Gas Chromatography for Fatty Acid Analysis

Jack McAlhany, Wofford College

Untitled

In applying to College of Charleston REU at Grice Marine Laboratory, I assumed I would be researching the well-being of a local fishery or the biomechanics of an organism in response to different variables, but this was not the case. My research to this point has not focused on a living organism, but rather tiny portions of blood plasma that we vaporize into a gas to determine the basic constituents of the blood. A solid understanding of chemical structures, or a wild imagination, is necessary for this research because unlike working with live samples, our desired product is not visible. To give you a better understanding, the fatty acids we are detecting have between 8 and 24 carbon atoms while a 70 kg human has approximately 7X10^26 carbon atoms (Kross, B.).

In order to detect such tiny fat molecules I am using a gas chromatography with a flame ionization detector (GC-FID). This instrument converts a liquid sample to a gas that then travels through a 100 meter column with an inert gas carrier. The time at which certain samples exit the column is reproducible, so the retention time in the column is a way to determine which fatty acids are in a blood plasma sample (E.T.S. Laboratories).

11

Figure 1: In a GC-FID, a sample (red) is vaporized in a vaporizing column (blue) and an inert gas carrier is the “liquid” phase that carries the vaporized fatty acid methyl ester (FAME) through a 100 meter column (gold). This column has a stationary phase that interacts differently with each FAME, causing the FAME’s to exit the column at different times. The detector responds to compounds that form ions when combusted in the hydrogen-air flame. This response is converted to electrical signals and produce a gas chromatogram we can analyze (E.T.S. Laboratories).

These retention times are represented on a gas chromatogram, which consists of peaks of varying sizes (Figure 2). Each fatty acid interacts in a unique, but constant, way with the stationary phase of the column, so fatty acid peaks are in the same order and have the relatively same retention time every trial. This constant retention time allows us to determine the fatty acid composition of a sample. Not only can we find the blood plasma fatty acid composition, but by analyzing the area under each peak, we can determine the concentration of each fatty acid in the blood. The concentration of each fatty acid or relationships in these concentrations hopefully will provide a biomarker that we can test in the field for the fatty acid disease, pansteatitis.

Figure5-16

Figure 2: Gas Chromatogram of 19 fatty acids. Each peak is labeled with its corresponding fatty acid (Christie W. 2011).

Acknowledgements

I would like to thank the College of Charleston for this internship, the National Science Foundation for funding, and Dr. John Bowden for his guidance as a mentor.

MUSC_TAG_4C  CofClogoUnknown-2 Unknown-4

Works Cited

Christie, W. 2011. Gas Chromatography and Lipids. The Oily Press” Chapter 10.http://lipidlibrary.aocs.org/GC_lipid/10_misc/index.htm

E.T.S. Laboratories https://www.etslabs.com/analysis.aspx?id=$GCP

Kross, B. Questions and Answers. Jefferson Lab. http://education.jlab.org/qa/mathatom_04.html.

Pansteatitis: A Fat Inflammatory Disease in South African Fishes

Jack McAlhany, Wofford College

  Untitled    

I applied for College of Charleston REU uncertain of the specific research I would partake in this summer. The email finally arrived containing my area of focus and I immediately told my parents “ This is perfect and will unify the topics I studied in school this year. I am researching a disease of the pancreas. My project is titled ‘Pancreatitis: an environmentally-induced inflammatory disease of South African fishes. ’ ” I arrived at Grice Marine Laboratory well-read on the etiology and symptoms of pancreatitis, only to find that an exchange of two little letters could make all the difference in my summer plans.

My research this summer was actually on panSTeatitis, a non-transmissible,  inflammatory disease of the fat tissue in South African fishes, particularly the tilapia. The interest for this research arose in 2008 when, as seen in Figure 2, 170 of the 600 Nile Crocodiles in Kruger National Park in South Africa died and the cause of death was attributed to pansteatitis (Ashton, 2010). The crocodiles are considered a sentinel species (Botha et al. 2011) as well as one of the main tourist attractions of the park, so this mobilized considerable interest into determining the causes of pansteatitis. After considerable research from various laboratories, the general consensus is that pansteatitis is the oxidation and eventual death of fat tissue seen in Figure 1 (Huchzermeyer, 2012), although the cause of the oxidation is still questioned. The proposed roots of pansteatitis are consumption of rancid, already oxidized fats, consumption of a high polyunsaturated to saturated fatty acids, which are more readily oxidized, or ingestion of metal pollutants that exacerbate the oxidation process (Huchzermeyer et al. 2013).

pansteatitis fat

Figure 1: Early pansteatitis lesion in mesenteric fat of sharp tooth catfish (left). Adipose tissue necrosis after developed pansteatitis in the same fish (right) (Huchzermeyer, 2012).

croc dead

Figure 2: Nile Crocodiles found dead in Kruger National Park due to pansteatitis (Rickrideshorses, 2011).

We will be researching the fatty acid composition of tilapia, which act as a model organism, and attempting to find a relationship among the fatty acids that will be able to act as a biomarker for determining whether an organism is affected with pansteatitis. This biomarker will allow for field testing using a simple blood test rather than the current method, which requires euthanasia to observe the fat tissue. A success in our research will facilitate sampling of organisms for pansteatitis and hopefully hone in on a potential cause of pansteatitis.

Acknowledgements

Funding for analysis of data is through the National Institute of Standards and Technology (NIST). Funding for sample collection is from Dr. Bowden, MUSC and Dr. Guillette, as well as collaborative South African laboratories.

NIST        MUSC

Bibliography

Ashton, P. 2010. The demise of the Nile crocodile (Crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in South Africa: The case of the Olifants River. Aquatic Conservation: Marine and Freshwater Ecosystems. 20: 489-493.

Botha, H., Van Hoven, W., Guillette, L. 2011. The decline of the Nile crocodile population in Loskop Dam, Olifants River, South Africa. Water SA. 37(1): 103-108.

Huchzermeyer, D. 2012. Prevalence of pansteatitis in African sharptooth catfish, Clarias gariepinus (Burchell), in the Kruger National Park, South Africa. Journal of the South African Veterinary Association. 83(1).

Huchzermeyer, D., Osthoff, G., Hugo, A., Govender, D. 2013. Comparison of the lipid properties of healthy and pansteatitis-affected African sharptooth catfish, Clarias gariepinus (Burchell), and the role of diet in pansteatitis outbreaks in the Olifants River in the Kruger National Park, South Africa. Journal of Fish Diseases. 36(11): 897-909.

Rickrideshorses. 2011. The Pansteatitis Pollution that Turned Crocodiles to Rubber. HubPages.