Introduction to Human Nutrition 2nd Edition ( PDFDrive )

6 Nutrition and Metabolism of Lipids Bruce A Griffin and Stephen C Cunnane Key messages • Dietary lipids (fats) are emulsified, lipolyzed (hydrolyzed), and solubilized in the upper small gut before they are absorbed in • Lipids are organic compounds composed of a carbon skeleton the ileum, entering enterocytes with the help of fatty acid-binding with hydrogen and oxygen substitutions. The most abundant proteins. lipids are sterols or esters of fatty acids with various alcohols such as glycerol and cholesterol. • Lipids are precursors to hormones such as steroids and eicosanoids, and dietary lipids are carriers for fat-soluble • Fatty acids are the densest dietary source of energy, but lipids vitamins. also have important structural roles in membranes. The processes controlling the synthesis, modification, and degradation of fatty • Lipids are transported in the blood circulation as lipoprotein acids contribute to the fatty acid profile of membrane and storage particles: the chylomicrons, very low-density, low-density, and lipids. high-density lipoproteins. • By enhancing the taste of cooked foods, some dietary lipids are • Some polyunsaturated fatty acids are vitamin like because they potentially significant risk factors for obesity and other chronic, cannot be synthesized de novo (linoleate, α-linolenate). degenerative diseases that influence human morbidity and mortality. 6.1 Introduction: the history of lipids in termed “amphipathic” by Hartley in 1936 and human nutrition renamed “amphiphilic” by Winsor in 1948. The term “lipid” was introduced by Bloor in 1943, by The first understanding of how fat was absorbed which time the existence of cholesterol had been emerged in 1879 when Munk studied fat emulsions known for nearly 200 years and individual fats for 130 and showed that lymph contained TAG after a fatty years. Cholesterol was named “cholesterine” (Greek meal, and even after a meal not containing TAG. In for bile-solid) by Chevreul in 1816, although he did 1905, Knoop deduced that fatty acid β-oxidation not discover it. Cholesterol’s association with aortic probably occurred by stepwise removal of two carbons plaques dates at least to Vogel’s work in 1843. Chevreul from the fatty acid. The probable role of two carbon isolated a mixture of 16- to 18-carbon saturated fatty units as building blocks in the synthesis of fatty acids acids in 1813 that was called margarine because he was recognized by Raper in 1907, but it took until the thought it was a single 17-carbon fatty acid, marga- 1940s for Schoenheimer, Rittenberg, Bloch, and others rate. The mixed triacylglycerol (TAG) of palmitate to confirm this, using tracers such as deuterated water (16:0) and stearate (18:0) was also called margarine, and carbon-13. The late 1940s was a seminal period whereas the triglyceride of oleate, stearate, and palmi- in our understanding of how fatty acid oxidation tate became known as oleomargarine. Phospholipids occurs. Green and colleagues discovered that ketones were discovered by Thudicum, who isolated and were fatty acid oxidation products, and Lehninger named sphingosine in 1884 and also lecithin (phos- demonstrated the role of mitochondria as the cellular phatidylcholine) and kephalin (phosphatidylethanol- site of fatty acid oxidation. Microsomal desaturases amine). The difference in polarity across phospholip- were shown to introduce an unsaturated bond into ids is a key attribute of these molecules and was long-chain fatty acids by Bloomfield and Bloch in 1960. © 2009 BA Griffin and SC Cunnane.

Nutrition and Metabolism of Lipids 87 In 1929, Mildred and George Burr discovered that Table 6.1 Classification of lipids the absence of fat in a diet otherwise believed to contain all essential nutrients impaired growth and Simple lipids (fatty acids Fats (fatty acids esterified with caused hair loss and scaling of the skin of rats. This esterified with alcohols) led to the isolation of the two primary “essential” glycerol) polyunsaturated fatty acids, linoleate (18:2n-6) and Complex lipids (fatty acids α-linolenate (18:3n-3). The prostaglandins are a sub- esterified with alcohols plus Waxes (true waxes, sterol esters, class of eicosanoids that were discovered in the early other groups) 1930s by Von Euler, who mistakenly believed that they vitamin A and D esters) originated from the prostate gland. The link between Derived lipids (obtained by Phospholipids (contain the eicosanoids and polyunsaturates, principally hydrolysis of simple or arachidonate, was established in the 1960s. complex lipids) phosphoric acid and, usually, 6.2 Terminology of dietary fats Miscellaneous lipids a nitrogenous base) Glycolipids (lipids containing a Lipids carbohydrate and nitrogen but Like other organic compounds, all lipids are com- posed of a carbon skeleton with hydrogen and oxygen no phosphate and no glycerol) substitutions. Nitrogen, sulfur, and phosphorus are Sulfolipids (lipids containing a also present in some lipids. Water insolubility is a key but not absolute characteristic distinguishing most sulfur group) lipids from proteins and carbohydrates. There are Lipoproteins (lipids attached to some exceptions to this general rule, since short- to medium-chain fatty acids, soaps, and some complex plasma or other proteins) lipids are soluble in water. Hence, solubility in a “lipid Lipopolysaccharides (lipids solvent”such as ether, chloroform, benzene, or acetone is a common but circular definition of lipids. attached to polysaccharides) Fatty acids (saturated, There are four categories of lipids, as classified by Bloor: simple, compound (complex), derived, and monounsaturated, or miscellaneous (Table 6.1). Simple lipids are esters of fatty acids with various alcohols such as glycerol or polyunsaturated) cholesterol. They include triacylglycerols (TAG = neutral fats and oils), waxes, cholesteryl esters, and Monoacylglycerols and vitamin A and D esters. Compound lipids are esters of fatty acids in combination with both alcohols and diacylglycerols other groups. They include phospholipids, glycolip- Alcohols (include sterols, ids, cerebrosides, sulfolipids, lipoproteins, and lipo- polysaccharides.Derived lipids are hydrolysis products steroids, vitamin D, vitamin A) of simple or compound lipids, including fatty acids, Straight-chain hydrocarbons monoacylglycerols and diacylglycerols, straight-chain Carotenoids and ring-containing alcohols, sterols, and steroids. Squalene Miscellaneous lipids include some wax lipids, carot- Vitamins E and K enoids, squalene, and vitamins E and K. RCOOH, where R is hydrogen in formic acid, CH3 in Saturated and unsaturated fatty acids acetic acid, or else a chain of one to over 30 CH2 groups terminated by a CH3 group. The various The main components of dietary fat or lipids are fatty names for individual fatty acids (common, official) acids varying in length from one to more than 30 and their abbreviations are complicated, and the use carbons. They are carboxylic acids with the structure of one or other form is somewhat arbitrary. The basic rule for the abbreviations is that there are three parts: number of carbons, number of double bonds, and position of the first double bond. Thus, the common dietary saturated fatty acid palmitate is 16:0 because it has 16 carbons and no double bonds. The common dietary polyunsaturated fatty acid linoleate is 18:2n-6 because it has 18 carbons, two double bonds, and the first double bond is at the sixth carbon from the methyl-terminal (n-6). Beyond six carbons in length, most fatty acids have an even number of carbons (Table 6.2). Older fatty acid ter- minology referring to saturated or unsaturated carbons in lipids that still occasionally appears includes: aliphatic (a saturated carbon), olefinic (an unsaturated carbon), allylic (a saturated carbon adja- cent to an unsaturated carbon), and doubly allylic carbon (a saturated carbon situated between two unsaturated carbons).

88 Introduction to Human Nutrition Table 6.2 Nomenclature of common fatty acids Saturates cis-Monounsaturates Saturated Monounsaturated Polyunsaturated cis-Polyunsaturates Formic (1:0) Lauroleic (12:1n-3) Linoleic (18:2n-6) Acetic (2:0) Myristoleic γ-Linolenic (18:3n-6) Propionic (3:0) (14:1n-5) Dihomo-γ-linolenic Palmitoleic (20:3n-6) Butyric (4:0) Valeric (5:0) (16:1n-7) Arachidonic (20:4n-6) Oleic (18:1n-9) Adrenic (22:4n-6) Caproic (6:0) Elaidic n-6 Docosapentaenoic Caprylic (8:0) (trans-18:1n-9) (22:5n1-6) Vaccenic (18:1n-7) Capric (10:0) α-Linolenic (18:3n-3) Petroselinic Lauric (12:0) (18:1n-12) Stearidonic (18:4n-3) Myristic (14:0) Gadoleic Eicosapentaenoic (20:1n-11) (20:5n-3) Palmitic (16:0) Gondoic (20:1n-9) n-3 Docosapentaenoic Margeric (17:0) (22:5n-3) Stearic (18:0) Euricic (22:1n-9) Arachidic (20:0) Docosahexaenoic Behenic (22:0) Nervonic (24:1n-9) (22:6n-3) Lignoceric (24:0) Lengthening of the chain and the introduction of Figure 6.1 Stick models illustrating the basic structural differences additional double bonds beyond the first one occur between saturated, cis-monounsaturated, and cis-polyunsaturated from the carboxyl-terminal. The presence of one or fatty acids. As shown in two dimensions, the increasing curvature more double bonds in a fatty acid defines it as “unsat- caused by inserting one or more double bonds increases the area urated,” compared with a saturated fatty acid which occupied by the fatty acid. The physical area occupied by unsaturated contains no double bonds. A saturated fatty acid gen- fatty acids is further accentuated in three dimensions because esteri- erally occupies less space than an equivalent chain fied fatty acids rotate around the anchored terminal. length unsaturated fatty acid (Figure 6.1). Double bonds allow for isomerization or different orientation ucts containing ruminant milk fat. Hence, although (cis or trans) of the adjoining carbons across the they are produced in relatively large quantities double bond (Figure 6.2). In longer chain fatty acids, from the fermentation of undigested carbohydrate in double bonds can also be at different positions in the the colon, as such, they do not become part of the molecule. Hence, unsaturation introduces a large body lipid pools. Medium-chain fatty acids (8–14 amount of structural variety in fatty acids and carbons) arise as intermediates in the synthesis of the resulting lipids. Further details about the features long-chain fatty acids or by the consumption of of the different families of fatty acids are given in coconut oil or medium-chain TAG derived from it. Sections 6.6 and 6.8. Like short-chain fatty acids, medium-chain fatty acids are present in milk but they are also rarely esterified Short- and medium-chain fatty acids into body lipids, except when consumed in large amounts in clinical situations requiring alternative Short-chain fatty acids (less than eight carbons) energy sources. Medium-chain fatty acids (8–14 are water soluble. Except in milk lipids, they are carbons) are rare in the diet except for coconut and not commonly esterified into body lipids. Short- milk fat. chain fatty acids are found primarily in dietary prod-

Nutrition and Metabolism of Lipids 89 cis-Monounsaturates long-chain saturates can probably be sustained without a dietary source of these fatty acids. Com- trans-Monounsaturates pared with all other classes of dietary fatty acid, espe- cially monounsaturated or polyunsaturated fatty Figure 6.2 Stick models comparing a cis- with a trans-unsaturated acids, excess intake or synthesis of long-chain satu- fatty acid. A cis-unsaturated double bond creates a U-shaped space rates is associated with an increased risk of cardiovas- and confers curvature to the molecule because, relative to the longi- cular disease. tudinal axis of the fatty acid, the two hydrogens at the double bond are on the same side of the molecule. A trans-unsaturated double bond The most common long-chain cis-monounsatu- does not confer curvature to the molecule because the hydrogens are rated fatty acids in diet and in the body are oleate on opposite sides of the double bond. A trans-double bond therefore (18:1n-9) and palmitoleate (16:1n-7), with the former tends to give the fatty acid physicochemical properties more like that predominating by far in both the body’s storage and of a saturated fatty acid. membrane lipids. As with stearate, most oleate in the human body appears to be of dietary origin. Hence, Long-chain saturated and although humans have the capacity to desaturate monounsaturated fatty acids stearate to oleate, dietary oleate is probably the domi- Long-chain fatty acids (>14 carbons) are the main nant source of oleate in the body. Only plants can constituents of dietary fat. The most common satu- further desaturate oleate to linoleate and again to α- rated fatty acids in the body are palmitate and stea- linolenate. As with saturates of >18 carbons in length, rate. They originate from three sources: directly from 20-, 22-, and 24-carbon monounsaturates derived the diet, by complete synthesis from acetyl-coenzyme from oleate are present in specialized membranes A (CoA), or by lengthening (chain elongation) of a such as myelin. pre-existing shorter-chain fatty acid. Hence, dietary or newly synthesized palmitate can be elongated Polyunsaturated fatty acids (PUFAs) within the body to form stearate and on to arachidate (20:0), behenate (22:0), and lignocerate (24:0). In Linoleate and α-linolenate are the primary dietary practice, little stearate present in the human body cis-polyunsaturated fatty acids in most diets. Neither appears to be derived by chain elongation of pre- can be synthesized de novo (from acetate) in animals existing palmitate. In humans, saturates longer than so are ‘essential’ fatty acids. They can be made 24 carbons do exist but usually arise only during by chain elongation from the two respective 16- genetic defects in fatty acid oxidation, as will be dis- carbon precursors, hexadecadienoate (16:2n-6) and cussed later. hexadecatrienoate (16:3n-3), which are found in common edible green plants at up to 13% of total Palmitate and stearate are important membrane fatty acids. Hence, significant consumption of green constituents, being found in most tissue phospholip- vegetables will provide 16-carbon polyunsaturates ids at 20–40% of the total fatty acid profile. Brain that contribute to the total available linoleate and membranes contain 20- to 24-carbon saturates that, α-linolenate. like palmitate and stearate, are synthesized within the brain and have little or no access to the brain from Linoleate is the predominant polyunsaturated fatty the circulation. The normal membrane content of acid in the body, commonly accounting for 12–15% of adipose tissue fatty acids. In the body’s lean tissues there are at least three polyunsaturates present in amounts >5% of the fatty acid profile (linoleate, ara- chidonate, docosahexaenoate). In addition, at least two other biologically active polyunsaturates are present in body lipids [dihomo-γ-linolenate (20:3n- 6) and eicosapentaenoate (20:5n-3)], although usually in amounts between 1% and 3% of total fatty acids. Marine fish are the richest source of 20- to 22-carbon polyunsaturates. α-Linolenate and its precursor, hexadecatrienoate (16:3n-3), are the only n-3 polyun- saturates in common terrestrial plants.

90 Introduction to Human Nutrition Hydrogenated and conjugated fatty from their lower proportion of saturated (straight- acid isomers chain) and higher proportion of unsaturated (bent- chain) fatty acids. Unsaturated fatty acids usually The introduction of unsaturation with one double have a lower melting point; this facilitates liquefaction bond creates the possibility of both positional and of the fats of which they are a component. TAGs of geometric isomers in fatty acids. Among long-chain animal origin are commonly fats, whereas those of unsaturated fatty acids, positional isomers exist fish or plant origin are usually oils. Animal fats and because the double bond can be introduced into fish oils frequently contain cholesterol, whereas plant several different locations, i.e., 18:1n-7, 18:1n-9, oils do not contain cholesterol but usually contain 18:1n-11, etc. Geometric isomers exist because the other “phyto” sterols. two remaining hydrogens at each double bond can be opposite each other (trans) or on the same side of the TAGs are primarily used as fuels, so dietary fats molecule (cis; Figure 6.2). Thus, there is cis-18:1n-9 (mostly TAGs) are commonly associated with energy (oleate) and trans-18:1n-9 (elaidate), and so on for all metabolism rather than with structural lipids unsaturated fatty acids, with the combinations found in membranes. However, membrane lipids as mounting exponentially as the number of double well as TAGs are extracted with lipid solvents used to bonds increases. determine the fat content of foods, tissues, or plant material. Hence, because organs such as brain are Trans isomers of monounsaturated or polyunsatu- rich in membrane phospholipids, when the total rated fatty acids arise primarily from partial hydroge- lipids are extracted to determine the organ’s chemical nation during food processing of oils, but some also composition, these organs are said to have a certain occur naturally in ruminants. The number of trans fat content. On a chemical basis this is true, but this isomers increases with the number of double bonds, description often misconstrues the nature of the lipid so there is only one trans isomer of oleate but there because the brain in particular contains virtually no are three trans isomers of linoleate and seven of α- TAG. linolenate. Virtually all naturally occurring polyun- saturated fatty acids have double bonds that are Phospholipids methylene interrupted, i.e., have a CH2 group between the two double bonds. However, methylene interrup- Phospholipids contain two nonpolar, hydrophobic tion between double bonds can be lost, again, through acyl tail groups and a single functional head group food processing, and the bonds moved one carbon that is polar and hydrophilic. Hence, they are rela- closer together, becoming conjugated. Thus, the tively balanced amphiphilic lipids and, in this capac- double bonds in linoleate are at the 9–10 and 11–12 ity, are crucial components of biological membranes. carbons, but in conjugated linoleate they are at the The head groups contain phosphorus and amino 9–10 carbons and the 11–12 carbons. Some degree of acids (choline, serine, ethanolamine), sugars (inosi- further desaturation and chain elongation can occur tol), or an alcohol (glycerol). Phosphatidylcholine in conjugated fatty acids, but much less than with (lecithin) is the most abundant phospholipid in methylene-interrupted polyunsaturates. Thus, conju- animal tissues but phosphatidylglycerols (glycosides) gated linoleate is the main conjugated fatty acid that predominate in plant lipids. Phospholipids contain- has attracted considerable attention with respect to its ing a fatty acid amide are sphingolipids. Various phos- potential role in nutritional health. pholipases can hydrolyze the acyl groups or head group during digestion or metabolism. Fats and oils One of the outstanding characteristics that make Fats are esters of fatty acids with glycerol (Table 6.1). phospholipids suitable as major constituents of bio- They usually occur as triesters or triacylglycerols logical membranes is that, in water, they naturally (TAGs), although monoacylglycerols and diacylglyc- aggregate into spherical or rod-like liposomes or ves- erols occur during fat digestion and are used in food icles, with the hydrophilic portion facing outwards processing. Most common dietary fats contain a and the hydrophobic portion facing inwards (Figure mixture of 16- to 18-carbon saturated and unsatu- 6.3). Changing the constituent acyl groups from satu- rated fatty acids. By convention, fats that are liquid at rated to polyunsaturated changes the fluidity of these room temperature are called oils, a feature arising aggregates because of the greater amount of space

Nutrition and Metabolism of Lipids 91 Outer surface Inner surface Trans-membrane Saturated Phospholipid head groups protein/receptor phospholipid (asymmetrically distributed) Ion channel Unsaturated phospholipid Carbohydrate unit Cholesterol Figure 6.3 Simplified schematic view of a membrane bilayer. The main components are proteins, free cholesterol, phospholipids, and carbohy- drates. There are many different proteins with a myriad of shapes, membrane distribution, and functions, of which three are illustrated. Membrane phospholipids principally help to create the bilayer. They have four types of “head groups” (choline, ethanolamine, serine, and inositol) that are located at or near the membrane’s two surfaces. The two fatty acids in phospholipids are mixtures of 16- to 22-carbon saturates, monounsaturates, and polyunsaturates in all combinations, with those rich in unsaturated fatty acids occupying more space; hence, their trapezoid shape compared with the narrower, rectangular shape of the more saturated phospholipids. Free cholesterol represents 30–40% of the lipid in most membranes. The many different carbohydrates are on the membrane’s surfaces and are bound to lipids and/or proteins in the membrane. occupied by more unsaturated fatty acids. At inter- 6.3 Lipids as components of the diet faces between non-miscible polar and non-polar sol- vents, phospholipids also form a film or monolayer. Food or dietary sources of lipids are listed in Table 6.3. Cholesterol is found only in animal lipids, while Sterols a variety of other phytosterols occur in plants. Soyabeans, leafy plants, and lean animal meat are rich The main sterol of importance in human nutrition is in dietary phospholipids. Animal fat and plant oils cholesterol. It has multiple roles including being: from seeds or nuts are rich in TAG. ● a vital component of biological membranes The leafy and fruit components of plants contain ● a precursor to bile salts used in fat digestion phospholipids and sterols, whereas seeds contain tri- ● a precursor to steroid hormones. glycerides. With rare exceptions such as flaxseed (linseed), edible green leaves are proportionally much Sterols are secondary alcohols belonging to the poly- richer in α-linolenate than are seeds. Seed oils are isoprenoids or terpinoids (terpenes), which have a usually rich in either linoleate or oleate. Common common precursor, isopentenyl diphosphate. Other plant sterols include β-sitosterol, β-sitostanol, and members of the terpinoids include squalene, carot- campesterol. Foods enriched with esters of plant enoids, and dolichols. Bacteria appear to be the only sterols are used widely to lower blood cholesterol via life forms not containing cholesterol. Sterols have the inhibition of cholesterol absorption in the gut. a common cyclopentano(a)perhydrophenanthrene skeleton with different substitutions giving rise to the Phospholipids and cholesterol constitute the multiple sterols and steroids. majority of lipids in tissues (gut, kidney, brain,

92 Introduction to Human Nutrition Table 6.3 Common food sources of lipids is also found in several types of edible seaweed. Tropi- cal fish generally have higher arachidonate than do Cholesterol Eggs, shellfish, organ meats cold-water fish. Phytosterols Soya products, olive oil Short-chain fatty acids Milk fat Partial hydrogenation is a common feature of unsaturated fatty acids in processed foods. Complete (1–6 carbons) Milk fat, coconut fat hydrogenation makes fats very hard and is more Medium-chain fatty expensive than partial hydrogenation. Depending on Saturates: animal fat, shortening, butter, the applications and the source of the original oil or acids (8–14 palm oil, peanuts fat, partial hydrogenation is an economical way to control the properties of fats or oils used in food carbons) Monounsaturates: olive, canola oils production. Dietary diacylglycerols and monoacyl- Long-chain fatty acids Linoleate: sunflower, safflower, corn oils, glycerols are used by the food industry for emulsifica- tion of water- and oil-based components in foods (16–20 carbons) soyabean such as ice cream and mayonnaise. α-Linolenate: flaxseed oil, canola, 6.4 Digestion, absorption, and transport soyabean oil, walnuts of dietary fat γ-Linolenate: evening primrose oil, borage The average daily intake of fat in a Western diet ranges oil, blackcurrant seed oil between 50 and 100 g and provides between 35% and Stearidonate: blackcurrant seed oil 40% of total energy. It consists mainly of TAG, which Arachidonate: lean meat and organ lipids forms the principal component of visible oils and fats, Eicosapentaenoate: marine cold-water and minor quantities of phospholipids and choles- terol esters (CEs). The physical properties of dietary fish, shellfish, some seaweeds fat, such as their hardness at room temperature Docosahexaenoate: marine cold-water (melting point) and subsequent metabolic properties once in the body, are determined by the number of fish, shellfish double bonds in their constituent fatty acids (degree Trans fatty acids: partially hydrogenated of saturation or unsaturation) and length of the fatty acid carbon chain (see Tables 6.2 and 6.3). As men- fats and oils tioned in Section 6.2, fats that are solid at room tem- perature tend to consist of long-chain saturated fats skeletal muscle, etc.) of lean, undomesticated animals. (>14 carbons, no double bonds), whereas oils consist By contrast, in domesticated animals, TAGs or non- of long-chain unsaturated fats with several double membrane lipids present in subcutaneous and intra- bonds. It has become conventional to refer to dietary muscular adipose tissue deposits are the dominant fats as “lipids” once they have been absorbed into the form of lipid on a weight basis. This is because domes- body via the small intestine, although it is not incor- tication usually involves rearing animals with minimal rect to refer to dietary fat as “dietary lipid.” exercise and on higher energy intakes, leading to more subcutaneous and visceral TAG obtained through Reception, emulsification, lipolysis, both fat synthesis and deposition of dietary fat. solubilization, and absorption Animal meat lipids are the main dietary source of arachidonate (20:4n-6), although it can also be The digestion of dietary fat takes place in three phases, obtained from tropical marine fish. known as the gastric, duodenal, and ilial phases. These involve crude emulsification in the stomach, lipolytic Lipoproteins are the main form of lipid in the breakdown by lipases and solubilization with bile salts blood (see Section 6.5). Like lipoproteins, milk lipids in the duodenum and, finally, absorption into the epi- also occur as globules consisting of a combination of thelial cells or enterocytes lining the walls of the small a mainly TAG core surrounded by a membrane con- intestine or ileum. Digestion may actually be initiated taining proteins, cholesterol, and phospholipids. in the mouth under the influence of a lingual lipase Phospholipids and cholesterol constitute the main lipids of undomesticated edible fish, which usually have low amounts of TAG or stored body fat. As in domesticated animals, it is likely that subcutaneous and intramuscular fat deposits of TAG will increase in commercially farmed fish. Cold-water marine fish are the main dietary source of the long-chain n-3 (omega-3) polyunsaturates eicosapentaenoate (20:5n- 3), and docosahexaenoate (22:6n-3), but the former

Nutrition and Metabolism of Lipids 93 secreted by the palate, although its contribution to fashion with the initial removal of a fatty acid from lipolysis in adults is questionable and thought to be position 1 and then position 3 from the glycerol back- more important in young suckling infants, in which bone, generating a 2,3-diacylglycerol, followed by a its release is stimulated by suckling and the presence 2-monoacylglycerol (2-MAG). of milk. It is possible that this lingual lipase is carried into the stomach, where it acts as a human gastric Solubilization of emulsified fat lipase (HGL) that has been shown to degrade up to 10% of ingested fat. Although these early products of With the notable exceptions mentioned previously fat digestion, fatty acids and monoacylglycerols, rep- (Section 6.2), fats are insoluble in water and must be resent a relatively minor component of fat digested, rendered soluble before they can be absorbed in the their entry into the duodenum is believed to supply a gut and transported within cells and in the circula- major stimulus for the production of the hormone tion. In each of these situations, this is achieved by cholecystokinin (CCK), which inhibits gut motility. the hydrophobic fat or lipid associating with mole- cules that are capable of interfacing with both hydro- The stomach serves mainly as an organ of mechan- phobic and hydrophilic environments. Molecules ical digestion and, by churning its contents, produces with these characteristics are called amphipathic mol- a coarse creamy emulsion known as chyme. The cir- ecules, examples of which are phospholipids, bile cular pyloric sphincter muscle that separates the salts, and specialized proteins known as apoproteins stomach from the duodenum and, with other factors, (Figure 6.5). In the small intestine emulsified fats are controls the rate of gastric emptying opens twice a solubilized by associating with bile salts produced in minute to release approximately 3 ml of chyme. Since the liver and stored and released from the gallbladder, emulsified fat in chyme is less dense than the aqueous and phospholipids to form complex aggregates known material, the two fractions separate with the fat col- as mixed micelles. Lipids within cells and in the cir- lecting above the aqueous layer. As a result, the entry culation are solubilized by combining with specific of emulsified fat into the duodenum is delayed, allow- proteins known as fatty acid-binding proteins (FABPs) ing sufficient time for the minor breakdown products and apolipoproteins (ApoA, B, C, E), respectively. to act on CCK. Further details of the structure and function of these specialized proteins are given in Section 6.5. The duodenal phase involves the breakdown of the emulsified fat by a process known as lipolysis and the The action of pancreatic lipase on TAG yields free solubilization of the products of lipolysis. The entry fatty acids and 2-MAG. Fatty acids of short- and of chyme containing minor lipolytic products into medium-chain length (≤14 carbons) are absorbed the duodenum stimulates the: directly into the portal circulation with free glycerol and transported bound to albumin to the liver, where ● release of CCK, which inhibits gut motility they are rapidly oxidized. In contrast, long-chain fatty ● secretion of bile acids from the gall bladder acids (LCFAs; >14 carbons) associate with bile salts in ● release of pancreatic juice containing a battery of bile juice from the gallbladder and are absorbed into the enterocyte for further processing and packaging lipases. into transport lipoproteins. The primary bile salts, cholic and chenodeoxycholic acids, are produced Lipolysis is an enzyme-catalyzed hydrolysis that from cholesterol in the liver under the action of the releases fatty acids from lipids (TAGs, phospholipids, rate-limiting enzyme 7-α-hydroxylase. These bile and CEs). It involves the hydrolytic cleavage of bonds salts act effectively as detergents, solubilizing lipids by between a fatty acid and the glycerol backbone of the formation of mixed micelles. These are spherical TAGs and phospholipids, and cholesterol in CEs, and associations of amphipathic molecules (with hydro- occurs not only in the digestive tract but also in cir- phobic and hydrophilic regions) with a hydrophilic culating and intracellular lipids (Figure 6.4). The surface of bile salts and phospholipids that encapsu- hydrolysis of emulsified dietary fat entering the duo- lates a hydrophobic core of more insoluble LCFAs denum is catalyzed by a battery of pancreatic enzymes and 2-MAG (see Figure 6.4). The micelle core will including a pancreatic lipase that acts chiefly on TAG also contain some lipid-soluble vitamins including and phospholipase A2 and a cholesterol ester hydro- tocopherols and carotenoids. The formation of mixed lase acting on phospholipids and CEs. The hydrolysis of TAG by pancreatic lipase occurs in a sequential

94 Introduction to Human Nutrition Plasma Thoracic duct Chylomicrons lymph Liver Short- and medium-chain Apoproteins fatty acids and Gall bladder phospholipids TAG Cholesterol OH esters OH ACAT Bile acids ؉ Mixed micelles Bile acid conjugates OH ؉ Unabsorbed material P Feces P Dietary PL Pancreatic fats lipase ؉OH TAGs OH Fatty acids Hydrophobic lipid core MAGs Duodenum Jejunum Ileum Figure 6.4 Reception, emulsification, lipolysis, solubilization, and absorption of fats. ACAT, acyl-CoA-cholesterol acyltransferase; MAG, mono- acylglycerol; TAG, triacylglycerol; PL phospholipid; P, phosphate. micelles increases the solubility of fat by 100- to 1000- ity for different types of LCFAs. Thus, the absorption fold. They create an acidic microenvironment for the of LCFAs and 2-MAG derived from dietary TAGs lipid core which, through protonation, facilitates the occurs by facilitated diffusion via FABP, which dissociation of LCFAs and 2-MAG from the micelle increases membrane permeation and promotes cel- and diffusion into the enterocyte. lular uptake of LCFAs and monoglycerides. An addi- tional factor that drives the diffusion gradient is the Absorption of solubilized fat rapid re-esterification of LCFAs into 2-MAG and 2- MAG into TAGs within the enterocyte by the enzyme The ilial or absorptive phase involves the transit of acyl-CoA-cholesterol acyltransferase (ACAT). The dietary fats from mixed micelles into the enterocyte. absorption of dietary TAGs in the small intestine is Although originally believed to be a purely passive extremely efficient, with up to 90% being absorbed. process, dependent on factors such as the rate of Dietary cholesterol also associates within mixed gastric emptying, extent of mixing, and gut motility, micelles and is absorbed in a similar manner by spe- the translocation of LCFAs and 2-MAG from the cific sterol-carrying proteins resident in the entero- micelle into the enterocyte is now known to be assisted cyte membrane. Thus, cholesterol is also absorbed by by the presence of FABPs within the cell membrane a protein-facilitated mechanism but, in contrast to and the cell. These maintain a diffusion gradient dietary TAGs, only about 40% of dietary cholesterol down which LCFAs and MAGs can flow into the cell. will be absorbed directly. FABPs have numerous roles within cells and specific-

Nutrition and Metabolism of Lipids 95 Surface lipids Free cholesterol Enterohepatic circulation Phospholipid The absorption of fat in the small intestine is depen- Protein (apoproteins) dent on the availability of bile acids from biliary secretions which also contain free cholesterol. Both Core lipids Cholesteryl esters bile acids (>95%) and biliary cholesterol are salvaged Triacylglycerols by an energy-dependent process in the terminal ileum. This active process of reabsorption via the Figure 6.5 General lipoprotein structure. (Reproduced from Dur- enterohepatic circulation is tightly controlled by a rington PN. Hyperlipidaemia Diagnosis and Management, 2nd edn. feedback mechanism that is sensitive to hepatic levels Elsevier Science, Oxford, copyright 1995 with permission of Elsevier.) of cholesterol. Thus, the reabsorption of cholesterol downregulates the activity of 7-α-hydroxylase in the liver, shutting down the further production of bile acids. Substances in the lumen of the gut that are capable of binding or competing with bound bile acids, such as naturally occurring plant sterols or soluble nonstarch polysaccharides (NSPs), prevent their reabsorption which, in effect, interrupts the enterohepatic circulation. This depletes the supply of cholesterol and accelerates the production of bile acids, depleting the liver of cholesterol (Figure 6.6). To replenish this loss, liver cells respond by increasing their uptake of cholesterol from circulating lipopro- teins in the blood, with the result of a decrease in blood cholesterol. Interruption of the enterohepatic circulation helps to explain the cholesterol-lowering action of some of the earliest known cholesterol-low- ering drugs, but also such dietary constituents as the phytosterols (sitosterol and stanol esters) and soluble fiber or NSPs. For further details of the control mech- anism see Section 6.5. Liver cell YY LDL HMG-CoA reductase YY Y Acetate Free cholesterol LDL receptors ؉ 7-␣-Hydroxylase x Excreted Figure 6.6 Interruption of the entero- in feces hepatic circulation. LDL, low-density ؉>؊ lipoprotein; HMG-CoA, 3-hydroxy-3-methyl- glutaryl-coenzyme A. Dietary cholesterol ϩ bile acids Anion exchange resins/soluble dietary fiber Intestine

96 Introduction to Human Nutrition Re-esterification of triacylglycerols in these molecules are rapidly acylated back to 1,2-dia- the enterocyte cylglycerol and finally TAGs by the sequential actions of three enzymes: CoA ligase, monoglycerol acyl- Once LCFAs have entered the cell they are activated transferase, and diacylglycerol acyltransferase. In a by acyl-CoA and are re-esterified with glycerol back similar fashion, lysophosphatidylcholine, produced into TAG and phospholipids by two distinct bio- by the action of pancreatic phospholipase ‘A’ on chemical pathways, the 2-MAG and glycerol-3- dietary phospholipids, is absorbed by the enterocyte phosphate (G-3-P) pathways. The difference between and re-esterified back to phosphatidylcholine in the these two pathways lies in: enterocyte by direct acetylation (Figure 6.7). The bulk of free cholesterol absorbed from the intestinal lumen ● their substrates of activation is also re-esterified in the enterocyte by the enzyme ● the former using 2-MAG and the latter ACAT. α-glycero-3-phosphate Lipoprotein assembly and secretion ● their location within different cellular organelles: Plasma lipoproteins are a family of spherical, macro- the 2-MAGs reside in the smooth endoplasmic molecular complexes of lipid and protein, the princi- reticulum and the G-3-P in the rough endoplasmic pal function of which is to transport endogenous reticulum lipids (synthesized in the liver) and exogenous ● the periods during which they are most active. lipids (synthesized in the gut from dietary fats) from these sites of production and absorption to The 2-MAG pathway is quantitatively of greater peripheral sites of utilization (e.g., oxidation in importance in the enterocyte of the intestine and thus muscle, incorporation in membranes, or as precur- predominates in the postprandial period, whereas the sors of biologically active metabolites) and storage G-3-P pathway is more active in the postabsorptive (e.g., adipose tissue). phase in tissues such as liver, muscle, and adipose tissue. Following the absorption of a fatty meal and uptake of 2-MAG into the enterocyte, up to 90% of Exogenous lipid transport pathway Endogenous lipid transport pathway Via lymph Liver VLDL Intestine CM Capillary Energy in Energy in Lipoprotein VLDL muscle muscle lipase FA FA Lipoprotein CM TAG in lipase TAG in adipose tissue adipose tissue Hepatic IDL lipase 50% 50% Liver CM Liver LDL Other tissues remnant 80% 20% Figure 6.7 Re-esterification of triacylglycerides in enterocytes. CM, chylomicron; FA, fatty acid; IDL, intermediate-density lipoprotein; LDL, low- density lipoprotein; TAG, triacylycerol; VLDL, very low-density lipoprotein. (Reproduced from Mangiapane EH, Salter AM, eds. Diet, Lipoproteins and Coronary Heart Disease. A Biochemical Perspective. Nottingham University Press, Nottingham, 1999, with permission of Nottingham University Press.)

Nutrition and Metabolism of Lipids 97 In the small intestine, the newly re-esterified TAGs chylomicrons containing lipids enriched with poly- and CEs associate with specific amphipathic proteins unsaturated fatty acids (PUFAs) are larger than and phospholipids in the enterocyte to form the chylomicrons enriched with saturated fat, since the largest and most TAG-rich lipoproteins, known as former occupy more space when packaged into a lipo- chylomicrons. The enterocyte is capable of synthesiz- protein. This has implications for the subsequent ing three different apoproteins (apo): apoA-I, apoA- metabolism and fate of these lipoproteins in the cir- IVs and apoB (B-48). The last apoprotein is expressed culation, since TAGs associated with larger chylomi- in two isoforms, the arbitrarily named apoB-100, crons are known to be hydrolyzed more rapidly. It is which is synthesized in the liver, and a shorter relative thought that apoB-48 is produced continuously in the of B-100, which is produced by the enterocyte and is enterocyte-forming pools of apoB-48 in readiness for approximately 48% of the size of B-100 and thus the sudden reception of dietary fat and production of appropriately named apoB-48. While both apopro- chylomicrons. Nevertheless, small chylomicrons can teins are products of the same gene, the mRNA be detected throughout the postabsorptive phase. undergoes post-transcriptional editing in the entero- cyte to produce a truncated polypepetide. ApoB-48 is The onset, duration, and magnitude of postpran- produced in the rough endoplasmic reticulum and dial lipemia can be monitored in the laboratory after transferred to the smooth endoplasmic reticulum, a standard fat-containing meal by making serial mea- where it combines with a lipid droplet, or nascent surements of serum TAG or more specifically TAG chylomicron, and then migrates to the Golgi appara- associated with TAG-rich lipoproteins over a post- tus. Here, the apoproteins (A-I, A-IV, and B-48) are prandial period of up to 8 or 9 hours (remnants of glycosylated before the chylomicrons eventually leave chylomicrons can be detected 12 hours after a meal). the enterocyte by exocytosis through the basement Alternatively, the levels of apoB-48 or retinyl esters in membrane, across the intracellular space between the serum act as useful markers or tracer molecules enterocyte and the lacteal, and are finally discharged for following the metabolism of chylomicrons in into the lymphatic vessels. the postprandial period. In normal subjects postpran- dial lipemia peaks between 3 and 4 hours and sub- Postprandial lipemia sides to baseline concentration after 5–6 hours. In some cases, postprandial TAG (mainly in chylomi- The turbidity or milkiness of serum or plasma follow- crons) can appear in the blood within 30 min and ing the ingestion of fat marks the arrival of dietary fat peak as early as 1 hour after the ingestion of fat. So now contained in chylomicrons in the blood. The rapid is this rise in TAG that it is believed to represent milky appearance of plasma or serum after the inges- preformed lipid in the enterocyte from the previous tion of fat arises from the chylomicrons, which are of meal that is shunted into the circulation by the incom- a sufficient size physically to scatter light and create ing fat load. Note that, in addition to the time taken the milky appearance of serum or plasma after a meal. to emulsify, hydrolyze, and absorb dietary fat, re- The size and composition of the chylomicrons pro- esterification of TAG in the enterocyte and lipopro- duced after a fatty meal are determined by the fat tein assembly alone takes about 15 min, although content of the meal. Hence, the nature of fatty acids shunting means that the first TAG can appear within in chylomicron TAG reflects the nature of fatty acid 30 min, with the first peak after 1 hour. This shunting in the meal. Each chylomicron particle carries a single phenomenon is particularly noticeable during the day molecule of apoB-48 which, unlike its other smaller and gives rise to two or even more peaks, whereas counterparts A-I and A-IV, remains with the chylomi- postprandial peaks following an overnight fast are cron throughout its life in the circulation. There is usually monophasic. little evidence to suggest that the production of apoB- 48, and thus the number of particles, increases in Chylomicrons are not the only TAG-rich lipopro- response to an increased flux of dietary fat. Instead, teins in the postprandial phase. Chylomicrons clearly the enterocyte incorporates more TAG into each chy- contribute significantly to the extent of postprandial lomicron and expands the size of each chylomicron lipemia, and the rate at which the TAGs in these lipo- to facilitate the transport of larger amounts of proteins are hydrolyzed is known to be a critical deter- absorbed dietary fat. There is evidence to suggest that minant of the extent and time-course of postprandial lipemia. The TAGs in circulating chylomicrons are

98 Introduction to Human Nutrition Table 6.4 Plasma lipoproteins: classes, composition, and distribution Mass (106 Da) Chylomicrons VLDLs LDLs HDLs Density (g/ml) 0.4–3.0 10–100 2–3.5 0.2–0.3 Particle diameter (nm) >0.95 <1.006 1.02–1.063 1.063–1.210 Apoproteins >90 30–90 22–28 5–12 Lipids % mass (molecules/particle) B-48, A-I, C-I, C-II, C-III, E B-100, E B-100 A-I, A-II Cholesterol 8 (60 000) 22 (10 000) 48 (2000) 20 (100) Triacylglycerols 83 (500 000) 50 (24 000) 10 (300) 8 (20) Ratio of particles Postabsorptive 1 40 1000 10 000 Postprandial 1 25 250 250 000 VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. lipolyzed by a rate-limiting lipase known as lipopro- about the same time, evidence emerged that TAG-rich tein lipase (LPL). LPL is tethered to the endothelial lipoproteins, and especially remnants of chylomi- lining of blood vessels in peripheral tissues, most crons, were directly atherogenic, meaning that they notably muscle and adipose tissue, by proteoglycan can damage the endothelial lining of arteries and fibers, and as such is known as an endothelial lipase. promote the deposition of cholesterol in coronary Several molecules of LPL can interact and lipolyze the arteries. For this reason, there is considerable research TAG from a single chylomicron particle to generate a interest in the metabolic determinants of postprandial chylomicron remnant which is removed by specific cell lipemia. This includes the mechanisms that underlie membrane receptors in the liver. The situation is com- the production and removal of TAG-rich lipoproteins, plicated by the fact that TAG-rich lipoproteins from not only in the intestine but also in the liver, since the the liver, known as very low-density lipoprotein production and removal of VLDL can clearly influ- (VLDL), also contribute to this postprandial lipemia ence postprandial events. The quality and, to a lesser to variable extents in health and disease states. These extent, quantity of dietary fat are extremely important VLDLs containing endogenously produced TAG are in this respect and have a major role to play in modu- similar in lipid composition to chylomicrons but con- lating lipid-mediated atherosclerosis. siderably smaller (Table 6.4). While chylomicrons carry up to 80% of measurable plasma TAG during the 6.5 Circulating lipids: lipoprotein postprandial period, VLDL particles can carry up to structures and metabolism 80% of the measurable protein (mainly as apo-B), and significantly outnumber chylomicrons at all times. Circulating blood lipids are insoluble in water and VLDL-TAG are also metabolized by LPL, which creates must be solubilized for transportation in the extracel- competition for the clearance of endogenously and lular fluid by combining with bipolar molecules with exogenously derived TAG carried by VLDLs and chy- charged and uncharged regions (apoproteins and lomicrons respectively. phospholipids). This property, known as amphipath- icity, enables these molecules to associate with aqueous Postprandial lipemia: relevance to (hydrophilic) and nonaqueous (hydrophobic) envi- atherosclerosis ronments and thus renders them perfect for enveloping insoluble lipids, chiefly TAG and CE, in It was suggested by Zilversmit in 1979 that atheroscle- macromolecular lipid–protein complexes called lipo- rosis was a postprandial phenomenon. This concept proteins. It is worth remembering that, in the absence was based on the finding that patients either with or at of lipoproteins, TAG would exist in aqueous blood as high risk of developing coronary heart disease (CHD) immiscible oil droplets, while free fatty acids liberated showed an impaired capacity to remove TAG-rich from TAG and phospholipids in the absence of the lipoproteins from the circulation after a meal. This blood protein albumin would act as detergents and resulted in enhanced postprandial lipemia, which also dissolve cell membranes. became known as the TAG intolerance hypothesis. At

Nutrition and Metabolism of Lipids 99 Lipoprotein structure: a shopping bag techniques permit the further resolution of VLDL, and groceries low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) into discrete subclasses, the dis- The general structure of a lipoprotein consists of a tribution of which relates to cardiovascular risk and central core of hydrophobic, neutral lipid (TAG and is determined by genetic and lifestyle factors. CE) surrounded by a hydrophilic coat of phospholip- ids, free cholesterol, and apoproteins. A useful analogy Lipoprotein transport pathways for this arrangement of molecules is that of a “shop- ping bag and groceries,” with the lipid core represent- Lipoprotein transport can be described in terms of ing the groceries and the outer coat the fabric of the the production, transport, and removal of cholesterol bag. The apoproteins weave in and out of the lipid or TAG from the circulation. In reality, these two core and outer surface layer and form the thread of processes are inseparable because both TAG and cho- the fabric which holds the bag together (see Figure lesterol are transported together in lipoproteins. 6.5). This clever arrangement of molecules renders Lipoproteins are in a constant state of change, with the hydrophobic lipids soluble for the purpose of lipids and apoproteins constantly shuttling between transport in blood. In addition to conferring struc- different lipoproteins that interrelate through inte- tural integrity on the lipoprotein particle, apoproteins grated metabolic pathways. A useful analogy here is have a vital role in controlling the metabolism of to think of lipoproteins as railway trains, transporting lipoproteins by acting as ligands for cell membrane passengers that represent lipids and apoproteins receptors and cofactors for key enzymes. within a complex rail network. The trains and passengers are in a constant state of flux within Plasma lipoproteins can be subdivided into distinct and between stations. Lipoprotein metabolism is con- classes on the basis of their physical properties and/or trolled by the activity of functional proteins (enzymes, composition, both of which reflect the physiological cell surface receptors, receptor ligands) that deter- role in the transport of lipids from sites of synthesis mine the rate at which lipoproteins enter and leave (endogenous lipids) and absorption (exogenous the system, and by the physicochemical properties of lipids, absorbed in the gut) to sites of storage (adipose the lipoprotein themselves. This corresponds to all of tissue) and utilization (skeletal muscle). The principal the rate-limiting features of a train journey, the classes of lipoproteins are traditionally defined by number of trains, and type of passengers. density, which is determined by the ratio of lipid to protein in the lipoprotein particle. Since lipids tend All lipoproteins, with the notable exception of to occupy a greater molecular volume than proteins, HDLs, begin life as TAG-rich particles The principal they are lighter and less dense. Thus, particles with transport function of these lipoproteins in the first high lipid content are larger and less dense (carry instance is to deliver fatty acids liberated from the more lipid groceries) than lipoproteins enriched with TAG to tissues. The enterocytes in the gut are the protein. This property relates directly to the transport producers of lipoproteins which deliver dietary fats function and metabolic interrelationships between into the blood as chylomicrons (exogenous lipid), lipoprotein classes in blood. It can also be used to whereas the liver is the central terminus for the pro- separate lipoproteins of different densities because duction of VLDLs and removal of their cholesterol- lipoproteins of different density have different flota- rich end-products, LDLs. VLDLs, although smaller tion characteristics in the ultracentrifuge (note that than chylomicrons, resemble the latter in many ways plasma lipoproteins will float when subjected to cen- and are often referred to as the liver’s chylomicrons. trifugal force, whereas pure proteins sink). Other clas- While the rate at which the gut produces chylomi- sification schemes for plasma lipoproteins have crons depends largely on the amount of absorbed exploited differences in their net electrical charge dietary fat, the rate of production of VLDL is deter- (electrophoretic mobility), particle size (exclusion mined by the supply of fatty acids in the liver that can chromatography, gradient gel electrophoresis), and be re-esterified back to TAG for incorporation into immunological characteristics conferred upon VLDL. These fatty acids are derived chiefly from the the lipoprotein by the types of apoproteins in each systemic circulation in the form of nonesterified fatty lipoprotein subclass (see Table 6.4). Some of these acids (NEFAs), and to a lesser extent from the uptake of circulating lipoprotein remnants. It is noteworthy

100 Introduction to Human Nutrition that, although the liver has the capacity to synthesize activation of LPL, are progressively depleted of their fatty acids, the amount synthesized by de novo lipo- TAG in a stepwise fashion by LPL to become choles- genesis is relatively small in humans on a mixed terol-rich remnants that are removed by specific, Western diet. However, the contribution of fatty acids high-affinity receptors found chiefly in the liver. from this source may increase in conditions associ- Several molecules of LPL may bind to a single chylo- ated with an overproduction of VLDLs, and has been micron or VLDL particle, although LPL shows greater shown to occur on low-fat, high-carbohydrate diets, affinity for chylomicrons in preference to VLDL. This and in metabolic disease. situation leads to competition between these TAG- rich lipoproteins and provides a mechanism to explain Metabolic determinants of how VLDL can influence the clearance of TAG in the lipoprotein metabolism postprandial period. The metabolism of serum lipoproteins and fate of Lipolyzed chylomicrons form chylomicron rem- their transport lipids is controlled by: nants which, during passage through the liver, bind to specific receptors on the surface of hepatocytes that ● the physical and chemical characteristics of the recognize apoE, an apoprotein that is also acquired at lipoprotein, such as its size and lipid and apopro- an early stage from HDLs. Remnant receptors are tein content maintained at a very high level of activity and are not downregulated through a feedback mechanism (see ● the activity of the endothelial LPL and hepatic low-density lipoprotein receptor pathway). This is lipase (HL), so called because they are attached to fortunate, since chylomicron remnants have been the surface of endothelial cells lining blood vessels shown to be capable of depositing their cholesterol in in peripheral tissues, such as adipose tissue and artery walls, thus promoting coronary atherosclerosis. skeletal muscle, and the liver, respectively The secretion of VLDL from the liver is again fol- lowed by the sequential lipolysis of TAG by LPL and ● lipid transfer proteins; cholesteryl ester and phos- generation of VLDL remnants or, in this case, the pholipid transfer proteins, (CETP and PLTP further lipolysis of these remnants into LDL. respectively). The remnants and LDLs bind to another receptor in the liver that recognizes both apoE exclusively in ● apoproteins that act as activators of enzymes and VLDL remnants and apoB-100 in LDLs, namely the ligands for specific lipoprotein receptors on the sur- LDL receptor. Approximately 60% of LDL is removed faces of cells (apoB-100 and apoE as ligands for by the LDL receptor. The remainder is internalized the LDLs and remnant receptors in the liver, into cells via scavenger receptors. This latter route has respectively) been associated with the development of atheroscle- rotic disease. ● the activity of specific lipoprotein receptors on cell surfaces. Whether a VLDL particle is removed as a remnant or transcends to LDL largely depends on its pedigree, Lipoprotein transport is traditionally described in i.e., its size and lipid composition. Experiments with terms of the forward and reverse transport of choles- radioactively labeled VLDL have shown that larger, terol. Forward transport encompasses the exogenous TAG-rich VLDL particles are less likely to be con- and endogenous pathways, which describes the arrival verted into LDL and are removed as partially delipi- of cholesterol in the blood from either the gut or the dated VLDL remnants, whereas smaller VLDLs are liver and carriage back to the liver for processing; the precursors of LDL. liver has the unique capacity to secrete cholesterol either as free cholesterol or as bile acids. Conversely, The low-density lipoprotein reverse transport describes the HDL pathway and the receptor pathway efflux of cholesterol out of peripheral tissues back to the liver. This directionality can be misleading because The incontrovertible link between plasma cholesterol each pathway can direct cholesterol back to the liver. and CHD is directly responsible for the rapid growth, Both the exogenous and endogenous pathways share and occasional quantum leaps, in our understanding a common saturable lipolytic pathway that consists of of cholesterol homeostasis in relation to diet and a delipidation cascade in which the TAG-rich lipopro- teins (chylomicrons and VLDLs), after receiving apo- C (C-II) from HDL, an essential cofactor for the

Nutrition and Metabolism of Lipids 101 Serum LDL Liver cell YY LDL HMG-CoA reductase Y YY Y Y Acetate Free cholesterol LDL receptors ؉ N-SREBP LDL receptor protein Transcription Figure 6.8 Low-density lipoprotein (LDL) factor receptor pathway. HMG-CoA, 3-hydroxy-3- methyl-glutaryl-coenzyme A; SREBP, sterol Transcription LDL receptor gene ؉ regulatory element binding protein. disease, the most prolific of which was the discovery The metabolic effects of intracellular free cholesterol of the LDL receptor pathway by the Nobel Prize are: winners Goldstein and Brown (1977) (Figure 6.8). All cells, most notably those in the liver, have a highly ● it decreases the production of LDL receptors via developed and sensitive mechanism for regulating SREBP intracellular and intravascular levels of cholesterol. Cells in the liver synthesize approximately 500 mg of ● it inhibits the synthesis of cholesterol by the cholesterol a day and, although they can import the enzyme 3-hydroxy-3-methylglutaryl (HMG)-CoA same quantity from the blood in the form of LDL, in reductase the complete absence of LDL, cells could theoretically manufacture sufficient cholesterol to meet their met- ● it increases the re-esterification of cholesterol for abolic needs. However, when stressed, cells will always storage as cholesterol esters. import cholesterol in preference to synthesizing it themselves as the former process takes less energy. Goldstein and Brown were aided in the discovery Cells acquire cholesterol from the blood by the uptake of the LDL receptor by studying a condition known and degradation of LDL particles. As the requirement as familial hypercholesterolemia, a genetic abnormal- for free cholesterol increases within the cell, it increases ity in the LDL receptor gene that produces defects in its production and thus activity of LDL receptors, so the LDL receptor pathway and considerably elevated that more LDL is extracted from the blood, lowering blood cholesterol concentrations in early life (15– blood cholesterol. Conversely, if the cell becomes 20 mmol/l) and premature cardiovascular disease. overloaded with cholesterol, it senses that it requires They also initiated pioneering studies on the influ- less cholesterol and produces fewer LDL receptors, ence of dietary fats on the activity of the LDL pathway, causing blood cholesterol to increase. Since the pro- which led to a widely accepted explanation for duction of LDL receptors is regulated by the intracel- the cholesterol-raising properties of saturated fatty lular level of free cholesterol, anything that increases acids. free cholesterol within the cell will inadvertently lower blood LDL cholesterol. Intracellular free cholesterol Reverse cholesterol transport (high-density represses the activity of a sterol regulatory element lipoprotein pathway) binding protein (SREBP), a positive nuclear tran- scription factor that promotes the transcription of the The removal of cholesterol from tissues back to the LDL receptor gene when free cholesterol levels fall. liver via HDLs represents the only route of elimina- tion for cholesterol from the body. This physiological role of HDLs explains, in part, the cardioprotective effects of these lipoproteins, as indicated by a strong inverse relationship between serum HDL cholesterol

102 Introduction to Human Nutrition Pre-␤-HDL Peripheral tissues Free LCAT (including lesions) cholesterol HDL3 Liver LPL Hepatic CE lipase LDL VLDL HDL2 Figure 6.9 Reverse cholesterol trans- CETP port. CE, cholesterol ester; CETP, choles- CE terol ester transfer protein; HDL, high-density lipoprotein; LCAT, lecithin– cholesterol acyltransferase; LDL, low- density lipoprotein; LPL, lipoprotein lipase; VLDL, very low-density lipoprotein. and CHD risk in prospective cohort studies. The phospholipid of HDL, where it effectively punches activity of the HDL pathway is influenced by genetic a hole in the surface coat to facilitate access to the and dietary factors that can interact to either increase lipid core and delivery of CE to the hepatocyte or reduce the efficiency of cholesterol removal. This, (Figure 6.9). in turn, may be reflected in changes in the concentra- tion of serum HDLs and their functional properties. Interrelationships among serum triacylglycerols and low- and HDLs are synthesized in the gut and liver, and high-density lipoproteins increase their particle size in the circulation as a result of the acquisition of cholesterol from two principal Lipids are constantly moving between lipoprotein sources: (1) surface material released from TAG-rich particles. This movement is not totally random but lipoproteins during lipolysis and (2) peripheral influenced by the relative lipid composition of the tissues. The particles, which are responsible for lipoproteins and by specific lipid transfer proteins removing cholesterol from cells, are very small pre- (LTPs) that act as lipid shuttles. In a normal, healthy HDLs and are disk-shaped particles composed of individual, TAG-rich lipoproteins transfer TAG to phospholipid and apoA-I (ApoA-I is capable of this LDL and HDL in equimolar exchange for CE. This is function on its own). The efflux of free cholesterol mediated through an LTP called cholesteryl transfer from tissue sites, including deposits of cholesterol in protein (CETP). In this way, CEs are transferred from the coronary arteries, is facilitated by the formation HDL to VLDL for passage back to the liver. Conversely, of a free cholesterol gradient from the cell across the when the concentration of serum TAG and thus TAG- cell membrane to pre-HDLs. The gradient is gener- rich lipoproteins is increased, for example by either ated by the re-esterification of free cholesterol by the the overproduction of TAG in the liver or the impaired enzyme lecithin–cholesterol acyltransferase (LCAT) removal of TAG by LPL, the result is a net transfer of and via the migration of these newly formed choles- TAG into LDL and HDL. As LDL and HDL are over- terol esters into the hydrophobic core of what becomes loaded with TAG they become favored substrates for mature, spherical HDL. The newly acquired choles- the action of HL and are remodeled into smaller and terol is transported back to the liver either directly by denser particles. While small, dense HDL is catabo- HDL or indirectly by transfer to apoB-containing lized rapidly in the liver, lowering serum HDL and lipoproteins VLDL and LDL. Blood vessels in the liver impairing reverse cholesterol transport, small, dense contain a close relative of LPL, i.e., HL. This enzyme LDLs are removed less effectively by LDL receptors acts on smaller lipoproteins and especially the surface and accumulate in serum. Small, dense LDL, by virtue

Nutrition and Metabolism of Lipids 103 of its size, has a much greater potential to infiltrate resistant conditions. Insulin also stimulates the the artery wall and deposit its cholesterol. Even a synthesis of cholesterol by activating HMG-CoA moderately raised concentration of serum TAG reductase and the activity of LDL receptors, although (>1.5 mmol/l) may be inversely associated with the overall effect on cholesterol homeostasis is small reduced HDL cholesterol (<1 mmol/l) and a predomi- in relation to the control of the LDL pathway described nance of small, dense LDL. This collection of findings above. is known as the atherogenic lipoprotein phenotype (ALP) and is a very common but modifiable source of Sex hormones increased CHD risk in free-living populations. The effect of sex hormones on serum lipoproteins is Endocrine control of best illustrated by the pronounced differences in lipid lipoprotein metabolism and lipoprotein profiles between adult men and pre- menopausal women. Men present with higher total All hormones exert an influence on lipoprotein serum and LDL cholesterol, higher serum TAG, and metabolism. However, with respect to diet and the lower HDL cholesterol concentrations than premeno- control of postprandial lipid metabolism, insulin has pausal women. This difference in lipid profiles confers by far the greatest impact. Although classically associ- protection against CHD on premenopausal women ated with carbohydrate metabolism and the uptake of so that their CHD risk lags some 10 years behind that glucose into cells, the actions of insulin are critical to of men of the same age. This applies until estrogen the control of postprandial lipid metabolism. Insulin failure at the menopause, when CHD risk in women is secreted in response to the reception of food in the overtakes that of men. Estrogen was the first com- gut and it: pound shown to stimulate LDL receptor activity in cell culture, an effect that not only accounts for lower ● stimulates LPL in adipose tissue LDL levels in women but also the sharp increase in ● suppresses the intracellular lipolysis of stored TAG LDL cholesterol after the menopause, to levels above those of men. Estrogens also stimulate the pro- in adipose tissue by inhibiting hormone-sensitive duction of TAG and VLDL, but any adverse effects lipase must be outweighed by the efficiency of TAG removal ● suppresses the release of VLDL from the liver. mechanisms that maintain lower serum TAG levels in women than in men until the menopause. In addition Insulin coordinates the lipolysis of dietary TAG and to these effects, estrogen selectively inhibits the activ- uptake of NEFA into adipose tissue. It achieves this ity of HL, which contributes to the HDLs in women. by minimizing the release of NEFA from TAG stores In direct contrast, the androgenic male hormone tes- in adipose tissue and TAGs produced in the liver by tosterone suppresses LDL receptor activity, raising suppressing the secretion of VLDL. The sensitivity of LDL cholesterol. It is also a powerful stimulant of HL the target tissues – liver, adipose tissue, and, perhaps activity, and is responsible for lowering HDL choles- to a lesser extent, skeletal muscle – to insulin is critical terol in men, most notably in male body builders on to the maintenance of these effects. Failure of insulin anabolic steroids, in whom serum HDL can be almost action, insulin resistance, in conditions such as obesity absent. and diabetes results in dyslipidemia characterized by an impaired capacity to lipolyze TAG-rich lipopro- The triacylglycerol hypothesis teins (TAG intolerance or enhanced postprandial lipemia). This effect is compounded by the failure of Dietary effects on serum cholesterol or LDLs alone insulin to suppress the mobilization of NEFA from provide an inadequate basis on which to explain the adipose tissue TAG, which increases the flux of NEFA relationship between diet and CHD within popula- to the liver and stimulates the overproduction of tions. The ability of humans to protect themselves VLDL (‘portal hypothesis’). The suppression of VLDL against an overaccumulation of cholesterol in their secretion is also abolished so that VLDL is released vascular system through nutritional changes depends into the postprandial circulation and is free to compete to a much greater extent on increasing the efficiency with chylomicrons, augmenting postprandial lipemia of the HDL pathway and utilization of TAG-rich still further. This series of events gives rise to a dys- lipoproteins. The latter represent the precursors of lipidemia or ALP, which is found frequently in insulin-

104 Introduction to Human Nutrition potentially harmful cholesterol-rich remnants and of proteins functioning as receptors, transporters, LDLs that contribute to coronary atherosclerosis. The enzymes, ion channels, etc. Some lipids, i.e., PUFAs, effects of diet, and in particular dietary fats, in modu- confer the feature of “fluidity” to membranes, whereas lating the clearance of TAG-rich lipoproteins in the others, i.e., cholesterol and saturated fatty acids, have postprandial period is of paramount importance in the opposite rigidifying effect. Membranes have preventing the accumulation of atherogenic remnants extraordinarily diverse fatty acid profiles and phos- and development of proatherogenic abnormalities in pholipid composition depending on their tissue and LDL and HDL. The actions of insulin coordinate the subcellular location. They are also the body’s reservoir metabolism of TAG-rich lipoproteins but can become of both fat-soluble vitamins and eicosanoid precur- defective through energy imbalance, weight gain, sors such as arachidonate. and ultimately obesity. As a consequence, the most common abnormalities in lipoproteins to increase Most of the body’s cholesterol is present in the risk in populations arise from a primary defect in the unesterified form in membranes, where it represents metabolism of TAG, induced through insulin resis- 35–45% of total lipids. Skin, plasma, and adrenal tance and not cholesterol per se. Equally important is cortex contain 55–75% of cholesterol in the esterified the fact that these metabolic defects originate, in part, form. Bile also contains free cholesterol and bile salts through nutrient–gene interactions and are thus derived from cholesterol. highly amenable to dietary modification. Storage lipid pool 6.6 Body lipid pools Triacylglycerols are the main energy storage form of Lipids in the human body exist in two major pools: lipids and they are the principal component of structural lipids in membranes and storage lipids in body fat. TAG-containing fatty acids destined for body fat. The lipid composition and metabolic fate of oxidation are also present in measurable but much these two pools are quite distinct, although many of lower amounts in all tissues that can oxidize long- the fatty acids occupying both pools are the same. The chain fatty acids, i.e., muscle and heart. TAG is syn- main components of both membrane and storage thesized by the intestine and liver, where it is subse- lipids are the long-chain (16–24 carbons) saturated, quently incorporated into lipoproteins (see Section monounsaturated, and polyunsaturated fatty acids. 6.4) for the transport of lipids to and from other Although several of the major long-chain fatty acids tissues. in the body are common to both membrane and storage lipids, namely palmitate, stearate, oleate, and The main fatty acids in the TAG of adult human linoleate, three important distinctions exist between body fat are palmitate (20–30%), stearate (10–20%), membrane and storage lipids. oleate (45–55%), and linoleate (10–15%). The fatty acid profile of adult body fat always reflects the profile 1 Membrane lipids are not usually hydrolyzed to of dietary fat. Only rarely would this result in other release free fatty acids for energy metabolism. fatty acids being more prevalent in body fat than the four listed here. At birth, the fatty acid profile of body 2 Membrane lipids contain a much higher propor- fat is unusual in having very low linoleate (<3%) and tion of long-chain PUFAs. α-linolenate (<1%) but a higher proportion of long- chain polyunsaturates than later in life. Body fat occu- 3 Membrane lipids are more diverse and rarely pies several discrete sites that expand and contract as include TAGs, which are the main component of needed. Body fat is about 82% by weight TAG, making storage lipids. it by far the main body pool of palmitate, stearate, oleate, and linoleate. Structural lipid pool The main sites of body fat are subcutaneous and Biological membranes surrounding cells and subcel- intravisceral, and they have different rates of response lular organelles exist primarily as lipid bilayers (Figure to stimuli for accumulation or release of fatty acids. 6.3). The lipids in both the inner and outer surfaces Within a given site, growing evidence suggests that of membranes are composed mainly of phospholip- PUFAs are more easily released from adipose tissue ids and free cholesterol, which interface with a myriad TAG than are saturated fatty acids, especially during fasting or longer term energy deficit.

Nutrition and Metabolism of Lipids 105 Plasma and milk lipids also rises rapidly in brain lipids, followed a little later by an increasing content of long-chain saturates and In a way, plasma and milk lipids are an exception to monounsaturates as myelin develops. Adipose tissue the general rule distinguishing membrane and storage contains very little linoleate or α-linolenate at birth lipids. Plasma and milk lipids are present mostly as but their content increases rapidly with milk feeding. lipoproteins, comprising mostly phospholipids and Plasma cholesterol is relatively low at birth and in cholesterol in the surrounding membrane and TAG infancy, but increases by more than twofold by in the core (see Section 6.5). Plasma lipids contain the adulthood. only significant pool of free fatty acids or NEFAs in the body. Free fatty acids are not components of lipo- In general, regardless of the profile of dietary fatty proteins but are transported bound to albumin. They acids, saturated and monounsaturated fatty acids pre- are liberated mostly from adipose tissue when plasma dominate in adipose tissue, whereas there is a closer glucose and insulin are low. Plasma also contains pro- balance between saturates, monounsaturates, and portionally more fatty acids esterified to cholesterol polyunsaturates in structural lipids. Long-chain (cholesteryl esters) than are found in tissues. PUFAs such as docosahexaenoate are present in high concentrations in specialized membranes, including Whole body content and organ profile of those of the retina photoreceptor, in synapses of the fatty acids brain, and in sperm. An estimate of the whole body content of lipids in a 6.7 Long-chain fatty acid metabolism healthy adult human is given in Table 6.5. Additional body fat is deposited during pregnancy, but the fatty Synthesis acid composition remains similar to that of nonpreg- nant adults and reflects dietary fat intake. The total lipid Synthesis of fatty acids occurs in the cytosol. It begins content of plasma rises in the third trimester, with a with acetyl-CoA being converted to malonyl-CoA by proportionally greater increase in saturated fatty acids acetyl-CoA carboxylase, an enzyme dependent on than PUFAs. This downward trend in the percentage of biotin. Malonyl-CoA and a second acetyl-CoA then PUFA towards term has led to some concern about the condense via β-ketothiolase. This is subsequently possible adverse consequences for the fetus of defi- reduced, dehydrated, and then hydrogenated to yield ciency of PUFA. However, the actual amount of PUFA a four-carbon product that recycles through the same in blood lipids rises but less so than for saturated fatty series of steps until the most common long-chain acids; resulting in a proportional decrease in PUFA. fatty acid product, palmitate, is produced (Figure 6.10). Acetyl-CoA is primarily an intramitochondrial Soon after birth, body lipid composition starts to product. Thus, the transfer of acetyl-CoA to the change. Brain cholesterol rises moderately from under cytosol for fatty acid synthesis appears to require its 40% to nearly 50% of brain lipids. Docosahexaenoate conversion to citrate to exit the mitochondria before being reconverted to acetyl-CoA in the cytosol. Table 6.5 Body fat content of major fatty acids in humans There are three main features of long-chain fatty Fatty acid Content (g) acid synthesis in mammals: Palmitic acid 3320 1 inhibition by starvation Stearic acid 550 2 stimulation by feeding carbohydrate after fasting Oleic acid 6640 3 general inhibition by dietary fat. Linoleic acid 1560 Arachidonic acid 80 Carbohydrate is an important source of carbon for α-Linolenic acid 130 generating acetyl-CoA and citrate used in fatty acid Eicosapentaenoic acid <10 synthesis. Enzymes of carbohydrate metabolism also Docosahexaenoic acid <10 help to generate the NADPH needed in fatty acid Total 12 300 synthesis. Acetyl-CoA carboxylase is a key control point in the pathway and is both activated and induced Data are based on a 70 kg adult human with 20% (14 kg) body fat. to polymerize by citrate. Acetyl-CoA carboxylase is Fat tissue contains about 88% actual fatty acids by weight, yielding about 12.3 kg fatty acids in this example.

106 Introduction to Human Nutrition C—C—CO (Malonyl SA) + C—CO (Acetyl SS) R—CH2—CH2—CH2—CO—CoA Condensation FAD CO2 + C—CO—C—CO (Acetoacetyl SA) FADH First reduction R—CH2—CH——CH—CO—CoA H2O C—COH—C—CO (β-Hydroxybutyryl SA) R—CH2—CHOH—CH2—CO—CoA NAD1 Dehydration H2O NADH R—CH2—CO—CH2—CO—CoA C—C——C—CO (Crotonyl SA) CoA Second reduction R—CH2—CO—CoA CH2—CO—CoA C—C—C—CO (Butyryl SA) Figure 6.10 Principal steps in fatty acid synthesis. The individual Figure 6.11 Principal steps in β-oxidation of a saturated fatty acid. steps occur with the substrate being anchored to the acyl carrier The steps shown follow fatty acid “activation” (binding to coenzyme protein. SA, S-acyl carrier protein; SS, S-synthase. A) and carnitine-dependent transport to the inner surface of the mito- chondria. Unsaturated fatty acids require additional steps to remove inhibited by long-chain fatty acids, especially PUFAs the double bonds before continuing with the pathway shown. FAD, such as linoleate. This is probably one important flavin adenine dinucleotide; FADH reduced flavin adenine dinucleo- negative feedback mechanism by which both starva- tide; R, 12 carbons. tion and dietary fat decrease fatty acid synthesis. High amounts of free long-chain fatty acids would also normal food intake can markedly alter tissue fatty compete for CoA, leading to their β-oxidation. acid profiles. Elongation of palmitate to stearate, etc., can occur in mitochondria using acetyl-CoA, but is more com- Oxidation monly associated with the endoplasmic reticulum where malonyl-CoA is the substrate. β-Oxidation is the process by which fatty acids are utilized for energy. Saturated fatty acids destined for Humans consuming >25% dietary fat synthesize β-oxidation are transported as CoA esters to the outer relatively low amounts of fat (<2 g/day). Compared leaflet of mitochondria by FABP. They are then with other animals, humans also appear to have a translocated inside the mitochondria by carnitine relatively low capacity to convert stearate to oleate acyl-transferases. The β-oxidation process involves and linoleate or α-linolenate to the respective longer repeated dehydrogenation at sequential two-carbon chain polyunsaturates. Hence, the fatty acid profiles steps and reduction of the associated flavoproteins of most human tissues generally reflect the intake of (Figure 6.11). Five ATP molecules are produced dietary fatty acids; when long-chain n-3 PUFAs are during production of each acetyl-CoA. A further 12 present in the diet, this is evident in both free-living ATP molecules are produced after the acetyl-CoA humans as well as in experimental animals. Neverthe- condenses with oxaloacetate to form citrate and goes less, fatty acid synthesis is stimulated by fasting/ through the tricarboxylic acid cycle. refeeding or weight cycling, so these perturbations in

Nutrition and Metabolism of Lipids 107 The efficiency of fatty acid oxidation depends on starvation, diabetes, and a very high-fat, low- the availability of oxaloacetate and, hence, concurrent carbohydrate “ketogenic” diet. carbohydrate oxidation. β-Oxidation of saturated fatty acids appears to be simpler than oxidation of Carbon recycling unsaturated fatty acids because, before the acetyl- CoA cleavage, it involves the formation of a trans Carbon recycling is the process by which acetyl-CoA double bond two carbons from the CoA. In contrast, derived from β-oxidation of one fatty acid is incor- β-oxidation of unsaturated fatty acids yields a double porated into another lipid instead of completing the bond in a different position that then requires further β-oxidation process to carbon dioxide. In principle, isomerization or hydrogenation. From a biochemical all fatty acids undergo this process to some extent but perspective, this extra step appears to make the oxida- it is most clearly evident for two PUFAs, linoleate and tion of unsaturated fatty acids less efficient than that α-linolenate. Carbon recycling captures the over- of saturated fatty acids. However, abundant in vivo whelming majority of α-linolenate carbon, i.e., about and in vitro research in both humans and animals 10 times more than is incorporated into docosahexae- clearly shows that long-chain cis-unsaturated fatty noate, which remains in the body of suckling rats 48 acids with one to three double bonds (oleate, linole- hours after dosing with uniformly 13C-labeled α-lino- ate, α-linolenate) are more readily β-oxidized than lenate. Carbon recycling of linoleate in the rat cap- saturated fatty acids of equivalent chain length, such tures similar amounts of the linoleate skeleton to as palmitate and stearate. The oxidation of PUFA those of arachidonate, the main desaturation and and monounsaturates in preference to saturates has chain-elongation product of linoleate. Hence, carbon potential implications for chronic diseases such as recycling appears to be a ubiquitous feature of the coronary artery disease because their slower oxida- metabolism of PUFA, although its biological signifi- tion implies slower clearance from the blood, thereby cance is still unclear. providing more opportunity for esterification to cho- lesterol and subsequent deposition in the vessel wall. Peroxidation In peroxisomes, fatty acid β-oxidation is a trun- Peroxidation (auto-oxidation) is the nonenzyme-cat- cated process by which long-chain PUFAs are chain alyzed reaction of molecular oxygen with organic shortened. This peroxisomal detour has been identi- compounds to form peroxides and related breakdown fied as an obligatory step in the endogenous synthesis products. PUFAs are particularly vulnerable to peroxi- of docosahexaenoate from eicosapentaenoate. dation at the double bonds. Initiating agents such as pre-existing peroxides, transition metals, or ultraviolet Odd-carbon long-chain fatty acids are relatively or ionizing radiation produce singlet oxygen. Singlet uncommon but, when β-oxidized, yield propionyl- oxygen can then abstract hydrogen at the double bonds CoA, the further β-oxidation of which requires biotin of polyunsaturates to produce free (peroxy) radicals, and vitamin B12 as coenzymes. which abstract further hydrogens from the same or different fatty acids and propagate the peroxidation Ketogenesis and ketosis process. Eventually, this leads to termination by the formation of stable degradation products or hydro- Large amounts of free fatty acids inhibit glycolysis peroxides (Figure 6.12). Trans isomers are frequently and the enzymes of the tricarboxylic acid cycle, formed during the process. Hydroperoxides can form thereby impairing production of oxaloacetate. When further hydroperoxy radicals or can be reduced by insufficient oxaloacetate is available to support the antioxidants, which contain thiol groups, i.e., glutathi- continued oxidation of acetyl-CoA, two acetyl-CoA one and cysteine. Peroxidation of dietary fats gives rise molecules condense to form a ketone, acetoacetate. to aldehydes, i.e., 2-undecenal, 2-decenal, nonanal, or Acetoacetate can be spontaneously decarboxylated to octanal, which have a particular odor commonly form acetone, a volatile ketone, or converted to a third known as rancidity. ketone, β-hydroxybutyrate. When glucose is limiting, ketones are an alternative source of energy for certain Since peroxidation is a feature of polyunsaturates, organs, particularly the brain. They are also efficient it is a potential hazard facing most membranes and substrates for lipid synthesis during early postnatal dietary lipids. Antioxidants such as vitamin E are development. Conditions favoring ketogenesis include usually present in sufficient amounts to prevent or

108 Introduction to Human Nutrition HH acyl chain into which the double bond is inserted. X• Although myristate (14:0) and palmitate can be XH converted to their monounsaturated derivatives, myristoleate (14:1n-5) and palmitoleate (16:1n-7) H• respectively, commonly it is only the fatty acids of 18 or more carbons that undergo desaturation. The Δ9 •H desaturases in all organisms, except for anaerobic bac- O2 teria, use oxygen and NADPH to introduce a cis double bond at carbons 9 and 10 of stearate. This is H O O• accomplished by an enzyme complex consisting of a series of two cytochromes and the terminal desatu- OO H OOH rase itself. The acyl-CoA form of fatty acids is the • usual substrate for the desaturases, but fatty acids esterified to phospholipids can also be desaturated Endoperoxide in situ. OO Hydroperoxide All mammals that have been studied can convert + R• stearate to oleate via Δ9 desaturase. However, in the absence of dietary oleate, young rats may have insuf- HH ficient capacity to sustain normal tissue oleate levels. Malondialdehyde Normal values depend on the reference, which can vary widely depending on the source and amount of Figure 6.12 Principal steps in peroxidation of a polyunsaturated fatty oleate in the diet. Nevertheless, it is important to dis- acid. tinguish between the existence of a given desaturase and the capacity of that pathway to make sufficient of block peroxidation in living tissues. Humans and the necessary product fatty acid. Hence, as with the animals readily detect peroxidized fats in foods by long-chain polyunsaturates and, indeed, with other their disagreeable odor and avoid them. However, nutrients such as amino acids (see Chapter 4), it is modeling the effects of peroxides produced in vivo important to keep in mind that the existence of a and in vitro is particularly challenging because lipid pathway to make a particular fatty acid or amino acid peroxidation undoubtedly is an important part of does not guarantee sufficient capacity of that pathway several necessary biological processes such as activa- to make that product. This is the origin of the concept tion of the immune response. of “conditional essentiality” or “indispensability.” Both plants and animals are capable of desaturating Desaturation, chain elongation, at the 9–10 carbon (Δ9 desaturase) of stearate, result- and chain shortening ing in oleate. However, only plants are capable of desaturating oleate to linoleate and then to α-lino- One important characteristic of long-chain fatty acid lenate. Once linoleate and α-linolenate are consumed metabolism in both plants and animals is the capacity by animals, their conversion to the longer chain to convert one to another via the processes of desatu- PUFAs of their respective families proceeds primarily ration, chain elongation, and chain shortening. by an alternating series of desaturation (Δ6 and Δ5 desaturases) and chain-elongation steps (Figure 6.13). Plants and animals use desaturases to insert a Sequential desaturations or chain elongations are also double bond into long-chain fatty acids. There are a possibility, resulting in a large variety, though low several desaturases, depending on the position in the abundance, of other PUFAs. During dietary deficiency of linoleate or α-lino- lenate, oleate can also be desaturated and chain elon- gated to the PUFA eicosatrienoate (20:3n-9). Hence, most but not all PUFAs are derived from linoleate or α-linolenate.

Nutrition and Metabolism of Lipids 109 w6 Polyunsaturates w3 Polyunsaturates Linoleic Δ6 Desaturation α-Linolenic γ-Linolenic Chain elongation Stearidonic Dihomo-γ-Linolenic Δ5 Desaturation ω3-Eicosatrienoic Arachidonic Chain elongation Eicosapentaenoic Figure 6.13 Conversion of linoleic (18:2n-6) and Adrenic ω3-Docosapentaenoic α-linolenic (18:3n-3) acids to their respective longer chain, more unsaturated polyunsaturates. In mem- Chain elongation, Docosahexaenoic branes, linoleic and arachidonic acids are the prin- peroxisomal cipal n-6 polyunsaturates, while docosahexaenoic acid is the principal n-3 polyunsaturate. Hence, chain shortening these two families of fatty acids have different affinities for the desaturation and chain-elongation ω6-Docosapentaenoic enzymes. This pathway is principally based in the endoplasmic reticulum but appears to depend on peroxisomes for the final chain shortening, which involves 24 carbon intermediates that are not illustrated. Chain elongation of saturated and unsaturated bacteria are the only organisms known to have this fatty acids occurs primarily in the endoplasmic retic- capability. As in chemical hydrogenation practiced by ulum, although it has also been demonstrated to the food industry, biohydrogenation in the rumen can occur in mitochondria. Unlike the desaturation steps be incomplete, resulting in the formation of small immediately before and after, the elongation steps do amounts of trans isomers, particularly of oleate, lino- not appear to be rate limiting in the metabolism of leate, and α-linolenate, which are found in milk fat. linoleate or α-linolenate. Eicosanoids Despite the capacity to insert at least three double bonds in both n-3 and n-6 polyunsaturates, there is Eicosanoids are 20-carbon, oxygen-substituted no proof that a Δ4 desaturase exists to insert the final cyclized metabolites of dihomo-γ-linolenate, arachi- double bond in docosapentaenoate (22:5n-6) or doc- donate, or eicosapentaenoate. They are produced via osahexaenoate (Figure 6.13). Rather, it appears that a cascade of steps starting with the cyclooxygenase or the precursors to these two fatty acids undergo a lipoxygenase enzymes present in microsomes. The second elongation, repeated Δ6 desaturation followed main cyclooxygenase products comprise the classical by chain shortening in peroxisomes. This unexpect- prostaglandins, prostacyclin and the thromboxanes. edly convoluted series of steps is corroborated by the The main lipoxygenase products are the leukotrienes docosahexaenoate deficiency observed in disorders (slow-reacting substances of anaphylaxis) and the of peroxisomal biogenesis such as Zellweger’s noncyclized hydroperoxy derivatives of arachidonate syndrome. that give rise to the hepoxylins and lipoxins (Figure 6.14). Hydrogenation Eicosanoids are considered to be fast-acting local Opposite to the desaturation process is hydrogena- hormones, the presence of which in the plasma and tion or removal of unsaturated bonds in lipids. Rumen urine is largely a spillover from localized production,

110 Introduction to Human Nutrition Membrane arachidonic acid Phospholipase A2 Figure 6.14 The arachidonic acid cascade is a fundamental component of cell signal- Free arachidonic acid ing during injury. Phospholipase A2 is immediately activated and the free arachi- Lipoxygenase Cyclooxygenase donic acid thus released is accessible to a controlled peroxidation process involving HPETEs PGG2 several cyclooxygenases (constitutive or Peroxidase inducible) and lipoxygenases. Over 50 metabolically active products are poten- Hepoxylins HETEs PGH2 tially produced, depending on the tissue Leukotrienes Lipoxins involved, the type of cell that has been stimulated, and the type of injury. Only the PGs PGI2 main classes of these metabolites are TXA2 shown. Before excretion, they are further metabolized to stable products that are not shown. Several of the cyclooxygenase products are competitive with each other, such as the platelet-aggregating and blood vessel wall-constricting effects of thromboxane A2 (TXA2) produced in plate- lets, versus the opposite effects of prosta- cyclin (PGI2) derived from the blood vessel wall. HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; PG, prostaglandin; TX, thromboxane. usually in response to an injury or a stimulus that derived from dihomo-γ-linolenate (1 series) and from releases the free precursor, most commonly arachido- eicosapentaenoate (3 series) often have effects that nate. The site of highest eicosanoid concentration oppose those derived from arachidonate (2 series) appears to be the seminal fluid, although some species (Figures 6.14 and 6.15). Thus, unlike prostaglandin have no detectable eicosanoids in semen. Eicosanoids E2, prostaglandin E1 has anti-inflammatory actions, are second messengers modulating, among other reduces vascular tone, and inhibits platelet aggrega- pathways, protein phosphorylation. The lung is a tion. Fourth, varying the ratio of the precursor fatty major site of eicosanoid inactivation. acids in the diet is an effective way to modify eico- sanoid production. Thus, eicosapentaenoate and Four important characteristics of eicosanoid action dihomo-γ-linolenate inhibit the synthesis of 2 series should be noted. First, individual eicosanoids often eicosanoids derived from arachidonate. This occurs have biphasic actions as one moves from very low by inhibiting arachidonate release from membranes through to higher, often pharmacological, concentra- by phospholipase A2 and its cascade through the tions. Thus, effects can vary dramatically depending cyclooxygenases and lipoxygenases. The overproduc- not only on the experimental system but also on the tion of 2 series eicosanoids is associated with higher eicosanoid concentration used. Second, several of the blood pressure, increased platelet aggregation, and more abundant eicosanoids arising from the same inflammatory processes, and can be effectively inhib- precursor fatty acid have opposite actions to each ited by dietary approaches using oils rich in eicosap- other. For instance, prostacyclin and thromboxane entaenoate and γ-linolenate (18:3n-6), the precursor A2 are both derived from arachidonate but the to dihomo-γ-linolenate. former originates primarily from the endothelium and inhibits platelet aggregation, while the latter orig- Stable analogues of some classical prostaglandins inates primarily from platelets and is a potent plate- have specialized medical applications, including the let-aggregating agent. Third, competing eicosanoids termination of pregnancy and the closing of a patent

Nutrition and Metabolism of Lipids 111 1 series 3 series PGs LTs Dihomo-γ- Figure 6.15 Arachidonic acid is not linolenic acid the only 20-carbon polyunsaturated fatty acid that can be metabolized via 2 series Cyclooxygenase Lipoxygenase 4 series the cyclooxygenases and lipoxygen- PGs LTs ases; both dihomo-γ-linolenic acid Arachidonic acid (20:3n-6) and eicosapentaenoic acid (20:5n-3) are well-established precur- Eicosapentaenoic sors as well, and produce prostaglan- acid dins (PGs) and leukotrienes (LTs) that are frequently competitive with those 3 series 5 series produced from arachidonate, thereby PGs LTs neutralizing the effects of the arachido- nate cascade (see Figure 6.14). This provides a critical balance in the overall reaction to cell injury. ductus arteriosus shortly after birth.Many anti-inflam- followed by refeeding a carbohydrate-rich meal is the matory and anti-pyretic drugs are inhibitors of eico- classic way to stimulate fatty acid synthesis. Insulin is sanoid synthesis.One potentially dangerous side-effect implicated in this process. When repeated, fasting/ of inhibiting eicosanoid synthesis is gastric erosion refeeding or weight cycling induces a gradual increase and bleeding. Receptor antagonists of leukotrienes are in the proportion of saturated and monounsaturated effective in reducing the symptoms of asthma. compared with PUFAs in tissues, especially body fat. This shift occurs because of the increase in fatty acid 6.8 Nutritional regulation of long-chain synthesis, easier oxidation of polyunsaturates, and the fatty acid profiles and metabolism inhibition of desaturation and chain elongation by fasting. The implications of such an alteration in tissue Phospholipids of all cellular and subcellular mem- fatty acid profiles have not yet been extensively studied, branes contain a diverse range of long-chain fatty but probably involve changes in insulin sensitivity and acids, the profile of which is subject to both dietary other hormone effects. Protein deficiency also inhibits influence and endogenous control. A few organs, desaturation and chain elongation of PUFAs. notably the brain, maintain extraordinarily strict control of their membrane composition. However, Copper supplementation increases Δ9 desaturase the fatty acid profile of most organs is usually respon- activity in animals, resulting in higher oleate levels. sive to the influence of changes in dietary fatty acid This effect was first observed when copper was used composition and other nutritional variables, yet to reduce gastrointestinal infection in pigs, but also maintains the vital “gatekeeper” functions of all mem- led to softer back fat. Opposite to the effects of copper branes. Hence, when changes in dietary fat alter supplementation, copper deficiency inhibits synthesis membrane fatty acid profiles, appropriate membrane of both oleate and docosahexaenoate. fluidity can be maintained by the addition or removal of other lipids such as cholesterol. Insufficient energy Polyunsaturated fatty acids intake and the presence of disease have important consequences for fatty acid synthesis, desaturation, There are four key features of the nutritional regula- and chain elongation and, consequently, tissue fatty tion of the profiles and metabolism of PUFAs. These acid profiles. attributes govern the effects of deficiency or excess of one or more of these families of fatty acids almost as Saturates and monounsaturates much as their level in the diet. These key features are: Inadequate energy intake increases macronutrient oxidation, including fatty acids. Short-term fasting 1 specificity within families 2 competition between families

112 Introduction to Human Nutrition 3 substrate and end-product inhibition NADH. This effect is severe enough that inherited 4 cofactor nutrients. forms of zinc deficiency such as acrodermatitis enteropathica cause a precipitous decline in plasma Specificity arachidonate, greater than usually observed with An n-6 PUFA cannot be converted to an n-3 or n-9 dietary deficiency of n-6 polyunsaturates. PUFA. Thus, deficiency of one family of polyunsatu- rates cannot be corrected by excess of those in a dif- 6.9 Nutritional and metabolic effects of ferent family and, indeed, is exacerbated by excess dietary fatty acids intake of the other families. Two types of issue exist in relation to the nutritional Competition and health implications of individual dietary lipids. The three families of PUFAs appear to use a common series of desaturases and chain elongases. The prefer- 1 Whether synthesized endogenously or only ence of these enzymes is for the more unsaturated obtained from the diet, what are the specific mem- fatty acids so, everything else being equal, more α- brane, precursor, or metabolic effects of dietary linolenate will be desaturated than linoleate or oleate. lipids beyond that of providing energy? However, in practice, more linoleate is consumed than α-linolenate and, as a result, more arachidonate 2 Whether synthesized endogenously or obtained is produced endogenously than eicosapentaenoate. from the diet, does an excess amount of a dietary Furthermore, this competition for desaturation and lipid have beneficial or deleterious implications for chain elongation between linoleate and α-linolenate health? can lead to exacerbation of symptoms of deficiency of one or other fatty acid family. Thus, as has been Short- and medium-chain fatty acids demonstrated both clinically and experimentally, excess linoleate intake using sunflower oil is a common Short-chain fatty acids (1–6 carbons) are mostly way to accelerate deficiency of n-3 PUFA. derived from carbohydrate fermentation in the large bowel and appear to be mainly used for energy, Inhibition although they are also substrates in several pathways. Excess linoleate or α-linolenate intake appears to Butyrate may have an important role as an energy inhibit production of the respective long-chain prod- substrate for enterocytes. Medium-chain fatty acids ucts in the same fatty acid family, i.e., high α-lino- (8–14 carbons) naturally appear in mammalian milk lenate intake inhibits synthesis of docosahexaenoate. and are almost exclusively used as energy substrates. Likewise, the main end-products of desaturation and They may also be chain elongated to palmitate. chain elongation tend to inhibit further metabolism through this pathway, so arachidonate inhibits its Saturated fatty acids own synthesis. Similarly, dietary deficiency of linole- ate increases activity of the Δ6 and Δ5 desaturases, Palmitate and stearate constitute a major proportion presumably to restore depleted levels of long-chain of the acyl groups of membrane phospholipids and n-6 polyunsaturates such as arachidonate. all mammals have the capacity to synthesize them. Hence, empirically, they presumably have an impor- Cofactors tant function in energy metabolism, cell structure, The cofactor requirements of the desaturation chain- normal development, and growth. The 20- to 24- elongation enzymes are not yet well understood, but carbon saturates are also important constituents of a few relationships are known. The desaturases are myelin. However, in any of these functions, it is metalloenzymes containing iron, and iron deficiency unlikely that a dietary source of saturates is necessary. therefore inhibits desaturase activity. Magnesium is In fact, the brain is unable to acquire saturated needed for microsomal desaturase activity in vitro. fatty acids from the circulation and relies on its Zinc deficiency inhibits Δ6 and Δ5 desaturation, appar- own endogenous synthesis for these fatty acids. ently by interrupting the flow of electrons from Furthermore, chronic excess intake and/or synthesis of palmitate and stearate is associated with an increased risk of diabetes and coronary artery disease.

Nutrition and Metabolism of Lipids 113 Monounsaturated fatty acids the diet of all mammals, including humans. Official dietary guidelines generally recommend a dietary Little is known about the nutritional or health impli- source of linoleate at 1–2% of energy intake. It has cations of palmitoleate (16:1n-7), but there is a bur- taken much longer to demonstrate that n-3 PUFAs geoning interest in the main dietary monounsatu- are required by humans. Although this now seems rated fatty acid, oleate, and the health implications of widely accepted among nutrition researchers, some olive oil. In the context of the same total fat intake, countries, including the USA, still do not yet officially the main benefit of higher oleate intake seems to be recognize that, as a minimum, α-linolenate is a that this reduces intake of palmitate and stearate and required nutrient. As with other nutrients, the require- that this helps to lower serum cholesterol. ment for polyunsaturates varies according to the stage of the life cycle, with pregnancy, lactation, and infancy Partially hydrogenated fatty acids being the most vulnerable. Symptoms of linoleate deficiency are virtually impossible to induce in healthy Partially hydrogenated fatty acids contain a large pro- adult humans, so the concept of “conditional indis- portion of trans fatty acids that are not naturally pensability or dispensability” of PUFAs has recently occurring but arise directly from food processing. emerged to replace the older but ambiguous term Hence, unlike saturates and cis-unsaturated fatty “essential fatty acid.” Linoleate appears to be condi- acids, they are not a necessary component of the diet tionally dispensable in healthy nonpregnant adults, except in the small amounts found in cow’s milk. but is not in pregnancy, lactation, or infancy. Their physical characteristics make them economi- cally suitable for inclusion in a wide variety of baked, Because of the competition between the two fami- fried, and oil-based foods, from which they can easily lies of PUFAs, deficiency of n-3 PUFA is commonly contribute up to 10% of dietary fat depending on induced by an excess of dietary linoleate. Hence, dis- food selection. Epidemiological evidence and some cussion of the requirements for linoleate and α-lino- experimental studies show that common dietary trans lenate has focused on their ratio in the diet. The ratio fatty acids raise LDL cholesterol and lower HDLs in of n-6 to n-3 polyunsaturates in human milk (5:1 to healthy adults, so the main nutritional concern is that 10:1) has been widely viewed as a suitable reference they may contribute to an increased risk of cardiovas- for this ratio in the general diet. In most affluent cular disease (see Section 6.11). countries, this ratio remains much higher, at about 20:1, and has been implicated in subclinical deficiency Trans fatty acids have also been experimentally of n-3 polyunsaturates. There is recent evidence to shown to compete with and impair the metabolism suggest that it is the absolute amounts of long-chain of other dietary long-chain fatty acids, but the rele- n-3 and n-6 fatty acids that are important in predict- vance of these observations in humans is unclear. ing health outcomes, and not the dietary ratio of these Trans fatty acids can be present in baby foods at rela- PUFAs. tively high concentrations but, so far, there is no evi- dence of deleterious effects on growth or development. Essential fatty acid deficiency Some information on the metabolism of trans fatty acids in humans has been gained from tracer studies, The first experimental model of deficiency of polyun- but fundamental information, such as the rate at saturates was total fat deficiency. The elimination of which they are oxidized, is still unknown. dietary fat had to be extreme because the traces of fat found in starch and dietary proteins were sufficient Polyunsaturated fatty acids to prevent reproducible symptoms of fat deficiency. The deficiency symptoms are now well known and Unlike saturates and monounsaturates, a dietary involve dry, scaly skin, growth retardation, and repro- source of n-6 and n-3 polyunsaturates is a necessity ductive failure. Most of these gross symptoms are for normal growth and development. As with other relieved by linoleate and arachidonate. Although α- essential nutrients, this has given rise to assessment of linolenate cannot be synthesized de novo, it has little the dietary requirements for polyunsaturates and the effect on these gross symptoms. However, careful implications of inadequate dietary intake of them. studies using a diet that is extremely deficient in n-3 polyunsaturates and contains an excess of n-6 poly- It has been accepted for over 50 years that n-6 polyunsaturates, particularly linoleate, are required in

114 Introduction to Human Nutrition unsaturates led to deficiency of n-3 polyunsaturates, an inherited disease is Zellweger’s syndrome. This characterized by delayed and impaired neuronal condition causes severe mental retardation and early development and impaired vision. These symptoms death. It is a disorder of peroxisomal biogenesis and have been traced in many species to the inadequate one outcome is markedly impaired synthesis of doco- accumulation of docosahexaenoate in the brain and sahexaenoate. Dietary supplementation with docosa- eye. Hence, the main function of n-3 polyunsaturates hexaenoate appears to partially restore neurological appears to hinge on synthesis of docosahexaenoate. development. In contrast, the function of n-6 polyunsaturates involves independent roles of at least linoleate and Epidemiological evidence shows that chronic arachidonate. degenerative diseases of affluence are directly associ- ated with the deficiency of n-3 PUFAs. Indeed, coun- Human cases of deficiency of PUFAs, usually tries with relatively high rates of these diseases usually involve a clinical disorder, often involving weight loss, have an adequate to perhaps unnecessarily higher trauma such as surgery, or a disease requiring paren- intake of linoleate. High intakes of linoleate have been teral nutrition. However, reports of these cases are implicated in death from coronary artery disease and uncommon and describe dissimilar characteristics, several types of cancer because these diseases are asso- leading one to question whether the same deficiency ciated with low intakes of n-3 polyunsaturates. Mental exists. Recent investigations into the amount of PUFA illnesses such as schizophrenia may also be associated in the whole body and the rate at which they can be with low intake of n-3 polyunsaturates and respond to oxidized suggest that traumatic or disease-related supplements of n-3 polyunsaturates. A more balanced processes leading to weight loss affect metabolism of ratio of intake of n-6 and n-3 polyunsaturates might polyunsaturates more severely than simple dietary achieve a reduction in the rate of these degenerative deficiency in a weight-stable, healthy individual. For diseases but has not yet been widely investigated. example, deficiency of linoleate has been long sus- pected but difficult to demonstrate in cystic fibrosis. Diets in Paleolithic times contained no processed Despite poor fat digestion, intake levels of linoleate food and probably balanced amounts of n-3 to n-6 may not be inadequate but its β-oxidation could well polyunsaturates and a lower level of saturates. Such be abnormally high owing to the chronic infectious diets would be predicted to lead to a lower incidence challenge. of degenerative disease. Since the brain has a very high energy requirement, it has also been speculated Clinical importance of polyunsaturates that human brain evolution beyond that of other pri- mates was dependent on a reliable and rich source of Infant brain and visual development is dependent on dietary energy and a direct source of long-chain poly- adequate accumulation of docosahexaenoate. The unsaturates, particularly docosahexaenoate. 1990s saw intense clinical and experimental assess- ment of the role of docosahexaenoate in early brain 6.10 Cholesterol synthesis and regulation development and a widespread concern that many infant formulae do not yet contain docosahexaenoate. Cholesterol and the brain Several clinical studies and extensive use of formulae containing docosahexaenoate and arachidonate have Mammalian brain function is dependent on special- shown that they are safe. Many but not all such studies ized membranes designed for signal transmission. show an improvement in visual and cognitive scores Greater cognitive sophistication in humans appears compared with matched formulae containing no to depend on a much greater number of connections docosahexaenoate or arachidonate. The infant brain and, consequently, greater potential for signal pro- and body as a whole clearly acquire less docosahexae- cessing. Like the membrane lipids of most other noate when only α-linolenate is given. As a whole, mammalian organs, brain lipids contain a relatively these data suggest that docosahexaenoate is a condi- high proportion of cholesterol, which increases from tionally indispensable fatty acid. about 40% of the lipid content in neonates to nearly 50% in adults. Aside from questionable deficiency of polyunsatu- rates in cystic fibrosis (see above), one of the most Unlike other organs, the mammalian brain is prob- graphic examples of their deficiency being caused by ably unique in being unable to acquire appreciable

Nutrition and Metabolism of Lipids 115 amounts of cholesterol from the circulation, i.e., from Over 30 years ago Keys and Hegsted made the land- the diet or from synthesis outside the brain. This has mark observation that variation in the concentration been extensively studied in the young rat and sup- of serum cholesterol across seven different countries porting, although inconclusive, evidence is also avail- was positively related to the amount of energy derived able for the pig. The brain has sufficient capacity to from saturated fat. Conversely, they found that intake synthesize cholesterol from acetyl-CoA derived pri- of dietary PUFA was inversely related to serum cho- marily from either glucose or ketones. Hence, it lesterol. From this finding they were able to formulate achieves the required level of cholesterol apparently equations that enabled them to predict the quantita- entirely by endogenous synthesis. In neonates, ketones tive effect of saturated and polyunsaturated fat on appear to play a greater role as substrates for brain serum cholesterol (Figure 6.16). In simpler terms, the cholesterol than in adults, in whom their main func- ratio of PUFAs to saturated fatty acids (SFAs), the tion seems to be as an alternative fuel to glucose. P : S ratio, was used with effect to predict changes in Among the common dietary long-chain fatty acids serum cholesterol. Although still effective today, the that would give rise to ketones during fat oxidation, equations and P:S ratio are being superseded by PUFAs, particularly linoleate and α-linolenate, appear advanced knowledge of the biological effects of indi- to be the best substrates for ketogenesis, since carbon vidual fatty acids. It is now well established that satu- from these fatty acids readily appears in brain choles- rated fats with between 12 and 16 carbon atoms, terol in suckling rats. namely lauric, myristic, and palmitic acids, are par- ticularly hypercholesterolemic, whereas stearic acid, 6.11 Effect of diet on serum lipids an extremely abundant SFA in most diets, is relatively and lipoproteins neutral in its effects on serum cholesterol. [Note that stearic acid is desaturated to monounsaturated fatty Diet and serum cholesterol acids (MUFAs) by Δ9 desaturase]. The cholesterol- raising effect of SFAs arises chiefly from an increase Diet exerts a profound influence on blood lipids and in LDL cholesterol and is about twice as potent as the lipoproteins and, as such, should always be a major hypocholesterolemic effect of dietary PUFAs. Para- component of strategies for the primary prevention doxically, SFAs actually increase serum HDL choles- of diseases in which lipids play an etiological role, terol. The polyunsaturates are divisible into two main such as CHD. Nevertheless, despite convincing epide- series on the basis of the position of the first double miological evidence and the existence of credible bond from the methyl end of the fatty acid chain, the biochemical mechanisms to support a relationship parent fatty acids being linoleic (C18:2n-6) and α- between dietary fat and serum cholesterol, the linolenic (C18:3n-3) acids. The cholesterol-lowering outcome of prospective intervention trials designed effects of dietary PUFA is largely attributable to the to test this relationship within populations has been effects of linoleic acid in lowering LDL. Historically, disappointing. LDL receptors Blood LDL-C Intracellular ACAT Cholesterol pool of free esters cholesterol Saturated fatty acids Intracellular ACAT Cholesterol Figure 6.16 Influence of dietary fatty pool of free esters acids on serum cholesterol through cholesterol differential effects on free cholesterol and low-density lipoprotein (LDL) receptor MUFAs and PUFAs activity. ACAT, acyl-CoA-cholesterol acyl- transferase; LDL-C, LDL cholesterol; MUFA, monounsaturated fatty acid; PUFA, poly- unsaturated fatty acid.

116 Introduction to Human Nutrition monounsaturated fat was considered to be neutral of intake these fats are unlikely to exert adverse effects with respect to its effects on lipids and lipoproteins on serum lipoproteins. and was omitted from the predictive formulae of Keys and Hegsted. However, further studies, prompted by Plant sterols and soluble interest in the role of MUFAs in the Mediterranean nonstarch polysaccharides diet, have shown that MUFA-enriched diets may decrease LDL cholesterol, although possibly to a lesser These compounds may be grouped together as they extent than linoleic acid, and increase HDL choles- share a similar mode of action on LDL cholesterol, terol. An additional benefit of MUFAs is thought to which is to reduce the availability of dietary and be conferred by the presence of a single double bond biliary cholesterol for absorption in the gut. This in MUFAs, which when incorporated into the mem- action interrupts the enterohepatic circulation and brane phospholipids of LDL, protect this lipoprotein upregulates the production and activity of LDL recep- from oxidative modification, an essential prerequisite tors (see Section 6.5). Plant sterols and their esters step in the deposition of cholesterol in the artery wall. such as those incorporated into margarines (stanols In this regard, there has been concern that increasing and stanol esters), despite being nearly identical in dietary PUFA will impose additional oxidative stress structure to cholesterol, are poorly absorbed and on LDL. While this idea forms part of the rationale interfere with the reabsorption of cholesterol origi- for limiting the amount of dietary PUFA to less than nating from bile (~1 g/day) and dietary sources 10% of energy intake, there is as yet no convincing (300 mg/day) by either coprecipitation or competi- evidence of adverse effects from increasing the level tion. Margarines or spreads (30–40 g/day) containing of PUFA in tissues and circulating lipoproteins. plant sterols or their derivatives have been shown to reduce LDL cholesterol by up to 14% in controlled Trans fatty acids trials. Soluble NSPs such as those found in gums and gelling agents from fruit (gum arabic and pectins) act While saturated fats consist of straight chains of in a similar way and have been shown to be equally carbon atoms which pack together tightly in the efficacious in reducing LDL cholesterol. phospholipids of cell membranes and lipoproteins, in contrast, carbon double bonds in the cis configura- Dietary cholesterol tion in MUFAs and PUFAs introduce a bend or kink into the carbon chain. This alters the physical proper- There is a popular misconception that dietary choles- ties of the phospholipids containing these fatty acids, terol correlates directly with serum cholesterol, when by, for example, increasing their packing volume, a in fact dietary cholesterol, within a range of normal physical property that contributes to an increase in dietary consumption (100–400 mg/day), has only a membrane fluidity. The partial hydrogenation of very small impact on blood cholesterol levels. Eggs MUFAs and PUFAs, most notably during the indus- represent the principal source of dietary cholesterol trial processing of foods for the purpose of solidifying in most diets (1 egg yolk = 150–250 mg cholesterol); unsaturated oils, results in a loss of this kink as the in their absence, most Western diets would contain fatty acid assumes a straighter, trans configuration considerably less than 100 mg cholesterol/day. The that resembles that found in SFAs. This likeness in classic but extreme egg-feeding studies showed that chemical structure is thought to account for the SFA- feeding of up to six eggs per day (900 mg cholesterol) like effects of trans fatty acids such as elaidic acid increased LDL cholesterol acutely. However, the body (trans isomer of oleic acid) on serum lipids (see effectively counters this effect with sensitive, compen- Figures 6.1 and 6.2). The results of prospective cohort satory mechanisms to deal with an increasing load of studies such as the Nurses’ Health Study showed that dietary cholesterol, one of which is to reduce the high levels of trans fats in excess of 7% energy amount of cholesterol absorbed in the gut. This com- increased serum LDLs and reduced HDLs (Willet et pensation effectively abolishes any dose–response al. 1993). However, despite the continued use of par- relationship between dietary cholesterol, over a prac- tially hydrogenated fats in food products, the average tically realistic range of intakes, and serum choles- intake of trans fatty acids in most Western diets does terol. Two factors that may influence the variability in not exceed 2% of total energy intake, and at this level response to dietary cholesterol are dietary saturated fatty acids, which have been shown to augment the

Nutrition and Metabolism of Lipids 117 cholesterol-raising effects of dietary cholesterol, and acid in the food industry, together with a widespread a phenomenon of increased susceptibility to dietary resistance to the consumption of fish, has increased cholesterol in some individuals for some, as yet, the ratio of n-6 to n-3 PUFAs in northern Europe and unknown reason. the USA since the 1970s. This situation has major implications for the development of abnormalities in To place these dietary influences on blood choles- circulating lipoproteins, since deficiency in eicosapen- terol in perspective with other cholesterol-lowering taenoic acid and docosahexaenoic acid could help to strategies, a metaanalysis of dietary intervention trials promote an increase in plasma TAG. This could occur undertaken by the World Health Organization through an overproduction of endogenous TAG (WHO) revealed that dietary modification could (VLDL) in the liver and intolerance to dietary (exoge- achieve reductions in serum cholesterol of between nous) fat, and lead to the development of dyslipidemia only 4% and 5%. This finding is in sharp contrast to known as an ALP (see Section 6.5). The frequency of the potent effects of cholesterol-lowering drugs, which this dyslipidemia is believed to be very rare in can reduce serum cholesterol by 30–40% and have Mediterranean countries that have a high intake of been shown, unequivocally, to reduce the incidence of dietary n-3 PUFA and an n-6:n-3 ratio closer to 1. death from CHD. It also highlights the need to address High-carbohydrate diets have been shown to increase other risk factors which are more responsive to dietary plasma TAG. Carbohydrate-induced hypertriacylglyc- change. erolemia is not, as was originally thought, a short- term adaptive response in the liver, as it changes its Fat quantity versus quality: importance pattern of oxidation from fat to carbohydrate, but a of the ratio of n-6:n-3 polyunsaturated real phenomenon associated with the overconsump- fatty acids tion of the non-milk extrinsic sugars, sucrose and fructose, most notably in individuals with insulin- The underlying principle for a reduction in total fat resistant dyslipidemia. There is evidence to suggest intake is to reduce energy intake from the consump- that this effect can be avoided by limiting the intake of tion of the most energy-dense macronutrient in order sucrose and increasing the intake of slowly absorbed to prevent weight gain and ultimately obesity. The carbohydrate with a low impact on blood glucose. current recommendation for the UK is to reduce energy derived from fat to 35% or less. Since weight The results of several metaanalyses of dietary inter- gain is associated with raised plasma TAGs and abnor- vention trials support dietary MUFAs as the most malities in circulating lipoproteins, reducing total fat favored substitute for dietary saturated fatty acids, intake should, in theory, reduce blood lipids. However, and even linoleic acid in areas of high intake. in practice there is little evidence to support such an effect within populations. Metaanalyses have revealed Effects of n-3 polyunsaturated fatty acids that little benefit is to be gained, at least in terms of from plants and fish changes in blood lipids, by reducing total fat without altering the composition of dietary fatty acids. The current dietary recommendation for the intake Metaanalyses have also helped to resolve the issue of of long-chain n-3 PUFAs (eicosapentaenoic acid/doc- what represents the most appropriate replacement osahexaenoic acid) in the UK is 450 mg/day (SACN, nutrient for SFAs. Since PUFAs, and specifically lin- 2004). This was to increase intake by consuming two oleic acid, were shown to counter the actions of SFAs portions of fish per week, one of which should be oily and were abundant in natural sources such as sun- (e.g., mackerel, sardines). This recommendation was flower and corn oils, they were an obvious first choice. based on evidence from a host of epidemiological and The alternative was to substitute fat with dietary car- intervention studies, which showed that regular fish bohydrate. There have been problems associated with consumption could reduce the risk of sudden cardiac both of these approaches. First, increasing dietary lin- death. Since this an acute end-point of CHD, the ben- oleic acid excludes the lesser abundant, but more met- efits of fish oil have been ascribed to the prevention abolically active, n-3 PUFA, and especially the longer of fatal cardiac arrhythmia, and, to a lesser extent, chain (C20–C22) members of this series derived from coronary thrombosis, but not to any favorable effects marine oils [C20:5 (eicosapentaenoic acid) and C22:6 on blood lipids. However, there is also convincing (docosahexaenoic acid)]. Overemphasis on linoleic evidence to show that fish oil supplementation (1 g/

118 Introduction to Human Nutrition day for 3 years) reduces the incidence of death from PUFA at the same time. However, in practice, this will CHD in healthy, free-living subjects. This longer term be difficult to achieve, not least because of a mass benefit may be linked to the effects of eicosapentae- resistance to the increased consumption of oily fish noic acid/docosahexaenoic acid on a host of other and diminishing fish stocks. An obvious alternative cardiovascular risk factors, including plasma TAGs would be to increase the intake of the shorter chain and lipoproteins. precursor of eicosapentaenoic acid/docosahexaenoic acid, α-linolenic acid (C18:3n-3). The latter is derived Long-chain n-3 PUFAs exert multiple effects on from plant seeds such as flax and rapeseed, and is lipid metabolism, the most notable of which is the desaturated and elongated to its longer chain relatives capacity to decrease postabsorptive plasma TAG levels in the body. Unfortunately, the rate of conversion to by 20–30%. Fish oil-enriched diets have also been eicosapentaenoic acid and especially docosahexaenoic shown to attenuate the magnitude and duration of acid is slow, and the efficiency of conversion is reduced postprandial lipemia following the ingestion of a fat- by high levels of linoleic acid, which competes more containing meal. These effects are frequently accom- effectively than α-linolenic acid for desaturation. panied by beneficial changes in circulating LDLs and There is, as yet, no evidence to suggest that the rate HDLs, and the correction of an ALP. of conversion of dietary α-linolenic acid to eicosa- pentaenoic acid and especially docosahexaenoic acid Widespread knowledge of the favorable effects of is sufficient to achieve fish oil-like effects on blood eicosapentaenoic acid and docosahexaenoic acid has lipids (Table 6.6). raised awareness of the need to increase intakes of these fatty acids, and to reduce the amount of n-6 Table 6.6 Effects on plasma lipids of substituting dietary saturated fats with polyunsaturated fatty acids, monounsaturated fatty acids, and carbohydrates LDL-C HDL-C TAG TC:HDL SFA a (C12–C16) n-6 PUFA Linoleic acid (sunflower/corn) n-3 PUFA b LC = EPA/DHA (fish oil) SC = linolenic acid (flax/soyabean/rape) n-9 MUFA Oleic acid (olive oil/rape) Carbohydrate Sucrose/fructose? a n-6 PUFA in excess of 10% energy. b Increase in response to redistribution of LDL subsclasses. PUFA: polyunsatruated fatty acids; MUFA: monounsaturated fatty acids. SFA: saturated fatty acids; LC: long chain; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; short chain; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TAG: triacylglycerols; TC: total cholesterol.

Nutrition and Metabolism of Lipids 119 How do dietary fatty acids influence serum TAG by inhibiting the enzymes phosphatidic acid cholesterol and triacylglycerols? phosphatase and diacylglycerol acyltransferase. They may also selectively increase the degradation of apoB- In common with other physiological systems, lipo- 100, further reducing the production of TAG-rich protein metabolism is coordinated by interplay VLDLs. (Note that apoB-100 is produced constitu- between the activity of specific genes and hormones tively, so that the production of VLDL is driven by the that determines the production and activity of func- supply of substrates for the synthesis of TAG). In tional proteins (enzymes, receptors, lipid transfer, and addition, long-chain n-3 PUFAs accelerate the clear- apoproteins). Effects on these functional proteins ance of TAG-rich lipoproteins from the circulation in ultimately regulate the quantity and quality of circu- the postprandial phase by stimulating the activity of lating lipids and lipoproteins. While there have been LPL. Together, these effects are thought to underlie significant advances in knowledge of the modulatory the ability of these fatty acids to correct the lipopro- effects of dietary fatty acids on hormones and gene tein abnormalities associated with an ALP. It is also expression, evidence for the effects of dietary fats on possible that many of the effects of eicosapentaenoic functional proteins is by far the most advanced. acid/docosahexaenoic acid on blood lipids and other cardiovascular risk factors are mediated through an Saturated fatty acids and low-density increase in the sensitivity of tissues to the action of lipoprotein cholesterol insulin. However, there is, as yet, no convincing evi- dence to support such an effect in adipose tissue, liver, The most well-elucidated mechanism to explain how or skeletal muscle. different dietary fats produce variable effects on LDL- cholesterol is through the LDL receptor pathway, the Nutrient–gene interactions control of which has already been described (see Section 6.5). The ability of the cell to regulate its pool It has been estimated that diet could account for up of free cholesterol depends to a large extent on the to 50% of the variation in blood lipids and lipopro- nature of the fatty acids available for esterification by tein levels between individuals. This would mean that the enzyme ACAT, an intracellular relative of LCAT. genetic differences must explain the remaining 50%. ACAT favors unsaturated fatty acids (MUFAs and In real terms, interactions between diet and genes PUFAs) as substrates for esterification, which utilizes represent a sizeable proportion of each of these unre- free cholesterol within the cell. The resulting reduc- alistically discrete fractions. tion in intracellular free cholesterol stimulates the transcription of the LDL receptor gene and produc- Fixed genetic polymorphisms tion of new LDL receptors through the SREBP mech- Variation in the structure of specific genes between anism, and fall in circulating LDL as already described. individuals (genetic heterogeneity) has been shown to Conversely, SFAs are poor substrates for ACAT, and give rise to differences in dietary responsiveness. A their presence in the cell exerts the opposite effect on few common polymorphisms have been identified in free cholesterol levels, thus increasing circulating LDL genes associated with lipoprotein metabolism, the cholesterol and total serum cholesterol (Figure 6.8). best example of which is apoE. ApoE facilitates the Fatty acids may also exert direct effects on the activity uptake of TAG-rich lipoproteins (chylomicron rem- of LDL receptors by altering the composition of nants and VLDL) via the remnant and LDL receptors membrane phospholipids and thus membrane fluid- and, thus, in part, determines the removal of TAG ity. Alternatively, there is evidence to suggest that from the circulation. The gene for apoE is polymor- dietary PUFA could upregulate LDL receptors indi- phic, which means that it exists in multiple forms rectly, by increasing the cholesterol content (litho- between individuals. This polymorphism generates genicity) of bile and in this way accelerate the excre- several isoforms of the protein that express variable tion of cholesterol. affinities for their receptors and thus variable poten- tial to remove TAG-rich lipoproteins from the circu- Long-chain n-3 polyunsaturated fatty acids lation. In this way, apoE genotype can modulate the and serum triacylglycerols response of an individual to any dietary fat that exerts an influence on TAG-rich lipoproteins, giving rise to Long chain n-3 PUFAs have potent effects in the liver, where they suppress the production of endogenous

120 Introduction to Human Nutrition Table 6.7 Effect of apoprotein E phenotype on serum cholesterol Phenotype (gene frequency) Receptor binding affinity Hepatic free cholesterol LDL receptor activity LDL-C E4 (ε4 15%) +++ ↑ (feedback) ↓ ↑ E3 (ε3 77%) ++ → → → E2 (ε2 8%) + ↓ (feedback) ↑ ↓ Carriage of ε4 allele is associated with increased risk of coronary heart disease and greater changes in low-density lipoprotein cholesterol (LDL-C) in response to increased dietary fat and cholesterol in men. the concept of genetic susceptibility to diet (Table for the “ideal” fat intake will continue, but this seems 6.7). misguided because humans in good health in differ- ent cultures and geographical locations ultimately Modulation of gene expression consume a wide range of total fat and fatty acid ratios; Dietary fatty acids and/or their intracellular (eico- their overall health is based on much more than their sanoid) derivatives can also influence the expression fat intake. Nevertheless, pursuit of an ideal fat intake of genes (rate of gene transcription) by interacting or composition will satisfy the thirst of many research- with specific nuclear receptors within the nucleus of ers, consumers, and government agencies but, in all cells. These nuclear receptors control the rate of gene likelihood, will not greatly alter the impact of fats on transcription by binding to specific regions of DNA disease processes. These would include: known as responsive elements. Genes associated with the production of functional proteins can be either ● the role of the quantity and quality of dietary fat on stimulated or repressed according to the nature of the postprandial lipemia, specific lipoproteins, and the nuclear transcription factor and its binding substrate risk of CHD (PUFAs or derivative). Peroxisome proliferator-acti- vated receptors (PPARs) represent examples of nuclear ● the metabolic roles of the short-chain fatty acids receptors that may utilize long-chain PUFAs as sub- (1–6 carbons) strates. PPARs can be found in all tissues of the body, but notably in the liver, where they control the syn- ● the effect of trans fatty acids in baby foods, and the thesis of lipid and apoproteins (PPAR-α), and in level of intake of trans fatty acids that will increase adipose tissue (PPAR-γ), where they control the dif- the risk of CHD in adults ferentiation of adipocytes and insulin-sensitive mobi- lization and synthesis of TAG. SREBPs represent ● the appropriate amounts of n-6 to n-3 polyunsatu- another example of nuclear transcription proteins rates to prevent, reverse, and/or treat chronic that control cholesterol and fatty acid metabolism degenerative diseases within the cell. ● the requirements of docosahexaenoate and other 6.12 Perspectives on the future long-chain PUFA in infant and enteral feeding Future research on fatty acids and their role in health ● the conditional indispensability of particular fatty and disease will be largely dictated by progress in acids throughout the life cycle (especially during intervention studies using fatty acid supplements in pregnancy and lactation, and in the aged) chronic diseases of inflammation, brain degeneration, cancer, and heart disease. Some of these studies will ● the effects of particular dietary fatty acids and com- be based on effects of dietary fats on gene expression binations of fatty acids on hormone and gene that have yet to be discovered; others will be based on expression. information that we already have. No doubt the search More knowledge in all of these areas will lead to a better understanding of the mechanisms through which dietary lipids influence blood lipids and lipo- proteins and therefore the risk of chronic diseases. It will also lead to improved recommendations regard- ing the quantity and quality of dietary fats that are commensurate with optimum human health.

Nutrition and Metabolism of Lipids 121 Acknowledgment Further reading This chapter has been revised and updated by Bruce Cunnane SC. Alpha-linolenate acid in human nutrition. In: Griffin based on the original chapter by Bruce Griffin Cunnane SC, Thompson LU, eds. Flaxseed in Human Nutrition. and Stephen Cunnane. AOCS Press, Champaign, IL, 1995: 99–127. References Dolecek TA. Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Bloomfield DK & Bloch K. The formation of Δ9-unsaturated fatty Risk Factor Intervention Trial. Proc Soc Exp Biol Med 1992; 200: acids. J Biol Chem 1960; 235: 337-345. 177–182. Burr MM & Burr GO. A new deficiency disease produced by the Durrington PN. Hyperlipidaemia Diagnosis and Management, 2nd rigid exclusion of fat from the diet. J Biol Chem 1929; 82: edn. Elsevier Science, Oxford, 1995. 345-367. Griffin BA The effects of n-3 PUFA on LDL subfractions. Lipids Goldstein JL & Brown MS. Atherosclerosis: the LDL receptor 2001; 36: S91–S97. hypothesis. Metabolism 1977; 26: 1257-1275. Griffin BA How relevant is the ratio of dietary n-6 to n-3 Hegsted DM, McGrandy RB, Myers ML et al. Quantitative effects polyunsaturated fatty acids to cardiovascular disease risk? of dietary fat on serum cholesterol in man. Am J Clin Nutr 1965; Evidence from the OPTILIP Study. Curr Opin Lipidol 2008; 19: 17: 281-295. 57–62. Keys A, Anderson JT & Grande F. Prediction of serum cholesterol Lands WEM. Impact of daily food choices on health promotion responses of man to changes in fats in the diet. Lancet 1977; 2: and disease prevention. In: Hamazaki H, Okuyama H, eds. Fatty 959-966. Acids and Lipids: New Findings. Karger, Basel, 2001: 1–5. Ponticorvo L, Rittenberg D & Bloch K. The utilisation of acetate Lee A, Griffin BA. Dietary cholesterol, eggs and coronary heart for the synthesis of fatty acids, cholesterol and protoporphyrin. disease in perspective. Br Nutr Found Bull 2006; 31: 21–27. J Biol Chem 1949; 179: 839-842. Mangiapane EH, Salter AM, eds. Diet, Lipoproteins and Coronary Scientific Advisory Committee on Nutrition (SACN) 2004. Report Heart Disease. A Biochemical Perspective. Nottingham University published for the Food Standards Agency and the Department Press, Nottingham, 1999. of Health by TSO. ISBN 0-11-243083-X. Simopoulos AP. Evolutionary aspects of diet and essential fatty Willet WC, Stamfer MJ, Manson JE et al. Intake of trans fatty acids acids. In: Hamazaki H, Okuyama H, eds. Fatty Acids and Lipids: and risk of coronary heart disease among women, Lancet 1993; New Findings. Karger, Basel, 2001: 18–27. 341: 581–585. Willett WC. Eat, Drink and Be Healthy: The Harvard Medical Zilversmit DB. Atherogenesis is a postprandial phenomenon. School Guide to Healthy Eating. Simon and Schuster, New York, Circulation 1979; 60: 473-485. 2001.

7 Dietary Reference Standards Kate M Younger Key messages • The methods used to determine requirements are discussed. These include deprivation studies, radioactive tracer studies, • This chapter discusses the development of terminology and the balance studies, factorial methods, measurement of nutrient change in conceptual approaches to setting nutrient recommen- levels in biological tissues, biochemical and biological markers, dations from adequate to optimum nutrition. and animal experiments. • The interpretation and uses of dietary recommendations are discussed. • The chapter describes how reference values can be used to assess the adequacy of the nutrient intakes of population groups. 7.1 Introduction past become obsolete as they are replaced by new figures based on new data or new interpretations of The first attempt to set standards for nutrient intakes existing data. was by the Food and Nutrition Board of the National Research Council of the USA in 1941, which pub- 7.2 Terminology and conceptual lished recommended daily allowances (RDAs) in 1943 approaches to setting nutrient to “provide standards to serve as a goal for good recommendations nutrition.” The first UK RDAs followed in 1950, pub- lished by the British Medical Association, and many From the time of their first issue in the 1940s and other countries and international agencies now throughout the next 50 years, the concepts and ter- publish dietary standards that are intended to allow minology of RDAs remained unchanged. The basis on the adequacy of the nutrient intakes of groups or which these RDAs were built was the statistical distri- populations to be assessed by comparison with the bution of individual requirements to prevent defi- standards. ciency criteria for the target nutrient. The peak of the curve of the Gaussian distributions of such require- As the amount known about human requirements ments is the “average requirement,” with half the and nutrient functions has increased, so too has the population having requirements above this value and size of the documents describing the recommenda- the other half having lower requirements. The RDA tions, from a mere six pages dealing with 10 nutrients was taken to be a point on that distribution that was in 1943 to the series of weighty books, each dealing equal to the mean or “average requirements” plus 2 with the dietary reference intakes (DRIs) of only a few standard deviations (SDs) (Figure 7.1). By setting the of more than 30 nutrients, published by the Institute recommendation close to the upper end of the distri- of Medicine of the USA. Furthermore, continuing bution of individual requirements, the needs of most research and the development of more informed of the population would be met. If the standard were interpretations of the expanding body of data avail- set to meet the apparent needs of almost everyone, able necessitate the regular revision and updating of the resultant value would be so high as to be unat- the recommendations; thus, the “standards” of the © 2009 KM Younger.

Dietary Reference Standards 123 2.5% 95% 2.5% However, since the 1980s, changes have occurred in both of these areas. Number of individuals Changes in terminology abc Two basic changes occurred with regard to terminol- Nutrient requirements ogy. The first was that the term “recommended dietary allowance” was altered and the second was that new Figure 7.1 Frequency distribution of individual requirements for a terms were introduced so that the adequacy of diets nutrient. (a) The mean minus a notional 2 standard deviations (SDs); could be evaluated from several perspectives. The intakes below this will be inadequate for nearly all of the population. reason for changing the terminology was in effect to (b) The mean; the midpoint of the population’s requirement. (c) The re-emphasize some of the basic concepts underlying mean plus a notional 2 SDs; the intake that is adequate for nearly all the term RDA. “Recommended” has a prescriptive air of the population. Note that, in practice, because insufficient data exist about it and there were concerns that consumers to establish reliable means and SDs for many nutrient requirements, might see this as something that had to be met daily the reference intakes describing the points a and c on the curve are and met precisely. The term “allowance” reinforces generally set, in the case of a, at the level that is judged to prevent the perception of a prescriptive approach. Thus, the the appearance of signs of deficiency (biochemical or clinical), and, in UK adopted the term dietary reference value (DRV), the case of c, at the level above which all individuals appear to be the EU introduced the term population reference adequately supplied. Thus, it is unlikely that even 2.5% of the popula- intake (PRI), the USA and Canada introduced the tion would not achieve adequacy at intake level c. term dietary reference intake (DRI), and Australia and New Zealand now use the term nutrient intake tainable at population level. If the standard were set value (NIV). All are precisely equivalent to the origi- at the point of the average of all individual require- nal concept of the RDA, a term that many countries ments, then half the population would have require- prefer to continue to use. ments in excess of the standard. In a normal distribu- tion, some 2.5% of points lie at the upper and lower Two new terms were introduced: a minimum tails outside that range of the mean plus or minus 2 requirement and a safe upper level. The minimum SDs. Thus, by setting the RDA to this point of the requirement represents the average requirement mean plus 2 SDs, we are setting the standard for minus 2 SDs (point a in Figure 7.1). A definition 97.5% of the population. The consumption of most describing this point is given in Figure 7.1 along with nutrients at levels somewhat greater than actually the various terms used to define this point (Box 7.1). required is generally not harmful; hence, setting rec- The concept of an upper safe limit of intake has ommendations at the population average require- gained importance in view of the increased opportu- ment plus a notional 2 SDs is logical if the aim is to nity for people to consume high levels of nutrients describe an intake that is adequate for almost every- from fortified foods or supplements. The recently one. However, this is spectacularly inappropriate in revised US DRI set “tolerable upper intake” levels the case of recommendations for energy intake, since (ULs) that are judged to be the highest level of nutri- even relatively small imbalances in energy intake over ent intake that is likely to pose no risk of adverse expenditure will lead, over time, to overweight and health effects in almost all individuals in a group. The ultimately obesity, an increasing problem in most current European and UK recommendations also populations. Recommendations for energy intake are address this concern in the case of those nutrients for therefore given only as the estimated population which toxic levels have been reported. The terms used average requirement. by different recommending bodies to describe the various points on the distribution of individual Thus, for almost half a century, these were the requirements for a nutrient are given in Box 7.2, while terms used and the underlying conceptual approaches. precise definitions may be found in the relevant pub- lications referred to. The World Health Organization (WHO) has taken a rather different approach, defining population safe ranges of intake. “Normative requirement” is now

124 Introduction to Human Nutrition Box 7.1 Terms used to describe points a, b, and c on the frequency distribution European Communities Scientific Committee for Food a b c Population reference intakes (1993) Lowest threshold intake Average requirement Population reference (LTI) (AR) intake (PRI) US Food and Nutrition Board, Recommended daily National Academy of Sciences, Lower reference Estimated average allowance (RDA) National Research Council nutrient intake (LRNI) requirement (EAR) Recommended daily allowances (1989) Recommended daily Estimated average allowance (RDA) US Food and Nutrition Board, requirement (EAR) Institute of Medicine, Reference nutrient intake National Academies of Health, Canada Estimated average (RNI) Dietary reference intakes (1997–2005) requirement (EAR) Average nutrient Recommended nutrient British Committee on Medical Aspects of Food Policy requirement (ANR) intake (RNI) (COMA) Recommended dietary Dietary reference values (1991) intake (RNI) World Health Organization/Food and Agriculture Individual nutrient level Organization (WHO/FAO) (INLx; in this case INL98) National Health and Medical Research Council (NHMRC), Australia and New Zealand Nutrient reference values (2006) United Nations University (UNU) Nutrient intake values (2007) Box 7.2 Additional terms used used to describe the population mean normative requirement (which would allow the maintenance of, European population Acceptable range of intakes or a desirable, body store or reserve); “maximum” to reference intake refer to the upper limit of safe ranges of population (1993) Safe intake and adequate intake mean intakes; and “basal” for the lower such limit, below which clinically detectable signs of inadequacy US recommended daily Adequate intake (AI) and tolerable would be expected to appear. These WHO require- allowance (1989) upper intake level (UL), acceptable ments are revised in groups of nutrients at different macronutrient distribution range times (see Further reading), and in those that date US dietary reference (AMDR) from 1974 the term “recommended intake” or “rec- intake (1997–2005) ommended nutrient intake” is used to describe the Safe and adequate intake average requirement plus an amount that takes into British dietary reference account interindividual variability and hence is con- value (1991) Recommended intake sidered to be sufficient for the maintenance of health in nearly all people. World Health Basal, normative, and maximum Organization (1974– population requirement ranges, More recently, the United Nations University 1996) mean intake goals (UNU) has published a suggested harmonized approach and methodologies for developing nutrient World Health Acceptable macronutrient recommendations, together with proposed terminol- Organization (1996–) distribution range (AMDR) and ogy (Box 7.1), that could be used worldwide to suggested dietary target (SDT) promote objectivity, transparency, and consistency National Health and among those setting and using nutrient recommen- Medical Research Upper nutrient level (UNL) Council (2006) United Nations University (2007)

Dietary Reference Standards 125 dations. Their preferred term, nutrient intake value In the USA, the reference value for calcium is based (NIV), refers to dietary intake recommendations on optimizing bone calcium levels, which is a move based on research data; the term“nutrient” was chosen away from the traditional approach of focusing on in order to distinguish these from dietary compo- preventing deficiency symptoms. An example of nents such as cereals, and the term “value” is intended attempts to set the reference standard for optimizing to emphasize the potential usefulness for both assess- a biochemical function is a level of folic acid that ing dietary adequacy (and hence dietary planning) would minimize the plasma levels of homocysteine, a and policy-making. The individual nutrient level potential risk factor for cardiovascular disease.Another (INLx) is flexible, in that x refers to the chosen per- might be the level of zinc to optimize cell-mediated centile of the population for whom this intake is suf- immunity. An example of a possible reference stan- ficient;for example 98% (mean or median requirement dard to optimize a risk factor for a disease is the level + 2 SDs), written as INL98, but it could be set lower in of sodium that would minimize hypertension or the the case of certain nutrients. level of n-3 polyunsaturated fatty acids (PUFAs) to lower plasma triacylglycerols (TAGs). The amount of Changes in conceptual approach folic acid to minimize the population burden of neural tube defect would be an example of a reference value When a committee sits to make a recommendation to minimize the incidence of a disease. At present, for a standard in relation to nutrient intakes, it begins there is much debate as to the best approach to choos- with a distribution of requirements. In the past, ing criteria for setting reference standards for minerals although the choice of criteria for requirement might and vitamins, and this is an area that is likely to con- vary between committees, the orientation was always tinue to court controversy. An important point to note the same: requirements were set at a level that should in this respect is that, while minimizing frank defi- prevent deficiency symptoms. More recently, the ciency symptoms of micronutrients is an acute issue concern for health promotion through diet has led to in many developing countries, any evolution of our the introduction of the concept of optimal nutrition, concepts of desirable or optimal nutrient require- in which the optimal intake of a nutrient could be ments must lead to a revision of the estimate of the defined as that intake that maximizes physiological numbers of those with inadequate nutrition. and mental function and minimizes the development of degenerative diseases. It should be borne in mind 7.3 Interpretation and uses of that, although this may appear simple enough to dietary recommendations define in the case of single nutrients, things clearly become more complex when considering all nutrients When using dietary recommendations, several impor- together, in all possible physiological situations. tant points need to be considered. Genetic variability may also, increasingly, be taken into account; for example, the requirement for folate The nutrient levels recommended are per person of those carrying certain variants of the MTHFR gene per day. However, in practice this will usually be (around 10% of the population tested thus far) might, achieved as an average over a period of time (days, arguably, need to be set higher than for the rest of the weeks, or months) owing to daily fluctuations in the population. diet. As stated above, the setting of a range of dietary recommendations should encourage appropriate It is now recognized that there are several levels for interpretation of dietary intake data, rather than the considering the concept of optimal nutrition, i.e., the inappropriate assumption that the value identified to level that: meet the needs of practically all healthy people is a minimum requirement for individuals. If an individ- ● prevents deficiency symptoms, traditionally used to ual’s nutrient intake can be averaged over a sufficient establish reference nutrient intakes period then this improves the validity of the compari- son with dietary recommendations. However, in the ● optimizes body stores of a nutrient case of energy intakes, such a comparison is still inap- ● optimizes some biochemical or physiological propriate: dietary reference values for energy are intended only for use with groups, and it is more function ● minimizes a risk factor for some chronic disease ● minimizes the incidence of a disease.

126 Introduction to Human Nutrition useful to compare an individual’s energy intake with milk compared with formula milks, but rather the some measure or calculation of their expenditure in reverse. order to assess adequacy. The dietary recommendations for infants post- In the case of a group, the assumption can be made weaning and for children and adolescents are gener- that the quality of the diet can be averaged across the ally based on less robust scientific evidence than those group at a given time-point, and therefore that appar- for adults, for whom much more good information is ently healthy individuals within a group may com- available. In the absence of reliable data, values for pensate for a relative deficiency on one day by a children are usually derived by extrapolation from relative excess on another. It should also be remem- those of young adults. The calculation of nutrient bered that allowances may need to be made for body requirements is generally based on energy expendi- size, activity level, and perhaps other characteristics ture because metabolic requirements for energy prob- of the individual or group under consideration, since ably go hand in hand with those for nutrients in the recommended intakes are designed for “reference” growing children. In the case of infants post-weaning populations. on mixed diets, values are obtained by interpolation between values known for infants younger than 6 Another assumption made when setting recom- months and those calculated for toddlers aged 1–3 mendations for a particular nutrient is that the intake years. Thus, the dietary recommendations for children of all other nutrients is adequate, which in an appar- and adolescents need to be approached with some ently healthy population eating a varied diet is prob- caution, being more suitable for planning and labeling ably reasonable. purposes than as a description of actual needs. Recommendations are not intended to address the Finally, assessment of the dietary adequacy of needs of people who are not healthy: no allowance is people at the other end of the population age range made for altered nutrient requirements due to illness is made difficult by the lack of data on healthy elderly or injury. For example, patients confined to bed may people. One of the normal characteristics of aging is require less energy owing to inactivity, and may that various body functions deteriorate to some require higher micronutrient intakes because of an extent, and disease and illness become more common illness causing malabsorption by the gut. Certain as people age. Until more data are available, the nutrients may also be used as therapeutic agents, for assumption is made that, except for energy and a few example n-3 fatty acids can have anti-inflammatory nutrients, the requirements of the elderly (usually effects. These clinical aspects are considered elsewhere defined as those over 65 years old) are no different in these texts. from those of younger adults. One complication arising in the formulation of Bearing the above points in mind, dietary recom- dietary recommendations is caused by the fact that mendations can be useful at various levels. various groups of people within a population may have different nutrient requirements. Therefore, the ● Governments and nongovernment organizations population is divided into subgroups: children and (NGOs) use dietary recommendations to identify adults by age bands, and by gender. For women, allow- the energy and nutrient requirements of popula- ances are also made for pregnancy and lactation. tions and hence allow informed decisions on food policy. This could include the provision of food aid Infants are recommended to be fully breast-fed for or supplements (or rationing) when the diet is the first few months of life. This poses a problem for inadequate, fortification of foods, providing appro- the bodies setting the dietary recommendations, priate nutrition education, introducing legislation which have to set standards for those infants who are concerning the food supply, influencing the import not breast-fed. The dietary recommendations for and export of food, subsidies on certain foods or formula-fed infants are based on the energy and for producers of food, and so on. nutrients supplied in breast milk, but, because the bioavailability of some nutrients is lower in formula ● The food industry requires this information in the than in breast milk, the amounts stated appear higher development and marketing of products. The than those that might be expected to be achieved by industry is aware of consumers’ increasing interest breast-feeding. This should not therefore be inter- in the nutritional quality of the food that they buy, preted as an inadequacy on the part of human (breast)

Dietary Reference Standards 127 and has responded by providing foods to address eat to meet their own perceived needs. At the very particular perceived needs, and more informative least, consumers should be able to compare prod- food labels. ucts to get their money’s worth. ● Researchers and the health professions need to assess the nutritional adequacy of the diets of 7.4 The use of reference values to assess groups (or, cautiously, of individuals) by compar- the adequacy of the nutrient intakes of ing dietary intake survey data with the dietary refer- population groups ence values (see below). Once the limitations of the dietary assessment data have been taken into Ideally, this is accomplished by discovering the distri- account (see Chapter 10), this information can be bution of intakes of a nutrient in the population used to attempt to improve people’s nutrient intakes group (e.g., by carrying out a dietary survey), and by bringing them more into line with the dietary comparing these intakes with the distribution of recommendations. The formulation of dietary requirements for that nutrient within the same popu- advice or guidelines depends on an appreciation of lation. In practice, reliable data with which to plot the the existing situation: the solution can only be second of these distributions have rarely been col- framed once the problem is characterized. lected, and therefore what must be used is an estima- ● Institutions and caterers use dietary recommenda- tion of the average requirement together with an esti- tions to assess the requirements of groups and mation of the variance in that requirement, i.e., the devise nutritionally adequate menus. This is a great standard deviation (based on whatever scientific evi- deal more easily said than done, mainly because of dence is available), that is used to plot the population the financial constraints involved and, often, the distribution of requirements as shown in Figure 7.1. food preferences of the population being catered for. When considering how to assess the adequacy of ● The public needs this information to help in the nutrient intakes of populations it is important interpretation of nutrition information on food to compare the intakes with the most appropriate labels that may describe nutrient content in both level of requirement as defined in dietary absolute terms (g, mg, etc.) and as a percentage of recommendations. the recommended dietary allowance (RDA) for that nutrient (usually per 100 g or per “serving”). It is It is not useful to compare usual intakes with the thought that the latter is more meaningful to con- RDA (PRI, RNI, i.e., the average requirement plus a sumers, even though the concepts involved in notional 2 SDs) at the population level since this setting the dietary recommendations are rather approach leads to overestimates of the prevalence of complex (making it difficult to judge which level of inadequacy. (It may, however, be justified to compare recommendation should be used as the standard) an individual’s intake with the RDA.) Furthermore, and they can be open to misinterpretation (see this approach might be seen to encourage the con- above). Since 1998, some UK manufacturers and sumption of higher intakes, which could be toxic in retailers have provided information about guide- the case of certain nutrients. line daily amounts (GDAs) for energy, some nutri- ents, salt, and fiber. These were developed by the Comparison of the population intake with the Institute of Grocery Distribution (IGD, a UK average requirement [AR; estimated average require- research and training body for the food and grocery ment (EAR)] is now considered to be the best estima- chain) and are derived from the DRVs [and the tion of dietary adequacy; if the average intake is less British Committee on Medical Aspects of Food than the average requirement, then it is clear that Policy (COMA) and Scientific Advisory Council there could be a problem in that population. Accord- on Nutrition (SACN) recommendations for salt ingly, using the average requirement as a cut-off point, intake], but are much simplified. Unless consumers the proportion of individuals in the group whose are provided with nutrition information in the usual intakes are not meeting their requirements can most appropriate form on food labels, they cannot be calculated, allowing the problem to be quantified. make informed choices as to what foods to buy and However, this approach cannot be used in the case of energy since energy intakes and requirements are highly correlated (the effects of an imbalance being quickly obvious to the individual).

128 Introduction to Human Nutrition The lowest defined intake level [lowest threshold US EAR 60 and 75 mg/day for women and men, intake (LTI), lower reference nutrient intake (LRNI), respectively). Similarly, variations in calcium recom- i.e., the average requirement minus a notional 2 SDs] mendations exist because some committees choose to is not regarded as being useful in the context of assess- use zero calcium balance as the criterion of adequacy, ing the adequacy of population nutrient intakes. This while others use maximum skeletal calcium reserves. is because it would identify only those individuals who were almost certainly not meeting their require- In some cases, one recommending body will include ment, and by the same token would omit to include a nutrient among its dietary recommendations while many in the population who would be at appreciable others will not; for example, vitamin E, the require- risk of nutrient inadequacy (in other words, those ment for which depends directly on the dietary intake whose intake was below the average requirement). and tissue levels of PUFAs, which are highly skewed. The vitamin E requirement corresponding to the Finally, the tolerable upper levels of intake defined highest levels of PUFA intake would be much higher for certain nutrients can also be used as cut-off points than that needed by those with much lower (but ade- to identify those individuals at risk of consuming quate) intakes. To set the high value as the recom- toxic levels of a nutrient. mendation might suggest to those with lower polyunsaturate intakes that they should increase their 7.5 Methods used to determine intake of vitamin E (unnecessarily). Thus, in Britain requirements and set dietary and Europe, only “safe and adequate” intakes have recommendations been set, based on actual intakes in healthy popula- tions, which should be at least 3 mg/day for women In order to derive the most accurate and appropriate and 4 mg/day for men. In contrast, the US RDA (DRI) dietary recommendations, committees of experts are has been raised to 15 mg/day as α-tocopherol, based established that look at the scientific evidence and use on induced vitamin E deficiency studies in humans their judgment to decide which nutrients to consider and measures of lipid peroxidation. and then, for each nutrient, make decisions in respect of the: There are even some examples of dietary compo- nents that have not traditionally been regarded as ● criterion by which to define adequacy essential nutrients having recommendations set for ● estimation of the average amount required to meet them, as in the case of choline. The US DRI defines an adequate intake for choline (of 450 and 550 mg/ that criterion of adequacy day for women and men, respectively), on the basis ● estimated standard deviation of requirement in the that endogenous synthesis of this compound is not always adequate to meet the demand for it (for population under consideration (i.e., the shape of the synthesis of acetylcholine, phospholipids, and the frequency distribution over the range of require- betaine). Dietary intake data for choline and the sci- ments: broad, narrow, skewed, etc.). entific evidence for inadequacy are limited; thus, dose–response studies would need to be done before The problem of different committees identifying an average requirement could be derived. It is proba- different criteria of adequacy is illustrated by vitamin ble that further dietary components will be included C (ascorbic acid). Experimental evidence (the Shef- in dietary recommendations as research data accu- field and Iowa studies) has shown that an intake of mulate. Potential candidates include the flavonoids approximately 10 mg/day is required to prevent the and some other antioxidant compounds. deficiency disease scurvy in adult men. At intakes below 30 mg/day, serum levels are negligible, rising 7.6 Methods used to steeply with intakes of between 30 and 70 mg/day, determine requirements after which they begin to plateau (and urinary excre- tion of the unmetabolized vitamin increases). The Deprivation studies question facing the committees drafting dietary refer- ence values is whether to choose a level of intake that This is the most direct method and involves removing allows some storage of the vitamin in the body pool the nutrient from the diet, observing the symptoms (e.g., EU AR 30 mg/day for adults), or one that more nearly maximizes plasma and body pool levels (e.g.,

Dietary Reference Standards 129 of deficiency, and then adding back the nutrient until in the gut (generally in the case of those nutrients of the symptoms are cured or prevented. Difficulties which the uptake is regulated) or the rate of excretion with this approach are as follows. First, that the exper- in the urine (in the case of very soluble nutrients) or iment may need to continue for several years owing feces, or both. However, there comes a point beyond to the presence of body stores of the nutrient, and which balance cannot be maintained; therefore, it can often requires a very limited and therefore boring be proposed that the minimum intake of a nutrient dietary regimen. Second, unpredicted long-term at which balance can be maintained is the subject’s adverse consequences may result. Third, such experi- minimum required intake of that nutrient. However, ments are not ethical in vulnerable groups such as this approach would need to be extended over time children (often the most relevant for study). In some to investigate possible adaptive responses to reduced cases, epidemiological data may be available; for intakes, e.g., absorption could eventually be increased. example, the deficiency disease beriberi occurs in In the case of calcium, the European consensus is that populations whose average thiamin intake falls below average daily losses are assumed to be 160 mg/day in 0.2 mg/4.2 MJ (1000 kcal). adults, and absorption is assumed to be 30%; thus, around 530 mg would need to be consumed to balance Radioactive tracer studies the losses. Adding or subtracting 30% to allow for individual variation (the notional 2 SDs explained This approach makes use of a known amount of the above) gives (rounded) dietary reference values of radioactively labeled nutrient, which is assumed to 400, 550 and 700 mg/day (LTI, AR, and PRI, disperse evenly in the body pool, allowing the estima- respectively). tion of the total pool size by dilution of the isotope in samples of, for instance, plasma or urine (i.e., if the Factorial methods body pool is large, then the dilution will be greater than if the body pool is small). Specific activity, that These are predictions, rather than measurements, of is radioactivity per unit weight of the nutrient in the the requirements of groups or individuals, taking into samples, can be used to calculate pool size as long as account a number of measured variables (factors, the total dose administered is known. The rate of loss hence “factorial”) and making assumptions where can then be monitored by taking serial samples, allow- measurements cannot be made. For example, the ing calculation of the depletion rate. In the case of increased requirements during growth, pregnancy, or vitamin C, the average body pool size of a healthy lactation are calculated by this method; this approach male was found to be 1500 mg, which, on a vitamin is necessitated by the lack of experimental data in C-free diet, depleted at a rate of approximately 3% (of these physiological situations owing to ethical prob- the body pool) per day. This fractional catabolic rate lems. The idea is that the rate of accumulation of was independent of body pool size, and symptoms of nutrients can be calculated and hence the amount scurvy appeared when the body pool fell below required in the diet to allow that accumulation can be 300 mg. The estimated replacement intake needed to predicted. In the case of pregnancy, the requirement maintain the body pool above 300 mg was therefore is estimated to be the amount of the nutrient needed 3% of 300 mg, i.e., 9 mg (similar to the 10 mg found to achieve balance when not pregnant plus the amount to be needed to prevent scurvy in the earlier Sheffield accumulated daily during the pregnancy, all multi- experiment). plied by a factor accounting for the efficiency of absorption and assimilation (e.g., 30% for calcium). Balance studies For lactation, the calculation for energy is based on the amount in the milk secreted daily, which is These rely on the assumption that, in healthy increased by a factor accounting for the efficiency of individuals of stable body weight, the body pool of conversion from dietary energy to milk energy (reck- some nutrients (e.g., nitrogen, calcium, and sodium) oned to be 95%), from which total is subtracted an remains constant. Compensation mechanisms equal- allowance for the contribution from the extra fat ize the intake and output of the nutrient over a wide stores laid down during pregnancy, which it is desir- range of intakes, thereby maintaining the body pool. able to reduce in this way. The difficulty with this Thus, day-to-day variations of intake are compen- approach is that the theoretical predictions do not sated for by changes in either the rate of absorption

130 Introduction to Human Nutrition necessarily take account of physiological adaptations reductase depends on riboflavin and, when activity is (e.g., increased efficiency of absorption in the gut) measured in both the presence and absence of excess that may reduce the predicted requirement. This riboflavin, the ratio of the two activities (the erythro- would apply particularly in the case of pregnancy, as cyte glutathione reductase activation coefficient, shown by the ability of women to produce normal EGRAC) reflects riboflavin status: if perfectly suffi- babies even in times of food shortage. cient, the ratio would be 1.0, whereas deficiency gives values greater than 1.0. Measurement of nutrient levels in biological tissues Biological markers Some nutrient requirements can be defined according These are measures of some biological function that to the intakes needed to maintain a certain level of is directly dependent on the nutrient of interest; the nutrient in blood or tissue. For many water- again, not always easy to find, hence the recent sug- soluble nutrients, such as vitamin C, blood levels gestion that some functional indices be considered reflect recent dietary intake, and the vitamin is not that are not necessarily directly dependent on the generally measurable in plasma at intakes less than nutrient. Iron status is assessed according to a battery about 40 mg/day. This level of intake has therefore of biological markers, including plasma ferritin been chosen as the basis for the reference in some (which reflects body iron stores), serum transferrin countries such as the UK. This approach is not, saturation (the amount of plasma transferrin in rela- however, suitable for those nutrients of which the tion to the amount of iron transported by it is reduced plasma concentration is homeostatically regulated, in deficiency), plasma-soluble transferrin receptor such as calcium. In the case of the fat-soluble vitamin (an index of tissue iron status), and the more tradi- retinol, the dietary intake required to maintain a liver tional tests such as blood hemoglobin (now consid- concentration of 20 μg/g has been used as the basis of ered to be a rather insensitive and unreliable measure the reference intake. To do this, the body pool size of iron status since it indicates only frank anemia, and needed to be estimated; assumptions were made as to also changes as a normal response to altered physio- the proportion of body weight represented by the logical states such as pregnancy). liver (3%) and the proportion of the body pool of retinol contained in the liver (90%). The fractional Vitamin K status is assessed by measuring pro- catabolic rate has been measured as 0.5% of the body thrombin time (the length of time taken by plasma to pool per day, so this would be the amount needing to clot), which is increased when vitamin K levels fall be replaced daily. The efficiency of conversion of since the synthesis of prothrombin in the liver depends dietary vitamin A to stored retinol was taken to be on vitamin K as a cofactor. This test is clinically useful 50% (measured range 40–90%), giving an EAR of in patients requiring anticoagulant therapy (e.g., around 500 μg/day for a 74 kg man. using warfarin, which blocks the effect of vitamin K), in whom the drug dosage must be closely Biochemical markers monitored. In many respects, biochemical markers represent the Animal experiments most satisfactory measure of nutrient adequacy since they are specific to the nutrient in question, are sensi- These are of limited use in defining human nutrient tive enough to identify subclinical deficiencies, and requirements because of species differences (e.g., rats may be measured precisely and accurately. However, can synthesize vitamin C, so it is not a “vitamin” for such markers are available for only a few nutrients, them), differences in metabolic body size (i.e., the mostly vitamins, at present. One well-established proportions of metabolically active tissue, such as example of a biochemical marker is the erythrocyte muscle, and less active tissue, such as adipose tissue, glutathione reductase activation test for riboflavin gut contents), and differences in growth rates (young status. Erythrocytes are a useful cell to use for enzyme animals generally grow far more rapidly than humans, assays since they are easily obtainable and have a e.g., cattle reach adult size in about 1 year). However, known life-span in the circulation (average 120 days), animals have provided much of the information on aiding the interpretation of results. Glutathione the identification of the essential nutrients, and their physiological and biochemical functions. Furthermore,

Dietary Reference Standards 131 animals can be used in experiments that would not Food and Agriculture Organization/United Nations University/ be possible in humans, such as lifelong modifications World Health Organization. Energy and Protein Requirements. in nutrient intake; it is merely the setting of human Report of a Joint FAO/WHO/UNU Expert Consultation. requirements for which they are inappropriate. Technical Report Series 724. WHO, Geneva, 1985. 7.7 Perspectives on the future Food and Agriculture Organization/World Health Organization. Human Vitamin and Mineral Requirements. Report of a joint As the amount known about human requirements FAO/WHO expert consultation. Bangkok, Thailand. FAO, Rome, and nutrient functions increases, so too will the com- 2002. plexity of dietary recommendations. It is probable that further dietary components will be included in Institute of Medicine (USA). Dietary Reference Intakes for Calcium, dietary recommendations as research data accumu- Phosphorus, Magnesium, Vitamin D and Fluoride. National late. Potential candidates include the flavonoids and Academy Press, Washington, DC, 1997. some other antioxidant compounds. Furthermore, continuing research and the development of more Institute of Medicine (USA). Dietary Reference Intakes for Thiamin, informed interpretations of the expanding body of Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic data available necessitate the regular revision and Acid, Biotin and Choline. National Academy Press, Washington, updating of the recommendations. DC, 1998. The general conclusion that can be drawn here is Institute of Medicine (USA). Dietary Reference Intakes for Water, that no single criterion of nutrient status can be used Potassium, Sodium, Chloride and Sulfate. National Academy to define human requirements for all nutrients. This Press, Washington, DC, 1998. is not surprising when one considers the range of roles that the different essential nutrients play in Institute of Medicine (USA). Dietary Reference Intakes for Vitamin humans. A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. Further reading National Academy Press, Washington, DC, 2000. Department of Health. Dietary Reference Values for Food Energy and Institute of Medicine (USA). Dietary Reference Intakes for Vitamin Nutrients for the United Kingdom. Report on Health and Social C, Vitamin E, Selenium and Carotenoids. National Academy Subjects 41. Committee on Medical Aspects of Food Policy. Press, Washington, DC, 2000. HMSO, London, 1991. Institute of Medicine (USA). Dietary Reference Intakes for Energy, Department of Health. Nutrition and Bone Health: with Particular Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Reference to Calcium and Vitamin D. Report on Health and Amino Acids (Macronutrients). National Academy Press, Social Subjects 49. Committee on Medical Aspects of Food and Washington, DC, 2005. Nutrition Policy. The Stationery Office, London, 1998. Institute of Medicine (USA). Dietary Reference Intakes: The Essential Dietary reference intake texts available online at: http://lab. Guide to Nutrient Requirements. National Academy Press, nap.edu/nap-cgi/discover.cgi?term=dietary%20reference% Washington, DC, 2006. 20intakes&restric=NAP. National Health and Medical Research Council. Nutrient Reference EC Scientific Committee for Food Report. Nutrient and Energy Values for Australia and New Zealand Including Recommended Intakes for the European Community. 31st Series. Director Dietary Intakes. Wickliffe Ltd, Wellington, 2006. Available online General, Industry, Luxembourg, 1993. at http://www.nhmrc.gov.au/publications/synopses/_files/n35. pdf. Expert Group on Vitamins and Minerals. Safe Upper Limits for Vitamins and Minerals. Food Standards Agency, London, National Research Council, Food and Nutrition Board, Commission 2003. on Life Sciences. Recommended Dietary Allowances, 10th edn. National Academy Press, Washington, DC, 1989. Food and Agriculture Organization/World Health Organization. Requirements for Vitamin A, Iron, Folate and Vitamin B12. Report United Nations University. International harmonisation of of a Joint FAO/WHO Expert Consultation. Food and Nutrition approaches for developing nutrient-based dietary standards. In: Series. FAO, Rome, 1988. King JC, Garza, C, eds. Food and Nutrition Bulletin, vol. 28, no. 1 (supplement). International Nutrition Foundation for The United Nations University, Tokyo, 2007. Available online at http://www.unu.edu/unupress/food/FNBv28n1_Suppl1_final. pdf. World Health Organization. Handbook on Human Nutritional Requirements. Monograph Series No. 61. WHO, Geneva, 1974. World Health Organization. Diet, Nutrition and the Prevention of Chronic Diseases. Technical Report Series 797. WHO, Geneva, 1990. World Health Organization. Trace Elements in Human Nutrition and Health. WHO in collaboration with FAO, AEA, Geneva, 1996.

8 The Vitamins David A Bender Key messages • Where relevant, this chapter will deal with each of the vitamins under the following headings: • The vitamins are a chemically disparate group of compounds with • vitamers a variety of functions in the body. • absorption and metabolism • metabolic functions and other uses • What they have in common is that they are organic compounds • deficiency that are required for the maintenance of normal health and meta- • requirements bolic integrity. • assessment of status • toxicity and drug interactions. • Vitamins are required in very small amounts, of the order of mil- ligrams or micrograms per day, and thus can be distinguished from the essential fatty acids and the essential amino acids, which are required in larger amounts of grams per day. 8.1 Introduction Two factors were found to be essential: one was found in the cream and the other in the watery part of milk. In order to demonstrate that a compound is a vitamin, Logically, they were called factor A (fat-soluble, in the it is necessary to demonstrate both that deprivation cream) and factor B (water-soluble, in the watery part of experimental subjects will lead to the development of the milk). Factor B was identified chemically as an of a more or less specific clinical deficiency disease amine, and in 1913 the name “vitamin” was coined and abnormal metabolic signs, and that restoration for these “vital amines.” of the missing compound will prevent or cure the deficiency disease and normalize metabolic abnor- Further studies showed that “vitamin B” was a malities. It is not enough simply to demonstrate that mixture of a number of compounds, with different a compound has a function in the body, since it may actions in the body, and so they were given numbers as normally be synthesized in adequate amounts to meet well: vitamin B1, vitamin B2, and so on. There are gaps requirements, or that a compound cures a disease, in the numerical order of the B vitamins. When what since this may simply reflect a pharmacological action might have been called vitamin B3 was discovered, it and not indicate that the compound is a dietary was found to be a chemical compound that was already essential. known, nicotinic acid. It was therefore not given a number. Other gaps are because compounds that were The vitamins, and their principal functions and assumed to be vitamins and were given numbers, such deficiency signs, are shown in Table 8.1; the curious as B4 and B5, were later shown either not to be vitamins, nomenclature is a consequence of the way in which or to be vitamins that had already been described by they were discovered at the beginning of the twentieth other workers and given other names. century. Early studies showed that there was some- thing in milk that was essential, in very small amounts, Vitamins C, D and E were named in the order of for the growth of animals fed on a diet consisting of their discovery. The name “vitamin F” was used at one purified fat, carbohydrate, protein, and mineral salts. time for what we now call the essential fatty acids; “vitamin G” was later found to be what was already © 2009 DA Bender.

The Vitamins 133 Table 8.1 The vitamins, their principal functions and deficiency diseases Vitamin Functions Deficiency disease A Retinol Visual pigments in the retina; cell differentiation; Night blindness, xerophthalmia; keratinization of skin D β-Carotene β-carotene is an antioxidant Calciferol Rickets (poor mineralization of bone); osteomalacia E Maintenance of calcium balance; enhances (demineralization of bone) K Tocopherols intestinal absorption of Ca2+ and mobilizes B1 Tocotrienols bone mineral Extremely rare: serious neurological dysfunction Phylloquinone B2 Menaquinones Antioxidant, especially in cell membranes Impaired blood clotting, hemorrhagic disease Niacin Thiamin B6 Coenzyme in formation of γ-carboxyglutamate in Peripheral nerve damage (beriberi) or central nervous Riboflavin enzymes of blood clotting and bone matrix system lesions (Wernicke–Korsakoff syndrome) Folic acid B12 Nicotinic acid Coenzyme in pyruvate and 2-keto-glutarate Lesions of corner of mouth, lips, and tongue; Nicotinamide dehydrogenases, and transketolase; poorly seborrheic dermatitis H Pyridoxine defined function in nerve conduction C Pyridoxal Pellagra: photosensitive dermatitis, depressive Pyridoxamine Coenzyme in oxidation and reduction reactions; psychosis prosthetic group of flavoproteins Cobalamin Disorders of amino acid metabolism, convulsions Coenzyme in oxidation and reduction reactions, Pantothenic acid functional part of NAD and NADP Megaloblastic anemia Pernicious anemia (megaloblastic anemia with Biotin Coenzyme in transamination and decarboxylation of amino acids and glycogen phosphorylase; degeneration of the spinal cord) Ascorbic acid role in steroid hormone action Peripheral nerve damage (burning foot syndrome) Coenzyme in transfer of one-carbon fragments Impaired fat and carbohydrate metabolism, dermatitis Coenzyme in transfer of one-carbon fragments Scurvy: impaired wound healing, loss of dental and metabolism of folic acid cement, subcutaneous hemorrhage Functional part of coenzyme A and acyl carrier protein Coenzyme in carboxylation reactions in gluconeogenesis and fatty acid synthesis Coenzyme in hydroxylation of proline and lysine in collagen synthesis; antioxidant; enhances absorption of iron known as vitamin B2. Biotin is still sometimes called in adequate amounts to meet requirements. These vitamin H. Vitamin K was discovered by Henrik Dam, include carnitine, choline, inositol, taurine, and in Denmark, as a result of studies of disorders of ubiquinone. blood coagulation, and he named it for its function: koagulation in Danish. Two compounds that are generally considered to be vitamins can be synthesized in the body, normally As the chemistry of the vitamins was elucidated, so in adequate amounts to meet requirements: vitamin they were given names as well, as shown in Table 8.1. D, which is synthesized from 7-dehydrocholesterol in When only one chemical compound has the biologi- the skin on exposure to sunlight, and niacin, which is cal activity of the vitamin, this is quite easy. Thus, synthesized from the essential amino acid tryptophan. vitamin B1 is thiamin, vitamin B2 is riboflavin, etc. However, both were discovered as a result of studies With several of the vitamins, a number of chemically of deficiency diseases that were, during the early related compounds found in foods can be intercon- twentieth century, significant public health problems: verted in the body, and all show the same biological rickets (due to vitamin D deficiency and inadequate activity. Such chemically related compounds are sunlight exposure) and pellagra (due to deficiency of called vitamers, and a general name (a generic descrip- both tryptophan and preformed niacin). tor) is used to include all compounds that display the same biological activity. 8.2 Vitamin A Some compounds have important metabolic func- Vitamin A was the first vitamin to be discovered, ini- tions, but are not considered to be vitamins, since, as tially as an essential dietary factor for growth. It has far as is known, they can be synthesized in the body

134 Introduction to Human Nutrition a role in vision, as the prosthetic group of the light- acid (preformed vitamin A); and a variety of caro- sensitive proteins in the retina, and a major role in the tenes and related compounds (collectively known as regulation of gene expression and tissue differentia- carotenoids) that can be cleaved oxidatively to yield tion. Deficiency is a major public health problem in retinaldehyde, and hence retinol and retinoic acid. large areas of the world, and prevention of vitamin A Those carotenoids that can be cleaved to yield retinal- deficiency is one of the three micronutrient priorities dehyde are known as provitamin A carotenoids. of the World Health Organization (WHO) (the other two are iron and iodine). Preformed vitamin A (mainly as retinyl esters) is found only in foods of animal origin. The richest Vitamers and international units source by far is liver, which may contain sufficient vitamin A to pose a potential problem for pregnant Two groups of compounds, shown in Figure 8.1, have women, since retinol is teratogenic in excess. Caro- vitamin A activity: retinol, retinaldehyde, and retinoic tenes are found in green, yellow, and red fruits and H3C CH3 CH3 CH3 CH2OH CH3 Retinol H3C CH3 CH3 CH3 H CO CH3 Retinaldehyde H3C CH3 CH3 CH3 H3C CH3 CH3 COO- CH3 all-trans-Retinoic acid CH3 9-cis-Retinoic acid H3C COO- H3C CH3 CH3 CH3 H3C CH3 CH3 CH3 H3C CH3 α-Carotene H3C CH3 CH3 CH3 H3C CH3 CH3 H3C CH3 CH3 β-Carotene Figure 8.1 The major vitamin A vitamers and vitamin A active carotenoids.

The Vitamins 135 vegetables, as well as in liver, margarine, and milk and absorbed, and even at high levels of intake this falls milk products. In addition to their role as precursors only slightly. However, in people with a very low fat of vitamin A, carotenoids have potentially useful anti- intake (less than about 10% of energy from fat), oxidant action, and there is epidemiological evidence absorption of both retinol and carotene is impaired, that diets that are rich in carotenoids (both those that and low-fat diets are associated with vitamin A are vitamin A active and those that are not) are associ- deficiency. ated with a lower incidence of cancer and cardio- vascular disease. However, intervention studies with Dietary retinyl esters are hydrolyzed by lipases in β-carotene have been disappointing, and it is not pos- the intestinal lumen and mucosal brush border mem- sible to determine desirable intakes of carotene other brane, then re-esterified to form retinyl palmitate than as a precursor of vitamin A. before release into the circulation in chylomicrons. Retinoic acid is a metabolite of retinol; it has Tissues can take up retinyl esters from chylomi- important biological activities in its own right and crons, but most retinol is in the chylomicron rem- will support growth in vitamin A-deficient animals. nants that are taken up by the liver. Here retinyl esters The oxidation of retinaldehyde to retinoic acid is irre- are hydrolyzed, and the vitamin may either be secreted versible. Retinoic acid cannot be converted in vivo to from the liver bound to retinol binding protein, or be retinol, and does not support either vision or fertility transferred to stellate cells in the liver, where it is in deficient animals. stored as retinyl esters in intracellular lipid droplets. Some 50–80% of the total body content of retinol is Some 50 or more dietary carotenoids are potential in the stellate cells of the liver, but a significant amount sources of vitamin A: α-, β-, and γ-carotenes and may also be stored in adipose tissue. cryptoxanthin are quantitatively the most important. Although it would appear from its structure that one The main pathway for catabolism of retinol is molecule of β-carotene will yield two of retinol, oxidation to retinoic acid (which, as discussed this is not so in practice. Nutritionally, 6–12 μg of β- below, has important biological activities in its carotene is equivalent to 1 μg of preformed retinol. own right, distinct from the activities of retinol). For other carotenes with vitamin A activity, 12–24 μg The main excretory product of both retinol and is equivalent to 1 μg of preformed retinol. retinoic acid is retinoyl glucuronide, which is secreted in the bile. Conventionally, the total amount of vitamin A in foods is expressed as μg retinol equivalents, calculated As the intake of retinol increases, and the liver con- from the sum of μg of preformed vitamin A + 1/6 centration rises above 70 μmol/kg, a different pathway × μg β-carotene + 1/12 × μg other provitamin A becomes increasingly important for the catabolism of carotenoids. Recent studies on the absorption of caro- retinol in the liver. This is a microsomal cytochrome tenes and their bioefficacy as vitamin A precursors P450-dependent oxidation, leading to a number of have led to the definition of retinol activity equiva- polar metabolites that are excreted in the urine and lents. 1 μg retinol activity equivalent = 1 μg preformed bile. At high intakes this pathway becomes saturated, retinol, 12 μg β-carotene or 24 μg other provitamin and excess retinol is toxic since there is no further A carotenoids. capacity for its catabolism and excretion. Before pure vitamin A was available for chemical Carotene dioxygenase analysis, the vitamin A content of foods was deter- Like retinol, carotenoids are absorbed dissolved in mined by biological assays and the results were lipid micelles. The biological availability and absorp- expressed in standardized international units (IU): tion of dietary carotene varies between 5% and 60%, 1 IU = 0.3 μg of retinol, or 1 μg of retinol = 3.33 IU. depending on the nature of the food, whether it is Although obsolete, IU are sometimes still used in cooked or raw, and the amount of fat in the meal. food labeling. As shown in Figure 8.2, β-carotene and other pro- Metabolism and storage of vitamin A and vitamin A carotenoids are cleaved in the intestinal pro-vitamin A carotenoids mucosa by carotene dioxygenase, yielding retinalde- hyde, which is reduced to retinol, then esterified and Retinol is absorbed from the small intestine dissolved secreted in chylomicrons together with retinyl esters in lipid. About 70–90% of dietary retinol is normally formed from dietary retinol.