FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / BMC Res Notes / Investigation of the solubility and the potentials for purif...
BMC research notes2013; 6; 152; doi: 10.1186/1756-0500-6-152

Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum–a pilot study.

Abstract: Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for purification of equine SAA based on biochemical properties.Freeze dried equine acute phase serum was dissolved in 70% 2-propanol, 8 M urea, and milli-Q water, respectively. Supercritical fluid extraction (SFE), size-exclusive chromatography (FPLC-SEC), and preparative isoelectric focusing (IEF) were performed in the attempt to purify. Immunostaining of IEF blots were used for isoform-specific detection of SAA in the preparations and purity was assessed by silverstained SDS-PAGE. Results: SAA was soluble in 70% 2-propanol, 8 M urea and Milli-Q water. SAA was not separated in the lipophilic or ampipathic fractions following SFE. SAA was included in a FPLC-SEC-fraction of 237 kDa, despite the molecular weight known to be much smaller, suggesting binding to other serum constituents. SAA precipitated following separation of other serum proteins by preparative IEF. Conclusions: No effective purification of SAA was achieved in the present study, but findings important for future investigations were made. The study suggested that SAA is not exclusively hydrophobic, but appears less hydrophobic when interacting with other serum components. These results suggest more complex aspects of solubility than previously believed, and indicate potentials for purification of native SAA.
Get Full Text
Publication Date: 2013-04-16 PubMed ID: 23590853PubMed Central: PMC3637563DOI: 10.1186/1756-0500-6-152Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study aimed to investigate the solubility (ability to dissolve) and potential purification methods of Serum Amyloid A (SAA), a marker of systemic inflammation in horses, from equine acute phase serum (a substance produced during an early stage of disease).

Purpose

  • This research was conducted to improve the accuracy of measurements detecting systemic inflammation in horses, particularly by using a purified version of SAA, a protein that responds during the early phase of inflammation or infection.
  • Currently, heterologous assays not specific to horses are used making measurements of equine SAA less accurate.
  • Understanding the biochemical properties of SAA may allow for the development of native species-specific calibration material, improving assay performance.

Methodology

  • The team used freeze-dried equine acute phase serum that was dissolved using 70% 2-propanol, 8 M urea, and milli-Q water.
  • They tried to purify SAA using supercritical fluid extraction (SFE), size-exclusion chromatography (also known as FPLC-SEC), and preparative isoelectric focusing (IEF).
  • They then used immunostaining of IEF blots for isoform-specific detection of SAA in the preparations and assessed purity using silver-stained SDS-PAGE, a common technique for protein analysis.

Results

  • The researchers found that SAA could dissolve in 70% 2-propanol, 8 M urea, and milli-Q water.
  • However, they also found that SAA didn’t separate out in lipophilic or amphipathic fractions after SFE.
  • During size-exclusion chromatography, SAA was part of a 237 kDa fraction, despite its molecular weight being significantly lower, suggesting it binds to other serum constituents.
  • During preparative IEF, other serum proteins separated but SAA precipitated.

Conclusions

  • Purification of SAA was not achieved in this study, but the research uncovered important findings for future investigations.
  • The study indicated that the properties of SAA are not only hydrophobic as previously believed, but also appear less hydrophobic (water repelling) when interacting with other serum components.
  • The newfound complexities of SAA’s solubility could offer potential pathways for its purification in future studies which would enhance its use as a diagnostic tool in equine medicine.

Cite This Article

APA
Christensen MB, Sørensen JC, Jacobsen S, Kjelgaard-Hansen M. (2013). Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum–a pilot study. BMC Res Notes, 6, 152. https://doi.org/10.1186/1756-0500-6-152

Publication

BMC research notes
ISSN: 1756-0500
NlmUniqueID: 101462768
Country: England
Language: English
Volume: 6
Pages: 152

Researcher Affiliations

Christensen, Michelle B
  • Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Groennegaardsvej 3, ground floor, Frederiksberg C 1870, Denmark. mic@sund.ku.dk
Sørensen, Jens Christian
    Jacobsen, Stine
      Kjelgaard-Hansen, Mads

        MeSH Terms

        • Acute-Phase Reaction / blood
        • Acute-Phase Reaction / veterinary
        • Animals
        • Calibration
        • Chromatography, Gel
        • Horses
        • Hydrophobic and Hydrophilic Interactions
        • Inflammation / blood
        • Inflammation / veterinary
        • Isoelectric Focusing
        • Pilot Projects
        • Serum Amyloid A Protein / isolation & purification
        • Silver Staining
        • Solubility

        References

        This article includes 38 references
        1. Eckersall PD, Bell R. Acute phase proteins: Biomarkers of infection and inflammation in veterinary medicine.. Vet J 2010 Jul;185(1):23-7.
          doi: 10.1016/j.tvjl.2010.04.009pubmed: 20621712google scholar: lookup
        2. Kjelgaard-Hansen M, Jacobsen S. Assay validation and diagnostic applications of major acute-phase protein testing in companion animals.. Clin Lab Med 2011 Mar;31(1):51-70.
          doi: 10.1016/j.cll.2010.10.002pubmed: 21295722google scholar: lookup
        3. Jacobsen S, Andersen P. The acute phase protein serum amyloid A (SAA) as a marker of inflammation in horses.. Eq Vet Education 2007;19:38–46.
        4. Jacobsen S, Jensen JC, Frei S, Jensen AL, Thoefner MB. Use of serum amyloid A and other acute phase reactants to monitor the inflammatory response after castration in horses: a field study.. Equine Vet J 2005 Nov;37(6):552-6.
          pubmed: 16295934doi: 10.2746/042516405775314853google scholar: lookup
        5. Hultén C, Sletten K, Foyn Bruun C, Marhaug G. The acute phase serum amyloid A protein (SAA) in the horse: isolation and characterization of three isoforms.. Vet Immunol Immunopathol 1997 Jul;57(3-4):215-27.
          doi: 10.1016/S0165-2427(97)00021-4pubmed: 9261960google scholar: lookup
        6. Jacobsen S, Niewold TA, Halling-Thomsen M, Nanni S, Olsen E, Lindegaard C, Andersen PH. Serum amyloid A isoforms in serum and synovial fluid in horses with lipopolysaccharide-induced arthritis.. Vet Immunol Immunopathol 2006 Apr 15;110(3-4):325-30.
          doi: 10.1016/j.vetimm.2005.10.012pubmed: 16337010google scholar: lookup
        7. Bausserman LL, Herbert PN, Forte T, Klausner RD, McAdam KP, Osborne JC Jr, Rosseneu M. Interaction of the serum amyloid A proteins with phospholipid.. J Biol Chem 1983 Sep 10;258(17):10681-8.
          pubmed: 6411718
        8. Benditt EP, Eriksen N. Amyloid protein SAA is associated with high density lipoprotein from human serum.. Proc Natl Acad Sci U S A 1977 Sep;74(9):4025-8.
          doi: 10.1073/pnas.74.9.4025pmc: PMC431828pubmed: 198813google scholar: lookup
        9. Carpintero R, Piñeiro M, Andrés M, Iturralde M, Alava MA, Heegaard PM, Jobert JL, Madec F, Lampreave F. The concentration of apolipoprotein A-I decreases during experimentally induced acute-phase processes in pigs.. Infect Immun 2005 May;73(5):3184-7.
          doi: 10.1128/IAI.73.5.3184-3187.2005pmc: PMC1087351pubmed: 15845530google scholar: lookup
        10. Ducret A, Bruun CF, Bures EJ, Marhaug G, Husby G, Aebersold R. Characterization of human serum amyloid A protein isoforms separated by two-dimensional electrophoresis by liquid chromatography/electrospray ionization tandem mass spectrometry.. Electrophoresis 1996 May;17(5):866-76.
          doi: 10.1002/elps.1150170508pubmed: 8783012google scholar: lookup
        11. Marhaug G, Husby G. Characterization of human amyloid-related protein SAA as a polymorphic protein: association with albumin and prealbumin in serum.. Clin Exp Immunol 1981 Jul;45(1):97-106.
          pmc: PMC1537261pubmed: 7307348
        12. Hocke G, Kaffarnik H, Münscher G, Steinmetz A. Purification of human serum amyloid A by anion-exchange fast protein liquid chromatography.. J Chromatogr 1990 Mar 16;526(1):203-9.
          pubmed: 2341533doi: 10.1016/s0378-4347(00)82499-8google scholar: lookup
        13. Ham D, Karska-Wysocki B. Purification and separation of hydrophobic serum amyloid A precursor isoforms by a one-step preparative method.. J Immunol Methods 2005 Aug;303(1-2):11-8.
          doi: 10.1016/j.jim.2005.05.013pubmed: 16039662google scholar: lookup
        14. Hultén C, Tulamo RM, Suominen MM, Burvall K, Marhaug G, Forsberg M. A non-competitive chemiluminescence enzyme immunoassay for the equine acute phase protein serum amyloid A (SAA) -- a clinically useful inflammatory marker in the horse.. Vet Immunol Immunopathol 1999 May;68(2-4):267-81.
          doi: 10.1016/S0165-2427(99)00027-6pubmed: 10438325google scholar: lookup
        15. Peeters H. The challenge of the lipoprotein molecule. In: Proceedings of the NATO Advanced Study Institute on the Lipoprotein Molecule: 8-20 May 1977; Belgium. Peeters H, editor. New York: Plenum Press; 1978; pp. 3–6.
        16. Yamada T. Serum amyloid A (SAA): a concise review of biology, assay methods and clinical usefulness.. Clin Chem Lab Med 1999 Apr;37(4):381-8.
          pubmed: 10369107doi: 10.1515/cclm.1999.063google scholar: lookup
        17. HAVEL RJ, EDER HA, BRAGDON JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.. J Clin Invest 1955 Sep;34(9):1345-53.
          doi: 10.1172/JCI103182pmc: PMC438705pubmed: 13252080google scholar: lookup
        18. Raynes JG, McAdam KP. Purification of serum amyloid A and other high density apolipoproteins by hydrophobic interaction chromatography.. Anal Biochem 1988 Aug 15;173(1):116-24.
          doi: 10.1016/0003-2697(88)90168-6pubmed: 3142296google scholar: lookup
        19. Soler L, Gutiérrez A, Martínez-Subiela S, Cerón JJ. Development and validation of a novel competitive ELISA for the detection of serum amyloid A in pigs.. Vet J 2011 Nov;190(2):e7-e11.
          doi: 10.1016/j.tvjl.2011.02.016pubmed: 21421332google scholar: lookup
        20. Hansen AE, Schaap MK, Kjelgaard-Hansen M. Evaluation of a commercially available human serum amyloid A (SAA) turbidimetric immunoassay for determination of feline SAA concentration.. Vet Res Commun 2006 Nov;30(8):863-72.
          doi: 10.1007/s11259-006-3373-6pmc: PMC7089358pubmed: 17139536google scholar: lookup
        21. Pepys MB, Baltz ML, Tennent GA, Kent J, Ousey J, Rossdale PD. Serum amyloid A protein (SAA) in horses: objective measurement of the acute phase response.. Equine Vet J 1989 Mar;21(2):106-9.
          doi: 10.1111/j.2042-3306.1989.tb02108.xpubmed: 2539996google scholar: lookup
        22. Christensen M, Jacobsen S, Ichiyanagi T, Kjelgaard-Hansen M. Evaluation of an automated assay based on monoclonal anti-human serum amyloid A (SAA) antibodies for measurement of canine, feline, and equine SAA.. Vet J 2012 Dec;194(3):332-7.
          doi: 10.1016/j.tvjl.2012.05.007pubmed: 22704135google scholar: lookup
        23. Jacobsen S, Kjelgaard-Hansen M. Evaluation of a commercially available apparatus for measuring the acute phase protein serum amyloid A in horses.. Vet Rec 2008 Sep 13;163(11):327-30.
          doi: 10.1136/vr.163.11.327pubmed: 18791207google scholar: lookup
        24. Jacobsen S, Kjelgaard-Hansen M, Hagbard Petersen H, Jensen AL. Evaluation of a commercially available human serum amyloid A (SAA) turbidometric immunoassay for determination of equine SAA concentrations.. Vet J 2006 Sep;172(2):315-9.
          doi: 10.1016/j.tvjl.2005.04.021pubmed: 15950503google scholar: lookup
        25. Eckersall PD, Duthie S, Toussaint MJ, Gruys E, Heegaard P, Alava M, Lipperheide C, Madec F. Standardization of diagnostic assays for animal acute phase proteins.. Adv Vet Med 1999;41:643-55.
          pubmed: 9890051doi: 10.1016/s0065-3519(99)80050-0google scholar: lookup
        26. Kjelgaard-Hansen M. Comments on measurement of C-reactive protein in dogs.. Vet Clin Pathol 2010 Dec;39(4):402-3; author reply 404.
          doi: 10.1111/j.1939-165X.2010.00276.xpubmed: 21198730google scholar: lookup
        27. Malle E, Hess H, Münscher G, Knipping G, Steinmetz A. Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing.. Electrophoresis 1992 Jul;13(7):422-8.
          doi: 10.1002/elps.1150130189pubmed: 1425555google scholar: lookup
        28. Strachan AF, de Beer FC, van der Westhuyzen DR, Coetzee GA. Identification of three isoform patterns of human serum amyloid A protein.. Biochem J 1988 Feb 15;250(1):203-7.
          pmc: PMC1148833pubmed: 3355511doi: 10.1042/bj2500203google scholar: lookup
        29. Soler L, Molenaar A, Merola N, Eckersall PD, Gutiérrez A, Cerón JJ, Mulero V, Niewold TA. Why working with porcine circulating serum amyloid A is a pig of a job.. J Theor Biol 2013 Jan 21;317:119-25.
          pubmed: 23073471doi: 10.1016/j.jtbi.2012.10.011google scholar: lookup
        30. Jacobsen S, Niewold TA, Kornalijnslijper E, Toussaint MJ, Gruys E. Kinetics of local and systemic isoforms of serum amyloid A in bovine mastitic milk.. Vet Immunol Immunopathol 2005 Mar 10;104(1-2):21-31.
          doi: 10.1016/j.vetimm.2004.09.031pubmed: 15661328google scholar: lookup
        31. Kjelgaard-Hansen M, Christensen MB, Lee MH, Jensen AL, Jacobsen S. Serum amyloid A isoforms in serum and synovial fluid from spontaneously diseased dogs with joint diseases or other conditions.. Vet Immunol Immunopathol 2007 Jun 15;117(3-4):296-301.
          doi: 10.1016/j.vetimm.2007.03.008pubmed: 17451811google scholar: lookup
        32. Chevallet M, Luche S, Rabilloud T. Silver staining of proteins in polyacrylamide gels.. Nat Protoc 2006;1(4):1852-8.
          doi: 10.1038/nprot.2006.288pmc: PMC1971133pubmed: 17487168google scholar: lookup
        33. Righetti P, Wenisch E, Faupel M. Preparative Protein-Purification in a Multi-Compartment Electrolyzer with Immobiline Membranes. J Chromatogr 1989;475:293–309.
          doi: 10.1016/S0021-9673(01)89684-9google scholar: lookup
        34. Righetti PG, Bossi A, Wenisch E, Orsini G. Protein purification in multicompartment electrolyzers with isoelectric membranes.. J Chromatogr B Biomed Sci Appl 1997 Oct 10;699(1-2):105-15.
          doi: 10.1016/S0378-4347(97)00156-4pubmed: 9392371google scholar: lookup
        35. Strachan AF, Shephard EG, Bellstedt DU, Coetzee GA, van der Westhuyzen DR, de Beer FC. Human serum amyloid A protein. Behaviour in aqueous and urea-containing solutions and antibody production.. Biochem J 1989 Oct 15;263(2):365-70.
          pmc: PMC1133438pubmed: 2597108doi: 10.1042/bj2630365google scholar: lookup
        36. Klime J, Sochor J, Kríz J. A study of the conditions of the supercritical fluid extraction in the analysis of selected anti-inflammatory drugs in plasma.. Farmaco 2002 Feb;57(2):117-22.
          doi: 10.1016/S0014-827X(01)01182-Xpubmed: 11902653google scholar: lookup
        37. Chaiyasut C, Tsuda T. Isoelectric points estimation of proteins by electroosmotic flow: pH relationship using physically adsorbed proteins on silica gel. Chromatography 2001;22:91–95.
        38. Nunokawa Y. Isolation, characterization and quantitative analysis of serum amyloid A protein from horses. Jap J Vet Res 1992;40:49.

        Citations

        This article has been cited 2 times.
        1. Jacobsen S, Vinther AM, Kjelgaard-Hansen M, Nielsen LN. Validation of an equine serum amyloid A assay with an unusually broad working range. BMC Vet Res 2019 Dec 19;15(1):462.
          doi: 10.1186/s12917-019-2211-3pubmed: 31856804google scholar: lookup
        2. Souto PC, Santos MR, Orozco AMO, Bento LD, Ramirez-Lopez CJ, Girardi FM, Machado JCA, de Oliveira LL, da Fonseca LA. Enzyme-Linked Immunosorbent Assay (ELISA) Development for Equine Serum Amyloid A (SAA) Determination Using Recombinant Proteins. Methods Protoc 2025 Apr 7;8(2).
          doi: 10.3390/mps8020037pubmed: 40278511google scholar: lookup
        Subscribe to our newsletter
        Join 99,006 horse owners like you who receive our email updates on horse health and nutrition.