Determination of Tolerable Fatty Acids and Cholera Toxin Concentrations Using Human Intestinal Epithelial Cells and BALB/c Mouse Macrophages

Summary

We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin that did not significantly and adversely affect cell survival by solubilizing the fatty acids and the toxin and using them in cell survival assays.

Abstract

The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages.

We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

Introduction

The health benefits of fatty acids, such as oleic, linoleic and linolenic acids have been and continue to be documented. For example, oleic acid helps facilitate penetration of lipophilic drugs in the body^1,2, reduces coronary heart disease by 24% when substituted for saturated fatty acids³, and is used to treat metabolic diseases such as Adrenoleukodystrophy⁴ which is an X-linked genetic disorder of fatty acid metabolism. While a necessary precursor for arachidonic acid in mammals, linoleic acid (unlike oleic acid) is not synthesized by the body and must be obtained through outside sources such as by flax seed consumption.

Protocol

1. Tissue Culture

1. Use Mus musculus macrophages (BALB/c mice) for cholera toxin determinations. Initially culture all of M. musculus cells following vendor's instructions.
2. Propagate BALB/c mice cells in Dulbecco's Modified Eagle's Medium with L-glutamine completed with 10% fetal bovine serum, and 1% antibiotic/antimycotic or RPMI 1640 base media completed with 10% fetal bovine serum, 5% L-glutamine, and 1% antibiotic/antimycotic reagent.
3. Grow cells in 75 cm² corning flasks with vented caps at 37 °C, 95% air and 5% CO₂.
4. Bring up human intestinal epithelial cells within 24 hr of deliver....

Results

Determination of the Optimum Concentration of Fatty Acids

The optimum concentration for fatty acids is defined as the maximum concentration at which cell growth is comparable to or exceeds that of control cells, with relatively low variability in results. To determine the optimum concentration of oleic, linoleic and linolenic acids cells were initially treated with varying concentrations of each fatty acid in large increments and later with smaller increments. Figure 1 shows the .......

Discussion

Suggestion of Concentration of Fatty Acids and Cholera Toxin

While the exact mechanism of how fatty acids enhance mucosal immunity is unknown, several studies have attempted to investigate their beneficial effects. Our study aims to provide methodology to determine the maximum concentration of fatty acids that cells can tolerate as well as the maximum concentration of cholera toxin that cells can tolerate without a significant influence on cell survival.

To determ.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cells/Reagent
Mus musculus macrophages	ATCC	ATCC RAW 264.7
Dulbecco's Modified Eagle's Medium	ATCC	30-2002
L-glutamine	ATCC	30-2115
Fetal bovine serum	Bio-west	S0250
Antibiotic/antimycotic	Hyclone	SV3007901
Human intestinal epithelial cells	ATCC	ATTC CCL-241
HybriCare media	ATCC	46-X
Oleic Acid	Sigma-Aldrich	O1008
Linoleic Acid	Sigma-Aldrich	L-1376
Linolenic Acid	Sigma-Aldrich	L-2376
Cholera toxin	Sigma-Aldrich	C8052
Equipment
BD Falcon 96-Well Cell Culture Plates	BD Biosciences	351172
Spectrophotometer with Dynex Revelations 4.22 software	Dynex	91000101