Full text of DOI:10.1007/s00417-002-0515-6

Graefe’s Arch Clin Exp Ophthalmol
2002) 240:666–671
(
C L I N I C A L I N V E S T I G AT I O N
DOI 10.1007/s00417-002-0515-6
Henrike Wüstemeyer
Cornelia Jahn
Andrei Nestler
Thomas Barth
A new instrument for the quantification
of macular pigment density: first results
in patients with AMD and healthy subjects
Sebastian Wolf
Abstract Background: There is
growing evidence that the develop-
ment of age-related macular degen-
subjects without ocular pathology
and 10 patients with dry AMD (geo-
graphic atrophy). Eyes of patients
Received: 4 February 2002
Revised: 20 March 2002
Accepted: 28 May 2002
Published online: 24 July 2002
eration (AMD) is related to oxidative with AMD had a significantly lower
©
Springer-Verlag 2002
damage. This makes macular pig-
ment (MP) an interesting target for
research, as antioxidative quality is
related to MP. As studies have
MPD (mean value 0.094±0.022 den-
sity units, DU) than healthy eyes
(mean value 0.184±0.023 DU).
Conclusions: It is possible to quanti-
fy MPD using a scanning laser oph-
Proprietary interest: none
shown that MP density (MPD) can
be increased by dietary supplementa- thalmoscope in a clinical setting.
tion, MP may also offer a therapeutic The modified SLO offers several im-
or preventive approach to AMD.
Methods: Using scanning laser tech- cial requirements of the clinical situ-
niques, we quantified MPD in heal-
thy subjects and patients with dry
AMD. We introduce a modified
scanning laser ophthalmoscope
provements with regard to the spe-
Presented in part at the annual meeting
of the Deutsche Ophthalmologische
Gesellschaft (DOG), Berlin, 2001
ation. Using the new instrument, we
were able to detect differences in
MPD between patients with dry
AMD and young healthy subjects.
H. Wüstemeyer · C. Jahn · A. Nestler
T. Barth · S. Wolf (
)
✉
Department of Ophthalmology,
(
SLO) which includes several advan- Further studies are needed to evalu-
University of Leipzig, Liebigstrasse 10–14,
tages for the use in the clinical rou-
tine compared to the standard SLO.
Results: We examined 10 healthy
ate the reliability and reproducibility
of the method.
0
4103 Leipzig, Germany
e-mail: wolfs@medizin.uni-leipzig.de
Fax: +49-341-9721509
ment consists of the two hydroxy carotenoids lutein (L)
and zeaxanthin (Z). Human MP is detectable throughout
Introduction
Age-related macular degeneration (AMD) is the leading the retina but the highest concentrations are found in the
cause of visual loss in the industrialized world for peo- fovea [1, 17].
ple over 65 years of age [7, 8]. Although the exact
The role of MP includes a high capacity to absorb
pathophysiology of AMD remains poorly understood, short-wavelength blue light. This can be considered a
there is growing evidence that the development of age- passive protection mode [19]. The peak of the MP absor-
related maculopathy (ARM) is related to oxidative dam- bance spectrum is at 460 nm and works as a broad-band
age [1, 9, 11, 27]. As many antioxidative properties are filter for the macula. Two advantages are achieved: (1)
attributed to the macular pigment (MP), it has been in- the macula’s optical accuracy is improved [10, 16] and
vestigated in respect to its role in the pathological con- (2) the damaging photo-oxidative influence on the neu-
cept of AMD [19, 20, 21, 23]. The first investigations rosensory retina is reduced. In the photoreceptor outer
into the function of MP were performed by Max segments, the antioxidant effect of L and Z is the essen-
Schultze, who concluded in 1866 that there is a func- tial mechanism [1, 22]. The antioxidant properties enable
tional connection between the “yellow spot” in the reti- the carotenoids to neutralize free radicals. This can be
na and the absorption of blue light. The macular pig- referred to as an active process.

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is available for measurements in clinical routine. This
prompted us to develop a method that allows fast and re-
liable MPD measurement. The purpose of this report is
to demonstrate that MPD can be quantified with a modi-
fied confocal scanning laser ophthalmoscope in a clinical
setting. Therefore, we examined a group of healthy sub-
jects and a group of patients in whom we expected low
MPD values.
Materials and methods
Fig. 1 Absorbance spectrum of macular pigment. Peak absor-
bance at 460 nm. Image recording MPD quantification at 488 nm
and 514 nm wavelengths
For quantification of MPD we used a modified confocal scanning
laser ophthalmoscope (SLO). This instrument was developed in col-
laboration with Heidelberg Engineering (Heidelberg, Germany). It
is optimized to record reflectance images at wavelengths of 488 nm
Several previous studies have demonstrated that mea- and 514 nm. It allows a fast switch between the different wave-
surement of MP density (MPD) by reflectometry tech-
niques using a scanning laser ophthalmoscope [3, 13]
can be used to quantify MPD and its distribution. How-
lengths and digital recording of the images, thus minimizing distur-
bances caused by movements of the patient’s eye. We obtain high-
resolution images at 488 nm and 514 nm wavelength with an argon-
laser. Since the absorption of macular pigment at 488 nm wave-
ever, currently no objective method for measuring MPD length is very high and at 514 nm wavelength close to zero (Fig. 1),
Fig. 2 A, B Images taken with
the modified SLO from a heal-
thy eye at wavelengths of
4
88 nm (B) and 514 nm (A).
C Functional image or density
map following digital process-
ing

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Fig. 3 A, B Images taken from
an eye with dry AMD at wave-
lengths of 488 nm (B) and
5
14 nm (A). C Functional im-
age or density map following
digital processing. D Fundus
photography of the macular re-
gion showing drusen and a
small area of geographic atro-
phy in the center
we can determine MPD by comparing foveal and parafoveal reflec- the International ARM Epidemiologic Study Group [4]. Geo-
tance at 488 nm and 514 nm. The MPD is given by Eq. 1:
graphic atrophy was affecting but not entirely covering the fovea
in eight of ten cases (Fig. 3D). In two eyes, geographic atrophy
was present in the macular area, sparing the fovea. The study fol-
lowed the tenets of the Declaration of Helsinki and was approved
where C is a constant depending on the absorption coefficients of by the local ethics committee.
λ
MP and the Ref values are the measured reflectances.
All subjects underwent comprehensive ocular examination.
For easy assessment of MPD we developed a program to create The pupil of the study eye was dilated with eye drops containing
density maps. In a first step this program corrects two reflection 0.5% tropicamide and 2.5% phenylephrine. Macular pigment mea-
images for eye movements. Thereafter, a density map or function- surements were obtained with the modified SLO. The patients
al image is created by digital subtraction of the log reflectance im- were positioned in front of the tabletop and instructed to look
ages (Figs. 2, 3). Since the difference in absorption by pigments straight and steady. We then obtained 20° reflectance images at
other than MP (e.g., retinal and choroidal blood, visual pigments, 488 nm and 514 nm wavelengths of the posterior pole. Density
RPE, and choroidal melanin) between the fovea and 14° outside maps were then processed and maximum MPD was calculated for
the fovea can be neglected, MPD can be determined by comparing a 2° diameter circle centered on the fovea.
foveal and parafoveal reflectance.
In the 10 healthy subjects we conducted two separate reflec-
We recruited ten healthy subjects (five males, five females) tance imaging sessions at wavelengths of 488 nm and 514 nm
aged from 16 to 43 years (mean 27±8 years). Subjects were in within 30 min in order to obtain reproducibility data for MPD
good general health and retinal pathology was present neither in measurements. For the assessment of the reproducibility of the
the examined nor in the fellow eye.
method we calculated the coefficient of variation cvintra from Eq. 2
Ten patients (three males, seven females) with dry AMD (geo- for test-retest variation [26]:
graphic atrophy present) in the study eye were recruited. Their age
ranged from 60 to 77 years (mean 69±6 years). Five patients had
geographic atrophy bilaterally, two had exudative AMD in the fel-
low eye, and three had ARM in the fellow eye. Diagnosis of ad-
vanced dry AMD was made according to the criteria published by

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lengths of light falls exponentially with decreasing
wavelength [14]. In primates, carotenoids cannot be syn-
thesized de novo and have to be gained from foodstuffs.
Studies showed a positive relation between dietary in-
take of xanthophylls, blood serum levels and MPD [15,
1
9] in humans. The variability of the MP initiated by nu-
tritional supplementation, however, makes it an interest-
ing target for future intervention with the aim of prevent-
ing or altering the course of AMD. Ex vivo techniques,
e.g., high-performance liquid chromatography (HPLC)
and microdensitometry, have been used to initiate the re-
search on MPD [5, 25]. Today, these are mainly used to
validate results from in vivo techniques. Bone et al. used
Fig. 4 Results of MPD in healthy eyes and AMD. Single results reversed-phase HPLC to quantify retinal L and Z in rela-
in the two groups. There is no paired association between the
groups. The mean values are indicated by the emphasized lines
tion to an internal standard in human donor eyes with
and without AMD [6]. There was a significantly higher
concentration of L and Z in eyes without AMD than in
where
are the values of the first series,
the values from the those with AMD [6]. The results imply an inverse asso-
ciation between the risk of AMD and MPD, although
there is no proof of a causal association. Microdensitom-
etry identifies the optical density profile of MP and
shows the spatial distribution of L and Z in the retina
second series, and n the number of cases.
Results
[
24]. Heterochromatic flicker photometry is a widely
Recording of reflectance images at 488 nm and 514 nm used in vivo technique on a psychophysical basis [2].
was easy in all subjects. Examination time varied be- The disadvantage of this method is the fact, that patients
tween 3 min and 5 min. Calculation of MPD maps from often need a training program to fulfill the requirements
the reflectance images was possible in all cases and was adequately. This is a barrier for the use in the clinical
the basis for further evaluation.
routine. Elsner et al. compared the distribution of cone
The mean relative MP in a 2° diameter test field, cen- photopigment and macular pigment using a standard
tered on the fovea, in healthy subjects was 0.184±0.023 SLO [13]. Other study groups used scanning laser oph-
DU (density units, ± standard deviation, SD). The lowest thalmoscopy for the measurement of MPD as well [3].
MPD in this group was 0.152 DU, measured in a woman The results were compared to those obtained by the
aged 43 years. The highest MPD in healthy eyes was foveal fundus reflectance measured with a fundus re-
0
.218 DU, measured in a man aged 24 years. The intra- flectometer. The results show a high reliability of the
individual variation was calculated and showed a value SLO measurements with fewer disturbing noises in the
of 6.2% for the MPD in the 10 healthy subjects.
measurements compared to the spectral analysis. They
In subjects with dry AMD the mean MPD was further showed a substantial and significant increase in
.094±0.022 DU. In this group the lowest MPD was MPD in the course of dietary supplementation with caro-
.047 DU (male, 71 years of age), the highest MPD was tenoids and also a significant correlation between plasma
.123 DU (female, 77 years of age), both measured in L levels and MPD. A new approach to in vivo measure-
0
0
0
patients with bilateral geographic atrophy. MPD in the ments has been proposed by Delori et al. [12]. The detec-
fovea was significantly lower in the patients with dry tion of the autofluorescence of lipofuscin being activated
AMD than in healthy subjects (P<0.001). The results for by two wavelengths, one well absorbed by MP and one
each subject are demonstrated in Fig. 4.
minimally absorbed, enables the examiner to obtain ac-
curate single-pass measurements of the MPD. The re-
sults correlate highly with the results taken from an alter-
native psychophysical method (heterochromatic flicker
photometry) and another optic method (fundus reflecto-
Discussion
The role of MP is based on its antioxidative activity and metry). The results indicate that the method leads to re-
a high capacity to absorb short-wavelength blue light producible data in repeated examinations, at both short
[
18, 19]. Since there is growing evidence that oxidative and long intervals.
damage contributes to the development of ARM [1, 9,
Previous studies using a SLO for MP measurements
1, 27] various efforts have been undertaken to quantify were performed using specially designed research instru-
1
MPD. Studies have examined the effect of cumulative ments [3, 13]. Currently, none of these instruments are
photochemical damage and it has been shown that the commercially available. The only standard SLO that al-
threshold for retinal damage induced by different wave- lows recording of reflectance images at 488 nm and

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5
14 nm is the Rodenstock. However, this instrument has ences [12]. However, our method for the calculation of
several limitations: (1) it takes a long time (e.g., 30 s) to test-retest reproducibility is slightly different form that
switch between the different wavelengths, resulting in used by Delori et al. Using the same calculation our test-
artifacts due to eye movements and misalignment; (2) retest differences would be 3.1%.
the linearity of the video amplifier cannot be achieved;
As expected, our measurements show that healthy
(
3) the signal to noise ratio is poor; and (4) the quality of subjects have a statistically significant higher MPD than
the video signal is poor (e.g., non-standard PAL or the studied subgroup of patients with dry AMD. In com-
NTSC). These limitations prompted us to develope a parison with the results Delori et al. obtained with the re-
new instrument for MP measurement. This instrument is flectance method, our mean MPD for healthy subjects is
based on the confocal SLO (HRA, Heidelberg Engineer- lower, but ranges within a comparable magnitude. Delori
ing). Compared with previous measurements with a stan- and colleagues found a MPD of 0.23±0.07 DU in a pop-
dard SLO (Rodenstock) the new instrument has several ulation of 159 subjects without ocular pathology aged
advantages: (1) The instrument allows a very fast switch 16–80 years (mean age 52±17 years) [12]. The compari-
between the different wavelengths (488 nm and 514 nm); son between the absolute numbers of Delori et al. and
(
2) the images are directly digitized, (3) the signal to our results presented here should be valid. However, an
noise ratio is improved, and (4) the linearity of the video accurate comparison is possible only after examining the
amplifier is assured. same patient with both tools. Points of differentiation
Our method to measure MPD noninvasively with a can be the definition of the fovea, resulting in a slightly
modified confocal SLO utilizes the differential absor- different positioning of the test field, and subtle differ-
bance of reflected light at 488 nm (well absorbed by MP) ences in the alignment of the instrument.
and 514 nm (minimally absorbed by MP) and thus obtain
Our findings demonstrate that measurement of MP
a double-pass measure of the optical density of the MP. density with the new instrument leads to results consis-
As with all methods to measure MP, we estimate the tent with previous studies. The modified confocal SLO
MPD from comparison of foveal and parafoveal mea- has shown to be a useful device for quantifying MPD
surements, minimizing the influence of spectral charac- and can in future be used in the clinical routine. No
teristics of the underlying tissues, of the ocular media, training sessions to make patients familiar with the tech-
and of the instrument. The intraindividual coefficient of nique’s requirements are necessary. In a few minutes we
variation of 6.2% demonstrates a good reproducibility of are able to obtain high-quality images to quantify MPD.
MPD measurements with our method. This variation This will allow us to perform large-scale prospective
shows that our method is at least as stable as the method studies in patients with different stages of ARM to eluci-
used by Delori et al., who found 9–22% test-retest differ- date the role of MPD in the pathogenesis of AMD.
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