Tocopheryl esters

Recent research has shown unequivocally that tocopherols are always present in vegetable oils in the free form and not as tocopheryl esters (18). From a biochemical point of view, this could be expected because only tocopherols with a free (and not esterified) hydroxyl group can act as an antioxidant. 

Because they lack the phenolic hydrogen, esters are more stable than free vitamers, but they are not antioxidants in vitro. Tocopheryl esters hydrolyze slowly under, eg., acidic aqueous systems, thus providing antioxidant activity for emulsions, soft drinks and some dairy products (Schuler, 1990). 

As mentioned before, tocopherols and tocotrienols are easily oxidized in the presence of light and metals and at high temperature or at alkaline pH, and they are sensitive to ionizing radiation, but tocopheryl esters are much more stable than tocopherols. 

Both normal-phase and reversed-phase HPLC have been applied in vitamin E analysis. Reversed-phase HPLC is unable to completely separate all tocopherols and toco-trienols. Because (1- and y-vitamers have very similar structures, their separation cannot be obtained with reversed-phase HPLC. It is, however, applicable when only tocopherols or a-tocopheryl esters are analyzed (Gimeno et al., 2000 Iwase, 2000). There are reversed-phase methods to analyze tocopherols together with other lipid constituents from biological and food samples such as carotenoids (Epler et al., 1993 Salo-Vaananen et al., 2000), ubiquinols and ubiquinones (Podda et al., 1996) or sterols (Warner and Mounts, 1990). 

Uekama, K., Horiuchi, Y., Kikuchi, M. et al. Enhanced dissolution and oral bioavailabiUty of a-tocopheryl esters by dimethyl-(3-cyclo-dextrin complexations. J. Inclusion Phenom. 1988, 6,167-174. 

With phosphomolybdic acid, free tocopherols give gray-blue spots even at room temperature. The tocopheryl esters react in the same way after prolonged heating. Seher (1960, 1961) was thus able to determine a-... 

Saponification. Lipid-rich samples such as oils, tissues, foods, and feeds may be saponified prior to double-phase extraction. In the course of this process not only the triglycerides and the phospholipids but also the possibly present tocopheryl esters are hydrolyzed. The procedure involves heating the sample with alcoholic potassium hydroxide, mostly in the presence of an antioxidant, followed by double-phase extraction, whereby the potassium salts of the liberated fatty acids and the glycerol remain in the aqueous phase. This decreases the overall organic load in the extract, which improves the selectivity and avoids contamination of the chromatographic column. 

Reversed-Phase. Reversed-phase chromatography continues to form the backbone of most assays of tocopherols and, rarely, tocotrienols in biological materials. Its popular status in the vitamin E area has been rationalized in ni.A.2. When the methods for the simultaneous determination of tocopherols and retinoids/carotenoids are also taken into account, reversed-phase systems outnumber their normal-phase counterparts by a factor 2. A survey of reversed-phase systems for the separation and quantitation of tocopherols, tocopheryl esters, tocotrienols, and a-tocopherolquinone is presented in Table 2. Methods specifically... 

Although all four tocopherols have been synthesized as their all-rac forms, the commercially significant form of tocopherol is i7//-n7i a-tocopheryl acetate. The commercial processes ia use are based on the work reported by several groups ia 1938 (15—17). These processes utilize a Friedel-Crafts-type condensation of 2,3,5-trimethylhydroquinone with either phytol (16), a phytyl haUde (7,16,17), or phytadiene (7). The principal synthesis (Fig. 3) ia current commercial use iavolves condensation of 2,3,5-trimethylhydroquiQone (13) with synthetic isophytol (14) ia an iaert solvent, such as benzene or hexane, with an acid catalyst, such as ziac chloride, boron trifluoride, or orthoboric acid/oxaUc acid (7,8,18) to give the all-rac-acetate ester (15b) by reaction with acetic anhydride. Purification of tocopheryl acetate is readily accompHshed by high vacuum molecular distillation and rectification (<1 mm Hg) to achieve the required USP standard. 

Tocopheryl)propionic acid (50) is one of the rare examples that the o-QM 3 is involved in a direct synthesis rather than as a nonintentionally used intermediate or byproduct. ZnCl2-catalyzed, inverse hetero-Diels-Alder reaction between ortho-qui-none methide 3 and an excess of <2-methyl-C,<9-bis-(trimethylsilyl)ketene acetal provided the acid in fair yields (Fig. 6.37).67 The o-QM 3 was prepared in situ by thermal degradation of 5a-bromo-a-tocopherol (46). The primary cyclization product, an ortho-ester derivative, was not isolated, but immediately hydrolyzed to methyl 3-(5-tocopheryl)-2-trimethylsilyl-propionate, subsequently desilylated, and finally hydrolyzed into 50. 

Belkner et al. [32] demonstrated that 15-LOX oxidized preferably LDL cholesterol esters. Even in the presence of free linoleic acid, cholesteryl linoleate continued to be a major LOX substrate. It was also found that the depletion of LDL from a-tocopherol has not prevented the LDL oxidation. This is of a special interest in connection with the role of a-tocopherol in LDL oxidation. As the majority of cholesteryl esters is normally buried in the core of a lipoprotein particle and cannot be directly oxidized by LOX, it has been suggested that LDL oxidation might be initiated by a-tocopheryl radical formed during the oxidation of a-tocopherol [33,34]. Correspondingly, it was concluded that the oxidation of LDL by soybean and recombinant human 15-LOXs may occur by two pathways (a) LDL-free fatty acids are oxidized enzymatically with the formation of a-tocopheryl radical, and (b) the a-tocopheryl-mediated oxidation of cholesteryl esters occurs via a nonenzymatic way. Pro and con proofs related to the prooxidant role of a-tocopherol were considered in Chapter 25 in connection with the study of nonenzymatic lipid oxidation and in Chapter 29 dedicated to antioxidants. It should be stressed that comparison of the possible effects of a-tocopherol and nitric oxide on LDL oxidation does not support importance of a-tocopherol prooxidant activity. It should be mentioned that the above data describing the activity of cholesteryl esters in LDL oxidation are in contradiction with some earlier results. Thus in 1988, Sparrow et al. [35] suggested that the 15-LOX-catalyzed oxidation of LDL is accelerated in the presence of phospholipase A2, i.e., the hydrolysis of cholesterol esters is an important step in LDL oxidation. 

It should be noted that pharmacological vitamin E is not a free natural RRR-a-tocopherol or synthetic All rac a-tocopherol but its acetate ester. a-Tocopheryl acetate has the phenolic hydroxyl group blocked and therefore, is not a genuine antioxidant, but this compound is very rapidly hydrolyzed in vivo into a-tocopherol. It is interesting that the biological activity of a-tocopheryl acetate is the same as that of a-tocopherol in humans but significantly lower in rats [30]. ( A man is not a rat Professor KU Ingold.)... 

GC analyses of the pupal secretion of E. borealis have indicated the presence of vitamin E acetate and other tocopherol derivatives [49,50]. However, in tests with ants, these compounds proved to be essentially inactive, whereas the secretion itself was potently deterrent. To find and identify the active components in the pupal Epilachna borealis secretion, NMR spectroscopic studies on the fresh secretion were carried out. One and two-dimensional NMR experiments revealed that the tocopheryl acetates account for only a relatively small percentage of the beetles5 total secretion (20%), whereas the major components represented a group of previously undetected compounds. By analysis of the COSY, HSQC and HMBC spectra of the mixture, these components were shown to be esters and amides derived from three (co-l)-(2-hydroxyethylamino)alka-noic acids 44-46. HPLC analyses coupled to a mass spectrometric detector revealed that the secretion contain a highly diverse mixture of macrocyclic polyamines, the polyazamacrolides (PAMLs) 47-52 (Fig. 8). 

The acetate ester of a-tocopherol, rather than the free alcohol, is used as a food supplement on account of its greater stability. Both 7 7 f -a-tocopheryl acetate and totally synthetic all-rac-a-tocopheryl acetate are commercially available, the former having a biological activity of 1.36 IU/mg and the latter 1.00 IU/mg (44). The / / / -a-tocopheryl acetate is obtained by extraction from vegetable oils. Since it is not isolated without some chemical processing, it cannot legally be called natural, but it can be described as derived from natural sources. 

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

Much less is known on the antioxidant activity of tocotrienols than tocopherols. Tocotrienols were shown to have similar reactivities to peroxyl radicals and antioxidant activities than tocopherols in solution and membranes (Yoshida et ah, 2003) also, in general, y-tocotrienol was a better antioxidant than a-tocotrienol, and tocotrienols were better than tocopherols in oil systems (Seppanen et ah, 2010). Recently, Muller et ah (2010) conducted a comparative study to investigate the four tocopherols, four tocotrienols, and a-tocopheryl acetate on their antioxidant activities in five different popular in vitro assays (FRAP, a-TEAC, DPPH, ORAC, and CL), which were adapted to nonpolar antioxidants. Most notably, they found that a-tocopheryl acetate, a popular ingredient in supplements, had no significant antioxidant activity in vitro. However, once ingested, tocol esters are hydrolyzed and antioxidant activities are retained. Overall, the eight tocols performed similarly in the five assays. The authors concluded that in vitro antioxidant assays performed in polar solvents are not a good way to predict in vivo antioxidant activity. 

Vitamin E is not very stable to storage. The stability can be greatly improved by esterjtying the vitamin via the hydroxyl group to acetic acid, The resulting molecule ot-tocopheryl acetate is used as a commercial form of vitamin E, The ester linkage is hydrolyzed in the body, liberating the vitamin in its active form. 

Many vitamins are quite stable under normal processing conditions and present little or no stability problems in finished pharmaceutical products. These include biotin, niacin, niacinamide, pyridoxine, riboflavin, and a-tocopheryl acetate. Others that can present problems are ascorbic acid, calciferol, calcium pantothenate, cyanocobalamin, fola-cin, and retinyl esters. Overages above label claim are customarily added to vitamin formulations as a means of maintaining the claimed level of each vitamin for the expected shelf life of the products. The percent overage for a particular vitamin such as L-ascorbic acid will vary... 

Tocopheryl acetate is the most stable ester of vitamin E. It is readily hydrolyzed by esterases into pure vitamin E after it has been absorbed by the skin. The advantage of the acetate form is that it forms a reservoir that gradually releases the vitamin E inside the skin. Vitamin E scavenges free radicals that could damage neighboring tissue. It blocks oxidative chain reactions by inhibiting the formation of lipoperoxides inside the cell, and in this way protects the nucleic acids and proteins. 

L.C.R DK dmar Labs] Erucyl oleate, squalane, wheat germ oil, avocado oil, ClO-30 cholesterol/lanosterol esters, tocopheryl acetate, retinyl palmitate. 

Commercially available vitamin E consists of either a mixture of namrally occurring tocopherols and tocotriraiols (from natural sources), RRR-a-tocopherol (formerly called d-a-tocopherol), synthetic a-tocopherol, consisting of the eight possible side-chain stereoisomers at eqnal amonnts (all roc-a-tocopherol, formerly called dl-a-tocopherol), or their esters (a-tocophrayl succinate, a-tocopheryl acetate, a-toco-... 

Lauridsen, C., Hedemann, M.S., and Jensen, S.K., Hydrolysis of tocopheryl and retinyl esters by porcine carboxyl ester hydrolase is affected by their carboxylate moiety and bile acids, J. Nutr. Biochem. 12 (4), 219-224, 2001. 

---