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Charles Saladino Introduction and Overview As a group, lipids, unflatteringly known as fats, are what I would describe as a rather heterogeneous group of biomolecules that serve a variety of cellular functions. Clearly, in chemical terms, the defining feature of lipids is their insolubility in water, which greatly impacts upon their major role in helping to achieve compartmentalization. This we shall detail later. In general, however, fats are the principle form of stored energy and major fuel source for many organisms. On the other hand, phospholipids and sterols are major structural components of membranes, whereas other lipids in relatively small quantities play critical roles as cofactors of enzymes, light-absorbing pigments, electron carriers, hydrophobic anchor points for proteins, emulsifying agents in the digestive tract, intracellular messengers, and hormones – among other functions. Therefore, we can already begin to see a structure/function relationship emerging as we examine various classifications and functions of lipids. First I will briefly discuss the main characteristics by which some of the more prominent lipids are classified. I will then describe some critical features of biological membranes, because these structures allow the great metabolic control that compartmentalization offers. I will then follow with the beginning stages of lipid metabolism, whereby fatty acids are activated and then transported into the mitochondria for next week’s discussion of beta oxidation. Classifications of Lipids Admittedly, different biochemistry text books do not classify lipids in exactly the same manner, with many not even covering all the classifications. For our purposes, we will focus on simple lipids (fatty acids, mono-, di-, and triaglycerols), and compound lipids, which include conjugated lipids (glycolipids & lipoproteins) and phospholipids. It is not possible or necessary to cover all the lipid types in this course. Therefore, I will focus on those lipids which are most relevant to nutrition, as well as the lipids that comprise cell membranes, which are the primary basis of intracellular compartmentalization. Thus, we will discuss fatty acids, triaglycerols, phospholipids, and some steroids.
Fatty Acids Of great importance is the fact that within living organisms, the fats used as stored forms of energy are derivatives of fatty acids. These hydrocarbon derivatives are about as highly reduced (low state of oxidation) as the hydrocarbons of fossil fuels. Just like burning fossil fuels, the complete oxidation of fatty acids to carbon dioxide and water is highly yielding of energy and is exergonic (negative ΔG thus and spontaneous). I will now be referring to the above table. Fatty acids contain the carboxyl group (hence the acidic portion) and anywhere from 4 to well over 20 carbons as a hydrocarbon chain. If we look at some of the most nutritionally relevant fatty acids (such as the essential - we need them in our diet - fatty acids, linolenic and linoleic acid), you will see there are two main ways of naming fatty acids, besides their more familiar common names. You are probably most familiar with the term “omega,” especially omega 3 fish oils. The omega refers to the first carbon encountered with a double bond, starting from the methyl end (i.e. not the carboxyl end). I personally don’t like this classification, because it tells nothing about how many carbons or how many double bonds are present. Now examine the table above, and notice in the left hand column a number - let’s take 18. That number 18 refers to the total number of carbons in the fatty acid, including the carboxyl carbon. So oleic, linoleic, and linolenic acids are all 18 carbon fatty acids. However, notice that there is a colon followed by a number. For example, linolenic acid is 18: 3 Δ9, 12, 15. This means that linolenic acid has 18 carbons, and it has three unsaturated bonds at carbons 9, 12, and 15, with the carbon count starting at the carboxyl end, including the carboxyl carbon. OK? Again, this nomenclature gives us the most information. I don’t wish to insult you by reminding you that the term saturated refers to the lack of any double bonds in the hydrocarbon chains, as in palmitic acid. On the other hand, unsaturated refers to one double bond present in the hydrocarbon chain (monounsaturated) or more than one double bond (polyunsaturated), as in linolenic acid. Also, a great deal of attention has been focused on the trans fats, which are ironically a cardiovascular risk factor, having been originally billed as the alternative to unhealthy saturated fats. We now know that these manufactured trans oils are not healthy, and steps are being taken by some food industrialists to remove them from their products. Please observe the structures in the above table to delineate the difference between the trans and cis fats. These are the same cis and trans criteria used in basic organic chemistry. Triaglycerols (referred to as triglycerides) (TG) This important group of lipids really represents the biomolecular form in which most fat is stored in the body, also accounting for about 95% of dietary fats. Refer to the figure below, and observe the formation of a TG from the dehydration synthesis reaction of three fatty acids and a glycerol molecule. This reaction requires the formation of three separate ester bonds (where an alcohol –glycerol is a tri-alcohol – and a carboxyl group are joined).
A given TG can be composed of the same or any combination of three different fatty acids. Although mono- or diaglycerols (glycerol plus one or two fatty acids, respectively) are important intermediates, their direct nutritional value is of no real consideration, although they can be found in processed foods.
As stated, triaglycerols are highly concentrated stores of energy, because they are both reduced and are anhydrous. Whereas completely oxidized carbohydrates and proteins can yield about 4 kcal/gram, the complete oxidation of lipids can produced approximately 9 kcal of energy per gram. As we shall see next week, the basis of this larger caloric yield from fats is because they are more highly reduced (which means they can be oxidized to a greater extent than their protein and carbohydrate counterparts.) Also, TG are relatively non-polar, which allows them to be stored in a more anhydrous form. What does this mean? Well, let’s cite an example. A gram of dried glycogen can bind nearly two grams of water. Thus, a gram of primarily anhydrous fat stores more than six times the energy as a gram of hydrated glycogen. This might explain why TG and not glycogen were designed as the major energy reserve. So let’s consider a 70 kg individual, who would have about 100,000 kcal of fuel reserve in TG, 25,000 kcal in mostly muscle protein, and 600 kcal in glycogen, with another 40 kcal in glucose. Think of this! It would take an additional 55 kg more weight in a 70 kg man if the amount of energy were to be stored in glycogen that would be equivalent to the 11 kg of TG that would be stored in that same person! Those glycogen and glucose stores could sustain a person for about 24 hours. Compare this to the several weeks of survival time that TG could provide. I am well aware that you all know that TG accumulate primarily in adipose tissue (fat cells). Most of the volume of the cell cytoplasm can be taken up by these fat globules. These cells are specialized for both the synthesis and the storage of TG, which can easily be mobilized into fuel molecules for transport though the blood to other tissues. (As an interesting aside, birds really illustrate the fuel value of TG for an energy source, whereby these birds can migrate great distances without eating. For example, the golden plover can migrate from Alaska to the southern tip of South America, covering 2400 miles of the journey over open water where they can not eat!
Steroids and Sterols. Page 1131 of your text shows the basis of a steroid nucleus – three six-member rings and one five-member ring. Below it is the structure of the sterol cholesterol (a steroid derivative). It is classified as a sterol, because the steroid nucleus contains an OH (alcohol) group. There is no question but that cholesterol is a cardiovascular risk factor regarding arterial plaque formation. However, it has several vital functions, including being a major component of bile, an important part of membrane structure, and it plays a critical role as a precursor in the biosynthesis of other steroids, including vitamin D, cortisol, and androgen- and estrogen-related compounds. The dietary intake of sterols is only a few percent of the lipid consumed each day. Please refer to page 723 of your text and become familiar with the overall picture of this steroid metabolism for the fourth exam, without specific details (just learn what compounds are inter-converted in general from steroids, being steroids themselves. Phospholipids These are the major class of lipids in all biological membranes. In order to construct a phospholipid, four components are required: fatty acids, a phosphate that is attached to an alcohol, and a platform to which the fatty acids attach. The fatty acids represent a hydrophobic barrier so characteristic of a membrane. The remainder of the molecule is hydrophilic in nature to allow interaction with the aqueous milieu of both the cytosol and the extracellular environment. This platform to which I refer and upon which phospholipids are built could be glycerol or sphingosine – a more complicated alcohol. I will only focus upon the role of phospholipids in membranes, remembering that these biomolecules serve many other functions, including their important role as second messengers in cell transignaling. Please find and read about in your text phospholipids, and be familiar with the general role of these molecules discussed for the fourth exam. Lipoproteins This is a subject discussed in more detail in Clinical Biochemistry 560D, both structureand function-wise. However, in general, lipids tend to be transported as conglomerates of lipids and proteins. Because of the general hydrophobic nature of lipids, we find them positioned primarily in the center of the lipoprotein particle, with the more hydrophilic portions of the proteins forming a shell around the lipids, because of the aqueous nature of the blood plasma and cytosolic environment. Chylomicrons, which transport lipids from the G.I. tract to the liver, HDL, LDL, and VLDL particles are common representative of this group. Biological Membranes It is certainly true that membranes are highly diverse in structure and, thus, in function. They do, however, have many features in common that are quite important.
1. Only two molecules thick, membranes are sheet-like organelles that form compartmentalized, closed boundaries. They run between 6 nm and 10 nm in thickness. 2. The lipids of membranes are fairly small molecules having both hydrophobic and hydrophilic components, which, in an aqueous environment, spontaneously form closed sheets. This lipid bilayer becomes a barrier to polar molecules. 3. Membranes are primarily lipids and proteins, also containing carbohydrate moieties that are linked to both the lipids and the proteins. These lipids and proteins are held together by numerous, non-covalent linkages. 4. The overall distribution of lipids across the membrane is asymmetrical. In other words, each layer of bilipid layer has a different sphingolipid and glycerophospholipid composition. 5. Membranes are fluid structures, with the lipids rapidly diffusing in the plane of the membrane, as do the proteins, unless they are specifically anchored. The degree of this fluidity is a function, at least in part, of the cholesterol content and the degree of saturation of the fatty acid components. The more saturated and the more cholesterol present, the less fluid the membrane. This is easily pictured by observing a stick of butter (very saturated) and a stick of margarine (highly unsaturated) melt at room temperature. Obviously, the margarine liquefies more rapidly than the butter. 6. Most cell membranes show electrical polarization, wherein the inside of the membrane is relatively negatively charged, when compared to the inside. This membrane electrical potential is a critical feature in transport, excitability, and energy conversion. Please refer either to Devlin or to the internet to refresh your memory regarding the structure of membranes. Please note the back-to-back positions of the non-polar lipid moieties within the inside of the membrane, with the hydrophilic areas (ex. phosphate groups) located on the inner and outer edges of the structure. Finally, let’s introduce a new biochemical term – amphiphatic. This means that membrane lipids contain both hydrophobic and hydrophilic regions. Digestion of Dietary lipids Although most dietary lipids are in the form of TG, they must be broken down into fatty acids for absorption into the intestinal mucosa. You might recall from an earlier reading that the TG are incorporated into micelles (you might want to review micelles in your text) that are formed with the help of bile salts. These bile salts are another example of amphipathic molecules which are synthesized by the liver from cholesterol and secreted from the gall bladder. By incorporating the lipids into the micelles in a manner where the ester bonds of the lipid are oriented toward the surface of the micelle, the bonds are more vulnerable to digestion by pancreatic lipases that are solution. Should bile salt production
be inadequate (say due to liver disease), as much as 30 grams of fats can be excreted into the feces – a condition known as steatorrhea. At any rate, the lipase action degrades the TG into free fatty acids and a glycerol. Please read in Devlin all regarding lipid digestion, and learn the main features for the fourth exam. Introduction to Fatty Acids as Fuels In order for the peripheral tissues to have access to fatty acids, so that might be utilized as fuels, three stages of processing are required. Mobilization of the lipids is the first phase. In this process, with the help of a hormone-sensitive lipase, TG are degraded into three fatty acids and glycerol. Second, as will be discussed later in this lecture, the fatty acids require activation for the subsequent transport into the mitochondria for oxidation. Third, the activated fatty acids are broken down to acetyl CoA in a step-wise process known as β-oxidation. The acetyl CoA can then enter the Kreb’s cycle. Let’s examine the initial event in the use of lipids as an energy source. That is, the TG are hydrolyzed by lipases in a process known as lipolysis. The adipose lipases are activated by hormones, such as epinephrine, norepinephrine, ACTH, and interestingly glucagon. This is because insulin inhibits lipolysis. (This will be reiterated in a later lecture.) Once the fatty acids are released into the blood, they bind to serum albumin, because they are not water soluble. The albumin then serves as a carrier. By these processes, fatty acids become available as fuel to the various tissues. The following figure illustrates this nicely. Please study and understand it for the fourth exam.
Note: The glycerol produced from the TG hydrolysis is taken up by the liver. It is then phosphorylated, followed by oxidation to dihydroxyacetone phosphate (DHAP), and is then isomerized to glyceraldehyde-3-P (GAP). You should remember that this molecule is an intermediate in both glycolysis and gluconeogenesis. In other words, the glycerol can be converted in the liver to either pyruvate or glucose. This is because the enzymes that are necessary for this conversion are present in the hepatocytes. Also note that this process is reversible, whereby DHAP can be reduced to glycerol-3-P and then, after a phosphate removal, back to glycerol. The bottom line then is that glycerol and some glycolytic intermediates are easily interconvertible. You will be reminded of this in a later lecture.
Fatty Acid Activation Before any fatty acid can be transported into and oxidized within the mitochondria, that lipid must first be activated. This means linking CoA to each fatty acid by an acyl CoA synthase enzyme system located on the outside of the outer mitochondrial membrane. However, this is a multi-step process culminating in a CoA being attached to the carboxyl end of the fatty acid, at the carbonyl carbon of that carboxyl, with water being released. We can now call this product a fatty acyl CoA. The fatty acyl CoA is the activated fatty acid, and this set of activating reactions has taken place in the free cytosol in conjunction with the mitochondrial membranes mentioned above. So the next logical question is, “why does this fatty acid need to be activated?” The answer is simple, the explanation involved. The answer is that it will not be able to enter the mitochondria to be oxidized if it is not activated. The details of this entry are complicated but quite understandable. When I finish giving the mechanisms for entry of the fatty acid into the mitochondria, you might take a few moments to ponder how amazing it is - once again, that all this is continuously going on most of the moments of our lives. The next step is to get the activated fatty acid into the mitochondria. The figure below illustrates this.
Now we will put the top portion of the diagram on hold for a moment and concentrate on the complex figure immediately above this sentence. You will notice that the fatty acid activation of which we just spoke takes place in the cytosol, but the enzyme for that process is located on the outer side of the outer mitochondrial membrane. Thus, the fatty acyl CoA (activated fatty acid) is now produced and able to penetrate the outer mitochondrial membrane to enter the space between the inner and outer membranes of that organelle. Now find carnitine palmitoyltransferase I (CPTI), which is localized in the outer membrane. The reaction that it catalyzes is shown in the equation at the very top of the above two-part diagram. Note the product, fatty acylcarnitine. The reaction is also shown in the more complex diagram immediately above. Also notice that the CoA is removed for the time being. The fatty acyl carnitine can now be translocated across the inner membrane with the help of a translocase. Once it is across the inner membrane and in the mitochondrial matrix, the CoA can be restored to the fatty acid (now activated again), while at the same time releasing carnitine. This reaction is catalyzed by CPT II located in the inner membrane. You will notice that the released carnitine can be recycled into the
inter-mitochondrial membrane space to be reused to synthesize more fatty acylcarnitine. What we have thus accomplished is to transport an activated fatty acid (meaning carrying a CoA) into the mitochondria, now ready for β-oxidation. This is a subject for the next lecture. However, I hope you are wondering why all these biochemical shenanigans must occur just to get a fatty acid into the mitochondrial. The answer lies in the fact that the activated fatty acid can not penetrate the inner mitochondrial membrane in its activated form, but rather requires carnitine to help bring it across the inner membrane. Once it is in the matrix, the carnitine is no longer required, whereas the re-addition of the CoA to the fatty acid is required, if that lipid is to be oxidized to produce energy. Interestingly, it has been known since 1949 that fatty acids are oxidized in the mitochondria. Clinical Comments To get a little jump on Clinical Biochemistry, a significant number of disorders have been traced to deficiencies in either the carnitine, the translocse, or the transferase. Carnitine deficiency can produce symptoms that could include muscle cramping all the way to severe weakness and even death. Primarily affected are the heart, kidney, and skeletal muscle. An important manifestation of carnitine acyl transferase deficiency is muscle weakness during prolonged exercise. We need to remind ourselves that muscle is dependent upon fatty acids as a long term energy source. The transport scheme described above is a requirement for long chain fatty acids to enter the mitochondria. However, medium chain fatty acids (8-C through 10-C) do not require carnitine to enter the mitochondria. Thus, they are oxidized in the usual manner in such patients as described above. So in quick summary, these disorders illustrate well the structure/function relationship we discussed in the first lecture, because they demonstrate that the impaired flow of a metabolite from one cellular compartment to another can result in a pathological condition. (See how compartmentalization keeps coming up again and again!) This closes our discussion for this week. What we have tried to accomplish in this lecture is to introduce lipids, their classification, structure, localization within membranes, and the preparation they undergo in order to be metabolized by a process called β-oxidation. This is for next week, when we sill see how much energy can be derived from the oxidation of a single fatty acid vs. that of carbohydrate.
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