Vitamin E concentrations in blood plasma of sheep and in sheep tissues after a single intraruminal or intraperitoneal administration of DL-α-tocopheryl acetate

Research in Veterinary Science 1990, 48, 158-161

Vitamin E concentrations in blood plasma of sheep and in sheep tissues after a single intraruminal or intraperitoneal administration of nt-e-tocopheryl acetate M. HIDIROGLOU, E. CHARMLEY, Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada KJA OC6

Twenty-five yearlin"g wethers, weighing" 40 to 45 kg Materials and methods were used in a trial designed to compare the effect of the route of administration of vitamin E upon plasma Animals Twenty-five yearling crossbred wethers, weighing" and tissue vitamin E status. Five control sheep without vitamin E administration were killed at the 40 to 45 kg, were used. All animals originated fr~m a beginning of the trial. Of the remaining 20 sheep, 10 flock born and raised in confinement at the Animal were given DL-a-tocopheryl acetate intraruminally Research Centre. The animals were offered feed ad and 10 by intraperitoneal injection. Of these, 10 libitum for several weeks before and during the wethers were killed three days after dosing (five from experiment. The diet consisted of lucerne/grass each treatment, IRJ and IP3) and the remaining silage, hay and corn silage (40:20:40). Groups of five wethers were killed eight days after dosing (IR8 and animals were randomly assigned to each of five treatments. The control sheep received no treatment and IP8). Blood samples were taken throughout the trial from sheep on the IR8 and IP8 treatments. Samples of were killed on day O. Sheep in treatments IR3 and IRS whole adrenal gland, heart, liver, kidney, brachio- were killed three and eight days after a single intracephalicus muscle, lung, pancreas and spleen were rumina! dose of 100 mg kg-I bodyweight of DL-ataken from all sheep at slaughter and were analysed tocopheryl acetate (Sigma) administered in gelatin for their vitamin E content. The blood plasma results capsules by an ovine balling gun. For intraperitoneal showed that the most important index of vitamin E administration (treatments IP3 and IPS), the injections bioavailability, the area under the plasma concentra- (100 mg kg-I bodyweight) were made according to tion versus time curve, was greater in the intra- Hurter (1987). The intraperitoneal preparations were peritoneally than intraruminally dosed sheep. There prepared in this laboratory as follows. An amount of was a higher concentration of vitamin E in the tissues DL-a-tocopheryl acetate, equivalent to the injected from the intraperitoneal group than the intraruminal dose was stirred slowly with 1 g"of Tween 80. To this group three days after the intraperitoneal injections. was added 30 ml diethyl ether and the mixture shaken The results suggest that the greatest" responses in for 20 minutes. The diethyl ether layer was evapovitamin E concentration in plasma and the tissues rated under nitrogen and 10 to 15 ml water added. were recorded in sheep following intraperitoneal Animals in treatments IP3 and IPS were killed three rather than intraruminal dosing with DL-a-tocopheryl and eight days after. the intraperitoneal administraacetate. tion of the aqueous solution. In treatments IRS "and IPS,S ml blood was collected ADMINISTRATION of vitamin E is one of the usual from a jugular vein into evacuated, heparinised tubes methods employed in the therapy and especially the and centrifuged. The plasma was removed and stored prophylaxes of vitamin E deficiencies in ruminants. It at - 20°C until assayed for D-Q'-tocopherol..Blood has previously been reported that dosage form and samples were taken before dosing, five, 10 and 30 route of administration of vitamin E have major minutes after dosing and thereafter at hourly intervals influences on the concentration of vitamin E in blood for the first day (seven hours) and then twice daily for plasma (Hidiroglou and Karpinski 1987, 1988) and eight days. Selected tissues were taken from all sheep after tissues (Hidiroglou 1986) of sheep. The objective of the research reported here was to evaluate the vitamin slaughter: adipose tissue, adrenal gland (whole), E status of sheep following intraruminal or intra- heart, kidney, liver, lung, brachiocephalicus muscle, peritoneal administration of a high dose of DL-a- pancreas and spleen. All the tissues were rinsed immediately with water and dried with filter paper. tocopheryl acetate. 158

159

Vitamin E in sheep

TABLE 1: Plasma a-tocopherol pharmacoklnetic values In sheep during the eight days after administration of a single dose of DL...• tocopheryl acetate 1100 mg kg -1 bodywelghtJ given either Intraperitoneally or Intrarumlnally

Route of administration

Time to Maximum maximum Initial concentration concentration concentration (C maxI (t maxI (Co) (,..g ml- 1) (h) (,..g ml- 1)

C max-Co (,..gml- 1)

Terminal observed concentration (Ct) (,..gml- 1)

Intraperitoneal Intraruminal

6·88 ±' 0·51 32·00 ± 10·23 1·28 ± 0·20 5·83 ± 0·39 28·00 ± 7·92 1·69 ± 0·15

5·59 ± 0'55 4·14± 0·42

2·n ± 0·20 2·02± 0'15

Area under curve after dosing (AUC)

([,..g ml- 1) x h) 1-48 ± 0·16 0·33± 0·21

720±45 590± 35

4·11 ± 0·39 3·81 ± 0·30

Values are least square means ± SEM Significance of difference between treatments: *P
Care was taken to dissect away superfluous adipose tissue. All tissues were frozen at - 20°C until analysed for n-e-tocopherol concentration. Analytical methods

The n-e-tocopherol content in the blood plasma and tissues was determined by high performance liquid chromatography (HPLC) using a fluorescent detector (McMurray and Blanchflower 1979). To monitor analytical recoveries at various stages of the assay, 5000 to 10,000 cpm of o-a-15-Me3Hl-tocopherol (Amersham International) were added to (the tissues, following homogenisation and heptane extraction (Burton et alI985). Also, a known amount of n-c-tocopherol in ethanolic solution was added to some tissue samples before homogenisation. Recovery of tocopherol was calculated by comparing the peak areas of the spiked samples to those of the standards. All sample analyses were carried out in duplicate or triplicate. Statistical methods

The indexes of bioavailability were analysed statistically by conventional least squares procedures (SAS Institute 1982). These indexes were: the maximum a-tocopherol plasma concentration (C max), the time of maximum a-tocopherol concentration (t max) and the area under the a-tocopherol plasma concentration versus time curve (AVC) (KochWeser 1976). The differences in the vitamin E concentration between corresponding tissues among the five groups were determined by analysis of variance.

larger (P
8

Intraruminal

7 6

5 4

3

~

2

CIl III

1

I

0

C.

E

Cl

3 '0

~

.c:

Intraperitoneal

8

87 0

1;

6

5

Results

Blood plasma The data in Table 1 show that the route of administration of oL-a-tocopherol acetate markedly influenced the plasma pharmacokinetic values. During the eight days after dosing, the most important index of bioavailability, the AVC, was

01-...-y-r-.--T"""'1---r--r--r-.......................-..----.-......-

o

20

40

60

80

100

120

140

160 180

Time after dose (hI

FIG 1: Blood plasma a-tocopherol concentration (least square mean ± S.EMI after either a single intraruminal or single intraperitoneal

dosinq of DL-a-tocopheryl acetate to five sheep

160

M. Hidiroglou, E. Charm/ey

TABLE 2: D-a-tocopherol /jAg g-1 fresh tissue. least square mean ± SEI concentl'lltlonsln shaep administered with DL-a·tocopheryl acetate (100 mg kg- 1 bodyweightllntl'llrumlnally or Intl'llperltoneally and killed three days or eight days aftar dosing

Adipose tissue Adrenal gland Heart Kidney Liver Lung Brachiocephalicusmuscle Pancreas Spleen

8d

3d

Control

IR

IP

IR

IP

1·8 ± 0·18b 9·8 ± l'54d 1·7 ± O'49d 1·2 ± 0'4Od 2·1 ± 1·74b 1·5 ± 2·12c 0·8 ± O'20d 9·7 ± l·30 d 1·6 ± l·23 d

1·9 ± O·22b 17·6 ± 1·81c 3·7 ± O·60c 2·9 ± O·44bc 4·8 ± 2'13 b 3·7 ± 2·60c 1·4 ± 0'25 bc 10·2 ± l·46 d 7·7 ± l·55 bc

2·8± 0.228 40·3 ± 2.188 16·3 ± 0.648 7·1 ± 0'49" 29·1 ± 2'13 8 B3·9± 2·608 3·8± 0.258 23·1 ± 1.468 14·7 ± 1'458

2·2 ± 0·228b 19·7± l'89 c 6·1 ± O·60b

2·0± 0'15 b 29·4± l·38 b 6·0± 0'42 b 4·1 ± O·34b 29·0± 1.568 19·5± l·B4b 2·1 ± 0·17b 14·0 ± l·Q3e 11·0± 1·018b

3·3 ± O·48 b

5·2 ± 2'13 b 4·6± 2·60c 1·6 ± 0'25 bc 18·1 ± l·46 b 7·1 ± l'55bc

Within rows means with the same letter are not significantly different (P>O·051

centrations in blood plasma and tissues. The intraperitoneal route supplied higher amounts of vitamin E to the body of the sheep than the intraruminal route. The increased availability of DL-a-tocopheryl acetate following intraperitoneal rather than intraTissues ruminal dosing could have been due to two factors. The results for n-e-tocopherol concentration (JAg One could be related to the numerous mesenteric g-I fresh tissue) are summarised in Table 2. Intra- lymph vessels in the peritoneum, which give easy peritoneal dosing of DL-a-tocopheryl acetate access of nt-e-tocopherol to the blood stream after increased a-tocopherol concentration in most tissues, intraperitoneal administration. According to Galloas evidenced by the significant differences between Torres (1980),DL-a-tocopherylacetate is deacetylated the control group and the intraperitoneal group three in the peritoneum before entering the lymphoid and eight days after dosing (P<0·05). Dosing with DL- tissues of the abdominal cavity. Alternatively, DL-ae-tocopheryl acetate intraruminally only increased a- tocopheryl acetate may enter the portal circulation tocopherol concentration in some tissues. In adipose (Upson 1985). Then it passes to the liver where it is tissue, liver and lung the effects were not significant. hydrolysed by the hepatic vitamin E esterase (Bai et al In the case of the pancreas the effect was significant 1972) before entering the general circulation. Three days after the intraperitoneal injection, a only eight days after dosing (P<0·05). Sheep receivvery large increase of a-tocopherol concentration ing DL-a-tocopheryl acetate and slaughtered three days later had higher vitamin E concentration occurred in the lung. According to Farrell (1979), (P<0'05) in all tissues following intraperitoneal evidence has accumulated indicating that vitamin E rather than intraruminal dosing. However, this plays a role in the respiratory s)item. Ehrenkranz et al difference in concentration between intraperitoneally (1978) reported that vitamin E deficiency enhances and intraruminally dosed sheep was less evident eight the toxic effects of oxygen and various oxidants on days after dosing when the vitamin E activity was the lungs, and vitamin E treatments can protect the higher only in the adrenal gland, liver and lung in the lungs of small animals against oxidative damage. In intraperitoneally compared with the intraruminally terms of concentration of a-tocopherol following the administered sheep (P<0·05). Generally, n-o- parenteral dosing, the liver and adrenal gland tocopherol concentration was higher in the tissues of contained a high level. According to Machlin and sheep three days rather than eight days after intra- Gabriel (1982), the liver concentration appears to reflect a relatively recent supply of vitamin E. As peritoneal dosing except for the liver and spleen. reported previously (Hidiroglou 1987), the variable pattern in tocopherol concentration in the various sheep tissues following vitamin E dosing may be Discussion considered a reflection of their different metabolic The results from blood concentrations showed that activities. This work illustrates that each tissue in the after intraruminal dosing there was a lag period animal body appears to exert its own selection of during the first few hours followed by a rapid absorp- vitamin E uptake. tion phase (Hidiroglou and Karpinski 1988). The current studies suggest that the peritoneum As was reported previously (Hidiroglou 1986, may be a feasible vitamin E delivery route, which Hidiroglou and Karpinski 1987), the route of could be used for pharmacological vitamin E therapy administration markedly influenced vitamin E con- in ruminants. tocopherol concentration in plasma increased very quickly and this increase continued until 32 hours after dosing.

Vitamin E in sheep Acknowledgements Contrib ution number 1566 of the Animal Research Centre. References BAI, J., KARTHA , V. N. R. & KRISHNAMURTHY, S. (1972) Current Science (Bangalore} 41. 566-567 BURTON, G. W., WEBB, A. & UNGOLD, K. U. (1985) Lipid 20, 29-39 EHRENK RANZ, R. A., BONTA, B. W., ABLOW, R. C. & WARSHA W, J. B. (1978) New England Journal ofMedicine 299, 565 FARRELL, P. M. (1979) Journal of Pediatrics 95,869-8 72 GALLO-T ORRES, H. E. (1980) Vitamin E, a Comprehensive Treatise. Ed L. J. Machlin. New York, M. Dekker. pp 170-192 HIDIROG LOU, M. (1986) International Journal for Vitamin and Nutrition Research 55, 247-252 HIDIROG LOU, M. (1987) International Journal for Vitamin and Nutrition Research 57, 381-384

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HIDIROG LOU, M. & KARPINSKI, K. (1987) British Journal of Nutrition 58.113-1 25 HIDIROG LOU, M. & KARPINSKI, K. (1988) British Journal of 'Nutrition 59. 509-518 HURTER , L. R. (1987) Onderstepoort Journal of Veterinary Research 54, 508 KOCH-WESER, J. (1976) New England Journal of Medicine 291. 233-237 MACHLI N, E. L. & GABRIEL , E. (1982) Vitamin E. Eds B. Lubin and L. J. Machlin. New York, New York Academy of Sciences. pp 48-60 -McMURRAY, C. H. & BLANCH FLOWER , W. J. (1979) Journal of Chromatography 178. 525-531 SAS INSTITU TE (1982) SAS User's Guide. New York, SAS Institute UPSON. D. W. (1985) Handbook of Clinical Veterinary Pharmacology. 2nd edn. Lenexa, Veterinary Medical Publications

Received October 5. 1988 Accepted June 14, 1989