BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS

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1 BLOOD PLASMA AND TISSUE CONCENTRATIONS OF VITAMIN E IN BEEF CATTLE AS INFLUENCED BY SUPPLEMENTATION OF VARIOUS TOCOPHEROL COMPOUNDS N. Hidiroglou, L.F. Laflamme and L.R. McDowell SUMMARY Twenty-four crossbred beef cows were used to investigate the concentration of vitamin E (α -tocopherol) in plasma and tissues following oral administration of four α tocopherol sources. Animals were allotted to the following treatments: DL-α -tocopherol, D-α - tocopherol, DL-α -tocopheryl acetate and D-α -tocopheryl acetate. Animals received a daily oral dose of 1,000 IU of the respective tocopherol treatment for 28 d and then were slaughtered. Blood samples were collected on d 0, 1, 7, 14 and 28 for tocopherol concentration assays, and samples from ten different tissues were collected from slaughtered cows. D-α -tocopherol and its acetate ester increased plasma tocopherol concentration faster than the racemic products, with the greatest response occurring with D-α -tocopherol. Across all treatments, the highest α -tocopherol concentrations were noted in the adrenal gland and liver with the lowest found in muscle and thyroid tissue. Tissue analyses confirmed that adrenal gland, kidney, liver and lung a-tocopherol concentrations were higher following D- α than DL-α -tocopherol supplementation. INTRODUCTION Biopotencies of various vitamin E (tocopherol) compounds are generally provided by the following relationship according to the National Formulary: 1 mg DL-α -tocopheryl acetate = 1.0 IU; 1 mg D-α -tocopherol = 1.1 IU; 1 mg D-α -tocopheryl acetate = 1.36 IU; 1 mg D-α -tocopherol = 1.49 IU. These values have been based largely on small animal bioassay (rat anti-sterility assay). Certain researchers reported that in humans, on a mg basis, D-α -tocopheryl acetate was 2.16 times more potent than D-α -tocopheryl acetate following a single oral dose. However, others confirmed in humans following a continuous oral dosing that 1 mg of DL-α -tocopheryl acetate (all racemic-a-tocopheryl acetate) has a biopotency of 1.0 IU whereas D-α -tocopheryl acetate (2R, 4'R, 8'R-α - tocopheryl acetate) has a biopotency of 1.36 IU (28 d). Data from these human studies used the elevation of plasma α -tocopherol as a measure of bioavailability. Although much is known about clinical signs of deficiency in calves and lambs very little attention is given to the different potencies of various vitamin E forms in cattle. Thus the purpose of this work was to compare plasma and tissue α -tocopherol concentrations following supplementation of various tocopherol compounds. MATERIALS AND METHODS Animals. Twenty-four Charolais Hereford crossbred beef cows ranging from 2 to 10 years of age were culled 6 months after calving. These unbred cows were stratified by age into four groups, and the six animals within each group were randomly assigned to four dietary tocopherol preparations. The treatments were D-α -tocopheryl acetate, D-α -

2 tocopherol, D-α -tocopherol and D-α -tocopheryl acetate. Cows received daily 1,000 IU (a dosage above a physiological level) of their respective vitamin E preparation. This amounted to 735 mg of D-α -tocopheryl acetate, 671 mg of D-α -tocopherol, 900 mg of DL-α -tocopherol and 1000 mg of DL-α -tocopheryl acetate, respectively. Each vitamin E preparation was diluted in alcohol to 3 ml and mixed with 25 g of dry molasses. All cows were individually fed 4.4 lb of average quality grass hay (8.7% CP, 35% ADF, 54% TDN,.51% Ca,.23% P..02 ppm Se and 12 ppm vitamin E) and had ad libitum access to a barley-soybean mixture (Table 1) with no added α -tocopherol. The vitamin Emolasses mixture was prepared daily and was top dressed on the grain portion for each individual cow. The experiment lasted 28 days after which time the cows were sacrificed at commercial facilities. Blood Collection. Blood samples (15 ml) were obtained via jugular venipuncture on day 0, 1, 7, 14 and 28 of the feeding period and plasma was analyzed for α -tocopherol concentration by a high pressure liquid chromatography (HPLC) method. Tissue Collection.On the last day of the experiment, cows were shipped to a local slaughter house and slaughtered the following day. At that time, g samples were collected from ten different tissues: heart, thyroid, liver, kidney, adrenal, pancreas, spleen, lung and neck muscle. These samples were frozen within 2 hours until analyzed for α -tocopherol concentration using high pressure liquid chromatography. Statistical Methods. Analysis of variance was used to estimate the difference in α tocopherol concentration among similar time blood samplings as well as among corresponding tissues in the four treatment groups. An additional two way analysis was carried out to test groups and days for blood plasma and for groups and tissue types. RESULTS Plasma. Supplementation over a 1 to 7 days period of time with the various vitamin E preparations increased (P<.01) plasma α -tocopherol concentration above the baseline level (Table 2). Plasma α-tocopherol concentrations at 1 day were higher (P <.01) than the original baseline values in the D-atocopherol group but not for the other groups (P<.05). To elevate plasma α-tocopherol concentration, D-α-tocopherol outranked all the other forms at all sampling dates except at 28 days, where its effect was not different (P >.05) from D-α-tocopherol acetate (Table 2). D-α-tocopherol supplementation during the 28 days experimental period resulted in the highest blood plasma α-tocopherol concentration followed by D-α -tocopheryl acetate; in contrast the racemic mixtures ranked lower (Table 2) despite being dosed at equal units. Blood plasma α-tocopherol increased continuously from 0 to 28 days (Table 2). The difference in plasma a- tocopherol concentration (g/ml) between day 28 and day O were as follows (Table 2). D- α-tocopheryl acetate = 6.16; D-α-tocopherol = 7.83; DL-α-tocopherol = 4.05; DL-αtocopheryl acetate = Consequently the relative bioavailabilities of the various tocopherols vs DL-α -tocopheryl acetate (1.0 IU/mg) could be calculated as follows: DL-α-tocopheryl = (4.05x1.1) / 3.25 = 1.37 IU/mg D-α-tocopheryl acetate (6.16x1.36) / 3.25 = 2.58 IU/mg D-α-tocopherol (7.83x1.1x1.36) / 3.25 = 3.60 IU/mg

3 Tissues. There were differences among treatments (P<.01) and types of tissue (P <.0001) in vitamin E tissue concentrations of vitamin E at slaughter 28 days after supplementation began. In the adrenalgland and liver concentrations (P <.01) of α-tocopherol were greater for cattle fed D-α-tocopherol or D-α-tocopheryl acetate than when fed racemic forms of vitamin E. The atocopherol concentrations were lower in the kidney (P <.05) and lung (P <.01) of the DL-α-tocopherol supplemented cattle than for cattle fed the other treatments. Higher concentrations (P <.05) of α-tocopherol were observed in the thyroid of cattle fed DL-α -tocopherol vs the cows receiving D-atocopheryl acetate, but the α-tocopherol concentrations were higher (P <.05) in the spleen of the D-α-tocopheryl acetate fed group than the DL-α-tocopherol group. No difference (P <.05) was found for heart and muscle as a result of feeding the various forms of vitamin E. Across all the treatments, the adrenal gland contained the highest α-tocopherol concentration, followed generally by the liver or heart, while the lowest concentrations were observed in the thyroid and muscle tissues (P <.05). DISCUSSION The data show that, in cattle, a greater elevation of plasma levels of α-tocopherol occurred over pre-treatment levels after ingestion of the naturally occurring D-αtocopherol than its ester forms or the L-epimer of α-tocopherol. These results agree with those of other researchers who reported in humans that plasma α -tocopherol was considerably higher when the free tocopherol was orally administered rather than its acetate ester. The higher blood plasma α-tocopherol attained following supplementation with α-tocopherol compared to α-tocopherol acetate suggests that hydrolysis of the acetate may be a limiting factor when high levels of the vitamin are administered. Certain researchers reported lower blood plasma α-tocopherol levels in humans dosed with the nonesterified rather than esterified forms. This is related to a diet-mediated oxidative destruction of the free form in the gut prior to its absorption. This does not seem to be the case with ruminants. Indeed, the stability of D-α -tocopherol in ruminal liquor is high. Vitamin E is not metabolized or oxidized to any great extent in the gastrointestinal tract of cattle. In pre-treatment cows, plasma levels of α-tocopherol concentrations were low. Levels less than 1.5 g/ml in blood plasma were considered by several workers as potentially hazardous for cattle. Following vitamin E administration, the levels increased progressively for all treatments during the 4 wk experimental period to the range (4 to 9 g/ml) reported acceptable for beef cattle. In humans and monkeys, it is advisable to treat subjects with high levels of α -tocopheryl for at least 1 to 3 wk to attain maximal plasma a-tocopherol levels. In order to evaluate the effectiveness of the various vitamin E preparations, most studies have relied on serum α -tocopherol concentrations and have paid little attention to tocopherol concentrations in tissue. In the present experiment, tissue analysis showed higher concentrations following D-atocopherol or D-α -tocopheryl acetate supplementation than with the racemic forms. These results suggest that D-α -tocopherol and its acetate form are more available to bovine tissue than the L-epimer. There is a wide variation in the distribution of α-tocopherol in the various tissues in the human body. In the present experiment, highest concentrations were detected in the

4 adrenal gland and liver. The liver is the major storage organ for α -tocopherol and (for a short time at least) helps maintain plasma tocopherol levels when the intake of vitamin E becomes inadequate. As indicated earlier, in rats, the adrenal gland accumulated more tocopherol per gram of tissue than other tissues, regardless of the source of vitamin E supplementation. In general, the apparent biopotency of various vitamin E sources for cattle do not correlate with the currently accepted national formulary. This research was supported, in part, by the US Department of Agriculture under CSRS special grant Number 86-CRSR managed by the Caribbean Advisory Group (CBAG). TABLE 1. COMPOSITION OF THE GRAIN PORTION OF THE DIET FED TO COWS FOR 28 D. Ingredient % as-fed basis Barley 90 Soybean meal 75 Calcium carbonate 1.0 Dicalcium phosphate 1.0 Vitamin Premix.25 Trace mineral salt.25 a Analyzed for vitamin E and selenium (8 ppm and.235 ppm, respectiviely). TABLE 2. PLASMA CONCENTRATIONS OF α-tocopherol (μg/ml) IN CATTLE FED VARIOUS PREPARATIONS OF VITAMIN E Day Group Item SE mean Treatment: D-a-tocopheryl 1.49 ag 2.46 bfg 3.67 bef 5.64 bde 7.65 abd a acetate D-a-tocopheryl 1.39 af 3.82 ae 5.25 ae 8.41 ad 9.22 ad b DL-a-tocopheryl 1.39 ag 2.40 bfg 3.l4 bef 3.98 be 5.44 bcd c DL-a-tocopheryl 1.37 af 2.65 bef 3.10 bde 4.02 bde 4.62 cd.52 3.l6 c acetate SE Day average 1.41 d 2.83 bef 3.80 f 5.51 g 6.73 h.31 a,b,c Means in the see column filth different superscripts differ (P<.01) d,e,f,g Means in the same row with different superscripts differ (P<.01)

5 TABLE 3. TISSUE α-tocopherol CONCENTRATIONS (μg/g FRESH TISSUE) IN CATTLE FED VARIOUS VITAMIN E PREPARATIONS Dietary form D- α- DL-αtocopheryl D-α- DL-α- tocopheryl Tissue acetate tocopherol tocopherol acetate SE Tissue mean mg/g fresh tissue Adrenal gland a a 27.9l b b d Heart a l8.7l a a l6.l2 a f Kidney l2.07 a l3.ll a 8.81 b a h Liver ab a l5.67 c bc e Lung l5.73 a a b a g Muscle 5.29 a 5.79 a 5.70 a 5.79 a i Spleen a l5.79 ab b ab g Thyroid 3.40 b 4.43 ab 5.63 a 4.35 ab i SE Group average l7.l8 a a b b.57 a,b,c Means in the same row with different letters in their superscripts differ (P <.05). d,e,f,g,h,i Means in the same column with different letters in their superscripts differ (P<.0001).