Anal Bioanal Chem (2004) 378 : 615–620 DOI 10.1007/s00216-003-2235-0 O R I G I N A L PA P E R Jing Shao · Guoqing Shi · Jingfu Liu · Guibin Jiang A rapid two-step chromatographic method for the quantitative determination of vitellogenin in fish plasma Received: June 2003 / Revised: August 2003 / Accepted: 18 August 2003 / Published online: 16 October 2003 © Springer-Verlag 2003 Abstract By combining anion-exchange membrane purification with high-performance size-exclusion chromatography (HPSEC) analysis, a two-step chromatographic method was developed for the determination of vitellogenin (Vtg) in fish plasma Most plasma protein interferences can be removed during anion-exchange membrane purification process Vtg is eluted from the size-exclusion chromatography column with a retention time of about and is characterized based on the native molecular weight, with a limit of quantification of 20 µg Vtg mL–1 plasma The spiked recovery and interassay variability were better than 80% and 4.8% This method was successfully applied to analyze the plasma Vtg levels of loach (Misgurnus angaillicaudatus) and sea catfish (Enchelyopus elongatus) In addition to all the female fish, Vtg is detected in 75% of male loaches and 100% of male sea catfish The result indicates that some chemicals or unknown factors with estrogenic activity have induced male fish to produce Vtg Keywords Vtg · HPSEC · Membrane purification · Loach · Sea catfish Introduction The presence of endocrine disrupting chemicals (EDCs) in the environment has recently become a major issue of concern from the perspectives of both human and ecosystem integrity [1, 2, 3] Most of those EDCs have the ability to induce responses similar to those caused by estrogens Vitellogenin (Vtg), a sexual protein inducted by estrogens, has become a popular biomarker for estrogenic effects of exposure to estrogen or estrogen mimics [4, 5, 6] There are a number of reports on the appearance of Vtg in male J Shao · G Shi · J Liu · G Jiang (✉) Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O Box 2871, 100085 Beijing, China e-mail: gbjiang@mail.rcees.ac.cn fish plasma [7, 8, 9, 10] In order to assess the potential estrogenicity of chemical substances and their effects on fish, it is important to accurately measure the plasma Vtg levels A number of methods have been developed for measuring Vtg levels in fish plasma [11, 12, 13, 14, 15, 16, 17, 18] Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) are the most commonly used approaches among them [12, 13, 14] These antibody-based methods have considerable merits of sensitivity and selectivity, but they suffer from problems related to antibody specificity while facing multiple fish species, because an antibody made against Vtg from one species is limited in its application as a probe for another [5, 19] Although cross-reaction was also found in some species, it is not possible to make an accurate quantification of low levels of Vtg from other species [20] because of the affinity difference Moreover, Vtg is very susceptible to proteolytic degradation [5], so it is important to measure Vtg as soon as possible However, as the immunoassay method normally need several hours or days, the degradation of Vtg is inevitable during this period, even with the present of antiproteolytic agents The degradation of Vtg may occur not only in the period of determination, but also during sample collection or storage As the sample degrades, some epitopes recognized by the antibody are destroyed and others normally buried inside the Vtg molecule are exposed, thus changing the overall reactivity of the antibody towards Vtg [5] For these reasons, it is desirable to develop a rapid and accurate method for Vtg determination High-performance liquid chromatography (HPLC) has been widely applied in the determination of proteins or other biological molecules [21, 22, 23] Recently, a rapid method for the detection fish plasma Vtg using high-performance anion-exchange chromatography column (POROS-HQ) was reported [18] The lowest detectable amount of Vtg was µg per assay Although Vtg was eluted as a single peak from the column, it should be further examined by molecular weight High-performance size-exclusion chromatography (HPSEC) is a simple and widely used technique for protein assay [24, 25, 26] Because the separa- 616 tion is based on the molecular weight, its potential for the rapid and non-destructive analysis of Vtg with both quantitative and qualitative was of interest Waagboe and Sandnes successfully used HPSEC for the determination of Vtg in the plasma of estrogen-treated rainbow trout The Vtg determined by this method was significantly correlated with the alkali-labile protein phosphorus assay in naturally maturing fish [25] However, owing to the interference of plasma proteins, HPSEC, for the time being, cannot separate and detect trace levels Vtg in fish plasma In our previous work, we found that Vtg could be rapidly separated from fish plasma by step-gradient elution with anion-exchange membrane chromatography [27] The Vtg fraction by this method shows almost a single peak when analyzed with HPSEC This indicates that the anion-exchange membrane can be used to extract Vtg from fish plasma before further analysis In the present study, we developed a two-step method for detecting Vtg with a commercially available anion-exchange membrane and HPSEC column The method was applied to analyze the plasma Vtg levels of loach (Misgurnus angaillicaud atus) and sea catfish (Enchelyopus elongatus) collected from a fish farm or estuary of the Haihe River during February to April 2003 Experimental Chemicals and instruments 17β-Estradiol (E2), thyroglobulin (640 kDa), ferritin (440 kDa), immunoglobulin (158 kDa), bovine serum albumin (BSA, 66 kDa), and heparin were obtained from Sigma (St Louis, MO, USA); aprotinin was from Boehringer (Mannheim, Germany) All other chemicals were reagent-grade compounds obtained from commercial sources Buffers and sample solutions were filtered through a 0.2-µm cellulose acetate filter The ready-to-use unit Sartobind MA Q15 (Sartorius, Goettingen, Germany) is a strongly basic anion exchanger with quaternary ammonium groups The membrane material of this unit is supported cross-linked regenerated cellulose with an effective adsorption area of 15 cm2 The HPLC system for protein analysis consists of Isopump (Agilent, G1310A, Germany), VWD detector (Agilent, G1314A, Japan), and high-performance size-exclusion chromatography column: TSK G3000SWXL column (30 cm×7.8-mm ID, TOSOH Biosep, Japan) with HPLC workstation (Agilent HP1100 system) An Allitech 426 HPLC Pump (Allitech, USA), which can achieve a higher flow rate, was used for sample cleaning Fish Adult loaches for method developing were cultured in the laboratory with an average weight of 15 g Another batch of loaches (20 males and females) with body weights of 10–25 g were caught from a fish farm where the water was polluted by industrial effluent Sea catfish (5 males and females) with body weights of 100–200 g were captured from the estuary of the Haihe River Plasma preparation The fish was anesthetized with quinaldine sulfate (40 mg L–1) Blood was then collected from the caudal vein with heparinized syringes, and transferred to 1.5-mL centrifuge tubes containing aprotinin (2.5 TIU) and heparin (30 USP units) Then, the blood was centrifuged at 3,000 g, 4°C for 20 min, and plasma was collected and stored at –80°C until analysis Preparation of vitellogenin standard curve Vitellogenin in plasma from E2-treated fish was purified and validated by SDS-PAGE according to the described method [27] The protein concentration was determined by following the method of Bradford at 595 nm [28] The Vtg standard curve was established with concentrations ranging from to 400 µg mL–1 Sample purification A commercially available ready-to-use anion-exchange membrane unit, Sartobind MA Q15, was connected to the high-flow-rate HPLC pump and the procedure for sample purification was as follows: 1) initialize the anion-exchange membrane with mL of buffer A (20 mM phosphate buffer, pH 6.5, containing 0.32 mol L–1 NaCl); 2) dilute 100 µL of fish plasma with mL of buffer A and pass through 0.2-µm filter, then load the solution on the anion-exchange membrane; 3) wash the membrane with 10 mL of buffer A; 4) elute Vtg with mL of buffer B (20 mM phosphate buffer, pH 6.5, containing 0.42 mol L–1 NaCl) and immediately analyze the fractions by HPSEC; 5) regenerate the membrane with mL of buffer C (20 mM phosphate buffer, pH 6.5, containing mol L–1 NaCl) The flow rate of the mobile phase was 10 mL min–1 High-performance size-exclusion chromatography analysis The mobile phase for high-performance size-exclusion chromatography analysis is 50 mM sodium phosphate buffer, pH 6.5, containing 300 mM NaCl After injecting 20 µL of sample, the separation was carried out at a flow rate of 0.7 mL min–1 at room temperature Absorbance at 210 nm was monitored and the concentration of Vtg was calculated based on the standard curve Results and discussion Selection of wavelength In order to find the optimum wavelength for Vtg, we measured the serum protein at three wavelengths: 210 nm, 254 nm, and 280 nm The results show that the absorbance of Vtg at 210 nm is much higher than that at 254 nm and 280 nm (Fig 1) It is well known that the absorption of proteins at 254 nm and 280 nm is mainly due to phenyl chromophores, while absorption at 210 nm is mainly due to carboxyl groups It can be expected that the Vtg contains more carboxyl groups than phenyl groups Experiments demonstrated that the detection limit for Vtg at 210 nm was much lower than that at 280 nm Therefore, in this study we choose 210 nm as the detective wavelength Analysis of Vtg HPSEC has the advantage of accomplishing quantitative and qualitative analysis simultaneously However, Vtg at low concentration levels in fish plasma cannot be detected by HPSEC because of the interference from non-Vtg proteins This drawback has been partly overcome by adding an anion-exchange membrane purification procedure before HPSEC analysis Figure compares chromatograms 617 obtained before and after membrane purification for the plasma from a female loach Figure 2a illustrates the HPSEC analysis of plasma without being treated by anion-exchange membrane Although a small Vtg peak could be observed, it cannot be quantification because the peak is severely overlapped with other plasma proteins After membrane purification, most of the plasma proteins are eliminated (Fig 2b), and a major Vtg peak with a retention time of 8.9 appeared as shown in Fig 2c The relative molecular mass of Vtg was calculated to be 419 kDa with thyroglobulin, ferritin, IgG, and BSA as molecular mass standard The result is in accordance with those obtained by size-exclusion chromatography from other fish species, for example, 440 kDa for Salmo gairdneri [25], 442, 435, and 424 kDa for Oncorhynchus mykiss, Gobio gobio, and Leuciscus cephalus, respectively [29] The anion-exchange membrane used in this study is a commercially available cartridge with three pieces of membrane inside; the total absorption area is 15 cm2 and the absorption capacity for Vtg is 12 mg The principle for protein separation on the anion-exchange membrane is similar to that on a conventional anion-exchange column However, because of the more efficient mass transfer characteristics of membrane adsorbers (MAs) relative to particle adsorbers that are used in conventional LC column, the MAs can be operated at higher flow rates without impeding the binding capacity and separation efficiency [30] In this study, Vtg can be separated from most other plasma proteins within at a flow rate of 10 mL min–1 Fig 1a–c Chromatograms of serum protein detected at different wavelengths: a 210 nm, b 254 nm, c 280 nm Arrows indicate the Vtg position Fig 2a–d Comparison of chromatograms of female loach plasma obtained before and after anion-exchange membrane purification Mobile phase: 50 mM PBS, pH 6.5, containing 300 mM NaCl; 20-µL injection volume a serum diluted with buffer A before membrane cleaning; b serum diluted with buffer A and passed through the membrane; c the Vtg fraction eluted with buffer B; d the fraction eluted with buffer C 618 According to the manufacturer’s information, the highest flow rate of mobile phase that the Sartobind MA Q15 can tolerate is 50 mL min–1 By connecting a SartobindMA Q15 unit to a syringe one can also carry out the purification procedure In our experiment, we found that the recovery of Vtg was still higher than 60% when we push the corresponding sample and buffer through the membrane gently (the flow rate was about 30 mL min–1) We also found that although the concentration of Vtg may vary greatly, the elution volume of Vtg fraction basically did not change Thus, under the protein capacity of the SartobindMA Q15 unit, it is expectable that one may load much more fish serum onto the membrane, which is helpful in increasing the Vtg concentration in the eluted fraction Because the maximum serum we can get from a loach is about 200–400 µL, we take 100 µL fish serum per assay for Vtg analysis The limit of quantification (LOQ) of Vtg Figure 3a is the HPSEC chromatogram of blank plasma after membrane purification The result indicates that there are still little interferential proteins that cannot be eliminated In order to examine the limit of quantification (LOQ) for real samples, various amounts of Vtg were added to the same elution fraction; the lowest amount of Vtg that can give a quantitative peak in the vicinity of is empirically identified as the LOQ for this method (see Fig 3b) For loach, the LOQ is 20 µg Vtg mL–1 of plasma Reproducibility and recovery Pooled plasma from female loach was analyzed five times to test the reproducibility of this method When 100 µL plasma was analyzed each time according to the abovedescribed procedure, the mean retention time of Vtg was 8.88 (RSD=0.6%, n=5) and the mean Vtg peak height was 14.9 (RSD=4.8%, n=5) at a concentration level of 1,532.4 µg mL–1 vitellogenin in the plasma In order to evaluate the assay recovery, different amounts of purified Vtg (10 µg and 30 µg) were added to 100 µL pooled female serum, and the assay was then performed The recovery of the added Vtg was found at 84.6% and 90%, respectively Measurement of plasma Vtg levels in loach and sea catfish A total of 28 loaches (20 males and females) from a fish farm where the water was polluted by industrial effluent, and 12 sea catfishes (5 males and females) captured from the estuary of the Tianjin Haihe River were analyzed using this method The results are shown in Fig For loach, Vtg was detected in the plasma of 15 male loaches with concentrations of 73–243 µg mL–1; 170–3,829 µg Vtg mL–1 was also detectable in all of eight female loaches The high Vtg level and high detection ratio (75%, 15 out of 20) in male loach indicates noteworthy EDC pollution in this fish farm The loach usually live at the bottom of water and sometimes in the sediment, so the route of exposure is also of interest Fig 3a,b Typical chromatograms of Vtg obtained by the proposed method: a blank plasma; b blank plasma spiked with µg of Vtg Fig 4a,b The Vtg levels of loach (a) and sea catfish (b), the number on the graph indicates the detection ratio of Vtg 619 Conclusion A two-step method based on the combination of anion-exchange membrane purification and high-performance sizeexclusion chromatography analysis was developed to determine Vtg in fish plasma This method can be used to determine Vtg levels greater than 20 µg Vtg mL–1 plasma, which is adequate to detect moderate to strong estrogenic effects The identification of Vtg was based on the retention time in the range 8.36–9.36 on a size-exclusion chromatography column; this retention time corresponds to 300–600 kDa in native molecular weight, which is generally agreed to be a characteristic of most teleost Vtg [5, 26] As HPLC is a widely used instrument, this study provides an alternative or reference method that is technically easy to carry out for the determination of Vtg in fish plasma Acknowledgements This work was jointly supported by the National Natural Science Foundation of China (20137010), the State High Tech Development Plan (2001AA640610), and the Chinese Academy of Sciences (KZCX2–414) References Fig 5a,b Chromatograms of sea catfish (a) female and (b) male For sea catfish, the Vtg was first identified based on a retention time of 8.7 min, which corresponds to the molecular weight of 474 kDa The chromatograms for a female and a male sea catfish are shown in Fig Plasma Vtg levels for the sea catfish were in the range 20–320 µg mL–1 (166±128 µg mL–1, mean±SD) in male and 1,370–5,570 µg mL–1 (1,377±1,218 µg mL–1, 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Vtg levels in loach and sea catfish A total of 28 loaches (20 males