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F2-isoprostanes in Fish mucus: A new, non-invasive method for analyzing a biomarker of oxidative stress
Chemosphere 239 (2020) 124797
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F2-isoprostanes in Fish mucus: A new, non-invasive method for analyzing a biomarker of oxidative stress Patrique Bulloch a, Sara Schur a, Dhasni Muthumuni a, Zhe Xia a, Wesley Johnson a, Mitchell Chu a, Vince Palace b, Guanyong Su c, d, Robert Letcher c, Gregg T. Tomy a, * a
Centre for Oil and Gas Research, University of Manitoba, Department of Chemistry, Winnipeg, Manitoba, R3T 2N2, Canada International Institute for Sustainable Development e Experimental Lakes Area, Winnipeg, Manitoba, R3B 0T4, Canada Environment and Climate Change Canada, Ecotoxicology and Wildlife Health Division, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada d Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, PR China b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
F2 isoprostanes (F2-isoPs) are biomarkers for oxidative stress in vivo in animals. LC/MS/MS was used to measure F2isoPs in fish mucus. Minimal sample processing was necessary to measure F2-isoPs. F2-isoPs readily detectable in several species of fish mucus from the wild. F2-isoPs in mucus has potential to be a non-invasive approach to assess fish health.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2019 Received in revised form 4 September 2019 Accepted 5 September 2019 Available online 6 September 2019
F2-isoprostanes (F2-isoPs) are a reliable biomarker class for oxidative stress in vivo in animals. These compounds are traditionally measured in matrices like liver and plasma, however social and environmental pressures warrant the development of non-lethal and non-invasive methods to assess animal health. Therefore, this study aimed to develop a high-performance liquid chromatography tandem mass spectrometry (HPLC-ESI-MS/MS) method to separate and detect F2-isoPs in fish mucus. The method was developed and validated for four native F2-isoP isomers using Northern pike mucus (Esox lucius). Linearity was observed between 5 and 1000 pg/mL. The limits of detection of the four F2-IsoP isomers ranged from 0.63 to 2.0 ng/g. Recoveries ranged from 78 to 95%, and matrix effects were small (<10%). The between-day and within-day repeatability for all target analytes was lower than 20% RSD. Endogenous F2-isoPs were measured in the pike mucus (5.3e28.8 ng/g). A preliminary study of baseline F2-isoP concentrations in lake trout (Salvelinus namaycush) captured from five lakes at the IISD-Experimental Lakes Area in Northwestern Ontario, Canada, was also conducted to test the interspecies applicability of the method. Endogenous F2-isoPs were quantified in lake trout (6.3e132 ng/g). Lake trout samples displayed large variability within and between the different lakes, which suggests sampling methods may require adjustment for this species. This work developed a sensitive analytical method for
Handling Editor: Keith Maruya
* Corresponding author. E-mail address: [email protected] (G.T. Tomy). https://doi.org/10.1016/j.chemosphere.2019.124797 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
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P. Bulloch et al. / Chemosphere 239 (2020) 124797
measuring F2-isoPs in fish mucus, however several further studies are required to determine its ability to accurately measure oxidative stress in fish species. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Contaminants are introduced daily into aquatic ecosystems through a variety of anthropogenic activities such as wastewater discharge, industrial effluents, agricultural runoff, and environmental incidents (e.g. oil spills) (Phillips et al., 2012; Leu et al., 2004; United States Environment, 2013; Debruyn et al., 2007). Many of these contaminants have toxic effects on aquatic organisms: notable examples are polycyclic aromatic hydrocarbons (PAHs), pesticides, pharmaceuticals, and transition metals. Oxidative stress has been shown to be a mechanism of toxicity for the previously mentioned classes of environmental contaminants (Morcillo et al., 2015; Lushchak, 2011; Dorval et al., 2003; Sturve et al., 2006). PAHs have been shown to induce oxidative stress responses at aqueous concentrations of 10 mg/L (Madison et al., 2015) Significant increases in lipid peroxidation was demonstrated in channel catfish (Clarias gariepinus) by Amin et al. (2012) upon exposure to low concentrations of the pyrethroid pesticide deltamethrin (Amin and Hashem, 2012). It has also been described as an important mechanism of mercury toxicity in fish (Cappello et al., 2016). Accordingly, studies routinely include analyses of oxidative stress biomarkers, such as thiobarbituric acid reactive substances (TBARS) and antioxidant enzymes activity assays as determinants of fish health post-exposure (Sturve et al., 2006; Pampanin et al., 2016; Secci et al., 2016; Santana et al., 2018). Prostaglandin F2-like derivatives, specifically the F2-isoprostanes (F2-isoPs), have emerged as reliable biomarkers for oxidative stress in visceral tissues of mammals. To date, F2-isoP have primarily been used as biomarkers of lipid peroxidation to investigate the pathogenesis of human diseases (Inonu et al., 2012; , 2010), but have seldom been studied in Konishi et al., 2006; Pratico fish (Janicka et al., 2013; Klawitter et al., 2011; Langhorst et al., 2010; Milne et al., 2005). However, orally administered high molecular weight PAH exposures of 7.82 mg/g fish resulted in increased muscle concentrations of F2-isoPs in rainbow trout (Oncorhyncus mykiss) after 50 days (Bravo et al., 2011). Chung et al (2013) also demonstrated that F2-isoP levels in medaka rise when exposed to a classic oxidant, hydrogen peroxide (Chung et al., 2013). F2-isoPs are the free radical catalyzed products of non-enzymatic lipid peroxidation of arachidonic acid (AA) (Lawson et al., 1999), a fatty acid found abundantly in brain tissue and cell membranes of all animals. Several factors make F2-isoPs advantageous as biomarkers of oxidative stress. They are chemically stable, and have been detected at relatively constant low levels in all biological tissues and in the fluid of healthy vertebrates, namely urine, plasma, and cerebrospinal fluid (Lawson et al., 1999; Montuschi et al., 2004). F2-isoPs are also more reliable indicators of lipid peroxidation than commonly used TBARS as biomarkers of oxidative stress, as the latter is a non-specific assay and is therefore susceptible to interactions that reduce the assay's accuracy (Moselhy et al., 2013). High performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) has become the preferred method of analysis for F2-isoPs, as it achieves the desired separation and sensitivity without time-consuming derivatization and yields more reproducible results than enzyme-linked immunoassay (ELISA) kits (Klawitter et al., 2011; Waugh et al., 1997; Lee et al., 2008; Taylor et al., 2006).
Many established methods for analyzing fish health, including isoprostanes, involve lethal tissue sampling prior to chemical analysis (Chung et al., 2013; Zananski et al., 2011; Stahl et al., 2014; Hicken et al., 2011; Olsen et al., 2012). To minimize the impact of repeated sampling on ecosystems e often with small fish populations e monitoring programs assessing the health of wild populations are increasingly moving toward non-destructive and noninvasive methods (European Union. European, 2010). Current techniques for F2-isoP quantification make use predominantly of blood and urine as matrices, and while these fluids can be collected non-lethally from fish, current methods are still invasive and stressful. More recently, fish skin mucus has been investigated as a potential biological matrix for the analysis of fish health (Meucci and Arukwe, 2005; Fekih-Zaghbib et al., 2013; Ekman et al., 2015). Mucus has important biological functions for fish, ranging from communication and reproduction to osmotic regulation (Shephard, 1994). It is composed mainly of glycoproteins, but also contains immunoglobulins, pheromones, lysozyme, and proteolytic enzymes (Shephard, 1994). Previous analyses using this matrix include hemoglobin, metabolomics studies, and antioxidant activity assays (Meucci and Arukwe, 2005; Fekih-Zaghbib et al., 2013; Ekman et al., 2015), but mucus is also known to contain proteins, large molecules such as egg yolk precursors (Meucci and Arukwe, 2005) as well as RNA and DNA material (Livia et al., 2006; Le Vin et al., 2011; Ren et al., 2015). Here we describe the development and optimization of a HPLC-MS/MS method to isolate and quantify F2-isoPs isomers in fish mucus from several lakes in northwestern Ontario and Manitoba, Canada (Magnusson, 2014). Sampling was non-invasive, and processing was simple and rapid as few modifications to the matrix were needed prior to analysis. 2. Methods and materials 2.1. Materials Methanol (Optima grade), hydrochloric acid (HCl), and water (HPLC grade) were purchased from Fisher (Fair Lawn, NJ). (±) 5-isoprostaglandin-F2a-VI, (±) 5-iso-prostaglandin-F2a-VI-d11 (±indicates a racemic mixture of two enantiomers), 8-iso-prostaglandin-F2a, and 8-iso-prostaglandin-F2a-d4, ent-iso-15(S)prostaglandin- F2a and ent-iso-15(S)-prostaglandin- F2a-d9 (all >98%) were procured from Cayman Chemicals (Ann Arbor, MI). The chemical structures can be found in Fig. S1. 2.2. Sample collection Lake trout (Salvelinus namaycush) were angled from five experimental lakes at the International Institute for Sustainable Development e Experimental Lakes Area (IISD-ELA): lake 223 (49.698194 , 93.707952 ), lake 224 (49.688778 , 93.719360 ), lake 260 (49.694108 , 93.767919 ), lake 373 (49.743863 , 93.799417 ), and lake 626 (49.753069 , 93.796672 ) (Fig. 1). More experimental lake parameters can be found in Table S2. Individuals of mixed sex (n ¼ 7) were sampled from each of the five lakes (35 total) at over a period of two weeks in autumn (September 26 e October 10, 2018) to attempt preliminary measurements of baseline F2-isoP
P. Bulloch et al. / Chemosphere 239 (2020) 124797
concentrations across a spectrum of lakes. Care was taken to minimize handling and to reduce the surface area denuded of mucus, to minimize impact on each individual fish. Fish were anaesthetized in lake water containing pH buffered MS-222 (0.4 g/ L), until all fin movement ceased (~3min), and then measured, weighed and their sex was determined using external characteristics. A small volume of mucus (10e50 mg, but variable among individuals) was scraped from the skin using methanol-rinsed forceps and aspirated using a sterile syringe. The sample was then dispensed into a 1.5 ml microcentrifuge tube and immediately frozen on dry ice for transport to the laboratory where they were stored at 80C until processing. The lake trout sampling procedures were conducted according to standards established by the Canadian Council on Animal Care and were approved under AUP #F16-018. Northern pike (Esox lucius) mucus samples collected from specimens angled in Rice River, MB (Latitude: 51.3364 N, Longitude: 96.4025 W), were provided by a recreational angler, though sample storage practices were still observed. These samples were used for method development and validation due to the large quantity available. 2.3. Sample preparation: fish mucus Northern pike mucus samples prepared from undiluted mucus (45 mL total) were used for the development of the extraction protocol. Lake trout mucus samples were prepared using the same method, but were not pooled. Samples were centrifuged at 5000 rpm for 15min. 50 mL of the supernatant was aliquoted into microcentrifuge tubes. Aliquots of 50 mL (n ¼ 10) were weighed, and found to have an average density of ~1.00 g/L. Deuterated isoprostanes (5 mL of 1 ng/mL stock) were spiked directly into the mucus. Sample volume was adjusted to 0.5 mL with methanol, acidified to pH 3 with 0.1 M HCl, and vortexed. Solid mucus components were removed by centrifuging for 5 min at 14,800 rpm. The supernatant was then transferred to 2 mL plastic microcentrifuge tubes, with two rinses of methanol. Samples were centrifuged again to assure full exclusion of particulates and precipitates, and transferred to fresh 2 mL vials to be blown down to 100 mL under a nitrogen stream, using an N-EVAP 111 (Organomation, Berlin, MA).
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compound. Entrance potential was 10 V. The quantitative transition for 8-isoPG and its enantiomer ent-8-iso-15(S)-PG was m/z 353/193. The m/z 357/197 and m/z 362/300 transitions were monitored for their respective internal standards. The m/z 353/115 and m/z 364/115 transitions were monitored for (±) 5-isoPG-VI and its deuterated internal standard. Confirmation transitions can be found in Table S3. 2.5. Confirmation of isoprostanes by high resolution time of flight MS An Agilent 1200 LC system, consisting of a degasser, binary highpressure gradient pump, and autosampler coupled to an Agilent 6520A Q-ToF-MS system (Agilent Technologies, Mississauga, ON, Canada), was used to confirm the identity of isoprostanes in fish mucus extracts. LC separation was carried out on a Luna C18(2) column (2.0 mm 50 mm, 3 mm particle size) (Phenomenex, Torrance, CA, USA). The LC mobile phases were water (A) and methanol (B), and both contained 0.1% of formic acid. The mobile phase flow rate was 0.3 mL/min and the gradient was as follows: 0 min, 5% B; 0e5 min, 30% B; 1e7 min, 60% B; 7e15 min 80% B; 15e15.5 min 100% B; post run, 10 min. A volume of 20 mL of isoprostane standards (50 ng/mL) or fish mucus extract was injected into the LC system, respectively. The Q-ToF was tuned and calibrated with tuning calibration solution (G1969-85000) provided by Agilent Technologies. The resolution of the MS was >20 000 at both m/z 301.9981 and 601.9790 when the ESI interface was operated in the negative mode and the capillary voltage was 3500 V. The fragmentor and skimmer voltages were 180 and 80 V, respectively. Nitrogen was used as the drying and nebulizing gas. The gas temperature was 350 C, dry gas 10 L/min, and nebulizer 40 psi. Fullscan data acquisition was performed by scanning from m/z 50 to 1700. For each run, the reagent containing TFA anion (m/z 112.9855) and HP-0921 formate adduct (m/z 966.0007) were consistently introduced into the Q-ToF-MS as reference masses. Potential F2-isoP isomers in fish mucus extract were confirmed by monitoring its theoretical [M H]- ion (m/z 353.2333); m/z values within 5 ppm of its theoretical value were considered as a positive identification. 3. Results and discussion
2.4. Separation and quantitation of isoprostanes by triple quadrupole tandem MS All sample analyses were performed using a Varian 1100HPLC system (Palo Alto, CA) equipped with a CTC PAL autosampler. A Phenomenex (Torrance, CA) Zorbax Eclipse XDB-C18 column (2.1 mm 50 mm x 3.5 mm particle size) was used and maintained at room temperature. The mobile phase consisted of methanol (A) and water (B), both containing 0.1% formic acid (v/v). Flow rate was 300 mL/min. A solvent gradient was used to resolve the F2-isoP isomers: 30% B for 1min, increased to 60% B by 7min, 80% B at 15min, to 100% B at 15.5min, held for 1 min, then back to initial conditions by 17min. Injection volume was 5 mL. The HPLC was coupled to a Sciex API 365 triple quadrupole mass spectrometer (QqQ-MS) equipped with an ESI source. The source was a custom designed hot source induced desolvation (HSID) source with orthogonal injection. The instrument was operated in negative ionization mode, with the source temperature set to 500 C. Spray voltage was maintained at 3500 V. Samples were analyzed using multiple reaction monitoring (MRM) mode. Each compound was monitored using two MRM transitions; one for quantitation and one for confirmation. The eight chemical standards (four native F2isoP and four deuterated F2-isoP) were optimized individually. Declustering potential (DP), focusing potential (FP), collision energy (CE), and cell exit potential (CXP) were optimized manually for each
3.1. Method development The LC-MS/MS method parameters were validated according to the Eurachem guidelines for analytical methods (Magnusson, 2014). Matrix effect, recoveries, limits of detection, linearity, and repeatability were assessed and found to comply within the accepted ranges (Note: as previously mentioned, the concentrations of 5-isoPG-VI, 5-epi-isoPG-VI and their d-analogs are halved due to being racemic mixtures). Only Northern pike mucus was used for method development. 3.1.1. Linearity The linear dynamic range was determined by injecting standards prepared in methanol from stock mixture solutions containing four F2-isoP and their d-analogs, at concentrations of 5, 10, 25, 50, 100, 500, and 1000 pg/mL. The concentrations of 5-isoPG-VI, 5-epi-isoPG-VI, and their deuterated internal standards are halved (2.5, 5, 12.5, 25, 50, 250, 500 pg/mL), since they are 50:50 in their racemic standard solution. All compounds tested showed linearity within this concentration range (R2 > 0.99). A matrix matched approach was additionally carried for the deuterated F2-isoPs, using extracted pike mucus samples. Linearity was observed over this range of concentrations (R2 > 0.999). Matrix matched external standard (MMES) linearity was not attempted for non-deuterated
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native F2-isoPs due to interfering endogenous F2-isoP.
Table 2 Internal standard recoveries at varying IS concentrations in Northern pike mucus. Sample
3.1.2. Matrix effect Interference from matrix components is common in HPLC-ESIMS analysis, and may result in addition or suppression of the signal. Matrix effect was therefore assessed to determine the impacts on this F2-isoP protocol. Matrix matched standards were prepared by spiking 8-isoP-d4, ent-iso-15(S)-isoPG-d9, and (±) 5isoPG-VI-d11 at concentrations of 5, 10, 25, 50, and 100 pg/mL into final mucus extracts. Integrated areas were then compared to those of d-F2-isoP standards in methanol to determine the matrix effect, by taking the ratio of MMES and solvent calibration curves. Both MMES and standards in solvent were run in triplicates at each concentration. As shown in Table 1, matrix interference was relatively small using 50 mL mucus, ranging from 1 to 7%. R2 values were all >0.993. Matrix effects were consistent for each of the four isomers.
3.1.3. Recoveries Acceptable extraction efficiency from the mucus matrix was achieved for all four isoprostane isomers. Determination of analyte recoveries was conducted using matrix matched standards, to account for any possible signal interference from the matrix constituents. Peak areas of mucus samples (n ¼ 6) spiked with d-isoPs at three different concentrations (5, 25 and 100 pg/mL) were compared to matrix matched standards of equivalent concentrations. Recoveries of the four isoprostane isomers ranged between 78 and 95%, with RSD of 15% at all three concentration levels. Student t-tests were used to determine if statistically significant differences in analyte recovery existed between concentrations: recoveries did not differ significantly for any of the four isomers. Recovery of Type III isomers was slightly higher than Type VI isomers. Detailed results can be found in Table 2.
3.1.4. Limits of detection Low limits of detection were achieved for all four isoprostane isomers, ranging between 0.44 and 2.0 ng/g. Water blanks (n ¼ 10) were spiked with 0.5 ng of each F2-isoP and extracted according to the protocol, giving a final concentration of 5 pg/mL in the injected sample. These samples were prepared in distilled water because endogenous isoprostanes observed in the mucus samples would interfere with the signal. The internal standards were not used, as they showed lower responses than the native isoprostanes. Better detectability of the native isoPs, paired with the small matrix effect, justified the use of water as a sample matrix. Limit of detection (LOD) calculations were based on the Eurachem specifications ffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 , where n is the number of replicates), and (LOD ¼ 3s* 1n þ nblank converted to concentration in ng/g (Magnusson, 2014). Similarly, limits of quantitation (LOQs) were determined using a 10-fold multiplication in the above equation. These results are tabulated below.
Spiked
Measured (mean þSD)a
pg/mL 8-isoPG-d4
5-isoPG-d11
5-epi-isoPG-d11
ent-iso-15(S)-isoPG-d9
5 25 100 2.5 12.5 50 2.5 12.5 50 5 25 100
4.5 ± 0.7 21.6 ± 1.6 89.6 ± 11.7 2.1 ± 0.2 10.2 ± 0.9 39.0 ± 1.8 2.1 ± 0.4 10.1 ± 0.8 41.7 ± 3.3 4.6 ± 0.2 22.3 ± 2.2 95.4 ± 10.4
Recovery
RSD
%
%
90.5 86.3 89.6 82.2 81.7 78.1 84.7 81.1 83.3 92.1 89.0 95.4
14.7 6.3 13.1 8.2 7.6 4.6 9.6 6.6 7.8 5.1 8.8 10.3
RSD: Relative standard deviation, in %. a Mean and standard deviation (n ¼ 6).
3.1.5. Repeatability Northern pike mucus samples showed acceptable variability in isoprostane signal (RSD < 20%) for same-day and inter-day batch precision analysis. Method precision was measured through repeatability measurements. Same-day and inter-day repeatability were measured using Northern pike (n ¼ 6). Pike samples were analyzed with IS concentrations of 5, 25 and 100 pg/mL. Day-to-day repeatability tests were carried out for 3 days. The peak area ratios of quantitation: confirmation ion transitions were also monitored during batch analyses. Fragmentation patterns of stable molecules should remain constant during analyses if conditions are not changed. The average Q/C ratios of all four F2-isoP isomers remained constant (<10%) throughout the entirety of the method validation process, indicating the analyte underwent no degradation over the analysis period (data not shown). 3.2. Quantitation of F2- isoprostanes in fish samples 3.2.1. Northern pike All four selected F2-isoP isomers were observable and resolved chromatographically or by mass in Northern pike samples (see Fig. S4). Isoprostane peak identities were confirmed with relative retention times of deuterated internal standards. Concentrations ranged from 5.3 to 28.8 ng/g e detailed results can be found in Table 4 below.
Table 3 Limits of detection and quantitation of four F2-isoprostane isomers. Analyte
LOD (ng/g)
LOQ (ng/g)
8-isoPG 5-isoPG-VI 5-epi-isoPG-VI ent-iso-15(S)-isoPG
1.4 0.44 0.63 2.0
4.7 1.5 2.1 6.5
LOD: Limit of detection. LOQ: Limit of quantitation.
Table 1 Matrix effect calibration lines of four deuterated F2-isoprostanes in Northern pike mucus.a Analyte
Curve equation Matrix matched
8-isoPG-d4 5-isoPG-VI-d11 5-epi-isoPG-VI-d11 ent-iso-15(S)-isoPG-d9
y y y y
a
¼ ¼ ¼ ¼
986:8:5x þ 1735 1024:2x þ 405:8 1481:8x þ 1840 713:3x þ 399:4
Curve equation Solvent matrix y y y y
¼ ¼ ¼ ¼
1012:4x 166 986:4x 381 1595x 1453 723:7x 1129
% difference in matrix 97 ± 2 104 ± 3 93 ± 5 99 ± 4
Matrix effect is determined by dividing the slopes of a matrix-matched standard calibration curve and a standard curve from analyte prepared in a pure solvent, methanol.
P. Bulloch et al. / Chemosphere 239 (2020) 124797 Table 4 Endogenous F2-isoprostanes measured in Northern pike and lake trout mucus.a. Species/Lake
Northern Pike LT L224 LT L260 LT L373 LT L626
Concentration (ng/g)
b
8-isoPG
ent-8-iso-15(S)-PG
5-isoPG-VI
5-epi-isoPG-VI
28.8 ± 2.5 11.3
5.3 ± 0.8 ND ND 7.2 < LOQ
9.9 ± 1.3 ND ND 8.0
LT: Lake trout. ND: Not detected.
In addition, several other isoprostane peaks were fully or partially resolved, eluting up to 2min later than our compounds of interest. These were confirmed to be isoprostanes by high resolution LC-qTOF-MS to 2.5 ppm accuracy (Table S1). During the F2-isoP formation process, the multiple unsaturated sites of arachidonic acid result in 4 possible classes of regioisomers: Type III, IV, V, and VI, categorized by the location of the backbone hydroxyl group. Class-specific enantiomers bring up the total isomer count to 64. Resolving regioisomers and enantiomers is therefore essential since the accurate quantitation of selected isomers is dependent on freedom from interference from other stereoisomers with common quantitative m/z transitions. This study focused on two regioisomer classes: Type III and Type VI (shown in Fig. S1). The presence of other unidentified isoprostane isomers could make this method more broadly applicable as additional commercial standards become available for other F2-isoP isomers. 8-isoprostaglandin-F2a, a Type III isomer, is the most researched of these compounds, and has been linked to several human diseases such as atherosclerosis (Milne et al., 2005). However, other isomers e also lipid peroxidation endpoints e have the same functions and are more abundant (Reilly et al., 1998). A more comprehensive and accurate analysis of lipid peroxidation would be achieved by identifying and quantifying more isoprostanes.
3.2.2. Lake trout F2-isoprostanes were detected in individual fish in four of the five lakes, the exception being lake 223: an example chromatogram is shown in Fig. 2. Measureable Type III isoprostane peaks were present in only one out of seven individuals in lake 224, and were detected but below the LOQ in one individual from lake 260. In
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contrast, three and four individuals showed measurable concentrations in lakes 373 and 626 respectively with concentrations ranging from 6.3 to 132 ng/g (see Table 4). The variability was large: the concentrations of Type III isoprostanes in lake trout individuals from L373 with measurable levels ranged from 7.5 to 132 ng/g. Similarly, in 4 individuals from L626, concentrations ranged from 6.3 to 70.7 ng/g, with an RSD of 91%. Parsons et al. (2009) demonstrated in rats and fathead minnows that natural variability is usually greater in biofluids such as mucus (~50%) than in tissues (~30%) (Parsons et al., 1039). The variability of metabolite concentrations in fathead minnow mucus was also shown to fall within the range of this natural variability typical of biofluids by Ekman et al. (2015) (Ekman et al., 2015). The 91% variation observed in our results may indicate that the collection method has to be modified. A potential solution could be to normalize isoprostane concentrations relative to protein and lipid content of the mucus, which may prove more accurate than total mass. Such normalization is common for other biomarker assays of exposure such as for ethoxyresorufin-O-deethylase (EROD) and TBARS (Sturve et al., 2006; Pampanin et al., 2016). Despite this, a qualitative comparison of isoprostanes can still be made on these preliminary results. A possible explanation for the higher isoprostane concentrations in trout from lakes 373 and 626 is the sampling dates, which coincide with the beginning of fall spawning season for lake trout (Plumb et al., 2014). Lake trout fall spawning typically begins when water temperature decreases closer to the species’ optimal temperature range of 8e12 C. Spawning is a stressful, energetically demanding process (Stewart et al., 1983), which could explain the heightened isoprostane concentrations observed in the samples from these lakes. Mean water temperatures (1 m depth) for the five experimental lakes (Fig. S5, Table S2) measured on the trout sampling days were within this range for the L373 sampling event (12.0 C), and just above for L626 sampling at 13.2 C. In comparison, the other three lakes range from 14.9 to 15.5 C e a significant difference of at least ~2 C. It is therefore possible that higher F2-isoprostane concentrations could be measured in the L223, L224, and L260 lake trout by sampling during spawning. However, further study is required to support this hypothesis, especially given the large variability observed in the data. The two Type VI isomers measured in this study, 5- and 5-epiisoprostanglandin-VI, were measurable only in one fish from L373 (see Table 4), though they were also present below LOQ in other individuals. Concentrations of both these compounds in the lake trout were within 2 ng/g (or < 30%) of those measured in the pooled Northern pike samples. Though encouraging, this result must be treated with caution as conclusions cannot be drawn before several more samples are analyzed to properly compare F2-isoP levels between the two species. Several chromatographic peaks with the m/z 353 / 115 transition were in evidence in most of the sampled individuals listed above. These peaks eluted up to 2.5min later than the four isomers selected for this study (see Fig. 3). These peaks cannot be identified with certainty because commercial standards for most isoprostane isomers are unavailable, however their high resolution molecular weights correspond to those in the samples who were previously identified as isoprostane isomers by HR-LC-MS/MS (Fig. 4, Table S1). Identifying and measuring these compounds is an important consideration for future studies. 4. Conclusion
Fig. 1. Location of IISD-Experimental Lakes Area research station in Northwestern Ontario, Canada (top right) and research lakes sampled for this study (main body). Credit: G. Gunn, IISD-ELA. Data sources: NRCAN and IISD-ELA.
In this study, four F2-isoprostane isomers were successfully separated and quantified in fish mucus of wild individuals from two species. Repeated F2-isoP measurements in individuals of a
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Fig. 2. HPLC-MS/MS elution of a) deuterated standards ent-8-iso-15(S)- prostanglandin-F2a-d9 (Peak 1) and 8-iso-prostanglandin-F2a-d4 (Peak 2) and b) endogenous ent-8-iso15(S)- prostanglandin-F2a (Peak 3) and 8-iso-prostanglandin-F2a (Peak 4) in a lake trout mucus sample. Other isoprostane peaks in the mucus are present but have yet to be positively identified.
Fig. 3. HPLC-MS/MS elution of a) endogenous 5-isoprostanglandin-F2a-VI-d11 (Peak 1) and 5-epi-isoprostanglandin-F2a-VI-d11 (Peak 2) and b) 5-iso-prostaglandin-F2a-VI (Peak 3) and 5-epi-iso-prostaglandin-F2a-VI in a lake trout mucus sample. Other isoprostane peaks in the mucus have not yet been posively identified.
P. Bulloch et al. / Chemosphere 239 (2020) 124797
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Fig. 4. High-resolution LC-qTOF-MS mass chromatogram and mass spectrum at 13.01 min (inset) of endogenous F2-isoprostane isomers in lake trout. The detailed information for all the peaks are provided in Table S1.
population over time could eventually prove to be an effective method for monitoring fish population health during and after an exposure to contaminants, as in vivo concentrations have been shown to relate with oxidative stress (Milne et al., 2005). Determining baseline levels of F2-isoP in fish populations is therefore a crucial first step in the implementation of this method, as baseline concentrations of F2-isoP likely vary between fish species and water bodies. Effects of water chemistry and temperature, temporal effects on physiology, and sexual dimorphism are all factors that require further study to better understand natural variability of F2isoPs in fish mucus. Several further studies are required to validate this method before it is deemed applicable for measuring oxidative stress in wild fish. Though it demonstrated that the method can be extended to species other than Northern pike, preliminary results showed large variability in isoprostane concentrations of individual lake trout, both within and between lakes. Concentrations need to be validated against more typical sampling matrices, such as plasma, under controlled laboratory conditions to establish if there is a reliable, reproducible relation between F2-isoP levels in these media. This will also provide a better understanding of natural variability. Additionally, the variability raises questions as to if normalizing F2-isoP concentrations to total lipid and protein content in each sample would provide a more accurate measure of lipid peroxidation. Another important consideration would be to determine if F2-IsoP concentrations differ when mucus is collected from different parts of the fish. Furthermore, determining a doseresponse relationship to chemical stressors and comparing response to other established biomarkers such as ratios of oxidized to reduced glutathione and malondialdehyde will determine the usefulness of this method as an ecological monitoring method. A study is currently underway to address these questions. Clearly, the validated method reported here demonstrates that quantifiable amounts of these robust oxidative stress biomarkers can be measured in very small mucus samples collected from fish in pristine environments and is a necessary prerequisite for future studies assessing the ecological significance of this new approach.
Note The authors declare no competing financial interest. Acknowledgment We thank the staff and students at the International Institute for Sustainable Development e Experimental Lakes Area (IISD-ELA) for providing data and assistance, especially Sonya Michaleski for her assistance in sample collection and processing. This work was supported in part by a Natural Science and Engineering Research Council of Canada Discovery Grant (Grant #: RGPIN/5303-2014) to G.T.T. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124797. References Amin, K.A., Hashem, K.S., 2012. Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol. BMC Vet. Res. 8 https://doi.org/ 10.1186/1746-6148-8-45. Bravo, C.F., Curtis, L.R., Myers, M.S., et al., 2011. Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture. Environ. Toxicol. Chem. 30 (3), 704e714. https:// doi.org/10.1002/etc.439. Cappello, T., Brand~ ao, F., Guilherme, S., et al., 2016. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining1H NMR metabolomics and conventional biochemical assays. Sci. Total Environ. 548e549, 13e24. https://doi.org/10.1016/j.scitotenv.2016.01.008. Debruyn, A.M.H., Wernick, B.G., Stefura, C., et al., 2007. In situ experimental assessment of lake whitefish development following a freshwater oil spill. Environ. Sci. Technol. 41 (20), 6983e6989. https://doi.org/10.1021/es0709425. Dorval, J., Leblond, V.S., Hontela, A., 2003. Oxidative stress and loss of cortisol secretion in adrenocortical cells of rainbow trout (Oncorhynchus mykiss) exposed in vitro to endosulfan, an organochlorine pesticide. Aquat. Toxicol. 63 (3), 229e241. https://doi.org/10.1016/S0166-445X(02)00182-0. Ekman, D.R., Skelton, D.M., Davis, J.M., et al., 2015. Metabolite profiling of fish skin mucus: a novel approach for minimally-invasive environmental exposure monitoring and surveillance. Environ. Sci. Technol. 49 (5), 3091e3100. https:// doi.org/10.1021/es505054f.
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F2-isoprostanes in Fish mucus: A new, non-invasive method for analyzing a biomarker of oxidative stress Patrique Bulloch a, Sara Schur a, Dhasni Muthumuni a, Zhe Xia a, Wesley Johnson a, Mitchell Chu a, Vince Palace b, Guanyong Su c, d, Robert Letcher c, Gregg T. Tomy a, * a
Centre for Oil and Gas Research, University of Manitoba, Department of Chemistry, Winnipeg, Manitoba, R3T 2N2, Canada International Institute for Sustainable Development e Experimental Lakes Area, Winnipeg, Manitoba, R3B 0T4, Canada Environment and Climate Change Canada, Ecotoxicology and Wildlife Health Division, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada d Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, PR China b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
F2 isoprostanes (F2-isoPs) are biomarkers for oxidative stress in vivo in animals. LC/MS/MS was used to measure F2isoPs in fish mucus. Minimal sample processing was necessary to measure F2-isoPs. F2-isoPs readily detectable in several species of fish mucus from the wild. F2-isoPs in mucus has potential to be a non-invasive approach to assess fish health.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 July 2019 Received in revised form 4 September 2019 Accepted 5 September 2019 Available online 6 September 2019
F2-isoprostanes (F2-isoPs) are a reliable biomarker class for oxidative stress in vivo in animals. These compounds are traditionally measured in matrices like liver and plasma, however social and environmental pressures warrant the development of non-lethal and non-invasive methods to assess animal health. Therefore, this study aimed to develop a high-performance liquid chromatography tandem mass spectrometry (HPLC-ESI-MS/MS) method to separate and detect F2-isoPs in fish mucus. The method was developed and validated for four native F2-isoP isomers using Northern pike mucus (Esox lucius). Linearity was observed between 5 and 1000 pg/mL. The limits of detection of the four F2-IsoP isomers ranged from 0.63 to 2.0 ng/g. Recoveries ranged from 78 to 95%, and matrix effects were small (<10%). The between-day and within-day repeatability for all target analytes was lower than 20% RSD. Endogenous F2-isoPs were measured in the pike mucus (5.3e28.8 ng/g). A preliminary study of baseline F2-isoP concentrations in lake trout (Salvelinus namaycush) captured from five lakes at the IISD-Experimental Lakes Area in Northwestern Ontario, Canada, was also conducted to test the interspecies applicability of the method. Endogenous F2-isoPs were quantified in lake trout (6.3e132 ng/g). Lake trout samples displayed large variability within and between the different lakes, which suggests sampling methods may require adjustment for this species. This work developed a sensitive analytical method for
Handling Editor: Keith Maruya
* Corresponding author. E-mail address: [email protected] (G.T. Tomy). https://doi.org/10.1016/j.chemosphere.2019.124797 0045-6535/© 2019 Elsevier Ltd. All rights reserved.
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P. Bulloch et al. / Chemosphere 239 (2020) 124797
measuring F2-isoPs in fish mucus, however several further studies are required to determine its ability to accurately measure oxidative stress in fish species. © 2019 Elsevier Ltd. All rights reserved.
1. Introduction Contaminants are introduced daily into aquatic ecosystems through a variety of anthropogenic activities such as wastewater discharge, industrial effluents, agricultural runoff, and environmental incidents (e.g. oil spills) (Phillips et al., 2012; Leu et al., 2004; United States Environment, 2013; Debruyn et al., 2007). Many of these contaminants have toxic effects on aquatic organisms: notable examples are polycyclic aromatic hydrocarbons (PAHs), pesticides, pharmaceuticals, and transition metals. Oxidative stress has been shown to be a mechanism of toxicity for the previously mentioned classes of environmental contaminants (Morcillo et al., 2015; Lushchak, 2011; Dorval et al., 2003; Sturve et al., 2006). PAHs have been shown to induce oxidative stress responses at aqueous concentrations of 10 mg/L (Madison et al., 2015) Significant increases in lipid peroxidation was demonstrated in channel catfish (Clarias gariepinus) by Amin et al. (2012) upon exposure to low concentrations of the pyrethroid pesticide deltamethrin (Amin and Hashem, 2012). It has also been described as an important mechanism of mercury toxicity in fish (Cappello et al., 2016). Accordingly, studies routinely include analyses of oxidative stress biomarkers, such as thiobarbituric acid reactive substances (TBARS) and antioxidant enzymes activity assays as determinants of fish health post-exposure (Sturve et al., 2006; Pampanin et al., 2016; Secci et al., 2016; Santana et al., 2018). Prostaglandin F2-like derivatives, specifically the F2-isoprostanes (F2-isoPs), have emerged as reliable biomarkers for oxidative stress in visceral tissues of mammals. To date, F2-isoP have primarily been used as biomarkers of lipid peroxidation to investigate the pathogenesis of human diseases (Inonu et al., 2012; , 2010), but have seldom been studied in Konishi et al., 2006; Pratico fish (Janicka et al., 2013; Klawitter et al., 2011; Langhorst et al., 2010; Milne et al., 2005). However, orally administered high molecular weight PAH exposures of 7.82 mg/g fish resulted in increased muscle concentrations of F2-isoPs in rainbow trout (Oncorhyncus mykiss) after 50 days (Bravo et al., 2011). Chung et al (2013) also demonstrated that F2-isoP levels in medaka rise when exposed to a classic oxidant, hydrogen peroxide (Chung et al., 2013). F2-isoPs are the free radical catalyzed products of non-enzymatic lipid peroxidation of arachidonic acid (AA) (Lawson et al., 1999), a fatty acid found abundantly in brain tissue and cell membranes of all animals. Several factors make F2-isoPs advantageous as biomarkers of oxidative stress. They are chemically stable, and have been detected at relatively constant low levels in all biological tissues and in the fluid of healthy vertebrates, namely urine, plasma, and cerebrospinal fluid (Lawson et al., 1999; Montuschi et al., 2004). F2-isoPs are also more reliable indicators of lipid peroxidation than commonly used TBARS as biomarkers of oxidative stress, as the latter is a non-specific assay and is therefore susceptible to interactions that reduce the assay's accuracy (Moselhy et al., 2013). High performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) has become the preferred method of analysis for F2-isoPs, as it achieves the desired separation and sensitivity without time-consuming derivatization and yields more reproducible results than enzyme-linked immunoassay (ELISA) kits (Klawitter et al., 2011; Waugh et al., 1997; Lee et al., 2008; Taylor et al., 2006).
Many established methods for analyzing fish health, including isoprostanes, involve lethal tissue sampling prior to chemical analysis (Chung et al., 2013; Zananski et al., 2011; Stahl et al., 2014; Hicken et al., 2011; Olsen et al., 2012). To minimize the impact of repeated sampling on ecosystems e often with small fish populations e monitoring programs assessing the health of wild populations are increasingly moving toward non-destructive and noninvasive methods (European Union. European, 2010). Current techniques for F2-isoP quantification make use predominantly of blood and urine as matrices, and while these fluids can be collected non-lethally from fish, current methods are still invasive and stressful. More recently, fish skin mucus has been investigated as a potential biological matrix for the analysis of fish health (Meucci and Arukwe, 2005; Fekih-Zaghbib et al., 2013; Ekman et al., 2015). Mucus has important biological functions for fish, ranging from communication and reproduction to osmotic regulation (Shephard, 1994). It is composed mainly of glycoproteins, but also contains immunoglobulins, pheromones, lysozyme, and proteolytic enzymes (Shephard, 1994). Previous analyses using this matrix include hemoglobin, metabolomics studies, and antioxidant activity assays (Meucci and Arukwe, 2005; Fekih-Zaghbib et al., 2013; Ekman et al., 2015), but mucus is also known to contain proteins, large molecules such as egg yolk precursors (Meucci and Arukwe, 2005) as well as RNA and DNA material (Livia et al., 2006; Le Vin et al., 2011; Ren et al., 2015). Here we describe the development and optimization of a HPLC-MS/MS method to isolate and quantify F2-isoPs isomers in fish mucus from several lakes in northwestern Ontario and Manitoba, Canada (Magnusson, 2014). Sampling was non-invasive, and processing was simple and rapid as few modifications to the matrix were needed prior to analysis. 2. Methods and materials 2.1. Materials Methanol (Optima grade), hydrochloric acid (HCl), and water (HPLC grade) were purchased from Fisher (Fair Lawn, NJ). (±) 5-isoprostaglandin-F2a-VI, (±) 5-iso-prostaglandin-F2a-VI-d11 (±indicates a racemic mixture of two enantiomers), 8-iso-prostaglandin-F2a, and 8-iso-prostaglandin-F2a-d4, ent-iso-15(S)prostaglandin- F2a and ent-iso-15(S)-prostaglandin- F2a-d9 (all >98%) were procured from Cayman Chemicals (Ann Arbor, MI). The chemical structures can be found in Fig. S1. 2.2. Sample collection Lake trout (Salvelinus namaycush) were angled from five experimental lakes at the International Institute for Sustainable Development e Experimental Lakes Area (IISD-ELA): lake 223 (49.698194 , 93.707952 ), lake 224 (49.688778 , 93.719360 ), lake 260 (49.694108 , 93.767919 ), lake 373 (49.743863 , 93.799417 ), and lake 626 (49.753069 , 93.796672 ) (Fig. 1). More experimental lake parameters can be found in Table S2. Individuals of mixed sex (n ¼ 7) were sampled from each of the five lakes (35 total) at over a period of two weeks in autumn (September 26 e October 10, 2018) to attempt preliminary measurements of baseline F2-isoP
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concentrations across a spectrum of lakes. Care was taken to minimize handling and to reduce the surface area denuded of mucus, to minimize impact on each individual fish. Fish were anaesthetized in lake water containing pH buffered MS-222 (0.4 g/ L), until all fin movement ceased (~3min), and then measured, weighed and their sex was determined using external characteristics. A small volume of mucus (10e50 mg, but variable among individuals) was scraped from the skin using methanol-rinsed forceps and aspirated using a sterile syringe. The sample was then dispensed into a 1.5 ml microcentrifuge tube and immediately frozen on dry ice for transport to the laboratory where they were stored at 80C until processing. The lake trout sampling procedures were conducted according to standards established by the Canadian Council on Animal Care and were approved under AUP #F16-018. Northern pike (Esox lucius) mucus samples collected from specimens angled in Rice River, MB (Latitude: 51.3364 N, Longitude: 96.4025 W), were provided by a recreational angler, though sample storage practices were still observed. These samples were used for method development and validation due to the large quantity available. 2.3. Sample preparation: fish mucus Northern pike mucus samples prepared from undiluted mucus (45 mL total) were used for the development of the extraction protocol. Lake trout mucus samples were prepared using the same method, but were not pooled. Samples were centrifuged at 5000 rpm for 15min. 50 mL of the supernatant was aliquoted into microcentrifuge tubes. Aliquots of 50 mL (n ¼ 10) were weighed, and found to have an average density of ~1.00 g/L. Deuterated isoprostanes (5 mL of 1 ng/mL stock) were spiked directly into the mucus. Sample volume was adjusted to 0.5 mL with methanol, acidified to pH 3 with 0.1 M HCl, and vortexed. Solid mucus components were removed by centrifuging for 5 min at 14,800 rpm. The supernatant was then transferred to 2 mL plastic microcentrifuge tubes, with two rinses of methanol. Samples were centrifuged again to assure full exclusion of particulates and precipitates, and transferred to fresh 2 mL vials to be blown down to 100 mL under a nitrogen stream, using an N-EVAP 111 (Organomation, Berlin, MA).
3
compound. Entrance potential was 10 V. The quantitative transition for 8-isoPG and its enantiomer ent-8-iso-15(S)-PG was m/z 353/193. The m/z 357/197 and m/z 362/300 transitions were monitored for their respective internal standards. The m/z 353/115 and m/z 364/115 transitions were monitored for (±) 5-isoPG-VI and its deuterated internal standard. Confirmation transitions can be found in Table S3. 2.5. Confirmation of isoprostanes by high resolution time of flight MS An Agilent 1200 LC system, consisting of a degasser, binary highpressure gradient pump, and autosampler coupled to an Agilent 6520A Q-ToF-MS system (Agilent Technologies, Mississauga, ON, Canada), was used to confirm the identity of isoprostanes in fish mucus extracts. LC separation was carried out on a Luna C18(2) column (2.0 mm 50 mm, 3 mm particle size) (Phenomenex, Torrance, CA, USA). The LC mobile phases were water (A) and methanol (B), and both contained 0.1% of formic acid. The mobile phase flow rate was 0.3 mL/min and the gradient was as follows: 0 min, 5% B; 0e5 min, 30% B; 1e7 min, 60% B; 7e15 min 80% B; 15e15.5 min 100% B; post run, 10 min. A volume of 20 mL of isoprostane standards (50 ng/mL) or fish mucus extract was injected into the LC system, respectively. The Q-ToF was tuned and calibrated with tuning calibration solution (G1969-85000) provided by Agilent Technologies. The resolution of the MS was >20 000 at both m/z 301.9981 and 601.9790 when the ESI interface was operated in the negative mode and the capillary voltage was 3500 V. The fragmentor and skimmer voltages were 180 and 80 V, respectively. Nitrogen was used as the drying and nebulizing gas. The gas temperature was 350 C, dry gas 10 L/min, and nebulizer 40 psi. Fullscan data acquisition was performed by scanning from m/z 50 to 1700. For each run, the reagent containing TFA anion (m/z 112.9855) and HP-0921 formate adduct (m/z 966.0007) were consistently introduced into the Q-ToF-MS as reference masses. Potential F2-isoP isomers in fish mucus extract were confirmed by monitoring its theoretical [M H]- ion (m/z 353.2333); m/z values within 5 ppm of its theoretical value were considered as a positive identification. 3. Results and discussion
2.4. Separation and quantitation of isoprostanes by triple quadrupole tandem MS All sample analyses were performed using a Varian 1100HPLC system (Palo Alto, CA) equipped with a CTC PAL autosampler. A Phenomenex (Torrance, CA) Zorbax Eclipse XDB-C18 column (2.1 mm 50 mm x 3.5 mm particle size) was used and maintained at room temperature. The mobile phase consisted of methanol (A) and water (B), both containing 0.1% formic acid (v/v). Flow rate was 300 mL/min. A solvent gradient was used to resolve the F2-isoP isomers: 30% B for 1min, increased to 60% B by 7min, 80% B at 15min, to 100% B at 15.5min, held for 1 min, then back to initial conditions by 17min. Injection volume was 5 mL. The HPLC was coupled to a Sciex API 365 triple quadrupole mass spectrometer (QqQ-MS) equipped with an ESI source. The source was a custom designed hot source induced desolvation (HSID) source with orthogonal injection. The instrument was operated in negative ionization mode, with the source temperature set to 500 C. Spray voltage was maintained at 3500 V. Samples were analyzed using multiple reaction monitoring (MRM) mode. Each compound was monitored using two MRM transitions; one for quantitation and one for confirmation. The eight chemical standards (four native F2isoP and four deuterated F2-isoP) were optimized individually. Declustering potential (DP), focusing potential (FP), collision energy (CE), and cell exit potential (CXP) were optimized manually for each
3.1. Method development The LC-MS/MS method parameters were validated according to the Eurachem guidelines for analytical methods (Magnusson, 2014). Matrix effect, recoveries, limits of detection, linearity, and repeatability were assessed and found to comply within the accepted ranges (Note: as previously mentioned, the concentrations of 5-isoPG-VI, 5-epi-isoPG-VI and their d-analogs are halved due to being racemic mixtures). Only Northern pike mucus was used for method development. 3.1.1. Linearity The linear dynamic range was determined by injecting standards prepared in methanol from stock mixture solutions containing four F2-isoP and their d-analogs, at concentrations of 5, 10, 25, 50, 100, 500, and 1000 pg/mL. The concentrations of 5-isoPG-VI, 5-epi-isoPG-VI, and their deuterated internal standards are halved (2.5, 5, 12.5, 25, 50, 250, 500 pg/mL), since they are 50:50 in their racemic standard solution. All compounds tested showed linearity within this concentration range (R2 > 0.99). A matrix matched approach was additionally carried for the deuterated F2-isoPs, using extracted pike mucus samples. Linearity was observed over this range of concentrations (R2 > 0.999). Matrix matched external standard (MMES) linearity was not attempted for non-deuterated
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native F2-isoPs due to interfering endogenous F2-isoP.
Table 2 Internal standard recoveries at varying IS concentrations in Northern pike mucus. Sample
3.1.2. Matrix effect Interference from matrix components is common in HPLC-ESIMS analysis, and may result in addition or suppression of the signal. Matrix effect was therefore assessed to determine the impacts on this F2-isoP protocol. Matrix matched standards were prepared by spiking 8-isoP-d4, ent-iso-15(S)-isoPG-d9, and (±) 5isoPG-VI-d11 at concentrations of 5, 10, 25, 50, and 100 pg/mL into final mucus extracts. Integrated areas were then compared to those of d-F2-isoP standards in methanol to determine the matrix effect, by taking the ratio of MMES and solvent calibration curves. Both MMES and standards in solvent were run in triplicates at each concentration. As shown in Table 1, matrix interference was relatively small using 50 mL mucus, ranging from 1 to 7%. R2 values were all >0.993. Matrix effects were consistent for each of the four isomers.
3.1.3. Recoveries Acceptable extraction efficiency from the mucus matrix was achieved for all four isoprostane isomers. Determination of analyte recoveries was conducted using matrix matched standards, to account for any possible signal interference from the matrix constituents. Peak areas of mucus samples (n ¼ 6) spiked with d-isoPs at three different concentrations (5, 25 and 100 pg/mL) were compared to matrix matched standards of equivalent concentrations. Recoveries of the four isoprostane isomers ranged between 78 and 95%, with RSD of 15% at all three concentration levels. Student t-tests were used to determine if statistically significant differences in analyte recovery existed between concentrations: recoveries did not differ significantly for any of the four isomers. Recovery of Type III isomers was slightly higher than Type VI isomers. Detailed results can be found in Table 2.
3.1.4. Limits of detection Low limits of detection were achieved for all four isoprostane isomers, ranging between 0.44 and 2.0 ng/g. Water blanks (n ¼ 10) were spiked with 0.5 ng of each F2-isoP and extracted according to the protocol, giving a final concentration of 5 pg/mL in the injected sample. These samples were prepared in distilled water because endogenous isoprostanes observed in the mucus samples would interfere with the signal. The internal standards were not used, as they showed lower responses than the native isoprostanes. Better detectability of the native isoPs, paired with the small matrix effect, justified the use of water as a sample matrix. Limit of detection (LOD) calculations were based on the Eurachem specifications ffi qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 , where n is the number of replicates), and (LOD ¼ 3s* 1n þ nblank converted to concentration in ng/g (Magnusson, 2014). Similarly, limits of quantitation (LOQs) were determined using a 10-fold multiplication in the above equation. These results are tabulated below.
Spiked
Measured (mean þSD)a
pg/mL 8-isoPG-d4
5-isoPG-d11
5-epi-isoPG-d11
ent-iso-15(S)-isoPG-d9
5 25 100 2.5 12.5 50 2.5 12.5 50 5 25 100
4.5 ± 0.7 21.6 ± 1.6 89.6 ± 11.7 2.1 ± 0.2 10.2 ± 0.9 39.0 ± 1.8 2.1 ± 0.4 10.1 ± 0.8 41.7 ± 3.3 4.6 ± 0.2 22.3 ± 2.2 95.4 ± 10.4
Recovery
RSD
%
%
90.5 86.3 89.6 82.2 81.7 78.1 84.7 81.1 83.3 92.1 89.0 95.4
14.7 6.3 13.1 8.2 7.6 4.6 9.6 6.6 7.8 5.1 8.8 10.3
RSD: Relative standard deviation, in %. a Mean and standard deviation (n ¼ 6).
3.1.5. Repeatability Northern pike mucus samples showed acceptable variability in isoprostane signal (RSD < 20%) for same-day and inter-day batch precision analysis. Method precision was measured through repeatability measurements. Same-day and inter-day repeatability were measured using Northern pike (n ¼ 6). Pike samples were analyzed with IS concentrations of 5, 25 and 100 pg/mL. Day-to-day repeatability tests were carried out for 3 days. The peak area ratios of quantitation: confirmation ion transitions were also monitored during batch analyses. Fragmentation patterns of stable molecules should remain constant during analyses if conditions are not changed. The average Q/C ratios of all four F2-isoP isomers remained constant (<10%) throughout the entirety of the method validation process, indicating the analyte underwent no degradation over the analysis period (data not shown). 3.2. Quantitation of F2- isoprostanes in fish samples 3.2.1. Northern pike All four selected F2-isoP isomers were observable and resolved chromatographically or by mass in Northern pike samples (see Fig. S4). Isoprostane peak identities were confirmed with relative retention times of deuterated internal standards. Concentrations ranged from 5.3 to 28.8 ng/g e detailed results can be found in Table 4 below.
Table 3 Limits of detection and quantitation of four F2-isoprostane isomers. Analyte
LOD (ng/g)
LOQ (ng/g)
8-isoPG 5-isoPG-VI 5-epi-isoPG-VI ent-iso-15(S)-isoPG
1.4 0.44 0.63 2.0
4.7 1.5 2.1 6.5
LOD: Limit of detection. LOQ: Limit of quantitation.
Table 1 Matrix effect calibration lines of four deuterated F2-isoprostanes in Northern pike mucus.a Analyte
Curve equation Matrix matched
8-isoPG-d4 5-isoPG-VI-d11 5-epi-isoPG-VI-d11 ent-iso-15(S)-isoPG-d9
y y y y
a
¼ ¼ ¼ ¼
986:8:5x þ 1735 1024:2x þ 405:8 1481:8x þ 1840 713:3x þ 399:4
Curve equation Solvent matrix y y y y
¼ ¼ ¼ ¼
1012:4x 166 986:4x 381 1595x 1453 723:7x 1129
% difference in matrix 97 ± 2 104 ± 3 93 ± 5 99 ± 4
Matrix effect is determined by dividing the slopes of a matrix-matched standard calibration curve and a standard curve from analyte prepared in a pure solvent, methanol.
P. Bulloch et al. / Chemosphere 239 (2020) 124797 Table 4 Endogenous F2-isoprostanes measured in Northern pike and lake trout mucus.a. Species/Lake
Northern Pike LT L224 LT L260 LT L373 LT L626
Concentration (ng/g)
b
8-isoPG
ent-8-iso-15(S)-PG
5-isoPG-VI
5-epi-isoPG-VI
28.8 ± 2.5 11.3
5.3 ± 0.8 ND ND 7.2 < LOQ
9.9 ± 1.3 ND ND 8.0
LT: Lake trout. ND: Not detected.
In addition, several other isoprostane peaks were fully or partially resolved, eluting up to 2min later than our compounds of interest. These were confirmed to be isoprostanes by high resolution LC-qTOF-MS to 2.5 ppm accuracy (Table S1). During the F2-isoP formation process, the multiple unsaturated sites of arachidonic acid result in 4 possible classes of regioisomers: Type III, IV, V, and VI, categorized by the location of the backbone hydroxyl group. Class-specific enantiomers bring up the total isomer count to 64. Resolving regioisomers and enantiomers is therefore essential since the accurate quantitation of selected isomers is dependent on freedom from interference from other stereoisomers with common quantitative m/z transitions. This study focused on two regioisomer classes: Type III and Type VI (shown in Fig. S1). The presence of other unidentified isoprostane isomers could make this method more broadly applicable as additional commercial standards become available for other F2-isoP isomers. 8-isoprostaglandin-F2a, a Type III isomer, is the most researched of these compounds, and has been linked to several human diseases such as atherosclerosis (Milne et al., 2005). However, other isomers e also lipid peroxidation endpoints e have the same functions and are more abundant (Reilly et al., 1998). A more comprehensive and accurate analysis of lipid peroxidation would be achieved by identifying and quantifying more isoprostanes.
3.2.2. Lake trout F2-isoprostanes were detected in individual fish in four of the five lakes, the exception being lake 223: an example chromatogram is shown in Fig. 2. Measureable Type III isoprostane peaks were present in only one out of seven individuals in lake 224, and were detected but below the LOQ in one individual from lake 260. In
5
contrast, three and four individuals showed measurable concentrations in lakes 373 and 626 respectively with concentrations ranging from 6.3 to 132 ng/g (see Table 4). The variability was large: the concentrations of Type III isoprostanes in lake trout individuals from L373 with measurable levels ranged from 7.5 to 132 ng/g. Similarly, in 4 individuals from L626, concentrations ranged from 6.3 to 70.7 ng/g, with an RSD of 91%. Parsons et al. (2009) demonstrated in rats and fathead minnows that natural variability is usually greater in biofluids such as mucus (~50%) than in tissues (~30%) (Parsons et al., 1039). The variability of metabolite concentrations in fathead minnow mucus was also shown to fall within the range of this natural variability typical of biofluids by Ekman et al. (2015) (Ekman et al., 2015). The 91% variation observed in our results may indicate that the collection method has to be modified. A potential solution could be to normalize isoprostane concentrations relative to protein and lipid content of the mucus, which may prove more accurate than total mass. Such normalization is common for other biomarker assays of exposure such as for ethoxyresorufin-O-deethylase (EROD) and TBARS (Sturve et al., 2006; Pampanin et al., 2016). Despite this, a qualitative comparison of isoprostanes can still be made on these preliminary results. A possible explanation for the higher isoprostane concentrations in trout from lakes 373 and 626 is the sampling dates, which coincide with the beginning of fall spawning season for lake trout (Plumb et al., 2014). Lake trout fall spawning typically begins when water temperature decreases closer to the species’ optimal temperature range of 8e12 C. Spawning is a stressful, energetically demanding process (Stewart et al., 1983), which could explain the heightened isoprostane concentrations observed in the samples from these lakes. Mean water temperatures (1 m depth) for the five experimental lakes (Fig. S5, Table S2) measured on the trout sampling days were within this range for the L373 sampling event (12.0 C), and just above for L626 sampling at 13.2 C. In comparison, the other three lakes range from 14.9 to 15.5 C e a significant difference of at least ~2 C. It is therefore possible that higher F2-isoprostane concentrations could be measured in the L223, L224, and L260 lake trout by sampling during spawning. However, further study is required to support this hypothesis, especially given the large variability observed in the data. The two Type VI isomers measured in this study, 5- and 5-epiisoprostanglandin-VI, were measurable only in one fish from L373 (see Table 4), though they were also present below LOQ in other individuals. Concentrations of both these compounds in the lake trout were within 2 ng/g (or < 30%) of those measured in the pooled Northern pike samples. Though encouraging, this result must be treated with caution as conclusions cannot be drawn before several more samples are analyzed to properly compare F2-isoP levels between the two species. Several chromatographic peaks with the m/z 353 / 115 transition were in evidence in most of the sampled individuals listed above. These peaks eluted up to 2.5min later than the four isomers selected for this study (see Fig. 3). These peaks cannot be identified with certainty because commercial standards for most isoprostane isomers are unavailable, however their high resolution molecular weights correspond to those in the samples who were previously identified as isoprostane isomers by HR-LC-MS/MS (Fig. 4, Table S1). Identifying and measuring these compounds is an important consideration for future studies. 4. Conclusion
Fig. 1. Location of IISD-Experimental Lakes Area research station in Northwestern Ontario, Canada (top right) and research lakes sampled for this study (main body). Credit: G. Gunn, IISD-ELA. Data sources: NRCAN and IISD-ELA.
In this study, four F2-isoprostane isomers were successfully separated and quantified in fish mucus of wild individuals from two species. Repeated F2-isoP measurements in individuals of a
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P. Bulloch et al. / Chemosphere 239 (2020) 124797
Fig. 2. HPLC-MS/MS elution of a) deuterated standards ent-8-iso-15(S)- prostanglandin-F2a-d9 (Peak 1) and 8-iso-prostanglandin-F2a-d4 (Peak 2) and b) endogenous ent-8-iso15(S)- prostanglandin-F2a (Peak 3) and 8-iso-prostanglandin-F2a (Peak 4) in a lake trout mucus sample. Other isoprostane peaks in the mucus are present but have yet to be positively identified.
Fig. 3. HPLC-MS/MS elution of a) endogenous 5-isoprostanglandin-F2a-VI-d11 (Peak 1) and 5-epi-isoprostanglandin-F2a-VI-d11 (Peak 2) and b) 5-iso-prostaglandin-F2a-VI (Peak 3) and 5-epi-iso-prostaglandin-F2a-VI in a lake trout mucus sample. Other isoprostane peaks in the mucus have not yet been posively identified.
P. Bulloch et al. / Chemosphere 239 (2020) 124797
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Fig. 4. High-resolution LC-qTOF-MS mass chromatogram and mass spectrum at 13.01 min (inset) of endogenous F2-isoprostane isomers in lake trout. The detailed information for all the peaks are provided in Table S1.
population over time could eventually prove to be an effective method for monitoring fish population health during and after an exposure to contaminants, as in vivo concentrations have been shown to relate with oxidative stress (Milne et al., 2005). Determining baseline levels of F2-isoP in fish populations is therefore a crucial first step in the implementation of this method, as baseline concentrations of F2-isoP likely vary between fish species and water bodies. Effects of water chemistry and temperature, temporal effects on physiology, and sexual dimorphism are all factors that require further study to better understand natural variability of F2isoPs in fish mucus. Several further studies are required to validate this method before it is deemed applicable for measuring oxidative stress in wild fish. Though it demonstrated that the method can be extended to species other than Northern pike, preliminary results showed large variability in isoprostane concentrations of individual lake trout, both within and between lakes. Concentrations need to be validated against more typical sampling matrices, such as plasma, under controlled laboratory conditions to establish if there is a reliable, reproducible relation between F2-isoP levels in these media. This will also provide a better understanding of natural variability. Additionally, the variability raises questions as to if normalizing F2-isoP concentrations to total lipid and protein content in each sample would provide a more accurate measure of lipid peroxidation. Another important consideration would be to determine if F2-IsoP concentrations differ when mucus is collected from different parts of the fish. Furthermore, determining a doseresponse relationship to chemical stressors and comparing response to other established biomarkers such as ratios of oxidized to reduced glutathione and malondialdehyde will determine the usefulness of this method as an ecological monitoring method. A study is currently underway to address these questions. Clearly, the validated method reported here demonstrates that quantifiable amounts of these robust oxidative stress biomarkers can be measured in very small mucus samples collected from fish in pristine environments and is a necessary prerequisite for future studies assessing the ecological significance of this new approach.
Note The authors declare no competing financial interest. Acknowledgment We thank the staff and students at the International Institute for Sustainable Development e Experimental Lakes Area (IISD-ELA) for providing data and assistance, especially Sonya Michaleski for her assistance in sample collection and processing. This work was supported in part by a Natural Science and Engineering Research Council of Canada Discovery Grant (Grant #: RGPIN/5303-2014) to G.T.T. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.124797. References Amin, K.A., Hashem, K.S., 2012. Deltamethrin-induced oxidative stress and biochemical changes in tissues and blood of catfish (Clarias gariepinus): antioxidant defense and role of alpha-tocopherol. BMC Vet. Res. 8 https://doi.org/ 10.1186/1746-6148-8-45. Bravo, C.F., Curtis, L.R., Myers, M.S., et al., 2011. Biomarker responses and disease susceptibility in juvenile rainbow trout Oncorhynchus mykiss fed a high molecular weight PAH mixture. Environ. Toxicol. Chem. 30 (3), 704e714. https:// doi.org/10.1002/etc.439. Cappello, T., Brand~ ao, F., Guilherme, S., et al., 2016. Insights into the mechanisms underlying mercury-induced oxidative stress in gills of wild fish (Liza aurata) combining1H NMR metabolomics and conventional biochemical assays. Sci. Total Environ. 548e549, 13e24. https://doi.org/10.1016/j.scitotenv.2016.01.008. Debruyn, A.M.H., Wernick, B.G., Stefura, C., et al., 2007. In situ experimental assessment of lake whitefish development following a freshwater oil spill. Environ. Sci. Technol. 41 (20), 6983e6989. https://doi.org/10.1021/es0709425. Dorval, J., Leblond, V.S., Hontela, A., 2003. Oxidative stress and loss of cortisol secretion in adrenocortical cells of rainbow trout (Oncorhynchus mykiss) exposed in vitro to endosulfan, an organochlorine pesticide. Aquat. Toxicol. 63 (3), 229e241. https://doi.org/10.1016/S0166-445X(02)00182-0. Ekman, D.R., Skelton, D.M., Davis, J.M., et al., 2015. Metabolite profiling of fish skin mucus: a novel approach for minimally-invasive environmental exposure monitoring and surveillance. Environ. Sci. Technol. 49 (5), 3091e3100. https:// doi.org/10.1021/es505054f.
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