Vitamin E, Retinyl Palmitate, and Protein in Rhesus
Monkey Retina and Retinal Pigment Epithelium-Choroid
Donald V. Crabtree,* Alice J. Adler,* and D. Max Snodderly*^
Purpose. To measure the amounts of vitamin E, retinyl palmitate, and protein in the primate
retina and its supporting tissues—the retinal pigment epithelium and choroid. To compare
the amounts and concentrations of these materials in the central retina with those in the
peripheral retina and to compare the concentration of vitamin E in the retina with that in
plasma. Finally, to compare these results in rhesus monkey with existing measurements in
humans.
Methods. Ocular tissues from rhesus monkeys {Macaca mulatto) were extracted with a twophase solvent system. Components in the extract were separated by reverse-phase high-pressure liquid chromatography. Two detectors in series monitored the effluent: Vitamin E was
quantified with an internal standard and fluorescence detection, whereas retinyl palmitate
was quantified with an external standard and ultraviolet light detection.
Results. Amounts of vitamin E, retinyl palmitate, and protein in tissues from rhesus monkey
compared reasonably well with those reported for humans. The content of vitamin E in the
peripheral neural retina was moderately correlated with its protein content and, to a greater
extent, with the concentration of vitamin E in the plasma; however, the content of vitamin
E in the central neural retina correlated only with the amount of protein in the central neural
retina.
Conclusions. These results are consistent with rhesus monkey as a model for the use of vitamin
E by human ocular tissues. The amount of vitamin E in the central neural retina appears to
be more closely regulated than the amount of vitamin E in the peripheral neural retina.
Invest Ophthalmol Vis Sci. 1996;37:47-60.
V itamin E has been shown to be important for good
health and a healthy retina.1'2 Its putative function is
that of a protective antioxidant for unsaturated lipids,
a task for which it is clearly well suited.3'4
Four common forms of vitamin E are: a-tocopherol (a-T), /5-tocopherol (fi-T), y-tocopherol (y-T),
and 6-tocopherol (6-T).5 All four forms have in common a phenolic functional group that is relatively stable after hydrogen atom abstraction (Fig. I).3'5 The
arrangement and number of methyl groups on the
chromanol ring account for the chemical differences
From the *Schepens Eye Research Institute and the *Departmenl of Ophthalmology
and f Program in Neuroscience, Harvard Medical School, Boston, Massachusetts.
Presented in part at the ARVO Annual Meeting, Sarnsota, Florida, May 1992, and
at the International Congress of Eye Research, Stresa, Italy, 1992.
Supported l/y National Institutes of Health grants EY04368 (AJA), EY04911
(DMS), and EY06591 (DMS), and by the Massachusetts Lions Eye Research Fund.
Submitted for publication March 13, 1995; revised August 7, 1995; accepted
September 11, 1995.
Proprietary interest category: N.
Reprint requests: Donald V. Crabtree., Schepens Eye Research Institute, 20 Slaniford
Street, Boston, MA 02114.
Investigative Ophthalmology & Visual Science, January 1996, Vol. 37, No. 1
Copyright © Association for Research in Vision and Ophthalmology
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that biologic systems use to distinguish among the
different forms of tocopherol.6 7 This study considered
a-T, (3-T, and y-T. Of the tocopherols, a-T has been
shown to be the most effective free-radical scavenger,3
the most biologically active tocopherol in humans,8'9
and the most predominant tocopherol in human retina10 and plasma." <5-T, which we did not consider, is
the least effective free-radical scavenger of the four
tocopherols.3 l2
Lipid makes up approximately 20% of the dry
weight of the vertebrate retina, and approximately two
thirds of that is phospholipid.13 Comparison of the
fatty acid composition of various rat organs indicates
that the mole percentage of fatty acids that are polyunsaturated fatty acids (PUFA) is very high in retina.14
Furthermore, rod outer segments, which are nearly
adjacent to the choriocapillaris and, hence, are subjected to high oxygen tension,15 are particularly enriched in the PUFA—docosahexaenoic acid.13 As a
consequence, one would anticipate that without ap-
47
Investigative Ophthalmology & Visual Science, January 1996, Vol. 37, No. 1
48
"2
T
O
CH 3
CH 3
Chromanol
Head
CH
CH,
CH,
Phytyl Tail
FIGURE 1. Structural diagram of the tocopherols. R is either
hydrogen or methyl. For a-T: Rl = R2 = methyl. For 0-T:
Rl = methyl; R2 = hydrogen. For y-T: Rl = hydrogen; R2
= methyl. For <5-T: Rl = R2 = hydrogen.
propriate antioxidant protectors, such as vitamin E,
the retina would be especially vulnerable to the ravages of lipid peroxidation.
Significant epidemiologic evidence supporting a
protective role for a-T in preventing age-related macular degeneration has been presented by West and colleagues.16 Consistent with those results, another epidemiologic study by the Eye Disease Case-Control Study
Group found that reduced serum levels of vitamin E
were associated with increased risk for neovascular
age-related macular degeneration.17 However, for that
study, there was not a clear trend because the highest
serum levels of vitamin E did not confer greater protection against neovascular age-related macular degeneration than the intermediate serum levels.17 This
makes us question how well serum levels of vitamin E
influence retinal levels. We have examined this question
using tissues from rhesus monkeys. Our results suggest
that the central retina more closely regulates its vitamin
E content than does the peripheral neural retina.
The development of efficacious treatments for human diseases is impaired without the benefit of an
appropriate animal model.18 In this article, we compare our measurements of vitamin E, retinyl palmitate,
and protein in the entire, peripheral, and central neural retina and the retinal pigment epithelium (RPE) choroid of rhesus monkey with the corresponding values for humans.1019"23 We also compare the plasma
levels of the different forms of vitamin E in humans"
and rhesus monkey. The similarity between results
from human eyes and from rhesus-monkey eyes suggest that the rhesus monkey may be a good animal
model for the use of vitamin E by human ocular tissue
and for understanding how vitamin E protects against
age-related macular degeneration.
19 years; there were 10 males and 6 females. Fourteen
of the monkeys had been housed in a local colony,
and tissue was shared with other investigators. Animal
quarters were illuminated by cool white fluorescent
lighting (12 hours on-12 hours off) and sunlight.
Ambient luminance levels ranged from 27 to 46 footcandles. Monkeys were fed Purina (Richmond, IN)
Monkey Chow #5038 ad libitum (stored at room temperature). Reported vitamin E and ascorbic acid content of this Chow was 55.0 IU/kg and 0.75 mg/g,
respectively. This nonpurified diet was supplemented
with apples, oranges, bananas, and carrots three times
per week. Although these supplements are low in vitamin E content,24 oranges may have a significant impact
on the plasma level of vitamin E.25
The other two animals (both 19 years of age) had
been transferred from other colonies. We were only
able to ascertain that for the preceding 21 months,
the lighting cycle had been 12 hours on-12 hours off
and that they had not been subjected to any procedures during that time.
For plasma analyses, blood was drawn onto ethylenediaminetetraacetic acid from the femoral vein, or
it was drawn from the heart just before whole body
perfusion. To remove blood from the tissues, all 16
monkeys were subjected to whole body perfusion. For
12 of them, the perfusate was Krebs-Henseleit buffer,
and for the other four, the perfusate was 4% paraformaldehyde in Krebs-Henseleit buffer. Eyecups from
two of the monkeys that had been perfused with
Krebs-Henseleit were immersed in half-strength Karnovsky'sfixative(2% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2 to 7.4) for
4 hours. Using fixed tissue facilitated sharing among
numerous other investigators and reduced the number of animals used. More specific details about individuals are tabulated in our companion article.26 All
experimental procedures conformed to the ARVO
Statement for the Use of Animals in Ophthalmic and
Vision Research.
Tissue Preparation
After death, heads were surrounded by crushed ice
within 35 ± 25 minutes (mean ± SD). Subsequently,
eyes were enucleated under ordinary fluorescent lighting at room temperature and put on ice (average postmortem time: 100 ± 50 minutes). Eyes were cleaved
anterior to the ora serrata, and the vitreous was removed with a Pasteur pipette. Each remaining eyecup
was flattened by making four cuts in the periphery
before pinning it to silicone rubber in the bottom of a
MATERIALS AND METHODS
Petri dish. Tissue was covered with ice-cold phosphateAnimals
buffered saline with 5 mM ascorbate (pH 7.2 to 7.4).27
Eyecups were dissected into a series of concentric
Retinas and RPE-choroids from 16 Old World rhesus
(Macaca mulatto) monkeys (coded: M05 to M32; M = rings, centered on the fovea. In some cases, the neural
retina was separated from the RPE-choroid. See our
Macaque) were studied. Their ages ranged from 2 to
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Vitamin £ in Macaque Retina and RPE-Choroid
companion article26 for details regarding the dissection of these two tissues and controls for possible crosscontamination. When the dissection was completed,
the pieces of tissue were stored at — 80°C until they
were analyzed for their content of tocopherols, retinyl
palmitate, and, in the case of fresh tissues, protein.
Average postmortem time until storage at — 80°C was
280 ± 120 minutes. A plot of the amount (nanomoles)
of a-T in neural retina versus postmortem time until
storage at — 80°C indicated that postmortem time did
not have a significant impact on the amount of a-T
in neural retina (data not shown; r = 0.22, P > 0.50
for slope different from zero). Average time for tissue
storage at —80°C before analysis was 4.0 ± 3.5 months.
The longest time any tissue was stored at — 80°C was
11.3 months, and 63% of the tissue was analyzed
within 4.0 months. Vitamin E is stable in plasma stored
at -70°C for 28 months.28
The focus of the current article is analysis of the
central retina (contained within an 8-mm diameter
disc centered on the fovea). The circumference of
the 8-mm disc approximately bisects the optic disc.29
(Although the type of macaque used in reference 29
was M. fascicularis, the distance from the center of the
fovea to the center of the optic disc is approximately
the same as for M. mulatto,.) For eyes in which the
central retina was microdissected,26 the amounts of
each component (for example, a-T) in the small constituent pieces of tissue were summed.
Chemical Analyses
Materials. dk*-Tocopherol (95%, lot #87F-0505)
was obtained from Sigma (St. Louis, MO). Formaldehyde and glutaraldehyde were electron-microscopic
grade (Polysciences, Warrington, PA). For use in protein assays, protein-binding dye and bovine serum albumin (BSA) were purchased from Bio-Rad Laboratories
(Melville, NY). Tocol was kindly supplied by Dr. Peter
Sorter of Hoffmann-La Roche (Nutley, NJ). /?-, y-, and
6-Tocopherols were a gift from Dr. V. Rozier-Martin of
Henkel Corporation (La Grange, IL).
Instrumentation. A Beckman model 344 (Beckman
Instruments, Fullerton, CA) high-pressure liquid chromatograph (HPLC) was used; it had a model 210 injector and two detectors connected in series. The column
effluent first flowed through a SpectroVision FD-300
fluorescence detector (Groton Technology, Concord,
MA) provided with an 8-//1flowcell. The effluent then
flowed through a Beckman model 160 fixedwavelength, dual-beam spectrophotometric detector
equipped with a mercury lamp, a 340-nm filter, and an
18.5-/41 flow cell with a 1.0-cm path length. The FD300 fluorescence detector had dual monochromators
(holographic gratings), a variable-rate pulsed xenon
lamp (set at 100 Hz), and a Hamamatsu R268 photomultiplier tube (Hamamatsu, Hamamatsu City, Japan) use-
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49
ful from 250 nm to 600 nm. The slit widths were set by
the manufacturer at 10 nm for excitation and 20 nm
for emission. For monkeys Mil to M32, the output from
the detectors was integrated by Beckman System Gold
data acquisition software. For the analysis of earlier samples (M05 to M10), the detectors were interfaced to a
Spectra-Physics (dual channel) SP-4250 integrator (Spectra-Physics, San Jose, CA). When using the SP-4250 integrator, when the baseline was extremely noisy, peak areas
were determined by weighing cut-out photocopies of the
peaks.
Tissue Extraction. Tissues in 1 ml methanol:ethanol
(9:1), containing the internal standard (tocol) and
500 fig BHT (2,6-ditert-butyl-p-cresol, an antioxidant),
were homogenized in a 3-ml all-glass homogenizer30
under ambient (fluorescent) lighting and temperature. The homogenizer was rinsed once with 1 ml of
methanolrethanol and once with 3 ml of 2,2,4-trimethylpentane (TMP, commonly called iso-octane). (We
chose to use TMP rather than hexane because of its
lower volatility.) The homogenate, along with the two
rinses, was poured into a glass 7-ml scintillation vial
with a Teflon-lined cap (Thomas Scientific, Swedesboro, NJ) that contained 200 fA of distilled, deionized
water. The mixture was vigorously vortexed and then
centrifuged at low speed to facilitate phase separation.
The TMP (top) layer (2.4 ml) was transferred with a
200-fA Gilson (Middleton, WI) Pipetteman to a second
7-ml scintillation vial. The TMP was evaporated with
a stream of argon (grade 4.8), and the residue was
redissolved by vigorous vortexing into 160 (i\ of methanol:ethanol. The solution was then filtered with a Microfilterfuge Tube with a 0.45-/Ltm nylon-66 membrane
(Rainin Instruments, Woburn, MA).
High-Pressure Liquid Chromatography Analysis. Usu-
ally, 20 fi\ of filtrate was injected into the HPLC injection port with a 25-//1 Hamilton (Reno, NV) syringe.
For samples derived from smaller pieces of tissue, a
larger syringe and injection volume were used when
necessary. Separation of the mixture was achieved at
room temperature with a 15 X 0.46 cm ODS (octadecylsilyl; reverse-phase) 3-/zm particle Econosphere column (Alltech, Deerfield, IL), with 100% methanol for
a mobile phase and aflowrate of 1 ml/minute. Tocol
(the internal standard), a-T, and /3+y-T were detected fluorometrically with excitation at 295 nm,
emission at 340 nm, and a time constant of 1 second." 31 Retinyl palmitate was detected by its absorbance at 340 nm and quantified with an external
standard. In comparison with previous results reported for tocopherols,11 our chromatographic conditions yielded similar resolution, faster analysis times,
and a better signal-to-noise ratio (S/N) (see Fig. 2).
Twenty microliters of 0.216 fjM a-T (4.56 pmol in
methanol:ethanol) yielded S/N = 3.7. Tocol was recovered with an efficiency of 95%.
50
Investigative Ophthalmology & Visual Science, January 1996, Vol. 37, No. 1
A
BHT
A
ft ^ / Tocol
II
a-T
8.11
0.00
Time (min)
la
CO
<D
o
c
o
V)
0)
0.00
Time (min)
8.79
FIGURE 2. Typical high-pressure liquid chromatograms, with
fluorescence detection, from fresh and fixed tissues. Flow
rate was 1 ml/minute. See our companion article26 for detailed description of dissections. Material eluting between
BHT and tocol was mostly contamination from the nylon66 filter. (A) Chromatogram obtained from the combined
left and right 1-mm central discs of neural retinas of monkey
18 (fresh tissue). To increase S/N, 1-mm discs were usually
pooled before the extraction procedure. Note that although
the signal from a-T was well above baseline, the signal for
P+y-T was close to its limit of detection. (B) Chromatogram
obtained from the ring of tissue between 2 mm and 4 mm
diameter, centered on the fovea of the right neural retina
of monkey 27 (fixed tissue).
Figure 2 shows chromatograms for both fresh
(Fig. 2A) and fixed (Fig. 2B) tissue. Comparison of
these chromatograms indicates that the fixative did
not interfere with chromatographic analysis. Note that
our reverse-phase column did not separate /3-T from
y-T.
Because of our choice of column and because we
did not know the ratio of /?-T to y-T in our samples,
we were confronted with three possible choices for a
standard curve for /?+y-T: use either our standard
curve for 0-T, or the one for y-T, or arbitrarily combine the two. With our conditions, we found that the
fluorescence yield of y-T was only slightly greater than
that of /3-T (a ratio of 1.17). Furthermore, humans
typically have a plasma /?-T concentration that is only
9% of the plasma concentration of y-T." As a conse-
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quence, but somewhat arbitrarily, we used the standard curve that we generated for y-tocopherol to calculate the quantity of tocopherol eluted in the fi+yT peak.
Retention times and Rr values of our commercially
obtained standards were used to identify tocopherols
and retinyl palmitate in our tissue samples. On our
liquid chromatographic system, the order of elution
of these standards was consistent with the literature."31'32 Furthermore, using thin-layer chromatography (TLC), we found that the Rf values of our standard tocopherols were in good agreement with the
reported Rf values.33
With the exception of retinyl palmitate, serial dilutions were made up by determining the weight of the
solute and solvent with a 5-place Mettler balance. That
yielded a standard curve good to three significant figures. Concentrations were corrected for impurities by
means of the suppliers' specifications. High-pressure
liquid and thin-layer chromatograms of these tocopherol standards were consistent with the reported percent-purity values. To generate the standard curve for
retinyl palmitate, we used its extinction coefficient34
and the measured absorbances at 325 nm of the standard solutions along with the extent of the impurities
that we observed on HPLC chromatograms. Although
retinyl stearate was separated easily from retinyl palmitate, retinyl oleate coeluted with it. In humans, retinyl
oleate is a minor component of RPE and neural retina.23 As a consequence, the coelution of retinyl oleate
and retinyl palmitate was ignored. Retinyl oleate was
synthesized from its corresponding acid chloride and
retinol.35 Retinyl stearate was synthesized from stearic
anhydride and retinol.36
Protein Assay. Protein amounts in tissues were measured by the Bio-Rad dye-binding protein assay; BSA
was used to generate the standard curves. The methanol:ethanol (bottom) layer from tissue extractions was
evaporated under argon, and the residue was redissolved in 0.1 N NaOH. In some samples in which the
tissue did not dissolve well, the protein content was
remeasured several weeks to several months later. Because solutions were translucent, their absorbance at
595 nm (without any reaction reagents) was subtracted from the absorbance that was determined after
the dye reaction.
Protein values obtained using the Bio-Rad assay
cannot be compared directly to literature values obtained by the method of Lowry.37 For comparison of
our data for neural retina with measurements made
by others, 10 samples of neural retina were analyzed
by the method of Lowry37 using BSA for a standard.
A Cartesian plot of the values obtained by the two
methods indicated that conversion of values obtained
by the Bio-Rad method to values compatible with the
Lowry method required multiplication of the Bio-Rad
51
Vitamin E in Macaque Retina and RPE-Choroid
values by 1.7. This conversion factor is valid only for
tissues from neural retina of rhesus monkey when BSA
was used as the standard for both assay types.
For a brief discussion on why we used protein
rather than lipid phosphorus as a tissue metric for
vitamin E, see our companion article.2'1
Statistical Analysis
Paired Wests and least-squares regression lines and
their associated parameters (r = correlation coefficient) were calculated with RS/1 (BBN Research Systems, Cambridge, MA). Linear regression by groups
was performed with the BMDP statistical software
package (Los Angeles, CA).
24
0
1
a.
o
20
D
V"—
a
^—*——»—.
P+r-Tocophero
Iso-octane/MeOH
CHCIj/MeOH
a-Tocopherol
O
16
^
•
•
•
•
—
O *
D
I
n >
250 ug
BHT
O
——
a
a
250 ug
BHT
O)
Comparison of Two Methods Used to Extract
Tocopherols
Before we began to measure tocopherols in difficult
to obtain primate retinal tissues, we compared two
methods (TMP-methanol versus chloroform-me thanolS8) of extracting tocopherols from tissues. Figure
3 summarizes data that led to our choice of TMPmethanol as the preferred method.
Chloroform-methanol has been reported to be
equivalent to 50% ethanol-hexane for extracting vitamin E from human retina and RPE-choroid.10 However, the comparison of the two methods (a group of
human eyes was arbitrarily divided into two groups by
measuring some eyes with one method and other eyes
with the other method; then, the average values of aT of each group were compared) was made after adding the quantity of vitamin E measured in neural retina to that measured in RPE-choroid. Unless the ages
of the two groups were similar, that comparison may
have been confounded because the amount of vitamin
E in human RPE increases with increasing age.1920
Because the nonpurified diet fed to most of the
monkeys was a good proxy for biologic tissues and
could be weighed accurately, we chose to analyze it
for comparison of the two methods. Furthermore, because it contained dl-a-tocopherol as a supplement,
the relative amounts of the different forms of tocopherol it contained were of interest. Because humans
who supplement with a-T have significantly depleted
plasma levels of y-T," if the nonpurified diet contained mostly a-T, possibly the plasma of the monkeys
would also have less y-T than typical for their species.
From the ^intercepts of the two least-squares lines
(Fig. 3), we calculated that a-T made up approximately 30% of the total amount of tocopherol in the
nonpurified diet. This low percentage of a-T relative
to the other forms of vitamin E probably can be attributed to corn and soybean content of the diet.24 Not
coincidentally, that is approximately the same percentage of a-T in a typical American diet.39
Throughout much of the time the pellet of non-
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o
SO
4
8
12
16
20
24
28
32
Time (days)
FIGURE 3.
Comparison of two methods for extraction of vitamin E. One method used chloroform-methanol, whereas
the other used TMP-methanol for extraction of tocopherols from the nonpurified diet fed to the monkeys in this
study. Comparison of these two methods was made during
a 29-day interval. Because the nonpurified diet was not kept
at ultralow temperatures during that interval, depletion of
tocopherols in the nonpurified diet became a confounding
variable. To eliminate depletion of tocopherols from the
comparison of methods, it was necessary to overlay comparison data on depletion data. Negatively sloping least-squares
lines show depletion of a-T (dashed) and /?+y-T (solid). Symbols indicate the two different methods. Circle = TMPmethanol. Square = chloroform-methanol. All measurements were made in the presence of 50 mg BHT (2,6-ditertbutyl-p-cresol), except where it is noted that 250 mg BHT
was used.
purified diet was being monitored, it was at 4°C. Even
so, its content of vitamin E declined during the 29
days when the two methods were being compared. As
a consequence, overlaid upon the comparison of the
two methods are the least-squares regression lines
showing this decline. Data points for a-T indicated
that when chloroform-methanol was used for extraction, all measurements were below the least-squares
line. Furthermore, for all data points above the line,
TMP-methanol:ethanol was the method of extraction. Even when the amount of BHT was five times
greater (250 mg) in the chloroform-methanol extraction than in the TMP/methanol:ethanol extraction,
the corresponding data point (at 28 days) was still
below the line and was lower than a measurement
made at a later time with TMP-methanol:ethanol containing 50 mg of BHT. This loss of a-T in the presence
of chloroform contrasts with the data for /?+y-T,
where the results obtained from the two extraction
methods were approximately the same.
Chloroform stimulates hydroperoxidation in rat
Investigative Ophthalmology 8c Visual Science, January 1996, Vol. 37, No. 1
52
l. Amounts of Vitamin E, Protein, and Retinyl Palmitate in
Neural Retina and RPE-Choroid of Rhesus Monkey (mean ± SD)
TABLE
Fresh Tissue
a-Tocopherol
nmol/eye
nmol/cm^l
nmol/mg protein§
fi+y-Tocopherol
nmol/eye
mol% a-tocopherol||
Protein§
mg/eye
mg/cm2|
Retinyl palmitate
nmol/eye
nmol/cm2|
nmol/mg protein§
nH
Fixed Tissue
Neural Retina
RPE-Choroid*
14.9
± 3.8
± 0.5
± 0.4
11.0 ± 4.5
1.4 ± 0.6
2.1 ± 0.8
25.2 ± 6.4
17.5 ± 3.3
82.4
± 0.6
± 3.1
2.4 ± 0.8
81.7 ± 3.3
5.4 ± 1.1
82.0 ± 3.2
4.0 ± 1.2
81.4 ± 4.0
7.1
0.9
± 1.2
±0.1
6.4 ± 3.8
0.8 ± 0.5
13.4 ± 4.2
1.8
2.1
3.1
Total
Total-f
0.75 ± 0.39
0.094 ± 0.05
0.11 ± 0.05
7.2 ± 2.7
0.89 ± 0.34
1.6 ± 1.5
7.9 ± 2.8
0.98 ± 0.38
2.9 ± 1.9
0.36 ± 0.23
9
8
8
5
RPE = retinal pigment epithelium.
* With the assumption that M26 was 7 yr and M32 was 7.5 yr, the average age of the eight monkeys
that contributed to the values for the RPE-choroid was 11.5 yr.
f Values for fixed tissue include both neural retina and RPE-choroid.
X All retinas were assumed to be three fourths of a sphere with a diameter of 18.5 mm.
§ Proteins were measured with the Bio-Rad assay.
|| Calculated as (a-T) (100)/(a-T + (3+y-T), where a-T = a-tocopherol and /3+y-T = /3+ytocopherol.
U n refers to the number of animals. When two eyes were available from the same animal, the values
for both eyes were averaged together.
liver microsomes,40 and autooxidizes in the presence
of oxygen to form phosgene.41 A free-radical mechanism was thought to be the most probable explanation
for both processes. Our data (Fig. 3) also are consistent with chloroform inducing the formation of free
radicals. When more than one antioxidant is present
in a system subjected to a free-radical initiator (chloroform in the current case), the most effective antioxidants are depleted more rapidly than the least effective antioxidants.27 Data in Figure 3 indicate that,
when chloroform was one of the extraction solvents,
a-T (the most effective free-radical scavenger) was depleted more rapidly than /?+y-T and our internal standard, tocol. Based on the comparison of these two
methods, we recommend that chloroform not be used
for extracting vitamin E from biologic samples.
RESULTS
Monkeys often are assumed to be appropriate models
for human biology and pathology.42 However, sometimes those assumptions cannot be validated. Although severe vitamin E deficiency in monkeys is associated with disruption of the photoreceptor outer segments, primarily in the macula,43 normal baseline
values of vitamin E have not been reported for ocular
tissues of these especially valuable animals. Therefore,
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one of the major objectives of this article was to compare data on the amounts of vitamin E and other key
components of monkey retina and RPE-choroid with
the corresponding data reported for humans.1019"23
Table 1 presents the average amounts of vitamin
E, retinyl palmitate, and protein in the entire neural
retina and RPE-choroid for fresh and fixed tissues.
When tissue had not been fixed, we could separate
neural retina from RPE-choroid efficiendy, consistent with what has been observed for cat, rabbit, and
rat.44 Table 2 shows the average vitamin E, retinyl palmitate, and protein contents of the 8-mm central discs
of neural retina and RPE-choroid from eyes that had
not been fixed.
Vitamin E in Neural Retina
For the entire neural retina, our value of 1.8 ± 0.5
nmol a-T/cm 2 is somewhat higher than that indicated
by Alvarez et al10'20 for humans (1.0 ± 0.4). Organisciak et al19'20 reported an average of 0.65 nmol a-T/
mg protein (Lowry assay) for human retina. Division
of our corresponding value (2.1 nmol/mg protein;
Bio-Rad assay) by 1.7 to adjust for the different methods used to measure protein (see Materials and Methods) yields 1.2 nmol/mg protein (Lowry assay). Once
again, our value for rhesus monkey was higher than
what has been reported for human retina. With minor
Vitamin E in Macaque Retina and RPE-Choroid
53
2. Amounts of Vitamin E, Protein, and Retinyl Palmitate in 8mm-Diameter Central Disk of Neural Retina and RPE-Choroid of
Rhesus Monkey (mean ± SD)*
TABLE
Neural Retina
a-Tocopherol
pmol/disk
pmol/sq mm2^
pmol/mm3§
pmol/mg protein||
/?+y-Tocopherol
pmol/disk
mol% a-tocopherolf
Protein||
mg/disk2
^g/mm' |
Retinyl palmitate
pmol/disk
pmol/mm 2 |
pmol/mg protein||
n#
RPE-Choroidf
Total
1740 ± 435
33.0 ± 8.2
120 ± 30
1900 ± 200
1280 ± 460
330 ± 70
83.8 ± 3.4
100 ± 25
83.4 ± 3.8
425 ± 80
84.7 ± 2.6
0.92 ± 0.22
17.4 ± 4.2
0.44 ± 0.12
8.3 ± 2.3
1.37 ± 0.3
150 ± 65
2.85 ± 1.2
170 ± 70
905 ± 450
17.1 ± 8.5
2220 ± 1210
1050 ± 455
19.8 ± 8.6
10
6
6
545 ± 220
10.3 ± 4.1
2350 ± 330
RPE — retinal pigment epithelium.
* Only fresh tissue was used for these measurements.
f With the assumption that M32 was 7.5 yr, the average age of the six monkeys that contributed to
the values for the RPE-choroid was 11.8 yr.
X All retinas were assumed to be three fourths of a sphere with a diameter of 18.5 mm. It was
assumed that the 8-mm-diameter disk had spherical curvature.
§ For details on how this was calculated see our companion article.211
|| Proteins were measured with the Bio-Rad assay.
f Calculated as (a-T) (100)/(«-T + /3+y-T), where a-T = a-tocopherol and P+y-T = P + ytocopherol.
# n refers to the number of animals. When two eyes were available from the same animal, the values
for both eyes were averaged together.
assumptions, we calculated that in humans, Nielsen et
al21 found 1.18 nmol a-T/mg protein in central retina
(a 3-mm trephined punch from within the temporal
arcades, but avoiding the fovea) and 1.44 nmol a-T/
mg protein in a 3-mm diameter disc from the area
just anterior to the temporal equator (using the Lowry
protein assay). These values compare well with our
(adjusted) values of 1.12 nmol/mg protein in the central 8-mm diameter disc (Table 2) and 1.25 nmol/mg
protein (entire periphery; SD = 0.26, n = 9; data not
shown in tables).
The average mole percent of a-T in the neural
retina (Table 1; 82.4%; range, 78.2% to 87%) is in
excellent agreement with reported values for humans.10 Somewhat surprisingly, although there appears to be a slight positive correlation, in neither the
central (n = 10) nor the peripheral (n = 9) neural
retina was the correlation between the amounts of aT and /3+y-T statistically significant (data not shown).
Table 2 indicates that the central 8-mm disc of
neural retina contains 120 pmol a-T/mm3. Although
it is not our intent to imply that the central retina is
a homogeneous solution, these units imply that if it
were, the concentration of a-T would be 120 /JM. Furthermore, if one assumes retinal tissue has a density
of 1 g/ml, then 120 pmol/mm 3 is equivalent to 120
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nmol (52 //g) per gram of tissue (wet weight). These
approximate conversions enable us to compare a-T
values in the central retina of rhesus monkey (which
is similar to human central retina) with tabulated values of human tissues.1 Although the central retina is
relatively high in a-T, adipose tissue (150 /^g/g) and
the adrenal gland (132 A^g/g) are much higher. The
vitamin E content of retinal tissue is similar to that of
testis (40 //g/g) and pituitary gland (40 Atg/g).
Vitamin E in Retinal Pigment Epithelium^noroid
Our data are consistent with previous measurements
in humans showing that the total amount of vitamin
E in RPE increases as a function of age, whereas the
total amount of vitamin E in neural retina remains
constant.19 (Least-squares analyses, with age as the independent variable and total vitamin E as the dependent variable, yielded: for RPE-choroid, n — 9, r =
0.51 (slope >0); for neural retina, n = 9, r = 0.15
(slope <0).) Although the average values of vitamin E
in neural retinas of rhesus monkeys can be compared
directly with the corresponding values for humans,
comparisons for RPE-choroid ideally should be adjusted in a species-specific manner for age. Nonetheless, our average value (1.4 ± 0.6 nmol/cm2) for the
Investigative Ophthalmology 8e Visual Science, January 1996, Vol. 37, No. 1
54
FIGURE 4. Comparison of tocopherols in left and right eyes.
{open circles) a-T. {filled circles) /3+y-T. {solid lines) Y = X
Entire Retina-RPE-Choroid
(A) Total amount of tocopherols (nmol) in entire neural
retina-RPE-choroid. Ages, to the nearest year, are shown
adjacent to values for a-T. When only a range for age was
known, the median for the range is shown. (B) Concentration (pmol/mg protein) of tocopherols in peripheral (exclusive of the 8-mm diameter disc) neural retina. (C) Concentration (pmol/mg protein) of tocopherols in 8-mm disc
of neural retina centered on fovea.
amount of a-T per unit area in the RPE-choroid of
rhesus monkey is within 15% of the reported value
for humans (1.2 ± 0.5 nmol/cm 2 ). 1 0
For one monkey (M08), the choroid detached,
leaving the neural retina plus RPE intact. This enabled
us to measure the vitamin E content of choroid without RPE (the retinyl palmitate content of the tissues
confirmed this). This choroid contained 0.97 nmol/
eye of a-T, compared with the average a-T content of
11.0 nmol/eye in the combined RPE-choroid (Table
1). This indicates that most of the vitamin E in the
macaque RPE-choroid is probably in the RPE, as reported for both cat and rabbit, but not rat.44
Right Eye (nanomoles)
Peripheral Neural Retina
3000
Retinyl Palmitate in Neural Retina and Retinal
Pigment Epithelium-Choroid
500
1000
1500
2000
2500
3000
Right Eye (picomoles/mg protein)
Central Neural Retina
2400
If the difference (a factor of 1.75) in retinal surface
areas between humans and rhesus monkeys is considered, our values for amounts of retinyl palmitate in
RPE-choroid and neural retina of rhesus monkey
(Table 1) were somewhat higher than previously reported values of vitamin A (mostly retinyl palmitate)
for humans of 7.9 nmol (RPE-choroid) and 1.1 nmol
(neural retina).23 As is the case in human ocular tissues,23 rhesus monkey also had a broad range of values
for retinyl palmitate in the entire RPE-choroid (2.8
to 10.2 nmol) and in the entire neural retina (0.31 to
1.6 nmol).
Protein in Neural Retina and Retinal Pigment
Epithelium - Choroid
Average amounts of protein in fresh neural retina and
RPE-choroid are shown in Table 1. After adjustment
to the Lowry assay, our protein value of 1.53 mg/cm2
for the entire neural retina can be compared with
values reported for human peripheral retina: 1.47
mg/sq cm21 and 1.1 mg/sq cm.22 Our adjusted value
of 29.6 //g/mm 2 for the central 8-mm diameter disc
(Table 2) also compares well with 25.8 //g/mm 2 for
central human retina.21
Interocular Comparisons—Vitamin E
400
800
1200
1600
Right Eye (picomoles/mg protein)
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2000
2400
Neural Retina Plus Retinal Pigment Epithelium-Choroid. Although it has been reported that the amounts
Vitamin E in Macaque Retina and RPE-Choroid
of vitamin E in left and right eyes of adult humans do
not show significant differences,10 it was unclear in
that study whether the same extraction method was
used to analyze both eyes from the same subject (see
Materials and Methods on comparison of extraction
methods). We show (Fig. 4A) close agreement between left and right eyes for the total amount of a-T
and the total amount of P+y-T in the retina-RPEchoroid complex of rhesus monkey. For both a-T and
P+y-T, a paired t-test indicated that the difference
between left and right eyes was not significant (P >
0.3). For the left-right pairs that had been fixed, the
total amounts of a-T and P+y-T in the retina-RPEchoroid complex were measured because the tissues
were not separated. For tissues that had not been
fixed, total amounts of tocopherols were calculated
by adding the amounts in the retina and the RPE—
choroid. In that way, we were able to maximize the
number of pairs (n = 9) used to compare the left and
right retinas inclusive of their supporting tissues.
Entire Neural Retina. For the eyes that had not been
fixed, we also examined the entire neural retina for
bilateral symmetry. The amount (nmol) of a-T in the
left eye was higher in 6 of 7 pairs by an average of
7.5% (data not shown). Furthermore, the concentration (per mg protein) of a-T in the left eyes was higher
in all seven pairs by an average of 17%. The same
trends and similar magnitudes were observed for P+yT. These results could not be correlated with the order
in which each pair of eyes was dissected. Furthermore,
because the magnitude of the difference between the
left and right retinas was similar for both a-T and
P+y-T, the difference could not be attributed to decay
of tocopherols in the right eyes because of free-radical
attack during processing. If that were so, because a-T
is a much better antioxidant, we would anticipate that
the difference between left and right eyes would be
much greater for a-T than for P+y-T (see Fig. 3). A
lack of bilateral symmetry in the vitamin E levels of
neural retina has been reported for preterm neonates45 and kittens.46
Peripheral Neural Retina. For both a-T and P+y-T
(per mg protein) in the peripheral (exclusive of the
central 8-mm disc) neural retina (Fig. 4B), the asymmetry between the two eyes was slighdy more pronounced than for the entire neural retina. For a-T
per mg protein, in all seven individuals examined, the
left eye was higher than the right eye by an average
of 20%. The corresponding value for P+y-T was 23%.
For both a-T and P+y-T, a paired £-test indicated that
the difference between left and right eyes was significant ( P < 0.02).
Central Neural Retina. In contrast, for both a-T and
P+y-T (per mg protein), the central retinas (8-mm
diameter discs) of the two eyes were well matched
(Fig. 4C), and the difference between the left and
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55
right eyes was not significant (P > 0.4, n = 7; paired
Mest). The absolute value of the deviation from the
line Y = X (Fig. 4C) averaged 4.2% for a-T and 3.7%
for P+y-T.
Correlation of Tocopherols With Protein in
Neural Retina
Because protein often is considered an index of tissue
mass,22 we tested whether the amount of a-T in the
neural retina correlated with the amount of protein
there. For the peripheral neural retina (Fig. 5A), protein and a-T were positively, but only modestly, correlated (n = 9, r = 0.60; P = 0.087 for slope different
from zero). However, in the central neural retina (Fig.
5B), protein and a-T were closely correlated (n = 10,
r= 0.93, P < 0.0001).
Correlations between P+y-T and protein for both
the peripheral neural retina and the central neural
retina exhibited trends similar to those for a-T (data
not shown). However, in neither instance was the leastsquares line for P+y-T statistically significant (n = 9,
r = 0.43, P > 0.2 for peripheral neural retina; n = 10,
r = 0.61, P > 0.05 for central neural retina).
Correlation Between Tocopherols in Neural
Retina and Tocopherols in Plasma
Figure 6A shows the correlation between a-T per mg
protein in the peripheral neural retina and the concentration of a-T in plasma (mean = 21.3 ± 5.2 //M,
n = 5). Comparison of previous data47 for rhesus monkey (a plasma level of 18.8 ± 2.9 fiM a-T) with our data
is consistent with the opinion that thefivemonkeys for
which we have complete data for both neural retina
and plasma had normal plasma levels of a-T. Although
the number of data points shown in Figure 6A was
small, the correlation coefficient (r) was 0.89, and P
< 0.05. A positive correlation between vitamin E in
plasma and retina is consistent with previous studies
on eye tissues44'48 showing correlations between the
amount of a-T in diet and a-T in various components
of rat eyes and in bovine rod outer segments. In addition, plasma levels of a-T in sheep closely correlate
with a-T concentrations of many of their tissues, including adrenal and adipose tissue.49
In contrast to data for othertissuesand for peripheral neural retina, Figure 6B indicates that the concentration (per mg protein) of a-T in the central 8-mm
disc of the neural retina did not significantly correlate
with the concentration of a-T in plasma (r = 0.44, P
> 0.4). The same five monkeys were used to generate
Figures 6A and 6B.
Concentrations of P+y-T in neither the peripheral neural retina nor the central neural retina correlated with the concentration of P+y-T in plasma (data
not shown).
Clearly, the number of data points in Figure 6 is
56
Investigative Ophthalmology 8c Visual Science, January 1996, Vol. 37, No. 1
ment, we must draw the most reasonable conclusions
from admittedly limited data.
Implications for Biologic Control. Examination of
Figures 5 and 6 suggests that the central neural retina
more closely regulates its content of a-T than does
the peripheral neural retina. The a-T content of the
peripheral neural retina (Fig. 5A) was only weakly cor-
Correlation between alpha-Tocopherol and
Protein in Periphery of Neural Retina
Correlation of alpha-Tocopherol in Peripheral Neural Retina
with alpha-Tocopherol in Plasma
0
1
2
3
4
5
6
7
Correlation of alpha-Tocopherol with Protein in
8-mm Central Disk of Neural Retina
3000
©M05R
® M06R
<^M10
2500
y
5
10
15
20
25
30
alpha-Tocopherol in Plasma
2000
O
yf
Correlation of alpha-Tocopherol in Central Neural
Retina with alpha-Tocopherol in Plasma
1500
1000
500
B
n
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Protein (mg)
5. Correlation of a-T in neural retina with protein
content. (A) Correlation between total amount of a-T
(nmol) in peripheral neural retina and total amount of protein (mg) in peripheral neural retina, n = 9, r = 0.601, P
> 0.05 (for slope different from zero). (B) Correlation of
total a-T (pmol) and total protein (mg) in central portion
(8-mm disc) of neural retina, n = 10, r= 0.930, P< 0.0001
(for slope different from zero), (square) M05R; (circle)
M06R; (diamond) M10.
FIGURE
small. A power analysis50 indicated that, if both the
trend and the scatter about the regression lines remained constant, we would need 51 monkeys to
achieve a minimum power of 0.80 to show that the
regression line in Figure 6A is different from the one
in Figure 6B. Because this is an impractical require-
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alpha-Tocopherol in Plasma
FIGURE 6. Correlation between a-T in neural retina and aT in plasma. (A) Correlation of the concentration (nmol/
mg protein) of a-T in peripheral neural retina with the
concentration (//M) of a-T in plasma; n — 5, r = 0.89, P <
0.05 (for slope different from zero). (B) Correlation of the
concentration (nmol/mg protein) of a-T in central portion
(8-mm diameter disc) of neural retina with the concentration (fiM) of a-T in plasma; n = 5, r = 0.44, P > 0.4 (for
slope different from zero); P < 0.05 for K-intercept.
Vitamin E in Macaque Retina and RPE-Choroid
related with protein (a common proxy for amount of
tissue22). In fact, the slope of the least-squares line was
not significantly different from zero (P > 0.05). A
stronger correlation was observed between a-T in the
peripheral neural retina and a-T in plasma (Fig. 6A).
Those observations contrast with the situation when
the central neural retina was considered. The protein
content of the central neural retina had a significant
impact on its content of a-T (Fig. 5B), whereas the concentration of a-T in plasma had litde effect (Fig. 6B).
Mole Percent of P+y-T in Neural Retina
Compared With Plasma
Figure 7 shows the correlation between the percentage of /?+y-T in neural retina and the percentage of
/?+y-T in plasma (expressed as mole percent of total
tocopherols). The percent in both peripheral (Fig.
7A) and central (Fig. 7B) neural retina was positively
correlated with the percent in plasma. Because the
percent of /?+y-T in tissues varies with dietary intake
(and type of tissue),39'51 it is convenient, for purposes
of comparing monkey and human values of tocopherols, that the monkeys' diets had approximately the
same mole percent of/?+y-T as typical American diets
(see Materials and Methods). We found that the mole
percent /3+y-T in rhesus monkey plasma averaged
12.5% (w = 6). That compared reasonably well with
what has been reported for rhesus-monkey plasma47
(11.0%, n = 5) and human plasma31 (9.4%, n = 6).
These values suggest that regulation by the liver6'7 of
the proportions of tocopherols is probably similar in
the two species. Our average value (17.6%) for the
mole percent of /?+y-T in the neural retina (calculated from Table 1) was almost identical to that for
humans (17.1%).10 Taking into consideration that
/?+y-T is a much poorer antioxidant than a-T,3 it is
somewhat unexpected that the mole percent of /3+yT is higher in neural retina than in plasma.
57
Correlation of Mole Percent fky-Tocopherol in Peripheral Neural
Retina with Mole Percent p+yTocopherol in Plasma
Q.
S
5
10
15
20
25
Mole Percent (J+y-Tocopherol in Plasma
Correlation of Mole Percent p-Hy-Tocopherol in 8-mm Central Disk
of Neural Retina with Mole Percent p+y-Tocopherol in Plasma
1
5
10
15
20
25
Mole Percent p+y-Tocopherol in Plasma
DISCUSSION
Our results show that vitamin E, retinyl palmitate, and
protein contents of retinas and supporting tissues of
rhesus monkeys are similar to those reported for humans. Furthermore, the mole percent of a-T in rhesus
monkey plasma was similar to that in humans. Because
one of the putative functions of vitamin E is inhibition
of free-radical damage that may be associated with
aging,52 these similarities between rhesus monkey and
humans indicate that rhesus monkeys may be useful
animal models for studying human diseases, such as
the atrophic form of age-related macular degeneration.53
The following results suggest that the central neural retina more closely regulates its vitamin E content
than does the peripheral neural retina: Although
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FIGURE 7. Correlation between mole percent /3+y-T in neural retina and mole percent /3+y-T in plasma. (A) Peripheral neural retina; n = 5, r = 0.91, P = 0.030 (for slope
different from zero). (B) Central portion of neural retina
(8-mm diameter disc); n = 5, r = 0.84, P= 0.074 (for slope
different from zero).
plasma levels of a-T had significant impact on the aT concentration of the peripheral neural retina, they
had little effect on the a-T concentration of the central retina. In addition, although the amount of a-T
in the peripheral neural retina did not correlate well
with its protein content (an index of tissue amount22),
the amount of a-T in the central neural retina closely
correlated with its protein content.
There is a precedent for a situation in which the
central portion of the primate retina exerts precise
58
Investigative Ophthalmology & Visual Science, January 1996, Vol. 37, No. 1
control over materials that must be obtained from
the diet. It was demonstrated (in New World squirrel
monkeys and Old World macaques) that the macularpigment carotenoids, lutein and zeaxanthin, which
differ only in the position of one double bond, are
each distributed in a distinctive manner in the macula.54'55 Furthermore, as with vitamin E in the central
retina, the macular pigments also display good
agreement between left and right eyes.56
It has been suggested that the vitamin E content
of a tissue might correlate positively with both the
PUFA content of the tissue and the oxygen tension to
which the tissue is subjected.57 The vitamin E distribution in the retina and its supporting tissues is consistent with that hypothesis. Although the choroid is subjected to high oxygen tension,15 in humans younger
than 50 years of age, PUFA content is low.58 In one
monkey (8 years of age) for which we were able to
measure vitamin E in the choroid without RPE, we
found it to be much lower in vitamin E content than
choroid plus RPE. (It might be of interest to know
whether the vitamin E content of Bruch's membrane
increases as its lipid content increases during aging.)
The RPE, which is subjected to almost the same high
oxygen tension as the choriocapillaris and contains a
high content of PUFA, has an exceedingly high concentration of vitamin E,19 as one might predict. In
humans, on a per milligram protein basis, the vitamin
E content of the RPE may be as much as four to eight
times higher than that of the neural retina.19'20 The
oxygen tension to which the photoreceptor outer segments are subjected is only slightly lower than that for
the RPE,15 and the outer segments of the rods have
an especially high content of both long-chain PUFA13
and vitamin E. In both rat30 and bovine retina,59 the
vitamin E per milligram protein of the rod outer segments is approximately three times higher than that
for the complete neural retina.2 In comparison to the
RPE and rod outer segments, the entire neural retina
averages a lower oxygen tension,15 a lower long-chain
PUFA content,13 and a lower vitamin E content. Although these correlations strongly suggest that these
tissues have a way of regulating their vitamin E content, the underlying biochemical mechanisms are unknown.
It would be useful to compare the depletion of
vitamin E in the central retina with that in the peripheral retina as plasma levels decline. Although an early
vitamin E deficiency study in monkeys indicates that
chronic low plasma levels of vitamin E correlate with
severe disruption of the outer segments of the photoreceptors in the macula lutea,43 the conclusions of
that study are confounded by the semipurified diet
used. Macular-pigment carotenoids were not provided
in either the +E or the —E groups.60'61 Furthermore,
because retinal levels of vitamin E were not reported,
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they could not be correlated with the extent or location of the disease. Because we found vitamin E in the
central neural retina and in the plasma to be weakly
correlated, transient declines in plasma levels of vitamin E may not alter the vitamin E content of the
central neural retina; however, chronically low plasma
levels may deplete the central retina of vitamin E and,
as a consequence, the risk of retinal disease leading
to loss in central vision may be significantly increased.
Key Words
macular degeneration, monkey, retina, retinyl palmitate, vitamin E
Acknowledgments
The authors thank Marita Sandstrom for providing the excellent technical assistance required to perform the microdissections, Dr. Billy Hammond for performing the power
analysis, Dr. Ronald J. Bosch for helping with the statistical
analysis of the data for Figure 4, Dr. Doug Rosene for sharing
tissue with us, Dr. Peter Sorter of Hoffmann-La Roche for
providing the tocol, and Dr. V. Rozier-Martin of Henkel
Corporation for providing the different forms of tocopherol. Chemical measurements and data analyses were performed by DVC. Experimental design was established after
discussion among all authors. DVC had the primary responsibility for carrying out the project and writing the manuscript.
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