A new spectrophotometric method for improved indirect measurement of low levels of vitellogenin using malachite green

Abstract

Vitellogenin (VTG) is a well-known biomarker for endocrine disruption and the measurement of alkali-labile protein bound phosphate (ALP) has been used as an indirect quantification method for VTG in fish, clam and mussel. One limitation of the ALP method has been the insufficient detection limit but with the method presented here this obstacle has been overcome, enabling measurement in fish species with very low background levels and hence a wider usage of ALP measurements in the future. In a previous publication, relying on spectrophotometric measurement with molybdate, we presented an improved protocol for ALP measurement in fish plasma. The sample preparation in that paper has here been combined with an improved spectrophotometric determination using malachite green as colour reagent. The spectrophotometric measurement was optimised with respect to acidity and reagent concentration. The validated method has a detection limit of 0.3 µg  ml⁻¹ plasma, which is 10 times lower than previous spectrophotometric methods using molybdate reagent, and an intra-assay variation of 7%. The new method was used for screening of endocrine effects by placing juvenile rainbow trout at three locations along the Vallkärra brook, Lund Sweden, which receives leachate water from a nearby covered landfill. In comparison with reference fish, ALP was significantly higher (66%) at the 99% confidence level in fish placed close to the landfill. ALP was also significantly higher (33%) at the 95% confidence level in fish from the group placed further downstream. Fish placed in a brook branch not receiving leachate were unaffected. The low levels of ALP in reference fish (5.5 ± 0.7 SD  µg/ml plasma) could not have been quantified with such precision with another method for ALP measurement. The indirect quantification of VTG as ALP is a more economic alternative compared to quantification with immunological methods.

Keywords:

• vitellogenin
• alkali-labile phosphate
• endocrine disruptors
• biomarker
• malachite green
• Oncorhynchus mykiss

Acknowledgements

We acknowledge Niklas Hanson and Åke Larsson from the Department of Plant and Environmental Sciences, Gothenburg University for collaboration during exposure studies in Vallkärra brook. We gratefully acknowledge financial support from Sparbanksstiftelsen, Skåne.