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Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine

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Abstract

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10−7 M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.

Lentil plant of ‘Rubia de la Armuña’ (Lens culinaris Medikus)/Determination of sarcosine

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Acknowledgements

This work was supported by Consejería de Educación (Ref.: SA129A07) and Instituto Tecnológico Agrario (Ref.: ITACYL/SA-06-00-0) de la Junta de Castilla y León (Spain).

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Authors and Affiliations

  1. Centro de Investigación y Desarollo Tecnológico del Agua, University of Salamanca, 37008, Salamanca, Spain

    Patricia Pérez Galende & Teresa Manzano Muñoz

  2. Dpto. Química Física, University of Salamanca, 37008, Salamanca, Spain

    Manuel G. Roig

  3. Dpto. Química Analítica, Nutrición y Bromatología, University of Salamanca, Plaza de la Merced s/n, 37008, Salamanca, Spain

    Cándido García de María

Authors
  1. Patricia Pérez Galende
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  2. Teresa Manzano Muñoz
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  3. Manuel G. Roig
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  4. Cándido García de María
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Correspondence to Cándido García de María.

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Pérez Galende, P., Manzano Muñoz, T., G. Roig, M. et al. Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine. Anal Bioanal Chem 404, 2377–2385 (2012). https://doi.org/10.1007/s00216-012-6343-6

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  • DOI: https://doi.org/10.1007/s00216-012-6343-6

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