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.
Aitken
R J
RJ
MRC Reproductive Biology Unit, Edinburgh, U.K.
Buckingham
D
D
D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase; D006801:Humans; D006834:Hydrazines; D006861:Hydrogen Peroxide / Q000378:metabolism; D008163:Luminescent Measurements; D008297:Male; D009281:Naphthalenes; D010100:Oxygen / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*
D005456:Fluorescent Dyes; D006834:Hydrazines; D009281:Naphthalenes; C045681:7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010100:Oxygen