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

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Author

  • Pär Hallgren
  • Lennart Mårtensson
  • Lennart Mathiasson

Summary, in English

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 mu g PO43- 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 Vallkarra 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 PO43- mu g/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.

Publishing year

2012

Language

English

Pages

894-908

Publication/Series

International Journal of Environmental Analytical Chemistry

Volume

92

Issue

7

Document type

Journal article

Publisher

Taylor & Francis

Topic

  • Analytical Chemistry

Keywords

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

Status

Published

ISBN/ISSN/Other

  • ISSN: 1029-0397