Serum Concentrations Vitamin E
and Cellular Uptake of
M. J. GONZALEZ Michigan State University, 48824, USA
Deparment
of Food Science and Human Nutrition,
East Lansing, Michigan
Abstract - The high affinity receptor for low density lipoprotein (LDL) is demonstrated to function as a mechanism for delivery of vitamin E to cells. It is suggested that LDL which serves as its carrier is specific for d-alpha tocopherol which in turn is the most active biological formof vitamin E in human adults; also, its concentration is the highest in human adults serum as compared to other forms such as beta, gamma, delta, alpha tocopherol succinate and acetate when serum concentrations of human adults were measured after ingestion of various alpha tocopherol preparations the highest concentration in serum was achieved by d-alpha tocopherol over other forms as alpha tocopherol acetate or alpha tocopherol succinate. This data pemits one to conclude that the most important biological form of vitamin E for absorption, transport and utilization is the d-alpha tocopherol. Specific receptor sites are apparent for d-alpha tocopherol in LDL, for which a high affinity receptor exists in the cells. This has been demonstrated to be a mechanism for cellular uptake of d-alpha tocopherol and cholesterol by cells.
Introduction
It is well established that alpha tocopherol is carried in blood lipoproteins primarily in low density lipoproteins (LDL), and/or high density lipoproteins (HDL) (l-4). Previous data has indicated that LDL and HDL are the main carriers of alpha tocopherol in both males and females. It has been suggested that this different distri-
Date received_ 20 FebruaJ~ 1989 Date accepted 18 August 1989
bution is due to the different levels of orotein . in the lipoprotein fractions (5). Furthermore, tocopherol can exchange between the different lipoproteins and it can exchange between lipoproteins and erythrocytes. In this regard, tocopherol is similar to cholesterol (4). LDL contains a large proportion of the plasma tocopherol and LDL has been demonstrated to enter cells via the specific, high affinity LDL receptor mechanism (5). The most abundant tocopherol in diet is gamma tocopherol but very little is known about its plasma transport. Gamma tocopherol con-
107
108 centration in the plasma and tissues is low in comparison to that of alpha tocopherol (6). Moreover, it has been suggested that the concentration of gamna tocopherol in plasma and tissues is determined by the relative concentration of alpha tocopherol (6). Thus, it has been assumed that the absorption, transport and tissue uptake of vitamin E is specific for alpha tocopherol. It has been determined that d-alpha tocopherol is the form of vitamin E that when ingested increases the most in the serum (8) as compared to other vitamin forms such as succinate and acetate. D-alpha tocopherol has been reported as the most biologically active form of vitamin E (2, 3, 9). Experiments with rats indicated that when the concentration of alpha tocopherol was relatively low, gamma tocopherol was absorbed, transported, and taken up by tissues in increased amounts (6, 7). This review will try to relate cellular uptake of the vitamin with serum concentrations in a way that will help in the understanding of this important physiological pathway of this lipid soluble nutrient. Discussion Traber and Kayden (4) tested whether the presence of LDL receptors was necessary for the transfer of tocopherol from LDL to cells by making a comparison of the tocopherol contents of cells which do or do not have LDL receptors, after incubation with LDL. Fibroblasts from either a normal subject or from a patient with a form of familial hyperhomozygous cholesterolemia were incubated overnight in lipoprotein deficient serum which results in the efflux of cellular cholesterol, which in turn stimulates the synthesis of the rate-limiting enzyme in cholesterol synthesis 3-hydroxy-3-methyl glutaryl coenzyme A, and the synthesis of LDL receptors. As fibroblasts from homozygotes for familial hypercholesterolemia do not have the capability of synthesizing LDL receptors, therefore, incubation with lipoprotein deficient serum (LPDS) does not result in LDL receptor activity in these cells. After incubation with 10% LDPS for 24 h, the fibroblasts were then incubated with or without LDL (100 rug of protein per ml) for 4 h. The cells were harvested and the cellular tocopherol content measured. The normal fibroblasts that had been incubated with LDL contained 45+18 ng/mg cell protein, twice as
MEDICAL HYPOTHESES
much tocopherol as normal fibroblasts not exposed to LDL, which contained 19+7. In contrast, irrespective of whether or not the medium contained LDL, the LDL receptor negative fibroblast contained the same amount of intracellular tocopherol (15+4 compared to ll+lO ng/mg cell protein, respectively). The level of intracellular tocopherol in the receptor negative cells was the same as that in the normal cells which had not been exposed to LDL. This data suggests that the tocopherol present in LDL is made available to cells via the LDL receptor mechanism, which is regulated by cellular cholesterol requirements. Interestingly, the LDL receptor negative fibroblasts from the patient with the homozygous form of familial hypercholesterolemia did contain a measurable amount of tocopherol under all incubation conditions. This data suggests that the LDL receptor mechanism is not the only mechanism for delivery of tocopherol to cells, which is consistent with the observation that patients with the homozygous form of hypercholesterolemia do not become vitamin E deficient. The form of vitamin more often found bound to LDL is alpha tocopherol (4). For cellular uptake as a means of transportation it is clearly estabhshed that vitaminE utihzes primarily LDL (l-4). Behrens and Madere (5) measured the alpha and gamma tocopherols and total lipid concentration in the serum of 451 healthy human male and female subjects ranging in age from 19 to 70 years with mean serum alpha tocopherol concentrations of 1.3.5kO.70. The concentration of alpha tocopherol increased significantly with age, as well as with total lipids; gamma tocopherol concentration remained unchanged with age. The data indicated that subjects with higher serum alpha tocopherol had lower gamma tocopherol levels and vice versa. A close interrelationship and competition appeared to exist between these two forms of vitamin E in subjects with high gamma tocopherol levels (5). The result of this study showed that alpha tocopherol is the major tocopherol in plasma of the adult human, accounting for nearly 87% of the total tocopherol concentration (4, 10-12). This finding is interesting in view of the fact that a typical North American diet contains two to four times more gamma tocopherol than alpha tocopherol (11). Experimental data suggest that the absorption, transport and tissue uptake of vitamin E are so specific for alpha tocopherol that a relatively small amount of it is sufficient to displace gamma tocopherol (6). This could
SERUM
CONCENTRATIONS
AND CELLULAR
UPTAKE
OF VITAMIN
indicate that gamma tocopherol does not contribute to the vitamin E activity when an adequate amount of alpha tocopherol is present in the diet. However, some data have suggested an interrelationship or competition between them could occur, especially in conditions where the amount of alpha tocopherol is relatively low in comparison to that of gamma, beta or delta forms of tocopherol (5). It has been proposed that the mechanisms regulating the absorption, plasma transport, and tissue uptake of vitamin E are determined by specific carriers and/or binding sites for alpha tocopherol (4, 6, 7). This statement is fully supported by the work of Traber and Kayden (4) who found alpha tocopherol mainly carried in LDL, although this view is contradictory to other investigators who postulate that plasma tocopherol concentrations are dependent on total lipid concentration of plasma or tissues (8, 13). A small difference in serum tocopherol levels found between males and females in Behrens and Maderes experiments (5) could be explained in terms of lipid concentration. Moreover, their results demonstrated that alpha tocopherol is correlated with serum total lipids. Whereas total lipid and alpha tocopherol rise with age, the relationship between alpha tocopherol and total lipid remains significant in the presence of other variables, indicating that other factors besides total lipids determine the concentration of alpha tocopherol is plasma. Thus, even if lipid concentration determines the amount of tocopherol in plasma or tissues, this does not explain why the gamma tocopherol concentration is lower in plasma and tissues than is alpha tocopherol (6, 7). Another important aspect pertaining to serum concentrations of vitamin E is which is the most predominant form founded. It is reasonable to suppose that the biological activity of any vitamin E compound is related. to the time it remains in the blood and tissues in which it exerts its antioxidant or other physiological function. This supposition is the basis for measuring serum concentrations of alpha tocopherols as a function of time after administration of vitamin E compounds. The mode of administration of the vitamin E compound is of great importance to determine potencies, i.e. the vehicle in which vitamin E is administered may have an effect on the rate of absorption. Horwitt et al. (8) studied serum concentrations after ingestion of d-alpha tocopherol, alpha tocopheryl acetate, all-rat-alpha tocopherylacetate. alpha tocopheryl succinate, alpha toco-
E
109
pheryl acetate plus apple pectin by 20 adult human subjects. Measurements were made at 0, 8, 24, 48 h after ingestion of 800 I.U. of the various preparations. The results at 24 h were representative of the differences observed. The main increase in concentration of alpha tocopherol (mg/g lipid) in 24 h was 72.2% after alpha tocopherol, 63.3% after tocopherol acetate plus apple pectin, 60.9% after tocopherol tocopherol acetate, 31.6% after all-rat-alpha acetate and 41.2% after alpha tocopherol succinate. This experiment indicates that d-alpha tocopherol is the most active form of vitamin E and also it is better absorbed and transported in the human adult which does not correlate with animal assays where the acetate form is favored (8). Summary The high affinity receptor for low density lipoprotein (LDL) is demonstrated to function as a mechanism for delivery of vitamin E to cells. It is suggested that LDL which serves as its carrier is specific for d-alpha tocopherol which in turn is the most active biological form of vitamin E in human adults; also, its concentration is the highest in human adult serum as compared to other forms such as beta, gamma, delta, alpha tocopherol succinate and acetate. When serum concentrations of human adults were measured after ingestion of various alpha tocopherol preparations, the highest concentration in serum was achieved by d-alpha tocopherol over such other forms as alpha tocopherol acetate or alpha tocopherol succinate. This data permits one to conclude that the most important biological form of vitamin E for absorption, transport and utilization is the dalpha tocopherol. Specific receptor sites are apparent for d-alpha tocopherol in LDL, for which a high affinity receptor exists in the cells. This has been demonstrated to be a mechanism for cellular uptake of d-alpha tocopherol and cholesterol by cells,
References 1. Lewis L A, Quaife M L and Page I H. Lipoproteins of serum, carriers of tocopherol. Am. J. Physiol. 1954; 178: 221-Z. 2. Davies T, Kelleher J and Losowsky M S. Interrelationship of serum lipoprotein and tocopherol levels. Clin. Chem. Acta 1969; 24: 431-6. 3. Bjornson L K, Kayden H J, Miller E, and Moshell A N.
110
4.
5.
6.
I. 8.
The transport of alpha tocopherol and beta carotene in human blood. J. Lipid Res. 1976; 17: 343-52. Traber M G and Kayden H J. Vitamin E is delivered to cells via the high affinitv receptor for low-densitv lipoprotein. Am. i. Clin. Nutr. 1984; 40: 747-51. . Behrens WA and Madere R. Alpha and gamma tocopherol concentrations in human serum. J. Am. Coil. Nutr. 1986; 5: 91-6. Behrens W A and Madere R. Interrelationship and competition of alpha and gamma tocopherol at the level of intestinal absorption, plasma transport and liver uptake. Nutr. Res. 1983; 3: 891-7. Behrens W A and Madere R. Studies on the absorption, plasma transport and tissue uptake and retention of alpha and gamma tocopherol. Fed. Proc. 1983; 42: 813. Horwitt M K, Elliott W H, Kanjahanggulpan P and Fitch C D. Serum concentrations of alpha tocopherol after ingestion of various vitamin E preparations. Am. J. Clin. Nutr. 1984; 40: 240-5.
,MEDICAL HYPOTHESES
9. Bieri J G, Corash L and Hubbard V S. Medical uses of vitamin E. N. Eng. J. Med. 1983; 308: 1063-71. 10. Chow F 1 and Omaye S T. Use of antioxidants in analysis of vitamins A, and E in mammalian plasma by high performance liquid chromatography. Lipids 1983; 11: 837-40. 11. Vatasserv G T, Krezowski A M and Eckfeldt J H. Vitamin
and Cellular Uptake of
M. J. GONZALEZ Michigan State University, 48824, USA
Deparment
of Food Science and Human Nutrition,
East Lansing, Michigan
Abstract - The high affinity receptor for low density lipoprotein (LDL) is demonstrated to function as a mechanism for delivery of vitamin E to cells. It is suggested that LDL which serves as its carrier is specific for d-alpha tocopherol which in turn is the most active biological formof vitamin E in human adults; also, its concentration is the highest in human adults serum as compared to other forms such as beta, gamma, delta, alpha tocopherol succinate and acetate when serum concentrations of human adults were measured after ingestion of various alpha tocopherol preparations the highest concentration in serum was achieved by d-alpha tocopherol over other forms as alpha tocopherol acetate or alpha tocopherol succinate. This data pemits one to conclude that the most important biological form of vitamin E for absorption, transport and utilization is the d-alpha tocopherol. Specific receptor sites are apparent for d-alpha tocopherol in LDL, for which a high affinity receptor exists in the cells. This has been demonstrated to be a mechanism for cellular uptake of d-alpha tocopherol and cholesterol by cells.
Introduction
It is well established that alpha tocopherol is carried in blood lipoproteins primarily in low density lipoproteins (LDL), and/or high density lipoproteins (HDL) (l-4). Previous data has indicated that LDL and HDL are the main carriers of alpha tocopherol in both males and females. It has been suggested that this different distri-
Date received_ 20 FebruaJ~ 1989 Date accepted 18 August 1989
bution is due to the different levels of orotein . in the lipoprotein fractions (5). Furthermore, tocopherol can exchange between the different lipoproteins and it can exchange between lipoproteins and erythrocytes. In this regard, tocopherol is similar to cholesterol (4). LDL contains a large proportion of the plasma tocopherol and LDL has been demonstrated to enter cells via the specific, high affinity LDL receptor mechanism (5). The most abundant tocopherol in diet is gamma tocopherol but very little is known about its plasma transport. Gamma tocopherol con-
107
108 centration in the plasma and tissues is low in comparison to that of alpha tocopherol (6). Moreover, it has been suggested that the concentration of gamna tocopherol in plasma and tissues is determined by the relative concentration of alpha tocopherol (6). Thus, it has been assumed that the absorption, transport and tissue uptake of vitamin E is specific for alpha tocopherol. It has been determined that d-alpha tocopherol is the form of vitamin E that when ingested increases the most in the serum (8) as compared to other vitamin forms such as succinate and acetate. D-alpha tocopherol has been reported as the most biologically active form of vitamin E (2, 3, 9). Experiments with rats indicated that when the concentration of alpha tocopherol was relatively low, gamma tocopherol was absorbed, transported, and taken up by tissues in increased amounts (6, 7). This review will try to relate cellular uptake of the vitamin with serum concentrations in a way that will help in the understanding of this important physiological pathway of this lipid soluble nutrient. Discussion Traber and Kayden (4) tested whether the presence of LDL receptors was necessary for the transfer of tocopherol from LDL to cells by making a comparison of the tocopherol contents of cells which do or do not have LDL receptors, after incubation with LDL. Fibroblasts from either a normal subject or from a patient with a form of familial hyperhomozygous cholesterolemia were incubated overnight in lipoprotein deficient serum which results in the efflux of cellular cholesterol, which in turn stimulates the synthesis of the rate-limiting enzyme in cholesterol synthesis 3-hydroxy-3-methyl glutaryl coenzyme A, and the synthesis of LDL receptors. As fibroblasts from homozygotes for familial hypercholesterolemia do not have the capability of synthesizing LDL receptors, therefore, incubation with lipoprotein deficient serum (LPDS) does not result in LDL receptor activity in these cells. After incubation with 10% LDPS for 24 h, the fibroblasts were then incubated with or without LDL (100 rug of protein per ml) for 4 h. The cells were harvested and the cellular tocopherol content measured. The normal fibroblasts that had been incubated with LDL contained 45+18 ng/mg cell protein, twice as
MEDICAL HYPOTHESES
much tocopherol as normal fibroblasts not exposed to LDL, which contained 19+7. In contrast, irrespective of whether or not the medium contained LDL, the LDL receptor negative fibroblast contained the same amount of intracellular tocopherol (15+4 compared to ll+lO ng/mg cell protein, respectively). The level of intracellular tocopherol in the receptor negative cells was the same as that in the normal cells which had not been exposed to LDL. This data suggests that the tocopherol present in LDL is made available to cells via the LDL receptor mechanism, which is regulated by cellular cholesterol requirements. Interestingly, the LDL receptor negative fibroblasts from the patient with the homozygous form of familial hypercholesterolemia did contain a measurable amount of tocopherol under all incubation conditions. This data suggests that the LDL receptor mechanism is not the only mechanism for delivery of tocopherol to cells, which is consistent with the observation that patients with the homozygous form of hypercholesterolemia do not become vitamin E deficient. The form of vitamin more often found bound to LDL is alpha tocopherol (4). For cellular uptake as a means of transportation it is clearly estabhshed that vitaminE utihzes primarily LDL (l-4). Behrens and Madere (5) measured the alpha and gamma tocopherols and total lipid concentration in the serum of 451 healthy human male and female subjects ranging in age from 19 to 70 years with mean serum alpha tocopherol concentrations of 1.3.5kO.70. The concentration of alpha tocopherol increased significantly with age, as well as with total lipids; gamma tocopherol concentration remained unchanged with age. The data indicated that subjects with higher serum alpha tocopherol had lower gamma tocopherol levels and vice versa. A close interrelationship and competition appeared to exist between these two forms of vitamin E in subjects with high gamma tocopherol levels (5). The result of this study showed that alpha tocopherol is the major tocopherol in plasma of the adult human, accounting for nearly 87% of the total tocopherol concentration (4, 10-12). This finding is interesting in view of the fact that a typical North American diet contains two to four times more gamma tocopherol than alpha tocopherol (11). Experimental data suggest that the absorption, transport and tissue uptake of vitamin E are so specific for alpha tocopherol that a relatively small amount of it is sufficient to displace gamma tocopherol (6). This could
SERUM
CONCENTRATIONS
AND CELLULAR
UPTAKE
OF VITAMIN
indicate that gamma tocopherol does not contribute to the vitamin E activity when an adequate amount of alpha tocopherol is present in the diet. However, some data have suggested an interrelationship or competition between them could occur, especially in conditions where the amount of alpha tocopherol is relatively low in comparison to that of gamma, beta or delta forms of tocopherol (5). It has been proposed that the mechanisms regulating the absorption, plasma transport, and tissue uptake of vitamin E are determined by specific carriers and/or binding sites for alpha tocopherol (4, 6, 7). This statement is fully supported by the work of Traber and Kayden (4) who found alpha tocopherol mainly carried in LDL, although this view is contradictory to other investigators who postulate that plasma tocopherol concentrations are dependent on total lipid concentration of plasma or tissues (8, 13). A small difference in serum tocopherol levels found between males and females in Behrens and Maderes experiments (5) could be explained in terms of lipid concentration. Moreover, their results demonstrated that alpha tocopherol is correlated with serum total lipids. Whereas total lipid and alpha tocopherol rise with age, the relationship between alpha tocopherol and total lipid remains significant in the presence of other variables, indicating that other factors besides total lipids determine the concentration of alpha tocopherol is plasma. Thus, even if lipid concentration determines the amount of tocopherol in plasma or tissues, this does not explain why the gamma tocopherol concentration is lower in plasma and tissues than is alpha tocopherol (6, 7). Another important aspect pertaining to serum concentrations of vitamin E is which is the most predominant form founded. It is reasonable to suppose that the biological activity of any vitamin E compound is related. to the time it remains in the blood and tissues in which it exerts its antioxidant or other physiological function. This supposition is the basis for measuring serum concentrations of alpha tocopherols as a function of time after administration of vitamin E compounds. The mode of administration of the vitamin E compound is of great importance to determine potencies, i.e. the vehicle in which vitamin E is administered may have an effect on the rate of absorption. Horwitt et al. (8) studied serum concentrations after ingestion of d-alpha tocopherol, alpha tocopheryl acetate, all-rat-alpha tocopherylacetate. alpha tocopheryl succinate, alpha toco-
E
109
pheryl acetate plus apple pectin by 20 adult human subjects. Measurements were made at 0, 8, 24, 48 h after ingestion of 800 I.U. of the various preparations. The results at 24 h were representative of the differences observed. The main increase in concentration of alpha tocopherol (mg/g lipid) in 24 h was 72.2% after alpha tocopherol, 63.3% after tocopherol acetate plus apple pectin, 60.9% after tocopherol tocopherol acetate, 31.6% after all-rat-alpha acetate and 41.2% after alpha tocopherol succinate. This experiment indicates that d-alpha tocopherol is the most active form of vitamin E and also it is better absorbed and transported in the human adult which does not correlate with animal assays where the acetate form is favored (8). Summary The high affinity receptor for low density lipoprotein (LDL) is demonstrated to function as a mechanism for delivery of vitamin E to cells. It is suggested that LDL which serves as its carrier is specific for d-alpha tocopherol which in turn is the most active biological form of vitamin E in human adults; also, its concentration is the highest in human adult serum as compared to other forms such as beta, gamma, delta, alpha tocopherol succinate and acetate. When serum concentrations of human adults were measured after ingestion of various alpha tocopherol preparations, the highest concentration in serum was achieved by d-alpha tocopherol over such other forms as alpha tocopherol acetate or alpha tocopherol succinate. This data permits one to conclude that the most important biological form of vitamin E for absorption, transport and utilization is the dalpha tocopherol. Specific receptor sites are apparent for d-alpha tocopherol in LDL, for which a high affinity receptor exists in the cells. This has been demonstrated to be a mechanism for cellular uptake of d-alpha tocopherol and cholesterol by cells,
References 1. Lewis L A, Quaife M L and Page I H. Lipoproteins of serum, carriers of tocopherol. Am. J. Physiol. 1954; 178: 221-Z. 2. Davies T, Kelleher J and Losowsky M S. Interrelationship of serum lipoprotein and tocopherol levels. Clin. Chem. Acta 1969; 24: 431-6. 3. Bjornson L K, Kayden H J, Miller E, and Moshell A N.
110
4.
5.
6.
I. 8.
The transport of alpha tocopherol and beta carotene in human blood. J. Lipid Res. 1976; 17: 343-52. Traber M G and Kayden H J. Vitamin E is delivered to cells via the high affinitv receptor for low-densitv lipoprotein. Am. i. Clin. Nutr. 1984; 40: 747-51. . Behrens WA and Madere R. Alpha and gamma tocopherol concentrations in human serum. J. Am. Coil. Nutr. 1986; 5: 91-6. Behrens W A and Madere R. Interrelationship and competition of alpha and gamma tocopherol at the level of intestinal absorption, plasma transport and liver uptake. Nutr. Res. 1983; 3: 891-7. Behrens W A and Madere R. Studies on the absorption, plasma transport and tissue uptake and retention of alpha and gamma tocopherol. Fed. Proc. 1983; 42: 813. Horwitt M K, Elliott W H, Kanjahanggulpan P and Fitch C D. Serum concentrations of alpha tocopherol after ingestion of various vitamin E preparations. Am. J. Clin. Nutr. 1984; 40: 240-5.
,MEDICAL HYPOTHESES
9. Bieri J G, Corash L and Hubbard V S. Medical uses of vitamin E. N. Eng. J. Med. 1983; 308: 1063-71. 10. Chow F 1 and Omaye S T. Use of antioxidants in analysis of vitamins A, and E in mammalian plasma by high performance liquid chromatography. Lipids 1983; 11: 837-40. 11. Vatasserv G T, Krezowski A M and Eckfeldt J H. Vitamin
