CHAPTER CONTENTS

Some definitions 97

Types of natural lipids and their functions 98 Structural lipids 99 Storage lipids 101 Metabolic lipids 102

Fats in foods 102 Food fats from adipose tissue 102 Milk fat 102 Eggs 103 Fish oils 103 Muscle meats 103 Vegetable oils 104 Plant leaves 104

Modification of fats during food processing 104

Determination of food fat content and composition 105 Extraction of fat 106 Determination of individual fat components 106 Measurement of energy value 106

Consumption of fats in diets 106

Basics of fat metabolism 106 Digestion 106 Absorption 107 Defects in fat digestion and absorption 107 Lipid transport 109 Fatty acid metabolism 112

Nutritional roles for fatty acids and lipids 114 Essential fatty acids 114 Fatty acids in growth and development 117 Role as energy source: storage and utilization 118

Summary 119

Fats M. I. Gurr

SOME DEFINITIONS Fats are important components of all diets for the reasons illustrated in Table 7.1.

This chapter is about substances, in the body and in food, that do not dissolve in water. Biochemists give the name 'lipids' to substances that are insoluble or poorly soluble in water but soluble in organic solvents. When applied to lipids in foods or in the body, this physical definition encompasses many widely different chemical structures (Gunstone et al 1994, Gurr 1992, Gurr & Harwood 1991). Of the different lipids whose structures are illustrated in Figure 7.1, the triacylglycerols are quan-titatively the most important, contributing more than 90% of food lipids. These are what we refer to when we speak about dietary fats. Familiar everyday examples are butter and other spreads, cooking oils and the fatty parts of meats. As we shall learn, however, fats are rarely present as pure triacylglycerols but almost always have other fat-soluble substances (for example fat-soluble vitamins, Ch. 13) dissolved in them or are present as emulsions with water.

In this chapter we will reserve the word 'fats' for the fatty components of foods and diets and use the word 'lipid' when discussing the metabolism of fats in the body. We think of fats as solid in texture as distinct from oils which are liquid at ambient temperature, although chemically there is no distinction between fats and oils; both are triacylglycerols. Edible oils should not be con-fused with the oils used in the petroleum industry, which are hydrocarbons.

The fatty nature of triacylglycerols is due to their fatty

Table 7.1 Roles of dietary fats

• Provision of metabolic energy, 38 kj /g (9 kcal/g) • Supply of essential nutrients

—Fat-soluble vitamins —Essential fatty acids

• Fats often improve flavour perception and imparl a pleasing texture to foods, thereby improving palatability

97

98 NUTRITIONAL SCIENCE

0 HJC.O.C.R. 1

R 2 . C . O — C — H

O I II

H 2 C . O . C . R 3

(a) Triacylglycerols

o O —CH2

I C H O C O R '

C H 2 O C O R 2

(c) Galactosyldiacylglycerols

C H ,

/

0 CHJ .O .C .R 1

R 2 . C . O — C — H O

CH2.0 — P — O — CH CH N(CH )

O-

\ C H ,

(b) Phosphatidyl choline (d) Cholesterol

Fig. 7.1 Structures of some important food and body lipids, (a) Triacylglycerols (sometimes called triglycerides) are the major storage lipids. R represents the hydrocarbon chain of the fatty acids. (b) Phosphoglycerides: a subclass of a broader group of lipids that contain phosphorus, generally called phospholipids. R represents the hydrocarbon chain of the fatty acids. Several low-molecular-weight bases can be esterified with the phosphate; the commonest is choline (OH.CH2.CH2.N+(CH3)3), in which case the phospholipid is phosphatidylcholine, sometimes called by its trivial name, lecithin. Other bases present in naturally occurring phospholipids are ethanolamine, serine, inositol, and glycerol; the positions occupied by fatty acids are numbered 1 and 2; the phophate is at position 3. When the hydroxyl at position 2 is free and not esterified with a fatty acid, the compound is known as a lysophospholipid (e.g. lysophosphatidylcholine). (c) Galactosyldiacylglycerols: here a sugar replaces the phosphate ester. R again represents fatty acid hydrocarbon chains. G may be hydrogen, in which case the lipid is monogalactosyldiacylglycerol, or another galactose molecule, in which case the lipid is digalactosyldiacylglycerol. (d) Cholesterol: this is the main sterol of animal tissues and is present as the free alcohol (in which group R is a hydrogen atom), or as cholesteryl esters (in which group R is a fatty acid).

acid components (Table 7.2, Fig. 7.2). The melting points of fats (and their degree of 'hardness') increase with the chain lengths of the constituent fatty acids and their degree of saturation. The geometry of the double bond (icis or trans; Fig. 7.2) also influences physical properties: trans isomers have higher melting points than the corre-sponding cis isomers. Thus the 'hard' fats contain a rela-tively high proportion of the long-chain saturated fatty acids (e.g. lard) or saturated and frans-monounsaturated acids (e.g. hard margarines), while oils, such as sunflower seed oil, contain a large proportion of highly unsaturated fatty acids. In nutrition and dietetics, a distinction is also often made between 'visible' and 'invisible' fats. Visible fats are those clearly apparent to the consumer: spreads, cooking oils and fat on meats. In contrast, much fat in foods is hidden, having been incorporated into the food during preparation or cooking. Examples are the fat in cakes, biscuits, potato crips or processed meats, or in emulsions like mayonnaise. These fats, like the visible fats, are mainly triacylglycerols, although hidden fats may also be present in the membranes of plant and animal

tissues and these are mainly the phospholipids, glyco-lipids and cholesterol. Hence the total fat in the diet is hard to measure, because different samples of the same foods may vary widely in fat content as well as fat type. Only approximate figures for fat content are usually provided by food tables or nutrition labels.

TYPES OF NATURAL LIPIDS AND THEIR FUNCTIONS Lipid molecules are important constituents of all living cells. It is convenient to divide them into three cate-gories: structural, storage and metabolic, although there is considerable overlap between these types.

The pioneering work on the chemistry of the natural fats was done by the Liverpool chemist, T.P. Hilditch. His classic book The chemical composition of natural fats (Hilditch & Williams 1964) makes fascinating reading, although the painstaking chemical techniques that he had to use have now been supplanted by modern chromatographic methods.

FATS 99

Table 7.2 Some important fatty acids in foods

Common name

Chemical name

Shorthand nomenclature

Saturated Short-chain Butyric Butanoic 4:0 Caproic Hexanoic 6:0 Medium-chain Caprylic Octanoic 8:0 Capric Decanoic 10:0 Long-chain Laurie Dodecanoic 12:0 Myristic Tetradecanoic 14:0 Palmitic Hexadecanoic 16:0 Stearic Octadecanoic 18:0

Monounsaturated Oleic c/s-9-Octadecenoic 18:1 (n-9) Elaidic frans-9-Octadecenoic 18:1 f Erucic cis-13-Docosenoic 22:1 (n-9)

Polyunsaturated Linoleic cis,cis- 9,12-

Octadecadienoic 18:2 (n-6) a-Linolenic all-c/s-9,12,15-

Octadecatrienoic 18:3 (n-3) Arachidonic all-c/s-5,8,11,14-

Eicosatetraenoic 20:4 (n-6) EPA all-c/s-5,8,11,14,17-

Eicosapentaenoic 20:5 (n-3) DHA all-c/s-4,7,10,13,16,19-

Docosahexaenoic 22:6 (n-3)

See the caption to Figure 7.2 for an explanation of the notation used here.

Structural lipids These contribute to cellular architecture, mainly as con-stituents of cell membranes. In animal membranes, the phosphoglycerides (Fig. 7.1b) are the major lipids, whereas in plant cells the glycosylglycerides predominate (Fig. 7.1c). Phosphoglycerides are mixed acid esters of glycerol with two fatty acids and one phosphoric acid moiety. The more general term 'phospholipid' will suffice to describe these components, since phospholipids other than phospho-glycerides are of minor importance in nutrition and metabolism. Students should consult specialist lipid text books such as Gurr & Harwood (1991) or textbooks of general biochemistry (e.g. Zubay 1993) for further details of lipid chemistry and biochemistry.

Phospholipids are called 'polar lipids' or, more techni-cally 'amphiphilic' lipids (from the Greek: 'liking both', because they possess chemical groups that associate with water (hydrophilic groups) and those that are lipid soluble (hydrophobic groups). For example, the most abundant animal phospholipid, phosphatidylcholine (Fig. 7.1b) contains a polar moiety consisting of the negatively charged phosphate group and the positively charged choline group. The fatty acids are responsible for the fat-like properties.

Fatty acids consist of hydrocarbon chains with a

Saturated 16 14 12 *10 6 4 2

V W W V A A COOH Palmitic acid Hexadecanoic acid

Monounsaturated

1

8 6 4 2

AA/\A=AAAAc 18 16 14 12 10 9 1

COOH 18 16 14 12 10 9 Oleic acid (n-9 family) cis-9-octadecenoic acid (c-9-18:1 or 18:1, n-9)

18 16 14 12 10 6 4 2

\ A A / V ^ / V \ A COOH 1

Elaidic acid trans-9-octadecenoic acid (t-9-18:1)

Polyunsaturated 18 16 14 18 16 14 11 8 6 4 2

W W V W \ , 13 12 10 9 1

COOH

Linoleic acid (n-6 family) cis,cis-9,12-octadecadienoic acid (c,c-9,12-18:2 or 18:2,n-6)

17 14 11

/ ^ A A A M A C O O H 18 16 15 13 12 10 9 1

a-Linolenic acid (n-3 family) all-cis-9,12,15-octadecatrienoic acid (c,c,c-9,12,15-18:3 or 18:3, n-3)

COOH

Arachidonic acid (n-6 family) all-cis-5,8,11,14-eicosatetraenoic acid (c,c,c,c-5,8,11,14-20:4

or 20:4, n-6)

Fig. 7.2 Some fatty acids occurring in food and body lipids. The zig-zag lines represent hydrocarbon chains, with a carbon atom at the intersection of lines; thus / \ y % / represents CH2-CH2-CH2-CH=CH-CH2 etc. According to a biochemical convention, the numbering of the carbon chain is from the carboxyl group (-COOH). Thus a substituent such as a methyl group on the 4th carbon from the carboxyl group of a 16-carbon saturated fatty acid would be 4-methyl-hexadecanoic acid, etc. If a double bond occurs between carbon atoms 9 and 10 of an 18-carbon acid, the fatty acid is called 9-octadecenoic acid. The nomenclature n-3, n-6, n-9 describes families of fatty acids in which the last double bond in the chain is 3, 6 or 9 carbon atoms from the methyl end of the molecule. The suffixes c and t below double bonds in the formulae denote the geometrical configuration of the double bond, cis or trans. The terms 'monounsaturated' and 'monoenoic' etc. are interchangeable. In subsequent figures and tables in this chapter, a common shorthand notation for fatty acids will be used. In this system, the fatty acid is identified by a number denoting the number of carbon atoms in its hydrocarbon chain, followed by a colon, followed by a number denoting the number of double bonds. Thus, stearic acid (18 carbon atoms with no double bonds) is represented as 18:0 and octadecenoic acid (18 carbon atoms with one double bond in an unspecified position and with an unspecified geometrical configuration) as 18:1, etc. If the unsaturated acid needs to be identified more precisely, it can be done by indicating the position(s) and configuration(s) of the double bonds, thus: oleic acid, c9-18:1; linoleic acid, c9,c12-18:2, etc. In some cases it is useful to specify the family to which the fatty acid belongs; thus, 18:3 may be either 18:3n-3 (a-linolenic acid, all-c/s-9,12,15-18:3) or 18:3n-6 (y-linolenic acid, all-c/s-6,9,12-18:3).

100 NUTRITIONAL SCIENCE

terminal carboxyl group and it is the latter that forms the ester linkage with glycerol hydroxyl groups in the phos-phoglycerides and triacylglycerols. Naturally occurring fatty acids normally have even-numbered carbon chains ranging from four to 22 carbon atoms in length, although chains with an odd number of carbon atoms do occur and it is possible to find chain lengths longer than 22. Sometimes the chains are branched or contain various constituents such as hydroxyl groups. Despite the bewildering variety of fatty acids found in nature, those of quantitative significance in human nutrition are limited in number (Table 7.2). The carbon chains may be completely saturated or contain one (monounsaturated) or several (polyunsaturated) ethylenic double bonds (Fig. 7.2).

Membranes provide barriers between the cell and its environment, or between different cell compartments. In biological membranes, the phospholipid molecules associate to form a bilayer throughout the membrane, with fatty acid chains pointing inwards towards each other and the polar headgroups on the surfaces (Gurr & Harwood 1991). In nervous tissue and in most plant membranes, glycolipids (those in which the polar moiety is a sugar rather than a phosphodiester) are important constituents of the bilayer. Protein molecules, which may have a mainly structural function or serve as enzymes or receptors, are inserted into the phospholipid bilayer,

interacting by non-covalent forces with both the polar and non-polar regions of the lipids (Fig. 7.3). They may be located at external or internal faces or project through from one side to the other. Lipid molecules are quite mobile along the plane of the membrane and there may be limited movement across it. Indeed the patterns of lipid molecules on each side of some membranes are quite different, a phenomenon known as 'membrane asymmetry' (Gurr & Harwood 1991). The fatty acid chains are in constant motion and the degree of molecular motion within the membrane (often referred to as 'fluidity') is influenced by the nature of the fatty acid chains, inter-actions between fatty acid chains, and interactions be-tween proteins and lipids. The degree of fluidity may be crucial to certain biological functions of the membrane.

The chemistry of individual fatty acids influences their shape (Fig. 7.4), which in turn determines the space they occupy in the bilayer. Straight-chain saturated fatty acids can pack together in an almost crystalline array and molecular motion tends to be minimized compared with unsaturated or branched acids, which occupy more space (Fig. 7.3) and are more mobile. A relatively high proportion of unsaturated fatty acids, and especially polyunsaturated fatty acids, seems to be an essential requisite for the proper functioning of cell membranes. Dietary fat can influence membrane composition but only within certain limits. Storage fats, by contrast, are

Region of saturated close packed chains

Lipid Bilayer

Phospholipid polar groups

. Surface proteins Transmembrane

protein

Potential pore

Cholesterol

Receptor

Surface glycoprotein with carbohydrate groups

Region of unsaturated mobi le chains

Fig. 7.3 Lipids in biological membranes. A schematic representation of what a biological membrane might look like. The small spheres are the polar head groups of phospholipid molecules which form a double layer (lipid bilayer) throughout the membrane, with fatty acid chains (represented by the tails) pointing inwards towards each other. Cholesterol and vitamin E are inserted between the fatty acid chains. Protein molecules (represented by large stippled shapes) are inserted at intervals into the bilayer, sometimes mainly at one face or the other, sometimes extending all the way through. Sugar groups are attached to the surface proteins.

Positions of w w rosmons or ^ ^ # % the double

bonds 0Y

i W i '

)

& Monounsaturated mmm

FATS 101

Fig. 7.4 Space-filling molecular models indicating the shapes of fatty acids.

far more variable in composition (see Table 7.3). Foods containing membranes are, therefore, important sources of certain polyunsaturated fatty acids in diets.

Cholesterol plays an important role in stabilizing the hydrophobic interactions within animal membranes by inserting itself between the fatty chains in the bilayer (Fig. 7.3). Animals consuming diets relatively rich in polyunsaturated fatty acids tend to accumulate a higher proportion of these acids in the membrane bilayer. Under these circumstances the proportion of cholesterol to phospholipid increases, maintaining the fluidity of the membrane constant. For reasons that are incompletely understood, cholesterol seems to be the only sterol that will allow the proper functioning of animal membranes. Plant membranes in contrast, have little or no cholesterol and contain mainly beta-sitosterol. Dietary cholesterol, therefore, comes almost entirely from animal foods.

Storage lipids These provide a long-term reserve of metabolic fuel.

Triacylglycerols (Fig. 7.1a) are by far the most important storage lipids. (In older biochemical literature and in current medical publications and material for the general reader, you will normally find 'triglyceride' being used. The term 'triacylglycerol' is the officially approved nomenclature as well as being more descriptive of the structure and will be used throughout this chapter. It is entirely synonymous with triglyceride.)

Storage fats tend to contain a higher proportion of saturated and monounsaturated and a lower proportion of polyunsaturated fatty acids than structural fats. How-ever, this is only a general guideline, since the compo-sition of storage lipids is influenced strongly by the fatty acid composition of the diet in simple-stomached animals or by the activities of the rumen micro-organisms in ruminants.

In man and animals that provide man's food, the biggest reservoir of fatty acids for supplying long-term needs for energy is the adipose tissue. In Western coun-tries, where the amount of fat in diets is generally high, human adipose tissue normally stores fatty acids derived from the diet rather than from fat synthesized in the body from carbohydrates. Other tissues, such as the liver of mammals, can accommodate some fat but only in the short term. The excessive accumulation of fat in mamma-lian liver is a pathological condition. Many species of fish that contribute to the human diet, however, normally store fat in the liver or flesh rather than in adipose tissue (Wiseman 1984).

Milk fat is also a form of energy store, for the benefit of the newborn, and like adipose tissue fat, it is mainly composed of triacylglycerols (Gunstone et al 1994). The mammary gland is unique in making short- and medium-chain fatty acids (chain length up to 10 or 12 carbon atoms) whereas all other tissues produce longer-chain fatty acids (mainly 16 and 18 carbon atoms). Finally, egg yolk lipids provide a store of fuel for the developing embryo.

Table 7.3 Fatty acid composition of some animal storage fats important in foods. (Values are g/100 g total fatty acids)

Food fat 4:0-12:0 14:0 16:0 16:1 18:0 18:1 18:2 20:1 LC Others 22:1 PUFA

Lard (1) 0 1 29 3 15 43 9 0 0 0 Lard (2) 0 1 21 3 12 46 16 0 0 1 Poultry 0 1 27 9 7 45 11 0 0 1 Beef suet 0 3 26 9 8 45 2 0 0 7 Lamb 0 3 21 4 20 41 5 0 0 6 Milk (cow) 13 12 26 3 21 29 2 0 0 4 Milk (goat) 21 11 27 3 10 26 2 0 0 0 Egg yolk 0 0 29 4 9 43 11 0 0 4 Cod-liver oil 0 6 13 13 3 20 2 18 20 5

LC PUFA, long-chain polyunsaturated fatty acids. The column 'Others' comprises the total of components not specifically identified. This fraction tends to be longer in fats of ruminant origin, which contain many branched-chain and other unusual fatty acids which are not individually identified. Lard (1) is the fat obtained from pigs given low-fat cereal-based diets, whereas Lard (2) is fat from pigs given a high-fat diet containing soyabean oil. Source Gurr (1992).

102 NUTRITIONAL SCIENCE

Metabolic lipids This term is used here to signify those lipids that, as indi-vidual molecules, undergo metabolic transformations to produce specific substances of physiological and nutri-tional significance. These molecules are derived from the bulk lipids stored in adipose tissue or in membranes. They are mobilized from those stores when needed to perform a metabolic function at a site remote from the store. Each aspect is summarized here and described in greater detail later.

• Energy. Fatty acids make a major contribution to the production of cellular energy but must first be released from storage fats and directed into metabolic pathways in the mitochondria of cells to generate usable chemical energy.

• Metabolic regulation. Specific types of polyunsaturated fatty acids that are stored in membrane phospholipids can be released on demand and converted into hormone-like substances called 'eicosanoids'. A new area of research is based on the finding that dietary fatty acids themselves can influence genes. The investigation of the regulation by fatty acids of gene transcription and expression for proteins involved in lipid metabolism has included fatty acid synthetase and desaturase (Clarke & Jump 1993), HMG-CoA reductase, low density lipoprotein receptor and lipoprotein lipase (Salter & White 1996). (See also Ch. 15.)

• Steroid hormones. Cholesterol is metabolized in the adrenal gland to a variety of steroid hormones and in the liver to bile acids. Conversion of cholesterol in the skin to 7-dehydrocholesterol is the beginning of a series of transformations leading to vitamin D (Ch. 13).

FATS IN FOODS More detailed descriptions can be found in Wiseman (1984), Chow (1992) and Gunstone et al (1994).

Food fats from adipose tissue These include visible fats attached to meats and processed cooking fats such as lard or suet. Although predomi-nantly composed of triacylglycerols, the fat also contains some cholesterol and fat-soluble vitamins, and may con-tain small traces of adventitious fat-soluble substances such as pesticides and antioxidants added to the animal feed.

Although palmitic and oleic acids are likely to be the major components whatever the source of the fat, the proportions of the different fatty acids are markedly affected by the diet of single-stomached animals, of which pigs and poultry are economically the most important. Thus, the inclusion of vegetable oils in pig and poultry

diets results in a higher proportion of linoleic acid and a lower proportion of palmitic and oleic acids than the inclusion of tallow, which tends to produce a body fat similar to that of animals given cereals alone (Table 7.3). Giving supplements that contain high proportions of lipids with unsaturated fatty acids, or including appre-ciable amounts of copper in pig diets, results in a soft backfat (Wiseman 1984). This may be useful to encourage a higher polyunsaturated to saturated fatty acid (P/S) ratio in the human diet but may be counterproductive if the purchaser is averse to the texture of the fat as some surveys show.

Ruminant adipose tissue fat (mainly beef and lamb fat in the UK) is less variable than that of pigs and poultry because 90% of the unsaturated fatty acids in the animals' diets are hydrogenated (i.e. converted into relatively more saturated fatty acids by the reduction of double bonds) by microorganisms in the rumen (Table 7.3). The fat, therefore, contains a higher proportion of saturated and monounsaturated fatty acids and a lower proportion of polyunsaturated fatty acids than pig fat (i.e. lard) and poultry fat. It also contains more frans-unsaturated and branched-chain fatty acids. Trans double bonds are formed by the isomerization of the more common cis double bonds during the hydrogenation process catalysed by enzymes in certain of the anaerobic rumen microorganisms (Gurr & Harwood 1991).

In traditional farming practice, ruminants feed on pasture or on cereal-based concentrates that have a low fat content. Variations in adipose tissue fatty acid com-position can occur, depending on the species of animal, the time of year and the type of feed. For example, giving sheep a diet rich in barley leads to a much higher propor-tion of branched-chain fatty acids and results in a softer carcass fat. A more recent trend to improve the efficiency of animal production, is to feed ruminants, especially dairy cows, fat supplements or crushed oilseeds (Wiseman 1984). In this way modest changes in adipose tissue composition can be effected, although not so extensively as by giving so-called 'protected fats', in which the fat particles are treated in such a way as to prevent hydro-genation in the rumen (McDonald & Scott 1977). It was thought that these products would be useful for people following lipid-lowering diets (see Chapter 42) but they have not been successful in the marketplace because of their cost and their susceptibility to oxidative deterio-ration, contributing to poor taste and a short shelf-life. It is more effective to approach the problem of lipid lowering by using lean meat (see p. 103).

Milk fat Cow's milk and the dairy products derived from it cur-rently contribute 23% of the fat in the UK diet, although this figure is slowly decreasing as sales of lower-fat

FATS 103

milks increase at the expense of full-fat milk. In 1983 only 3% of liquid milk sold in the UK was of the lower-fat varieties, whereas in 1991 and 1995 the figures were 42% and 62% respectively. In Norway this figure is 70%. Cow's milk is the only milk of quantitative importance in the UK, but in some countries goat and sheep milks are of considerable importance, and there is increasing interest in these milks in the UK.

Milk fat is composed mainly of triacylgycerols (Gunstone et al 1994), which are present as an emulsion in which the fat globules are stabilized by a surrounding membrane composed of proteins, phospholipids and cholesterol. The fat globules also contain small amounts of cholesteryl esters and fat-soluble vitamins and pro-vitamins, mainly A, D and beta-carotene. The fatty acid composition of ruminant milk fat is characterized by a high proportion of short- and medium-chain saturated fatty acids, long-chain saturated and monounsaturated fatty acids, and very small proportions of polyunsaturated fatty acids (Table 7.3). They also contain small quantities of a wide variety of branched- and odd-chain fatty acids. About 5% of the double bonds in the unsaturated acids have the trans configuration.

Butter is a common food fat, containing 15% water as an emulsion in oil. The fat is derived entirely from cow's milk fat and its composition does not normally vary greatly, compared with margarines which have a wide range of compositions. Experimentally it has been shown that giving cattle supplements of varieties of oats with a relatively high fat content or crushed oil seeds, can increase the proportion of oleic acid in milk fat (Table 7.4). This could confer a nutritional advantage (Ch. 42) and provide a product that was conveniently more spreadable after storage in the refrigerator.

Cream is an example of an emulsion of milk fat in water. Fat emulsions markedly affect the palatability of foods.

Eggs Eggs provide a significant source of fat in many human diets. One egg on average provides 6-7 g triacylglycerols and phospholipids and 250-300 mg cholesterol. The fatty acid composition is shown in Table 7.3. The importance of eggs in foods lies not only in the nutritive value of the fats but also in the contribution made by the egg yolk lipoproteins to food structure, for example to the textural quality of cake after baking.

Fish oils Fish are classified broadly into 'lean' fish that store their reserve fats as triacylglycerols in the liver (e.g. cod), or 'fatty' or 'oily' fish' (e.g. mackerel, herring) where the fat is located in the flesh. The oils have a high content of fatty acids with 20 or more carbon atoms which are either predominantly monounsaturated or polyunsaturated with five or six double bonds belonging to the 'n-3' family (Tables 7.2 and 7.3). These are broad generalizations; wide variations in fatty acid composition occur depend-ing on the species of fish, its diet and the season of the year.

Muscle meats The fats eaten in muscle meats comprise the structural phospholipids and free cholesterol, although in many meat animals the muscles are infiltrated with triacyl-glycerols (marbling). Five fatty acids account for over 85% of the total muscle fatty acids: palmitic, stearic, oleic, linoleic and arachidonic acids (Table 7.5). The compo-sition of the marble fat tends to resemble more the com-position of the adipose tissue. Lean meat, which provides mainly structural fat with a high P/S ratio, has been found to be useful as part of a lipid-lowering regimen (Watts et al 1988).

Table 7.4 The fatty acid composition of some familiar spreading fats. (Values are g/100 g total fatty acids, unless indicated otherwise)

Spread Average fat content of product (g/100 g product)

4:0-12:0

14:0 16:0 18:0 20:0 22:0 16:1 18:1 20:1 22:1 18:2 18:3 20:4 +20:5

22:5 +22:6

Others

Butter* 85 13 12 26 11 0 0 3 28 0 0 2 tr 0 0 5 Butter* 85 9 7 20 14 0 0 2 35 0 0 5 1 0 0 7 Hard margarine 85 trace 6 20 8 2 2 6 22 9 9 5 tr 7 4 0 Soft margarine 85 1 5 16 5 2 3 6 25 7 9 9 tr 7 5 0 Polyunsaturated

margarine 85 3 1 11 9 1 1 trace 18 1 1 53 1 trace trace 0 Monounsaturated

margarine 85 Blended low-fat

spread 50 12 9 24 9 0 0 2 21 0 0 14 tr 0 0 9

Compiled from Gurr (1992). 'Normal butter from cows grazed on pasture. Gutter from cows grazed on pasture with supplement of crushed soybeans.

104 NUTRITIONAL SCIENCE

Table 7.5 Fatty acid composition of some structural fats important in foods. (Values are g/100 g total fatty acids)

Food 16:0 16:1 18:0 18:1 18:2 18:3 20:4 LC Others PUFA

Beef (muscle) 16 2 11 20 26 1 13 0 11 Lamb (muscle) 22 2 13 30 18 4 7 0 4 Lamb (brain) 22 1 18 28 1 0 4 14 12 Chicken (muscle) 23 6 12 33 18 1 6 0 1 Chicken (liver) 25 3 17 26 15 1 6 6 1 Pork (muscle) 19 2 12 19 26 0 8 0 14 Cod (flesh) 22 2 4 11 1 trace 4 52 4 Green leaves 13 3 trace 7 16 56 0 0 5

For explanation of 'Others' see footnote to Table 7.3. Source Gurr (1992).

Vegetable oils Most oil-bearing plants store their fat as triacylglycerols in the seed endosperm (e.g. rape, sunflower, soybean) or the fleshy fruit mesocarp (e.g. avocado). Some, like the palm, store oil both in the mesocarp (palm oil) and the endosperm (palm kernel oil). Seed oils vary widely in their fatty acid compositions (Table 7.6 and Gunstone et al 1994). Seed oils that are of dietary importance are generally those in which the predominant fatty acids are the common ones: palmitic, stearic, oleic and linoleic acids. The exceptions are coconut and palm kernel oils, which are unusual in containing saturated fatty acids of medium-chain length (C8-C14). Elsewhere in nature, only milk fat contains appreciable amounts of these acids.

Seed oils can also be important sources of carotenoids, tocopherols and plant sterols, such as beta-sitosterol, although the latter is not absorbed from the human gut. Some may contain unusual fatty acids, which, if ingested in large amounts, may have toxic or otherwise undesir-able metabolic effects. A good example is the old variety of rapeseed, which contained the 22-carbon mono-unsaturated fatty acid erucic acid. When toxicity studies with experimental animals indicated that high dietary concentrations of rapeseed oil could cause lipid infiltration

into heart muscle followed by necrosis of the tissue, the plant breeders embarked on a breeding programme to eliminate the erucic acid, thought to be the toxic con-stituent. Most varieties now grown are of the 'zero erucic' type. It was never demonstrated scientifically, however, that toxic effects would occur in man and indeed, much rapeseed oil consumed in China still has a high erucic acid content.

Plant leaves The structural lipids of plant leaves, provided by such foods as lettuce, cabbage and other green vegetables, pro-vide most of the alpha-linolenic acid in the diet. The fatty acid composition is very simple and varies little between different types of leaves. Five fatty acids account for over 90% of the total: palmitic, hexadecenoic, oleic, linoleic and alpha-linolenic (Table 7.5; Gunstone et al 1994).

MODIFICATION OF FATS DURING FOOD PROCESSING Margarine is the oldest example of a fat manufactured to simulate a natural product - butter - although there

Table 7.6 The fatty acid composition of some vegetable oils important in foods, (g/100 g total fatty acids)

Oil 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3 Others

Avocado 0 0 0 0 20 1 60 18 0 1 Cocoa butter 0 0 0 trace 26 34 35 3 trace 2 Coconut 8 7 48 16 9 2 7 2 0 1 Corn 0 0 0 1 14 2 30 50 2 1 Olive 0 0 0 trace 12 2 72 11 1 2 Palm 0 0 trace 1 42 4 43 8 trace 2 Palm kernel 4 4 45 18 9 3 15 2 0 0 Peanut 0 0 trace 1 11 3 49 29 1 6 Rape* 0 0 0 trace 4 1 54 23 10 8 Soyabean 0 0 trace trace 10 4 25 52 7 2 Sunflower 0 0 trace trace 6 6 33 52 trace 3

For explanation of 'Others' see footnote to Table 7.3. 'This gives the composition of the low-erucic rape variety; high-erucic rape is no longer widely used in food manufacture. Adapted from Gurr (1992) and Gunstone et al (1994).

FATS 105

are now many more fat spreads available with different compositions and physical characteristics (Table 7.4).

Modern margarine, like butter, is an emulsion of water in fat. The aqueous component is skimmed milk (or some-times an artificial 'milk' made by adding dried protein to water) which has been incubated briefly with lactic acid bacteria. These organisms partly break down the lactose, proteins and fats in the milk releasing low-molecular-weight compounds that contribute to flavour. All margarines are made by blending oils and fats from different animal and vegetable sources, the blend depending on the physical and nutritional properties desired for a particular branded product (e.g. hard, soft, polyunsaturated, monounsaturated, etc.) and the supply and cost of oils at a given time.

Some natural oils (e.g. fish, soybean) have melting points that are too low for the successful formulation of many products because of their high degree of un-saturation. They also oxidize readily, so that the product would have too short a shelf-life before becoming inedible. This problem is normally overcome by catalytic hydrogenation (Gunstone et al 1994), which results in a decrease in the number of double bonds, an increase in the proportion of trans double bonds and a random-ization of double-bond positions along the chain. Other processing techniques include interesterification, which randomizes the position of the double bonds on the triacylglycerol molecules (in the natural oils they are specifically distributed), or fractionation, which separates out fats of different melting points. These alternative ways of obtaining fats with firmer texture are now being used more widely as concerns about the safety of trans unsaturated fatty acids, produced during hydrogenation, are increasingly expressed because of their cholesterol-raising properties (see p. 111). Fractionation can also be used to create softer spreads from traditionally harder ones, as for example in the modern spreadable butters. It can also be used to remove certain constituents, for example cholesterol, to provide a wider range of products for consumers desiring particular nutritional attributes.

Formerly, the only way to achieve a solid margarine using a polyunsaturated seed oil was to hydrogenate the oil. The solid physical characteristics of modern 'high polyunsaturates' margarines are achieved, not by hydro-genation, but by the clever use of emulsion technology. Margarine and butter are known technically as 'water-m-oil' emulsions, that is, the predominant phase is oil with water droplets dispersed in it. Cream, by contrast, is an 'oil-in-water' emulsion. So that the oil and water phases do not separate (as happens, for example, when French dressing is left standing), the emulsion must be stabilized and this is achieved by the use of an emulsifier, a chemical that has both hydrophilic (water-attracting) and lipo-philic (fat-attracting) parts of the molecule. The emulsifier molecules form a coat around the droplets of oil or water

and keep them in suspension. Examples of natural emul-sifiers are the bile salts (see section below on Basics of fat metabolism) and the apolipoproteins. Emulsifiers used in the food industry include phospholipids and lysophos-pholipids, monoacylglycerols and a number of approved synthetic emulsifiers. By blending carefully selected oils and fats and choosing approportiate emulsifiers, even highly polyunsaturated oils can be incorporated into a solid, albeit soft, margarine. Even these need the presence of a small amount of high melting fat ('hardstock') to provide a crystalline lattice and achieve the required physical properties.

Examples of margarine compositions are given in Table 7.4. Even within a particular margarine brand, the com-position may change within defined limits from batch to batch because fats and oils are blended to give a least-cost formulation. Therefore these compositions should be regarded as rough guides only.

Modern emulsifier technology has now reached a stage where the aqueous phase can be increased from about 15% in margarines to around 70% or more to produce 'low-fat' spreads which may find application in energy-reduced diets. Other manufactured fats for frying or for use as shortenings, whose role is to 'shorten' or tenderize baked foods by preventing the cohesion of wheat gluten strands, do not contain an aqueous phase. They are made from mixtures of partly hydrogenated vegetable oils with or without animal fats, and are found in biscuits, cakes, doughnuts, pastries and breads.

Recent important developments include fats that have energy values less than the 38 kj/g of triacylglycerols com-posed of the normal long-chain fatty acids (Gurr 1996). For example 'Caprenin' and 'Salatrim' are triacylglycerols that contain mixtures of very long-chain (22:0) with medium-chain (8:0,10:0) or stearic acid (18:0) with short-chain (4:0) fatty acids respectively. These fats have about half the metabolizable energy of normal fats owing to the poorer digestibility of the long chain fatty acids combined with the intrinsically lower energy value per gram of the shorter chain fatty acids. 'Olestra' is an example of a sub-stance with fat-like properties (sucrose polyester) which is not digested by pancreatic lipase and therefore has zero energy value. The United States Food and Drug Adminis-tration has recently allowed its limited use in foods.

DETERMINATION OF FOOD FAT CONTENT AND COMPOSITION Nutritionists need to be able to quantify, easily and accu-rately, the total fat content of a food or of a whole diet and to determine the amounts of the different types of fats present. One of the ways in which consumers can be helped to identify fats in foods is by sensible product labelling, and to supply this information the manu-facturer needs an appropriate analytical facility. It is

Duodenum Jejunum Ileum

GALL BLADDER

' Short and medium chain

fatty acids

Dietary Fats

Unabsorbed material

• Faeces

Bile acid conjugates

Amphiphiliccoat

r \AA/n

OH

Phospholipids

Cholesterol Esters

Proteins

vesicles

ENTEROCYTE

Chylomicrons

vw wv wv

IVW [WAA L ®

v w W

pOH AW LQH

4. /vwv

IWvA kOH L ®

+ W\A

+ AAA

Plasma Thoracic

duct lymph

Mixed micelles

Fig. 7.6 Digestion and absorption of lipids in the small intestine. The enzyme pancreatic lipase catalyses the breaking of ester bonds in positions 1 and 3 of triacylglycerols, releasing 2 moles of fatty acid per mole of triacylglycerol, with 1 mole of 2-monoacylglycerol remaining. Bile salts aid the emulsification of the fat droplets, while co-lipase anchors the enzyme to the surface of the fat droplets. Phospholipids are split by a pancreatic phospholipase A2, releasing the fatty acid in position 2 and leaving a lysophospholipid. A cholesterol ester hydrolase splits the fatty acid from cholesterol esters. Lipolysis products are stabilized in mixed micelles comprising surface-active components, i.e. monoacylglycerols, long-chain fatty acids and bile salts, with unhydrolysed triacylglycerols, cholesterol and fat-soluble vitamins in the hydrophobic interior. Short-chain fatty acids are absorbed as free acids into the portal blood. Monoacylglycerols and long-chain fatty acids from mixed micelles are transported across the brush border and re-esterified in the enterocytes. Reformed triacylglycerols are stabilized with phospholipids and apoproteins and exported into plasma as chylomicrons.

106 NUTRITIONAL SCIENCE

important to decide what information is required, because although it is now possible to supply detailed analyses of most lipid mixtures, this undoubtedly adds to the cost of products and the consumer seldom requires the same amount of detail as the research nutritionist.

Extraction of fat (Gunstone et al 1994, Gurr 1992)

A commonly used older method (Soxhlet) was to extract the finely divided dried food by continuous refluxing with light petroleum. This method determines only the readily extractable storage fats and generally gives values lower than more recent methods (e.g. Weibull, Roese-Gottlieb, Bligh & Dyer; see Gurr 1992). The latter use treatments with acids, alkalis or more polar solvents (e.g. methanol) to break down the chemical bonds that bind the lipids into the structure of the food, before extraction with hydrocarbons. Extraction with mixtures of chloro-form and methanol is the method of choice when the analyst wishes to determine all the individual lipid components in the food.

Determination of individual fat components (Christie 1982, 1989, Gunstone et al 1994)

Phospholipids and cholesterol in the extract can be deter-mined by specific colorimetric reactions, while triacyl-glycerols and cholesterol can be determined by enzymic methods for which there are readily available commercial kits. More detailed information on individual lipids and fatty acids is best obtained by chromatographic procedures such as thin-layer (TLC), gas-liquid (GLC) or high performance liquid (HPLC) chromatography. These methods can now be highly automated for rapid, specific and highly reproducible quantitative and qualitative analysis.

Measurement of energy value A principal characteristic of fat is its high energy density and the research worker investigating dietary fats will need to be familiar with bomb calorimetric determination of energy (Ch. 3). The practical nutritionist or dietitian may need to go no further than food composition data-bases (Ch. 16) to find the fat content of a food and apply the standard energy value for fat of 38 kj/g (9 kcal/g). However, with the growing interest in specialized fats that have lower energy densities (see p. 105) the student should be aware that the standard conversion factor may not always provide an accurate value for the energy content of these foods.

CONSUMPTION OF FATS IN DIETS There are large differences between different cultures in the part that fat plays in the diet. The Ho tribe in India make little or no use of fat in their food preparation

and their fat intake has been estimated to be no more than 2-4 g/d or 2% of their dietary energy intake. Certain Inuit communities, by contrast, may consume as much as 80% of their energy as fat. In the UK, people eat on average about 100 g fat daily and this contributes just over 40% of dietary energy. Accurate figures are difficult to obtain. The most reliable consumption figures (Chapter 17) until recently excluded foods eaten outside the home and we know less about how the fat profile of foods eaten in restaurants and canteens compares with that eaten at home. Individuals differ not only in the average amounts of fat they eat but also in their variability from day to day; it may be necessary to record food intakes over 10 days to get a reliable estimate of an individual's fat intake. Many studies have relied only on a 24-hour recall or at best weighed intakes recorded over 7 days (Chapter 17). Finally, while food composition tables are convenient, it should be remembered that foods analysed by their compilers were not precisely those that you may be dealing with. For reliability, there is no substitute for direct analysis.

BASICS OF FAT METABOLISM Digestion Triacylglycerols, which form the bulk of the fats in the diet, must be broken down into monoacylglycerols and fatty acids by a pancreatic lipase in the small intestine before they can be absorbed efficiently. Hydrolytic release of fatty acids from lipids catalysed by lipases is called lipolysis; this is the process of fat digestion. In most adults, fat digestion and absorption are very efficient and more than 95% of the 100 g or so of fat consumed daily by individuals in the UK are probably digested and absorbed. Much larger quantities, up to 250 g/day or even more, can sometimes be digested and absorbed if the body is short of energy. Arctic explorers and lumber-jacks frequently consume such large amounts.

The newborn baby has to adapt to the relatively high fat content in breast milk after relying mainly on glucose as an energy substrate in fetal life. He can digest fat, but not as efficiently as the older child or adult, because his pancreatic and biliary secretions are not fully devel-oped (Ch. 28). Neonatal fat digestion is aided by a lipase secreted from the lingual serous glands and carried into the stomach, where hydrolysis occurs without the need for bile salts, at a pH of around 4.5-5.5 (Carey et al 1983). The activity is probably stimulated by the action of sucking and the presence of fat in the mouth. The pro-ducts of lipolysis are mainly 2-monoacylglycerols, diacyl-glycerols and non-esterified fatty acids (NEFA), the latter being richer in medium-chain length acids than the original triacylglycerols. There is also evidence that a lipase in human breast milk contributes to fat digestion in the newborn (Carey et al 1983).

FATS 107

H Cholesterol 7 a-OH cholesterol Cholic acid 3a,7a, 12a -OH

Chenodeoxycholic acid 3a, 7a - OH

Fig. 7.5 Metabolic conversion of cholesterol into bile acids. Note the preponderance of hydrophobic groups on one side of the planar molecule, and hydrophilic groups on the other.

Later in life, the process of fat digestion also begins in the stomach, where a churning action helps to form a coarse fat emulsion. This is not hydrolysed but enters the small intestine and is modified by mixing with bile and pancreatic juice (Carey et al 1983). The biliary secretion which is enhanced as the amount of dietary fat increases, contains bile acids that are formed in the liver from cholesterol (Fig. 7.5). The first and rate-limiting step is hydroxylation at C7, followed by oxidation of the side chain with the formation of a carboxyl group to produce chenodeoxycholic acid. Further hydroxylation at C12 yields cholic acid. These two primary bile acids are excreted in the bile as conjugates with taurine or glycine. The polar groups and the shape of the molecules make the bile acids effective detergents and enable them to help emulsify the fats present in the small intestine.

Digestion takes place in the duodenum, catalysed by a pancreatic lipase (Carey et al 1983, Nutrition Society 1996). The main products of triacylglycerol digestion are 2-monoacylglycerols and non-esterified fatty acids. Phos-pholipid digestion yields a lysophosphoglyceride and a fatty acid released from position 2 by pancreatic phos-pholipase. Cholesterol esters in the dietary fat must be hydrolysed by a pancreatic cholesterol esterase before ab-sorption can occur. Normally more than 95% of ingested triacylglycerols are absorbed and only 40% of cholesterol.

As digestion progresses, the oil phase decreases in volume as the lipolytic products pass into 'mixed micelles': large molecular aggregates consisting of monoacyl-glycerols, fatty acids longer than CI2, bile salts and phospholipids (Fig. 7.6). The mixed micelles are able to draw into their hydrophobic core the less water-soluble molecules such as cholesterol, carotenoids, tocopherols and some undigested triacylglycerols.

Absorption Lipid absorption occurs mainly in the jejunum. The digestion products pass from the mixed micelles into the enterocyte membrane by passive diffusion (Carey et al

1983, Nutrition Society 1996). A diffusion gradient is maintained by the presence of a fatty acid binding pro-tein, which immediately binds to fatty acids entering the cell, and the rapid re-esterification of fatty acids to monoacylglycerols (Fig. 7.6). Cholesterol absorption is completed by re-esterification catalysed by acyl-CoA: cholesterol acyltransferase, or by the reversal of choles-terol esterase. The former enzyme is induced by high concentrations of dietary cholesterol.

The resynthesis of triacylglycerols, cholesterol esters and phosphoglycerides in the enterocyte employs fatty acids with chain lengths greater than 12 carbon atoms. Short (C4-C6) and medium- (C8-C10) chain length fatty acids are absorbed directly into the portal blood and carried to the liver as albumin complexes, where they are rapidly oxidized (Fig. 7.6). They do not, therefore, contri-bute to plasma lipids, nor are they deposited in adipose tissue in significant quantities. About 4 g/d are absorbed from dairy products; further small amounts may be derived from food products that incorporate coconut and palm kernel oils.

Defects in fat digestion and absorption Failure to assimilate lipids of dietary origin into the body may arise from defects in digestion (maldigestion) or ab-sorption (malabsorption). Maldigestion can occur when the pancreas fails to secrete sufficent lipase, as in pan-creatitis, the presence of a pancreatic tumour, or in mal-nutrition. Alternatively, there may be enough functional lipase to effect lipolysis but a failure to produce enough bile, due to biliary disease with obstruction of the bile duct, or chronic liver disease. The commonest cause of biliary insufficiency in affluent societies, however, is due to surgical resection of the ileum, where active transport of bile salts occurs (Ch. 35). Bile salt deficiency results in inability to solubilize lipolysis products into the mixed micelles. Gastric disturbances can also sometimes affect digestive efficiency.

Malabsorption may occur even when digestion is

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FATS 109

CE = cholesteryl ester LCAT = lecithin cholesterol acyltransferase

Fig. 7.7 The metabolism of lipoproteins. The 'exogenous pathway' is concerned with the transport and metabolism of fat coming from the diet. The fats are packaged as chylomicrons, which circulate and are removed from the plasma by the enzyme lipoprotein lipase, mainly in the adipose tissue. The chylomicrons are not entirely consumed by this enzyme but are degraded to smaller particles called remnants, which are removed by the liver. Parts of the chylomicrons also go into making HDL which, in conjunction with the enzyme LCAT, remove excess cholesterol from membranes and other lipoprotein particles, converting it into cholesteryl esters. This process involves the interconversion of two forms of HDL: HDL2 and HDL3 and cholesteryl esters are taken to the liver for further processing. The 'endogenous pathway' is concerned with the transport and metabolism of fats made in the body itself. The products are the very low density lipoproteins (VLDL), which are metabolized in a manner analogous to the chylomicrons. Their remnants are usually called intermediate density lipoproteins (IDL), and are further metabolized to low density lipoproteins (LDL) which are taken up by the specific LDL receptor.

functioning normally, because of defects in the absorptive surfaces of the small intestine. Common causes are bacterial invasion or sensitization of the gut to dietary components such as gluten, as in coeliac disease. Malab-sorption syndromes (often called 'sprue') are charac-terized by dramatic changes in the morphology of the intestinal mucosa. The epithelium is flattened and irre-gular and atrophy of the villi reduces the absorbing surface area. A common feature of all fat malabsorption syndromes is a massively increased excretion of fat in the faeces (steatorrhoea), which arises not only from un-absorbed dietary material but also from the bacterial overgrowth in the gut. Patients with poor fat absorption are at increased risk of deficiencies of energy, fat-soluble vitamins and essential fatty acids. The clinical manage-ment of fat malassimilation is facilitated by replacing normal dietary fats by medium-chain triacylglycerols (MCT) (Gurr & Harwood 1991, Nutrition Society 1996). This product, which is available as a cooking oil or spread, is refined from coconut oil and consists of the fraction containing mainly C8 and CIO saturated fatty acids, which are more efficiently digested and absorbed directly into the portal blood, bypassing the normal absorptive route. For other dietetic approaches to steatorrhoea and diarrhoea see Chapter 35.

Lipid transport (Fig. 7.7)

Lipids that have been absorbed from a meal or, alter-natively, that have been formed within the body, are transported in the blood to tissues where they are needed as sources of energy, as components of membranes or as precursors of biologically active metabolites (British Nutrition Foundation 1992, Gurr & Harwood 1991, Hunt & Grof 1990, Nutrition Society 1996). Nature has solved the problem of how to stabilize water-insoluble lipids in an aqueous environment by coating the lipid particles with a layer of amphiphilic lipids and proteins to form aggregates called lipoproteins (Table 7.7). The protein components (apolipoproteins, Table 7.8) have another function. They confer specificity on the lipoproteins and determine the way they are metabolized as well as the tissues in which they are metabolized. The apoproteins recognize and interact with specific receptors on cell sur-faces, following which the receptor-lipoprotein complex is taken into the cell by a process of endocytosis. There are several apolipoprotein peptides, identified by the letters A-E, but as research continues subclasses are being discovered. Thus apoC is now divided into apoC-I, II and III. While many of these peptides are involved in receptor recognition, some are also involved in the

110 NUTRITIONAL SCIENCE

Table 7.7 Composition and characteristics of human plasma lipoproteins

Chylomicrons VLDL LDL HDL

Protein (% particle mass) 2 7 20 50 Triacylglycerols (% particle mass) 83 50 10 8 Cholesterol (free + esterified) 8 22 48 20

(% particle mass) Phospholipids (% particle mass) 7 20 22 22 Particle mass (daltons) 0.4-3.0 x 106 10-100 x 106 2-3.5 x 106 1.75-3.6 x 10s

Density range (g/ml) >0.95 0.95-1.006 1.019-1.063 1.063-1.210 Diameter (nm) >70 30-90 22-28 5-12 Apolipoproteins A-l, B-48 B-100, E B-100 A-l, A-l I

C-l, C-ll, C-lll

Source Gurr & Harwood (1991). VLDL, very low density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins.

Table 7.8 Characteristics ot human apolipoproteins

Shorthand Molecular Amino-acid Function Major name weight

(daltons) residues sites of

synthesis

A-l 28 000 243 Activates LCAT*

Liver Intestine

A-l I 17 000 154 Inhibits LCAT? Activates hepatic lipase

Liver

B-48 Cholesterol clearance

Intestine

B-100 350-550 000 Cholesterol clearance

Liver

C-l 6 605 57 Inhibits LCAT?

Liver

C-ll 8 824 79 Activates LPL

Liver

C-lll 8 750 79 Inhibits LPL Inhibits VLDL uptake via B/E receptor

Liver

E 34 000 279 Cholesterol clearance

Liver

Source, Gurr & Harwood (1991). LCAT, lecithin-cholesterol acyltransferase; LPL, lipoprotein lipase.

functioning of the enzymes of lipoprotein metabolism; thus apoC-II is needed for the activation of lipoprotein lipase and apoA-I for an enzyme involved in cholesterol esterification (Table 7.8). An individual's apolipoprotein profile is genetically determined and variants are now recognized that result in specific metabolic disorders.

During active fat absorption, the triacylglycerols re-synthesized in the enterocytes acquire a stabilizing coat of phospholipids and apolipoproteins (apoA and apoB-48). The resultant large spherical lipoprotein particles, 75-600 nm in diameter (chylomicrons), are secreted into the lymphatic vessels (Fig. 7.6) and pass via the thoracic duct to the jugular vein. In the bloodstream, they acquire apolipoproteins C and E from high density lipoproteins (HDL) (Fig. 7.7).

As they circulate, the chylomicrons enter the capillaries

of the skeletal muscles, heart, mammary glands and adipose tissues, where they can interact with the enzyme lipoprotein lipase (LPL). This enzyme catalyses the lipolysis of triacylglycerols and the fatty acids that are released are taken up into the cells of the target tissue. After a meal, an elevated insulin concentration directs most chylomicron breakdown to adipose tissue by acti-vating the adipose tissue LPL. (However, recent research suggests that in some individuals, a considerable propor-tion of the NEFA released by the adipose tissue LPL are not taken up by the tissue but circulate in the plasma as albumin complexes. Very high concentrations of NEFA are characteristic of obese subjects and patients with non-insulin-dependent diabetes mellitus (NIDDM) and may be a marker for abnormalities in lipid metabolism associated with these metabolic diseases: see Chs 34, 37, 41.)

During a period of fasting, the hormonal balance activates muscle LPL so that fatty acids can be used as a fuel by that tissue. During lactation, prolactin causes the activity of LPL in mammary glands to be elevated to provide a supply of substrates for milk fat synthesis.

About half of the chylomicron triacylglycerols are hydrolysed in 2-3 minutes but the particles are not com-pletely degraded. Remnant particles, containing propor-tionately less triacylglycerol and more cholesterol are poor substrates for LPL and are taken up, via the apoE receptor, by the liver, where the cholesterol is used for membrane or new lipoprotein biosynthesis, or converted into bile acids.

The rate at which the liver is able to remove remnants depends on the type of apoE receptor present (there are three genetically determined apoE phenotypes), the activity of the liver triacylglycerol lipase that hydrolyses remnant triacylglycerols, and the close integration with high density lipoprotein metabolism (see below). The ability to metabolize circulating triacylglycerols, which derive from a meal rich in fat, varies considerably between individuals as a result of differences in apoE phenotype, LPL or hepatic lipase activities. Some people

FATS 111

clear triacylglycerols rapidly, whereas others maintain high circulating concentrations of triacylglycerol-rich lipoprotein remnants. Since most people eat about three main meals spaced throughout the day, they remain in the postprandial state for much of the time and a state of almost continual hyperlipidaemia may exist in people with a relative 'triacylglycerol intolerance'. Such indivi-duals may be at increased risk of developing vascular diseases (see Ch. 42).

Whereas chylomicrons are the major carriers of fat from the diet, very low density lipoproteins (VLDL, Table 7.7) are involved in transporting lipids that are synthesized endogenously, mainly in the liver. They are degraded by a mechanism similar to that described for chylomicrons. The VLDL remnant is normally called an 'intermediate density lipoprotein' (IDL). The name arises because further degradation yields a particle called a low density lipoprotein (LDL, Table 7.7). This class of lipoprotein is the major carrier of cholesterol in human beings. Thus, in a person with a plasma cholesterol concentration of 5 mmol/1, about 70% is carried on LDL. Their role is to deliver cholesterol to tissues for the vital functions of membrane synthesis and repair. The discharge of choles-terol can occur by passive endocytosis or by a specific receptor-mediated uptake process (Brown et al 1981) in which the receptor recognizes the apoB-100 component of LDL and binds to it. The LDL-receptor complex is taken into the cell and the LDL degraded by lysosomal enzymes. The liberated cholesterol interacts with the endoplasmic reticulum membranes, in which are located the enzymes of cholesterol biosynthesis, and inhibits hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the sequence. In this way, endo-genous cholesterol biosynthesis is regulated by the amount available from the diet. Familial hypercholesterolaemia, an inherited disorder characterized by high circulating concentrations of LDL, results from the absence of a functional LDL receptor (Brown et al 1981).

The remaining important class of lipoprotein is the type known as high density lipoproteins (HDL, Table 7.7). Their role is to carry cholesterol from peripheral cells to the liver, where it is degraded or repackaged, a process known as 'reverse cholesterol transport'. This pathway is particularly important since the rate of LDL transport to peripheral tissues generally exceeds tissue cholesterol requirements except during periods of active growth and tissue repair.

HDL have attracted a great deal of research attention since it was realized that a low plasma concentration may be indicative of increased risk of cardiovascular disease and it has been proposed (but not conclusively confirmed; see Ch. 42) that they have a protective role. A key step in reverse cholesterol transport is catalysed by the enzyme lecithin cholesterol acyltransferase (LCAT). A fatty acid is transferred from phosphatidylcholine (lecithin) to

Fig. 7.8 The lecithin-cholesterol acyltransferase reaction (LCAT). The reaction is responsible for the formation of cholesteryl esters in plasma and involves the participation of high density lipoprotein (HDL) particles (see also Fig. 7.7).

cholesterol to form a cholesterol ester, as illustrated in Fig. 7.8. In human plasma, LCAT is associated with HDL and the phospholipid substrate is also present in HDL, having been transferred from chylomicron remnants or IDL during the degradation of the triacylglycerol-rich lipoproteins. The cholesterol substrate is derived from the surfaces of plasma lipoproteins or the plasma membranes of cells. LCAT, by consuming cholesterol, promotes its net transport from cells into plasma. Molecules of choles-terol ester transferred to remnant lipoproteins containing apoB or apoE are taken up by the liver, thereby completing the process of reverse cholesterol transport (Fig. 7.7).

During transfer of cholesteryl ester to triacylglycerol-rich lipoproteins, there is a reciprocal enrichment of HDL with triacylglycerols. This exchange is catalysed by an enzyme cholesteryl ester transfer protein.

The amounts and proportions of lipoproteins in the plasma, especially the chylomicrons and VLDL, respond to the influx of digestion products of dietary lipids and carbohydrates after the consumption of the meal. The average long-term concentration of VLDL, LDL and HDL may also to some extent be determined by habitual intakes of fats and carbohydrates (British Nutrition Foundation 1992, Grundy & Denke 1990, Salter & White 1996).

• Increasing the dietary intake of cholesterol increases plasma cholesterol by 0.5 mmol/1 for every 100 mg cholesterol consumed daily and this is mainly in the LDL fraction.

• The substitution of dietary saturated fatty acids by monounsaturated or n-6 polyunsaturated fatty acids leads to a reduction in the concentration of LDL-cholesterol. HDL cholesterol is not reduced unless linoleic acid provides more than about 12% of dietary energy.

• Exchanging as- with irans-monounsaturated fatty acids leads to an increase in total and LDL-cholesterol and a small decrease in HDL-cholesterol. The cholesterol-raising effect of trans unsaturated fatty acids is similar to that of the cholesterol raising saturated fatty acids.

• The addition of long-chain n-3 polyunsaturated fatty acids (mainly 20:5 and 22:6) to diets leads to a reduction in the concentration of VLDL but not LDL or HDL.

• The replacement of dietary saturated fatty acids with carbohydrates leads to decreased concentrations of LDL and HDL and in some people increased concentrations of VLDL.

• The replacement of dietary n-6 polyunsaturated

112 NUTRITIONAL SCIENCE

fatty acids with carbohydrates leads to increased LDL cholesterol concentrations with little change in HDL and, in some people, increased concentrations of VLDL.

The mechanisms by which these changes occur are poorly understood. In part they may result from reduc-tions in the rate at which the apoB receptors in the liver remove LDL, brought about by some saturated fatty acids (12:0, 14:0,16:0) and reversal of this effect by some unsaturated fatty acids. Some research suggests that the sensitivity of the apoB receptors depends on:

1. the chain length of the saturated fatty acid (14:0 more inhibitory than 16:0 or 12:0);

2. the amount of cholesterol in the diet (more inhibition by saturates at high cholesterol intakes), and

3. the level of dietary linoleic acid (less effect of 18:2 above a 'threshold' of about 6% of dietary energy) (Hayes & Khosla 1992).

As an alternative to a mechanism that involves the apoB receptor, different types of fatty acids and carbo-hydrates may influence the rates of biosynthesis of LDL from VLDL or the conversion of cholesterol into bile salts (Grundy & Denke 1990).

Fatty acid metabolism Biosynthesis

Most body tissues contain enzymes for the biosynthesis of fatty acids and their esterification in triacylglycerols, phospholipids and other body lipids (Gurr & Harwood 1991). This is known from experiments with samples of biopsied tissue, limited tracer studies in vivo, or by inference from studies with other species. In most mammals, when the fat content of the diet is low, rates of fatty acid synthesis are high, particularly in the liver, to supply the needs of structural and storage fats, the main products being palmitic and oleic acids. However, this rarely occurs in Western man, whose diet generally contains a high proportion of its energy as fat. At most times, the enzymes of fat biosynthesis are 'switched off' and the needs for storage and structural fats are satisfied from dietary intakes.

Diet may regulate fatty acid biosynthesis in two main ways. First, the activities of several enzymes on the fatty acid biosyntheic pathway are dependent on the presence of coenzymes that are derived from vitamins in the B-group, including pantothenic acid and biotin. Other low-molecular-weight compounds, such as citrate, con-trol enzyme activity by influencing the conformation of the protein molecules. Secondly, the levels of the macro-nutrients, protein, carbohydrate and fat, and the balance between them, influence the concentrations of circulating hormones which induce or suppress the synthesis of some enzymes of fatty acid biosynthesis. The former is

short-term regulation, affecting the minute-by-minute activity of enzymes; the latter is longer term regulation, affecting the total amount of enzyme protein present. Fatty acids themselves may influence the expression of genes for proteins involved in their own metabolism, by mechanisms which are still poorly understood.

Fatty acid biosynthesis in human adipose tissue (mea-sured in isolated adipocytes and subcellular fractions) increases up to 11-fold when people change from a high-fat to a high-carbohydrate diet (Sjostrom 1973). Under these conditions, the liver is probably the dominant human organ for lipid biosynthesis and the newly synthesized fat is exported into the blood as VLDL. Nevertheless, Sjostrom (1973) found that fatty acid biosynthesis de novo was of little quantitative importance in human beings, even when carbohydrates were the main source of dietary energy.

High rates of fatty acid biosynthesis can occur in the mammary gland during lactation. The medium-chain fatty acids caprylic (octanoic, 8:0) and capric (decanoic, 10:0) are produced specifically in the mammary gland and can, therefore, act as a marker for endogenous fatty acid synthesis. When the amount of fat in the human diet is very low, there is a marked elevation of milk medium-chain fatty acids compared with the concentrations in the milk of women consuming a high-fat diet. Dietary and non-dietary factors affecting human milk fat composition are extremely complex (Jensen 1996).

Desaturation

To achieve the desired physical properties of lipids in cells, a high degree of unsaturation is required (British Nutrition Foundation 1992). Virtually all tissues contain enzymes (desaturases) that insert double bonds into satu-rated fatty acids, normally at position 9. Thus palmitic and stearic acids, arising either from the diet or from biosynthesis in the tissues, are desaturated to palmitoleic (cz's-9-hexadecenoic) and oleic (ris-9-octadecenoic) acids respectively. Human tissues also contain desaturases that catalyse the introduction of further double bonds to pro-duce polyunsaturated fatty acids. These desaturations, alternating with chain elongations by two carbon atoms at each step, give rise to several families of fatty acids, depending on the structure of the precursor fatty acids (Fig. 7.9). The most important families are n-3, n-6 and n-9 (see legend to Fig. 7.2).

Esterification

Fatty acids in the unesterified form are toxic to cells in other than trace amounts. They may be attached to various binding proteins (for example NEFA are carried in the plasma as albumin complexes) but the overwhelming majority are esterified in complex lipids: triacylglycerols,

FATS 113

First Source Family member Desaturation

Diet or n-9 endogenous synthesis

Diet only

n-6

Elongation +C,

oleic A6desaturase

9,12-18:2 linoleic

~ J

Desaturation

n-3 9,12,15-18:3-a-linolenic

• 6,9,12-18:3 y-linolenic

• 6,9,12,15-18:4

8,11,14-20:3

8,11,14,17-20:4

5,8,11,14-20:4 arachidonic

5,8,11,14,17-20:5

Accumulates in EFA deficiency

1. Components of membranes

2. Precursors of eicosanoids

Fig. 7.9 The metabolism of three different families of unsaturated fatty acids. The first member of the n-9 family, oleic acid, can be taken in from the diet or can be formed in body tissues, whereas linoleic and a-linolenic acids, the first members of the n-6 and n-3 families respectively, can only come from the diet. The first step in their metabolism is the introduction of a new double bond at position 6 by an enzyme called 6-desaturase. All three fatty acids compete for this enzyme. There follows a series of alternate competitive elongation and desaturation steps. The products formed from oleic acid accumulate only when linoleic acid is absent from the diet or present only in very small amounts. They are therefore markers of essential fatty acid deficiency. Arachidonic acid (n-6 family), is the major long-chain polyunsaturated fatty acid in membranes of most human tissues; docosahexaenoic acid (DHA; n-3 family) is a characteristic component of nervous tissue.

phosphoglycerides or cholesteryl esters. Esterification needs the coenzyme-A thiolester of the fatty acid as a substrate and is catalysed by a variety of acyltransferases. Sometimes these have wide specificity but in some cases they may catalyse the esterification of specific positions in the lipid molecule or specific fatty acids.

Oxidation

Fatty acids may be degraded by several oxidative path-ways. With regard to the utilization of fatty acids as sources of metabolic energy (see p. 118) the process of beta-oxidation is quantitatively the most important. This process also requires the coenzyme A thiolester of the fatty acid as the substrate. Before entry into the mito-chondrion, the fatty acyl-CoA must be converted into the corresponding acyl carnitine. Beta-oxidation occurs mainly in the mitochondria of cells (see Gurr & Harwood 1992, Hunt & Grof 1990, Zubay 1993). Some fatty acids, notably those with very long chain lengths, may undergo initial degradation to short chain lengths in specialized subcellular particles called peroxysomes, before being handed on to mitochondria for complete degradation. Beta-oxidation involves stepwise reduction of the fatty acid chain by two carbon atoms in each cycle of the oxida-tion, by a complex of four enzymes. At each step of the cycle, acetyl-CoA is released and fed into the tricarboxylic acid cycle, where it is further degraded to carbon dioxide and water. The reduced pyridine nucleotides generated in the tricarboxylic acid cycle enter the mitochondrial electron transport chain to fuel the generation of ATP as a source of metabolic energy. One mole of the commonly occurring fatty acid, palmitic acid (16:0) yields 129 moles of ATP (Table 7.9).

Another type of oxidation is known as lipid peroxi-dation. This occurs when oxygen forms a bond with

Table 7.9 The ATP yield of palmitate oxidation

Reaction ATP yield

A. Beta-oxidation CH3 (CH2)14COSCOA + 7FAD + 7H 20 + 7NAD+ + 7CoA

> 7FADH2 + 7NADH + 7H+ + 8CH3COSCoA (B) (C) (D)

B. Oxidation of FADH2 in the mitochondrial electron transport chain 14H+ + 7FADH2 + 14Pi + 14ADP + 3.502

> 7FAD + 21H20 + 14ATP 14

C. Oxidation of NADH in the mitochondrial electron transport chain 7NADH + 28H+ + 21 Pi + 21ADP + 3.502

> 7NAD+ + 28H20 + 21 ATP 21

D. Oxidation of acetyl-CoA in the tricarboxylic acid cycle 96H+ + 8CH3COSCoA + 1602 + 96Pi + 96ADP

> 8CoA + 104H20 + 16C02 + 96ATP 96

Overall equation 131H+ + CH3(CH2)14COSCOA + 131 Pi + 131ADP + 230 2

> 131 ATP + 16C02 + 146H20 + CoA 131

Less 2ATP required for initial activation of palmitate (formation of CoA thiolester) - 2

129

the particularly reactive carbon atoms between pairs of double bonds in polyunsaturated fatty acids. The initi-ating step is the formation of a free radical, a very reactive chemical species owing to the presence of an unpaired electron. Free radical formation may be catalysed by trace metal ions (such as Fe3+), haem compounds or enzymes. Oxygen attacks to form a peroxide free radical which converts into a hydroperoxide. The peroxide and hydro-peroxide intermediates may either break down into small molecular weight compounds or polymerize as in Figure 7.10.

114 NUTRITIONAL SCIENCE

-CH=CH-CH2-CH£CH- c/s,c/s-methylene interrupted polyunsaturated fatty acid chain

+Fe3+

-CH=CH-CH-CH=CH- lipid free radical

+02

-CH=CH-CiCH- lipid peroxide free radical I 00"

-CH=CH-C=CH- lipid hydroperoxide I OOH

Carbonyl compounds Polymeric compounds

Fig. 7.10 Lipid peroxidation; c, c/s; f, trans.

Lipid peroxidation is nutritionally significant in at least two ways. First, it can occur in foods, eventually making them unpalatable and unfit to eat. The carbonyl products may be toxic (Chow 1992). Secondly, it can also occur in vivo if the body's antioxidant defences are in-sufficient (Rice-Evans & Burdon 1993). This leads to tissue disruption and initiates disease processes, including atherosclerosis and cancer (see Chs 42, 46, 52). Fruits and vegetables, containing vitamin E and other antioxidant substances provide a useful contribution to the body's antioxidant defences (Chs 13, 25).

NUTRITIONAL ROLES FOR FATTY ACIDS AND LIPIDS Essential fatty acids In the course of evolution, human beings lost the ability to make enzymes that catalyse the introduction of double bonds between positions 12-13 and 15-16, as present in linoleic and alpha-linolenic acids, which are formed in plants (Fig. 7.2). Yet these fatty acids are essential to life and must, therefore, be supplied by the diet.

Historical background

In a classic paper in 1929, two American nutritionists, Burr & Burr, described how acute deficiency states could be produced in rats by feeding fat-free diets, and how these deficiencies could be eliminated or prevented only by adding specific fatty acids to the diet. It was shown that fatty acids related to linoleic acid were the most effective, and the term vitamin F was coined for them, although they are now always referred to as essential fatty acids (EFA). EFA deficiency can be produced in a variety of animals, including man, but the condition is best documented in the laboratory rat. The disease is characterized by skin symptoms such as dermatosis, and the skin becomes 'leaky' to water. Growth is retarded, reproduction is impaired and there is degeneration or

impairment of function in many organs of the body. Bio-chemically, EFA deficiency is characterized by changes in the fatty acid composition of many tissues, especially biological membranes whose function is impaired, and in the mitochondria the efficiency of production of meta-bolic energy by the oxidation of fatty acids is reduced.

Well documented EFA deficiency in man is rare but was first seen in children given virtually fat-free diets (Hansen et al 1958). Four hundred infants were given milk formulas containing different amounts of linoleic acid. When the formulas contained less than 0.1% of dietary energy as linoleic acid, clinical and biochemical signs of EFA deficiency ensued. The skin abnormalities were similar to those seen in rats and these and other signs of EFA deficiency disappeared when more linoleic acid was added to the diet.

In 1971, the first unequivocal case of EFA deficiency in an adult was reported (Collins et al 1971). The patient, a man of 44, had undergone surgical removal of all but 60 cm of his bowel. He was then given intravenous feeding only, with preparations containing no fat, and after 100 days he developed scaly dermatitis. A bio-chemical test that allows early diagnosis of EFA defi-ciency before the appearance of skin lesions depends on the failure of tissues to produce sufficient arachidonic acid (all-ris-5,8,11,14-20: 4; AA) and at the same time to produce an excess of all-ris-5,8,11-20:3 (Fig. 7.9). The ratio of 20:3 n-9/20:4 n-6 (the triene/tetraene ratio) measured in plasma phospholipids is used as a bio-chemical index of EFA status. In health, the ratio is about 0.1 or less, rising to 1.0 in severe EFA deficiency. A ratio of 0.4 has been used conventionally as indicating EFA deficiency, and using this ratio as a diagnostic criterion, three patients with chronic disease of the small bowel who had been treated with low fat diets but not given intravenous feeding were found in a London hospital (Press et al 1974). They responded successfully to the application to the skin of lipids containing a high proportion of linoleic acid, demonstrating that EFA need

FATS 115

not necessarily to be absorbed through the conventional route to be effective.

Long-chain metabolites of EFA

Figure 7.9 illustrates the pathways for the metabolism of three families of unsaturated fatty acids, n-9, n-6 and n-3, beginning with their parent fatty acids, oleic, linoleic and alpha-linolenic acids respectively (see also British Nutrition Foundation 1992, Gurr & Harwood 1991). Each pathway involves alternate desaturation and elongation by two carbon atoms to produce a variety of long-chain more highly polyunsaturated fatty acids. Because the same desaturases are involved in the metabolism of the different families, the substrates in the different pathways compete with each other for the desaturases, thus influ-encing the proportions of the end-products formed. The affinity of the 6-desaturase for its substrates is in the order: 18:3 > 18:2 > 18:1. Normally, conditions are such that the pathway beginning with linoleic acid (the n-6 series) predominates and arachidonic acid is the main end product. If the amount of linoleic acid in the diet is very small compared with oleic acid, the n-9 pathway will begin to predominate, giving rise to an excessive pro-duction of 5,8,11-20:3 (n-9). This explains why the ratio of 20:3 «-9/20:4 n-6 is an indicator of EFA deficiency as described in the last section. Other fatty acids, such as the isomeric fatty acids formed during hydrogenation, can also compete with linoleic acid and diets with excessive amounts of these isomers and limiting amounts of linoleic acid could also give rise to EFA deficiency as defined by these biochemical criteria, without any clinical signs neces-sarily being apparent. For most individuals this rarely occurs, but some people eating strange diets in which hardened fats provide much of the energy could be at risk.

If the diet contains a large contribution from fish oils rich in n-3 polyunsaturated fatty acids, the balance of polyunsaturated fatty acid metabolism can be tipped towards the n-3 family. The main end products are eicosa-pentaenoic (all-cz's-5,8,11,14,17-20: 5; EPA) and docosa-hexaenoic (all-ris-4,7,10,13,16,19-22: 6; DHA) acids and this has implications for the production of eicosanaoids as described later.

Role of EFA in membranes

During EFA deficiency, changes occur in the properties of membranes, for example permeability to water and small molecules, which can be correlated with changes in fatty acid composition of the membrane (British Nutri-tion Foundation 1992). Membranes of liver mitochondria isolated from EFA-deficient animals have smaller pro-portions of linoleic and arachidonic acids and larger proportions of oleic (n-9) and all-ds-5,8,11-20: 3 n-9 than those of healthy animals. Beta-oxidation and oxidative

phosphorylation are less efficient. These changes at the molecular level are reflected in the animal's poorer per-formance in converting food energy into metabolic energy for growth and maintenance of body function. It seems that the stability and integrity of the membrane and its ability to provide an environment for the efficient func-tioning of the enzymes, receptors and other proteins em-bedded in the lipid bilayer, can only be supported by the presence of lipids with a certain pattern of polyunsatu-rated fatty acids, in ways that are as yet incompletely understood.

Eicosanoids

Arachidonic acid and other C20 and C22 polyunsaturated fatty acids of the n-3 and n-6 families can be metabolized to a range of compounds that exert a multitude of physio-logical activities at concentrations down to 10"9 g per gram of tissue (British Nutrition Foundation 1992). These include the ability to contract smooth muscle, to inhibit or stimulate the adhesion of blood platelets and to cause constriction or dilation of blood vessels, with related influence on blood pressure. They also act upon cells of the immune system thereby influencing immune re-sponses and inflammatory reactions. The range of physio-logical functions and their nutritional significance is summarized in Table 7.10.

As early as the 1930s, physiologists knew that fatty acid-like substances in seminal plasma could cause a contraction or relaxation of smooth muscle and the active factor was called prostaglandin. Further work in Sweden and the Netherlands demonstrated that the activity was associated with oxygenated unsaturated fatty acids which could be shown by radiotracer techniques to be derived directly from arachidonic acid. In the intervening years, several more classes of compounds have been dis-covered: prostacyclins, thromboxanes and leukotrienes (British Nutrition Foundation 1992). These are now col-lectively called 'eicosanoids' because they are derived from 20-carbon precursor fatty acids, the eicosenoic acids (Fig. 7.11). Similar substances are derived from the n-3 family of polyunsaturated fatty acids EPA and DHA. (Compounds derived from DHA should strictly be called 'docosanoids' but for convenience are normally all included under the umbrella term 'eicosanoids'.)

Two groups of these metabolites, the prostacyclins and the thromboxanes have essentially opposing physio-logical effects. Prostacyclins, formed in arterial walls, are among the most powerful known inhibitors of platelet aggregation. They relax the arterial walls and promote a lowering of blood pressure. Thromboxanes, formed in platelets, stimulate platelets to aggregate (an important component of wound healing), contract the arterial wall and promote an increase in blood pressure. The balance between these activities is important in maintaining

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Table 7.10 Biological activities of eicosanoids derived from polyunsaturated fatty acids

Platelet aggregation

n-6 n-3

20:3 PGE, 20:5 PGE3 _ PGD, - PGD3 -

20:4 PGE2 + TXA3 + PGD2 - - PGI3 -

TXA2 +++ PGI2

20:3 PGE, + 20:5 PGE3 + LTB5 +

20:4 PGE2 +++ LTB4 +++

The immune system, inflammation

Cardiac electrical activity An increase in the ratio of n-3/n-6 polyunsaturated fatty acids in cardiac membrane phospholipids (which can be influenced by dietary intake) increases TXA3 and PGI3 at the expense of TXA2 and PGI2 and reduces risk of ventricular fibrillation and sudden cardiac death.

Activities in relation to muscles and blood vessels PGI2 and PGI3 are both vasodilators and therefore can influence blood pressure. Renal PGE2 and PGF2 counteract vasoconstrictor substances and help to regulate renal blood flow; they stimulate renin release and are involved in regulation of salt secretion. Prostaglandins stimulate contractions in various muscles. In the gut they are involved in peristaltic activity and in the uterus they are involved in labour contractions.

PGD, PGE, PGF, types of prostaglandins; PGI, prostacyclins; TXA, thromboxanes; LTB, leukotrienes. - , — , or , weakly, moderately or strongly antiaggregatory; +, or +++, weakly or strongly aggregatory/inflammatory.

normal vascular function. Several studies have demon-strated that altering the amounts and types of n-6 and n-3 polyunsaturated fatty acids in the diet can influence the spectrum of eicosanoids produced. For example, the substitution of fish oils in which n-3 polyunsaturated fatty acids predominate for diets in which linoleic acid (n-6) is the main polyunsaturated fatty acid (as typified by the average UK diet) results in changes in plasma and platelet fatty acid profiles from arachidonic acid to EPA as the predominant polyunsaturated fatty acid, a reduction in the formation of thromboxane A2 and an increase in the formation of thromboxane A3 in platelets. In general the potency of eicosanoids derived from n-3 polyunsaturated fatty acids is less than that of those de-rived from n-6 polyunsaturated fatty acids. Thus, blood

platelets are stimulated to aggregate less strongly by the 3-series thromboxanes than by those of the 2-series and the thrombotic tendency is reduced. Moreover the n-3 polyunsaturated fatty acids also tend directly to inhibit the cyclo-oxygenase enzyme responsible for eicosanoid biosynthesis.

Polyunsaturated fatty acids can also be metabolized to leukotrienes (Fig. 7.11). Arachidonic acid (n-6) gives rise to leukotriene B4/ which has considerable inflammatory potential, while EPA (n-3) gives rise to leukotriene B5, which is one to two orders of magnitude less inflam-matory. Dietary n-3 polyunsaturated fatty acids may inhibit the formation of leukotriene B4, thus reducing inflammatory responses.

The physiological effects of eicosanoids are so powerful

Arachidonic acid 20 :4 n-6

Cyclic Eiidoperoxides

Hydroperoxy acids

Prostacyclins Thromboxanes Leukotrienes Hydroxy-fatty acids

Prostaglandins

Fig. 7.11 The two major pathways of arachidonic acid metabolism to eicosanoids. (NSAID, non-steroidal antiinflammatory drugs, e.g. aspirin.)

FATS 117

that these substances are generated locally and destroyed immediately they have produced their effect. The excre-tion of eicosanoid breakdown products in urine has been used to estimate daily eicosanoid production and require-ments. The essential fatty acid precursors of eicosanoids are released from membrane phospholipids, which can therefore be regarded as a vast body store of EFA that are immediately available for eicosanoid biosynthesis. As they are depleted, they must be replaced by new poly-unsaturated fatty acids produced by the pathways illus-trated in Fig. 7.9. The mechanisms by which the relative proportions of the different eicosanoids are regulated, particularly how diet influences this regulation, and the quantitative relationship between the daily requirements for EFA, which are measured in grams, and the daily production of eicosanoids, which is measured in micro-grams, are subjects for future research.

Requirements for essential fatty acids

There is no question that linoleic acid, 9c,12c-18:2 (n-6), is essential for man. Overt EFA deficiency is seen only when it provides less than 1-2% of dietary energy, or 2-5 g daily for an adult. Rather more may be needed in pregnancy and lactation, although we cannot be precise because the body contains considerable stores in the adipose tissue and little is known about its ability to adapt to lower intakes. The argument that an even higher intake, say 10% of energy, is desirable in view of its effects in lowering the concentration of cholesterol in the blood, has nothing to do with the essentiality of linoleic acid in its strictest sense.

The essentiality of alpha-linolenic acid, 9c,12c,15c-18:3 (n-3), for man has remained in doubt (British Nutrition Foundation 1992). A case has been described of a young girl displaying neurological symptoms 4-5 months after being on total parenteral nutrition, in which the fat com-ponent contained mainly linoleic acid and only a minute quantity of alpha-linolenic acid. When safflower oil was replaced by soybean oil, containing much more alpha-linolenic acid, the neurological symptoms disappeared. Evidence has also been provided for alpha-linolenic acid deficiency in elderly patients fed by gastric tube. Like linoleic acid, alpha-linolenic acid cannot be made by the human body and must come from the diet. Since a large proportion of the lipids of the brain, and of specialized tissues such as the retina, are composed of long-chain polyunsaturated fatty acids of the n-3 family, ingested as such or derived from dietary alpha-linolenic acid, it is reasonable to assume its essentiality. It seems quite cer-tain, however, that only quite small amounts are required in human diets (British Nutrition Foundation 1992). The UK Department of Health (1991) dietary reference values for EFA are at least 1% of total energy as 18:2 n-6 and at least 0.2% as 18:3 n-3.

It can be argued that linoleic and alpha-linolenic acids are essential dietary components but that their longer chain more highly unsaturated derivatives (Fig. 7.9) are not, since they can be formed readily in the body from their precursor EFA. There is no doubt that the long-chain derivatives are essential metabolites since they are required in membranes and for eicosanoid production as described in earlier sections. Nevertheless, there is accumulating evidence that the activities of the human enzymes in-volved in the elongation and further desaturation of 18:2 n-6 and 18:3 n-3 are normally low (compared with the most studied animal, the rat) and that sometimes their activities are insufficient to produce the amounts of long-chain polyunsaturated products required. In such cases there may be an additional requirement for the metabolic products, gamma-linolenic and dihomo-gamma-linolenic acids in the n-6 family and EPA and DHA in the n-3 family. Such conditions may occur in early infant devel-opment, especially when babies are born prematurely (see below) or in diseases such as diabetes.

Fatty acids in growth and development Biological membranes are vital constituents of all cells in the body, and lipid (mainly phospholipid and choles-terol) is an important constituent of all biological membranes. Different membranes have widely different proportions of lipid to protein and the fatty acid compo-sition of the phospholipids also differs widely. Brain and nervous tissue membrane lipids contain a particularly high proportion of arachidonic acid (AA) and DHA and low concentrations of their 18-carbon precursors. The photoreceptor in the retina of the eye also contains a high proportion of the phospholipids containing two DHA moieties.

Human fetal brain experiences a rapid growth spurt in the last trimester of pregnancy, increasing four- or fivefold in weight during this time (British Nutrition Foundation 1992). Evidence from animal studies (and limited evidence from studies of human infants) suggests that learning ability and retinal function are permanently impaired if there is a failure to accumulate sufficient DHA during development. In progressing from maternal circulation to placenta to fetal liver and finally to fetal brain there is an increase in the concentration of AA and DHA and a decrease in their 18-carbon precursors. The present understanding is that the elongation and further desaturation of 18:2 n-6 and 18:3 n-3 that can occur in the placenta is a slow and inefficient process that cannot meet the demands for AA and certainly not for DHA at this stage of life. Selective incorporation of preformed long-chain polyunsaturated fatty acids from the mother's diet is thought be the main contributor to the accumu-lation of these fatty acids in nervous tissue. There are significant associations between dietary intakes of AA

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and DHA and birthweight, head circumference and placental weight. Intakes of these fatty acids are also posi-tively correlated with their content in the phospholipids of maternal and cord blood.

Postnatally, human milk provides a source of these long-chain polyunsaturated fatty acids (PUFA). Term human milk has an AA content of about 0.5% and a DHA content of about 0.3% of total fatty acids; the mean con-centrations in colostrum and preterm milk are somewhat higher. A dietary supply of long-chain polyunsaturates is particularly desirable for preterm infants who convert the 18-carbon precursors into long-chain polyunsaturates even less efficiently than term infants. The long-chain poly-unsaturates are not normally present in infant formulas, although some specialized formulas that contain them are now available for preterm babies. The need to incor-porate these fatty acids into commercial term infant feed products is currently under active investigation. Some research suggests that supplementing formulas with DHA improved infants' DHA status and visual acuity.

There is a tendency to emphasize the role of EFA in tissue growth and membrane formation but it should not be overlooked that saturated fatty acids are important, indeed vital, constituents of membranes. Thus, 57% of the fatty acids of the phosphatidylcholine of human brain grey matter are saturated, mainly located at position 1. The composition of myelin sheath is similar. Saturated fatty acids are clearly important in the structure of nervous tissue (Gunstone et al 1994). In contrast to the EFA, saturated fatty acids do not give rise to biologically active products like eicosanoids and it is not obligatory that they are supplied by the diet, although we cannot be sure what proportion of the body's structural fatty acids are derived from the diet and what proportion from biosynthesis de novo.

Role as energy source: storage and utilization Adipose tissue stores

Triacylglycerols represent a very concentrated form of fuel with a gross energy value of 38 kj/g.

The body's fat reserves are stored in the white adipose tissue which is comprised mainly of mesenchymal con-nective tissue cells called adipocytes or more commonly 'fat cells'. Fat cells have an enormous potential to expand as more fat is available to be accommodated. Adipose tissue begins to develop in the fetus, and the newborn baby weighing 3.5 kg has about 560 g. In a healthy man, about 15% of his body weight will be adipose tissue, about 85% of which is triacylglycerol and the rest fat-free cellular material. A healthy woman carries relatively more adipose tissue, amounting to about 25% of her body weight; normally about 15 kg or so. In a very emaciated person this is reduced to about 1 kg, while some very

obese people can carry around 100 kg or more. The health problems associated with excessive adipose tissue mass are described in Chapter 34.

Fetal adipose tissue has to synthesize most of its fat from glucose; fat cells therefore possess all the pathways for the de novo biosynthesis of lipids. The activities of these enzymes are largely suppressed by high fat intakes later in life (see p. 112).

The lipolysis of fatty acids from fat cell triacylglycerols is catalysed by the so-called 'hormone-sensitive lipase'; this enzyme is important when fatty acids need to be mobilized from fat cells as an energy source (see below). Even when the adipose tissue mass is undergoing little or no expansion or contraction, there is a small 'turnover' of fatty acids in fat cells as a result of continual esterifica-tion and lipolysis. It has been estimated, for example, that the time taken to replace half the linoleic acid in adult human adipose tissue is between 350 and 750 days. Because of this slow turnover, the fatty acid composition of adipose tissue tends to reflect the fat composition of the diet for some time in the past. Because linoleic acid is derived entirely from the diet and none from body syn-thesis, its concentration in adipose tissue makes an ideal marker for its dietary intake. Small pieces of fat tissue are taken by needle biopsy and analysed for fatty acid composition by gas-liquid chromatography. The method is a more reliable measure of habitual fatty acid intake than dietary surveys which have a large random error (Ch. 17). It can also provide a good estimate of relative trans fatty acid intakes but a simple relationship between dietary intake and adipose tissue composition does not hold true for all fatty acids (e.g. arachidonic, alpha-linolenic and medium-chain fatty acids) because they may be selectively metabolized in other tissues rather than being stored. Nevertheless, adipose tissue fatty acid composition is increasingly being used as a marker of intake in epidemiological studies of the association between dietary fats and disease (see Ch. 17).

The body obtains a considerable proportion of its meta-bolic energy by oxidizing fatty acids in the mitochondria (see p. 113). A low circulating insulin concentration limits the activity of adipose tissue LPL and signals the activation of hormone-sensitive lipase to mobilize fatty acids from adipose tissue. Overall, fat mobilization is stimulated under conditions when adrenergic activity predominates (e.g. in exercise or starvation); it is sup-pressed when insulin predominates (e.g. after meals, in the postprandial state). The conditions favouring mobili-zation are also those that stimulate the uptake of fatty acids into tissues, such as muscle, that need to utilize fatty acids as energy sources.

The long-held belief that the energy derived from dietary carbohydrate is equivalent, in terms of utilization and storage, to the energy derived from fat has been challenged by research that indicates that a unit of energy

FATS 119

from fat may be 'more fattening' than a unit of energy from carbohydrate (Ch. 34). There are several reasons why this may be so: the capacity to store carbohydrate (as glycogen) in the body is limited whereas fat storage in adipose tissue is virtually unlimited; the human body's capacity to convert carbohydrate into fat is limited and the body adapts much more rapidly to oxidizing excess carbohydrate than excess fat. It is hoped that the devel-opment of reduced fat products and fats with lower energy values (see p. 105) will encourage reduced fat intakes in the population.

Specialized storage depots

Adipose tissue is organized into distinct depots, ranging in size from up to 30% to less than 1% of its total mass.

REFERENCES

British Nutrition Foundation 1992 Unsaturated fatty acids: nutritional and physiological significance. Report of the British Nutrition Foundation's Task Force. Chapman and Hall, London

Brown M S, Kovanen P T, Goldstein J L 1981 Regulation of plasma cholesterol by lipoprotein receptors. Science 212:628-635

Some depots are closely associated with individual organs, such as the mammary glands, kidneys and lymph nodes. The selective depletion and expansion of some of these depots is difficult to explain in terms of routine needs for metabolic energy and it is likely that they have developed specialized functions. Thus the adipose tissue associated with lymph nodes may play a role in nourishing and regulating the cells of the immune system (Nutrition Society 1996). Mammary adipose tissue sup-plies substrates for milk fat synthesis. Brown adipose tissue, which is conspicuous in newborn babies in the thorax and between the scapulae, is specialized for the production of heat to maintain body temperature. In this tissue, heat is generated as a result of the uncoupling of the oxidation of substrates in the mitochondrial electron transport chain from ATP synthesis.

Burr G O, Burr M M 1929 A new deficiency disease produced by rigid exclusion of fat from the diet. Journal of Biological Chemistry 82:345-367

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SUMMARY

• Fats in the body play a key role in membrane structure and are stored in adipose tissue as a fuel reserve. Fats in foods provide a concentrated form of metabolic energy. They often improve flavour perception and impart a pleasing texture to foods thereby increasing palatability. Dietary fats also supply essential nutrients: fat-soluble vitamins (Ch. 13) and essential fatty acids. The latter perform vital body functions but cannot be made in the body and so are essential in the diet.

• Two primary essential fatty acids, linoleic and alpha-linolenic acids, are converted in the tissues into longer chain length, more highly unsaturated fatty acids that are located principally in biological membranes. These contribute to membrane structure and are also converted into oxygenated fatty acids, the eicosanoids. These hormone-like substances are locally produced and have many functions in cellular communication. They are involved in the regulation of blood coagulation, blood pressure, muscular contraction, immune function and inflammatory responses and diverse aspects of the regulation of cellular metabolism.

• Fats are normally efficiently digested and

absorbed; when normal fat absorption is impaired, fats containing short- and medium-chain length fatty acids can be effectively utilized. The products of fat digestion are resynthesized into lipids that are transported in the bloodstream as lipoproteins. The protein components of lipoproteins interact with specific receptors on cell surfaces to allow efficient clearance and utilization of the products of fat digestion. Malfunction of these receptors, as a result of gene defects, dietary imbalance or ineffective energy expenditure results in aberrations in lipoprotein metabolism that can lead to chronic diseases (Chs 37, 42). Overconsumption of fats without concomitant increases in the utilization of the energy leads to excessive storage in adipose tissue and finally obesity (Ch. 33). Tailor-made fats with zero or reduced energy value are now being introduced into foods.

• Different foods supply different amounts of fat with widely differing fatty acid compositions. Improvements in food composition databases (Ch. 16) and better information on food labels now allow selection of foods to help consumers regulate the amount of fat eaten and ensure an appropriate balance of fatty acids.

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