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

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 amino-naphthalin-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 less than 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 chemiluminescence system could give linear standard curves with hydrogen peroxide concentrations into the nmol l-1 range. In contrast, the exponential rise in chemiluminescence recorded with luminol was not observed until hydrogen peroxide concentrations exceeded 10 mumol l-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.