Abstract
purpose. Macular pigment (MP) may protect against age-related macular
degeneration. This study was conducted to determine the extent of
changes in the macular pigment density as a consequence of oral
supplementation with lutein. A second purpose was to compare two
objective measurement techniques.
methods. In the first technique, reflectance maps were made with a scanning
laser ophthalmoscope. Digital subtraction of log reflectance maps and
comparison between the foveal area and a 14° temporal site provided
MP density estimates. In the second technique, spectral fundus
reflectance of the fovea was measured with a fundus reflectometer and
analyzed with a detailed optical model, to arrive at MP density values.
Eight subjects participated in this study. They took 10 mg lutein per
day for 12 weeks. Plasma lutein concentration was measured at 4-week
intervals.
results. After 4 weeks, mean blood level of lutein had increased from 0.18 to
0.90 μM. It stayed at this level throughout the intake period and
declined to 0.28 μM 4 weeks after termination. Measurement of the
density of MP showed a within-subject variation of 10% with MP maps
and 17% with spectral reflectance analysis. MP density showed a mean
linear 4-week increase of 5.3% (P < 0.001) and 4.1%
(P = 0.022), respectively.
conclusions. Supplementation with lutein significantly increased the density of the
MP. Analyzing reflectance maps with a scanning laser ophthalmoscope
provided very reliable estimates of MP.
Macular pigment (MP), concentrated in the central area of the
retina, contains the carotenoids lutein and zeaxanthin. It protects the
macular region by its capability of filtering blue light, thereby
possibly decreasing photochemical light damage.
1 In
addition, MP is capable of scavenging free radicals.
2 Cross-sectional studies have observed an inverse association between a
diet with a high content of the carotenoids lutein and zeaxanthin and
the prevalence of age-related macular degeneration
(AMD).
3 4 Although for a definite proof of this
relationship a follow-up study is needed, it is of interest to know
whether MP density can be modified. Two studies have been published
showing a change in MP density, one by a dietary modification
(consumption of spinach and/or corn) on 11 subjects
5 and
one by supplementing lutein in two subjects.
6 Hammond et
al.
5 had some subjects fail to show a change in MP
density. Both studies used heterochromatic flicker photometry to
determine MP density in experienced subjects. This psychophysical
method has disadvantages. It is time consuming, and its reliability
depends on the understanding by the subject of the task involved. This
makes the test less suitable for a naive target population. Recently,
Bernstein et al.
7 demonstrated in an animal model
resonance Raman scattering as an objective method that may have future
use in studying human MP density. The purpose of the present study was
twofold. First, we wanted to establish the extent of changes in the MP
density as a consequence of oral supplementation with lutein. Second,
we wanted to compare two objective techniques for measuring MP on the
basis of fundus reflectance.
Eight male, nonsmoking volunteers between the ages of 18 and 50
years (mean age, 40.6 years) were recruited for the study. To assure
homogeneity in lutein plasma levels at baseline, a simple
food-frequency questionnaire was used to exclude subjects with a high
intake of lutein–that is, subjects who had eaten vegetables high in
lutein, such as spinach and kale, more than four times in the 4-week
period preceding the beginning of the study. Furthermore, only subjects
with a relatively normal (Dutch) dietary pattern and not taking vitamin
or mineral supplements were selected. The subjects took a daily dose of
10 mg of lutein, in the form of lutein diesters derived from marigolds,
for a period of 12 weeks. The study was conducted according to good
clinical practice, approved by the local medical ethics committee, and
was conducted in accordance with the tenets of the Declaration of
Helsinki. Informed consent was obtained from all subjects before
participation in the study.
Blood was sampled after an overnight fast at baseline and at
4-week intervals during the study. Four weeks after the end of the
lutein supplementation period, a final blood sample was taken. For
lutein analyses 1 ml plasma was mixed with 1 ml ethanol (containing 16
to 32 micromoles tocopheryl acetate per liter as internal standard).
After 10 minutes, 2 ml hexane was added, and the sealed tubes were
vortexed for 4 minutes. After centrifugation for 10 minutes at
3000g at 4°C, the hexane layer was separated and
evaporated under nitrogen at room temperature. The residue was
dissolved in 0.4 ml high-performance liquid chromatography (HPLC)
solvent and transferred into brown HPLC injection vials. Lutein was
quantified by HPLC with a hyperchrome 3-μm column Nucleosil
120. The mobile phase consisted of acetonitrile-methylene
chloride-methanol (70:15:10, vol/vol/vol), and the flow rate was 1
ml/min. An absorbance detector was used at 445 nm for detection of
lutein. Limit of detection of lutein was 3.0 nM plasma. The amount of
lutein present in the plasma sample was quantified by calculating the
ratio of the peak height of lutein to that of the internal standard.
MP Maps from SLO.
Spectral Analysis.
A daily dose of 10 mg lutein supplementation induced an increase
in mean plasma lutein by a factor of 5 and a linear 4-week increase in
relative MP density of 4% to 5%. To our knowledge, this is the first
study in which the effects of intake of lutein have been assessed with
objective measurement techniques. In particular, the SLO-based
technique provided very reliable results. With this technique all
subjects showed a significant increase in MP density. The spectral
reflectance analysis provided noisier results that did not allow such a
conclusion for all individuals.
Figure 4 shows a comparison between our
results on naive subjects and heterochromatic flicker photometry data
from literature. Landrum et al.
6 published results
of a study of 20 weeks of supplementation of 30 mg lutein per day
derived from marigolds in two skilled subjects who were measured four
to five times week. The noise in their results seems comparable to that
in our technique. They found a 4-week increase of 4.2% in relative MP
density, which also compares well with our results. Hammond et
al.
5 presented results of a dietary intake study for 15
weeks in 11 subjects who consumed spinach and/or corn (10.8 mg of
lutein). Eight subjects showed an increase in MP density, but two
showed a slight decrease. We estimated a 4-week increase in relative
density of 3.5% for all subjects in their study. Their mean results
suggest a considerably higher noise level than that in the other
studies, partly due to a temporary return to baseline MP density at 8
weeks, which they attribute to interaction with other tissues that
accumulate lutein. This was neither observed in this study, nor in
Landrum et al. It could be due to the use by Hammond et al. of dietary
intake of lutein modification (consumption of spinach and/or corn),
compared with supplementing lutein in the form of lutein diesters
derived from marigolds in this study and the study by Landrum et al.
Hammond et al. also studied both men and women, whereas in this study
and that of Landrum et al., only men were involved. In women, there is
no significant correlation between MP density and lutein in the diet,
and the correlation between MP density and lutein in the blood is much
weaker than in men.
13 A division between responders and
nonresponders, as proposed by Hammond et al., also could be seen in the
spectral analysis of the fundus reflectance presented in the current
study. We wonder whether this may be an artifact of the measurement
techniques. With the SLO-based technique, all subjects showed a
significant increase.
Heterochromatic flicker photometry is a rather demanding technique for
subjects—in particular the task of adjusting flicker at a peripheral
location. This may be the cause for noisier results than those obtained
with the present SLO technique. A significant improvement in
heterochromatic flicker photometry, however, was recently described in
a psychophysical setup that avoids Maxwellian view.
14
In Landrum et al.
6 lutein intake was three times as high
and plasma lutein concentration was twice as high as in the present
study
(Fig. 2) . However, their increase in MP density was similar to
ours. Apparently, a dose of 10 mg per day is sufficient to provide a
4-week increase of 4% to 5%. This conclusion is supported by the
still-elevated plasma lutein concentration a month after the end of
supplementation, compared with baseline, accompanied with a
still-increasing MP density between weeks 12 and 16 (
P = 0.03 for the MP maps,
P = 0.42 for the reflectance
analysis). Thus, a high plasma lutein level seems to elevate MP density
gradually over time. This is further corroborated by the high
correlation between plasma lutein concentration and MP density at
baseline in our (all male) subject group
(Fig. 5) . Considering the
study design, we cannot exclude that this effect was due to factors
other than plasma lutein alone. For a definitive answer, a double-blind
randomized controlled trial is needed.
Because we obtained maps of MP distribution we were able to look for
changes in MP distribution with lutein supplementation. An exponential
decay of MP density as a function of eccentricity fitted our data well
up to 4°. No changes were found between the different measurements in
time.
Our results show that fundus reflectance, in particular as obtained
with our custom-built SLO, can be used as a fast and objective test to
obtain reliable estimates of MP density. To reduce the effect of
differences in pupil size, our SLO has a well-defined exit pupil,
whereas commercially available SLOs use the whole pupil plane. Further,
to minimize the influence of head and eye movements on the reflectance
maps, our SLO allows reflectance maps at different wavelengths to be
grabbed within a few video frames, which may be difficult in other
SLOs. However, both adaptations may be of minor influence, because MP
density is determined by a relative comparison of two reflectance maps.
We opted for using a bite board, which is costly and time consuming. It
may be possible to avoid using it in larger scale studies. The Utrecht
retinal densitometer uses a spot with a rather low intensity to measure
spectral fundus reflectance, because it has been optimized for retinal
densitometry. Therefore, to obtain an adequate signal-to-noise ratio,
densitometer outputs were averaged over a 2-minute interval.
Nevertheless, one subject, the fourth in
Figure 5 , had such low fundus
reflectance in the bluish wavelength region, that the MP density,
determined by analyzing this wavelength region, showed an SD of 0.40 OD
and 0.28 OS. All others showed a mean SD of 0.12. This may explain the
discrepancy for this particular subject between the results obtained
with the MP maps and with the spectral analysis. Severe cataract lowers
the intensity and gives rise to similar problems. Increasing the
intensity of the measuring light may shorten the time interval for data
acquisition and improve the performance of the reflectance analysis. At
baseline, mean values for MP density (single pass) were 0.26 for the
SLO technique and 0.47 for the reflectance analysis. Reflectance at the
ILM, anterior to the MP, may lower the apparent MP density measured.
This ILM reflectance is corrected for in the model for spectral
analysis. Other sources of light scatter, such as floaters, could also
introduce an underestimate of MP density, both in the SLO technique and
spectral analysis. However, influence of light scatter is minimized by
the confocal optics and by the spatial separation of entrance and exit.
Determination of MP density by either comparing foveal and peripheral
reflectance with the SLO or by analyzing the spectral content of the
reflectance assumes a retinal structure as in normal subjects. It will
be of interest to measure MP density in various stages of AMD. Drusen,
present in early stages of AMD, act as a neutral reflector at the level
of the retinal pigment epithelium. Their presence is not accounted for
in the model. However, their reflectance shows up as an enhancement of
the reflectance at the discs in the outer segments of the
photoreceptors, which does not affect the determination of MP density.
Only in late stages of AMD, in case of neovascularization or atrophy,
would the methods fail.
Supported in part by the Henkel Nutrition and Health Group.
Submitted for publication February 7, 2000; revised April 21, 2000; accepted April 26, 2000.
Commercial relationships policy: C2.
Corresponding author: Tos T. J. M. Berendschot, UMC Utrecht, Department of Ophthalmology, PO Box 85500, NL-3508 GA Utrecht, The Netherlands.
[email protected]
The authors thank Hélène G. de Vries,
Wilfred Sieling, Itta M. Minderhout and Anneloes L. Berends
for their assistance and the Henkel Nutrition and Health Group for
providing the lutein.
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