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1 From the Department of Physics, Florida International University, Miami, Florida 2 Department of Chemistry, Florida International University, Miami, Florida 3 Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut.
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Abstract |
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METHODS. Retinas from 56 donors with AMD and 56 controls were cut into three concentric regions centered on the fovea. The inner, medial, and outer regions covered the visual angles 0° to 5°, 5° to 19°, and 19° to 38°, respectively. The amounts of lutein (L) and zeaxanthin (Z) extracted from each tissue sample were determined by high-performance liquid chromatography.
RESULTS. L and Z levels in all three concentric regions were less, on average, for the AMD donors than for the controls. The differences decreased in magnitude from the inner to medial to outer regions. The lower levels found in the inner and medial regions for AMD donors may be attributable, in part, to the disease. Comparisons between AMD donors and controls using the outer (peripheral) region were considered more reliable. For this region, logistic regression analysis indicated that those in the highest quartile of L and Z level had an 82% lower risk for AMD compared with those in the lowest quartile (age- and sex-adjusted odds ratio = 0.18, 95% confidence interval = 0.05–0.64).
CONCLUSIONS. The results are consistent with a theoretical model that proposes an inverse association between risk of AMD and the amounts of L and Z in the retina. The results are inconsistent with a model that attributes a loss of L and Z in the retina to the destructive effects of AMD.
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Introduction |
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As far as we are aware, no studies have attempted to evaluate the possible association between L and Z concentrations in the retina and risk of AMD. There are two general approaches for measuring L and Z in the retina: direct analytical measurements on autopsy eyes, and indirect MP optical density measurements using, for example, heterochromatic flicker photometry.6 7 8 For subjects who have advanced forms of AMD, flicker photometry may prove difficult, if not impossible, and could lead to ambiguous results. To our knowledge, the technique has been validated only in subjects without retinal disease.19 We therefore adopted an analytical approach, using high-performance liquid chromatography (HPLC), to quantify the distribution of L and Z in human autopsy retinas. The study was designed to compare the amounts of L and Z in donors diagnosed with AMD to those in a control group without the disease. The purpose of the analysis was to evaluate a possible association between the amounts of L and Z found in the retina and the risk of AMD.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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0° to 5°, and had an area of 7.1 mm2. The
medial and outer portions were concentric annuli, covering the ranges
5° to 19° (area 93 mm2), and 19° to 38°
(area 343 mm2). The positions of the two smaller
trephines relative to the retina’s architecture are shown in Figure 1
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10 ng of lutein monopropyl ether as an
internal standard. The homogenate was transferred to a large culture
tube and the tissue-grinder rinsed with three 2 ml aliquots of
ethanol/water and two 5 ml aliquots of hexane, the rinses being added
to the culture tube. Thorough extraction of the carotenoids was
accomplished by placing the culture tube first in an ultrasonic bath
for 1 minute, then on a vortex mixer for 1 minute. The
carotenoid-containing hexane layer was separated by centrifuging for 3
minutes at 1400g. The hexane layer was then
transferred to a pear-shaped flask and dried under a stream of
N2.
HPLC Analysis
Quantification of the L and Z content in each sample was
accomplished by reversed-phase HPLC using a 250x2 mm Ultracarb ODS 3
µm column (Phenomenex, Torrance, CA). The mobile phase was
acetonitrile/methanol (85:15) with the addition of 0.1% triethylamine
to inhibit degradation of carotenoids. The flow rate was 0.2 ml/min and
detection was at 451 nm. This wavelength was chosen because the
extinction coefficients of L, Z, and the internal standard at 451 nm
were determined to be equal for the particular solvent mix that was
used. Therefore, the amounts of L and Z in a sample could be determined
by comparing their chromatogram peak areas with that of the internal
standard.
Statistical Analysis
Demographic characteristics of the cases and controls were
compared using Students’ t-test or
2 analysis, as appropriate. All significance
tests were 2-sided, with a P value of 0.05 or less
considered statistically significant. The cases were significantly
older than controls and had a greater proportion of women, so these
variables were considered in all multivariate analyses. Mean levels of
L and Z were calculated in the inner, medial, and outer regions, with
case-control differences evaluated using Students’ t-test.
For each concentric region, L and Z levels were categorized into
quartiles, based on the control distribution for that region. Logistic
regression analysis was then used to estimate the odds ratios and 95%
confidence intervals, with adjustment for age (continuous) and sex. The
lowest quartile was set as the referent category. The test for linear
trend was evaluated by modeling the quartile values as a continuous
variable in a logistic regression model, again adjusting for age and
sex.
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Results |
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In the inner region, the AMD cases were found to have, on average, 62% of the L and Z found in the controls. Based on a 2-sided t-test, the difference was significant at P = 0.0002. In the medial and outer regions, the figures were 73% (P = 0.014), and 79% (P = 0.05), respectively (Fig. 2) . In gender-specific analyses, mean differences were more notable for females (case-control differences statistically significant for inner, medial and outer L + Z) compared with males (case-control differences significant only for inner L + Z).
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The amounts of carotenoid per unit area in the outer annulus for cases and controls were divided into quartiles according to the distribution of these amounts among control subjects only. Table 2 displays the median value for each quartile, together with the odds ratios for AMD, adjusted for age and sex. Corresponding results for the inner disc and medial annulus are presented for comparison. In Figure 3 , the odds ratios for the outer annulus are plotted as a function of the median L and Z per unit area for each quartile. There was a significant trend for reduction in risk for AMD with increasing amount of carotenoid in the outer annulus. Those in the highest quartile of L and Z per unit area had an 82% lower risk for AMD, compared with those in the lowest quartile (OR = 0.18, 95% CI = 0.05 to 0.64, P for linear trend = 0.027).
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It is unlikely that high levels of L and Z in the periphery could have a protective effect against a disease that characteristically affects the macula. However, a high level of L and Z in the periphery has been found to be indicative of a correspondingly high level in the macula,21 where it might have the potential to be protective. With the current data, we performed a linear correlation analysis between the amounts of L and Z in the inner disc and outer annulus, using control eyes only. The results shown in Figure 4 (open circles, solid line) indicate a positive correlation between the two quantities (r = 0.69, P < 0.0001). Therefore, AMD subjects in the lowest quartile of L and Z in the outer annulus would generally be among the lowest for L and Z in the macula, disregarding any possible loss of L and Z due to the disease. For comparison purposes, Figure 4 also displays the corresponding results for the AMD subjects (filled circles, dashed line, r = 0.69, P < 0.0001). The majority of the data points, together with their regression line, are seen to lie below the regression line for the control subjects, consistent with the possibility that AMD may be causing some loss of L and Z in the inner region. As another way to examine this, the inner-to-outer region ratio of L and Z was compared in cases versus controls. The mean ratio was less in cases than controls (mean 64.0 vs. 75.5), although the means were not significantly different (P = 0.13).
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Discussion |
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The differences that we have observed between the amounts of L and Z in the retinas of donors with and without AMD are possibly underrepresented in this study as a result of the paucity of donor information that the NDRI was able to provide. For example, the appearance of drusen in a patient’s retina may have prompted an incorrect or premature diagnosis of AMD. Similarly, an undiagnosed individual who served as a control subject, may have had the disease. Thus it is likely that some of the AMD cases should have been assigned to the control group, and vice versa. If AMD is indeed associated with lower-than-average levels of L and Z in the retina, such misclassification of disease status would have the effect of decreasing the differences between the case and control groups. It must also be noted that the L and Z levels were measured at the time of the donor’s death and may not be representative of the levels earlier in life when their postulated protective influence would be relevant. Given the magnitude of the observed odds ratios in Table 2 , however, neither of these considerations is likely to be operating to any significant extent.
There is a crucial question to consider: Do our results indicate that
individuals with low amounts of L and Z in their retinas are at greater
risk of acquiring AMD, or that low amounts of L and Z are merely a
consequence of the disease? To help resolve this question, we have
developed two theoretical models that generate odds ratios
corresponding to each of these possibilities. For the first model, we
began with a set of 1000 random numbers having a pseudonormal
distribution covering the range of carotenoid levels that we observed
in the outer annulus ( 3–60 pmol). The set was characterized by
approximately the same mean and SD that characterized the experimental
data. It was obtained by removing equal, small numbers of numbers from
either end of an appropriate normal distribution. This set was assumed
to represent a sample of carotenoid levels in the human population. The
numbers were ranked and divided into quartiles, and the median for each
quartile was determined. We then assigned a level of risk for AMD for
each quartile that was inversely proportional to the median, and used
this number as a P value to generate a corresponding set of
250 Bernoulli random variables. (These variables are obtained from a
set of random numbers with a uniform distribution in the range 0 to 1.
If the number is less than or equal to P, the variable is
assigned the value 1; otherwise, it is assigned the value 0.) In our
model, the "subject" acquired AMD if the variable was equal to 1;
if equal to 0, the "subject" did not. To generate odds ratios, new
quartiles were established according to the distribution of carotenoid
levels among the non-AMD "control subjects" (as was done with the
experimental data). Figure 5
(open circles) shows the odds ratios plotted against the median values
for these quartiles. As the risk for AMD in this model is inversely
proportional to the median carotenoid level, the characteristic
hyperbolic shape is to be anticipated.
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3–60 pmol) and was
characterized by the same mean value and SD. The first of these columns
represented the carotenoid levels among "controls", and the second
represented carotenoid levels among "AMD cases" before the
destructive effects of AMD. The interpretation of our experimental
results (Fig. 2)
that is consistent with this model, is that AMD is
responsible for an approximately 20% loss of L and Z in the periphery.
We therefore generated a third column of 1000 random numbers, with a
pseudonormal distribution having a mean value of 0.8, to represent the
fraction of carotenoid surviving the destructive effects of AMD.
Reduced carotenoid levels among "AMD cases" were obtained by
multiplying this column by the second column. Odds ratios, shown in
Figure 5
(filled circles), could then be calculated from an analysis of
these numbers and the "control" numbers in the first column. The two models produce distinctly different trends. If prevalence of AMD is inversely associated with L and Z levels, the biggest jump in odds ratios occurs between the lowest and second lowest quartiles. Conversely, if AMD causes loss of L and Z, the smallest jump in odds ratios occurs between these two quartiles. This is a general result and not sensitive to the specific choice of parameters in the two models. For example, using uniform, rather than pseudonormal, distributions of random numbers in the two models did not produce a substantially different result. The experimentally determined odds ratios in Figure 3 follow a similar trend to the lower curve in Figure 5 , consistent with the proposal that individuals with low amounts of L and Z in their retinas are at greater risk of acquiring AMD. Our results are inconsistent with the hypothesis that AMD causes loss of L and Z in the peripheral retina between 19° and 38° eccentricity. Although emphasis has been placed on the outer region, for reasons given previously, Table 2 indicates that the odds ratios for all three regions behaved in a similar fashion. Thus according to our model, loss of L and Z due to AMD was not the major factor anywhere in the retina.
Is there any evidence suggesting that AMD does cause loss of
carotenoid-containing tissue in the peripheral part of the retina? In
transverse sections through the central macula, the carotenoids are
visible and have been mapped by microspectrophotometry.22
They are found in abundance in the receptor axon layer and inner
plexiform layer, but their localization in specific cells has not been
established. In the peripheral retina, concentrations of carotenoids
are very low, precluding this technique. However, a recent study by
Sommerburg et al., involving HPLC analysis of isolated rod outer
segments (ROS), has revealed the presence of L and Z in these
structures.23
Even more recently, Rapp et al. have
confirmed this observation in both the perifoveal and peripheral retina
but have found that the total mass of L and Z in ROS is less than in
the residual (ROS-depleted) retinal membranes.24
In the
parafoveal retina, between approximately 1.5° and 10° eccentricity
from the foveal center, there is an increasing loss of rods with
age.25
This loss has been estimated to be undetectable 8
mm ( 28°) from the foveal center. The loss of rods in the
parafoveal retina is compensated to some extent by the remaining rods
that expand to fill the space vacated by the dying rods. Possibly, as a
result, there is no net loss of the fraction of L and Z that is
associated with parafoveal rods. This suggestion is supported by our
observation here and elsewhere20
that there is no
age-related decline in L and Z levels anywhere in the retina. In
another study, photoreceptor losses were compared in the eyes of AMD
donors and controls.26
In nonexudative AMD, receptor
densities were normal at eccentricities greater than 10°. In
exudative AMD, preferential rod loss (as opposed to cone loss) was
reported up to 2.5 mm from the margin of the disciform scar. However,
no data were available for the peripheral retina. The two studies by
Sommerburg et al.23
and Rapp et al.24
also
reported the presence of low concentrations of L and Z in the retinal
pigment epithelium (RPE). In both the macular and peripheral RPE, the
amounts of L and Z were approximately 15% of those found in the
adjacent regions of the retina.23
Histopathologic changes
occur with age in the peripheral RPE, as well as Bruch’s membrane and
the choriocapillaris, but are not correlated with the presence or
absence of AMD.27
Similarly peripheral retinal function
(>15°) is affected by age, but has been reported to be no worse in
the majority of AMD cases.27
28
For these reasons, the
hypothesis, that AMD does not cause loss of L- and Z-containing tissues
in the peripheral retina, remains tenable, if unproven.
The strength of this study is that it is the first, to our knowledge, to estimate the association between low levels of L and Z concentrations in human retinas and risk of AMD. Although our results show an inverse association between the risk of AMD and MP density, they do not, by themselves, imply a causal association. It is quite possible that some other factor both increases the likelihood of a person developing AMD, and leads to low levels of L and Z in the retina. As we were unable to obtain data from these donors on some important potential confounding variables, such as tobacco use, we cannot adjust our estimates for this type of confounding. Future studies will need to replicate these findings, with better evaluation of possible confounding, particularly by smoking. Such studies would be greatly strengthened by including detailed pathologic analysis in the protocol to provide assessment and classification of the donors’ eye disease.
Our results reinforce those of earlier epidemiologic studies that show associations between low levels of L and Z in the diet or serum and increased risk of neovascular AMD, even after adjustment for smoking.2 3 The assumption would be that, in general, individuals having these low levels of L and Z in the diet or serum would have correspondingly low levels in their retinas. But this may not always be the case, and could be the reason for the insignificant associations that have been observed in other studies.4 5 Future epidemiologic studies would be strengthened by measuring L and Z levels directly in the retina rather than by proxy in the diet or serum.
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Footnotes |
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Submitted for publication May 30, 2000; revised August 17, 2000; accepted September 22, 2000.
Commercial relationships policy: N.
Corresponding author: Richard A. Bone, Department of Physics, Florida International University, 11200 SW 8th Street, Miami, FL 33199. bone{at}fiu.edu
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