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Improved Chemiluminescence Assay for Measuring Antioxidant Capacity of Seminal Plasma

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Spermatogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 927))

Abstract

An improved enhanced chemiluminescence antioxidant assay utilizes horseradish peroxidase conjugate and luminol to produce a cell-free oxygen radical generating system. We introduce the use of a peroxidase enzyme stabilizer to prolong the production of oxygen radicals at a steady rate. Addition of antioxidants temporarily interrupts oxygen radical generation, resulting in an inhibition curve. A linear relationship exists between the area of the inhibition curve and the molar quantity of added antioxidant used to quantify total nonenzymatic antioxidant capacity (TAC) in biological fluids including seminal plasma. We streamline the existing enhanced chemiluminescence technique by using a microtiter plate luminometer. A plate luminometer is as accurate as a tube luminometer in measuring TAC, using identical reaction volumes. As little as 1–50 μL of sample may be analyzed. A plate luminometer can detect molar Trolox equivalents as low as 12.5 μM, compared to 25 μM in tube luminometer, using identical volumes. The plate luminometer assay is made even more rapid with use of an injector.

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Acknowledgments

The authors thank Ashok Agarwal, Ph.D. of the Cleveland Clinic, Cleveland, Ohio, for sending us his TAC protocol, which we modified for this study. We also thank Professor Seymour Klebanoff, M.D. Ph.D., of the Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, for allowing us to use his tube luminometer and for illuminating discussions. The Paul G. Allen Foundation for Medical Research supported this research.

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Authors and Affiliations

  1. Male Fertility Lab, Department of Urology, University of Washington School of Medicine, Seattle, WA, USA

    Charles H. Muller, Tiffany K. Y. Lee & Michalina A. Montaño

Authors
  1. Charles H. Muller
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  2. Tiffany K. Y. Lee
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  3. Michalina A. Montaño
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Corresponding author

Correspondence to Charles H. Muller .

Editor information

Editors and Affiliations

  1. Andrology and IVF Laboratories, Department of Surgery (Urology), University of Utah School of Medicine, Arapeen Dr. 675, Salt Lake City, 84108, Utah, USA

    Douglas T. Carrell

  2. , Andrology and IVF Laboratories, University of Utah School of Medicine, Arapeen Way 675, Salt Lake City, 84108, Utah, USA

    Kenneth I. Aston

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Muller, C.H., Lee, T.K.Y., Montaño, M.A. (2013). Improved Chemiluminescence Assay for Measuring Antioxidant Capacity of Seminal Plasma. In: Carrell, D., Aston, K. (eds) Spermatogenesis. Methods in Molecular Biology, vol 927. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-038-0_31

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  • DOI: https://doi.org/10.1007/978-1-62703-038-0_31

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-037-3

  • Online ISBN: 978-1-62703-038-0

  • eBook Packages: Springer Protocols

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