Improved spectrophotometric vitellogenin determination via alkali-labile phosphate in fish plasma - a cost effective approach for assessment of endocrine disruption

摘要

Vitellogenin (VTG) is a well known protein biomarker for exposure to environmental estrogens and possible endocrine disruption in fish. VTG is very dominant in plasma after the onset of vitellogenesis and the protein is heavily phosphorylated. This enables indirect quantification through measurement of alkali-labile protein bound phosphate (ALP) as an alternative to the more expensive enzyme linked immunosorbent assay. Good correlation has previously been shown between ALP and actual VTG levels but little effort has been made to investigate the method in an analytical way e.g., to assure the origin of the measured phosphate. During this method development care has been taken to rule out non-VTG sources of phosphate such as phospholipids and free phosphate in the blood plasma. Sample preparation has been simplified and unnecessary steps have been omitted. The common spectrophotometric measurement for ALP involves measurement at two wavelengths and calculation of corrected absorbance values. With a quick phase separation step the spectrophotometric phosphate determination using molybdic acid and ascorbic acid has been improved and all matrix interference has been eliminated. The final ALP method presented here has a detection limit of 3.2 μg PO_4~(3-)/ml plasma which is six times lower than similar methods and it also has less variability. A high sample throughput in comparison to previous ALP methods is possible after scaling down sample and reagent volumes to fit in a 96 well microtiter plate. The cost for buying all chemicals and plastic consumer goods for setting up the indirect protocol for the analysis of 1000 samples is only circa 350 euro. This is only 1% of the material cost for buying commercially available test kit for direct quantification of VTG in the same number of samples. The ALP method should thus be of interest also for applied scientists outside advanced research laboratories.