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Clinical and Epidemiologic Research
Circulating Omega-3 Fatty Acids and Neovascular
Age-Related Macular Degeneration
B´
en´
edicte M. J. Merle,
1
Pascale Benlian,
2,3
Nathalie Puche,
1
Ana Bassols,
4
C´
ecile Delcourt,
5,6
and Eric H. Souied,
1
for the Nutritional AMD Treatment 2 Study Group
1
Ophthalmology Department, Hˆ
opital Intercommunal de Cr´
eteil, University Paris Est Cr´
eteil, Cr´
eteil, France
2
CHRU Lille, Biochemistry and Molecular Biology Institute, Molecular Medicine of Metabolic Diseases (U4M), Lille, France
3
Lille2 University, School of Medicine, Department of Biochemistry and Molecular Biology, Lille, France
4
Laboratoire Bausch & Lomb, Montpellier, France
5
INSERM, Centre INSERM U897-Epidemiologie-Biostatistique, Bordeaux, France
6
University Bordeaux, ISPED, Bordeaux, France
Correspondence: B´
en´
edicte M.J.
Merle, Service d’ophtalmologie
CHIC Cr´
eteil, 40 avenue de Verdun,
94000 Cr´
eteil, France;
benedicte.merle@u-bordeaux.fr.
See the appendix for the members of
the Nutritional AMD Treatment 2
Study Group.
Submitted: January 9, 2014
Accepted: February 12, 2014
Citation: Merle BMJ, Benlian P, Puche
N, Bassols A, Delcourt C, Souied EH.
Circulating omega-3 fatty acids and
neovascular age-related macular de-
generation. Invest Ophthalmol Vis
Sci. 2014;55:2010–2019. DOI:
10.1167/iovs.14-13916
PURPOSE.We assessed the associations of serum, red blood cell membranes (RBCM) and
dietary long-chain n-3 polyunsaturated fatty acids (LC-PUFAs) with neovascular age-related
macular degeneration (AMD).
METHODS.We included 290 patients of the Nutritional AMD Treatment 2 Study (NAT2) with
neovascular AMD in one eye and early AMD lesions in the other eye, and 144 normal vision
controls without AMD. Dietary intake of seafood was estimated by food frequency
questionnaire. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) composition
in serum and RBCM were determined by gas chromatography from 12-hour fasting blood
samples and was expressed as percentages of total fatty acids profile. Logistic regressions
estimated associations of neovascular AMD with dietary intake of seafood and circulating n-3
LC-PUFAs.
RESULTS.Dietary oily fish and seafood intake were significantly lower in AMD patients than in
controls. After adjustment for all potential confounders (age, sex, CFH Y402H,ARMS2 A69S,
and ApoE4 polymorphisms, plasma triglycerides, hypertension, hypercholesterolemia, and
family history of AMD), serum EPA was associated significantly with a lower risk for
neovascular AMD (odds ratio [OR] ¼0.41; 95% confidence interval [CI], 0.22–0.77; P¼
0.005). Analysis of RBCM revealed that EPA and EPAþDHA were associated significantly with a
lower risk for neovascular AMD (OR ¼0.25; 95% CI, 0.13–0.47; P<0.0001 and OR ¼0.52;
95% CI, 0.29–0.94; P¼0.03, respectively).
CONCLUSIONS.The RBCM EPA and EPAþDHA, as long-term biomarkers of n-3 dietary PUFA
status, were associated strongly with neovascular AMD and may represent an objective
marker identifying subjects at high risk for neovascular AMD, who may most benefit from
nutritional interventions. (http://www.controlled-trials.com/isrctn number,
ISRCTN98246501.)
Keywords: age-related macular degeneration, omega-3 fatty acids, epidemiology, case-control
study
Age-related macular degeneration (AMD) is the leading cause
of irreversible vision loss in industrialized countries.
1
It
comprises two late forms associated with severe visual
impairment (neovascular and atrophic AMD), generally preced-
ed by early, asymptomatic, retinal abnormalities (drusen,
pigmentary abnormalities). Treatments for neovascular AMD
have been available for a few years. Although they stabilize
vision, they are not curative, supporting the need for a targeted
prevention toward high-risk asymptomatic subjects, identified
by relevant biomarkers.
The condition of AMD is a multifactorial disease, involving
genetic and environmental factors (in particular smoking and
nutrition).
1
Omega-3 long-chain polyunsaturated fatty acids (n-3
LC-PUFAs), mainly eicosapentaenoic acid (EPA) and docosahex-
aenoic acid (DHA), have important structural and protective
functions in the retina.
2
The DHA reaches its highest
concentration in the membranes of photoreceptors, and is
important in photoreceptor differentiation and survival, as well
as in retinal function.
2
The anti-inflammatory properties of EPA
and DHA
2,3
are of particular interest in AMD, since inflamma-
tion appears to have a pivotal role in this condition.
4
Moreover,
n-3 LC-PUFAs may increase the retinal density of macular
pigment, which filters blue light, and has local antioxidant and
anti-inflammatory activities.
5
Finally, derivatives of dietary n-3
LC-PUFAs, exhibit antiangiogenic properties in the retina.
6
In 2008, a meta-analysis
7
of nine epidemiologic studies
8–16
showed a significantly reduced risk for AMD in subjects with
high dietary intake of n-3 PUFAs and fish, the main food source
of n-3 PUFAs. Since then, 10 additional studies have shown
similar and consistent results.
17–26
Dietary assessment methods rely on the subjects’ memory
and perceptions, and face the difficulties of the extreme day-to-
Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
www.iovs.org jISSN: 1552-5783 2010
day variability of human diet, the hidden nature of many fats
used for dressing and cooking, the bias in reporting due to
social standards and nutritional recommendations, and the
estimation of the nutritional content of foods. Because of the
multiple difficulties of dietary assessment, circulating biomark-
ers may represent a more objective alternative for the
assessment of nutritional status.
27
A better assessment of n-3
nutritional status could help identify high-risk subjects, who
may benefit most from nutritional intervention. Such biomark-
ers also might be used to follow the efficacy of nutritional
interventions in restoring adequate nutritional status.
Over the last 20 years, a number of biomarkers have been
developed to assess the nutritional status in fatty acids according
to different source tissues. Because of very limited capacity of
endogenous synthesis, the body status of n-3 LC-PUFA mainly
reflects dietary intake of these essential fatty acids. The shortest-
term biomarkers of n-3 LC-PUFA body status are serum or
plasma measurements, reflecting dietary intakes of the past few
hours for triglycerides or of the past few days for cholesterol
ester and phospholipid fatty acids carried within circulating
lipoproteins. Red blood cell membranes (RBCM) and platelets
are of particular interest, since they reflect longer-term overall
dietary intake of n-3 LC-PUFA, incorporated within membrane
phospholipids of bone marrow–derived cell lines during the
past few months.
28
Because n-3 fatty acids may undergo variable
interconversion after intestinal absorption, the omega-3 index
(i.e., RBCM EPAþDHA) appears as an interesting long-term
integrator of n-3 LC-PUFA body status.
29
Circulating n-3 PUFAs have been evaluated in numerous
studies, showing good correlation with dietary intake, and
sensitivity to changes in dietary supplementation studies.
27
They have been used widely in association studies of n-3 PUFAs
with a variety of health outcomes (cardiovascular diseases,
obesity and diabetes, chronic inflammatory or neuro-psychiat-
ric disorders, cancers, and so forth).
30–34
However, with regard
to AMD, while many studies have reported associations with
dietary intakes of n-3 PUFAs, very few data are available on
associations of AMD with circulating biomarkers of n-3 PUFA
status. Recently, we have shown that high plasma n-3 LC-PUFAs
were associated significantly with a decreased risk for late AMD
in elderly subjects from South of France.
35
This study used a
single plasma measurement that represented a crude estimate
of body fatty acid status. Measurement of n-3 PUFAs in RBCM
may represent a better biomarker for longer term status, with a
half-life of 120 days.
28
In the present study, we reported the associations of dietary
intake of seafood, and serum and RBCM n-3 LC-PUFAs with
neovascular AMD in a French case-control study.
METHODS
Study Population
Cases. The 290 cases of neovascular AMD were included
from Nutritional AMD Treatment 2 Study (NAT2) baseline
examination.
36
The NAT2 study is a randomized, placebo-
controlled, double-blind, parallel, comparative study. Patients
were enrolled from December 2003 to October 2005 in a
single center at the Department of Ophthalmology, Hˆ
opital
Intercommunal de Creteil, France. The study was reviewed and
approved by the relevant institutional review board (CPP, Paris-
Ile de France 5, Paris, France).
Eligible patients were affected by neovascular AMD in one
eye and early AMD (any drusen or reticular pseudodrusen with
or without pigmentary changes) in the other eye. Neovascular
AMD was defined on the basis of fundus color pictures and
fluorescein angiography examination. Inclusion criteria were
age 55 years or older and younger than 85 years, and visual
acuity better than þ0.4 logarithm of minimum angle of
resolution units in the study period.
36
The main exclusion
criteria were: choroidal neovascularization (CNV) in both eyes
or no CNV in either eye, wide central subfoveal atrophy of the
study eye, and progressive ocular diseases (severe glaucoma or
other severe retinopathy).
36
Eye examination included best-corrected visual acuity, slit-
lamp examination, fundus photography, and fluorescein
angiography (Topcon501A; Topcon, Tokyo, Japan). The study
was registered on the International Standard Randomized
Controlled Trial Number Register and was allocated registra-
tion number ISRCTN98246501.
Controls. Controls were enrolled through local-newspa-
pers calls for collaboration. A total of 144 men and women,
aged 55 years or more, with normal visual acuity, no history of
ocular diseases, and normal fundus examination and fundus
photography, was recruited and examined at the Department
of Ophthalmology of Creteil between 2002 and 2008. Controls
were from the same geographical area as the AMD cases.
Written informed consent was obtained for all participants
(cases and controls), as required by the French bioethical
legislation and local ethic committee (CPP Henri Mondor). This
study followed the tenets of the Declaration of Helsinki.
Biological Measurements of Fatty Acids
Overnight fasting blood samples were delivered to a single
clinical chemistry laboratory (Hˆ
opital Saint Antoine, APHP,
Paris, France) within five hours and processed immediately as
described.
36
For cases, blood samples collected at baseline
examination (before any supplementation) were used for the
present study. For controls, blood samples were obtained at the
time of eye examination.
Fatty acid composition in serum and RBCM was determined
by gas chromatography after they were transmethylated by
diazomethane following a modified Dole’s procedure.
37
Results
for EPA and DHA content were expressed as a percentage of
the total fatty acid profile in serum and RBCM, and were
available for all participants (n¼434).
Other Biomarkers
Biological samples were collected in the same conditions and
at time of fatty acid measurements. They included serum lipids
and lipoproteins, and genetic polymorphisms validated as
genetic markers of exudative AMD.
Serum total, high (HDL) and low (LDL) density lipoprotein-
cholesterol, and triglycerides, were measured by enzymatic
colorimetric and electrophoretic methods as described previ-
ously.
38
Genomic DNA was extracted from 10 mL blood
leukocytes as described previously in AMD patients
39
and
using the Illustra kit according to the manufacturer’s protocol
(GE Healthcare, Little Chalfont, Buckinghamshire, UK) in
controls. Genotyping of CFH rs1061170, ARM2/HTRA1
rs10490924, and Apolipoprotein E2, 3, 4 alleles were
performed by quantitative polymerase chain reaction allelic
discrimination using reagents and conditions from Custom
Taqman Single-Nucleotide Polymorphism Genotyping Assays
(Applera, Corp., Saint Aubin, France), using ABI 7900HT
(Applied Biosystems, Carlsbad, CA). Quality control of
genotyping by Sanger sequencing and bioinformatics analysis
were performed as described.
39
Dietary Data
Dietary data were collected using a validated food frequency
questionnaire (FFQ) that recorded the usual food intakes for
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2011
the last year.
16,40,41
The interview was conducted by trained
technicians, by telephone, and lasted 45 to 60 minutes. The
FFQ consists of 165 items and portions were estimated using a
validated set of photographs. The set of photographs was given
to the patient before the telephone interview. It was arranged
by food type and meal pattern. In the analysis, the intakes were
expressed in daily consumption in grams. The food composi-
tion table was REGAL
42
(Ciqual, Edinburgh, UK) expanded
with carotenoid and fatty acid contents from the SU.VI.MAX
table.
43
Total dietary intake of seafood is the sum of oily fish,
white fish, and other seafood, and total dietary intake of fish is
the sum of oily fish and white fish. Dietary data were available
for 423 participants (97.4%).
Covariates
Socio-demographic factors and medical history were collected
through face-to-face, standardized interviews at the same time
as eye examination. They included age, sex, body mass index
(BMI; weight [kg]/height
2
[m
2
]), smoking status (never smoker
or ever smoker), self-reported history of hypercholesterolemia,
hypertension, diabetes, and family history of AMD, circulating
biomarkers (serum total, HDL- and LDL-cholesterol, and
triglycerides), and genetic biomarkers (CFH rs1061170,
ARM2/HTRA1 rs10490924, and Apolipoprotein E2, and E4
alleles). All covariates were available for all participants (n¼
434).
Statistical Analyses
Comparisons between neovascular AMD patients and controls
were performed using the Pearson v
2
for sex, Student’s t-test
for age, and logistic regression adjusted for age and sex for
other variables.
Associations of circulating n-3 PUFAs and fish intake with
socio-demographic factors, medical history, dietary intake of
seafood, and genetic polymorphisms were performed using
Kruskal-Wallis ANOVA and Wilcoxon tests.
Associations of neovascular AMD with dietary intake of
seafood and circulating n-3 PUFAs were estimated using logistic
regression. Potentials confounders retained in the final
multivariate model were factors associated significantly with
neovascular AMD or n-3 PUFAs in our study (hypercholester-
olemia, hypertension, family history of AMD, plasma triglycer-
ides, and CFH,ARMS2, and ApoE4 polymorphisms; P<0.05).
Dietary intake of seafood and circulating n-3 PUFAs variables
were used as tertiles of distribution, the first tertile being the
reference.
We also analyzed potential gene-environment interactions,
and potential age- and sex-circulating n-3 PUFAs interactions.
Interactions were introduced independently in the fully
adjusted model and retained if they were significant (P<0.05).
For all analyses, differences were considered significant at P
<0.05. All statistical analyses were performed using SAS
version 9.3 (SAS Institute, Inc., Cary, NY).
RESULTS
Neovascular AMD patients were older than controls (P<
0.0001), but were not different regarding sex, smoking status,
and BMI (Table 1). After adjustment for age and sex,
neovascular AMD patients declared more frequently a family
history of AMD (P¼0.004), hypercholesterolemia (P¼0.004),
or hypertension (P¼0.001), both latter conditions being under
stable corrective therapy. Frequency of self-declared diabetes
did not differ between neovascular AMD patients and controls.
Regarding genetic polymorphisms, CFH Y402H (P<0.0001),
ARMS2 A69S (P<0.0001), and ApoE4 (P¼0.03) polymor-
phisms were associated significantly with neovascular AMD.
Neovascular AMD patients had lower plasma triglycerides than
controls (P¼0.0009), while they had similar plasma total, HDL-
and LDL-cholesterol (Table 1). Neovascular AMD patients had
lower serum EPA (P¼0.03), RBCM EPA (P<0.001), RBCM
DHA (P¼0.03), and omega-3 index (RBCM EPAþDHA, P¼
0.001) than controls, while they had serum DHA and EPAþDHA
similar to controls after adjustment for age and sex (Table 1).
Neovascular AMD patients had lower dietary intake of oily fish
(P¼0.02) and total seafood (P¼0.03) than controls, but were
not different regarding dietary intake of total fish, white fish,
and other seafood (Table 1).
Table2presentstheassociationsoffishintakeand
circulating n-3 fatty acids with socio-demographic factors,
medical history, and genetic polymorphisms. Younger partic-
ipants had a higher dietary intake of oily fish than older
participants (P¼0.0003). Men had a higher dietary intake of
total and oily fish (respectively, P¼0.002 and P¼0.005).
Participants who declared hypertension had lower dietary
intake of oily fish (P¼0.003). Participants with at least one
allele E4 for ApoE polymorphism had higher dietary intake of
total fish and oily fish (respectively, P¼0.03 and P¼0.03).
Other socio-demographic factors, lifestyle, and AMD-related
genetic polymorphisms were not associated with dietary intake
of fish or seafood. Remarkably, none of the circulating n-3 LC-
PUFAs appeared influenced by any of the socio-demographic,
medical, or genetic risk factors for AMD analyzed herein.
As shown in Table 3, serum EPA, DHA, and EPAþDHA were
associated significantly with all items of dietary intake of
seafood (total fish, oily fish, white fish, other seafood, and total
seafood). Subjects in the third tertile, for all seafood items had
higher serum EPA, DHA, and EPAþDHA. The same trend was
observed with RBCM EPA, DHA, and EPAþDHA, and reached
statistical significance for all items of dietary intake of seafood
except for RBCM DHA and white fish (P¼0.08). Of note, the
median omega-3 index (i.e., RBCM EPAþDHA) was constantly
>4, in subjects from the third tertile, for all seafood items.
As shown in Table 4, after adjustment for age and sex,
dietary intake of total seafood and of total fish was associated
inversely with neovascular AMD (respectively, P¼0.05 and P¼
0.04). After adjustment for all potential confounders (age, sex,
CFH Y402H, ARMS2 A69S, and ApoE4 polymorphisms, plasma
triglycerides, hypertension, hypercholesterolemia, and family
history of AMD), these associations were no longer statistically
significant. With regard to dietary intake of oily fish, white fish,
or other seafood, associations were in the same direction, but
did not reach statistical significance.
Associations of neovascular AMD with circulating n-3 PUFAs
are shown in Table 5. After adjustment for age and sex, serum
EPA was significantly associated with a lower risk for
neovascular AMD (odds ratio [OR] ¼0.59, P¼0.04), while
serum DHA and EPAþDHA were not significantly associated
with neovascular AMD. This association remained significant
after adjustment for all potential confounders (P¼0.005).
With regard to RBCM n-3 PUFAs, after adjustment for age
and sex, EPA and EPAþDHA were associated strongly with a
lower risk for neovascular AMD (OR ¼0.33, P<0.0001 and
OR ¼0.44, P¼0.002, respectively) and after adjustment for all
potential confounders, these associations remained significant
(OR ¼0.25, P<0.0001 and OR ¼0.52, P¼0.03, respectively).
As in serum, DHA in RBCM was not associated significantly
with neovascular AMD.
There was no detectable interaction between dietary intake
of seafood or circulating n-3 PUFAs with CFH,ARMS2 or ApoE
genetic polymorphisms, age, or sex.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2012
TABLE 1. Characteristics of Neovascular AMD Patients and Controls
Characteristics Controls, n¼144 Neovascular AMD Patients, n¼290 Adjusted P*
Sociodemographic factors
Age, y, mean 6SD 67.7 68.2 70.8 67.59 <0.0001
Sex, n(%)
Male 55 (38.2) 105 (36.2) 0.69
Female 89 (61.8) 185 (63.8)
Smoking status, n(%)
Never smoker 91 (63.2) 165 (56.9) 0.12
Ever smoker 53 (36.8) 125 (43.1)
BMI, kg/m
2
, mean 6SD 25.2 63.7 25.7 63.97 0.17
Self–reported medical history
Hypercholesterolemia, n(%)
No 102 (70.8) 147 (51.4) 0.0004
Yes 42 (29.2) 143 (49.3)
Hypertension, n(%)
No 102 (70.8) 149 (51.0) 0.001
Yes 42 (29.2) 141 (48.6)
Diabetes, n(%)
No 131 (91.0) 266 (91.7) 0.59
Yes 13 (9.0) 24 (8.3)
Family history of AMD, n(%)
No 125 (86.8) 222 (76.6) 0.004
Yes 19 (13.2) 68 (23.5)
Genetic polymorphisms
CFH Y402H,n(%)
TT 56 (38.9) 63 (21.7) <0.0001
CT 68 (47.2) 134 (46.2)
CC 20 (13.9) 93 (32.1)
ARMS2 A69S,n(%)
GG 93 (64.6) 81 (27.9) <0.0001
GT 46 (31.9) 133 (45.9)
TT 5 (3.5) 76 (26.2)
ApoE,n(%)
At least 1 allele E2 18 (12.5) 53 (18.3) 0.12
At least 1 allele E4 39 (27.1) 48 (16.6) 0.03
Plasma lipids, mmol/L, median (fifth–95th percentiles) or mean 6SD
Triglycerides 1.14 (0.57–2.30) 0.98 (0.48–2.17) 0.0009
HDL–cholesterol 1.83 60.56 1.79 60.55 0.48
LDL–cholesterol 3.91 (2.51–5.30) 3.64 (2.30–5.59) 0.29
Total cholesterol 5.85 60.93 5.68 61.04 0.16
Circulating omega 3 PUFA, % of fatty acids, median (fifth–95th percentiles)
Serum EPA 0.74 (0.24–1.96) 0.60 (0.30–1.40) 0.03
Serum DHA 1.25 (0.63–2.00) 1.30 (0.60–2.40) 0.1
Serum EPAþDHA 1.99 (1.08–3.53) 1.90 (1.00–3.70) 0.78
Red blood cell membranes EPA 0.78 (0.29–1.47) 0.60 (0.30–1.20) <0.0001
Red blood cell membranes DHA 3.51 (2.13–5.03) 3.20 (1.80–5.10) 0.03
Red blood cell membranes EPAþDHA 4.32 (2.63–6.48) 3.80 (2.10–5.90) 0.001
Dietary intake of seafood, g/d, median (fifth–95th percentiles) n¼139 n¼284
Total fish 19.9 (7.4–51.1) 17.1 (4.9–41.9) 0.05
Oily fish 8.2 (0.0–31.4) 5.5 (0.0–22.9) 0.02
White fish 9.9 (0.0–19.7) 9.9 (0.0–34.0) 0.68
Other seafood 1.8 (0.0–17.1) 0.7 (0.0–15.7) 0.16
Total seafood 22.7 (9.9–64.0) 20.4 (5.3–51.1) 0.03
*PStudent’s t-test for age, Pearson v
2
for sex, and logistic regression adjusted for age and sex for other variables.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2013
TABLE 2. Variations of Circulating n-3 PUFAs and Dietary Intake of Fish According to Socio-Demographic Factors, Lifestyle, and AMD-Related
Genetic Polymorphisms
Characteristics n
Serum EPAþDHA
(% of Fatty Acids)
Median
(Fifth–95th
Percentiles)
RBCM EPAþDHA
(% of Fatty Acids)
Median
(Fifth–95th
Percentiles) n
Total Fish
(g/d)
Median
(Fifth–95th
Percentiles)
Oily Fish
(g/d)
Median
(Fifth–95th
Percentiles)
White Fish
(g/d)
Median
(Fifth–95th
Percentiles)
Sociodemographic factors
Age, y
<70 203 2.04 (1.15–3.70) 4.10 (2.47–5.83) 199 19.7 (5.3–51.1) 8.2 (0.0–31.4) 9.9 (2.5–19.7)
‡0 231 1.90 (0.90–3.60) 3.86 (2.11–6.02) 224 17.0 (4.9–42.6) 5.0 (0.0–22.9) 9.9 (0.0–38.4)
P* 0.11 0.20 0.05 0.0003 0.84
Sex
Men 160 1.91 (1.05–3.70) 4.00 (2.45–5.86) 157 19.9 (4.9–58.3) 7.9 (0.0–31.4) 9.9 (0.0–39.4)
Women 274 1.91 (1.00–3.70) 4.00 (2.10–6.20) 266 15.7 (5.0–41.3) 5.4 (0.0–22.9) 9.9 (0.0–26.4)
P0.61 0.71 0.002 0.005 0.25
Smoking status
Never smoker 256 1.93 (1.11–3.70) 4.00 (2.20–6.40) 248 16.6 (5.0–42.6) 5.7 (0.0–21.4) 9.9 (2.5–24.1)
Ever smoker 178 1.91 (0.90–3.70) 4.00 (2.40–5.80) 175 19.7 (4.9–53.4) 7.9 (0.0–31.4) 9.9 (0.0–39.4)
P0.32 0.72 0.06 0.17 0.31
BMI, kg/m
2
<25 218 2.00 (1.02–4.10) 4.05 (2.30–5.83) 211 19.7 (4.9–51.1) 5.7 (0.0–25.7) 9.9 (0.0–34.0)
‡25 214 1.90 (1.00–3.53) 4.00 (2.30–6.20) 212 17.8 (4.9–42.6) 7.5 (0.0–25.7) 9.9 (0.0–26.4)
P0.30 0.61 0.71 0.67 0.31
Medical history
Hypercholesterolemia
No 245 1.90 (1.00–3.53) 4.03 (2.40–5.90) 237 18.4 (5.3–50.9) 7.9 (0.0–25.7) 9.9 (2.5–38.4)
Yes 189 2.00 (1.05–3.70) 4.00 (2.20–6.00) 186 21.0 (5.0–50.7) 5.5 (0.0–25.7) 19.0 (4.9–42.6)
P0.95 0.62 0.44 0.31 0.70
Hypertension
No 251 2.00 (1.00–3.60) 4.07 (2.40–6.20) 246 18.9 (5.3–45.4) 7.9 (0.0–25.7) 9.9 (0.0–34.0)
Yes 183 1.90 (1.05–3.70) 4.00 (2.20–5.90) 177 17.7 (4.9–47.2) 5.0 (0.0–22.9) 9.9 (0.0–26.9)
P0.26 0.35 0.14 0.003 0.89
Diabetes
No 397 2.00 (1.02–3.70) 4.03 (2.20–6.00) 386 18.9 (5.0–47.2) 7.5 (0.0–25.7) 9.9 (0.0–28.6)
Yes 37 1.60 (0.90–3.20) 3.50 (2.47–6.29) 37 15.7 (2.5–42.6) 5.4 (0.0–31.4) 9.9 (0.0–24.1)
P0.05 0.09 0.47 0.90 0.40
Family history of AMD
No 347 1.90 (1.02–3.60) 4.07 (2.39–5.90) 337 19.5 (4.9–45.4) 7.5 (0.0–25.7) 9.9 (0.0–34.0)
Yes 87 2.00 (1.00–3.90) 3.90 (2.20–6.20) 86 16.5 (7.1–47.3) 5.0 (0.0–25.7) 9.9 (2.5–23.3)
P0.47 0.21 0.51 0.17 0.67
Genetic polymorphisms
CFH Y402H
CC 113 1.90 (1.10–4.00) 3.80 (2.20–6.29) 109 19.7 (4.9–42.6) 5.7 (0.0–25.7) 9.9 (2.5–34.0)
CT 202 1.96 (1.08–3.90) 4.10 (2.40–6.02) 198 19.7 (5.7–50.9) 7.9 (0.0–27.9) 9.9 (0.0–39.4)
TT 119 1.90 (0.90–3.00) 4.07 (2.10–5.70) 116 15.6 (3.6–48.3) 5.5 (0.0–22.9) 9.9 (0.0–19.7)
P0.40 0.49 0.13 0.86 0.09
ARMS2 A69S
GG 174 1.90 (1.02–3.90) 4.14 (2.50–6.02) 169 19.7 (4.9–51.1) 7.9 (0.0–31.4) 9.9 (0.0–34.0)
GT 179 2.00 (1.00–3.70) 4.03 (2.00–6.20) 176 17.9 (4.9–41.1) 5.7 (0.0–22.9) 9.9 (0.0–19.7)
TT 81 1.90 (1.20–3.10) 3.80 (2.60–5.62) 78 19.5 (5.0–58.0) 6.6 (0.0–31.4) 9.9 (0.0–39.4)
P0.63 0.27 0.66 0.77 0.65
ApoE
At least 1 E2 allele 71 1.90 (0.90–3.50) 3.75 (2.00–5.80) 69 19.9 (7.1–50.9) 7.9 (0.0–22.9) 9.9 (2.5–39.4)
No E2 allele 363 1.95 (1.10–3.70) 4.07 (2.40–6.00) 354 17.8 (4.9–45.1) 5.7 (0.0–25.7) 9.9 (0.0–26.4)
P0.21 0.10 0.16 0.80 0.10
At least 1 E4 allele 87 1.90 (0.90–3.70) 4.10 (2.00–6.02) 84 19.8 (7.3–58.0) 7.9 (0.0–31.4) 9.9 (2.5–39.4)
No E4 allele 347 1.91 (1.10–3.70) 4.00 (2.30–6.00) 339 17.8 (4.9–42.6) 5.7 (0.0–25.7) 9.9 (0.0–24.1)
P0.88 0.96 0.03 0.03 0.18
*Pfor Wilcoxon test or Kruskal-Wallis ANOVA.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2014
DISCUSSION
In the present study, a high RBCM EPAþDHA index (omega-3
index) was significantly associated with a 48% reduction of the
odds of neovascular AMD. The associations of neovascular
AMD with EPA status also appeared particularly strong (OR ¼
0.25, P<0.0001 for RBCM EPA and OR ¼0.41 P¼0.005 for
serum EPA).
In the present study, the results of seafood consumption
are consistent with previous dietary studies. Although AMD
patients had significantly lower oily fish and seafood intake
than controls, associations did not reach statistical significance
after adjustment for all potential confounders. Among
published case-control studies reporting associations between
fish consumption and AMD, one found a significant associa-
tion,
18
whereas 3 studies, including the Age-Relate Eye Disease
Study (AREDS), showed no significant association.
11–13
More-
over, in 2008, a meta-analysis estimated that the risk for late
AMD was reduced by 38% in participants with high dietary
intakes of n-3 LC-PUFAs.
7
Since then, 4 large prospec-
tive
20,21,24,26
and 4 large cross-sectional
18,19,23,25
dietary
studies published consistent and similar results.
The present results for serum EPAþDHA are consistent
with the only published study on plasma n-3 LC-PUFAs in
AMD, from the population-based Alienor Study.
35
This study
showed a 33% decreased risk for neovascular AMD in subjects
with high plasma n-3 LC-PUFAs; however, not reaching
statistical significance (OR ¼0.67, P¼0.08).
35
Interestingly,
AMD risk was found here, in a new and independent sample
of the French population, in the same range (OR ¼0.74, P¼
0.35) for serum EPAþDHA. In the Alienor study, plasma EPA
was not associated with neovascular AMD (P¼0.51), while
plasma DHA was borderline with neovascular AMD (P¼0.06).
In the present study, we found a significant association with
serum EPA (P¼0.005), but not with serum DHA (P¼0.81).
To our knowledge, the present study is the first case-
control study reporting associations of RBCM n-3 long-chain
fatty acids with neovascular AMD. We showed significant and
strong associations of neovascular AMD with RBCM EPA and
RBCM EPAþDHA. As expected, association with AMD was
stronger for RBCM than serum measurements, because EPA or
DHA measured in RBCM are more stable and longer-term
biomarkers of body LC-PUFAs homeostasis and less influenced
by lifestyle or other endogenous factors than EPAþDHA in
serum or plasma.
28
In the present study, associations of neovascular AMD with
circulating EPA (in serum and RBCM) were markedly stronger
than with circulating DHA. This could reflect differences in
endogenous metabolism of n-3 LC-PUFA, which could be
visible more readily through circulating EPA than through
circulating DHA. For example, there is high interindividual
variability with different tissue-specific rates of EPA/DHA
interconversion, depending on age, sex, nutritional, or
metabolic conditions.
29
Moreover, although DHA is quantita-
tively more abundant than EPA in serum or cell membranes,
changes in serum and RBCM EPA are more pronounced than
serum or RBCM DHA, with changes in dietary intakes of
EPAþDHA, even in subjects taking n-3 LC-PUFA oral supple-
ments exclusively enriched in DHA.
29
Alternately, the protec-
tive role of EPA is supported by oxidative metabolism by
cyclooxygenases and lipoxygenases to produce eicosanoids
with vasoregulatory and anti-inflammatory properties in the
retina.
2
The EPA also is the precursor of docosapentaenoic
acid (DPA), which is known to be the potential precursor of n-
3 very long chain PUFAs (VLC-PUFAs), including 24:5 n-3 fatty
acid, the most abundant VLC-PUFA present in the retina.
44
A
recent study has observed a decreased of some n-3 VLC-PUFAs
(notably 24:5 n-3) in early and intermediate AMD retinas as
TABLE 3. Variations of Circulating n-3 PUFAs According to Dietary Intake of Seafood
Dietary
Intake
of
Seafood Tertile (Range, g/d)
SERUM (% of Fatty Acids) Median (Fifth–95th Percentiles) RBCM (% of Fatty Acids) Median (Fifth–95th Percentiles)
EPA P* DHA PEPAþDHA PEPA PDHA PEPAþDHA P
Total fish 1, n¼151 (0–12.8) 0.60 (0.22–1.20) <0.0001 1.20 (0.60–2.20) 0.0004 1.77 (0.90–3.10) <0.0001 0.60 (0.29–1.00) <0.0001 3.00 (1.70–5.10) <0.0001 3.70 (1.90–5.70) <0.0001
2, n¼147 (12.8–23.0) 0.70 (0.20–1.60) 1.30 (0.63–2.40) 2.00 (1.00–3.52) 0.60 (0.30–1.18) 3.22 (2.00–4.90) 3.92 (2.40–5.83)
3, n¼125 (23.0–139.0) 0.76 (0.40–2.20) 1.40 (0.73–2.38) 2.20 (1.20–4.77) 0.80 (0.40–1.60) 3.70 (2.37–5.30) 4.50 (2.90–6.68)
Oily fish 1, n¼198 (0–5.4) 0.60 (0.23–1.34) 0.0008 1.20 (0.60–2.20) 0.02 1.80 (1.00–3.40) 0.002 0.60 (0.24–1.12) <0.0001 3.00 (1.60–5.10) 0.0001 3.70 (2.00–5.77) <0.0001
2, n¼125 (5.4–12.0) 0.75 (0.20–2.00) 1.30 (0.60–2.60) 2.00 (0.90–4.00) 0.79 (0.40–1.40) 3.40 (2.20–5.00) 4.29 (2.60–6.20)
3, n¼100 (12.0–100.0) 0.70 (0.30–2.10) 1.37 (0.80–2.31) 2.20 (1.24–4.65) 0.71 (0.31–1.60) 3.71 (2.28–5.30) 4.55 (2.81–6.70)
White fish 1, n¼156 (0–9.0) 0.60 (0.22–1.23) 0.004 1.20 (0.60–2.10) 0.002 1.82 (0.90–3.10) <0.0001 0.60 (0.30–1.10) 0.0002 3.20 (1.81–5.10) 0.08 3.86 (2.20–5.80) 0.01
2, n¼135 (9.0–14.0) 0.70 (0.24–1.70) 1.30 (0.70–2.40) 1.90 (1.00–3.70) 0.60 (0.29–1.20) 3.30 (1.90–4.80) 3.90 (2.20–5.80)
3, n¼132 (14.0–69.0) 0.70 (0.25–2.15) 1.40 (0.70–2.38) 2.20 (1.10–4.00) 0.70 (0.40–1.60) 3.55 (1.80–5.30) 4.30 (2.39–6.40)
Other
seafood
1, n¼254 (0–2.6) 0.60 (0.20–1.40) 0.05 1.27 (0.63–2.20) 0.01 1.90 (1.0–3.41) 0.002 0.60 (2.29–1.16) 0.008 3.20 (1.80–5.32) 0.03 3.80 (2.10–6.29) 0.003
2, n¼86 (2.6–7.0) 0.67 (0.29–1.82) 1.23 (0.60–2.30) 1.90 (1.00–4.13) 0.61 (0.33–1.40) 3.32 (2.00–4.96) 4.10 (2.60–5.70)
3, n¼83 (7.0–62.9) 0.73 (0.30–2.00) 1.40 (0.80–2.40) 2.20 (1.20–4.00) 0.70 (0.33–1.56) 3.67 (2.20–4.90) 4.50 (2.60–5.80)
Total
seafood
1, n¼142 (0–15.7) 0.60 (0.25–1.10) <0.0001 1.17 (0.60–2.20) <0.0001 1.70 (1.00–3.10) <0.0001 0.57 (0.28–0.98) <0.001 3.00 (1.80–5.10) 0.001 3.65 (2.10–5.70) <0.0001
2, n¼142 (15.7–26.0) 0.60 (0.18–1.42) 1.29 (0.60–2.10) 1.90 (0.90–3.41) 0.60 (0.30–1.12) 3.29 (1.80–4.94) 3.91 (2.30–5.83)
3, n¼139 (26.0–155.4) 0.80 (0.40–2.20) 1.40 (0.71–2.40) 2.26 (1.20–4.40) 0.80 (0.40–1.60) 3.70 (2.20–5.10) 4.50 (2.60–6.40)
*Pfor Kruskal-Wallis ANOVA.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2015
compared to age-matched control.
44
Finally, two randomized,
prospective, placebo-controlled, clinical trials have tested the
efficiency of oral n-3 LC-PUFAs supplementation on late AMD
development.
36,45
First, the NAT2 study found no effect of a
three-year oral EPAþDHA (1:3, EPA:DHA [mg/mg ratio] from
fish-oil) on progression from early AMD to neovascular AMD, in
the second eye of patients with unilateral neovascular AMD at
baseline.
36
Second, AREDS2 primary analyses showed that
addition of luteinþzeaxanthin, EPAþDHA (2:1, EPA:DHA [mg/
mg ratio] from ethyl esters) or both to the AREDS formulation
did not further reduce the 5-year risk of progression from early
to late AMD (geographic or neovascular AMD).
45
Remarkably,
in placebo groups from both trials, incidence of late AMD at
follow-up was lower than that expected from observational
studies, suggesting that trial-effects (e.g., healthy lifestyle,
unreported self-supplementation in LC-PUFA, and so forth)
might have reduced statistical study power in both randomized
trials. Therefore, these two recent clinical trials, may not
challenge more than one decade of observational studies in
favor of a protective effect of dietary n-3 PUFAs on AMD. The
AREDS study recently published that 5 years after the clinical
trial end, the beneficial effects of the AREDS formulation
persisted for development of neovascular AMD, suggesting a
potential long-term effect of nutritional factors involved in
TABLE 5. Associations of Circulating n-3 PUFAs With Neovascular AMD.
Tertile
Range,
% of Fatty Acids
Model 1* Model 2†
OR 95% CI Pfor Trend OR 95% CI Pfor Trend
Serum
EPA 1 0–0.5 1.00 Ref 0.04 1.00 Ref 0.005
2 0.5–0.9 0.61 0.37–1.00 0.50 0.27–0.91
3 0.9–3.7 0.59 0.36–0.98 0.41 0.22–0.77
DHA 1 0–1.1 1.00 Ref 0.46 1.00 Ref 0.81
2 1.1–1.5 0.66 0.40–1.07 0.69 0.39–1.24
3 1.5–3.9 1.23 0.74–2.04 1.10 0.60–2.01
EPAþDHA 1 0–1.7 1.00 Ref 0.87 1.00 Ref 0.35
2 1.7–2.4 1.10 0.67–1.80 0.95 0.53–1.72
3 2.4–7.5 0.96 0.58–1.59 0.74 0.40–1.38
RBCM
EPA 1 0–0.5 1.00 Ref <0.0001 1.00 Ref <0.0001
2 0.5–0.8 0.63 0.37–1.09 0.46 0.24–0.87
3 0.8–3.4 0.33 0.20–0.55 0.25 0.13–0.47
DHA 1 0–2.9 1.00 Ref 0.09 1.00 Ref 0.37
2 2.9–3.9 0.51 0.31–0.83 0.59 0.33–1.07
3 3.9–7.3 0.64 0.38–1.07 0.76 0.41–1.39
EPAþDHA 1 0–3.5 1.00 Ref 0.002 1.00 Ref 0.03
2 3.5–4.6 0.53 0.32–0.89 0.60 0.33–1.10
3 4.6–9.3 0.44 0.27–0.74 0.52 0.29–0.94
* Model 1, OR estimated using logistic regression adjusted for age and sex; AMD patients, n¼290; controls, n¼144.
† Model 2, OR estimated using logistic regression adjusted for age, sex, CFH Y402H,ARMS2 A69S, and ApoE4 polymorphisms, plasma
triglycerides, hypertension, hypercholesterolemia and family history of AMD; AMD patients, n¼290; controls, n¼144.
TABLE 4. Associations of Dietary Intake of Seafood With Neovascular AMD
Dietary Intake
of Seafood Tertile Range, g/d
Model 1* Model 2†
OR 95% CI Pfor Trend OR 95% CI Pfor Trend
Total fish 1 0–12.8 1.00 Ref 0.04 1.00 Ref 0.21
2 12.8–23.0 0.63 0.38–1.05 0.55 0.30–1.00
3 23.0–139.0 0.57 0.34–0.97 0.69 0.37–1.29
Oily fish 1 0–5.4 1.00 Ref 0.13 1.00 Ref 0.56
2 5.4–12.0 0.85 0.52–1.39 0.99 0.55–1.80
3 12.0–100.0 0.67 0.40–1.12 0.82 0.44–1.53
White fish 1 0–9.0 1.00 Ref 0.34 1.00 Ref 0.17
2 9.0–14.0 1.00 0.60–1.67 1.25 0.68–2.29
3 14.0–69.0 0.79 0.47–1.29 0.63 0.34–1.15
Other seafood 1 0–2.6 1.00 Ref 0.10 1.00 Ref 0.64
2 2.6–7.0 0.60 0.36–1.01 0.59 0.32–1.11
3 7.0–62.9 0.71 0.42–1.20 0.98 0.52–1.86
Total seafood 1 0–15.7 1.00 Ref 0.05 1.00 Ref 0.22
2 15.7–26.0 0.60 0.36–1.01 0.50 0.27–0.92
3 26.0–155.4 0.59 0.35–0.99 0.68 0.36–1.28
* Model 1, OR estimated using logistic regression adjusted for age and sex; AMD patients, n¼284; controls, n¼139.
† Model 2, OR estimated using logistic regression adjusted for age, sex, CFH Y402H,ARMS2 A69S, and ApoE4 polymorphisms, plasma
triglycerides, hypertension, hypercholesterolemia, and family history of AMD; AMD patients, n¼284; controls, n¼139.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2016
AMD pathogenesis.
46
Moreover, in the NAT2 study, the 3-year
incidence of CNV was reduced significantly (hazard ratio [HR],
0.32; 95% confidence interval [CI], 0.10–0.99; P¼0.047) in
patients achieving the highest RBCM EPAþDHA (omega-3 index
>8) over 3 years.
36
From these combined results, it seems to
be relevant to analyze n-3 RBCM EPAþDHA status in AMD.
Biological status of n-3 PUFAs could help identify those
subjects at risk for AMD, and RBCM n-3 PUFAs appear more
relevant as a biomarker of AMD.
Strength of our study was the combined use of biological
data, mainly EPAþDHA RBCM measurements with dietary
assessment of n-3 PUFA status, in the same groups of
individuals affected or not with AMD. Indeed, from differences
in well-established risk factors (age, medical history, CFH,
ARMS2, and APOE polymorphisms) found with a group of
normal vision/normal fundus individuals, the AMD group
seemed as typical of a population of patients with exudative
AMD. Although apparently paradoxical, that triglycerides were
found significantly lower in AMD patients despite them being
more numerous with dyslipidemia, may be somewhat expect-
ed since the whole population had plasma triglyceride
concentrations within the normal range, including AMD
patients regularly taking lipid-lowering medications. Finally,
the omega-3 index (EPAþDHA index) measured in RBCM is a
very good biomarker of n-3 PUFAs status in humans and
recognized as a risk factor in cardiovascular diseases.
47
In the
future, it may prove useful in the clinical setting, for the
identification of AMD patients deficient in n-3 LC-PUFAs, which
may benefit the most from nutritional intervention.
Selection of controls always is a concern in case-control
studies, selection bias being difficult to avoid.
48
In the present
study, controls were selected from the general population, in
the same geographic area as cases. They were not aware of the
specific objectives of the study, before the interview and blood
sample. When we compared cases and controls, they were not
different for sex, smoking, BMI, diabetes, and plasma
cholesterol. However, cases were older than controls. Also,
hypercholesterolemia and hypertension were more frequent in
cases, which is partially consistent with previous studies.
49
Our two groups also were comparable for dietary intakes. To
limit the potential bias due to differences in age, hypertension,
or hypercholesterolemia, we used multivariate modeling.
However, despite that we adjusted our analyses for these
potential confounders, as well as major AMD-related genes, we
cannot exclude residual confounding as in all epidemiologic
studies.
Also, as our study focused on neovascular AMD cases only,
our results can be generalized only to this type of AMD.
In conclusion, from the present report, elderly individuals
with high RBCM levels of EPAþDHA, a long-term marker of
intracellular LC-PUFAs, have a strongly reduced risk for
neovascular AMD. This suggests the RBCM EPAþDHA index
to be considered as added to the list of clinically relevant
biomarkers of AMD.
Acknowledgments
The authors thank all physicians, nurses, and patients from Creteil
University Eye Clinic; all biologists, in particular Claude Wolf, for
scientific advice and support, all laboratory technicians, particu-
larly Dominique Farabos, Myriam Mahe, and Dominique Labaud,
for excellent technical assistance, from Biochimie B Laboratory
from Saint Antoine Hospital.
Supported by Laboratoire Bausch & Lomb, Clinical Research,
Montpellier, France, and by a grant from F´
ed´
eration des aveubles et
handicaps visuels de France and from Fondation Nestl´
e France
(BMJM).
Disclosure: B.M.J. Merle,F
´
ed´
eration des aveugles et handicap´
es
visuels de France (F), Fondation Nestl´
e France (F), Laboratoires
Th´
ea (R), Bausch & Lomb (R); P. Benlian, Bausch & Lomb (R); N.
Puche, None; A. Bassols, Bausch & Lomb (E); C. Delcourt,
Laboratoires Th´
ea (C), Bausch & Lomb (C, R), Novartis (C),
Laboratoires Th´
ea (R); E.H. Souied, Bausch & Lomb (F, C, R),
Laboratoires Th´
ea (C), Laboratoires Th´
ea (R)
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APPENDIX
Nutritional AMD Treatment 2 Study Group (alphabetic
order):
Catherine Allaire, MD, Laboratoires Bausch & Lomb, Montpel-
lier, France; Ana Bassols, MD, Laboratoires Bausch & Lomb,
Montpellier, France; Khaldia Belabbas, APHP, Hˆ
opital Saint
Antoine, Laboratoire de Biochimie B, Paris, France; Dominique
Brault, Laboratoires Bausch & Lomb, Montpellier, France; Yves
Brouquet, Laboratoires Bausch & Lomb, Montpellier, France;
St´
ephanie Castagnet, APHP, Hˆ
opital Saint Antoine, Laboratoire
de Biochimie B, Paris, France; Antoine Cri´
e, APHP, Hˆ
opital Saint
Antoine, Laboratoire de Biochimie B, Paris, France; Isabelle
Gaudino, APHP, Hˆ
opital Saint Antoine, Laboratoire de Bio-
chimie B, Paris, France; Patricia Gawrilow, MD, Department of
Ophthalmology, NYU School of Medicine, New York, NY;
Mich`
ele Lablache-Combier, PhD, Laboratoires Bausch & Lomb,
Montpellier, France; Nicolas Leveziel, MD, PhD, Service
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2018
d’Ophtalmologie, Hˆ
opital Intercommunal de Cr´
eteil, Universit´
e
Paris Est Cr´
eteil, Cr´
eteil, France; Nadja Mechai, PhD, MSc,
Laboratoires Bausch & Lomb, Montpellier, France; Gilles
Morineau, PharmD, PhD, Laboratoires Bausch & Lomb,
Montpellier, France; Natasa Orlic-Pleyer, MD, Laboratoires
Bausch & Lomb, Montpellier, France; Brigitte Paccou, Service
d’Ophtalmologie, Hˆ
opital Intercommunal de Cr´
eteil, Universit´
e
Paris Est Cr´
eteil, Cr´
eteil, France; Nicole Pumariega, Depart-
ment of Ophthalmology, NYU School of Medicine, New York,
NY; Giuseppe Querques, Service d’Ophtalmologie, Hˆ
opital
Intercommunal de Cr´
eteil, Universit´
e Paris Est Cr´
eteil, Cr´
eteil,
France; Rapha¨
ele Siou-Mermet, MD, MS, Laboratoires Bausch &
Lomb, Montpellier, France; Isabelle Turquois, Laboratoires
Bausch & Lomb, Montpellier, France.
Circulating Omega-3 Fatty Acids and AMD IOVS jMarch 2014 jVol. 55 jNo. 3 j2019
