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"Determination. Apart from photometric methods, chromatographic methods are primarily employed for analysis of vitamin E today.
An older method used to determine vitamin E in biological material is the Emmerie –Engel reaction, which is based on the colorimetric measurement of colored Fe(II) complexes with bipyridine [*] or 4,7-diphenyl-1,10-phenanthroline [*]. Spectrofluorometry is a very sensitive analytical method for free and esterified tocopherols. For instance, this method is used for vitamin E determinations in the spinal cord and in erythrocytes [*]. Photometric determination of tocopherol with the 2,2-diphenyl-1-picrylhydrazyl radical is also possible [*].
Of the chromatographic methods, gas chromatography (GC) and high performance liquid chromatography (HPLC) allow the most accurate determinations [*]. HPLC permits the simultaneous determination of vitamin E homologues [*], even directly in plasma and tissue samples [*].
The measurement of the optical rotation of K3Fe(CN)6 oxidation products can be used to differentiate the individual stereoisomers of a-tocopherol (see Table (*)). However, only the configuration at C-2 can be determined with reasonable reliability. The diastereomers can be differentiated by gas chromatography [*] and 13C NMR spectroscopy [*]. All eight stereoisomers of (all-rac)-a-tocopherol can be separated by using a combination of chiral HPLC and GC [*].
Standardization of Biological Activity. The relative biological activity of vitamin E is expressed in International Units (I.U.), 1 I.U. corresponding to the activity of 1 mg of racemic (2RS,4'RS,8'RS )-a-tocopherol acetate [7695-91-2]. The relative biological activities were mainly derived from fetus resorption tests on rats. They have been established by the National Formulary as follows [*]:
1 mg (2RS,4'RS,8'RS )-a-tocopherol acetate = 1.00 I.U.
1 mg (2RS,4'RS,8'RS )-a-tocopherol = 1.10 I.U.
1 mg (2R,4'R,8'R)-a-tocopherol acetate = 1.36 I.U.
1 mg (2R,4'R,8'R)-a-tocopherol = 1.49 I.U.
According to [*], 2R,4'R,8'R-a-tocopherol has a higher biological activity than that established by the National Formulary. As far as farm animals and humans are concerned, the biological activity of the different forms of vitamin E is a controversial subject.
The relative biological activity of the eight a-tocopherol stereoisomers, determined by fetus resorption tests in rats [*] is as follows:
2R,4'R,8'R 100
2R,4'R,8'S 90
2R,4'S,8'S 73
2R,4'S,8'R 57
2S,4'S,8'S 60
2S,4'R,8'R 31
2S,4'S,8'R 21
2S,4'R,8'S 37
The R configuration at C-2 of the chromane ring seems to be important for high biological activity.
Biotests. Data on the relative biological activities of the tocopherol stereoisomers are obtained in animal experiments. The biological activity is determined by the ability of the test substances to prevent or remedy symptoms of vitamin E deficiency in comparison with a standard substance. The following test systems (bioassays) are preferentially used:
1) The fetus resorption test [*] in pregnant rats with vitamin E deficiency is the classical method for the determination of the biological activity of tocopherols.
2) The erythrocyte hemolysis test in the rat [*] is an in vitro process which measures the protective function of vitamin E against peroxide-induced hemolysis of erythrocytes. This test is also applied to humans [*].
3) In the muscular dystrophy test in the rat, rabbit, and chicken, muscular damage due to vitamin E deficiency is measured either directly [*] or via the determination of enzyme activities, e.g., plasma pyruvate kinase activity [*] as a measure of the course of muscular dystrophy.
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