Investigative Ophthalmology & Visual Science, Vol. 29, No.

6, June 1988
Copyright Association for Research in Vision and Ophthalmology

Analysis of the Macular Pigment by HPLG

Retinal Distribution and Age Study
Richard A. Done,* John T. Landrum.t Lilio Fernandez, f and Sara L. Tarsis*
High performance liquid chromatography (HPCL) has been employed to study the distribution
throughout the human retina of zeaxanthin and lutein, the two major components of the macular
pigment. Differences between individuals have also been studied with a view to uncovering possible
age-related effects. Both pigments were detected in prenatal eyes ( ~ 2 0 weeks gestation) but did not
form a visible yellow spot. Generally they were not easily discernible until about 6 months after birth.
For 87 donors between the ages of 3 and 95, no dependence on age was observed in the quantity of
either pigment. For ~90% of these, zeaxanthin was dominant. For the remaining 10%, as well as for
the seven youngest donors, all below the age of 2, and in prenatal eyes, lutein was the major pigment.
In individual retinas, the lutein:zeaxanthin ratio increased from an average of approximately 1:2.4 in
the central 0-0.25 mm to over 2:1 in the periphery (8.7-12.2 mm). The variation in this ratio with
eccentricity was linearly correlated with the corresponding rodxone ratio. A selective mechanism of
uptake, which results in cones and rods preferentially acquiring zeaxanthin and lutein, respectively,
could explain this correlation. Invest Ophthalmol Vis Sci 29:843-849, 1988

The role of the macular pigments may be two-fold:

to improve visual acuity1 and to protect retinal tissue
against photodegradation.2 While feeding studies
using monkeys demonstrate the dietary origin of the
macular pigments,3 there are no reports on the mechanism of their uptake by the neural retina. Is the
mechanism highly discriminating, or are the pigments transported nonselectively into retinal cells?
Our recent observations, that the isomeric dihydroxy-carotenoids, zeaxanthin and lutein, constitute
the macular pigment,4 might favor the latter. Alternatively, a selective uptake mechanism might deliver
the two pigments to different target cells within the
retina. To explore these possibilities, we have applied
high performance liquid chromatography (HPLC) to
examine the variation in pigment density and composition with retinal location and age. The rationale
for this approach was that the dependence of the cell
populations on retinal eccentricity5 and age6'7 might
be reflected in the macular pigments.
The variability in pigmentation among individuals, including the possible contributing factor of age,

has been addressed in a number of psychophysical

investigations.8"11 One such study,9 involving subjects in the age range 10 to 90, uncovered wide variations in the optical density of their pigment, but no
significant dependence on age. Optical densities obtained by psychophysical methods, such as sensitivity
measurements," however, rest upon the validity of a
number of assumptions. (See Pease et al10 for review
as well as means of circumvention.) Chemical analysis, on the other hand, is relatively straightforward
and, being independent of observer skill, may be used
equally reliably on all age groups, including prenatal.
Materials and Methods
Sample Preparation
Frozen donor eyes were provided by the Florida
Lions Eye Bank and the National Disease Research
Interchange, and were stored in sealed containers at
-100C until needed. Fetal eyes, supplied through
the latter organization, were shipped on ice and analyzed immediately. Dissection of the thawed eye was
performed in 0.9% saline solution. After the neural
retina had been separated, it was trimmed by cutting
around the equator, and inspected for signs of damage such as holes, tears or separation of retinal layers.
If such damage occurred in the retinal area intended
for analysis, the macula was put aside for future use.
About 40% of all retinas were found to be unsuitable
for the present study.

From the Departments of *Physics and fChemistry, Florida International University (The State University of Florida at Miami),
Miami, Florida.
Presented in part at the 1986 and 1987 meetings of ARVO,
Sarasota, Florida.
Supported by NIH/MBRS Grant RR-O82O5-O2A3.
Submitted for publication: June 30, 1987; accepted December 7,
1987.
Reprint requests: Richard A. Bone, PhD, Department of Physics,
Florida International University, Miami, FL 33199.

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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1988

l_Microscope

Fig. 1. Apparatus used for dissecting retinas into annuli concentric with the fovea.

A lucite sphere, approximately matching the curvature of the retina (1 inch diameter for adults), was
placed in the saline-filled dissecting dish, and the retina maneuvered into position above it. Upon lifting
the sphere from the solution, the retina could be
placed smoothly over it without folds or wrinkles.
The sphere, with the retina uppermost, was then
seated in a rubber-lined ring in the apparatus shown
in Figure 1.
Co-axial with the ring was a low-power microscope
with cross-wires on which the operator centered the
fovea by rotation of the sphere. In order to analyze

the distribution of macular pigments as a function of

retinal eccentricity, the microscope tube was replaced
in the upper aluminum block (Fig. 1) by a set of
spaced, concentric, tubular cutters, designed to slide
telescopically relative to one another and to the upper
block itself. By bearing down with these onto the
sphere, the retina could be cut into six annuli, concentric with the fovea. Only adult eyes were used for
this part of the study. The ranges of linear surface
distance, from the center of the fovea to the inner and
outer edges of the annuli, were 0-0.75, 0.75-1.6,
1.6-2.5, 2.5-5.8, 5.8-8.7, and 8.7-12.2 mm. A single
cutter, 0-0.25 mm, was also available, but could not
be used in conjunction with this set. For the age
study, a single cutter covering the range 0-2.3 mm

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Vol. 29

was employed, this being sufficiently large to include

the largest macula lutea. By placing the retina on a
spherical surface, selected to match as closely as possible the curvature of the retina, problems of tears,
folds or stretching were virtually eliminated. For the
very small prenatal eyes, however, this technique was
not feasible. Instead, extracts from the entire neural
retina were analyzed for the presence of carotenoids.
Each annulus or disk of retinal tissue was transferred to a glass tissue homogenizer, to which was
added a known mass (~ 10 ng) of lutein monomethyl
ether, as an internal standard, and about 2 to 3 ml of
acetone for extraction of the carotenoids. This ether is
readily prepared from lutein,12 and its HPLC retention time corresponds to a window in the retinal
chromatogram. Furthermore, its similarity of structure to lutein and zeaxanthin should render it equally
sensitive to decomposition during work-up and chromatography (see Results). The acetone solutions were
centrifuged at low speed,filtered(0.2 nm nylon-mesh
syringe-filters), and dried in 5 ml pear-drop flasks
under a stream of pure nitrogen. With extracts from
larger tissue samples, greasy contaminants were
clearly visible on the walls of the flask. It was possible
to preferentially dissolve the carotenoids by briefly
swirling cold (-20C) acetone around the prechilled
flask and quickly transferring it to a cleanflaskwhere
it was dried prior to injection on the HPLC.
Quantification by HPLC
HPLC was conducted on an LDC/Milton Roy system (LDC/Milton Roy, Riviera Beach, FL) including
a Spectromonitor D UV-visible detector (0-0.001
absorbance units full-scale) set at 450 nm. At this
wavelength, absorption by zeaxanthin is maximum;
that of lutein is ~92% of maximum. A 250 X 4.6 mm
column with C18 reversed-phase support (5 nm
Spherisorb ODS1) was supplied by Keystone Scientific (State College, PA). Essentially baseline separation was achieved isocratically with an eluent composed of 92% methanol and 8% water/acetonitrile
(3:1 v/v) and a flow rate of 1 ml min~'.
Results
Age Study
A typical chromatogram, labeled a), representing
the pigments found in the central portion of the retina (0-2.3 mm), is shown in Figure 2. The dominant
zeaxanthin peak and the secondary lutein peak were
found in the great majority of samples analyzed, with
the reversed situation occurring only rarely. The four
minor peaks have not yet been identified; however,

No. 6

845

MACULAR PIGMENT: RETINAL DISTRIBUTION AND AGE STUDY / Done er ol.

Fig. 2. Representative
chromatograms of pigments
extracted from retinal tissue. L = lutein, Z = zeaxanthin, detector wavelength
= 450 nm. Chromatogram
a), for a 47-year-old donor,
was obtained from a central
disk (0-2.3 mm), as used in
the age study. Chromatograms b), c), and d), for a
65-year-old donor, illustrate
the dramatic change in the
lutein:zeaxanthin ratio with
retinal eccentricity. For the
sake of clarity, the internal
standard peak (retention
time ~24 min), has been
omitted.

2.5-5.8mm

1.6-2.5 mm

0 - 2.3 mm A

masses of zeaxanthin and lutein varied considerably

among donors, as illustrated by the frequency distribution of Figure 3, neither this nor the composition
of the pigment showed any significant variation with
age after 2 years. This can be seen in Figure 4, where

13-

20SJOUOP

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Age study

30

10
20
Time, minutes

their spectra indicate that they too are carotenoids

and it is conceivable that they are cis-trans isomers of
lutein or zeaxanthin.13 Their presence raises the question of which pigments constitute the macular pigment. In what follows, we restrict ourselves to the two
major compounds, with the realization that in so
doing, we may be slightly underestimating the total
quantity of what may legitimately be called the macular pigment.
The masses of zeaxanthin and lutein in each sample were determined from their peak areas relative to
that of the internal standard, the mass of which was
known. The peak areas were suitably weighted by the
corresponding extinction coefficients of the compounds at 450 nm. From HPLC analyses of mixtures
of spectroscopically determined amounts of the three
compounds, the accuracy of this method was found
to be better than 96%.
For the age study, ten donors were available in each
decade of life apart from the second and tenth, for
which the numbers were eight and six, respectively.
For approximately 40% of the donors, results were
obtained from both eyes and an average was recorded. For the remainder, reliable data were obtained for one eye only. Although the combined

| Retinal
distribution
study

n\o5-

10

20

30

40

1 ^601 ^701

50

Lutein 4 zeaxanthin, ng

1 '

80

90

Fig. 3. Frequency distribution showing the number of donors

having a given mass of lutein and zeaxanthin in the central region
of the retina (0-2.3 mm). The average value was determined to be
27 SD 11 ng.

846

INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1988

Vol. 29

size. With the same diameter cutter, tissue samples

from smaller eyes would span a greater range of angular eccentricities. By recalculating lutein:zeaxanthin
ratios within equal ranges of angular eccentricity, we
have concluded that donors below the age of 2 are
still significantly different from older donors. Also for
donors less than a year old, the total amount of pigment was generally well below the average for older
donors, resulting in an imperceptible yellow spot.
In the case of prenatal eyes (17 to 22 weeks gestation), both lutein and zeaxanthin were detectable by
HPLC (but not by visual inspection), again with the
former in greater abundance. The lutein:zeaxanthin
ratio for the entire neural retina was 2.51 SD 0.94
compared with 1.14 SD 0.34 for seven postnatal
donors (mean age 61). The average abundance in the
prenatal eye, of ca. 3 ng of combined carotenoids, was
comparable, on the basis of mass per unit area, with
that found in postnatal donors.
20

40
60
Age, years

Retinal Distribution

Fig. 4. Age distributions showing for each decade the total mass
of lutein and zeaxanthin (lower) and the lutein/zeaxanthin ratio
(upper) in the central region of the retina (0-2.3 mm).

the average results and associated standard deviations

are plotted for each decade. The only instance in
which we noticed any age effect was in the 0 to 2 age
group, for which the dominant pigment was consistently lutein. This can be seen in Table 1, the results
obtained from donors in the first decade, where the
average lutein:zeaxanthin ratio for donors below age
2 was 1.44 SD 0.16 compared with 0.77 SD 0.20
for all other donors. We have taken into account the
possibility that this might be a result of smaller eye

Table 1. Macular pigment data for donors

in the first decade
Age
(years)

Lutein + zeaxanthin*
(ng)

Lutein/zeaxanthin*
(mass ratio)

o.ot

7.0
4.5
24.6
4.8
66.2
41.5
28.6
25.1
41.0
27.6

.59
.25
.41
.67
.75
.24
.49
0.67
0.53
0.87

0.4
0.5
0.8
1.3
1.6
1.7
3.0
7.0
9.0

* Extracted from 4.7 mm diameter disk of tissue centered on the fovea.

t One day old.

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Three sample chromatograms, labeled b), c), and

d), of the six derived from concentric annuli, are
shown in Figure 2. The important feature to be noted
is the change from zeaxanthin being the dominant
pigment in the central 0 to 0.75 mm of the retina to
lutein taking that role at eccentricities exceeding approximately 2.5 mm. The quantitative variation in
the lutein:zeaxanthin ratio with eccentricity may be
seen in Table 2, where the average results from seven
donors are given. The table includes the total mass of
pigment per unit area, a quantity which decreased
from the macula to the peripheral retina by a factor of
nearly 300. Also presented in the first row of Table 2
are the average results, obtained using the 0-0.25 mm
cutter, for six donors of average age 49.
A number of earlier eluting peaks were found in
chromatogram d) (Fig. 2), as well as those obtained at
greater eccentricities. Their apparent absence at
smaller eccentricities (chromatograms a), b), and c))
is thought to be due largely to the correspondingly
smaller areas of tissue available for pigment extraction. The largest of the earlier peaks, and that immediately preceding it, have been examined spectroscopically. Their spectra are characterized by a broad
peak at about 390 nm.
Discussion
HPLC has proven its effectiveness in our studies of
the macular pigment, beyond merely aiding in its
identification.4 The results of our age study not only
reinforce psychophysical findings, that macular pig-

No. 6

847

MACULAR PIGMENT: RETINAL DISTRIBUTION AND AGE STUDY / Done er ol.

Table 2. Average macular pigment data and photoreceptor data f within different ranges
of linear surface distance from the fovea
Distance from
fovea (mm)
0-0.25
0-0.75
0.75-1.6
1.6-2.5
2.5-5.8
5.8-8.7
8.7-12.2

Lutein + zeaxanthin
per unit area
(ng mm~2)
13.3
5.7
2.9
0.81
0.143
0.067
0.047

4.3*
1.5
1.1
0.25
0.027
0.024
0.018

Optical densityX
0.33
0.14
0.073
0.020
0.0036
0.0016
0.0012

0.11*
0.04
0.028
0.006
0.0007
0.0006
0.0005

Lutein/zeaxanthin
(mass ratio)
0.42 0.04*
0.50 0.10
0.73 0.11
1.04 0.24
1.85 0.57
2.24 0.47
2.22 0.55

Rod/cone\
(# ratio)
0.12
1.3
5.5
10.4
21.7
26.6
25.2

0.03

0.1
0.1
0.2
4.7
5.1
5.6

* Mean data for six donors. All other data in these columns are mean
values for a different set of seven donors.

t Calculated from Osterberg's" data for a single 16-year-old donor eye.

t Calculated from the data in the second column.

mentation shows no significant variation from age 10

to 90,9 but have extended the age range to include
newborn and fetal eyes. Thus we are able to answer
the outstanding question of Nussbaum et al14 concerning the presence or absence of the macular pigment in the newborn. In all of our studies, we have
never encountered a retina in which zeaxanthin and
lutein were not present. This is not to suggest that the
pigments were always observable by visual inspection. In prenatal retinas, a yellow spot was never visible. In postnatal cases, the pigments were discernible
only if their combined masses in the macula exceeded
about 5 ng.
In many instances, reliable samples were obtained
from both left and right eyes of the donor, thereby
permitting a comparison to be made. Of the 36
donors in this category, the percentage difference in
total pigment ranged from 0 to 67% with an average
value of 29 SD 19%. In contrast, Snodderly15 reported that among ten squirrel monkeys, pigment
density distributions in both eyes were very similar.
As far as the lutein:zeaxanthin ratios are concerned,
we found a higher consistency between left and right
eyes, with differences ranging from 0 to 43% and averaging 13 SD 10%.
Chromatography has also proven a valuable tool in
allowing us to quantify the macular pigment as a
function of retinal eccentricity. In studies involving
spectral sensitivity measurements to assess the optical
density of the pigment, results are usually calculated
by assuming negligible absorption by the pigment at
some eccentric location. Eccentricities of 5, 7, and
8 have been used,8"10 for example, corresponding to
linear surface distances of approximately 1.5,2.0 and
2.3 mm. The validity of this assumption may be examined by calculating the variation in the average
optical density of the pigment from the areal densities
given in Table 2. The third column in this table gives

the results. For the three references noted above,

average peak optical densities were reported to be
0.39,9 0.77,10 and 0.53.8 By interpolating between the
data in Table 2, we have estimated the corrections to
these figures to be about +14%, +3%, and +3%, respectively. Therefore eccentricities of 7 or more
would appear to be appropriate locations in the retina
where absorption by the macular pigment can be assumed to be practically zero. In psychophysical studies, however, increasing eccentricity generally involves added difficulty to the visual task which is
being performed, and a compromise must be sought.
The smallest cutter used in our distribution study
had a radius of 0.25 mm, corresponding in the adult
eye, to a visual field of approximately 1.8 in diameter. Within this region, the average optical density
was calculated to be 0.33 SD 0.11 (see Table 2).
This may be compared with the somewhat higher
values obtained in psychophysical studies, in which a
smaller visual field (eg, 1 in diameter) is often used.
The results are consistent with the radial density gradient which has been observed by microspectrophotometry within this central region.1617
Perhaps the most thought provoking discovery
emerging from this investigation is the increase in the
lutein:zeaxanthin ratio with eccentricity, a variation,
incidentally, found in preliminary studies to be duplicated in nonhuman primates (M. mulatta, M. fascicularis, C. ethiops). Had these pigments maintained
a constant ratio throughout the retina (as well as between both eyes), it might have been assumed that
this merely reflected their relative abundance in the
diet. It might have been argued further that the similarity in structure of these isomers rendered them indistinguishable to the uptake mechanism responsible
for their presence in the retinal tissue. However, the
dramatic change in the ratio with eccentricity and, to
a lesser extent, the differences often observed between

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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1988

848

2.5-,

2.0-

8 1.5o
0)
N

c
<u 1.0-

0.5-

10

15

20

25

30

Rod:cone
Fig. 5. Linear relationship between the average lutein:zeaxanthin
ratio and the corresponding rodxone ratio within different ranges
of linear surface distance from the fovea. The rodxone data is due
to Osterberg." The straight line is a least squaresfityielding a linear
correlation at the >99.9% confidence level.

left and right eyes, effectively nullifies these arguments and suggests, in addition, that each pigment
may be associated with a specific cell.
From microspectrophotometry studies,1617 we
know that a major portion of the macular pigment, at
least in the macaque, is seen in the Henle fibers, a
layer which consists largely of the internal fibers of
photoreceptor cells. This observation finds support in
studies of Haidinger's polarization brushes.18 A possible interpretation of the decrease in pigmentation
with eccentricity is that it is simply related to the
corresponding change in dimensions, particularly a
decrease in length, of these fibers. We have considered the possibility that lutein and zeaxanthin may be
associated primarily with each of the two photoreceptor types, rods and cones, respectively. Owing to this
variation in receptor geometry with eccentricity, it
would be inappropriate to explore this possibility by
examining, for example, the extent to which cone
density and the areal density of zeaxanthin are linearly correlated. On the other hand, the geometrical
factor may be largely eliminated by comparing the
average luteinrzeaxanthin ratio as a function of eccentricity with the corresponding rod:cone ratio. This
comparison has been made using 0sterberg's19 rod/
cone data (See Table 2) for a 16-year-old donor. Our
own macular pigment data were obtained from older
donors whose receptor populations were therefore
probably different.6 In spite of this, a graph of the

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Vol. 29

ratios (Fig. 5) yielded a linear correlation (r = 0.998)

at the >99.9% confidence level, sufficiently high to
warrant further investigation. It should be noted,
however, that for the first data point, representing
very nearly the rod-free area, the lutein:zeaxanthin
ratio is still finite. This suggests that lutein and zeaxanthin may not be associated exclusively with rods
and cones respectively. In addition, the slope of the
graph would suggest that the amount of zeaxanthin
associated with each cone may be considerably
greater that the amount of lutein associated with
each rod.
The predominance of zeaxanthin over lutein in the
central 0-2.3 mm of the retinas of the majority of
donors aged 3 and above is clearly evident from Figure 4 and Table 1. For about 10% of these donors, the
situation was reversed, as also was the case in postnatal eyes from donors below the age of 2 and in prenatal eyes. In light of our speculation above, there exists
the possibility that the rodxone ratios for these
groups are higher than in the normal adult. A comparison of this rodxone ratio between very young and
adult eyes is a problem which should be addressed,
assuming the correlation with the lutein:zeaxanthin
ratio, revealed in the present study, can be shown to
be truly causal.
Key words: macular pigment, zeaxanthin, lutein, age,
cones, rods
Acknowledgments
The authors gratefully acknowledge the National Disease
Research Interchange and the Florida Lions Eye Bank for
supplying human donor eyes. We also thank the Bowman
Gray School of Medicine for supplying monkey eyes
through their Tissue Request Program, supported by NIH
Grant #RR00919. J. Martinez provided valuable technical
assistance.

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