DOI: 10.4231/ga9m-7080
title: Lipid profile of Zebrafish larvae exposed to low concentrations of lead (Pb) during the development period

description: &lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;This study was based on the hypothesis that low doses of lead (Pb) during embryonic development can trigger behavioral, biochemical, and lipid alterations. To test this, the detrimental effects of Pb were investigated in zebrafish (&lt;i&gt;Danio rerio&lt;/i&gt;) larvae. Wild-type (5D strain) adult zebrafish were bred, and fertilized eggs were collected and transferred to Petri plates (50 embryos per plate). Embryos were exposed to 0, 0.01, 0.1, or 1 ppb (ng/L) of Pb in embryo water solution (20 mL) from 1 to 72 hours post-fertilization (hpf). At 72 hpf, the Pb solution was removed by rinsing the fish three times with embryo water only.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;For lipidomic analysis, one larva from each plate was collected at 72 hpf and transferred to an Eppendorf tube. The remaining larvae were allowed to develop until 120 hpf, when another larva from each plate was collected for lipidomic analysis. The remaining individuals were used for behavioral and biochemical assessments. In total, eight larvae per group were collected from eight independent replicates (n = 8).&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Lipid profiling was performed using multiple reaction monitoring (MRM) profiling (Reis et al., 2023; Xie et al., 2021). For each concentration group, eight individual whole larvae were processed and evaluated. At 72 or 120 hpf, a single larva was collected, transferred to a 1.5 mL tube, and placed on ice to reduce activity. After movement ceased, residual water was removed, and 50 &micro;L of ultrapure water was added. Tubes were flash-frozen in liquid nitrogen and stored at &ndash;80 &ordm;C for up to one week until processing.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;For lipid extraction, samples were thawed and homogenized in ultrapure water for 30 s using pellet pestles (Kimble Kontes). Lipids were then extracted following the Bligh &amp; Dyer protocol as previously described (Lima et al., 2018). Briefly, 90 &micro;L of methanol (HPLC grade) and 50 &micro;L of chloroform (HPLC grade) were added sequentially, with mixing by pipetting after each addition to generate a single-phase solution. Samples were incubated at room temperature for 15 min, followed by the addition of 50 &micro;L of ultrapure water and mixing. Next, 50 &micro;L of chloroform was added and mixed again. Samples were centrifuged at 5000 &lt;i&gt;&times; g&lt;/i&gt; for 5 min at room temperature, and the lower (organic) phase was transferred to a new tube and dried in a SpeedVac for 12 h at room temperature. The dried extracts were resuspended in 200 &micro;L of acetonitrile:methanol:ammonium acetate (3:6.65:0.35, v/v/v) with a final ammonium acetate concentration of 10 mM.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; text-indent:.5in; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;For data acquisition, samples were diluted seven times using the same solution in which they were resuspended. The total detected lipids in the MRM profiling exploratory experiment and conditions were the same as previously described (Edwards et a., 2021). In summary, data acquisition was performed using flow-injection (no chromatographic separation) from 10 &mu;L of the diluted lipid extract stock solution which was delivered using a micro-autosampler (G1377A) to the ESI source of an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). A capillary pump was connected to the autosampler and operated at a flow rate of 8 &mu;L/min and pressure of 150 bar. The capillary voltage on the instrument was 5 kV and the gas flow was 5.1 L/min at 300&deg;C.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt; &lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;b&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Notes:&lt;/span&gt;&lt;/b&gt;&lt;span new="" roman="" style="font-family:" times=""&gt; Data were produced using an Agilent 6410 QQQ mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Raw MRM mass spectrometry data was processed using an in-house script and MRM transition. Samples that had a sample ion intensity 30% higher than the blank were selected for further analysis. Each ion intensity was normalized by the Total Ion Intensity (TIC) in the respective class that it belong.&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;br /&gt;
 &lt;/p&gt;

&lt;p style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;b&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;References&lt;/span&gt;&lt;/b&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;Reis LG, Casey TM, Sobreira TJ, Cooper BR, Ferreira CR, 2023. &lt;/span&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Step-by-Step approach to build multiple reaction monitoring (MRM) profiling instrument acquisition methods for class-based lipid exploratory analysis by mass spectrometry. Journal of Biomolecular Techniques: JBT 34&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Xie Z, Ferreira CR, Virequ AA, Cooks RG, 2021. Multiple reaction monitoring profiling (MRM profiling): Small molecule exploratory analysis guided by chemical functionality. Chemistry and Physics of Lipids 235, 105048&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span lang="PT-BR" new="" roman="" style="font-family:" times=""&gt;de Lima CB, Ferreira CR, Milazzotto MP, Sobreira TJP, Vireque AA, Cooks RG, 2018. &lt;/span&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Comprehensive lipid profiling of early stage oocytes and embryos by MRM profiling. Journal of mass spectrometry: JMS 53, 1247&ndash;1252&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p class="MsoBibliography" style="text-align:justify; margin-bottom:11px"&gt;&lt;span style="font-size:12pt"&gt;&lt;span style="line-height:115%"&gt;&lt;span style="font-family:Aptos,sans-serif"&gt;&lt;span new="" roman="" style="font-family:" times=""&gt;Edwards ME, Marasco Jr CA, Schock TB, Sobreira TJ, Ferreira CR, Cooks RG, 2021. Exploratory analysis using MRM profiling mass spectrometry of a candidate metabolomics sample for testing system suitability. International Journal of Mass Spectrometry 468, 116663&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/span&gt;&lt;/p&gt;
description: Target-MRM profile of whole-zebrafish larvae at 72 hours post-fertilization (hpf) and 120 hpf after developmental exposure (1 – 72 hpf) to Pb at low concentrations of 0, 0.01, 0.1, and 1 ppb.