Download WHAT YOU EAT - Montana State University Extended University

e
r
a
u
AT
yo
E
u
o
y
what
metabolomics
by Dr. Edward Dratz
Department of Chemistry and Biochemistry, Montana State University
INTRODUCTION This module provides a basic introduction to the biochemistry of three major nutritional components of your diet: carbohydrates, proteins and fats. You probably have had repeated advice to avoid fat in your diet—but, your body urgently needs certain essential fats and if you avoid fats you will probably get too much carbohydrate (which tends to make you fat, and more importantly-­‐-­‐make your liver fat, unless you do extreme exercise). As we shall discuss, carbohydrates include sugars and starches, while fat moderates blood sugar swings and increases satiation (the sense that you have eaten enough). It has recently been found that adapting your body to fat burning by cutting excess carbohydrates can increase athletic performance and greatly increase endurance. We will introduce two groups of essential fats called omega-­‐3 and omega-­‐6 fatty acids. It is extremely important for the best health to obtain a balance of omega-­‐3 and omega-­‐6 fatty acids and most modern diets have a very great excess of omega-­‐6 fatty acids, which greatly increases the risk of poor brain function and a wide variety of chronic diseases. This information will give you the background knowledge to better understand the material in this BioScience Montana learning module and understand the processes you are using and concepts you are exploring. You are what you eat This saying, handed down through the ages, is more true than you may realize. Your diet has a profound impact on your health and well being. Food provides the source of the molecular building blocks that are needed to build, maintain and repair your cells and connective tissue that make up your entire body. Your body regularly replaces most all of its cells with fresh ones (different cell types at different rates—many every day or two—e.g., the lining of your intestines is replaced every two days = tennis court area) and needs lots of “spare parts” to make the fresh cells. Food also provides fuel for all your body’s cellular processes, which allows you to do all the things that you do, whether it’s working on a project, sleeping, sitting on the couch watching TV, reading a book, playing with your dog, or riding your bike. The choices you make in your diet effect the type of building blocks you give your cells and the energy sources that you supply them. The balance of essential fats, essential protein (amino acid) components, and the amounts of different kinds of carbohydrates have a major impact on the quality of your life and health. Therefore, it is important to translate knowledge you gain in Activity 3 into terms and concepts that are easy to understand, so that you and your family and friends can benefit from the cutting-­‐edge research that is being done at labs across the world and here at Montana State University. Vital life molecules: carbohydrates, proteins, and fats You can’t begin to delve into the beauty of the inner workings of biology without understanding at least a little bit of the chemistry behind it, so let’s learn a little about the chemicals that do so much in your body. The three groups of “macro” nutrients in food that we will discuss here are carbohydrates, proteins, and fats. The periodic table of the chemical elements contains 92 naturally occurring elements, but of these only six (carbon, hydrogen, oxygen, nitrogen, sulfur and phosphorous) make up most molecules of life. The carbon, hydrogen, oxygen and nitrogen elements arranged in foods can be “burned” for fuel in our cells to make 1 energy, largely in the form of ATP (adenosine triphosphate). By far the most efficient means of making ATP in our cells is a process called cellular respiration, which also requires the oxygen that we breathe. ATP powers your muscles, the pumps that keep your brain and other cells electrically active, drives switches that control metabolism, and drives many of the transformations that convert food building blocks into body parts for growth and repair. Respiration is the main way your cells get energy, most all the cells in your body prefer to burn fat and respiration is required for fat burning. Carbohydrates Carbohydrates are a nutrient most people are familiar with because they are also known as starches and sugars. Simple carbohydrates have a basic chemical framework of carbon, hydrogen, and oxygen, usually in a ratio of 1:2:1. In chemical formulas, subscript numbers after each atom symbol tell you how many of that particular atom are in the molecule of interest. For example, the chemical formula of water is H20, which means that there are two hydrogens and one oxygen atom in each water molecule. Below are diagrams of some of the different ways the water molecule is represented in chemical symbols. One of the most important carbohydrates in the body is glucose, which is the chemical name for blood sugar. The chemical formula for glucose is C6H12O6, meaning that there are six carbon atoms, twelve hydrogen atoms, and six oxygen atoms in each glucose molecule. Carbohydrates are named because they contain carbon (C) and water (H20)—you can test for carbohydrates by dropping sulfuric acid on them and you get black carbon and steam (water) released (carbo-­‐hydrates). In each glucose molecule, atoms from six molecules of water are combined with the six atoms of carbon. The Illustration below shows three different depictions of a molecule of glucose. The “framework” diagram on the left best shows the skeleton, the ball and stick model in the middle shows the shape and bond angles, whereas the “space filling” model on the right is more realistic, but harder to understand. The carbon atoms (black) and one oxygen atom (red in the middle and the right models) are arranged in a ring with the hydrogen atoms (H) and oxygen atoms arranged around the ring. 2 Blood sugar provides “back-­‐up” energy to all cells in the body, but all your cells, except the brain, prefer to burn fat-­‐-­‐if there is enough oxygen in your tissues. If your tissues are short of oxygen (usually because of high exertion or if you are holding your breath for a long time) your cells burn blood sugar very inefficiently, but quickly to make lots of ATP to keep you going (or to respond to an emergency, if your adrenalin levels stimulated by a scare or ??). That’s why, if someone wants to lose fat they should use low intensity exercise to burn fat, not high intensity exercise that depletes tissue oxygen and burns sugar. Or, if you eat a lot of carbohydrate (more than you can burn in a short time), the elevated blood sugar elevates insulin and shuts off fat burning by the tissues (so the body can reduce the blood sugar spike caused by eating the carbohydrates) and usually much of the sugar is turned into fat in your body to help bring down the blood sugar level. So high carbohydrate diets tend to add fat to your body (which you can use, if you are too skinny). If you exert yourself a lot, sugars can provide easy-­‐to-­‐use energy that is stored in the bonds between the carbon, hydrogen, and oxygen atoms. Your blood sugar level is also crucial for optimum brain function, and taking in a lot of sugar can crash blood sugar in response. (Incidentally, the brains of many older people lose the ability to take up sugar and as a result, lose brain function, which can be restored in many cases by a low carb, high saturated fat diet). Energy in sunlight is used by plants to make chemical bonds in carbohydrates (starch), proteins and fats, and when the bonds are broken in respiration or other metabolism, that energy is released to make ATP. Proteins Proteins are amazing and complex molecules that are involved in nearly every part of cellular life, that we will discuss only briefly. Proteins are used to carry out thousands of chemical reactions in the body, send and receive hormone signals between cells, build cell and tissue structures, fight disease, move molecules in and out of cells, and produce muscle movement. Your DNA codes for instructions on how to make tens of thousands of different proteins. Like carbohydrates, proteins are made up of carbon, hydrogen, and oxygen, but they also contain nitrogen, some sulfur and a bit of phosphorous. Proteins are made up of smaller molecules, called amino acids, which are linked together in long chains by peptide bonds. The picture on the left, below shows two of the simplest amino acids, glycine, being linked in a peptide bond. The picture on the right shows amino acids with different “R” groups linked into a peptide bond (see below about the “R” groups). Many more amino acids are linked together to form long proteins with many different amino acids. 3 When we eat proteins, we have to digest them (hydrolyze = cut with water) back to individual amino acids so that our bodies can use the amino acids to build back into new proteins-­‐-­‐with amino acid chains sequences that are coded by our genes. There are 20 different types of amino acids (different chemical “R” groups above), that are used to make the proteins found in your body, and some of these amino acids are essential (8 essential, 11 sometimes essential) in your diet (thus the concern for the “quality” of proteins and combinations of proteins). The order of the amino acids in the protein chains are coded by your DNA, and the order of the amino acids determine the “shapes” that the proteins fold into. The “shapes” of particular proteins determines their activity and thus the function of that protein. The tens of thousands of different types of proteins all have different jobs to do. The illustration below shows a space filling model of a small protein molecule, called insulin, that regulates blood sugar and fat burning. Structural proteins give shape and strength to cells and tissues. Examples include collagen, which is the most abundant protein in our bodies. Collagen forms fibers in your bones and essential connective tissue in your body (incidentally, we need a lot of vitamin C to make mature collagen and so vitamin C is crucial for wound healing and for general health) and keratin, which makes up things like hair, fingernails, hooves and feathers. Signal receptor proteins receive messages from one cell to another cell or from a cell to a group of tissues. Some proteins are themselves signals that act between different cells. Insulin, which regulates blood sugar in our bodies, is a small but very vital signaling protein, shown above, which controls a wide range of activities in our bodies including carbohydrate, protein and fat metabolism. 4 Enzymes are an extremely important protein type. Enzymes can accelerate the speed of chemical reactions, up to millions of times faster than the reaction would progress without the enzyme. This means that a reaction that would take a hundred years without an enzyme could be completed in much less than a second by using the right enzyme! Your body uses thousands of different types of enzymes to carry out thousands of different chemical transformations—like digestion, respiration and synthesis of cellular building blocks. Enzymes also play key roles in the metabolism of omega-­‐3 and omega-­‐6 fatty acids, which you will be learning about in the on-­‐campus portion of this module. Fats and lipids Fats are the last, and probably the least appreciated, of the three classes of life molecules that we will be exploring. Fats can be combined to form molecules called phospholipids, which are used by cells to form membranes that surround all cells. Fats are also key sources of energy for animals (recall that most of the cells in our bodies prefer to burn fat as fuel virtually all of the time). And, energy for seed germination comes from stored fat in the seeds. Fat in the body is also used for insulation and to pad and protect organs, and tissues called “brown fat” provide body heat. You may have previously learned that the cell membrane, the outer layer of a cell that separates it from its external environment, is made up of a double layer of lipids called the phospholipid bilayer. Phospholipids also insulate and protect nerve fibers, together with high levels of cholesterol. Cholesterol is an important molecule that is classed as a lipid. Cholesterol is essential for the function of cell membranes, bile detergents that are essential for digestion of fat and absorption of fat soluble vitamins (A, D, E and K), and for the synthesis of sex hormones. Our body makes at least ten times as much cholesterol as we typically can eat in our diets, because it is so important for health. There is good evidence that the only people who should reduce cholesterol in their diets are a relatively small number of individuals (less than 1%) who have a defective LDL receptor and extremely high blood cholesterol. But this is a rather complex topic that we won’t have time or space to cover. Lipids and fats are truly vital life molecules, because of their many important roles in providing energy, providing structure for all cells, and being the material made into many hormones. Fats are the molecules that you will be exploring the most, as you progress through this BioScience Montana module. Fats consist of carbon, hydrogen, and oxygen atoms bonded together in particular ways. Most lipids are made up of at least two parts, and the two separate parts of a very simple lipid, called a “monoglyceride” are shown on the left below. The part on the far left of the illustration is a three carbon and three oxygen molecule called glycerol, which is a type of alcohol, and a building block for many lipids. The part to the right of the glycerol is a long, chain-­‐
like structure that is called a fatty acid. Fatty acids play a major role in most fats, and the types of fatty acids in your diet are crucial for health and minimization of disease. On the right a simpler diagram shows mono, di and tri glyceride backbones with no details. 5 Fatty acids have two structural parts that are designated as the “head” and “tail.” The “head” is made of a group of oxygen-­‐containing atoms called a carboxyl group (shown on the right-­‐side of the Illustration below). The arrangement of the hydrogen (white), carbon(black) and oxygen(red) atoms in the carboxyl group that make up the heads of the fatty acids are on the right side of both the “ball and stick” and the “space filling” models below. The lines between the C, H, and O atoms on the upper diagram below represent chemical bonds, which will be discussed in more depth in the next section. The “tail” of the fatty acid (on the left sides of a and b below) is made up of long chains of carbon atoms with hydrogen atoms sticking off of them. Because the tail is made out of hydrogens and carbons, it is often called a hydrocarbon chain. Hydrocarbons are oily and do not dissolve in water. Oil and water do not mix and oily compounds are often called “hydrophobic” (literally water-­‐fearing). Saturated, unsaturated, and polyunsaturated fats You have probably heard of saturated, unsaturated and polyunsaturated fats, which are often discussed in relation to the foods that you eat. But what do these terms mean? The terms saturated, unsaturated and polyunsaturated are actually referring to the chemical structures of the fatty acid molecules, so let’s explore that a little more. In order to understand the chemistry of fats, you need to understand a bit about how atoms bond together. Carbon atoms can bond to one another in single or in double bonds. Saturated fatty acids contain all carbon-­‐carbon single bonds. Unsaturated fatty acids contain a carbon-­‐carbon double bond. And polyunsaturated fatty acids contain two or more carbon-­‐carbon double bonds. The concepts of double and single bonds will be explained a bit more below. 6 Atoms consist of protons and neutrons in the positively-­‐charged nucleus and negatively-­‐charged electrons that orbit around the nucleus. The electrons orbit the atomic nucleus in distinct orbital shells and each shell can hold a specific number of electrons. Atoms can bond in different ways by giving or taking electrons from other atoms or by sharing electrons with other atoms. Atoms gain, lose or share electrons to try to fill up their outermost orbital shell, called their valence shell—to reach a stable state. When atoms share electrons, this type of bond is called a covalent bond. Such shared electron bonds are the type of bonds found in fats. A single covalent bond is present when two atoms share one pair of electrons, and a double covalent bond is when the two atoms share two pairs of electrons. Recall that the “tail” of a fatty acid, (shown in Illustration above) consists of a chain of carbon atoms bonded together. The next illustration shows a molecule of the simplest hydrocarbon, called methane (chemical formula CH4) with one carbon atom and four hydrogen atoms. Hydrocarbons are molecules that contain hydrogen and carbon, not surprisingly. Carbon has four electrons in its outermost shell, but it has eight spots in its outermost shell in which to put electrons. Having eight electrons in its outermost shell would make it full. Hydrogen, on the other hand, has only one electron in its first shell, as shown below. The outer shell of hydrogen would like to have two electrons to be full, but atoms can “share” electrons to effectively fill their shells when they make chemical bonds. So as diagrammed above, each hydrogen atom shares one electron with the carbon atom, and the carbon atom shares its four electrons with one of each of the hydrogens to form the molecule of methane (CH4). Each shared pair of electrons form a covalent bond and there are four single bonds between the carbon atom and the four hydrogens. The illustration above middle is a type of diagram that represents the electrons as dots or “x”es. However, it is more common to show molecules in terms of the atoms and the types of bonds present, but not to show each electron (for example, as in all the illustrations before methane). The most common way to show the structure of methane, in terms of the bonds present, is shown in on the left above. This diagram represents single bonds (containing one pair of shared electrons) as single connectors between the atoms or in other illustrations as lines between the letters that represent the atoms. The “space filling” model of methane on the right is more realistic, but it doesn’t show the bonds or electrons. Double bonds form when two atoms share four electrons (two pairs) between them. An example of a double bond using carbon is in carbon dioxide, CO2, which you probably have heard quite a lot about. Again, carbon has four electrons in its outermost shell, but a filled outer C shell would have eight electrons. Oxygen, in contrast, has six electrons in its outermost shell and its filled outer shell would also have eight electrons. If oxygen gets or shares two electrons, it will have a filled outer shell. Thus, if carbon shares electrons with two oxygen atoms, both oxygens and carbon can have full outer shells, as diagrammed below. 7 When two pairs of electrons are shared between each oxygen and carbon, these form double bonds, as diagrammed on the right above. The diagram on the left is drawn with more standard notations, where each covalent bond (formed from a shared pair of electrons) is shown as a solid line. The double bonds (sharing two pairs of electrons) are shown as two parallel lines on the bottom right of the left panel above. Incidentally, all of the carbons in carbohydrates and fats end up as carbon dioxide (that we breathe out) when we “burn” the carbs and fat for energy by respiration. The carbon dioxide is the limiting nutrient for plant growth under most conditions (if there is enough water) and so CO2 stimulates plant growth. Now that you have seen a little basic chemistry and have seen the difference between a single and a double bond, you can understand the difference between a saturated and unsaturated fat. Look again at the fatty acids illustrated at the start of this section on fats. The “tail” of that fatty has chain of carbons bonded together with single bonds, and two hydrogens also bonded to each carbon (except for the right hand end carbon, which had three hydrogens bound). We mentioned earlier that a carbon atom has four locations where it can bond with single electrons with other atoms, as we saw with the methane example. This is because carbon needs to share four electrons from other atoms to attain a full outer (valence) shell, and it has four electrons to share with other atoms. Carbon can also bond with two single bonds and a double bond to fill its outer shell. Alternatively, carbon has two spots where it can bond in double bonds (again because it wants to gain four electrons and it has four to share, as in the CO2 example). If you look at the fatty acid tail of the lipid at the start of this section on fats, each carbon (other than the end carbons) is bonding at all four spots in single bonds: with the carbon to its left, with the carbon to its right, with the hydrogen above it, and with the hydrogen below it. The carbon on the far left of the fatty acid is bonded to three hydrogens and only one carbon on its right in the diagrams at the start of this section. The carbon farthest to the right in the fatty acid is participating in two single bonds and one double bond to oxygen (carbons in stable molecules always form four bonds to other atoms). The right-­‐most fatty acid carbon is part of the carboxyl group, which makes up the acid “head” of the fatty acid molecule. The illustration below compares the chemical structures of saturated and unsaturated fatty acids. The carbon atoms in a saturated fatty acid are bonded to each other with single bonds, which allows them to bond with a hydrogen both above and below (since carbon forms four bonds). The single bonded carbons are full or “saturated” with hydrogen, meaning they have the maximum possible number of hydrogens bonded to the carbon chain. 8 An example of an unsaturated fatty acid is shown in the bottom of the diagram on the left above. The unsaturated fatty acid, has a double bond between two of the carbons in the chain. As a result, the carbons can’t hold as many hydrogens. Therefore, the fatty acid with a double bond is said to be unsaturated. Polyunsaturated fatty acids have two or more double bonds between carbons, as shown on the bottom of the diagram above on the right, and we shall see more of these later in this unit. This fatty acid, called linoleic acid, is the most abundant polyunsaturated fatty acid (often abbreviated PUFA) in most of our diets—rich in most types of grain and nut oils, and we tend to get way too much of this fatty acid in modern diets, as we will discuss in more detail. As shown in the diagram above, the double bonds makes fewer sites for hydrogens to bond on the hydrocarbon chain, so the chain is less saturated with hydrogen = unsaturated. Double bonds between carbon atoms also produce kinks or bends in the fatty acid chains. These seemingly subtle differences in chemistry on the atomic scale actually produce dramatic differences between the nutrition and health effects of the different types of fats. Longer chain saturated fats are solid at room temperature. Animal fats are largely saturated, but contain quite a bit of monounsaturated fatty acids and some polyunsaturated fatty acids, depending on what the animals have been eating. Two classic examples of largely saturated animal fats are butter and fats in meats (with some unsaturated and polyunsaturated fatty acids, depending on what the animals have been eating). Two examples of almost entirely saturated plant fats are coconut oil and palm oil (which turn out to have important health-­‐promoting effects in many situations). Unsaturated and polyunsaturated fats, on the other hand, are liquid at room temperature and, as a result, we call them oils. Oils generally, but not always, come from plant sources. Some examples of oils are olive oil, peanut oil, canola oil, corn oil, and fish oil. Olive oil contains mostly monounsaturated fatty acids and solidifies in the refrigerator. [Incidentally, our bodies can and do make a lot of the fatty acids in olive oil out of saturated animal fats by putting in a single double bond in the right place!]. Fish oil is rich in polyunsaturated omega-­‐3 essential fatty acids, which will be a major topic of this unit. Remember the kinks in the unsaturated fats? It turns out that those kinks prevent the molecules from packing closely enough together to solidify at room temperature. Saturated fats don’t have the kinks, so they can pack together more easily and form solids at higher temperatures. There are some notable exceptions to this. You have probably heard of hydrogenated and partially hydrogenated oils. Hydrogenation is a way to force hydrogen, through a chemical process, into the fatty acid chain in order to reduce the number of double bonds and increase the shelf life of products that contain these oils. Partial hydrogenation only adds hydrogen to some of the double bonds, but as we shall see later, partial hydrogenation kills off the omega-­‐3 fatty acids first. The omega-­‐3 fatty acids—which are crucial for health and are already very scarce in most of our diets—tend to be wiped out by hydrogenation. Partial hydrogenation 9 also changes the chemical structure of the remaining double bonds (producing “trans” fats) which we will discuss a bit later. Fatty acids and essential fatty acids (EFAs) As you have probably figured out by now, fatty acids are the key components of fats and lipids, and lipids are vital components of cell membranes. Membranes are important: They serve as the boundaries and chemical barriers around cells and organelles within the cell, they carry pumps and signal receptors, and they are surfaces where many important chemical reactions occur. If not for membranes, photosynthesis and respiration couldn’t take place, muscles couldn’t contract, and electrical conduction of nerve impulses essential for brain and muscle function would not occur. If not for properly functioning membranes, your body quite simply wouldn’t work. Your body does an excellent job of manufacturing a wide array of fatty acids, but it can’t make them all. You need an important group of fatty acids called the essential fatty acids or EFA’s. EFA’s are fatty acids that are necessary for normal growth and development, but your body can’t make them. You can only get them from food. This is one of the key reasons that it is so important to have a healthy diet. There are two families of EFAs called the omega-­‐3 fatty acids and omega-­‐6 fatty acids. You may have heard about the omega-­‐3 group of important nutrients in the news, as higher levels of omega-­‐3 fatty acids have been linked to many significant health benefits. Omega-­‐6 fatty acids are also essential for health, but our typical modern diets provide far too much omega-­‐6 and not enough omega-­‐3, and the imbalance is quite harmful. Omega-­‐3 and omega-­‐6 fatty acids are polyunsaturated fats which, as you recall, means that they have a several double bonds between pairs of carbons in the fatty acid chains. The omega-­‐6 and omega-­‐3 fatty acids get their name from the location in chain where the first double bond occurs. You already know that all fatty acid molecules have a carboxyl head and a fatty acid tail. The head of the fat is said to be the alpha or beginning of the molecule, and the far end of the tail is referred to as the omega end of the molecule. These terms are applied because alpha (α) and omega (ω) are the first and last letters of the Greek alphabet. Sometimes you will see these Greek letters used in notations with other names for the omega-­‐3 and omega-­‐6 molecules, and sometimes you may see them referred to as n-­‐3 or n-­‐6 fatty acids, which are a different way of denoting the same thing. The illustration below is a simplified diagram of examples of omega-­‐3 fatty acid and omega-­‐6 fatty acid molecules (note the hydrogens are not shown for simplicity). In this illustration, you can see the head of the molecule on the right and the tail on the left for both the omega-­‐3 and omega-­‐6 examples. The top diagram shows an example of an omega-­‐3. By starting at the omega (left) end of the molecule and counting the atoms, you can see that the first double bond starts at the third carbon atom. This bond closest to the omega end is the third bond from the omega end of the molecule, thus this example is referred to as an omega-­‐3 fatty acid. 10 The bottom part of Illustration shows an omega-­‐6 fatty acid. If you count bonds from the omega (left) end of the lower molecule, you will see that the sixth atom is the starting point of the first double bond closest to the omega end, thus its name, omega-­‐6. The particular omega-­‐3 shown in the illustration above is known as alpha linolenic acid (also named α linolenic acid, or ALA) and the particular omega-­‐6 fatty acid shown is known as linoleic acid (or LA). ALA is found in green plants or algae and in animals that eat green plants or algae (such as grass-­‐fed animals and fish) and in a few seeds including flax, camelina, and salba. Most seed oils and nuts (such as corn, soy, sunflower, safflower, and peanut) are rich in Linoleic acid (LA) and very low in ALA. Both the omega-­‐3 ALA and omega-­‐6 LA fatty acids are essential fatty acids (EFAs). Our bodies can make other, longer and more unsaturated omega-­‐3 and omega-­‐6 fatty acids out of the EFAs. Linoleic acid and alpha linolenic acid compete for the same enzymes in our bodies, and if we have too much LA, it will inhibit the beneficial metabolism of ALA. The longer omega-­‐3 and omega-­‐6s are converted in our bodies into powerful hormones and compounds that effect our brain function and other important processes in our bodies. Considerable research indicates that the ratio of omega-­‐6 to omega-­‐3 in a person’s diet can have an impact on several critical aspects of health. Diets high in omega-­‐6 and low in omega-­‐3 greatly increase a person;s risk of experiencing poor brain function and ill health conditions such as headaches, ADHD, depression, PMS and diseases such as cancer, cardiovascular, and autoimmune diseases. There is a lot of evidence that diets higher in omega-­‐3 and lower in omega-­‐6 help to prevent these types of disease and health problems. It appears that supplements of the longer omega-­‐3 fatty acids in fish oils or products made from algae can overcome excessive omega-­‐6 to a substantial extent. The average American diet has a omega-­‐
6/omega-­‐3 ratio of 15-­‐20 to 1 or higher and a desirable level is thought to be 4-­‐8 to one. Where do you think you are? You’re about to find out. 11 Heart Disease Mortality and n-­‐3 LCPUFA status
Lands,Lipids, 2003, 38: 317-321
12 As you learn and explore nutrition, diet, and the complex science behind it all in this BioScience Montana learning module, we hope that you will think about how your newly acquired knowledge can impact your life and the lives of your families and friends. We hope that you will be able to use this knowledge and experience to take responsibility for your health, help build a better community, create leadership opportunities, develop life skills, and… Enjoy, explore, learn, and discover! 13 The big picture We can make major improvements in our health, our brain function and the health and brain function of our family and friends—by better balancing EFAs in our food. But how do we do this? In this module, you will learn to carry out an analysis of your own blood for markers of your essential fatty acid nutrition & EFA balance. You will also learn a computer program to analyze foods for essential fatty acid balance, as well as for percent of target calories and other important nutrients that are often low in many American diets. The American health paradox: Amazingly, America ranks lowest of 13 industrialized nations in health. Yet, as shown in Illustration 1, America spends much more on health care than any other country (twice as much as Switzerland, which is #2). And, Americans have the third lowest cholesterol level (high cholesterol is essentially unimportant for health in most people!), and have lower smoking and alcohol consumption than most of the 13. In Illustration 1, that will be presented in an introduction, the US is almost off the chart for both health care expenditure, while citizens of the US are much less healthy than they could be. This is NOT a good deal! What is wrong? American nutrition is a mess 14 Americans tend to be over-­‐medicated with expensive pharmaceuticals that treat symptoms rather than underlying causes. • Probably the most serious problem is an excess of omega-­‐6 fatty acids and too low a level of omega-­‐3 fatty acids. • Probably second is excessive fructose, which is as harmful as excessive alcohol and is driving great increases in type 2 diabetes • American diets also tend to be low in a wide range of important nutrients (especially magnesium, zinc, folate, many B vitamins, copper and selenium), that lead to decreased health. What are EFAs? EFAs are fats that are essential for life and health, but that our bodies cannot make out of simpler parts. Your body needs dozens of different fatty acids, and your body can produce all of them by itself—except two: • Linoleic acid (an omega-­‐6) • Alpha-­‐linolenic acid (an omega-­‐3) These two nutrients are called Essential Fatty Acids (EFAs). Illustration 2 shows the chemical structures of EFAs. (Pages 1-­‐8 have more detailed information explaining the chemistry.) Why are EFAs important? EFA’s are important because they cannot be synthesized by our bodies. Omega-­‐6 and omega-­‐3 fatty acids must be obtained through the diet. A healthy balance of omega-­‐6 to 3 is about 1:1 up to about 4:1. However, a typical, modern American diet has around a 20:1 ratio! All fatty acids contain a long, oily chain and a “carboxylic acid” end: -­‐COOH, that can lose an H+ to form –COO. Why is the low O-­‐3/O-­‐6 imbalance so bad for health? Dietary “short” Omega-­‐3’s produce DHA, EPA “long” O-­‐3s and other vital nutrients for the brain and mild hormones that oppose inflammation. • Omega-­‐6’s and omega-­‐3’s utilize the same processing proteins to do their jobs. 15 • There are a limited number of processing proteins that O-­‐6’s and O-­‐3’s can use, so a higher amount of either will cause one to outcompete the other. What does the O-­‐3/O-­‐6 imbalance cause? With far too many O-­‐6’s compared to O-­‐3’s, the body has an excess of inflammatory response hormones. This leads to an increased risk for numerous health problems such as asthma, allergies, headaches, PMS, arthritis, heart disease, male infertility and 2/3 of all cancers. Furthermore, the brain is starved of key nutrients required for proper function (to avoid anxiety, depression, ADHD, etc.) Illustrations 4 through 7 show correlations between health problems and O-­‐3/O-­‐6 imbalances. 16 17 Research on increased omega-­‐3 intake Increasing intakes of omega-­‐3 improves the working memory of young adults who are at the peak of their cognitive abilities. New research published in PLoS One investigated the effects of omega-­‐3 supplementation on the cognitive functions of healthy young adults who are at the top of their ‘cognitive game’. In the first study of its kind, the US-­‐
based researchers supplemented 11 participants with high dose omega-­‐3 for six months [750 mg docosahexaenonic acid (DHA) and 930 mg eicosapentaenoic acid (EPA) per day], finding that the supplementation substantially improved working memory. “We found that members of this population can enhance their working memory performance even further, despite their already being at the top of their cognitive game,” explained project leader, Professor Moghaddam from the University of Pittsburgh. “What was particularly interesting about the pre-­‐supplementation cognitive ability test was that it correlated positively with plasma Omega-­‐3,” said Moghaddam. “This means that the Omega-­‐3s they were getting from their diet already positively correlated with their working memory.” DHA supplementation improved both memory and reaction time in healthy young adults, according to a randomized controlled trial that appeared in March 2013. * O-­‐3/O-­‐6 balance scores As a general rule, food that is derived from the chloroplasts of plants and algae and animals that eat them contain omega-­‐3’s. Food that comes from non-­‐green plants and animals who eat them, as well as more processed foods, tend to have more omega-­‐6’s. • Good: most fish and leafy green vegetables; grass-­‐fed meats and omega eggs. 18 • Neutral to positive: Many vegetables; grass fed dairy products; free range eggs. • Negative: Excessive common food oils, including salad dressings; corn-­‐fed meats, dairy and eggs from corn-­‐
fed animals. Need O-­‐3 Large increase in US life expectancy from 1900 to now ~ 30 years • ~25 of those years in increased life expectancy are attributed to advances in “public health” = mostly due to improved sanitation and cleaner water. • Tuberculosis was the leading cause of death in 1900, but death from TB down 90% (main drop came well before antibiotics—due to public health). • Current preventative care (e.g. blood pressure screening, cancer screening, counseling about weight loss, aspirin to decrease heart attacks) is good, but adds only about 18 months to our lives. What is next? On Day 1 of our on-­‐campus project, you will learn how to process your blood in the biochemistry and organic chemistry labs for fatty acid analysis. Next, you will learn to analyze the results of your blood composition and how to analyze foods in your diet that improve or degrade your fatty acid balances. 19 1.
Warm hands in hot water and dry. Obtain finger-­‐stick blood sample (Lab assistants will do this) ASSISTANTS NOTE: put about 0.5 ml of osmotically balanced, anticoagulant solution in each syringe BEFORE blood collection. 2.
Mix the collection syringe (no needle) to mix in anticoagulant and gently transfer the blood to a 1.5 mL snap top conical tube, rinse the syringe with osmotically balanced 0.15M NaCl solution and write your initials on the tube. 3.
Centrifuge your conical tube with your diluted blood for 1 minute on highest setting to pellet the red blood cells and leave the major portion of the yellow plasma on top. 4.
Using a fresh pipet, remove the yellow plasma top layer from conical tube and discard into bleach bucket. This top plasma layer may also be referred to as the supernatant (a liquid overlaying a solid). NOTE: This will leave you with a small red cell pellet at the bottom of your conical tube. 5.
Use a fresh plastic pipet and add 0.15 M NaCl (sodium chloride) up to the 1.5 mL line on your conical tube. 6.
Vortex (or shake vigorously) the conical tube for 10 seconds to resuspend the red blood cells.. 7.
Centrifuge your conical tube for 1 minute on highest setting to repellet the red cells and wash off plasma that was between the red cells in the initial red cell pellet. 8.
Using a fresh pipet, remove the top layer (plasma/supernatant wash) from conical tube and discard pipet into bleach container. NOTE: Again, this will leave you with a small red cell pellet at the bottom of your conical tube. 9.
Add deionized (DI) water using a fresh pipet to your conical tube containing the red blood cell (RBC) pellet up to the 1.5mL mark and cap the tube! NOTE: This step and the next will lyse (burst) the red blood cells, releasing the hemoglobin and other proteins. 10. Set vortex to max and vortex conical tube (or shake vigorously) for 10 seconds to resuspend the red cells and encourage their bursting. 20 11. Set centrifuge to max and centrifuge conical tube for 5 minutes. (This step will create a pellet of RBC “ghost” membranes.) NOTE: The RBC ghost membrane pellet is very small and may be hard to observe. 12. Obtain a fresh plastic pipet and remove the red hemoglobin supernatant by slowly pipetting down to the 0.3 mL mark on your conical tube. Discard the red hemoglobin supernatant into the bleach container. NOTE: Be careful not to remove all of the red hemoglobin supernatant, as you may lose your RBC membranes. Now you have obtained your RBC membranes and are ready to analyze them to determine your red blood cell fatty acid composition and the omega-­‐3/omega-­‐6 fatty acid ratio. You will conduct the next portion of the lab with the help of a teaching assistant. Please wait for their assistance. 21 1.
2.
With help of TAs, use Pipetman pipette (automatic pipet) to add 1.0 mL of 2:1 DCM (dichloromethane):methanol solution, containing a known amount of C15:0 fatty acid internal standard, to the conical tube containing your RBC membranes and cap tightly. DCM is similar to “dry cleaning fluid.” NOTE: This step will extract the fatty lipids from the RBC membranes. Set vortex to maximum and vortex conical tube (or shake vigorously) for 10 seconds to re-­‐suspend the red cell membrane pellet. 3.
Set centrifuge to highest setting and centrifuge conical tube for 2 minutes to separate the lower DCM-­‐rich and upper water-­‐rich layers. The DCM-­‐rich layer contains the oily lipids and the water-­‐rich layer contains the more water-­‐soluble compounds. 4.
Use Pipetman (with guidance from TAs) to remove lower clear liquid layer from conical tube and transfer it to a small glass vial with your initials on vial (this is probably the trickiest step). Dispose of conical tube. NOTE: The lower layer (DCM layer) contains the fatty lipids. 5.
Go to the organic chemistry lab down the hall to dry the sample in glass vial using a stream of nitrogen gas in a fume hood. Slowly and gently start nitrogen flow over sample and let stand for 5-­‐10 minutes or until completely dry. NOTE: This part of procedure is conducted under a ventilation hood in the organic chemistry lab to pull the DCM and methanol solvents out of the lab. 6.
TAs will add 340 uL of 10% BF3 (Boron Trifluoride) in MeOH (methanol) using Pipetman to convert all the fatty acids into methyl-­‐esters (will be discussed on the white board in the lab). Set vortex to maximum and vortex sample (or shake vigorously) for 10 seconds. 7.
Check to make sure cap is tightly on glass vial containing sample. Place vial in oven set at 60°C for 15 minutes to foster the reaction for formation of the methyl esters. 8.
Remove glass vial from oven (will be warm) and cool samples in cold water for 10 seconds. NOTE: Cooling sample prevents MeOH from boiling when cap is opened. 22 9.
Add 200 uL of deionized (DI) water to vial using Pipetman NOTE: The DI water will kill off any excess BF3 catalyst in the reaction mixture. 10. Set vortex to maximum and vortex sample (or shake vigorously) for 10 seconds. 11. Use a Pipetman to add 200 uL of hexane (similar to gasoline) to glass vial. Cap vial tightly. Set vortex to maximum and vortex sample (or shake very vigorously) for 10 seconds. 12. Let glass vial sit undisturbed for 2 minutes. Note: The liquid in the vial will separate into two layers. 13. Remove 50 uL of hexane (top liquid layer) using Pipetman and transfer to glass insert in a new glass sample vial. Put your initials on the new glass vial and dispose of old vial. 14. DONE! TAs will measure the fatty acids in your sample using the Gas Chromatography/Mass Spec machine and report the results to you when we carry out the final parts of this unit.
23 1.
Calculate the amount of each (major) fatty acid in your RBC Amount of fatty acid A = Amount of C15:0 standard in your sample (100 ug) X (GCMS signal strength for fatty acid A) / (GCMS signal for C15:0) Calculate this for each major fatty acid that is tabulated in your RBC sample 2.
Calculate the weight percent of each (major) fatty acid in your RBC Weight % of fatty acid A = Amount of fatty acid A / Total of all major fatty acids Multiply by 100. Calculate this for each major fatty acid in your RBC sample 3.
Calculate percent of n-­‐3 in HUFA % n-­‐3 in HUFA = 100 x [ n-­‐3 HUFA (20:5+22:6)] n-­‐3 HUFA (20:5+22:6) + n-­‐6 HUFA (20:4+22:4) 4.
Calculate the DHA/AA ratio = 22:6/20:4 This ratio is a good measure of the ratio of brain active omega-­‐3 and the most active omega-­‐6 5.
Calculate the EPA/AA ratio = 20:5/20:4 6.
This ratio is a good measure of the ratio of anti-­‐inflammatory omega-­‐3 and the pro-­‐inflammatory omega-­‐6
24 1.
Keep track of the foods you eat for a week or two and enter them in the Cronometer program on line: https://cronometer.com/ (We hope you will learn which foods improve Om-­‐3/Om-­‐6 Balance Scores from this!) 2.
Examine other foods on the Balance Scores and see if any palatable and practical changes could be made to improve your Balance. 3.
Read a couple of other scientific papers on health effects of omega-­‐3/omega-­‐6 that will be sent to you. • Keep note of questions that you might have that you can discuss with the TAs by Google+ or e-­‐mail. • Discuss these readings and video + questions at the next virtual lab meeting 4.
Set up an arrangement with one or more relative(s) who does not live with you (and does not eat with you), with whom you can easily keep in contact. • Help them to keep track of the foods they eat for a week or two. • Enter these in Chronometer and see what you can conclude about the likely health effects of their diets. • If you can suggest ways that they might make dietary changes to improve their risk of disease, that could be good! Discuss this project at the third virtual lab meeting 5.
Write or film a brief report about what you learned and what happened in your activities above. 25 GLOSSARY Adenosine Triphosphate-­‐ATP: The main molecule used by cells for energy. It is produced very efficiently by the process of cellular respiration in the mitochondria if there is sufficient oxygen or inefficiently but rapidly by process of glycolysis in the cytoplasm of the cell if oxygen is low or if blood sugar is too high. Alpha-­‐linolenic acid (ALA): A short chain omega-­‐3 fatty acid that is essential in our diets. It is found in small amounts in green leaves and algae, and is concentrated by animals that eat grass and fish that eat algae, and is also found in a few plant seed oils such as flax, camelina and salba. Our bodies can convert ALA into longer, more biologically active omega-­‐3 fatty acids, but this process is inhibited by high levels of linoleic acid (LA = main dietary omega-­‐6). Anti-­‐inflammatory: A substance that has the effect of preventing or reducing inflammation, such as omega-­‐3 fatty acids or aspirin type compounds. Carbohydrate: A type of molecule commonly referred to as a sugar or starch and composed of carbon, hydrogen, and oxygen bonded together in particular ways. The simplest carbohydrate is glucose, C6H12O6, which is blood sugar. C6H12O6 is made by plants from six atoms of carbon and six molecules of water, H2O. Carboxyl group: A common component of molecules that is composed of one carbon, two oxygens, and one hydrogen atom bonded together. Carboxyl groups are acidic and make up the “heads” of fatty acids. The carboxyl end is commonly referred to as the alpha end of the fatty acid molecule. Cellular respiration: Vital cellular process that involves a complex, multi stage process where oxygen “burns” fats, glucose, or amino acids (at body temperature of course) which produces Adenosine Triphosphate (ATP). This process occurs in the mitochondria of animal and plant cells and in the outer membrane of bacteria if there is sufficient oxygen present. Covalent bond: A type of chemical bond formed by sharing two electrons (one pair of electrons) between atoms. Double covalent bond: A type of covalent bond in which two atoms are sharing four electrons (two pairs of electrons). Enzyme: A type of protein that speeds up (catalyzes) chemical reactions. There are thousands of different enzymes that catalyze thousands of different reactions in our bodies. Essential fatty acid: Fatty acids that cannot be manufactured by the body and can only be supplied through diet. ALA (alpha-­‐linolenic acid) is the essential omega-­‐3 and LA (linoleic acid) is the essential omega-­‐6 fatty acid. It appears that the optimum ratio of LA and ALA in our diets is close to one, but typical modern diets are in the range of 15-­‐20 to one. Ester: A molecule formed by the reaction between an acid and an alcohol which gives off one molecule of water in the formation process. Fat: Most commonly molecules composed of glycerol linked by ester bonds to one, two or three fatty acid tails. Fatty acid: A molecule consisting of a carboxyl group bonded to a saturated, unsaturated or poly unsaturated hydrocarbon chain. Fatty acids are commonly bonded to a glycerol to make up fats or phospholipids. Glycerol: A type of alcohol that is the molecule that commonly binds to the alpha end, or head, of a fatty acid molecule. Inflammation: The reaction of the immune system to injury and/or infection which may be detected by swelling, 26 redness, heat, and/or pain. The process includes increased blood flow with an influx to the area of white blood cells and other substances that assist in killing bacteria and fungi and help to heal the body. Inflammatory: A substance that has the effect of causing or increasing inflammation in the body. Linolenic acid: A short chain omega-­‐6 fatty acid that is essential in our diets in small amounts and is found in most plant seed oils and nuts (such as corn, soy, sunflower). Our bodies can convert LA into longer, more biologically active omega-­‐6 fatty acids. Lipid: A group of organic molecules that are insoluble in water and soluble in oily solvents. They include fatty acids, fats, oils, and waxes. They are very important nutrients that form a major component of cell membranes, are formed into highly active hormones, and are a source of stored energy. Omega-­‐3 fatty acid: An unsaturated fatty acid whose carbon chain has its first double bond three carbons from the omega end of the molecule. The shortest common omega-­‐3 fatty acid is called alpha-­‐linolenic acid (sometimes written as α-­‐linolenic acid or ALA), and can be made longer and more unsaturated in our bodies to form highly bioactive substances. Omega-­‐6 fatty acid: An unsaturated fatty acid whose carbon chain has its first double bond six carbons from the omega end of the molecule. The shortest common omega-­‐6 fatty acid is called linolenic acid and can be made longer and more unsaturated in our bodies to make highly bioactive substances. Protein: A large, complex, multi-­‐purpose molecule composed of long chains of amino acids. There are 20 different types of amino acids, and different proteins have distinctive sequences of amino acids that are coded in the genes. Saturated fatty acid: A fatty acid in which all the carbons that make up the fatty acid tail are bonded together with carbon-­‐carbon single bonds. Single covalent bond: A type of covalent bond in which two electrons are shared between two atoms. Unsaturated fatty acid: A fatty acid in which one or more of the carbons that make up the fatty acid tail are bonded together with a double bond. Polyunsaturated fatty acids contain two or more double bonds (up to six double bonds). 27