Antioxidants and lipid peroxidation

[michelle409]

Hi,
I am trying to narrow down my ideas to formulate a science fair experiment worthy of advancing to ISEF. Right now, I am looking into antioxidants and how they can be used to combat lipid peroxidation in yeast or other organisms such as algae. I want to know the best method of exposing the organisms to free radicals and how to qualitatively measure maybe the respiration rate to see whether the free radicals and antioxidants are affecting the mitochondria as suggested in the theory of aging.

I would also like to know If there is a more novel way at looking at this situation. As of now, I have access to the following equipment: http://biocurious.org/about/. I can also use a spectrophotometer and can find ways to access more equipment if need be.

Thank you greatly.

[SciB]

Hi Michelle,
Sorry for taking so long getting back to you, but I was doing some background reading on lipid peroxidation in yeasts and algae as I am only familiar with studies in mammals. Plants and fungi are very different from humans!

One thing of interest, at least to me, was the fact that certain species of algae are producers of antioxidants. Here’s a paper I found in which the researchers tested the effects of nutrients and salinity in the algal growth medium, light intensity and temperature on the growth and antioxidant capacity of a marine algae called Dunaliella salina. The authors’ hypothesis appears to be that this algae can be grown in saltwater, which is available for free in many countries, with the addition of some inexpensive N-P-K fertilizer.

World J Microbiol Biotechnol. 2013 Jul 3. [Epub ahead of print] Growth, antioxidant capacity and total carotene of Dunaliella salina DCCBC15 in a low cost enriched natural seawater medium. Tran D, Doan N, Louime C, Giordano M, Portilla S. School of Biotechnology, International University, VNU, Thu Duc Dist., Ho Chi Minh, Vietnam, [email protected].

Something like this would, I think, make an excellent science fair project with lots of possibilities. Algae can be grown and harvested cheaply as a source of medicine or food, or both, and countries with limited resources could use this method to improve the health of their population.

If you google ‘antioxidants algae’ you will see a lot of information about using algae as a source of antioxidants and other nutraceuticals. Maybe you can get some good ideas there for a project.

Post back to Scibuddies when you have narrowed down your project choices and we will help you with the planning and design angles. I think you are on to something really important here.

Best wishes,

SciB

[michelle409]

Hi SciB,

Thank you so much for these suggestions! After reading through a few more articles, I found a type of algae called Aphanizomenon flos-aquae which has antioxidant properties. This is the article from which it is from: http://www.ncbi.nlm.nih.gov/pubmed/15350832. The article explains how the nutrient enriched pigment was extracted and added to blood cells. Do you think it would be a good idea if I extracted the pigment and added it to Saccharomyces cerevisiae (yeast)? The yeast would be exposed to hydrogen peroxide or another element that would induce free radicals. I could also use Dunaliella salina, and if I were to use algae, how would I extract the pigments/antioxidants?

[SciB]

Hi Michelle,

I read the abstract of the paper you found on the A. flos-aquae cyanobacterial extract and the phycocyanin sounds like an effective antioxidant. However, upon doing a search for Aphanizomenon, I found that cultures of the organism can produce neurotoxins (http://www.ncbi.nlm.nih.gov/pubmed/23792258) so it is probably not a good choice for a science fair project. In another paper (http://www.ncbi.nlm.nih.gov/pubmed/23726538) an extract of a different species of Aphanizomenon, A. ovalisporum, was toxic to the green algae Chlorella vulgaris, so while A. flos-aquae may have antioxidant properties, its toxicity would prohibit its use in humans.

Your idea to use algae or cyanobacteria as a source of antioxidants is a good one, but be careful to pick a source organism that is harmless to humans.

I looked for a way to make an extract of Dunaliella and I found this paper that is open access and may have some information about the process http://www.ncbi.nlm.nih.gov/pubmed/19475448. I would think that you might be able to simply disrupt the cells using a Dounce homogenizer or something similar (http://www.douncehomogenizer.com/) and centrifuge them to remove the debris. This would leave you with a semi-purified extract that you could use to treat the yeast. The homogenizers are expensive, but many biology labs do have them.

Good luck and keep me posted on your ideas and progress.

Best wishes

[michelle409]

Hi SciB,

I think the idea of extracting the carotenoids from the Dunaliella is great and I am pretty sure I can get access to a dounce homogenizer.
For my experiment, I think I will choose two or three commonly used antioxidants such as vitamins B and C and compare their effects to the antioxidants extracted from the dunalliela. Another variable I am looking to add is either different amounts of a single oxidative stress or different types of oxidative stresses. So far, I know that hydrogen peroxide is commonly used, but I'm looking into other options as well. One problem I am having is trying to find the best method of inducing the stress into the yeast and finding a way to measure the effects qualitatively.

I've also been trying to find recent research papers that associate with my project, so if you come across any, please let me know. I found this one article that talks about the antioxidant effects of Dunalliela in lab mice, and it uses CCl4 as the oxidative stress, but it is not as recent: http://www.ncbi.nlm.nih.gov/pubmed/15670617

Thank you again for your help!

[SciB]

Hi Michelle,

I was wondering how you planned to induce and measure oxidative stress in yeast. You should not use carbon tetrachloride as it is extremely toxic. Stick with hydrogen peroxide. It is easy to get and relatively harmless. What other oxidants were you thinking of using?

A simple way to measure the protective effects of antioxidants is by counting the yeast cells with a hemocytometer. The peroxide will kill some of the yeast cells and if your hypothesis is correct, the D. salina extract should protect them. You can do an initial count of the yeast cells, expose them to various concentrations of peroxide with or without antioxidant and count them after a certain period of time. The protected cultures should contain more yeast cells.

Most biology labs have hemocytometers and someone there can show you how to use one. Here are a couple of links that explain what it is and the correct method for doing dilutions, counting and calculating the number of cells per ml: http://www.ruf.rice.edu/~bioslabs/metho ... nting.html http://www.youtube.com/watch?v=pP0xERLUhyc

I know there are many details to work out in culturing the marine algae, growing the yeast and exposing it to the oxidants and antioxidants, so when you need help, just send us another post. We're here to help.

SciB

[michelle409]

Hi SciB,

I like the idea of using a hemocytometer, and I have already contacted many professors to see if I can use theirs. As for measuring the oxidative stress, I haven't come across a method that is doable for my level, but I am still looking. If you find anything please let me know.

For the oxidants, I looked into a couple different chemicals and I think I want to use hypochlorous acid (HClO), hydroxyl radical (·OH), and the superoxide anion (O2−) in addition to hydrogen peroxide.

After I figure out the basic methods and procedure of my experiment, I will start to look into the small details.

Thank you!

[michelle409]

Hi Scib,

I was doing some more research and I came across this abstract in which the theobarbituric acid test is performed to detect for lipid peroxidation: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC178803/.

In this paper, the yeast is analyzed on a more molecular level which I want to try to do. As I cannot access the full paper, I cannot read all the methods, but are there any methods that you think I can perform?

Also, I am still looking in to how to induce the oxidants into the yeast.

Thanks again!

[SciB]

Hi Michelle,

It sounds like you have made a lot of progress on your antioxidant project but are having trouble with the details. That is very common in science! Let me see if I can help you.

Your experiments involve both chemistry and biology so are a bit more difficult than most. If I understood you correctly, you are planning to isolate carotenoids from Dunaliella, so you will have to learn how to grow this alga and make the antioxidant extract. You are going to test the extract on yeast so you need to know how to consistently grow the yeast and measure its numbers using the hemocytometer. Then you have to produce the oxidants that cause the lipid damage that you are trying to protect against, and lastly you have to develop a method for quantitating the changes in the level of lipid damage to the yeast. That’s a pretty ambitious undertaking!

In regard to which compounds to use as oxidants, I would definitely go with hydrogen peroxide although you will have to do some searching to determine the range of concentrations to test. You can buy H2O2 at the grocery store. The same for sodium hypochlorite (Clorox) which will provide the hypochlorous acid, HOCl, although there again I have no idea what concentrations you should use. Also, when I looked up hypochlorous acid (http://en.wikipedia.org/wiki/Hypochlorous_acid), it said that it does not cause lipid peroxidation but rather adds a chloride atom across the double bond which alters the lipid’s structure. If you are assaying for lipid peroxidation then HOCl might not be a good choice.

Superoxide radicals are produced naturally by cells of the immune system as part of their attack of bacteria and parasites, but making superoxide chemically is not easy. I found one compound, abbreviated SOTS-1, that can be used to generate superoxide radical anions available from Cayman Chemical Co. https://www.caymanchem.com/app/template ... g/10009642 It is not very expensive, but you would have to get your school or mentor to order it.

Cayman also sells a thiobarbituric assay kit for lipid peroxides, but it IS expensive--$185, and you would need to have a spectrophotometer or plate reader to do the assay. You could but it from Sigma-Aldrich http://www.sigmaaldrich.com/catalog/sea ... partialmax I attached a copy of the article you mentioned in your previous email so you can read about the TBA assay method.

I’m sure you will have more questions! We’ll be here to help.

All the best,

Sybee