Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world and mainly affects people over 50 years of age. The role of specific dietary nutrients, such as, polyunsaturated fatty acids (PUFA), and carotenoids (lutein, zeaxanthin and beta-carotene) plus vitamins C and E, zinc and copper are effective in reducing the risk of development and progression from preclinical AMD to a more advanced form of AMD owing to their antioxidant and photo-protective properties [1
]. However, unlike the current study, these investigations did not isolate the effect of the carotenoids or showed a prospective protection by PUFAs [2
The most important PUFA in humans are the omega-3 PUFA, which include α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). ALA is a precursor of EPA, which is a precursor of DHA in the omega-3 PUFA metabolism. DHA is an important primary structural component of the retina. Humans cannot synthesize PUFA, therefore, marine fish in particular, is the main source of EPA and DHA. EPA and DHA have anti-inflammatory properties, and a reduced level is implicated in AMD [4
]. A diet rich in meat and dairy has high omega-6 PUFA levels and in contrast to omega-3 PUFA, these are sources of pro-inflammatory eicosanoids and have competitive interactions with omega-3 PUFAs in the metabolism [4
]. In addition, carotenoids (i.e., lutein, zeaxanthin and beta-carotene) are important antioxidants, and being fat-soluble, they interact with PUFA metabolism. Moreover, lutein and zeaxanthin are the main components of the macular pigment.
Increased dietary intake of low-glycemic index foods, omega-3 PUFA and lutein/zeaxanthin is considered useful in the prevention of AMD or in retarding its progression [1
]. A number of large-scale trials have investigated the effects of nutritional supplements in preventing disease progression. The Age-Related Eye Disease Study (AREDS) in particular, has resulted in the development of evidence-based supplements for preventing such progression [2
]. Subsequently, several randomized prospective studies have shown beneficial effects of omega-3 PUFA, lutein and zeaxanthin in reducing the development of AMD and increasing macular pigment optical density [3
]. In the landmark AREDS2 study, although there was no additional benefit of adding DHA and EPA on top of supplements originally proposed in the AREDS, the subgroup with low lutein and zeaxanthin level had the biggest benefit from these supplements [3
Most studies to date have focused on the effects of antioxidants and omega-3 PUFA supplements. There are very few studies looking at the relationship of circulatory levels of fatty acids and carotenoids, and AMD, especially in the Asian population where dietary intake of lipids is different compared to Western populations. We hypothesized that the AMD patients will have a worse PUFA profile (lower omega-3 PUFA and higher omega-6 PUFA) and lower antioxidant level (carotenoids, including lutein, zeaxanthin and beta-carotene) and that there will be a close association between those levels and dietary habits. The aim of the current study is to study the relationship between the blood levels of multiple fatty acids and antioxidants (carotenoids), dietary habits and AMD in our local population.
A total of 99 (62 males and 37 females) exudative AMD patients and 198 (97 males and 101 females) age-matched controls were recruited in the study period. The mean age was 73.7 ± 10.2 years old in AMD and 67.1 ± 9.3 years old in control. There was no significant difference in BMI between controls (24.48 ± 3.81 kg/m2
) and AMD (24.17 ± 3.22 kg/m2
). The mean difference of all tested carotenoids and fatty acids between AMD cases and controls after adjustment for age and sex are shown in Table 1
Of all tested carotenoids, including lutein and zeaxanthin combined, beta-carotene and lycopene, they were significantly lower in AMD subjects compared to controls (p
= 0.013, 0.036 and 0.001 respectively) (Figure 1
). For omega-3 PUFAs, EPA and DHA were significantly lower in AMD subjects than the control group (p
< 0.0005 and p
= 0.002 respectively) (Table 2
). In contrary, the eicosatrienoic acid was significantly higher in AMD group. Other omega-3 PUFAs including ALA and docosapentaenoic acid were inconsistently lower in AMD subjects (0.05 < p
< 0.1). For omega-6 PUFAs, arachidonic acid (ARA) level (p
< 0.0005) and eicosadienoic acid level (p
= 0.004) were significantly higher in AMD subjects, but dihomo-γ-linolenic acid was lower (p
= 0.002). For the monounsaturated fatty acids (MUFAs), the oleic acid level and heptadecenoic acid level were significantly higher in AMD group. Most saturated fatty acids (SFAs) were significantly higher in AMD group, except for stearic acid and arachidic acid (Table 2
We analyzed the blood level of all tested carotenoids and fatty acids and divided the levels into four quartiles. Using the highest quartile as a reference, we calculated these subjects with blood levels at the lowest quartile for the odds ratio in developing AMD. Those with a statistically significant results are summarized in Table 3
. When the subjects had low carotenoid levels (when comparing the bottom quartile to top quartile), the odds ratio of having AMD was 2.78 for beta-carotene, 6.02 for lycopene, and 4.82 for combined lutein and zeathanxin. A low level of omega-3 PUFA increased the odds ratio of developing AMD by 6.33 for DHA, 7.79 for EPA and 3.11 for ALA. In contrary, a low level of ARA and eicosatrienoic acid were protective, with odds ratio being 0.11 and 0.16 respectively.
We then used the lowest quartile as a reference and calculated the subjects’ blood fatty acid levels at the highest quartile for the odds ratio in developing AMD. Those with statistically significant results are summarized in Table 4
. When the subjects had high levels of eicosadienoic acid (omega-6 PUFA), oleic acid (MUFA), palmitic acid (SFA) and pentadecylic acid (SFA), when comparing the top quartile to bottom quartile, the odds ratio of having AMD were 2.63, 14.10, 11.48 and 33.75 respectively.
Using the PCA, we summarized multiple fatty acids into eight PC groups of high correlation. Each had different representations of multiple fatty acids. We then applied a logistic regression analysis using the PC 1 to PC 8 to understand which component was associated to AMD cases. We found that the PC 1, consisting of C15:0 (pentadecylic acid), C16:0 (palmitic acid), C17:0 (margaric acid), C18:1n9 (oleic acid) and C20:3n3 (eicosatrienoic acid), was significantly associated with developing AMD, while PC 3, consisting of C18:0 (stearic acid), C20:2n6 (eicosadienoic acid) and C20:3n6 (dihomo-γ-linolenic acid) as well as PC 5, which consisted of C16:1n9 (palmitoleic acid), C20:5n3 (EPA) and C22:6n3 (DHA), were significantly protective.
For the questionnaire, a statistically significant difference was found in the median score and interquartile range between the two groups in eight items (Table 5
): AMD group had higher intake frequency in Chinese preserved vegetable (p
= 0.045) and preserved meats (p
= 0.025). For vegetables, the AMD group had a lower serving size of both green leafy vegetables (Choi Sum, Pok Choi, Chinese kale, broccoli, spinach) (p
< 0.001) and other vegetables (turnip, celery cabbage, cabbage and potatoes) (p
< 0.001) when compared with control. The AMD group also had higher frequency in eating red meat including pork, beef and lamb (p
= 0.012) and poultry (p
= 0.031) compared with controls, although controls had higher serving size in poultry each time (p
= 0.030). For seafood, fish intake did not show significant difference between both groups, but AMD group had significantly less frequency of intake in other seafood (prawn, shrimp, crab, mussels, scallop) than in controls (p
Population-based studies indicate that AMD is more prevalent in Caucasians than in Asian origin people [18
]. However, in Asian populations, the more severe form of AMD i.e., exudative (or wet) type is prevalent [21
]. Some investigators have hypothesized that the lower incidence of the disease in Japan may be related to higher antioxidant content of the typical Japanese diet [20
]. In a recent study in Hong Kong, the age-standardized prevalence of early AMD was 17.9% and late AMD was 0.1% [23
]. Most studies to date have focused on the effects of supplements, and there are very few studies looking at the relationship of the circulatory levels of these nutrients and AMD. Among the limited data available, a few Western studies did show a lowered risk of AMD with a high combination of EPA plus DHA levels in the diet or in circulation [7
]. Also, most of the observations on the omega-3 PUFA and carotenoid supplementation focused on Caucasians, and there are scarce data whether these supplements are equally associated with reduced risk of AMD in Asian populations where dietary intake of lipids are different compared to Western populations. In addition, whilst numerous studies have reviewed the association between diet and AMD condition in Western societies and in other Asian countries, there has been insufficient scientific-based report to allow validation of such an association in the Hong Kong Chinese population. Therefore, our study results could serve as a benchmark and reference for future reviews of the factors influencing AMD occurrence amongst the local Chinese.
In this current study, the role of EPA and DHA, as well as the carotenoids were consistent with the literature [16
]. In addition, we have assessed a wide panel of omega-3 and omega-6 PUFAs, MUFAs as well as SFAs. We found that the blood level for the omega-3 PUFAs namely DHA, EPA and dihomo-γ-linolenic acid were significantly lower in AMD subjects than in the controls. In addition, a low level of DHA and EPA, as well as ALA, increased the odds ratio of developing AMD. Logistic regression also found a protective association against AMD. But for the omega-3 PUFA eicosatrienoic acid, the blood level was significantly higher in AMD, and was associated with AMD in logistic regression. ALA, EPA and DHA are attained through food sources (fish, nuts, seeds) and a good dietary habit is fundamental to maintain healthy omega-3 PUFA levels in human. The high eicosatrienoic acid and low EPA and DHA in AMD suggests a loss of Δ5
- and Δ6
-desaturase enzyme efficacy in the PUFA metabolism [24
]; these enzymes are required to convert eicosatrienoic acid to EPA and DHA. For omega-6 PUFAs, the ARA and eicosadienoic acid were significantly higher in AMD compared with controls. High level of eicosadienoic acid also increased the risk of AMD, while a low ARA level was protective. Although not much is known about eicosadienoic acid, it is posed to be a metabolite of linoleic acid that may modulate the synthesis of inflammatory mediators [26
]. Linoleic acid is also an essential fatty acid that is acquired from food sources such as red meat, poultry and nuts.
For MUFA, oleic acid was the most important in this study, as high-level increased the odds of AMD, and logistic regression also demonstrated its association with AMD. Moreover, for the first time, we report high SFAs levels in AMD. Most of the SFAs, except stearic acid were again associated with AMD in logistic regression, and a high blood level of these increased the odds of developing AMD. Indeed, SFAs are synthesized through de novo lipogenesis, mainly via dietary palmitic acid [27
]. Our observation suggests desaturation of palmitic acid to stearic acid was rapid and eventually converted to oleic acid. It is postulated high SFA is contributed by the poor dietary habit of the AMD who had higher frequency of red meat intake, which is a rich source of SFA.
Currently, there are some studies that look at the effect of nutrient supplementation in AMD. AREDS2, which was a multi-center 5-year randomized trial designed to examine the effects of oral supplementation of macular xanthophylls (10 mg lutein and 2 mg zeaxanthin) and/or omega-3 PUFAs (EPA 650 mg and DHA 350 mg) on the progression to advanced AMD. There was no additional benefit from adding the omega-3 PUFAs or a mixture of lutein and zeaxanthin to the formulation. Although the addition of omega-3 to the AREDS formulation was not shown to be advantageous, it is believed that higher doses of EPA and DHA may have a desirable effect [3
]. A small observational study reported by Georgiou and Prokopiou found dry AMD patient with severe vision loss had significant visual improvement when supplemented with EPA and DHA for up to 6 months [28
]. Another recent large prospective cohort reported higher intakes of EPA and DHA (350 mg/d or fatty fish of ≥ 2 servings/week) showed moderate reduction risk of AMD and potentially be the primary prevention or method to delay the occurrence of visually significant intermediate AMD for Americans [29
]. The conflicting findings in these intervention studies demonstrate that more understanding regarding the circulating levels of these nutrients are necessary. The ideal source, dosage, timing of intervention and route of omega-3 PUFA supplements were also unknown. Thus, in our current study, we focused on the actual difference in the blood omega-3 PUFA levels. To date, this was the only comprehensive case-control study that investigated the full fatty acid profiles in Chinese patients. In the Western countries, the relationship with macular pigment density and circulating levels of DHA and EPA has been studied [30
]. Limpia study recently found an association between increased macular pigment optical density (MPOD) with circulating levels of omega-3 docosapentaenoic acid as well [31
], but nutrient supplementation did not change the MPOD [32
Whether the circulating levels of these micronutrients were related to the dietary habit was not well reported in the literature. However, our study confirmed the carotenoid levels were significantly lower in AMD cases, and a low blood level of these increased the odds of developing AMD. We found AMD group had significantly less intake of both green leafy vegetables as well as other plant-based food (e.g., nuts, turnip, celery, cabbage, and potatoes). These foods are rich in carotenoids, and also ALA a plant-based omega-3 PUFA where a low level of such, increased the odds ratio of having AMD. Insofar, of the omega-3 PUFA, only poor fish oil intake (EPA and DHA) were implicated in AMD but not from plants. The meat intake, including preserved meats, red meats (pork, beef and lamb), and poultry were also significantly higher in the AMD group. These foods are rich in omega-6 PUFA which are sources of pro-inflammatory eicosanoids, especially ARA, and linoleic acid, as well as the SFAs, such as palmitic acid. In our study, a high level of SFAs was found to increase the odds ratio of having AMD, and our logistic regression model also found SFAs to be associated with AMD. As for seafood, although there was no difference in the frequency intake of fish between the control and AMD, other seafood (prawn, shrimp, crab, mussels, scallop) that has lesser EPA and DHA content than fish [33
], were consumed less frequently in the AMD group. Nonetheless, our study did not investigate the type of fish consumed where oily fish (mackerel, herring, salmon) contain higher omega-3 PUFA than white fish (cod, tilapia, sea bass); the latter is popular in Hong Kong Chinese diet. Nevertheless, results of the dietary analysis were not decisive because the difference in responses between cases and controls was small. This may be due in part to the subjectivity of the FFQ and therefore, the accuracy of the patient response in the questionnaire.
One of the strengths of this study was in fact the quantitative analysis of fatty acids and carotenoids of the collected blood samples from AMD subjects and age-matched controls. It has been suggested that these circulating biomarkers of lipid and the type of dietary lipid are important in the management of AMD [34
]. Utilizing these data, we could examine the patterns of subjects’ dietary habits and their relationships with the aforementioned blood levels and other risk factors for AMD.