Plasma sampling and freezing procedures influence vitellogenin measurements by enzyme-linked immunoassay in the fathead minnow (Pimephales promelas).
نویسندگان
چکیده
The present study compared three different methods for measuring plasma vitellogenin (VTG) in fathead minnow (FHM; Pimephales promelas): A procedure using liquid chromatography with electrospray ionization combined with tandem mass spectrometry (LC/ESI-MS/MS), and two commercial enzyme-linked immunoassay (ELISA) kits using either anti-carp or anti-FHM antibodies. The influence on plasma VTG measurements of using the protease-inhibitor aprotinin during blood sampling and of submitting the plasma samples to a freeze-thaw cycle before analysis also was evaluated. The addition of aprotinin to the blood during sampling significantly reduced the plasma VTG concentrations measured by ELISA, whereas the VTG values measured after plasma samples were submitted to a freeze-thaw cycle were significantly higher than those measured before freezing. This inflating effect of freezing on VTG measurements made by ELISA could be prevented if plasma samples were frozen diluted in citrate buffer containing 16 mg/ml of polyethylene glycol (PEG). In contrast, measurements of VTG made by LC/ESI-MS/MS were unaffected by freezing and, conceptually, are independent from enzymatic degradation. Although the use of aprotinin and PEG effectively reduced the influence of enzymatic and physical degradation caused by freezing and thawing on VTG measurements made by ELISA, it did not improve agreement between the three analytical techniques evaluated. More information is needed regarding the molecular structure and the existence of possible multiple forms of VTG before this protein can be measured adequately in FHM.
منابع مشابه
Oestrogenic Activity of Tropical Fish Food Can Alter Baseline Vitellogenin Levels in Male Fathead Minnow (Pimephales promelas)
Vitellogenin (VTG) is a precursor of egg-yolk protein and is therefore present at high concentrations in the plasma of female fish. In male fish, VTG concentrations are usually undetectable or low, but can be induced upon exposure to oestrogenic substances either via the water or the diet. This work was carried out to determine the reason for apparently elevated VTG concentrations in unexposed ...
متن کاملAn in Vivo Testing System for Endocrine Disruptors in Fish Early Life Stages Using Induction of Vitellogenin
An enzyme-linked immunosorbent assay (ELISA) based on carp (Cyprinus carpio) vitellogenin (c-VTG, an estrogendependent precursor of egg yolk) was developed to quantify VTG in the fathead minnow (Pimephales promelas) and was employed to establish an in vivo testing system for estrogens in fish early life stages. The working range of the c-VTG ELISA was between 1 and 50 ng/ml (20%–80% binding). T...
متن کاملEvaluation of the aromatase inhibitor fadrozole in a short-term reproduction assay with the fathead minnow (Pimephales promelas).
Cytochrome P450 aromatase (CYP19) is a key enzyme in vertebrate steroidogenesis, catalyzing the conversion of C19 androgens to C18 estrogens such as beta-estradiol (E2). The objective of this study was to assess effects of the CYP19 inhibitor fadrozole on fathead minnow (Pimephales promelas) reproductive endocrinology and physiology in a short-term reproduction assay proposed for identifying sp...
متن کاملLinkage of biochemical responses to population-level effects: a case study with vitellogenin in the fathead minnow (Pimephales promelas).
A challenge in the field of ecotoxicology is the linkage of alterations at molecular and biochemical levels of organization to adverse outcomes in individuals and populations. In the present study, a predictive relationship between plasma vitellogenin (VTG) concentration and fecundity in female fathead minnows (Pimephales promelas) was derived from 21-d laboratory toxicity tests with five chemi...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Environmental toxicology and chemistry
دوره 25 2 شماره
صفحات -
تاریخ انتشار 2006