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A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography

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Abstract

L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations.

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References

  1. Hill JM, Roberts J, Loeb E, Khan A, MacLellan A, Hill RW. L-asparaginase therapy for leukemia and other malignant neoplasms. JAMA. 1967;202:882–8.

    Article  CAS  Google Scholar 

  2. Vimal A, Kumar A. Biotechnological production and practical application of L-asparaginase enzyme. Biotechnol Genet Eng Rev. 2017;8725:1–22.

    Google Scholar 

  3. Aiswarya R, Baskar G. Enzymatic mitigation of acrylamide in fried potato chips using asparaginase from Aspergillus terreus. Int J Food Sci Technol. 2018;53:491–8.

    Article  CAS  Google Scholar 

  4. Lanvers C, Paulo J, Pinheiro V, Hempel G, Wuerthwein G, Boos J. Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum. Anal Biochem. 2002;309:117–26.

    Article  CAS  Google Scholar 

  5. Nath CE, Dallapozza L, Eslick AE, Misra A, Carr D, Earl JW. An isocratic fluorescence HPLC assay for the monitoring of L-asparaginase activity and L-asparagine depletion in children receiving E. coli L-asparaginase for the treatment of acute lymphoblastic leukaemia. Biomed Chromatogr. 2009;23:152–9.

    Article  CAS  Google Scholar 

  6. Kudryashova EV, Sukhoverkov KV. “Reagent-free” L-asparaginase activity assay based on CD spectroscopy and conductometry. Anal Bioanal Chem. 2016;408:1183–9.

    Article  CAS  Google Scholar 

  7. Broome JD. Factors which may influence the effectiveness of L-asparaginases as tumor inhibitors. Br J Cancer. 1968;22:595–602.

    Article  CAS  Google Scholar 

  8. Grossowicz N, Wainfan E, Borek E, Waelsch H. The enzymatic formation of hydroxamic acids from glutamine and asparagine. J Biol Chem. 1950;187:111–25.

    CAS  PubMed  Google Scholar 

  9. Shifrin S, Parrott CL, Luborsky SW. Substrate binding and intersubunit interactions in L-asparaginase. J Biol Chem. 1974;249:1335–40.

    CAS  PubMed  Google Scholar 

  10. Scheiner D. Determination of ammonia and kjeldahl nitrogen by indophenol method. Water Res. 1976;10:31–6.

    Article  CAS  Google Scholar 

  11. Ylikangas P, Mononen I. A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy. Anal Biochem. 2000;280:42–5.

    Article  CAS  Google Scholar 

  12. Cooney DA, Handschumacher RE. L-asparaginase and L-asparagine metabolism. Annu Rev Pharmacol. 1970;10:421–40.

    Article  CAS  Google Scholar 

  13. Handschumacher RE, Bates CJ, Chang PK, Andrews AT, Fischer GA. 5-Diazo-4-Oxo-L-norvaline: reactive asparagine analog with biological specificity. Science. 1968;161:62–3.

    Article  CAS  Google Scholar 

  14. Drainas D, Drainas C. A conductimetric method for assaying asparaginase activity in Aspergillus nidulans. Eur J Biochem. 1985;593:591–3.

    Article  CAS  Google Scholar 

  15. Chinese Pharmacopoeia Commission. General monographs part 1. In: Pharmacopoeia of the People’s Republic of China. 2010. pp. 35–38.

  16. Food and Drug Administration Guidance for Industry Acrylamide in Foods. FDA Food Guid. 2018. http://www.fda.gov/FoodGuidances. Accessed 16 April 2018.

  17. Yao H, Vancoillie J, D’Hondt M, Wynendaele E, Bracke N, Spiegeleer BD. An analytical quality by design (aQbD) approach for a L-asparaginase activity method. J Pharm Biomed Anal. 2016;117:232–9.

    Article  CAS  Google Scholar 

  18. Meister A. Glutaminase, asparaginase, and α-keto acid-ω-amidase. Methods Enzymol. 1955;2:380–5.

    Article  CAS  Google Scholar 

  19. Cooney DA, Capizzi RL, Handschumacher RE. Evaluation of L-asparagine metabolism in animals and man. Cancer Res. 1970;30:929–35.

    CAS  PubMed  Google Scholar 

  20. Miller GL, Miller EE. Determination of nitrogen in biological materials. Anal Chem. 1948;20:481–8.

    Article  CAS  Google Scholar 

  21. Thompson JF, Morrison GR. Determination of organic nitrogen control of variables in the use of nessler’s reagent. Anal Chem. 1951;23:1153–7.

    Article  CAS  Google Scholar 

  22. Morrison GR. Microchemical determination of organic nitrogen with Nessler reagent. Anal Biochem. 1971;43:527–32.

    Article  CAS  Google Scholar 

  23. Wehner A, Harms E, Jennings MP, Beacham IR, Derst C, Bast P, et al. Site-specific mutagenesis of Escherichia coli asparaginase II: none of the three histidine residues is required for catalysis. Eur J Biochem. 1992;208:475–80.

    Article  CAS  Google Scholar 

  24. Gentili D, Zucchetti M, Conter V, Masera G, D’Incalci M. Determination of L-asparagine in biological samples in the presence of L-asparaginase. J Chromatogr B Biomed Sci Appl. 1994;657:47–52.

    Article  CAS  Google Scholar 

  25. Jayaram HN, Cooney DA, Jayaram S, Rosenblum L. A simple and rapid method for the estimation of l-asparaginase in chromatographic and electrophoretic effluents: comparison with other methods. Anal Biochem. 1974;59:327–46.

    Article  CAS  Google Scholar 

  26. Khushoo A, Pal Y, Singh BN, Mukherjee KJ. Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II. Protein Expr Purif. 2004;38:29–36.

    Article  CAS  Google Scholar 

  27. Zhu J, Yan X, Chen H, Wang Z. In situ extraction of intracellular L-asparaginase using thermoseparating aqueous two-phase systems. J Chromatogr A. 2007;1147:127–34.

    Article  CAS  Google Scholar 

  28. Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K. Production, purification and characterization of L-asparaginase from Streptomyces gulbargensis. Brazilian J Microbiol. 2010;41:173–8.

    Article  CAS  Google Scholar 

  29. Kumar NSM, Ramasamy R, Manonmani HK. Production and optimization of L-asparaginase from Cladosporium sp. using agricultural residues in solid state fermentation. Ind Crop Prod. 2013;43:150–8.

    Article  Google Scholar 

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Funding

This work was developed under the scope of the Young Researcher Project financed by FAPESP (São Paulo Research Foundation Brazil) through the process 2014/16424-7. The authors also acknowledge the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, processes 163292/2015-9, and 445442/2014-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES and CAPES-PROEX). In addition, P. S. Barber acknowledges Earlham College for financial support.

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Authors and Affiliations

  1. Department of Bioprocesses and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville, Araraquara, São Paulo, 14800-903, Brazil

    Agnes Magri, Matheus F. Soler, André M. Lopes & Jorge F. B. Pereira

  2. Biochemistry and Technology Chemistry Department, Chemistry Institute, São Paulo State University (UNESP), Araraquara, São Paulo, 14800-900, Brazil

    Eduardo M. Cilli

  3. Department of Chemistry, Earlham College, 801 National Road West, Richmond, IN, 47374, USA

    Patrick S. Barber

  4. Department of Biochemical-Pharmaceutical Technology, Pharmaceutical Biotechnology Laboratory, University of Sao Paulo (USP), São Paulo, 05508-000, Brazil

    Adalberto Pessoa Jr

Authors
  1. Agnes Magri
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  2. Matheus F. Soler
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  3. André M. Lopes
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  4. Eduardo M. Cilli
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  5. Patrick S. Barber
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  6. Adalberto Pessoa Jr
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  7. Jorge F. B. Pereira
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Correspondence to Jorge F. B. Pereira.

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Magri, A., Soler, M.F., Lopes, A.M. et al. A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography. Anal Bioanal Chem 410, 6985–6990 (2018). https://doi.org/10.1007/s00216-018-1326-x

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  • DOI: https://doi.org/10.1007/s00216-018-1326-x

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