J. G. Bieri,2
and polyunsaturated in human tissues’
Ph.D.,
and
R. Poukka
ABSTRACT were
Five
analyzed
and
1958,
and
adipose
times 1949
the
or
three than
1958.
remarkable.
tissues and
The
from
presumably The from
tissue molar
calculated and its possible 28: 717-720, 1975.
three
cancer
ratio
American
Journal
of Clinical
Nutrition
had
were
of the
28: JULY
heart
no lower
contained
vitamin
cancer
victims
two
reports
in 1949
in liver,
twice
as high
was
considerably than
linoleic
acids
adult
with
in a-tocopherol
on a tissue fatty
three
concentrations was
in all tissues
expressed
in evaluating
and
compared
a-tocopherol
six subjects
subjects,
suddenly When
but
of -y-tocopherol
of polyunsaturated use
died
acids.
as earlier,
patients
three these
who
fatty
subjects
range
content
from in
same
Evaluation of nutritional status for vitamin E currently depends on either the plasma concentration of tocopherols or the in vitro hemolysis of red cells. The latter test gives an imprecise differentiation between deficiency and adequacy and has been criticized for its lack of specificity (1). Plasma levels of total tocopherols above 0.5 mg/dl are assumed to indicate an adequate intake of vitamin E (2) but in view of the known relationship between plasma tocopherols and plasma lipids (3), the significance of the blood tocopherol concentration alone has become uncertain (4). Even if a ratio between plasma tocopherols and lipids is accepted as a better expression of vitamin E status, there is essentially no quantitative information on the relationship between these plasma parameters and the deposition of tocopherols in body tissues. It would appear that periodic analyses of tocopherols and polyunsaturated fatty acids (PUFA) in tissues would provide the most accurate characterization of vitamin E status and permit detection of changes in dietary intake of these nutrients. Human tissue tocopherols were determined in two subjects by Quaife and Dju in 1949 (5) and in a larger number by Dju et al. in 1958 (6), and adipose fatty acids have been reported periodically (7-9). In this report, we compare tocopherols and polyunsaturated fatty acids in tissues of The
adults and
normal
the
earlier.
Tocopherols
D.V.M.
-y-tocopherols
in about
Tissues
Adipose acids.
a-
tissue
higher
subjects. fatty
for
Evarts,3
acid
weight
or tissue
and
muscle was
higher
tissues lipid in heart
is discussed.
Am.
three than
from
exceeding
to a-tocopherol
E status
lung
in
normal
15% of total basis,
were
not
and
lung
was
J.
Chin.
Nutr.
six adult subjects who died in the United States in 1973 with similar information reported 15 or more years ago. Materials
and methods
Tissues were obtained from autopsies performed within 18 hours following death (see Table I for description of subjects). After removal of adhering fat, tissues were frozen at - 20 C until analyzed, usually within 7 days. Tocopherols were separated from the unsaponifiable fraction by thin-layer chromatography (TLC) and measured colorimetrically as previously described (10). Two distinct spots were taken from the TLC plates, a-tocopherol and the spot containing both fi-and y-tocopherols. Since the amount of ‘y-tocopherol in mixed U.S. diets greatly exceeds that of -tocopherol, this spot ids
is and
reported fatty
as acids
‘y-tocopherol. were
described previously (II) found by gas chromatography
Results
Weights
determined
and
fatty (II).
acid
of
total
lip-
gravimetrically
as
distribution
was
and discussion
Concentrations of a- and -y-tocopherols in five tissues from three presumably normal subjects and three cancer patients are shown in Table 1. Generally there was at least a ‘From the Section on Nutritional Biochemistry, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Metabolism and Digestive Diseases. National Institutes of Health, Bethesda, Maryland 20014. 2 Chief, Section on Nutritional Biochemistry. Visiting Scientist.
1975.
pp.
7 17-720.
Printed
in U.S.A.
717
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/717/4716471 by University of Western Sydney Library user on 10 January 2019
Tocopherols fatty acids
718 TABLE
BIERI
AND
y-tocopherol
in
POUKKA
I of a-tocopherol
Concentrations
and
human
Age
death1
a
‘y
I 2 3
M F M
39 48 52
AMI CVA AMI
18.0 14.9 22.2
Blank
spaces accident: ‘ Psoas
Lu ng
Mus dc’
a
y
a
14.0 6.4 -
15.6 10.0 21.2
from cancer l7.3 4.l 18.8 1.4 28.9 7.7
10.8 32.7 26.4
Sudden
#{176}
tissue” Adip
ose”
35 37 54 indicate OC muscle.
=
OC BC
82.0’
5.1 11.9 10.6 Death 14.4
TC
30.4
12.3
tissues were not metastatic ovarian d Subcutaneous
available. cancer: fat.
=
a
‘y
a
‘y
2.3 4.5 6.6
l4.l 8.2 16.8
3.2 4.5 5.5
III 74.9 275
32.0 70.6 103
2.0 3.4 6.0
8.9 3.9 16.3
l.3 0.9 5.2
342 354 629
144 86.0 l48
death
27.0 12.7
AMI BC Tissue
twofold variation in the concentration of a-tocopherol in each tissue of the normal subjects. It was surprising to find values in cancer patients at least as high as in normals since two of the patients (nos. 4 and 6) had lost considerable weight prior to death. These subjects were continuously hospitalized for several months prior to death and it is highly unlikely that they received supplementary a-tocopherol during this time. An earlier study (6) did not find any consistent lowering of values in subjects dying from various illnesses when compared with accidental death victims. Comparison of these data with similar information in the literature is difficult because of the different methodology used in earlier studies (5, 6). Dju et al. (6) suggested that the expression of tocopherols onthe basis of tissue lipid content rather than on a whole tissue basis tended to reduce the variation among individuals for any given organ. This expression for five of our subjects is shown in Table 2. A twoor threefold variation still exists for each tissue, however, the rank order of concentrations is quite different from that in Table 1. Certain marked differences from the values found by Dju et al. (6) are the much higher a-tocopherol content in heart (Dju et al. (6) reported a range of 135-406, avg 269 tg/g lipid), and also in lung (Dju et al. (6) reported a range of 100-300, avg 200 g/g lipid). Values for liver and muscle in the two studies do not show any appreciable differences. Underwoodetal.(12)in l970analyzed liver of adult accident victims for a-tocoph=
y
= acute myocardial infarction: CVA metastatic breast cancer: TC = metastatic had abnormally high lipid content, I95.
=
=
cerebral thyroid
TABLE
2 Concentration human tissues,
in lipid
from
Liver
Heart
Muscle
I 2 3 4 5
65 180 400 440
795 400 910 780
280 330 425 400 555
880 455 990 1,110 975
Mean
271
721
398
882
described
in Table
Subj#{176}
a
no
of a-tocopherol g/g lipid
Subjects
I . Blank
Lung
spaces
indicate
analysis.
erol. using a similar analytical method, and found an average (260 g/g lipid) about the same as that reported here. Adipose tissue in earlier studies, as also found here, ranged very widely so that average values may have little meaning. It is of interest that of our three normal subjects, no. 3 had consistently higher values for a-tocopherol on a whole tissue basis than the other two, and subject no. 1 was consistently higher than no. 2 (Table 1 ). These results appear to be contrary to the observation of Dju et al. (6), who found no consistent relationship between high and low levels in tissues from the same individual. Of particular interest are the levels of -y-tocopherol since earlier analyses did not have the refinement of tocopherol separation by thin-layer chromatography but did determine #{176}y+ #{244}-tocopherols chemically. Quaife and Dju (5) found ‘y-tocopherol only in the fat tissue of a male subject but in all tissues from
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/717/4716471 by University of Western Sydney Library user on 10 January 2019
Heart
Sex
vascular cancer.
ig/g
of
Subj
F F F
tissues.
Li ver
Cause .
4 5 6
EVARTS
TOCOPHEROLS
AND
POLYUNSATURATED
ses
of
polyunsaturated
fatty
acids
in
tissues
from the subjects in this study are shown in Table 3 (linolenic acid is omitted for brevity). It would appear that several of these subjects (nos. 2, 3, and 5) consumed relatively more polyunsaturated fat than the others, as noted especially in the adipose linoleate content (15.8-17.4%), but apparent in other tissues also. The tocopherol content of tissues from these subjects does not appear to bear any consistent relationship with their generally higher polyunsaturated fatty acid content. In a previous study rats fed 20% corn or soybean oil as the sole source of dietary fat and vitamin E were judged to have a satisfactory vitamin E status (15). Calculations were made for the molar ratio of total PUFA to ct-tocopherol in tissues and it was also suggested that the molar ratio of the more highly unsaturated fatty acids (those with three or more double bonds) to a-tocopherol may give a more meaningful estimate of the tissue adequacy of vitamin E than the ratio which included linoleic acid (11). These ratios for three tissues from four subjects from whom sufficient material was available were
719
ACIDS
calculated (11) and are shown in Table 4. The ratios of total PUFA to a-tocopherol for heart in three subjects were lower (more favorable) than those found in rats (810) but the ratios of PUFA > 18:2 to a-tocopherol were similar (350 for rat). Lung ratios of total PUFA to a-tocopherol were about onethird those in rat lung (1,470), but PUFA> 18:2 to a-tocopherol ratios were about the same (300 for rat). Due to a much lower linoleic acid content and considerably greater a-tocopherol concentration in human adipose tissue, the ratios of total PUFA to a-tocopherol were very much lower than those for rats (25,000-55,000). The difference in ratios of PUFA > 18:2 to a-tocopherol were not as marked, being about 2 to 10 times greater for rat adipose tissue. It is of interest that human adipose tissue had ratios similar to those for heart and lung, particularly the ratio using PUFA > 18:2. If this relationship can be shown for a larger population it may provide a useful estimate of body vitamin E status. TABLE
3
Polyunsaturated fatty acids in human total fatty acids as determined by gas
tissues (percent chromatOgraphy)”
of
Subject Fatty acid6
I
2
18:2 20:4 >20:4
9.6 0.9 0.3
13.3 3.5 2.4
18:2 20:4 >20:4
14.8 ll.2 3.7
18.3 13.5 4.5
18:2 20:4 >20:4
10.3 13.0 5.2
18:2 20:4 >20:4
11.8 1.7 1.1
18:2 20:4 >20:4
9.8 0.4
and polyunsaturated in human tissues’
Ph.D.,
and
R. Poukka
ABSTRACT were
Five
analyzed
and
1958,
and
adipose
times 1949
the
or
three than
1958.
remarkable.
tissues and
The
from
presumably The from
tissue molar
calculated and its possible 28: 717-720, 1975.
three
cancer
ratio
American
Journal
of Clinical
Nutrition
had
were
of the
28: JULY
heart
no lower
contained
vitamin
cancer
victims
two
reports
in 1949
in liver,
twice
as high
was
considerably than
linoleic
acids
adult
with
in a-tocopherol
on a tissue fatty
three
concentrations was
in all tissues
expressed
in evaluating
and
compared
a-tocopherol
six subjects
subjects,
suddenly When
but
of -y-tocopherol
of polyunsaturated use
died
acids.
as earlier,
patients
three these
who
fatty
subjects
range
content
from in
same
Evaluation of nutritional status for vitamin E currently depends on either the plasma concentration of tocopherols or the in vitro hemolysis of red cells. The latter test gives an imprecise differentiation between deficiency and adequacy and has been criticized for its lack of specificity (1). Plasma levels of total tocopherols above 0.5 mg/dl are assumed to indicate an adequate intake of vitamin E (2) but in view of the known relationship between plasma tocopherols and plasma lipids (3), the significance of the blood tocopherol concentration alone has become uncertain (4). Even if a ratio between plasma tocopherols and lipids is accepted as a better expression of vitamin E status, there is essentially no quantitative information on the relationship between these plasma parameters and the deposition of tocopherols in body tissues. It would appear that periodic analyses of tocopherols and polyunsaturated fatty acids (PUFA) in tissues would provide the most accurate characterization of vitamin E status and permit detection of changes in dietary intake of these nutrients. Human tissue tocopherols were determined in two subjects by Quaife and Dju in 1949 (5) and in a larger number by Dju et al. in 1958 (6), and adipose fatty acids have been reported periodically (7-9). In this report, we compare tocopherols and polyunsaturated fatty acids in tissues of The
adults and
normal
the
earlier.
Tocopherols
D.V.M.
-y-tocopherols
in about
Tissues
Adipose acids.
a-
tissue
higher
subjects. fatty
for
Evarts,3
acid
weight
or tissue
and
muscle was
higher
tissues lipid in heart
is discussed.
Am.
three than
from
exceeding
to a-tocopherol
E status
lung
in
normal
15% of total basis,
were
not
and
lung
was
J.
Chin.
Nutr.
six adult subjects who died in the United States in 1973 with similar information reported 15 or more years ago. Materials
and methods
Tissues were obtained from autopsies performed within 18 hours following death (see Table I for description of subjects). After removal of adhering fat, tissues were frozen at - 20 C until analyzed, usually within 7 days. Tocopherols were separated from the unsaponifiable fraction by thin-layer chromatography (TLC) and measured colorimetrically as previously described (10). Two distinct spots were taken from the TLC plates, a-tocopherol and the spot containing both fi-and y-tocopherols. Since the amount of ‘y-tocopherol in mixed U.S. diets greatly exceeds that of -tocopherol, this spot ids
is and
reported fatty
as acids
‘y-tocopherol. were
described previously (II) found by gas chromatography
Results
Weights
determined
and
fatty (II).
acid
of
total
lip-
gravimetrically
as
distribution
was
and discussion
Concentrations of a- and -y-tocopherols in five tissues from three presumably normal subjects and three cancer patients are shown in Table 1. Generally there was at least a ‘From the Section on Nutritional Biochemistry, Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Metabolism and Digestive Diseases. National Institutes of Health, Bethesda, Maryland 20014. 2 Chief, Section on Nutritional Biochemistry. Visiting Scientist.
1975.
pp.
7 17-720.
Printed
in U.S.A.
717
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/717/4716471 by University of Western Sydney Library user on 10 January 2019
Tocopherols fatty acids
718 TABLE
BIERI
AND
y-tocopherol
in
POUKKA
I of a-tocopherol
Concentrations
and
human
Age
death1
a
‘y
I 2 3
M F M
39 48 52
AMI CVA AMI
18.0 14.9 22.2
Blank
spaces accident: ‘ Psoas
Lu ng
Mus dc’
a
y
a
14.0 6.4 -
15.6 10.0 21.2
from cancer l7.3 4.l 18.8 1.4 28.9 7.7
10.8 32.7 26.4
Sudden
#{176}
tissue” Adip
ose”
35 37 54 indicate OC muscle.
=
OC BC
82.0’
5.1 11.9 10.6 Death 14.4
TC
30.4
12.3
tissues were not metastatic ovarian d Subcutaneous
available. cancer: fat.
=
a
‘y
a
‘y
2.3 4.5 6.6
l4.l 8.2 16.8
3.2 4.5 5.5
III 74.9 275
32.0 70.6 103
2.0 3.4 6.0
8.9 3.9 16.3
l.3 0.9 5.2
342 354 629
144 86.0 l48
death
27.0 12.7
AMI BC Tissue
twofold variation in the concentration of a-tocopherol in each tissue of the normal subjects. It was surprising to find values in cancer patients at least as high as in normals since two of the patients (nos. 4 and 6) had lost considerable weight prior to death. These subjects were continuously hospitalized for several months prior to death and it is highly unlikely that they received supplementary a-tocopherol during this time. An earlier study (6) did not find any consistent lowering of values in subjects dying from various illnesses when compared with accidental death victims. Comparison of these data with similar information in the literature is difficult because of the different methodology used in earlier studies (5, 6). Dju et al. (6) suggested that the expression of tocopherols onthe basis of tissue lipid content rather than on a whole tissue basis tended to reduce the variation among individuals for any given organ. This expression for five of our subjects is shown in Table 2. A twoor threefold variation still exists for each tissue, however, the rank order of concentrations is quite different from that in Table 1. Certain marked differences from the values found by Dju et al. (6) are the much higher a-tocopherol content in heart (Dju et al. (6) reported a range of 135-406, avg 269 tg/g lipid), and also in lung (Dju et al. (6) reported a range of 100-300, avg 200 g/g lipid). Values for liver and muscle in the two studies do not show any appreciable differences. Underwoodetal.(12)in l970analyzed liver of adult accident victims for a-tocoph=
y
= acute myocardial infarction: CVA metastatic breast cancer: TC = metastatic had abnormally high lipid content, I95.
=
=
cerebral thyroid
TABLE
2 Concentration human tissues,
in lipid
from
Liver
Heart
Muscle
I 2 3 4 5
65 180 400 440
795 400 910 780
280 330 425 400 555
880 455 990 1,110 975
Mean
271
721
398
882
described
in Table
Subj#{176}
a
no
of a-tocopherol g/g lipid
Subjects
I . Blank
Lung
spaces
indicate
analysis.
erol. using a similar analytical method, and found an average (260 g/g lipid) about the same as that reported here. Adipose tissue in earlier studies, as also found here, ranged very widely so that average values may have little meaning. It is of interest that of our three normal subjects, no. 3 had consistently higher values for a-tocopherol on a whole tissue basis than the other two, and subject no. 1 was consistently higher than no. 2 (Table 1 ). These results appear to be contrary to the observation of Dju et al. (6), who found no consistent relationship between high and low levels in tissues from the same individual. Of particular interest are the levels of -y-tocopherol since earlier analyses did not have the refinement of tocopherol separation by thin-layer chromatography but did determine #{176}y+ #{244}-tocopherols chemically. Quaife and Dju (5) found ‘y-tocopherol only in the fat tissue of a male subject but in all tissues from
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/7/717/4716471 by University of Western Sydney Library user on 10 January 2019
Heart
Sex
vascular cancer.
ig/g
of
Subj
F F F
tissues.
Li ver
Cause .
4 5 6
EVARTS
TOCOPHEROLS
AND
POLYUNSATURATED
ses
of
polyunsaturated
fatty
acids
in
tissues
from the subjects in this study are shown in Table 3 (linolenic acid is omitted for brevity). It would appear that several of these subjects (nos. 2, 3, and 5) consumed relatively more polyunsaturated fat than the others, as noted especially in the adipose linoleate content (15.8-17.4%), but apparent in other tissues also. The tocopherol content of tissues from these subjects does not appear to bear any consistent relationship with their generally higher polyunsaturated fatty acid content. In a previous study rats fed 20% corn or soybean oil as the sole source of dietary fat and vitamin E were judged to have a satisfactory vitamin E status (15). Calculations were made for the molar ratio of total PUFA to ct-tocopherol in tissues and it was also suggested that the molar ratio of the more highly unsaturated fatty acids (those with three or more double bonds) to a-tocopherol may give a more meaningful estimate of the tissue adequacy of vitamin E than the ratio which included linoleic acid (11). These ratios for three tissues from four subjects from whom sufficient material was available were
719
ACIDS
calculated (11) and are shown in Table 4. The ratios of total PUFA to a-tocopherol for heart in three subjects were lower (more favorable) than those found in rats (810) but the ratios of PUFA > 18:2 to a-tocopherol were similar (350 for rat). Lung ratios of total PUFA to a-tocopherol were about onethird those in rat lung (1,470), but PUFA> 18:2 to a-tocopherol ratios were about the same (300 for rat). Due to a much lower linoleic acid content and considerably greater a-tocopherol concentration in human adipose tissue, the ratios of total PUFA to a-tocopherol were very much lower than those for rats (25,000-55,000). The difference in ratios of PUFA > 18:2 to a-tocopherol were not as marked, being about 2 to 10 times greater for rat adipose tissue. It is of interest that human adipose tissue had ratios similar to those for heart and lung, particularly the ratio using PUFA > 18:2. If this relationship can be shown for a larger population it may provide a useful estimate of body vitamin E status. TABLE
3
Polyunsaturated fatty acids in human total fatty acids as determined by gas
tissues (percent chromatOgraphy)”
of
Subject Fatty acid6
I
2
18:2 20:4 >20:4
9.6 0.9 0.3
13.3 3.5 2.4
18:2 20:4 >20:4
14.8 ll.2 3.7
18.3 13.5 4.5
18:2 20:4 >20:4
10.3 13.0 5.2
18:2 20:4 >20:4
11.8 1.7 1.1
18:2 20:4 >20:4
9.8 0.4
