Individual tocopherols and tocotrienols in human milk, mother’s milk substitutes and other infant formulas have been determined by an HPLC method. 107 human milk samples (23 colostral, 22 transitional and 62 mature) obtained from six healthy mothers throughout the lactation were found to contain all the tocopherols, although delta-tocopherol occurred only in traces. A high content of alpha-tocopherol was found in colostrum (average 1.90 +/- 1.62 (SD) mg/100 g), as compared with transitional (0.65 +/- 0.22 mg/100 g) and mature milk (0.47 +/- 0.16 mg/100 g). The content of beta-tocopherol averaged 0.05 +/- 0.03, 0.02 +/- 0.01 and 0.02 +/- 0.01 and gamma-tocopherol 0.11 +/- 0.09, 0.07 +/- 0.04 and 0.07 +/- 0.04 mg/100 g in colostral, transitional and mature milk respectively. The alpha-tocopherol equivalents thus were 1.93, 0.66 and 0.49 mg/100 g; their ratios to the contents of polyunsaturated fatty acids meet the nutritional need of the newborn and young infant: 5.7, 2.1 and 1.4 mg/g in colostral, transitional and mature milk. Mother’s milk substitutes and gruel and porridge powders are enriched with tocopherol acetate to vitamin E levels similar to or higher than those in human milk: substitutes contained on average 1.4 mg alpha-tocopherol equivalents/100 g and reconstituted powders 1.1 mg/100 g. The ratio of vitamin E to polyunsaturated fatty acids of these infant formulas was higher than the recommended value of 0.6 mg/g. The average values for alpha-tocopherol equivalents in fruit-berry and meat-vegetable infant formulas were 0.46 and 0.38 mg/100g.
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The composed one-day diets and plasma of 40 Finnish men screened for a selenium supplementation study were analyzed for tocopherols and tocotrienols. The men were divided into a low-Se group (in the screening phase plasma Se levels less than 70 micrograms/l and plasma alpha-tocopherol levels less than 1.2 mg/100 ml) and a high-Se group (plasma Se greater than 70 micrograms/l, plasma alpha-tocopherol not determined before the study). In the low-Se group plasma levels of alpha-tocopherol averaged 0.97 +/- 0.18 mg/100 ml. The daily dietary intake of alpha-tocopherol was 6.1 +/- 2.7 mg and that of total vitamin E 7.3 +/- 3.1 mg of alpha-tocopherol equivalents. In the high-Se group the corresponding average values were 1.16 +/- 0.21 mg of alpha-tocopherol/100 ml of plasma, 8.8 +/- 4.3 mg of alpha-tocopherol/day and 10.3 +/- 5.1 mg of alpha-tocopherol equivalents/day. The overall average for the contribution of alpha-tocopherol to the total dietary tocopherols was 44.6 +/- 11.0%. In the plasma samples alpha-tocopherol accounted for 92.0 +/- 2.1%, beta-tocopherol for 2.7 +/- 0.7% and gamma-tocopherol for 5.3 +/- 2.1% of the total amount of tocopherols.
A HPLC Method is described for the determination of tocopherols and tocotrienols in human diets and plasma. After a room-temperature saponification diet samples were extracted with n-hexane. A direct hexane extraction was used for plasma samples. Using a normal-phase column at elevated temperature and a fluorescence detector complete separation of all four tocopherols, alpha-, beta-, gamma-tocotrienols and BHA and good reproducibility and sensitivity were obtained. The recovery of tocopherols added to diet samples was 99% for alpha-tocopherol, 95% for beta-tocopherol, 99% for gamma-tocopherol and 80% for delta-tocopherol. The recovery of alpha-tocopherol added into plasma was 99%.
The vitamin E group includes tocopherols and tocotrienols and their isomers, esters, and derivatives. They differ not only in biopotencies as antisterility agents but also in activities in other physiological and chemical relationships. Unlike vitamins A and D, foods (vegetable oils) are among the richest sources of vitamin E, and assay methods for vitamin E include food applications more often than for the former vitamins. Physicochemical methods are replacing bioassays for vitamin E and tocopherol wherever possible because of greater specificity and less variability, time, and, sometimes, expense. Unless careful purifications and isolations are carried out and some of the relative vitamin E activities of components are calculated, bioassays are still required for total vitamin E activity. The vitamin E group is separated by column, paper, thin-layer, gas-liquid, and high-pressure liquid chromatography (HPLC). Gas-liquid chromatography has been more successfully used for vitamin E than for other fat-soluble vitamins. Recently developed HPLC methods for vitamin E are sensitive and apparently require less cleanup of extracts and less time than former methods; HPLC may prove to be the most useful technique for vitamin E in foods, especially if other fat-soluble vitamins can be determined simultaneously on the same sample extract.
The biological activity of the tocopherols and tocotrienols has been re-examined by the rat resorption-gestation test. The following values have been obtained (with d,l-alpha-tocopheryl acetate = 100%): d-alpha-tocopherol 80%; d,l-alpha-tocopherol 59%; d-alpha-tocopheryl acetate 136%; d-alpha-tocotrienol 13%; d-beta-tocopherol 45%; d-beta-tocotrienol 4%; d-gamma-tocopherol 13%; d-delta-tocopherol less than 0.4%. The possibility of alpha- and gamma-tocopherol being synergists has been tested, but no significant effect was found. The antioxidants BHT and ethoxyquin were without effect on the utilization of alpha-tocopherol by the rat. After chemical determination of the tocopherols and tocotrienols in foods and mixed feeds, these biological activities were used to calculate the vitamin E activity. For two samples of margarine and two samples of mixed feed, the calculated value of the vitamin E activity after chemical determination of the tocopherols and tocotrienols was compared with the value found by direct bioassay, and reasonably good agreement was found. The authors suggest that determination of vitamin E in foods and feeds as a rule should be carried out as a chemical determination of the individual tocopherols and tocotrienols followed by a calculation of the vitamin E activity from the biological activity of the tocopherols and tocotrienols.
The relative stabilities of selected individual tocols and tocotrienols and of equimolar mixtures of either alpha- plus gamma- or alpha- plus delta- tocopherols were determined in methyl myristate and methyl linoleate during autoxidation and photolysis. Solutions containing 0.05% of the appropriate tocopherol(s) or tocotrienols were subjected to UV light (254 nm) or to a flow of 4.3 ml/min of oxygen, both at 70 C. Tocopherols (T) andtocotrienols (T-3) were determined by gas chromatography without preliminary separation or purification. Under photolytic conditions, stabilities in increasing order in methyl myristate were gamma-T-3 less than alpha-T-3 less than delta-T less than alpha-T less than gamma-T less than 5,7-T less than beta-T and in methyl linoleate were alpha-T less than alpha-T-3 less than or equal to gamma-T-3 less than or equal to beta-T less than or equal to 5,7-T less than gamma-T less than delta-T. A solvent effect on the initial rate of photolysis was observed for 5-methyl substituted tocols but not for the tocols with an unsubstituted 5-position or for the tocotrienols. Under autoxidative conditions, stabilities in increasing order in methyl myristate were alpha-T = alpha-T-3 less than beta-T-3 less than gamma-T-3 less than delta-T-3 less than gamma-T less than delta-T = beta-T and in methyl linoleate were alpha-T less than alpha-T-3 less than gamma-T-3 less than beta-T less than gamma-T less than delta-T. Tocopherols were much more stable during autoxidation in methyl myristate than they were in methyl linoleate. In mixtures, there was no significant protection of alpha-tocopherol by either gamma- or delta-tocopherol under any of the conditions used. However, alpha-tocopherol was highly effective in protecting gamma- and delta-tocopherols in methyl myristate during both photolysis and autoxidation and in methyl linoleate during photolysis. During autoxidation in methyl linoleate, alpha-tocopherol protection of gamma- and delta-tocopherols after 24 hr was slight tough measurable.