Enhanced detection of reactive oxygen species produced by human spermatozoa with 7‐dimethyl amino‐naphthalin–1, 2‐dicarbonic acid hydrazide

Abstract

A new chemiluminescence technique has been assessed for the detection of reactive oxygen species generated by purified populations of human sperm. This revised protocol involves the use of horse‐radish peroxidase (HRP) in combination with a luminol analogue, 7‐dimethyl aminornaphthalin‐1,2‐dicarbonic acid hydrazide (DNDH), that exhibits two‐three times the quantal efficiency of luminol itself. The chemiluminescent signal generated with these reagents was significantly (P < 0.001) greater than that obtained with the conventional luminol‐based methodology for both the steady‐state situation and following stimulation of the sperm with PMA and A23187. Dose‐response analyses indicated that the DNDH/HRP chemi‐luminescence system could give linear standard curves with hydrogen peroxide concentrations into the nmol I‐1 range. In contrast, the exponential rise in chemiluminescence recorded with luminol was not observed until hydrogen peroxide concentrations exceeded 10 umol I‐1. It is concluded that the enhanced sensitivity of the DNDH/HRP system to low levels of hydrogen peroxide should facilitate the application of chemiluminescent techniques to the diagnosis of oxidative stress in cases of male infertility. Copyright © 1992, Wiley Blackwell. All rights reserved