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Absorption and Distribution of Vitamin E in the Tissues
Absorption and Distribution of Vitamin E in the Tissues OSWALD WISS, RAYMOND H. BUNNELL, Research Deparlnients
(1s
AND
URS GLOOR
F . Hoffvtlann-La Koche & Co., L i d . , Hasle, Switzerland, arid N u t l e y , New J e r s e y
Page 441
I. Absorption and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Absorption Studies with Vitamin E in Chicks.. . . . . . . . . 11. Distribution in the Tissues.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Studies on the Distribution of C14-dZ-a-Tocopheryl Acetate and EMQ in the Organs and Cell Particlefi of the R a t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
451 454 455
I. ABSORPTIONAND STORAGE The mechanism of the absorption of vitamin E is undoubtedly very similar to that of the other fat soluble vitamins, but compared to vitamin A, for example, its efficiency appears to be much lower. The specific site of absorption is not too well established, but it is probably linked with fat and oil absorption and is facilitated by bile. Work by Sternberg and Pascoe-Dawson (1959), however, indicates that the stomach may play an important role in the absorption of vitamin E. It has also been noted that the difference in the efficiency of absorption of an oil solution and an aqueous emulsion of vitamin E is not a great as the difference encountered with vitamin A. It is apparent from the work of several investigators that a high percentage of a test dose of tocopherol is excreted in the feces. Using chemical methods of tocopherol determination, Klatskin and Molander (1952) found that 64.4% of the calculated daily intake in man is excreted in the feces. Simon et al. (1956) using C14-labeled d-a-tocopheryl succinate reported that rabbits given a single test dose of 10-15 mg excreted in the feces about 65 % of the dose in 3 days and 80 % after 6 days. Dju et al. (1950) reported that hens maintained on high intakes of tocopherol excreted about three-fourths in the feces, but, Pudelkiewicz and. Matterson (1960) found that chicks receiving 8-16 mg of a-tocopheryl acetate per pound of diet excreted only about 23 % of their intake. The data from these balance studies and the considerable data collected on blood and liver levels of 441
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WISS, BUNNELL AND GLOOR
tocopherol, however, strongly indicate that thc absorption of vitamin E is incomplctc. The quantitativc and mathematical rcltttionships that exist between the intake of tocopherol and the levels in the blood plasma and liver havc interested a numbcr of investigators. At the Vitamin E Congress in Venice, Bolligcr and Uolliger-Quaifc (1955) reported an excellent study of these quantitative relationships, using the rat as the test animal. Fccding graded levels of tocopherol up to as high as 500 mg per rat per day they demonstrated that thcre is a linear relationship between thc logarithm of the liver tocopherol and thc logarithm of the a-tocopherol dosage. Their data also showcd that above an intake of about 32 mg per day the plasma tocopherol content was linearly related to the logarithm of thc dosage. These relationships also applied to d-y and dl-{-tocopherols, and using a slope-ratio typc of assay the potency ratio of dl-a-tocopherol t o dl-[- to d-7-tocopherol mas found to be 100:50:8. As a rcsult of this work they proposed that comparison of the uptake in liver and blood of various tocopherols can hclp in assessing the relative biopotencies of the various tocopherols. Biinnell (1955, 1957) also demonstrated n linear relationship between liver tocophcrol and levels of tocophcrol in the diet and used this technique t o demonstrate thc reduced availability of the tocopherol in alfalfa to the chick. With the establishment of the quantitative relationship between tocopherol intake and liver storage together with the availability of precise methods of rneasuremcnt of the tocopherol in the liver, a method for the bioassay of tocopherols based on livcr storage was quick to develop. Workers at the Univcrsity of Connecticut have becn particularly artive in this field. Pudelkiewicz et al. (19GO) and Dicks and Matterson (1961) working with chicks confirmed the linear relationship between dietary intakc and liver tocopherol and reported a very precise method of bioassay based on this technique. Using this systcm of bioassay, they found the relative potency of d- and dl-a-tocopheryl acetate to be 1.31, which is in excellent agreement with the accepted valuc of 1.36 based on the rat-resorption bioassay. Griffiths (1959) determined the relative rates of liver storage of pure a-, p-, y,and &tocopherol in the growing chick and found these ratios to be 100:41:19:nil, which is in good agreement with previous determinations. In a later study Griffiths (1960) found a linear relationship between log of serum tocopherol and log of dictary vitamin E levels for the chick. Rousseau ~t al. (1957) working with calves, lambs, and pigs found that plasma tocopherol increascd with diminishing rates with tocopherol intake whercas liver tocophcrol increased a t constant rates with tocopherol intake. This led to linear relationships between log plasma tocophcrol and log-log tocopherol intake and between log livcr tocopherol and log tocoph-
ABSORPTION AND DISTRIBUTION OF VITAMIN E
443
erol intake. Rousseau et al. also suggested that liver tocopherol storage could be estimated from plasma tocopherol by the linear regression of log liver tocopherol on plasma tocopherol. This suggestion was confirmed for certain ranges of tocopherol intake by Eaton et al. (1958). Although there is agreement concerning a linear relationship between tocopherol intake and liver tocopherol storage, there is some controversy over the relationship between tocopherol intake and plasma tocopherol. While Holliger and Bolliger-Quaife (1955) suggest a linear relationship between plasma tocopherol and the log of the dose a t higher intakes, the findings of Gray (1960) are not in agreement. The plasma tocopherol levels in rats which were obtained by Gray, however, were significantly lower on the higher intakes than the levels reported by Bolliger and Bolliger-Quaife. This fact probably explains the discrepancy in their work.
A. ABSORPTIONSTUDIES WITH VITAMINE
IN
CHICKS
Some work has recently been completed in our laboratory which further explores the absorption characteristics of the tocopherols in the chick. Two aspects were of interest, namely, to determine the rate of absorption of a-tocopherol and a-tocopheryl acetate after a single dose a t physiological levels and to determine the effect on the quantitative relationships of intake versus plasma and liver tocopherols on increasingly higher levels of vitamin E intake. 1. Rate of Absorption f r o m a Single Dose
Two experiments were run using a single dose a t physiological levels, one with 8 I.U. and one with 16 I.U. of vitamin E. I n the first experiment, dl-a-tocopheryl acetate and d-a-tocopheryl acetate were compared for rate of absorption in 9-week-old cockerels. Both products were assayed just prior to testing. On the basis of these assay values, 8 I.U. of vitamin E of each product was weighed into gelatin capsules and force-fed to each of 10 birds per group. Blood samples (by heart puncture) were taken a t 1, 2, 3, 434, 6, 9, and 12 hours after dosing. The blood from 5 birds was merged, providing two samples for vitamin E determination. The birds were sacrificed after bleeding a t each time interval, and the liver was removed. These livers were also merged into groups of five and frozen for later vitamin I3 determination. I n the second experiment 16 I.U. of pure samples of dl-a-tocopheryl acetate, d-a-tocopheryl acetate, dl-a-tocopherol and d-a-tocopherol were forc.e-fcd in gelatin capsules to 4-week-old White Leghorn cockerels. I n this experiment the feeding of the dose of vitamin E was so scheduled that all the birds would be sacrificed at the same time of day (3 P.M.) so as to minimize the effect of differences in fullness of crop and intestine on the
444
WISS, BUNNELL AND GLOOR
rate of absorption. Two other groups of birds were given 16 I.U. of dl-atocopheryl acctate and dl-a-tocopherol by intramuscular injcction of oil solutions containing Tween 80. The response in liver tocopherol and plasma tocopherol after a singlc 8 I.U. dose is shown in Pig. 1. The curves for liver storagc show a smooth rise and fall, peak storage occurring a t 6 hours. The plasma tocopherol wrve is more erratic, a rise a t 1 hour being followed by a decline and then a maximum value for thc dl-a-tocophcryl acctate a t 445 hours and the d-a-tocopheryl acetate a t 6 hours. These differences arc probably biological variations. The plasma tocopherol levels obtained on the 16 I.U. dosc arc shown in Fig. 2 . Since these tocopherols were given in a single oral dose it was felt that a more realistic comparison \vould be obtained if the potency values obtained by rat bioassay were used. These values and the amounts fed are given in the accompanying tabulation. Compound dl-a-Tocopheryl acet ate d-a-Tocopheryl acetate &-Tocopherol dl-a-Tocopherol
I.U./mg 1
.oo
1.36 0.92 0.68
Mg/l6 I.U. 16.00 11.76
17.35 23.53
On this basis, however, it ran be seen from the responses obtained that the blood levels obtained for the free tocopherols are much higher and are reached more rapidly than the acetate forms. The response to the two acetate forms is quite uniform, a result indicating the correctness of the potency ratios. The two free tocopherol curves are, however, much higher t,hari would he expected, even using the chemical cquivalcncics of 1.10 I.U. for dl-a-tocopherol and 1.49 I.U. for d-a-tocopherol. On thesc potency bascs the actual dosage for the free tocopherols was approximately 26 I.I.., ~ h i c his still not enough difference to account for the much higher plasma valurs. The probable explanation for this effcct is the likelihood that tocophcryl acetate is partially saponified in the gut and partially absorbed as the intact ester. The ester is then slowly hydrolyzed in the tissues so that blood levels do not show a sharp rise. Rindi and l'erri (1958) demonstrated that thc ptlrcnternl administration of tocopheryl acetate caused a rapid rise in plasma esterified tocopherol but plasma free tocopherol increased very slowly. In this work the tocopherols administered by intramuscular injection were absorhed slomly, increases not occurring until 12 hours after dosage. The liver tocopherol obtained on the 16 I.U. dose is shown in Fig. 3.
445
ABSORPTION AND DISTRIBUTION OF VITAMIN E
EL
\
\
\
b
HOURS AFTER DOSAGE d1-a-TOCOPHERYL
4-c
2,
“
b
i
1
1
3
b
J
&-
-
Q
4
d-a-TOCOPHEllYL
-8
1
0
1
1I
ACETATE ACETATE
1 l
2
l
HOURS AFTER DOSAGE
FIQ.1. Tocopherol absorption in the chick after a single dose of 8 I.U. of vitamin E.
446
I m
/
I
\
J
-5
WISS, BUNNELL AND GLOOR
.-e
0
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Q
447
ABSORPTION AND DISTRIBUTION OF VITAMIN E 20
-
0L-a-TOCOPHERYL
ACETATE
1)-a-TOCOPHEKYL ACETATE DL-a-TOCOPHEROL
15
O
-
D-CI-TOCOPHEROL
W
1
1
3I
1
b HOURS
4
I 12
AFTER DOSAGE
FIG.3. Tocopherol absorption in the chick after a single dose of 1G I.U. of vitamin E.
418
WISB, BUNNELL AND GLOOR
Here again the free tocopherols produce much higher liver storage than the csterified tocopherols. I n addition maximum storage in the liver occurs sooner with the free tocopherols than with the acetate forms. Whcn tocophcrols or tocopheryl ar*etatcs are fed continuously over a period of time, however, equilibrium occurs so that the end result according to liver storage is in line with the actual biopotency. This was demonstrated by Pudelkicwicz et al. (1960), who found relative potencies by liver bioassay for dl-a-tocopherol and d-a-tocopherol of 0.99 and 1.30, respectively, when fed in the diet in a stabilized form; these values are not too far from their chemical equivalencies of 1.10 and 1.49, respectively. 2. Effect of High Levels of Tocopherol Intake on 7’ocopherol Storage
I n order t o determine the effect of very high levels of tocopherol feeding on plasma and liver tocopherol. groups of 3-week-old White Leghorn TABLE I E INTAKE ON PLASMA LEVELSI N TEIE CHICK
1’:I”FECT O F I N C R E A S I N G V I T A M I N
Vitamin k !adAverage dition t o feed vitamin I< take (I.U./pourid) ( I . U . / h i r d / d n y )
Plasma tocopherol (mg% f S.D.)
-
AND
LIVERTOCO~~HEROL Liver tocopherol (mg% f S.11.)
. .
0 10 100 1 ,000 10,000
0.84 8.8F 84.7 901.
0.51 0.72 2.18 11.4 22.3
-I 0.135
f 0.235 0.127 f 0.424 -I 1.36
0.86 1.24 3.86 26.6 97.4
f f f f f
0.175 0.110 0.253 1.81 14.7
cockerels were maintained for 1 week on diets supplemented with 0, 10, 100,1000,and 10,000 I.IJ. of vitamin E per pound as d-a-tocopheryl acetate. At the end of the 1 w e k period they were sarrificed for blood and liver samplcs. The rcsults of the plasma and liver tocopherol assays are presentcd in Table I. The high plasma levels of tocopherol obtained on the 1000 and 10,000 I.U. per pound feeding levels offered some analytical difficulties because a micromethod of assay was used. Severalfold dilutions of the plasma extracts were required, and these dilutions may have introduced some error. The fact that the linear relationship of log liver tocopherol vcrsus log vitamin l? intake holds even at very high levels of tocopherol intake is dcmonstratcd in the graph of Fig. 4. Thc relationship between log vitamin Ti: intakc vcrsus plasma tocopherol is apparently lincar from 100 to 10,000 I.U. per pound of feed, as illustrated in the graph of Fig. 5 . This observation confirms the observation of Bolliger and Bolliger-Quaife
ABSORPTION AND DISTRIBUTION O F VITAMIN E
449
VITAMIN E IN FEED (LOG I . U . /LB) Flu. 4. Tocopherol storage in livers of chicks with increasing levels of vitamin I3 in the feed.
(1955) with rats. The fact that there is no significant deviation from the linear relationship between log vitamin E intake and log liver tocopherol indicates that a simple mass effect is operative and that there are probably no selectivity characteristics to the absorption of vitamin E from the digestive tract.
450
WISS, BUNNELL AND GLOOR
4-
PLASMA TOCOPHEROL MG
Z
FIG.5 . Plasina tocopherol levels i n t h e chick with increasing levels of vitamin E in the feed.
ABSORPTION AND DISTRIBUTION O F VITAMIN E
451
TISSUES The discussion up to now has been concerned primarily with the absorption of vitamin I3 and its storage in the liver. Although the liver is an important storage site which quickly reveals changes in the intake of vitamin E, this vitamin is also widely distributed in other organs and tissues of the body. I n the work just reported as well as in previous publications the occurrence of vitamin E in various organs of the animal body has been described; chemical methods used for its determination (Quaife et al., 1949; Swick and Baumann, 1952; Edwin et al., 1961). High concentrations were particularly found in the adrenals, nerves, heart, and uterus. By fractionating the cell particles of liver, it could be shown that vitamin E is enriched in the mitochondria and microsomes (Cowlishaw et al., 1957; Draper and Alaupovic, 1959). The use of C14-labeled a-tocopherol offers obvious advantages over chemical methods in determining the distribution of a-tocopherol in the tissues. By measuring the total radioactivity, metabolic products of atocopherol are included whereas they may be excluded if the a-tocopherol is determined by chemical methods. On the other hand, if chemical determinations are used, Emmerie-Engel positive compounds not related to a-tocopherol may falsify the picture. Several investigators (Martius and Costeli, 1957; Alaupovic et al., 1961) have used this technique to follow the isolation and separation of metabolites from the liver. Other workers (Niedner, 1957; Sternberg and Pascoe-Dawson, 1959) have reported on the distribution of radioactivity in the tissues after administration of C14labeled a-tocopherol. The mechanisms whereby several chemical antioxidants appear to substitute for vitamin E in several physiological situations has interested many investigators. The antioxidant ethoxyquin EM& (6-ethoxy-1 ,2dihydro-2,2,4-trimethyl quinoline) has attracted particular attention in recent years, particularly since its approval for use in animal feeds in the United States. Here also the use of carbon labeling should provide a useful tool for studying this antioxidant in biological systems. 11. DISTRIBUTION IN
THE
A. STUDIESON THE DISTRIBUTION OF Cl4 dl-a-TOCOPHERYL ACETATE AND EM& IN THE ORGANS AND CELLPARTICLES OF THE RAT The use of C14-labeled dl-a-tocopheryl acetate with a high specific activity should enable the measurement of increases or decreases in the tissues exactly even after administration of physiological doses. Although absorption and distribution of carbon-labeled EM& has been investigated previously (Wilson et al., 1959), similar experiments were included in our studies. It was hoped to get some information on the dif-
452
WISS, BUNNELL AND GLOOR
ferences of the biological activity between vitamin E and EM& by comparing the amounts of both substances in the various organs and after various time intervals following the administration. 1. Experimental
The dl-a-tocopheryl acetate was labeled in the methyl group in 8-position of the chromane ring and had a specific activity of 3.5 pclmg. The EM& was ring-labeled in 2- and 4-position with a specific activity of 5.15 ccc/mg. Both substanres were dissolved in Tween 80 and diluted with saline solution to a content of 4 mg of substance per milliliter containing 2 % Tween. These solutions were given orally to rats which had had no access to food 14 hours before and 4 hours after administration. Rats of 150-200 gm body weight received 2 mg of dl-a-tocopheryl acetate ( = 5.74 X loe cpm) or 2 mg of EM& (= 8.46 X lo6 cpm). On account of the higher specific activity, the figures obtained after EM& application have been reduced accordingly. All measurements of the radioactivity were made in a Tri-Carb liquid scintillation counter. The animals were killed by decapitation, the blood was collected, and the various organs were removed and weighed immediately. Either the whole or parts of the organs (100-200 mg) were directly burned in an atmosphere of oxygen to COZ and water, according to Kalberer and Rutschmann (1961). The intestine was removed, weighed, and ground in a mortar together with sea sand and the double amount of a 5 741 Tween 80 solution in water. An aliquot of this suspension-emulsion was burned as stated above. Blood was burned in essentially the same manner. Two rats were used for each compound and time, and duplicate runs of burnings were made for each organ. The cell particles of the livers were prepared by fractioned centrifugation according to the standard method of Schneider (1948).
2. Results and Discussion The results on the distribution of a-tocopherol and EM& in various organs and cell particles are compiled in Tables I1 to IV. As it appears from Table 11, a rapid increase of EM& takes place in all organs. Maximum values are obtained already within 30 minutes after dosing, a result which indicates that this substance is readily absorbed. EM& is also rapidly eliminated, most of the substance being excreted within 24 hours. Probably owing t o the excretion function of the kidneys, the EM& concentration in this organ remains relatively high over a longer
453
ABSORPTION AND DISTRIBUTION O F VITAMIN E
period. The highest levels are found in the liver, a fact which may indicate that this organ is the main site of metabolism of EM&. Table I11 shows that the increase of the a-tocopherol content in the various organs is much slower; maximum concentrations are found only
I)ISTRIBLTION
TABLE I1 (EM&) I N
OF C' 4- ET€I OXYQUI N
VARIOUS ORGANS O F THE
RAT^
Time in hours after administration of a single dose (P.O.)
Organ
f.5
2
4
24
96
~~
230,000 19,300 166,000 5,700 65,000 15,800 9,800 4,600 3,500
Intestine Mood Liver Mu sc1es Kidney Adrenals Heart Testes Brain
134,200 5,600 48,400 1,050 24,080 7,410 2,940 1,080 670
246,000 10,400 70,000 2,500 36,500 12,700 5,000 2,300 1,100
9,400 2,900 9,600 370 9,200 2,500 800 370 290
1,200 1,200 2,700 150 3,700 700 400 140 140
a Two milligrams of labeled EM& was administered to rats of 100-160 gm body weight. The values are counts per minute per gram of fresh tissue or per milliliter of blood.
TABLE I11 UISTRIBUTION OF C'4-ol-TOCoPHERO~ I N VARIOUS ORGANS OF T H E R A T 5 ~~~
Organ Intestine Blood Liver Mu sc1e s Kidney Adrerials Heart Testes Brain
Time in hours after administration of a single dose (P.O.) -
$6
2
4
24
96
775,000 730 890 150 420 1,500 640 180 170
410,000 3,700 28,200 700 1,600 10,000 2,100 400 600
233,000 6,150 54,000 880 2,800 39,200 3,400 400 540
70,000 7,@30 23,900 1,900 8,000 203,000 6,000 3,000 1,300
4,600 1,250 4,900 1,300 3,600 34,300 4,900 1,400 1,000
a T w o mllligruns of labeled dl-u-tocopheryl acetnte was administered to rats of 100-150 gm body weight The values are counts per minute per gram of fresh tissue or per milliliter of blood.
several hours after administration. High tissue levels, however, remain over a longer period. In several organs the decrease between 24 and 96 hours is insignificant, indicating that a level is reached which may remain even much longer. After 96 hours the concentrations of a-tocopherol are on the average about ten times above those of EM&. Comparing the
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WISS, BUNNELL AND GLOOR
a-tocopherol levels in the various organs, the exceedingly high concentrations in the adrenals is remarkable. After 24 and 96 hours a-tocopherol levels in the adrenals are about 50-100 times higher than that of EM&. The distribution studies in the cell particles revealed that EM& is predominantly present (about 60% of the total amount) in the soluble fraction, whereas about 80% of a-tocopherol is located in the structural parts of the cell, and especially concentrated in the mitochondria and microsomes (Table 1V). The increase of the vitamin E content in certain tissues and the maintenance of a certain level over a period of a t least several days, reflects its biological function in these places. EM& absorption, distribution, and TABLE I V DISTRIBUTION OF C 1 4 - a - T ~ (TO) ~ ~ A~N D~ Ci4-EM& ~ ~ o ~I N PARTICLES OF 1,IVER CEI,I,S"
Time in hours aft,er ad1ninist)rationof a single dose (P.O.) Liver
55
particles
Nuclei Mitochondria Micrommes Supernatant
2
4
24
96 EMQ TO EMQ
TO
EMQ
TO
EMQ
TO
EMQ
TO
0 0.3
183 132
88 185
63 98
101 250
75 101
110 177
31 45
51 83
12 17
3
377
340
188
387
210
297
96
99
21
0
1121
124
445
147
522
99
105
47
44
Two milliersms of labeled dl-a tocopheryl acetate or E M Q was adniinistrred to rats of 10[t200 gm body weight. T h e vnlues are counts per minute per milligram of protein.
elimination, however, follow the pattern of unphysiological substances which the body trim to cxcrete rapidly. This may explain why EM& has to be administered continuously in rather high dosage in order to produce such tissue levels as are required to maintain its antioxidative function.
111. SUMMARY The absorption and storage of vitamin E are similar to those of the other fat-soluble vitamins but does not appear to be as efficient as in vitamin A. Storage in the liver follows mathematical relationships which can he used as a method of bioassay which is convenient and precise, Maximum storage in the liver appears to occur about G hours after a single dose of tocopherol. The linear relationship between tocopherol content in the diet and livcr storage holds even for very high intakes of tocopherol. Studies with C14-laheleddl-a-tocopheryl acetate reveal that absorpt,ion occurs rather slowly, reaching maximal concentrations only after a few
ABSORPTION AND DISTRIBUTION OF VITAMIN E
455
hours. High levels, however, remain for longer periods. Of particular interest is thc high lcvcls attained in the adrenals. In liver cell particlcs, the hulk of the tocopherol is located in t>hcstructural part of the cell, especially in the mitochondria and microsomes. Thc antioxidant cthoxyquin is absorbed rapidly, reaching high concent,rations in t,he liver and kidneys; t,hen it is rapidly eliminated, following the pattern of an unphysiological substance. In the liver cell particles it is located primariIy in the supernatant rather than in the structural parts of the cells. REFEREXCES Alaupovic, P., Johnson, B. C., Crider, Q., Bhogawtn, H . N . , a n d Johnson, T3. J. 1961. Am. J . Clin. Rrutrition 9, Part 11, 76-88. Bolliger, H. It., and Bolliger-Quaife, M . L . 1956. Vitamin E . Atti conyr. i n t e r n . 3rd Venezia, 1956, pp. 30-45. Burinell, R. H . 1955. The effect of fish oil and diphenyl-p-phenylenediamineon the vitamin E metabolism of the chick. 1’h.T). Thesis, University of Connecticut, Storrs. Bunnell, R . H. 1957. Poultry Sci. 36, 413-516. Cowlishaw, 13., SZndergaard, E., Prange, I., and Dam, H . 1957. Riochim. et Biophys. Acta 26, 644-645. Dicks, M. W., and Matterson, L. D. 1961. J . Nutrition 76, 165-174. Dju, M. Y., Quaife, M. L., arid Harris, P. L. 1950. A m . J . Physiol. 160, 259-263. Draper, H. H., and Alaupovic, 1’. 1959. Federation Proc. 18, 218. Eaton, H . D., Rousseau, J. E., Jr., Diclts, M. W., Teichman, R., and Grifo, A. P., Jr. 1958. J . Dairy Sci. 41, 1456-1459. Edwin, E. E., Diplock, A. T., Bunyan, J., and Green, J. 1961. Biochem. J . 79, 91-105. Gray, D. E. 1960. J . Vitamind. (Osaka)6, 155-157. GriKiths, T . W. 1959. h7ature 183, 1061-1062. Griffiths, T . W. 1960. Brit. J . Nntrition 14, 269-280. Kalberer, F., and Rutschmann, J . 1961. Helv. Chim. Acta 44, 1956-1966. Klatskin, G., and Molandcr, D. W. 1952. J . Lab. Clin. Med. 39, 802-813. Martius, C., and Costeli, J . 1957. Biochem. Z . 329, 449. Niedner, A. It. B. 1957. Dissertation Abstr. 17, 2794. Pudelkiewicz, W. J., and Matterson, L. D . 1960. J . Nutrition 71,143-148. Pudelkiewicx, W. ,J., Matterson, L. D., Potter, L. M., Webster, L., and Singsen, E. P. 1960. J . Nutrition 71, 115-121. Quaife, M. L., Swanson, W. J., I>ju, M. Y., and Harris, P . I,. 1949. A n n . N . Y . Acad. S C ~62, . 300-305. Rindi, G., and I’erri, V. 1958. Intern. Z. Vitaminjorsch. 28, 274-281. Rousseau, J . E. Jr., Dicks, M. W., Teichman, R., Helmboldt, C. F., Bacon, E. L., Prouty, It. M., IIoIge, K. L., Eaton, H. D., dungherr, E. I,., and B e d , G. 1957. J . Animal 9 c i . 16, 612-622. Schneider, W. C. 1948. J . B i d . Chem. 176, 259-266. Simon, E . J., Gross, C. S., andMilhorat, A. T . 1956. J . Biol.Chem. 221, 797-805. Sternberg, J., and Pascoe-Dawson, E. 1959. Can. Med. Assoc. J. 80, 266-275. Swick, R . W., and Baumann, C. A. 1952. Anal. Chern. 24, 758-760 Wilson, R . H., Thomas, J. O., Thompson, C. R., Launer, H. F., and Kohler, G. 0. 1959. J . Sci. Food Agr. 10, Abstracts, P a r t 11, 143.
(1s
AND
URS GLOOR
F . Hoffvtlann-La Koche & Co., L i d . , Hasle, Switzerland, arid N u t l e y , New J e r s e y
Page 441
I. Absorption and Storage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Absorption Studies with Vitamin E in Chicks.. . . . . . . . . 11. Distribution in the Tissues.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Studies on the Distribution of C14-dZ-a-Tocopheryl Acetate and EMQ in the Organs and Cell Particlefi of the R a t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111. Summary.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
451 454 455
I. ABSORPTIONAND STORAGE The mechanism of the absorption of vitamin E is undoubtedly very similar to that of the other fat soluble vitamins, but compared to vitamin A, for example, its efficiency appears to be much lower. The specific site of absorption is not too well established, but it is probably linked with fat and oil absorption and is facilitated by bile. Work by Sternberg and Pascoe-Dawson (1959), however, indicates that the stomach may play an important role in the absorption of vitamin E. It has also been noted that the difference in the efficiency of absorption of an oil solution and an aqueous emulsion of vitamin E is not a great as the difference encountered with vitamin A. It is apparent from the work of several investigators that a high percentage of a test dose of tocopherol is excreted in the feces. Using chemical methods of tocopherol determination, Klatskin and Molander (1952) found that 64.4% of the calculated daily intake in man is excreted in the feces. Simon et al. (1956) using C14-labeled d-a-tocopheryl succinate reported that rabbits given a single test dose of 10-15 mg excreted in the feces about 65 % of the dose in 3 days and 80 % after 6 days. Dju et al. (1950) reported that hens maintained on high intakes of tocopherol excreted about three-fourths in the feces, but, Pudelkiewicz and. Matterson (1960) found that chicks receiving 8-16 mg of a-tocopheryl acetate per pound of diet excreted only about 23 % of their intake. The data from these balance studies and the considerable data collected on blood and liver levels of 441
442
WISS, BUNNELL AND GLOOR
tocopherol, however, strongly indicate that thc absorption of vitamin E is incomplctc. The quantitativc and mathematical rcltttionships that exist between the intake of tocopherol and the levels in the blood plasma and liver havc interested a numbcr of investigators. At the Vitamin E Congress in Venice, Bolligcr and Uolliger-Quaifc (1955) reported an excellent study of these quantitative relationships, using the rat as the test animal. Fccding graded levels of tocopherol up to as high as 500 mg per rat per day they demonstrated that thcre is a linear relationship between thc logarithm of the liver tocopherol and thc logarithm of the a-tocopherol dosage. Their data also showcd that above an intake of about 32 mg per day the plasma tocopherol content was linearly related to the logarithm of thc dosage. These relationships also applied to d-y and dl-{-tocopherols, and using a slope-ratio typc of assay the potency ratio of dl-a-tocopherol t o dl-[- to d-7-tocopherol mas found to be 100:50:8. As a rcsult of this work they proposed that comparison of the uptake in liver and blood of various tocopherols can hclp in assessing the relative biopotencies of the various tocopherols. Biinnell (1955, 1957) also demonstrated n linear relationship between liver tocophcrol and levels of tocophcrol in the diet and used this technique t o demonstrate thc reduced availability of the tocopherol in alfalfa to the chick. With the establishment of the quantitative relationship between tocopherol intake and liver storage together with the availability of precise methods of rneasuremcnt of the tocopherol in the liver, a method for the bioassay of tocopherols based on livcr storage was quick to develop. Workers at the Univcrsity of Connecticut have becn particularly artive in this field. Pudelkiewicz et al. (19GO) and Dicks and Matterson (1961) working with chicks confirmed the linear relationship between dietary intakc and liver tocopherol and reported a very precise method of bioassay based on this technique. Using this systcm of bioassay, they found the relative potency of d- and dl-a-tocopheryl acetate to be 1.31, which is in excellent agreement with the accepted valuc of 1.36 based on the rat-resorption bioassay. Griffiths (1959) determined the relative rates of liver storage of pure a-, p-, y,and &tocopherol in the growing chick and found these ratios to be 100:41:19:nil, which is in good agreement with previous determinations. In a later study Griffiths (1960) found a linear relationship between log of serum tocopherol and log of dictary vitamin E levels for the chick. Rousseau ~t al. (1957) working with calves, lambs, and pigs found that plasma tocopherol increascd with diminishing rates with tocopherol intake whercas liver tocophcrol increased a t constant rates with tocopherol intake. This led to linear relationships between log plasma tocophcrol and log-log tocopherol intake and between log livcr tocopherol and log tocoph-
ABSORPTION AND DISTRIBUTION OF VITAMIN E
443
erol intake. Rousseau et al. also suggested that liver tocopherol storage could be estimated from plasma tocopherol by the linear regression of log liver tocopherol on plasma tocopherol. This suggestion was confirmed for certain ranges of tocopherol intake by Eaton et al. (1958). Although there is agreement concerning a linear relationship between tocopherol intake and liver tocopherol storage, there is some controversy over the relationship between tocopherol intake and plasma tocopherol. While Holliger and Bolliger-Quaife (1955) suggest a linear relationship between plasma tocopherol and the log of the dose a t higher intakes, the findings of Gray (1960) are not in agreement. The plasma tocopherol levels in rats which were obtained by Gray, however, were significantly lower on the higher intakes than the levels reported by Bolliger and Bolliger-Quaife. This fact probably explains the discrepancy in their work.
A. ABSORPTIONSTUDIES WITH VITAMINE
IN
CHICKS
Some work has recently been completed in our laboratory which further explores the absorption characteristics of the tocopherols in the chick. Two aspects were of interest, namely, to determine the rate of absorption of a-tocopherol and a-tocopheryl acetate after a single dose a t physiological levels and to determine the effect on the quantitative relationships of intake versus plasma and liver tocopherols on increasingly higher levels of vitamin E intake. 1. Rate of Absorption f r o m a Single Dose
Two experiments were run using a single dose a t physiological levels, one with 8 I.U. and one with 16 I.U. of vitamin E. I n the first experiment, dl-a-tocopheryl acetate and d-a-tocopheryl acetate were compared for rate of absorption in 9-week-old cockerels. Both products were assayed just prior to testing. On the basis of these assay values, 8 I.U. of vitamin E of each product was weighed into gelatin capsules and force-fed to each of 10 birds per group. Blood samples (by heart puncture) were taken a t 1, 2, 3, 434, 6, 9, and 12 hours after dosing. The blood from 5 birds was merged, providing two samples for vitamin E determination. The birds were sacrificed after bleeding a t each time interval, and the liver was removed. These livers were also merged into groups of five and frozen for later vitamin I3 determination. I n the second experiment 16 I.U. of pure samples of dl-a-tocopheryl acetate, d-a-tocopheryl acetate, dl-a-tocopherol and d-a-tocopherol were forc.e-fcd in gelatin capsules to 4-week-old White Leghorn cockerels. I n this experiment the feeding of the dose of vitamin E was so scheduled that all the birds would be sacrificed at the same time of day (3 P.M.) so as to minimize the effect of differences in fullness of crop and intestine on the
444
WISS, BUNNELL AND GLOOR
rate of absorption. Two other groups of birds were given 16 I.U. of dl-atocopheryl acctate and dl-a-tocopherol by intramuscular injcction of oil solutions containing Tween 80. The response in liver tocopherol and plasma tocopherol after a singlc 8 I.U. dose is shown in Pig. 1. The curves for liver storagc show a smooth rise and fall, peak storage occurring a t 6 hours. The plasma tocopherol wrve is more erratic, a rise a t 1 hour being followed by a decline and then a maximum value for thc dl-a-tocophcryl acctate a t 445 hours and the d-a-tocopheryl acetate a t 6 hours. These differences arc probably biological variations. The plasma tocopherol levels obtained on the 16 I.U. dosc arc shown in Fig. 2 . Since these tocopherols were given in a single oral dose it was felt that a more realistic comparison \vould be obtained if the potency values obtained by rat bioassay were used. These values and the amounts fed are given in the accompanying tabulation. Compound dl-a-Tocopheryl acet ate d-a-Tocopheryl acetate &-Tocopherol dl-a-Tocopherol
I.U./mg 1
.oo
1.36 0.92 0.68
Mg/l6 I.U. 16.00 11.76
17.35 23.53
On this basis, however, it ran be seen from the responses obtained that the blood levels obtained for the free tocopherols are much higher and are reached more rapidly than the acetate forms. The response to the two acetate forms is quite uniform, a result indicating the correctness of the potency ratios. The two free tocopherol curves are, however, much higher t,hari would he expected, even using the chemical cquivalcncics of 1.10 I.U. for dl-a-tocopherol and 1.49 I.U. for d-a-tocopherol. On thesc potency bascs the actual dosage for the free tocopherols was approximately 26 I.I.., ~ h i c his still not enough difference to account for the much higher plasma valurs. The probable explanation for this effcct is the likelihood that tocophcryl acetate is partially saponified in the gut and partially absorbed as the intact ester. The ester is then slowly hydrolyzed in the tissues so that blood levels do not show a sharp rise. Rindi and l'erri (1958) demonstrated that thc ptlrcnternl administration of tocopheryl acetate caused a rapid rise in plasma esterified tocopherol but plasma free tocopherol increased very slowly. In this work the tocopherols administered by intramuscular injection were absorhed slomly, increases not occurring until 12 hours after dosage. The liver tocopherol obtained on the 16 I.U. dose is shown in Fig. 3.
445
ABSORPTION AND DISTRIBUTION OF VITAMIN E
EL
\
\
\
b
HOURS AFTER DOSAGE d1-a-TOCOPHERYL
4-c
2,
“
b
i
1
1
3
b
J
&-
-
Q
4
d-a-TOCOPHEllYL
-8
1
0
1
1I
ACETATE ACETATE
1 l
2
l
HOURS AFTER DOSAGE
FIQ.1. Tocopherol absorption in the chick after a single dose of 8 I.U. of vitamin E.
446
I m
/
I
\
J
-5
WISS, BUNNELL AND GLOOR
.-e
0
JO
c
g
.3 +
Q
447
ABSORPTION AND DISTRIBUTION OF VITAMIN E 20
-
0L-a-TOCOPHERYL
ACETATE
1)-a-TOCOPHEKYL ACETATE DL-a-TOCOPHEROL
15
O
-
D-CI-TOCOPHEROL
W
1
1
3I
1
b HOURS
4
I 12
AFTER DOSAGE
FIG.3. Tocopherol absorption in the chick after a single dose of 1G I.U. of vitamin E.
418
WISB, BUNNELL AND GLOOR
Here again the free tocopherols produce much higher liver storage than the csterified tocopherols. I n addition maximum storage in the liver occurs sooner with the free tocopherols than with the acetate forms. Whcn tocophcrols or tocopheryl ar*etatcs are fed continuously over a period of time, however, equilibrium occurs so that the end result according to liver storage is in line with the actual biopotency. This was demonstrated by Pudelkicwicz et al. (1960), who found relative potencies by liver bioassay for dl-a-tocopherol and d-a-tocopherol of 0.99 and 1.30, respectively, when fed in the diet in a stabilized form; these values are not too far from their chemical equivalencies of 1.10 and 1.49, respectively. 2. Effect of High Levels of Tocopherol Intake on 7’ocopherol Storage
I n order t o determine the effect of very high levels of tocopherol feeding on plasma and liver tocopherol. groups of 3-week-old White Leghorn TABLE I E INTAKE ON PLASMA LEVELSI N TEIE CHICK
1’:I”FECT O F I N C R E A S I N G V I T A M I N
Vitamin k !adAverage dition t o feed vitamin I< take (I.U./pourid) ( I . U . / h i r d / d n y )
Plasma tocopherol (mg% f S.D.)
-
AND
LIVERTOCO~~HEROL Liver tocopherol (mg% f S.11.)
. .
0 10 100 1 ,000 10,000
0.84 8.8F 84.7 901.
0.51 0.72 2.18 11.4 22.3
-I 0.135
f 0.235 0.127 f 0.424 -I 1.36
0.86 1.24 3.86 26.6 97.4
f f f f f
0.175 0.110 0.253 1.81 14.7
cockerels were maintained for 1 week on diets supplemented with 0, 10, 100,1000,and 10,000 I.IJ. of vitamin E per pound as d-a-tocopheryl acetate. At the end of the 1 w e k period they were sarrificed for blood and liver samplcs. The rcsults of the plasma and liver tocopherol assays are presentcd in Table I. The high plasma levels of tocopherol obtained on the 1000 and 10,000 I.U. per pound feeding levels offered some analytical difficulties because a micromethod of assay was used. Severalfold dilutions of the plasma extracts were required, and these dilutions may have introduced some error. The fact that the linear relationship of log liver tocopherol vcrsus log vitamin l? intake holds even at very high levels of tocopherol intake is dcmonstratcd in the graph of Fig. 4. Thc relationship between log vitamin Ti: intakc vcrsus plasma tocopherol is apparently lincar from 100 to 10,000 I.U. per pound of feed, as illustrated in the graph of Fig. 5 . This observation confirms the observation of Bolliger and Bolliger-Quaife
ABSORPTION AND DISTRIBUTION O F VITAMIN E
449
VITAMIN E IN FEED (LOG I . U . /LB) Flu. 4. Tocopherol storage in livers of chicks with increasing levels of vitamin I3 in the feed.
(1955) with rats. The fact that there is no significant deviation from the linear relationship between log vitamin E intake and log liver tocopherol indicates that a simple mass effect is operative and that there are probably no selectivity characteristics to the absorption of vitamin E from the digestive tract.
450
WISS, BUNNELL AND GLOOR
4-
PLASMA TOCOPHEROL MG
Z
FIG.5 . Plasina tocopherol levels i n t h e chick with increasing levels of vitamin E in the feed.
ABSORPTION AND DISTRIBUTION O F VITAMIN E
451
TISSUES The discussion up to now has been concerned primarily with the absorption of vitamin I3 and its storage in the liver. Although the liver is an important storage site which quickly reveals changes in the intake of vitamin E, this vitamin is also widely distributed in other organs and tissues of the body. I n the work just reported as well as in previous publications the occurrence of vitamin E in various organs of the animal body has been described; chemical methods used for its determination (Quaife et al., 1949; Swick and Baumann, 1952; Edwin et al., 1961). High concentrations were particularly found in the adrenals, nerves, heart, and uterus. By fractionating the cell particles of liver, it could be shown that vitamin E is enriched in the mitochondria and microsomes (Cowlishaw et al., 1957; Draper and Alaupovic, 1959). The use of C14-labeled a-tocopherol offers obvious advantages over chemical methods in determining the distribution of a-tocopherol in the tissues. By measuring the total radioactivity, metabolic products of atocopherol are included whereas they may be excluded if the a-tocopherol is determined by chemical methods. On the other hand, if chemical determinations are used, Emmerie-Engel positive compounds not related to a-tocopherol may falsify the picture. Several investigators (Martius and Costeli, 1957; Alaupovic et al., 1961) have used this technique to follow the isolation and separation of metabolites from the liver. Other workers (Niedner, 1957; Sternberg and Pascoe-Dawson, 1959) have reported on the distribution of radioactivity in the tissues after administration of C14labeled a-tocopherol. The mechanisms whereby several chemical antioxidants appear to substitute for vitamin E in several physiological situations has interested many investigators. The antioxidant ethoxyquin EM& (6-ethoxy-1 ,2dihydro-2,2,4-trimethyl quinoline) has attracted particular attention in recent years, particularly since its approval for use in animal feeds in the United States. Here also the use of carbon labeling should provide a useful tool for studying this antioxidant in biological systems. 11. DISTRIBUTION IN
THE
A. STUDIESON THE DISTRIBUTION OF Cl4 dl-a-TOCOPHERYL ACETATE AND EM& IN THE ORGANS AND CELLPARTICLES OF THE RAT The use of C14-labeled dl-a-tocopheryl acetate with a high specific activity should enable the measurement of increases or decreases in the tissues exactly even after administration of physiological doses. Although absorption and distribution of carbon-labeled EM& has been investigated previously (Wilson et al., 1959), similar experiments were included in our studies. It was hoped to get some information on the dif-
452
WISS, BUNNELL AND GLOOR
ferences of the biological activity between vitamin E and EM& by comparing the amounts of both substances in the various organs and after various time intervals following the administration. 1. Experimental
The dl-a-tocopheryl acetate was labeled in the methyl group in 8-position of the chromane ring and had a specific activity of 3.5 pclmg. The EM& was ring-labeled in 2- and 4-position with a specific activity of 5.15 ccc/mg. Both substanres were dissolved in Tween 80 and diluted with saline solution to a content of 4 mg of substance per milliliter containing 2 % Tween. These solutions were given orally to rats which had had no access to food 14 hours before and 4 hours after administration. Rats of 150-200 gm body weight received 2 mg of dl-a-tocopheryl acetate ( = 5.74 X loe cpm) or 2 mg of EM& (= 8.46 X lo6 cpm). On account of the higher specific activity, the figures obtained after EM& application have been reduced accordingly. All measurements of the radioactivity were made in a Tri-Carb liquid scintillation counter. The animals were killed by decapitation, the blood was collected, and the various organs were removed and weighed immediately. Either the whole or parts of the organs (100-200 mg) were directly burned in an atmosphere of oxygen to COZ and water, according to Kalberer and Rutschmann (1961). The intestine was removed, weighed, and ground in a mortar together with sea sand and the double amount of a 5 741 Tween 80 solution in water. An aliquot of this suspension-emulsion was burned as stated above. Blood was burned in essentially the same manner. Two rats were used for each compound and time, and duplicate runs of burnings were made for each organ. The cell particles of the livers were prepared by fractioned centrifugation according to the standard method of Schneider (1948).
2. Results and Discussion The results on the distribution of a-tocopherol and EM& in various organs and cell particles are compiled in Tables I1 to IV. As it appears from Table 11, a rapid increase of EM& takes place in all organs. Maximum values are obtained already within 30 minutes after dosing, a result which indicates that this substance is readily absorbed. EM& is also rapidly eliminated, most of the substance being excreted within 24 hours. Probably owing t o the excretion function of the kidneys, the EM& concentration in this organ remains relatively high over a longer
453
ABSORPTION AND DISTRIBUTION O F VITAMIN E
period. The highest levels are found in the liver, a fact which may indicate that this organ is the main site of metabolism of EM&. Table I11 shows that the increase of the a-tocopherol content in the various organs is much slower; maximum concentrations are found only
I)ISTRIBLTION
TABLE I1 (EM&) I N
OF C' 4- ET€I OXYQUI N
VARIOUS ORGANS O F THE
RAT^
Time in hours after administration of a single dose (P.O.)
Organ
f.5
2
4
24
96
~~
230,000 19,300 166,000 5,700 65,000 15,800 9,800 4,600 3,500
Intestine Mood Liver Mu sc1es Kidney Adrenals Heart Testes Brain
134,200 5,600 48,400 1,050 24,080 7,410 2,940 1,080 670
246,000 10,400 70,000 2,500 36,500 12,700 5,000 2,300 1,100
9,400 2,900 9,600 370 9,200 2,500 800 370 290
1,200 1,200 2,700 150 3,700 700 400 140 140
a Two milligrams of labeled EM& was administered to rats of 100-160 gm body weight. The values are counts per minute per gram of fresh tissue or per milliliter of blood.
TABLE I11 UISTRIBUTION OF C'4-ol-TOCoPHERO~ I N VARIOUS ORGANS OF T H E R A T 5 ~~~
Organ Intestine Blood Liver Mu sc1e s Kidney Adrerials Heart Testes Brain
Time in hours after administration of a single dose (P.O.) -
$6
2
4
24
96
775,000 730 890 150 420 1,500 640 180 170
410,000 3,700 28,200 700 1,600 10,000 2,100 400 600
233,000 6,150 54,000 880 2,800 39,200 3,400 400 540
70,000 7,@30 23,900 1,900 8,000 203,000 6,000 3,000 1,300
4,600 1,250 4,900 1,300 3,600 34,300 4,900 1,400 1,000
a T w o mllligruns of labeled dl-u-tocopheryl acetnte was administered to rats of 100-150 gm body weight The values are counts per minute per gram of fresh tissue or per milliliter of blood.
several hours after administration. High tissue levels, however, remain over a longer period. In several organs the decrease between 24 and 96 hours is insignificant, indicating that a level is reached which may remain even much longer. After 96 hours the concentrations of a-tocopherol are on the average about ten times above those of EM&. Comparing the
454
WISS, BUNNELL AND GLOOR
a-tocopherol levels in the various organs, the exceedingly high concentrations in the adrenals is remarkable. After 24 and 96 hours a-tocopherol levels in the adrenals are about 50-100 times higher than that of EM&. The distribution studies in the cell particles revealed that EM& is predominantly present (about 60% of the total amount) in the soluble fraction, whereas about 80% of a-tocopherol is located in the structural parts of the cell, and especially concentrated in the mitochondria and microsomes (Table 1V). The increase of the vitamin E content in certain tissues and the maintenance of a certain level over a period of a t least several days, reflects its biological function in these places. EM& absorption, distribution, and TABLE I V DISTRIBUTION OF C 1 4 - a - T ~ (TO) ~ ~ A~N D~ Ci4-EM& ~ ~ o ~I N PARTICLES OF 1,IVER CEI,I,S"
Time in hours aft,er ad1ninist)rationof a single dose (P.O.) Liver
55
particles
Nuclei Mitochondria Micrommes Supernatant
2
4
24
96 EMQ TO EMQ
TO
EMQ
TO
EMQ
TO
EMQ
TO
0 0.3
183 132
88 185
63 98
101 250
75 101
110 177
31 45
51 83
12 17
3
377
340
188
387
210
297
96
99
21
0
1121
124
445
147
522
99
105
47
44
Two milliersms of labeled dl-a tocopheryl acetate or E M Q was adniinistrred to rats of 10[t200 gm body weight. T h e vnlues are counts per minute per milligram of protein.
elimination, however, follow the pattern of unphysiological substances which the body trim to cxcrete rapidly. This may explain why EM& has to be administered continuously in rather high dosage in order to produce such tissue levels as are required to maintain its antioxidative function.
111. SUMMARY The absorption and storage of vitamin E are similar to those of the other fat-soluble vitamins but does not appear to be as efficient as in vitamin A. Storage in the liver follows mathematical relationships which can he used as a method of bioassay which is convenient and precise, Maximum storage in the liver appears to occur about G hours after a single dose of tocopherol. The linear relationship between tocopherol content in the diet and livcr storage holds even for very high intakes of tocopherol. Studies with C14-laheleddl-a-tocopheryl acetate reveal that absorpt,ion occurs rather slowly, reaching maximal concentrations only after a few
ABSORPTION AND DISTRIBUTION OF VITAMIN E
455
hours. High levels, however, remain for longer periods. Of particular interest is thc high lcvcls attained in the adrenals. In liver cell particlcs, the hulk of the tocopherol is located in t>hcstructural part of the cell, especially in the mitochondria and microsomes. Thc antioxidant cthoxyquin is absorbed rapidly, reaching high concent,rations in t,he liver and kidneys; t,hen it is rapidly eliminated, following the pattern of an unphysiological substance. In the liver cell particles it is located primariIy in the supernatant rather than in the structural parts of the cells. REFEREXCES Alaupovic, P., Johnson, B. C., Crider, Q., Bhogawtn, H . N . , a n d Johnson, T3. J. 1961. Am. J . Clin. Rrutrition 9, Part 11, 76-88. Bolliger, H. It., and Bolliger-Quaife, M . L . 1956. Vitamin E . Atti conyr. i n t e r n . 3rd Venezia, 1956, pp. 30-45. Burinell, R. H . 1955. The effect of fish oil and diphenyl-p-phenylenediamineon the vitamin E metabolism of the chick. 1’h.T). Thesis, University of Connecticut, Storrs. Bunnell, R . H. 1957. Poultry Sci. 36, 413-516. Cowlishaw, 13., SZndergaard, E., Prange, I., and Dam, H . 1957. Riochim. et Biophys. Acta 26, 644-645. Dicks, M. W., and Matterson, L. D. 1961. J . Nutrition 76, 165-174. Dju, M. Y., Quaife, M. L., arid Harris, P. L. 1950. A m . J . Physiol. 160, 259-263. Draper, H. H., and Alaupovic, 1’. 1959. Federation Proc. 18, 218. Eaton, H . D., Rousseau, J. E., Jr., Diclts, M. W., Teichman, R., and Grifo, A. P., Jr. 1958. J . Dairy Sci. 41, 1456-1459. Edwin, E. E., Diplock, A. T., Bunyan, J., and Green, J. 1961. Biochem. J . 79, 91-105. Gray, D. E. 1960. J . Vitamind. (Osaka)6, 155-157. GriKiths, T . W. 1959. h7ature 183, 1061-1062. Griffiths, T . W. 1960. Brit. J . Nntrition 14, 269-280. Kalberer, F., and Rutschmann, J . 1961. Helv. Chim. Acta 44, 1956-1966. Klatskin, G., and Molandcr, D. W. 1952. J . Lab. Clin. Med. 39, 802-813. Martius, C., and Costeli, J . 1957. Biochem. Z . 329, 449. Niedner, A. It. B. 1957. Dissertation Abstr. 17, 2794. Pudelkiewicz, W. J., and Matterson, L. D . 1960. J . Nutrition 71,143-148. Pudelkiewicx, W. ,J., Matterson, L. D., Potter, L. M., Webster, L., and Singsen, E. P. 1960. J . Nutrition 71, 115-121. Quaife, M. L., Swanson, W. J., I>ju, M. Y., and Harris, P . I,. 1949. A n n . N . Y . Acad. S C ~62, . 300-305. Rindi, G., and I’erri, V. 1958. Intern. Z. Vitaminjorsch. 28, 274-281. Rousseau, J . E. Jr., Dicks, M. W., Teichman, R., Helmboldt, C. F., Bacon, E. L., Prouty, It. M., IIoIge, K. L., Eaton, H. D., dungherr, E. I,., and B e d , G. 1957. J . Animal 9 c i . 16, 612-622. Schneider, W. C. 1948. J . B i d . Chem. 176, 259-266. Simon, E . J., Gross, C. S., andMilhorat, A. T . 1956. J . Biol.Chem. 221, 797-805. Sternberg, J., and Pascoe-Dawson, E. 1959. Can. Med. Assoc. J. 80, 266-275. Swick, R . W., and Baumann, C. A. 1952. Anal. Chern. 24, 758-760 Wilson, R . H., Thomas, J. O., Thompson, C. R., Launer, H. F., and Kohler, G. 0. 1959. J . Sci. Food Agr. 10, Abstracts, P a r t 11, 143.