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Learning Objectives
Apply appropriate experimental protocols to quantify fat (lipid) content in foods
Differentiate among different types of lipids using appropriate technology
Discuss how lipids can be characterized based on electric charge (polarity) of the fatty acid backbone
Explain how thin-layer chromatography works
Discuss how chemical reagents can be used to distinguish the degree of unsaturation in lipids
Monounsaturated Fatty Acids and Polyunsaturated Fatty Acids
MUFA and PUFA sound like names of characters that might appear in a version of Disney’s Lion King. In the lipid story of
nutrition, MUFAs and PUFAs are the ‘good guys.’ MUFA is an abbreviated way to say monounsaturated fatty acid while
PUFA refers to a polyunsaturated fatty acid, the heroes in the tale of the 20-35% daily requirement of fat in the human
diet. Fatty acids come in many shapes and sizes, and these differences are important to our health and can be studied
using a simple experimental design.
Meet your Lipid Heroes
It is commonly accepted that salmon is a healthy fish to eat and dietary intake recommendations include 3-7 servings per
week of salmon or other wild-caught fatty fish from cold northern oceans around the world 1 These fish are sources of
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It is commonly accepted that salmon is a healthy fish to eat and dietary intake recommendations include 3-7 servings per
week of salmon or other wild-caught fatty fish from cold, northern oceans around the world.1 These fish are sources of
PUFAs, one of our fatty acid diet heroes that have anti-inflammatory properties and even protect DNA from attack by
mutagenic agents.2 In the early 20th century, researchers discovered that fat, particularly from these cold-water fish, was
75% of the native Alaskan Inuit diet. Additional research revealed that the Inuits did not show any signs of heart disease,
joint dis s, or skin afflictions, which were ailments that plagued citizens of the lower 48 states in the United States of
America.3 In the lower 48 states, more than 79 million U.S. citizens have three or more risk factors for cardiovascular
disease and Type 2 diabetes, whereas none of the Inuits showed signs of cardiovascular disease.4 To address this
public health epidemic, the United States Department of Agriculture’s 2015-2020 Dietary Guidelines for Americans and
the National Academies Institute of Medicine set detailed dietary reference intake recommendations for fat consumption.
Total fat intake each day should be no more than 20-35% of daily calorie levels for adults; this range is referred to as the
Acceptable Macronutrient Distribution Range (AMDR). In addition to the range of fat consumption, the guidelines state
that the amount of daily saturated fat should be less than 10% of fat consumed each day, while the essential fatty acids
omega-3 and omega-6 should be in the range of 1.1-1.6 grams per day and 11-17 grams per day, respectively.
Omega-3 and omega-6 fatty acids are PUFAs and can be found in cold-water fish as well as in walnuts, pecans, soybean
oil, and flaxseed oil.5 The other hero in the nutrition lipid story, MUFAs, can be found in olive oil, sunflower oil, canola oil,
peanuts, walnuts, avocados, whole grains, and green leafy vegetables. Unlike a Disney movie, the lipid good guys in the
nutrition story tend to mingle inextricably with many of the other lipid players. Scientific evidence demonstrates that olive
6
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oil, and flaxseed oil.5 The other hero in the nutrition lipid story, MUFAs, can be found in olive oil, sunflower oil, canola oil,
peanuts, walnuts, avocados, whole grains, and green leafy vegetables. Unlike a Disney movie, the lipid good guys in the
nutrition story tend to mingle inextricably with many of the other lipid players. Scientific evidence demonstrates that olive
oil is one of the healthiest choices-if not the healthiest choice- for cooking oil and salads.6 However, even a superhero
like olive oil is a diverse collection of lipids rather than a single, ‘good guy’ mono-lipid. Olive oil is made up of several
different fatty acids. The distribution of these lipids in olive oil is roughly 72% MUFAs, 10% PUFAs, and about 14%
saturated fat.7 Let’s take a look at the distribution of lipid types in a few other foods. See Table 1 below.8 As you can see,
irrespective of whether the source is animal or plant, MUFAs, PUFAs, and saturated fats have been found to be present.
In this laboratory, you will explore how researchers learn about the composition of lipids.
Table 1. Lipid Types as a Percentage of Total Fat in Selected Foods
Food % Saturated Fat % MUFAs % PUFAs % Cholesterol
Olive oil 14 72 10 0
Shrimp 29 29 29 13
Venison 45 29 21 4
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Venison 45 29 21 4
Hamburger (¼ lb) 47 49 3 0.5
Mango 30 50 20 0
Avocado 16 71 13 0
Oatmeal (in H2O) 21 38 41 0
Soybeans 15 24 61 0
Lipids and Technology: How Do We Know the Composition and Health Effects of Fats?
The composition of olive oil, other lipids, and all food sources are studied using tools that have been engineered to
separate and extract the building blocks of these foods. Because the building blocks of foods are essentially organic
chemicals, food technology makes use of chemical properties of macronutrients such as reactivity and electronegativity
9,10 to study how the organic chemical components of food relate to health outcomes For example an experiment can
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chemicals, food technology makes use of chemical properties of macronutrients such as reactivity and electronegativity
9,10 to study how the organic chemical components of food relate to health outcomes. For example, an experiment can
be conducted over several years (these type studies are referred to as longitudinal studies) to study the health benefits
or harm of eating meals cooked using cold-pressed extra virgin olive oil versus meals cooked using chemically extracted
olive oil. In fact, several studies have been conducted involving olive oil and have shown repeatedly that using extra
virgin olive oil, which retains all the natural lipids and phytochemicals (antioxidants) of natural olive oil, is associated with
better health outcomes than chemically extracted olive oils. As a regular part of diets and in cooking, cold-pressed olive
oil reduces the percentage of heart attacks, leads to a decrease in the number of genetic mutations in DNA, improves
endothelial function (which directly improves cardiovascular circulation), lowers levels of glucose and lipids in the blood,
and confers several additional other health benefits.11 Equipped with the outcomes from longitudinal studies, nutrition
science researchers can use technology to isolate and characterize the composition of cold-pressed olive oil and
chemically extracted olive oil, determine how the two oils differ in composition, and draw inferences (correlations) about
how the differing composition of the oils contributed to the health outcomes data obtained in the longitudinal study.
Chromatography Technique
Nutrition science relies heavily on biotechnology to gain insights about the composition, function, and health impacts of
lipids. Research studies use similar technology to study nutrition in human participants as well as animal subjects.
Techniques in lipid analysis often include separation of lipids into constituent parts and chemical extraction.12 A subset of
lipids are separable based on chemical polarity such as the presence of alcohols, e.g., glycerol in triglycerides.
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Separation of constituent parts is straightforward for polar molecules such as salts and alcohols and can be slightly more
complex for lipids. Consider digestion: During lipid digestion in the gastrointestinal tract, fatty foods are emulsified and
shuttled into the blood supply by a distinct mechanism that is separate from processing stream for water-soluble foods
like carbohydrates and proteins. Fats are oily and do not mix well with water. This ‘oily’ characteristic makes fats nearly
immiscible in water (unable to be mixed with water), but there are techniques to increase immiscibility. For example, the
digestive system uses emulsification which is a way to break up fats into tiny fat globules that can then float about in
water, suspended and flowing in the aqueous environment.
Researchers prepare fats for analysis by exposing them to specialized reagents such as emulsifiers (e.g. lecithin) and
solvents (e.g. an acid-alcohol mixture) that first break up the different fats in each test food, e.g. a researcher might want
to know about the constituent lipids in a piece of hamburger and in a comparable amount of avocado, and, to begin the
analysis, each of these foods would be exposed to a solvent first to aid in separating all the different fatty acids and lipid
subtypes in each test lipid.13
After chemical separation, the constituents of each fat can be introduced to a number of other popular lipid analysis
techniques including thin-layer chromatography (TLC), high-performance liquid chromatography, capillary
electrophoresis, mass spectrometry, nuclear magnetic resonance, and more. In this laboratory, you will have the
opportunity to use TLC to conduct your lipid analysis.
A chromatograph can be as simple as a sheet of paper or a paper towel. The main idea is that the chromatograph serves
as a surface along which fluid and components of the samples can flow The chromatograph is referred to as the
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as a surface along which fluid and components of the samples can flow. The chromatograph is referred to as the
stationary surface while the flowing liquid is the moving phase.14 Lipids that tend to be more polar (more hydrophilic) will
be more closely associated with the polar stationary chromatographic plate (stationary phase), while less polar (more
hydrophobic) lipid constituents move along with the nonpolar solution (moving phase).
To conduct the lipid analysis, a small sample of each lipid is placed at the bottom edge of a chromatographic plate as
shown in Figure 1 below. Each constituent in the lipid sample retains its own properties such as size and polarity
(whether polar (hydrophilic) or nonpolar (hydrophobic)). The sheet is then placed in a container that has a small amount
of the moving phase solution (which can be a solvent that will aid in separating the constituent lipids). As shown in Figure
1 below for three separate samples, each of the original samples contains two separate components, red and blue, that
have separated over time and in space.
4
3
2
1
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Figure 1. Sequence of Thin-Layer Chromatography.
The content on a TLC plate also provides clues about the relative abundance of different constituents in the test
samples. The intensity and thickness of bands on a TLC plate are representative of the relative amounts of a constituent
in the sample (see Figure 2 below). In research studies, separated bands are often subjected to additional techniques
such as mass spectrometry to further characterize the content in each band. Essentially, the bands are exposed to
additional chemicals and solvents to precisely extract each of them from the TLC plate. New techniques that use
spectroscopy and digital imaging processing are capable of quantitative analysis (intensity, size, and content) of the
constituent bands on a TLC plate without extraction.15
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Figure 2. Intensity differences of bands on a TLC plate indicate the relative amounts of a constituent in a sample.
Saturation, Carbon Bonds, and Chemical Reactivity
In addition to chromatography, another popular lipid research tool is one that uses chemical elements called halogens to
serve as colorimetric indicators of the number of double bonds (degree of unsaturation) in lipids. Interestingly, halogens,
such as iodine and bromine, can also be used to visualize the bands of separated lipids on thin-layer chromatographic
plates due to the color characteristics of halogens.16 Halogens are a highly reactive chemical species due to their
tendency to strongly attract electrons to completely fill their outermost (valence) electron shell. Halogen reactivity has
been used in lipid analysis research to distinguish between saturated and unsaturated fatty acids. Double bonds in
unsaturated fatty acids chemically bind to iodine.17
Iodine reacts with double bonds in unsaturated oils and fats. The colorimetric test for iodine and binding to bonds in
unsaturated fats and oils works, say, similar to absorption. Iodine reacts with the double bonds and can be regarded as
absorbed into the lipid chemical structure. For example, when a color halogen solution is added to a solution containing
unsaturated lipids, the color will appear initially but will fade due to binding between the halogen and the double bonds in
the unsaturated lipid. The halogen becomes part of the lipid. After adding the color halogen solution to the unsaturated
lipid many times the color ceases to fade an indication that the iodine has bound with all available double bonds in the
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p g p p g g
lipid many times, the color ceases to fade, an indication that the iodine has bound with all available double bonds in the
lipid solution. If halogens are added to a solution containing saturated lipids, the lipid solution remains the color of the
halogen solution, i.e., no fading occurs. The iodine test is said to be a test of unsaturation.
To quantify the unsaturation test, one approach is to count the number of drops of halogen solution, e.g., iodine or
bromine, needed to retain the color intensity of the solution; this serves as an objective measurement that all the double
bonds in the test lipid (oil or fat) have reacted with the iodine.
Orientation to the Lipids Lab Activities
Procedure I Overview
In this procedure you will analyze lipid composition of samples using thin-layer chromatography (TLC). TLC allows for the
separation of lipid components based on differences in lipid component polarity. Less polar lipid components will travel
farther as the nonpolar solvent moves up the chromatography plate. Known lipid standards (in lanes 1-3 of the
chromatography plate) will be compared to fish and nut samples. Comparisons between the standards and samples
allows for identification of lipid components found in the samples. Please see the Activity Form for details.
Procedure II Overview
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