Blood steroid levels in the goldfish: Measurement of six ovarian steroids in small volumes of serum by reverse-phase high-performance liquid chromatography and radioimmunoassay

Abstract

A reliable and rapid technique for the measurement of estradiol-17β, testosterone, progesterone, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and cortisol by reverse-phase high-performance liquid chromatography and radioimmunoassay in single female goldfish serum samples of 50 μl was developed. The steroids were extracted with Sep-Pak C18 cartridges after heat treatment. While estradiol-17β was assayed directly after extraction, the other steroids were separated on a μBondapak C18 stainless-steel column with acetonitrile:water (53:47, v/v) in 17 min under isocratic conditions, and then quantitated by specific radioimmunoassays. The technique was validated and was shown to be highly accurate, precise, sensitive, and specific for measuring those particular ovarian steroids in goldfish serum. The steroid levels measured in this way were not significantly different from those measured after separation on a Nova-Pak C18 column with methanol:water (56:44, v/v), where all the individual steroids could be completely resolved during a 40-min run. With this technique, the steroid levels in the serum of the goldfish during the secondary yolk stage, the tertiary yolk stage, and at 0 hr after ovulation were determined. While estradiol-17β was found to be significantly higher in the tertiary yolk stage, testosterone was significantly higher in the secondary yolk stage than in the other two stages. There was no significant difference in the levels of cortisol, 17α-hydroxyprogesterone, 17α,20β-dihydroxy-4-pregnen-3-one, and progesterone among the three stages. We conclude that the technique is particularly useful for assaying multiple steroids in very small volumes of biological fluids.