Methods for the Manatees

Kaylie Anne Costa, University of Miami

IMG_1217

The Approach: In my previous post, I described cold stress syndrome (CSS) in Florida manatees and the major threat it poses to the survival of this integral species. To expand the current scientific knowledge of CSS, I will be analyzing the lipids (aka fats) and metabolites, which are the products remaining after biological processes such as digestion, respiration, and maintenance of homeostasis, in 12 healthy and 21 CSS-affected manatee plasma samples in hopes of learning more about the metabolism of this condition and potential avenues for therapeutic applications.

In order to study the lipids and metabolites in manatee blood, I will be using liquid chromatography and mass spectrometry (LC/MS) with an electrospray ionization source. Metabolomics and lipidomics will be separately analyzed. After a chemical extraction is performed to selectively separate either the lipids or metabolites in the plasma, each extract will be individually injected into the chromatographic column to separate the chemical compounds present so that only similar compounds are analyzed in any moment of time (methodology proposed by Bligh & Dyer, 1959 and Cambridge Isotope Laboratories, Inc.). Once the separated compounds reach the end of the column, they are

css manatee

Rescued manatee showing signs of cold stress syndrome. (Photo from: https://savethemanateenewtech.weebly.com/endangerment.html)

transferred to the electrospray ion source where a high temperature and voltage will be applied to evaporate the solvent and give the compounds a charge to form ions that are then directed into the mass spectrometer. Within the mass spectrometer, the ions will first be filtered by electric fields to remove anything other than either lipids or metabolites and then detected by mass to charge ratio. The most abundant ions will be fragmented and the mass to charge ration of the fragments will also be detected using an MS/MS scan. To see an animation of the flow of ions through the mass spectrometer, please click the following hyperlink: https://www.youtube.com/watch?v=_A6NBBBcdts

As a result of the above processes, retention times for each ion are displayed in a graphical form called a chromatogram and the mass spectrum is recorded. Since the masses and retention times will not change between scans, these parameters for each ion can be matched to known databases of known lipids and metabolites. By applying multivariate statistics, we can determine if there is a difference in the lipids and/or metabolites in the plasma of manatees with CSS compared to healthy manatees.

flow chart

The top left graph shows a chromatogram. The highlighted peak is then shown on the mass spectrum below with a mass to charge ratio (m/z) of 760.58607. By locating this m/z and the m/z of its fragments in the mass spectrum of a MS/MS scan and matching the values with a database, we know the original peak represents Phosphatidylcholine (16:0_18:1).                                        (Graphic by Dr. Mike Napolitano)

The goal of my project is to see if CSS alters the lipid and metabolite contents of manatee plasma. If differences exist, I will study them to learn more about the progression of cold stress syndrome in manatees and the particular systems and metabolic pathways that are affected. It is our hope that this information leads to developing both diagnostic and treatment options for these animals thereby reducing the impacts of this syndrome.


A huge thank you goes to my mentor Dr. John Bowden and co-mentor Dr. Mike Napolitano as well as everyone at NIST, HML, and Fort Johnson for all of their help and guidance. I would also like to thank the National Science Foundation for funding and the Fort Johnson REU program for making this research possible (NSF DBI-1757899).


References:

Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and

purification.Canadian journal of biochemistry and physiology, 37(8), 911-917.

Cambridge Isotope Laboratories, Inc. Metabolomics QC Kit For Untargeted/Targeted Mass

Spectrometry: User’s Manual. Tewksbury, MA: Author.

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