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Enhanced Chemiluminescence Assay vs Colorimetric Assay for Measurement of the Total Antioxidant Capacity of Human Seminal Plasma

SURESH C. SIKKA

EDWARD MASCHA

From the ^* Center for Advanced Research in Human Reproduction, Infertility and Sexual Function, Glickman Urological Institute, Cleveland Clinic Foundation, Cleveland, Ohio; Department of Urology, Tulane University, New Orleans, Louisiana; and Department of Biostatistics and Epidemiology, Cleveland Clinic Foundation, Cleveland, Ohio.

Correspondence to: Dr Ashok Agarwal, Director, Center for Advanced Research in Human Reproduction, Infertility, and Sexual Function, Glickman Urological Institute, The Cleveland Clinic Foundation, 9500 Euclid Ave, Desk A19.1, Cleveland, OH 44195 (e-mail: Agarwaa{at}ccf.org).

Although the enhanced chemiluminescence assay is commonly used to measure the nonenzymatic total antioxidant capacity (TAC) of the human seminal plasma, it is cumbersome, expensive, and time-consuming. We describe herein an alternate method to measure TAC that is based on the ability of antioxidants in seminal plasma to interfere with a reaction between 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] and metmyoglobin with H₂O₂. This reaction produces a relatively stable blue-green color with absorbance maxima at 600 nm. We compared this colorimetric assay with our established chemiluminescence method and assessed quality control parameters (ie, intra-assay and interassay variabilities) in addition to intraobserver and interobserver differences. Our results show that the colorimetric assay was fairly predictive of antioxidant capacity similar to the chemiluminescence assay (P < .001). Furthermore, there was a high level of agreement between the duplicate measures by the same observer (intraobserver) and intra-assay variability, with a concordance correlation coefficient of 0.99. The interassay coefficient of variation was 4.7% (overall). The mean ± SD of the difference between the 2 observers was 2.98% ± 4.1%. In conclusion, we found that the colorimetric assay is a reliable and accurate method to evaluate seminal TAC, and it could be used as a simpler, rapid, and cheaper alternative to the chemiluminescence assay.

     Key words: Luminometer, semen, spectrophotometer, Trolox, reactive oxygen species

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