Name: quantification of ethanol levels in zebrafish embryos using head space gas chromatography
Uploaded: 2020-02-11T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

The research presented in this article was supported by previous grants from National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR) R01DE020884 to J.K.E. and National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA) F32AA021320 to C.B.L. and by the current grant from National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L. We thank Rueben Gonzales for providing and assisting with gas chromatograph analysis. We thank Tiahna Ontiveros and Dr. Gina Nobles writing assistance.

All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols27. These protocols were approved by the University of Texas at Austin and the University of Louisville.
NOTE: The zebrafish line Tg(fli1:EGFP)y1 was used in this study28. All water used in this procedure is sterile reverse osmosis water. All statistical analyses were performed using Graphpad Prism v8.2.1.
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+concentration+in+56+refillable+electronic+cigarettes+liquid+formulations+determined+by+headspace+gas+chromatography+with+flame+ionization+detector+(HS-GC-FID).">Ethanol concentration in 56 refillable electronic cigarettes liquid formulations determined by headspace gas chromatography with flame ionization detector (HS-GC-FID).</a> Drug Testing and Analysis. 9 (10), 1637-1640 (2017).</li><li>Heit, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Neural+Ethanol+and+Acetaldehyde+Using+Headspace+GC-MS.">Quantification of Neural Ethanol and Acetaldehyde Using Headspace GC-MS.</a> Alcoholism: Clinical and Experimental Research. 40 (9), 1825-1831 (2016).</li><li>Chun, H. J., Poklis, J. L., Poklis, A., Wolf, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Validation+of+a+Method+for+Alcohol+Analysis+in+Brain+Tissue+by+Headspace+Gas+Chromatography+with+Flame+Ionization+Detector.">Development and Validation of a Method for Alcohol Analysis in Brain Tissue by Headspace Gas Chromatography with Flame Ionization Detector.</a> Journal of Analytical Toxicology. 40 (8), 653-658 (2016).</li><li>Schlatter, J., Chiadmi, F., Gandon, V., Chariot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+methanol,+acetaldehyde,+acetone,+and+ethanol+in+human+blood+by+gas+chromatography+with+flame+ionization+detection.">Simultaneous determination of methanol, acetaldehyde, acetone, and ethanol in human blood by gas chromatography with flame ionization detection.</a> Human and Experimental Toxicology. 33 (1), 74-80 (2013).</li><li>Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography.">Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.</a> Journal of Visualized Experiments. (67), e4142 (2012).</li><li>Adalsteinsson, E., Sullivan, E. V., Mayer, D., Pfefferbaum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+quantification+of+ethanol+kinetics+in+rat+brain.">In vivo quantification of ethanol kinetics in rat brain.</a> Neuropsychopharmacology. 31 (12), 2683-2691 (2006).</li><li>Quertemont, E., Green, H. L., Grant, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+ethanol+concentrations+and+ethanol+discrimination+in+rats:+effects+of+dose+and+time.">Brain ethanol concentrations and ethanol discrimination in rats: effects of dose and time.</a> Psychopharmacology. 168 (3), 262-270 (2003).</li><li>Flentke, G. R., Klinger, R. H., Tanguay, R. L., Carvan, M. J., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionarily-conserved+mechanism+of+calcium-dependent+neurotoxicity.">An evolutionarily-conserved mechanism of calcium-dependent neurotoxicity.</a> Alcoholism: Clinical and Experimental Research. 38 (5), 1255-1265 (2014).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. 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As a developmental model system, zebrafish are ideally suited to study the impact of environmental factors on development. They produce large numbers of externally fertilized embryos, which allows for precise timing and dosage paradigms in ethanol studies. This, combined with the live imaging capabilities and the genetic and developmental conservation with humans, make zebrafish a powerful model system for teratology studies. Described is a protocol for measuring embryonic ethanol concentrations in developing zebrafish e...

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of neurological impairments and craniofacial dysmorphologies associated with embryonic ethanol exposure1. Multiple factors, including timing and dosage of ethanol exposure and genetic background, contribute to the variation of FASD2,3. In humans, the complex relationship of these variables makes studying and understanding the etiology of FASD challenging. Animal models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity. A wide variety of animal model systems has been used to study multiple aspects of FASD and results have been remarkably consistent with what is found in exposure in humans4. Rodent model systems are used to examine many aspects of FASD, with mice being the most common5,6,7. The majority of this work has focused on developmental defects to early ethanol exposure8, though later exposure to ethanol has been shown to cause developmental anomalies as well9. Moreover, the genetic capabilities of mice have greatly aided in our ability to probe the genetic underpinnings of FASD10,11. These studies in mice strongly suggest that there are gene-ethanol interactions with the sonic hedgehog pathway, retinoic acid signaling, Superoxide dismutase, Nitric oxide synthase I, Aldh2 and Fancd28,10,11,12,13,14,15,16,17,18,19,20,21. These studies show that animal models are critical to advancing our understanding of FASD and its underlying mechanisms.
The zebrafish has emerged as a powerful model system to examine many aspects of ethanol teratogenesis22,23. Due to their external fertilization, high fecundity, genetic tractability, and live imaging capabilities, zebrafish are ideally suited to study factors such as timing, dosage, and genetics of ethanol teratogenesis. Ethanol can be administered to precisely staged embryos and the embryos can then be imaged to examine the direct impact of ethanol during developmental processes. This work can be related directly to humans, because the genetic programs of development are highly conserved between zebrafish and humans and can therefore help guide FASD human studies24. While zebrafish have been used to examine ethanol teratogenesis, a lack of consensus in reporting embryonic ethanol concentrations makes comparison to humans difficult25. In mammalian systems, blood-alcohol levels correlate directly to tissue ethanol levels26. Many of the zebrafish studies treat embryos before complete formation of their circulatory system. With no maternal sample to examine, a process to assess ethanol concentrations is required to quantify ethanol levels within the embryo. Here we describe a process to quantify ethanol concentrations in a developing zebrafish embryo using head space gas chromatography.

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			<td>Air</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>10204-962</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoSampler, CP-8400</td>
			<td>Varian</td>
			<td></td>
			<td>Gas Chromatograph Autosampler</td>
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		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td></td>
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		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph vial with polytetrafluoroethylene/silicone septum and plastic cap 2 mL</td>
			<td>Agilent</td>
			<td>8010-0198</td>
			<td>Can reuse the vials after cleaning, but not the caps/septa</td>
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			<td>Gas Chromatograph, CP-3800</td>
			<td>Varian</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Helium</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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		<tr>
			<td>HP Innowax capillary column</td>
			<td>Agilent</td>
			<td>19095N-123I</td>
			<td>30 m x 0.53 mm x 1.0 &#956;m film thick</td>
		</tr>
		<tr>
			<td>Hyrdogen</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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			<td>Magnesium Sulfate (Heptahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>2682002</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 10 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 1000 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611127</td>
			<td></td>
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		<tr>
			<td>Micropipette tips 200 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611112</td>
			<td></td>
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			<td>Petri dishes 100 mm</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
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			<td>Pipetman L p1000L Micropipette</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p200L Micropipette</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p2L Micropipette</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene/silicone septum and plastic cap</td>
			<td>Agilent</td>
			<td>5190-7021</td>
			<td>Replacement caps/septa for gas chromatograph vials</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Dibasic)</td>
			<td>VWR</td>
			<td>BDH9266-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>VWR</td>
			<td>97062-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads .5 mm</td>
			<td>Biospec Products</td>
			<td>11079105z</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads 1.0 mm</td>
			<td>Biospec Products</td>
			<td>11079110z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>VWR</td>
			<td>BDH9280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid-phase microextraction fiber assembly Carboxen/Polydimethylsiloxane</td>
			<td>Millipore Sigma</td>
			<td>57343-U</td>
			<td>Replacement fibers</td>
		</tr>
		<tr>
			<td>Star Chromatography Workstation</td>
			<td>Varian</td>
			<td></td>
			<td>Chromatography software</td>
		</tr>
		<tr>
			<td>Thermogreen Low Bleed (LB-2) Septa</td>
			<td>Millipore Sigma</td>
			<td>23154</td>
			<td>Replacement inlet septa</td>
		</tr>
	</tbody>
</table>

quantification of ethanol levels in zebrafish embryos using head space gas chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

Blood ethanol levels cannot be determined in early embryonic zebrafish, because they lack a fully formed circulatory system. To determine the level of ethanol concentration in the zebrafish embryos, the ethanol levels are measured directly from homogenized embryonic tissue. To properly measure the embryonic ethanol concentrations, the embryonic volume has to be taken into account. The embryo (yolk attached) sits inside the chorion (eggshell) surrounded by extraembryonic fluid (Figure 1). Any...

Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Research

JoVE Journal

Developmental Biology

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Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

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Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

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Isolation and Characterization of Single Cells from Zebrafish Embryos

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Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

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Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

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Induction of Hypoxia in Living Frog and Zebrafish Embryos

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Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

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Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...