Tocopherols in Milk

Erickson, D. R. and Dunkley, W. L. 1964. Spectrophotometric determination of tocopherol in milk and milk lipides. Anal. Chem. 36, 1055-1058. 

AOAC official method 992.03. Vitamin E activity (all-rac-a-tocopherol) in milk-based infant formula. Liquid chromatographic method. In MP Bueno, ed. Official Methods of Analysis of AOAC International. 16th ed. Arlington, VA AOAC International, 1995, p. 50-4-50-5. 

Vitamin E is an effective scavenger of lipid peroxy radicals and is efficient at protecting unsaturated fatty acids against lipid peroxidation. The chemistry of vitamin E is rather complex as there are eight compounds, four tocopherols and four tocotrienols, which exhibit vitamin E activity. The relative bioactivity of the various compounds varies considerably, from 1.0 for a-tocopherol to 0.03 for 8-tocopherol. In milk, a-tocopherol accounts for virtually all of vitamin E, although very small amounts of (3-tocopherols and y-tocopherols are present. Also, the concentration of a-tocopherol in milk fat varies widely, with the level in samples of commercial butter ranging from 18 to 35 pg/g fat (MAFF, 1999). These concentrations equate to a low level of vitamin E bioactivity, 0.025 to 0.05 IU/g fat. 

Clearly, more research is required to clarify the somewhat confused picture regarding the role of enzymes in the oxidation of milk lipids. However, the key factor affecting the susceptibility of milk to oxidation appears to be its relative content and distribution of pro-oxidants and antioxidants. Bruhn and Franke (1971) reported that spontaneous oxidation is directly proportional to the copper content and inversely proportional to the a-tocopherol content of milk. Charmley et al. (1991) showed that intramuscular injection of cows with a-tocopherol may overcome a spontaneous oxidized flavor problem caused by low levels of a-tocopherol in milk. In general, milk from pasture-fed cows is less susceptible to oxidation due to a higher content of tocopherols than milk from cows given dry feed (Bruhn and Franke 1971 Urbach, 1989, 1990). 

Tocopherols (vitamin E), which are discussed more fully in Chapter 6. The principal function of tocopherols in vivo is probably to serve as antioxidants. The concentration of tocopherols in milk and meat products can be increased by supplementing the animal s diet. 

Since the tocopherol content of feeds varies with the season and since the amount of tocopherol in milk is a reflection of the dietary intake, it is to be expected that the tocopherol content of butter will vary throughout the year. Anglin, Mahon, and Chapman (1955) estimated the tocopherol content of 329 butter samples collected throughout the year from 28 towns in 8 provinces in Canada. The average values by months are presented in Kg. 5. Highest values were noted during the late summer months and lowest values during the late winter months. A 100% variation occurred,... 

Azeredo, V. B. and N. M. Trugo (2008). Retinol, carotenoids, and tocopherols in the milk of lactating adolescents and relationships with plasma concentrations. Nutrition 24(2) 133-139. 

Romeu-Nadal, M., Morera-Pons, S., Castellote, A. 1., and Lopez-Sabater, M. C. (2006). Determination of gamma- and alpha-tocopherols in human milk by a direct high-performance liquid chromatographic method with UV-vis detection and comparison with evaporative Ught scattering detection. /. Chromatogr. A 1114,132-137. 

Erickson, D. R., Dunkley, W. L. and Ronning, M. 1963. Effect of intravenously injected tocopherol on oxidized flavor in milk. J. Dairy Sci. 46, 911-915. 

In cow s milk, nearly all of the vitamin E is a-tocopherol and the level can vary with the cow s feed and the season of the year (Lampert 1975). For example, summer milk can contain five times more vitamin E (1.1 mg a-tocopherol per quart) than winter milk (0.2 mg/quart) (Hertig and Drury 1969 McLaughlin and Weihrauch 1979). It is suggested that vitamin E, due to its antioxidant properties, may have some effect in retarding the development of oxidized flavor in milk (Lampert 1975). 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

W Kneifel, F Ulberth, U Winkler-Macheiner. HPLC methods for the simultaneous determination of retinol and tocopherol in butter and whole-milk powder. Deutsche Lebensm Rundschau 83 137-139, 1987. (In German). 

Vitamin E is widespread in foods and is stored in the body so that deficiency states are very rare. A possible exception may be premature infants with very low fat stores. The concentration of a-tocopherol in cows milk ranges from 3.0 to 5.0 mg/L and is present at about the same level in human milk. While vitamin E has been shown to be essential for normal fertility in rats and other animals, it has never been proven to be necessary for human fertility. However, in recent years there has been renewed interest in the antioxidant function of vitamin E [e.g., in protecting the cardiovascular system (Sytkowski et al., 1990 Gurr, 1994)]. 

The superoxide radical (O ) is another reactive oxygen species. Although the superoxide radical may not react readily with unsaturated fatty acids, it is capable of oxidizing phenolic compounds such as tocopherols, thiols and ascorbic acid (Korycka-Dahl and Richardson, 1980). This, in turn, may lead to earlier oxidation of lipids. However, the significance of superoxide as a pro-oxidant in milk is unclear. Superoxide rapidly dismu-tates in water, yielding hydrogen peroxide which, itself, may be involved in oxidative reactions (Korycka-Dahl and Richardson, 1980) ... 

The feasibility of increasing the a-tocopherol concentration of milk by supplementation of the feed has been investigated in many studies (Dunkley et al., 1966, 1967 King et al., 1966 St. Laurent et al., 1990 Barrefors et al., 1995 Focant et al., 1998 Granelli et al., 1998). These studies showed that when feed was supplemented with varying levels of a-tocopheryl acetate, the a-tocopherol content of the milk was increased with consequent increased resistance to spontaneous and copper-induced oxidation. King et al. (1967) reported that when feed was supplemented to achieve an intake of 1 g a-tocopherol per day per cow, oxidation was effectively controlled in milk... 

Only about 2% of ingested a-tocopherol is actually transferred to the milk (King et al., 1966 Dunkley et al., 1968b Schingoethe et al., 1979). Consequently, the economics of direct supplementation of feed with a-tocopherol are unfavourable (Bruhn et al., 1976). If protected supplements are fed, however, the potential for transfer to milk is much greater. Goering et al. (1976) fed protected safflower supplement to cows and reported a 200% increase in the a-tocopherol content of the milk. Control of oxidized flavor by direct addition of emulsified a-tocopherol to milk can be achieved with only 1% of the amount required by ration supplementation (Weihrauch, 1988). 

St. Laurent, A M., Hidiroglou, M., Snoddon, M., Nicholson, J.W.G. 1990. Effect of a-tocopherol supplementation to dairy cows on milk and plasma a-tocopherol concentrations and on spontaneous oxidized flavor in milk. Can. J. Anim. Sci. 70, 561-570. 

Chauveau-Duriot, B. Doreau, M. Noziere, P. Graulet, B. 2010. Simultaneous quantification of carotenoids, retinol, and tocopherols in forages, bovine plasma, and milk validation of a novel UPLC method. Anal. Bioanal. Chem. 397 777-790. 

Kadioglu, Y. Demirkaya, F. Demirkaya, A.K. 2009. Quantitative determination of underivatized alpha-tocopherol in cow milk, vitamin and multivitamin drugs by GC-FID. Chromatographia 70 665-670. 

The composition of milkfat is somewhat complex. Although dominated by triglycerides, which constitute some 98% of milkfat (with small amounts of diglycerides, monoglycerides, and free fatty acids), various other lipid classes are also present in measurable amounts. It is estimated that about 500 separate fatty acids have been detected in milk lipids it is probable that additional fatty acids remain to be identified. Of these, about 20 are major components the remainder are minor and occur in small or trace quantities (4, 5). The other components include phospholipids, cerebrosides, and sterols (cholesterol and cholesterol esters). Small amounts of fat-soluble vitamins (mainly A, D, and E), antioxidants (tocopherol), pigments (carotene), and flavor components (lactones, aldehydes, and ketones) are also present. 

It has been proposed that spontaneous milks have a high content (10 times normal) of xanthine oxidase (XO). Although addition of exogenous XO to non-susceptible milk induces oxidative rancidity, no correlation has been found between the level of indigenous XO and susceptibility to oxidative rancidity. The Cu-ascorbate system appears to be the principal pro-oxidant in susceptible milk. A balance between the principal antioxidant in milk, a-tocopherol (Chapter 6), and XO may determine the oxidative stability of milk. The level of superoxide dismutase (SOD) in milk might also be a factor but there is no correlation between the level of SOD and the propensity to oxidative rancidity. 

Oxidative lipid deterioration in milk and dairy products occurs mainly during storage. The rate of oxidation depends on the concentration of dissolved oxygen, the storage temperature, and the presence of antioxidants (a-tocopherol, p-carotene)... 

As you all know, research on vitamin E goes back quite a long way. It is forty-two years since it was first observed by Matill and Conklin that disturbances in reproduction occurred when rats were fed on a certain milk diet, and twenty-six years have passed since H. M. Evans, 0. H. Emerson, and G. A. Emerson isolated two E vitamins, a- and /3-tocopherol, in the form of the crystalline allophanatcs. After that, research on the vitamin E factor proceeded somewhat more rapidly, and in the next two years, 1937 and 1938, the constitution of a-tocopherol was established, and here in Zurich the first synthesis of dl-a-tocopherol was accomplished. How-... 

That one cannot rely on the tocopherol in stored human milk is indicated in Table IV. As can be seen, the mean scrum tocopherol level for a group of infants fed partially skimmed cows milk was 0.17 mg%, and for infants fed stored human milk, which on occasion was reenforced with skimmed lactic acid milk powder, was 0.29 mg%. The difference of 0.12 mg% is not statistically significant (p< 0.05). The tocopherol level in the group of premature infants fed stored human milk is only approximately 40 % that of full-term infants nursed by their mothers. This may be due to loss of vitamin E in storage of human milk (Harris et al., 1952) diminished fat absorption (Gordon and McNamara, 1941) or a combination of both factors and cannot be judged from our data. In contrast, the... 

The tocopherol content of milk varies with the season primarily because the diet of the cow is richer in tocopherols in the summer than in the winter (Abderhalden, 1947 Harris et al., 1950). For instance, early cut (47 mg % dry weight) and late cut (10 mg % dry weight) silages produced milks containing 3.09 and 2.61 mg tocopherol per 100 gm of butterfat (Val dman and Rezevskaya, 1955). Cows in pasture produced 0.155 rag % tocopherol in their milk and similar cows in stall feeding produced 0.084 mg % toeoph-... 

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