosphate (NADPH)-oxidase-dependent mechanism during monocytes differentiation into macrophages. Free. Radical. Biol. Med. 37:2052–2063.

    Article  CAS  Google Scholar 

  21. Shiner M, Fuhrman B, Aviram M (2007) Macrophage paraoxonase 2 (PON2) expression is up-regulated by pomegranate juice phenolic anti-oxidants via PPARgamma and AP-1 pathway activation. Atherosclerosis. 195:313–321.

    Article  CAS  PubMed  Google Scholar 

  22. Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, Shih DM, Lusis AJ, Navab M, Fogelman AM (2001) Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler. Thromb. Vasc. Biol. 21:542–547.

    CAS  PubMed  Google Scholar 

  23. Draganov D, Stetson PL, Watson C, Billecke S, La Du BN (2000) Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. J. Biol. Chem. 275:33435–33442.

    Article  CAS  PubMed  Google Scholar 

  24. Shih DM, Xia YR, Wang XP, Wang SS, Bourquard N, Fogelman AM, Lusis AJ, Reddy ST (2007) Decreased obesity and atherosclerosis in human paraoxonase 3 transgenic mice. Circ. Res. 100:1200–1207.

    Article  CAS  PubMed  Google Scholar 

  25. Ng. CJ, Bourquard N, Hama SY, Shih D, Grijalva VR, Navab M, Fogelman AM, Reddy ST (2007) Adenovirus-mediated expression of human paraoxonase 3 protects against the progression of atherosclerosis in apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 27:1368–1374.

    Article  CAS  PubMed  Google Scholar 

  26. Gan KN, Smolen A, Eckerson HW, La Du BN (1991) Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug. Metab. Dispos. 19:100–106.

    CAS  PubMed  Google Scholar 

  27. Aharoni A, Gaidukov L, Yagur S, Toker L, Silman I, Tawfik DS (2004) Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc. Natl. Acad. Sci. USA 101:482–487.

    Article  CAS  PubMed  Google Scholar 

  28. Khersonsky O, Tawfik DS (2005) Chromogenic and fluorogenic assays for the lactonase activity of serum paraoxonases (PONs). ChemBiochem 7:49–53.

    Article  Google Scholar 

  29. Garin MCB, Abbott C, Messmer S, Mackness M, Durrington P, Pometta D, James RW (1994) Quantification of human serum paraoxonase by enzyme-linked immunoassay: population differences in protein concentrations. Biochem J. 304:549–554.

    Google Scholar 

  30. Eckerson HW, Wyte CM, La Du BN (1983) The human serum paraoxonase/arylesterase polymorphism. Am. J. Hun. Genet. 35: 1126–1138.

    CAS  Google Scholar 

  31. Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol. 52:302–310.

    Article  CAS  PubMed  Google Scholar 

  32. El-Saadani M, Esterbauer N, El-Sayed M, Goher M, Nassar AY, Jurgens G (1989) Spectrophotometric assay for lipid peroxides in serum lipoproteins using commercially available reagent. J. Lipid Res. 30:627–630.

    CAS  PubMed  Google Scholar 

  33. Aviram M (1983) Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients. Biochem. Med. 30:111–118.

    Article  CAS  PubMed  Google Scholar 

  34. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.

    CAS  PubMed  Google Scholar 

  35. Dobrian AD, Lazar V, Sinescu C, Mincu D, Simionescu M (2000) Diabetic state induces lipid loading and altered expression and secretion of lipoprotein lipase in human monocytes-derived macrophages. Atherosclerosis 153:191–201.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lipid Research Laboratory, Technion Faculty of Medicine and Department of Laboratory Medicine, Rambam Medical Center, Haifa, Israel

    Michael Aviram

  2. The Lipid Research Laboratory, Rambam Medical Center, Haifa, Israel

    Mira Rosenblat

Authors
  1. Michael Aviram
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mira Rosenblat
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University at Buffalo, Buffalo, NY, USA

    Donald Armstrong

  2. Showa University School of Medicine, Tokyo, USA

    Donald Armstrong

  3. University of Florida, Gainesville, FL, USA

    Donald Armstrong

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Aviram, M., Rosenblat, M. (2008). Paraoxonases (PON1, PON2, PON3) Analyses In Vitro and In Vivo in Relation to Cardiovascular Diseases. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress I. Methods In Molecular Biology, vol 477. Humana Press. https://doi.org/10.1007/978-1-60327-517-0_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-517-0_20

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-218-6

  • Online ISBN: 978-1-60327-517-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation