Chapter 5. Vitamin E

Vitamin E 5.1 Background Vitamin E is represented by eight vitamers of varying biological potency: four tocopherols and four tocotrienols.. Vegetable oils are highly unsaturated and cont

Vitamin E

5.1 Background

Vitamin E is represented by eight vitamers of varying biological potency: four tocopherols and four tocotrienols The vitamin functions as a biologi-cal antioxidant by protecting the vital phospholipids in cellular and subcellular membranes from peroxidative degeneration

A deficiency of vitamin E in animals results in a variety of pathological con-ditions that affect the muscular, cardiovascular, reproductive, and central nervous systems, as well as the liver, kidney, and red blood cells The diver-sity of these disorders is attributable to secondary effects of the widespread damage caused to the membranes of muscle and nerve cells by lipid peroxidation There is a marked difference between animal species in their susceptibility to different deficiency disorders A complex biochemical inter-relationship exists between vitamin E and the trace element selenium Unsaturated fat, sulfur-containing amino acids, and synthetic fat-soluble antioxidants are also implicated in some disorders It is well documented that a diet rich in polyunsaturated fat, but which does not contain a corre-spondingly high amount of vitamin E, induces deficiency signs in animals Aside from instances of fat malabsorption or genetic abnormalities of lipid metabolism, clinical vitamin E deficiency is rare in adult humans and no recognizable deficiency syndrome has been demonstrated This

is due to the occurrence of the vitamin in a wide variety of foods, its wide-spread storage distribution throughout the body tissues, and the conse-quent extended period required for depletion However, various symptoms have been reported in preterm infants; these include hemolytic anemia, oedema, colic, and failure to thrive

Vitamin E, being fat-soluble, accumulates in the body, especially in the liver and pancreas Unlike vitamins A and D, however, vitamin E is essentially nontoxic

A possible role for vitamin E as a preventative factor for cardiovascular disease, cancer, Alzheimer’s disease, and other disease states involving oxidative stress is under intensive investigation

5.2 Chemical Structure, Biopotency, and

Physicochemical Properties

5.2.1 Structure

Tocopherols are methyl-substituted derivatives of tocol, which comprises

a chroman-6-ol ring attached at C-2 to a saturated isoprenoid side chain Tocotrienols are analogous structures whose side chains contain three trans double bonds In nature, there are four tocopherols and four corresponding tocotrienols; these are designated as alpha- (a), beta- (b), gamma- (g) and delta- (d) according to the number and position of the methyl substituents in the chromanol ring (Figure 5.1) The b- and

Tocotrienols possess only the chiral center at C-2

is produced by the condensation of trimethylhydroquinone with synthetic phytol or isophytol This method of synthesis results in

mixture of all eight possible diastereoisomers in virtually equal pro-portions The four enantiomeric pairs are RRR/SSS, RRS/SSR, RSS/SRR, and RSR/SRS

O

O HO

HO

CH3

CH3 CH3 CH3 CH3

CH3

H3C H H3C H

1 2 3 4 5 6

7 8

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′12′

3′

4′

7′

8′

11′

12′

(a)

(b)

Tocopherol or Tocotrienol 5,7,8-Trimethyl

5,8-Dimethyl 7,8-Dimethyl 8-Methyl

α β γ δ FIGURE 5.1

Stereochemical structures of tocol and tocotrienol: (a) RRR-tocol and (b) 2R,30-trans,

70-trans-tocotrienol.

The RS system of nomenclature for some a-tocopherols is given in

The principal commercially available forms of vitamin E used in the food, feed, and pharmaceutical industries are the acetate esters

of RRR-a-tocopherol and all-rac-a-tocopherol In commercial circles,

refer-red to by the trivial name of dl-a-tocopheryl acetate RRR-a-tocopheryl acetate is obtained by extraction from vegetable oils Since it is not isolated without chemical processing, it cannot legally be called natural, but it can

be described as derived from natural sources Another commercial preparation, not commonly used, is the hydrogen succinate of RRR-a-tocopherol

The term “vitamin E” is the generic descriptor for all tocol and tocotrienol derivatives that exhibit qualitatively the biological activity of a-tocopherol The term “tocopherol” refers to the methyl-substituted derivatives of tocol and is not synonymous with the term vitamin

E The tocopherols and tocotrienols may be referred to collectively as tocochromanols In food and clinical analysis, commonly used methods

do not distinguish the stereoisomers of vitamin E and therefore the toco-chromanols are referred to without their stereochemical designation

5.2.2 Biopotency

Only the naturally occurring RRR-a-tocopherol and the 2R-stereoisomeric forms of a-tocopherol (RRR-, RRS-, RSS-, and RSR-a-tocopherol) obtained from synthetic all-rac-a-tocopherol ester are maintained in plasma and delivered to tissues This is because of the discriminating ability of the hepatic a-tocopherol transfer protein (Section 5.4.3) and the fact that the vitamin E vitamers are not interconvertible in the human body There-fore, when establishing recommended intakes, the definition of vitamin E

is limited to the 2R-stereoisomeric forms of a-tocopherol [1] On the basis of this definition, all-rac-a-tocopherol has one-half the activity of

TABLE 5.1

Nomenclature for Some a-Tocopherols

Configuration

Designated Name

Trivial Name Description 2R,40R,80R RRR-a-Tocopherol d-a-Tocopherol The only isomer of

a-tocopherol found

in nature 2S,40R,80R 2-epi-a-Tocopherol I-a-Tocopherol C-2 epimer of RRR form 2RS,40RS,80RS

(mixture of four

enantiomeric pairs)

all-rac-a-Tocopherol dl-a-Tocopherol Totally synthetic

(produced from synthetic phytol

or isophytol)

RRR-a-tocopherol This 2:1 activity ratio for natural and synthetic vitamin

E has been demonstrated in human studies [2] and is more relevant to human needs than the officially accepted 1.36:1.00 ratio that is based on the rat resorption –gestation assay [3]

Deuterium-labeling of RRR-a-tocopherol and its acetate and succinate esters in healthy humans showed that these compounds are absorbed

to an equal extent overall, although the initial rate or absorption is higher from the acetate ester than from the succinate ester [4] These compounds can therefore be accorded equal potency on a molar basis

5.2.3 Physicochemical Properties

5.2.3.1 Appearance and Solubility

In the pure state, tocopherols and tocotrienols are pale yellow, nearly odorless, clear viscous oils which darken on exposure to air a-Tocopheryl acetate is of similar appearance The hydrogen succinate ester is a white granular powder

Nonesterified tocopherols and tocotrienols are insoluble in water and readily soluble in ethanol, other organic solvents (including acetone, chloroform, and ether), and in vegetable oils The vitamin E acetates are less readily soluble in ethanol than the unesterified vitamers

5.2.3.2 Stability in Nonaqueous Solution

Tocopherols and tocotrienols are destroyed fairly rapidly by sunlight and artificial light containing wavelengths in the UV region The vitamers are slowly oxidized by atmospheric oxygen to form mainly biologically inac-tive quinones; the oxidation is accelerated by light, heat, alkalinity, and certain trace metals The presence of ascorbic acid completely prevents the catalytic effect of iron(III) and copper(II) on vitamin E oxidation by maintaining these metals in their lower oxidation states [5] The tocotri-enols, by virtue of their unsaturated side chains, are more susceptible to destruction than the tocopherols The vitamers can withstand heating in acid or alkaline solution provided that oxygen and UV radiation are excluded Because a-tocopheryl acetate lacks the reactive hydroxyl group, air and light have practically no destructive effect

5.2.3.3 In Vitro Antioxidant Activity

stabil-ize animal fats, which have a much lower vitamin E content than veg-etable oils In the absence of an antioxidant, unsaturated fats undergo autoxidation to produce hydroperoxides These break down further to give a variety of volatile compounds such as aldehydes and ketones, which produce the disagreeable odors and flavors of rancidity

The order of in vitro antioxidant activities of the tocopherols conforms

to their oxidation potentials and parallels their biological activities, that

in detailed studies, although there was no significant difference between the b and g positional isomers [6]

5.3 Vitamin E in Foods

5.3.1 Occurrence

The important plant sources of vitamin E are the cereal grains and those nuts, beans, and seeds that are also rich in high-potency oils The vegetable oils extracted from these plant sources are the richest dietary sources of vitamin E Cereal grain products, fish, meat, eggs, dairy products, and green leafy vegetables also provide significant amounts Major sources

of vitamin E in the United States include margarine, mayonnaise and salad dressings, fortified breakfast cereals, vegetable shortenings and cooking oils, peanut butter, eggs, potato crisps, whole milk, tomato pro-ducts, and apples [7]

Vegetable oils are highly unsaturated and contain a correspondingly high concentration of vitamin E to maintain the oxidative stability of their constituent polyunsaturated fatty acids (PUFAs) The distribution

of tocopherols and tocotrienols in different plant oils varies greatly, as shown in Table 5.2 [8] In some vegetable oils, notabley soybean oil, g-tocopherol is the major vitamer present and in palm oil g-tocotrienol predominates Thus measurement of total tocopherols does not accurately

TABLE 5.2

Distribution of Tocopherols and Tocotrienols in Selected Vegetable Oils

Oil a-T a b-T a g-T a d-T a a-T3 a b-T3 a g-T3 a d-T3 a

Corn (maize) 25.69 0.95 75.23 3.25 1.50 — 2.03 — Olive 11.91 — 1.34 — — — — — Palm 6.05 — tr — 5.70 0.82 11.34 3.33 Peanut 8.86 0.38 3.50 0.85 — — — — Rapeseed 18.88 — 48.59 1.20 — — — — Safflower 44.92 1.20 2.56 0.65 — — — — Soyabean 9.53 1.31 69.86 23.87 — — — — Sunflower 62.20 2.26 2.67 — — — — — Note: —, Not detected; tr, trace.

a

Mean values (6–10 determinations) of each oil purchased from 3 to 5 different manu-facturers in mg/100/g.

Source: Syva¨oja, E.-L et al., J Am Oil Chem Soc., 63, 328, 1986 With permission.

represent the vitamin E biological activity of vegetable oils or food products containing them

Table 5.3 shows the vitamin E content of a selection of Finnish foods determined using high-performance liquid chromatography (HPLC)

TABLE 5.3

Distribution of Tocopherols and Tocotrienols in Selected Finnish Foods

Item a-Ta b-Ta g-Ta d-Ta a-T3a b-T3a g-T3a d-T3a Ref.

1.2 –1.4% ash b 1.6 0.8 — — 0.3 1.7 — nd

ca 0.7% ash c 0.4 0.2 — — 0.2 1.5 — nd

ca 0.5% ash d 0.2 0.1 — — 0.1 1.4 — nd Wheat bran 1.6 0.8 — — 1.5 5.6 — nd [9] Wheat germ 22.1 8.6 — ,0.1 0.3 1.0 — nd [9] Peanut 10.89 0.27 8.39 0.17 — nd — nd [10] Broccoli 0.68 tr 0.14 — — nd — nd [10] Lettuce 0.63 tr 0.34 — — nd — nd [10] Spinach 1.22 — — — — nd — nd [10] Tomato 0.66 tr 0.20 tr — nd — nd [10] Sweet pepper 2.16 0.11 0.02 tr — nd — nd [10] Orange 0.36 tr tr — — nd — nd [10] Banana 0.21 tr tr — — nd — nd [10] Peach (flesh only) 0.96 tr 0.05 — — nd — nd [10] Raspberry 0.88 0.15 1.47 1.19 — nd — nd [10] Blackcurrant 2.23 tr 0.83 tr — nd — nd [10]

summer 0.11 — — nd tr nd nd nd winter 0.06 — — nd tr nd nd nd

summer 2.00 — — nd 0.07 nd nd nd winter 1.01 — — nd 0.11 nd nd nd Egg, whole e 1.96 0.04 0.08 nd 0.25 nd nd nd [11]

spring 0.34 — 0.01 nd 0.05 — nd nd autumn 0.60 tr 0.01 nd 0.04 tr nd nd

spring 0.25 — 0.01 nd 0.01 tr nd nd autumn 1.37 — 0.01 nd tr — nd nd Pork, raw, shoulder 0.47 — 0.01 nd 0.05 tr nd nd [12] Chicken, raw 0.70 tr 0.06 nd 0.03 tr nd nd [12] Cod, raw 1.05 — — nd nd nd nd nd [13] Salmon, raw 2.02 — 0.02 nd nd nd nd nd [13] Note: —, Not detected; nd, not determined; tr, trace.

a Pooled samples in mg/100 g.

b

Milled mainly from the aleurone tissue.

c

Extraction rate ca 78%.

d

Extraction rate ca 74%.

e

Eggs from hens fed with vitamin supplements containing a-tocopheryl acetate.

with fluorometric detection Among the cereal grains, wheat, maize, barley, rye, rice, and oats are important plant sources of vitamin E The vitamin E content of cereal grains is influenced by plant genetics and is adversely affected by too much rain and humidity during harvest [14] The germ fraction of the cereal grains contains a far higher proportion

of tocopherols, and therefore a greater vitamin E activity, than the endo-sperm and other nongerm fractions in which most of the tocotrienol content of the grain in found [15,16] Thus flour, which is derived from endosperm, has a low vitamin E activity compared with milling fractions containing germ and aleurone tissue Wheat germ is the richest source of vitamin E among the various milling products

Most of the common nontropical vegetables and fruits contain less than

1 mg a-tocopherol equivalents/100 g fresh weight, a-tocopherol being the predominant vitamer present Green leafy vegetables are included among the richer vegetable sources of vitamin E The mature dark green outer leaves of brassicae, which are usually discarded, contain more vitamin

E than the lighter green leaves which are consumed The almost colorless heart of white cabbage and the florets of cauliflower contain practically no vitamin E, the determined tocopherol values for these vegetables being attributable to the green parts included [17] Paradoxically, yellow senescent leaves that have lost their chlorophyll contain much more a-tocopherol than fresh leaves [18] Presumably a-tocopherol, which resides in the chloroplasts, protects chlorophyll from destruction by the action of oxygen produced by photosynthesis, and is used up during high photosynthetic activity In apples and pears, the concentration of vitamin E is greater in the skin than in the flesh Green cooking apples contain more tocopherol than red or yellow types [17]

The concentration of vitamin E in animal tissues depends on the amount of vitamin in the animal’s diet In raw muscle, fat, and organs from mammals and birds, most of the vitamin E is in the form of a-tocopherol The a-tocopherol content of mammalian muscle is generally less than 1 mg/100 g There is a marked seasonal variation in the a-tocopherol content of beef and mutton, the values being about twice as high in the autumn as in the spring Cow liver shows a much greater seasonal variation The feeding of grass or fresh silage during the summer and dry forage and concentrates during the winter explains the higher autumn values observed in ruminants During the same season, the tocopherol concentration in different meat cuts of a given animal species increases with increasing fat content The a-tocopherol content of cow’s milk is higher

in summer than in winter owing to the changes in the animal’s diet In eggs, all the vitamin E is in the yolk; the concentration varies greatly depending

on the level of supplemental a-tocopheryl acetate (if any) contained in the chicken feed Contrasting values of 0.46 and 1.10 [19], 0.70 [20], and 1.96 [11]

mg a-tocopherol/100 g whole egg have been reported

In general, fish is a better source of vitamin E than meat Tuna and salmon canned in water contained, respectively, 0.53 [21] and 0.7 [22]

mg a-tocopherol/100 g and sardines canned in tomato sauce contained 3.9 mg/100 g [22]

Vitamin E is sometimes added to whole milk powder and breakfast cereals to supplement dietary requirements The vitamin E requirement increases with an increased intake of PUFA and hence several types of high-quality dietetic margarines are enriched with vitamin E The acetate ester of a-tocopherol, rather than the free alcohol, is used as a food supplement on account of its greater stability

5.3.2 Stability

The effects of processing on vitamin E retention have been reviewed in Ref [23] During processing, the food is exposed to the destructive influ-ences of oxygen, light, heat, and metal ions Therefore, refined and processed foods are variable and usually less predictable sources of vitamin E than whole fresh foods Frozen vegetables retain much of their vitamin E content, but losses in the canning of beans, peas, and sweetcorn can be as high as 70 – 90% [24] Frozen foods which have been fried in vegetable oil suffer a great loss of vitamin E during freezer storage This loss is presumably due to destruction by hydroperoxides, which are more stable at low temperatures than at high temperatures, and hence accumulate [25] Vitamin E is not destroyed during the normal cooking of meat and vegetables Little loss of the vitamin occurs during deep-fat frying in fresh vegetable oil, but shallow-pan frying is destructive

The thermal stabilities of the vitamin E vitamers in food vary with the heating time, heating method, and food composition The order of vitamer stability in rice bran heated in a microwave for 12 min is

In the production of white wheat flour from whole-grain wheat, the vitamin E content is reduced by about 50% due to the removal

of bran and germ [28] This reduction of vitamin E content is not usually compensated for by fortification Storage of wholewheat flour at 208C for 1 yr resulted in the following losses of constituent toco-chromanols: a-T, 44%; a-T3, 41%; b-T, 23%; b-T3, 22% Corresponding losses for stored white flour were: a-T, 42%; a-T3, 42%; b-T, 27%; b-T3, 29% [29]

In the making of French bread by the Chorleywood process, doughmaking was the only stage which resulted in major loss of

vitamin E (30 – 40%) Preparation of sour dough in the making of wheat/ rye bread decreased the vitamin E content of whole rye flour by about 60% [29]

Among various industrial processes for the manufacture of cereal pro-ducts such as breakfast cereals, drum drying and extrusion cooking of wheat flour result in a 90% loss of vitamin E [28] Vitamin E destruction during these two processes is due partly to the lipid degradation caused

by endogenous enzymes Polyunsaturated fatty acids originating from the lipase-catalyzed hydrolysis of cereal lipids are readily peroxidized by lipoxygenase, and peroxidizing lipids cause loss of vitamin E due to the vitamin’s antioxidant action Peroxidation begins immediately after water

is added to the cereal, as in the first stage of drum drying, and is enhanced

in the presence of copper and iron Nonenzymatic oxidation of vitamin E may also take place when the process temperature has passed the point at which the enzymes are heat-inactivated (about 608C) Ha˚kansson and Ja¨gerstad [30] reported that the steam flaking of whole-grain wheat inactivated lipoxygenase with no loss of vitamin E After drum drying, about 50% of the vitamin E of the steam-flaked flour was retained compared with 10–15% in the untreated flour Microwave treatment was another effective way of inactivating enzymes and improving vitamin E retention

Shin et al [31] studied the effect of variations in extrusion temperature (110, 120, 130, and 1408C) and holding time (0, 3, and 6 min) on the con-centration of individual of vitamin E vitamers in rice bran Rice bran oil contains a relatively high proportion of tocotrienols compared with other vegetable oils Increasing the extrusion temperature from 110 to 1408C with 0-min holding time resulted in a 5–10% loss of total vitamin

E At an extrusion temperature of 1108C, increasing the holding time from 0 to 6 min resulted in a 3 –6% loss of total vitamin E Storage losses of the raw and extruded rice bran were also determined Raw rice bran lost 44% of total vitamin E after 35 days of storage at ambient temperature, the least stable vitamers being a-tocopherol and a-tocotrienol (ca 57% loss) After 1 yr of storing raw bran, 73% of the total vitamin E was lost, the least stable vitamers being g-tocotrienol (78% loss) and a-tocopherol (75% loss) Rice bran extruded at 1108C with 0-min holding time lost 21 and 46% of its initial total vitamin E after 7 and 105 days of storage, respectively, with no difference in degra-dation rates among vitamers Lipoxygenase activity probably accounted for the destruction of vitamin E in raw bran Extrusion temperature, exposure to trace metals, and damage to the grains would account for the vitamin E losses in extruded bran

Vitamin E is the most radiation-sensitive of the fat-soluble vitamins The

losses: 46, 62, and 74%, respectively, of total tocopherols and 51, 69, and

85%, respectively, of total tocotrienols The order of loss of individual

as meats are a poor source of vitamin E, this is of little nutritional significance

Wyatt et al [34] measured the cooking losses of vitamin E (determined

as a- and g-tocopherols on a dry weight basis) from selected foods com-monly consumed in the Mexican diet Among grains, oats lost 22% of vitamin E, while rice, corn, and wheat lost 42 –55% Among legumes, gar-banzo beans (chick peas) and black and pinto beans gave low (9–17%) losses compared with bayo and faba beans, lentils and split peas (38 –59%) Legumes giving low cooking losses contained predominantly

destruction of the vitamin when the corn is steeped in lime water prior

to dough-making

5.3.3 Expression of Dietary Values

Vitamin E activity is commonly expressed as milligrams of a-tocopherol equivalents using data from rat fetal resorption assays to calculate the equivalencies [3] However, in 2000, the Institute of Medicine [1] redefined vitamin E in human physiology as solely the 2R-stereoisomers

of a-tocopherol (Section 5.2.2) In the light of the new findings in humans,

it is necessary to reevaluate the relative biological potencies of the vitamin

E vitamers Therefore, it is best to measure and report the actual concen-trations of each vitamer in food

5.3.4 Applicability of Analytical Techniques

HPLC is ideally suited for the measurement of the individual tocopherols and tocotrienols For the analysis of those animal products known to contain predominantly a-tocopherol, only this vitamer need be deter-mined In vitamin E-fortified foods, it is usually sufficient to determine either the added a-tocopheryl acetate or the total a-tocopherol (natural plus added vitamin)

5.4 Intestinal Absorption and Transport

The following discussion of absorption and transport is taken from a more detailed account in a book by Ball [35] published in 2004