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Macular pigment: New clinical methods of detection and the role of carotenoids in age-related macular degeneration
Optometry (2008) 79, 266-272
Macular pigment: New clinical methods of detection and the role of carotenoids in age-related macular degeneration Ivan Y-F. Leung, Ph.D., M.Phil. Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland. KEYWORDS Age-related macular degeneration; Macular pigment; Carotenoids; Zeaxanthin; Lutein; Dietary supplementation
Abstract Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over the age of 65. The Age-Related Eye Disease Study (AREDS) suggests antioxidants may delay the advance of age-related macular degeneration. The macular pigments zeaxanthin and lutein may serve as antioxidants as well as blue filter to protect the retina. In this review, the general characteristics of macular pigment are described. The nutritional value of zeaxanthin/lutein and methods to assess macular pigment are discussed. Several emerging instruments to assess macular pigment, including heterochromatic flickering photometer, motion detection photometer, fundus reflectance spectroscope, Raman spectrometer, and autofluorescence spectrometry, are introduced and reviewed. Optometrists should be aware that they may play a role to assess and monitor the risk of AMD. There is an opportunity to incorporate measurement of macular pigment in optometric practice. Optometry 2008;79:266-272
Age-related macular degeneration Age-related macular degeneration (AMD) is one of the leading causes of adult blindness in the United States.1 It is a degenerative eye disease that affects the macula of people over the age of 65. Consequently, central vision is affected, and daily tasks such as reading, writing, driving, and sewing can be impaired. Degenerative changes of AMD occur primarily in the photoreceptor-retinal pigment epithelium (RPE) complex. Because the photoreceptor cells cannot regenerate, current treatment strategies focus on delaying or preventing the advancement of the disease process. Currently, there are several treatment options for AMD, but satisfactory and efficient therapy in the majority of AMD patients is lacking.2,3
Corresponding author: Ivan Y-F Leung, Ph.D., M.Phil., 633 Tower 1, Grand Central Plaza, 138 Shatin Rural Committee Road, Shatin, NT, Hong Kong. E-mail address: [email protected]
By 2030, about 72 million people will be 65 years or older in the United States. This older population will be twice as large as in 2000.4 The number of individuals with AMD is estimated to increase by more than 50% from 1.75 million to 2.95 million between 2000 and 2020.1 Therefore, AMD will have enormous social and economic impact on the health care system.
Anatomic features of the human macula The adult macula is approximately 5.5 mm in diameter and the fovea approximately 1.5 mm in diameter. The numbers of cone cells and RPE cells attain highest densities at the floor of the fovea (foveloa), and the high density of cells in the fovea decrease toward the periphery.5-8 The rod cells, however, are relatively sparse in the foveloa for about 0.2 mm and then gradually increase with eccentricity.6,7 Ganglion cells and the inner retinal layers are displaced away from the foveloa, and this area is avascular. Although the
1529-1839/08/$ -see front matter © 2008 American Optometric Association. All rights reserved. doi:10.1016/j.optm.2007.03.017
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Figure 1
267
Chemical structures of zeaxanthin, meso-zeaxanthin, and lutein.
fovea may be exposed to high oxidative stress and strong light exposure,9-10 the morphologic features allow for maximal sensitivity and resolution of vision in the macular region. The specific morphologic characteristics of the fovea coexist with another unusual feature of the retina—the macular pigment (MP). The existence of the yellow macular pigment has drawn the attention of medical practitioners for hundreds of years.11 The biological functions of the yellow macular pigment, however, are still under investigation. Several lines of evidence suggest that the macular pigment plays a unique role in the central retina.
Macular pigment MP contains a mixture of carotenoids consisting of zeaxanthin, meso-zeaxanthin, and lutein (see Figure 1).12 All 3 of these carotenoids are stereoisomers with the same molecular size but are not distributed randomly in the macula. Both zeaxanthin and lutein decrease in density with increased eccentricity. However, zeaxanthin is higher in amount than lutein in the central fovea. Because zeaxanthin and lutein cannot be synthesized in our body, these molecules must be absorbed from our diet. Meso-zeaxanthin is found only in the central macula but it is neither found in the diet nor in the human blood stream.12 Recently, a biochemical study in monkey retinas found that meso-zeaxanthin is converted
from lutein in the primate retina.13 These specific distributions of the stereoisomers suggest that zeaxanthin and/or meso-zeaxanthin may have a specific function in the center of the macula.
Nutritional role of carotenoids in macular diseases Zeaxanthin and lutein are members of the carotenoid family. Some common foods contain different types of carotenoids in a way shown by their color (see Table 1). One common carotenoid, -carotene, is known for its conversion to vitamin A, whereas zeaxanthin and lutein cannot be converted to vitamin A. Although there are more than 600 carotenoids found in nature, only 13 of them are found in the human body.14,15 Zeaxanthin and lutein are the only 2 transported from serum into the retina. Thus, the retina exhibits a specific predilection for uptake of zeaxanthin and lutein. Because of the potential therapeutic effect of zeaxanthin and lutein in macular degeneration, many dietary supplementation studies have been carried out in different laboratories.16-21 Recently, no adverse effect has been reported or found in both monkey and human supplementation studies,22-25 and so lutein and zeaxanthin are safe for human
268 Table 1
Optometry, Vol 79, No 5, May 2008 Examples of carotenoids in various foods
Food
Major carotenoid
Color appearance
Carrot Maize Tomato Spinach Egg yolk Orange pepper Shrimp Salmon
Beta-carotene Lutein/zeaxanthin Lycopene Lutein Lutein Zeaxanthin Astaxanthin Astaxanthin
Orange Yellow Red Green* Yellow Orange Pink/red Orange/pink
* The yellow color of lutein is covered by the green-colored chlorophylls in the spinach leaves.
large-scale randomized clinical trial to show the protective role of nutritional vitamins and minerals to the eye. The next phase of the study is the AREDS II study. The formulation of the oral supplements has been revised according to current knowledge in eye research.39 The macular carotenoids zeaxanthin and lutein are added with or without omega-3 polyunsaturated fatty acids. The formulations are also modified with/without the addition of -carotene or a lower dosage of zinc. This phase III clinical trial started to recruit subjects in late 2006, and eligible participants will be followed up for 5 years.
Detection of MP in the retina consumption. Some studies found the increase of zeaxanthin and lutein in both serum and retina after dietary supplementation.16,17,19 A few studies also suggested improvement of visual functions in normal subjects after supplementation.26,27 A depletion study of zeaxanthin and lutein in monkeys has also shown the reduction of RPE density in the fovea.28 Although the biological functions of the MP are not fully understood, some evidence has suggested that zeaxanthin and lutein absorb blue light, which is most damaging to the retina.10 In addition, scattering and chromatic aberration of blue light may be minimized by the macular pigment.29 In vitro and in vivo studies have found that these carotenoids are also potent antioxidants.9,10,30,31 Hence, zeaxanthin and lutein may protect the retina against oxidative damage in the macula where free radicals may be generated by lengthy light exposure, high oxygen tension, and high metabolic rate. This hypothesis is supported by observational studies that showed that high dietary intake in zeaxanthin/lutein32 and high serum zeaxanthin33,34 lower the risk of AMD. Several small-size clinical trials suggested that zeaxanthin and/or lutein supplementation improve human visual function or visual performance.27,35,36 However, the biological effects of zeaxanthin/lutein supplementation need to be fully addressed in large clinical trial. In 1992, the National Eye Institute initiated a multicenter, prospective clinical trial known as the Age-Related Eye Disease Study (AREDS).37 One of the objectives of AREDS was to investigate the potential benefit of high levels of vitamin C, vitamin E, -carotene, and zinc in AMD patients. From 1992 to 1998, 4,757 participants age 55 to 80 were recruited and followed up for at least 7 years. During the process of selecting several antioxidants for the AREDS formulation, zeaxanthin and lutein were strong candidates for inclusion in AREDS. However, these macular carotenoids were not commercially available when AREDS started.37 In 2001, AREDS reported the protective role of vitamins C, E, zinc, and -carotene in advanced AMD.38 The AREDS formulation reduced the risk of development of advanced AMD by 25% for certain categories of AMD. The oral supplement also reduced the vision loss by 19% caused by advanced AMD. This study is the first
Because low serum zeaxanthin/lutein and low MP may be associated with high risk of AMD,32,40 assessment of MP may serve as an early screening tool to evaluate the risk of AMD. Although it may take years for the AREDS II study to determine the protective effects of zeaxanthin and lutein for AMD subjects, optometrists should be aware of the new development in this area and prepare for the opportunity to measure MP in their practice. Two methods have been established to measure MP in the human retinal tissues. High-pressure liquid chromatography (HPLC) is the standard method to identify the quantity and components of the MP in the retina.40-42 The second method, microspectrophotometry, measures optical density of the MP in the retinal sections.43,44 Both methods provide the basic data of the distribution of MP in the postmortem human retina. However, these methods cannot be applied to the living eye. Several clinical methods of assessing MP are being developed for patient evaluation, including (1) psychophysical photometry, (2) fundus reflectance spectroscopy, (3) Raman spectroscopy, and (4) autofluorescence spectrometry. In this review, 2 specific methods are emphasized: heterochromatic flickering photometry and autofluorescent spectrometry. These techniques have potential to become clinical procedures to assess MP in the next decades. The advantages and disadvantages of different methods are summarized in Table 2.
Heterochromatic flickering photometer Heterochromatic flickering photometer (HFP) is a well-established method to measure the concentration of MP psychophysically.45-46 Test stimuli from 0.5° to 1° in diameter are routinely used in different laboratories to measure MP at the fovea. The principle of measurement is based on the absorption properties and the distribution of the MP. Two sources of monochromatic light, which are substantially absorbed (e.g., blue light at 460 nm) or not absorbed (e.g., green light at 550 nm) by the MP, are presented to the testing eye at the fovea (point of fixation) alternatively. Because of the MP absorption of the blue light at 460 nm, the blue light becomes dimmer
Ivan Y-F. Leung Table 2
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269
The advantages and disadvantages of different methods to assess macular pigment Nature of the test
Postmortem/ in vivo
Cost
Pupil dilation
Light scatter
Availability
Reproducibility
Comment
Microspectrophotometry
Objective, optical
Postmortem
Moderate-High
N/A
Mild
Yes
Good
High-pressure liquid chromatography (HPLC) Heterochromatic flicker photometry Motion detection photometry Fundus reflectance spectroscopy Raman spectroscopy
Biochemical separation and optical detection Subjective psychophysical test Subjective psychophysical test Objective optical imaging Objective Raman signal detection Objective optical imaging
Postmortem
Moderate-High
N/A
N/A
Yes
Good
Standard optical method Standard biochemical method
In vivo
Low
No
N/A
Yes
Fair to good
In vivo
Low
No
N/A
No
Fair to good
In vivo
High
Yes
Mild-moderate
Yes
Fair
In vivo
High
Yes
Mild-moderate
Yes
Good
In vivo
High
Yes
Mild
Yes
Good
Autofluorescence spectrometry
when compared to green light at 550 nm. Thus, the subject will perceive flickering of blue and green light. The flicker will be eliminated when the intensity of the blue light is increased to match with that of the green light. This is considered the endpoint of measurement, and the intensity of the blue light is recorded. The procedure will be repeated at a reference point, for example at 5° to 7° from fixation, where MP is optically undetectable. The MP optical density (MPOD) is determined by calculating the ratio of the blue light intensity at the fixation point and the reference point.47 Once the minimal flicker is achieved at both the fixation and reference point, the intensity of the blue light absorbed by the photoreceptors at both the measured locus and the reference point are presumably the same. It can be expressed as: Bm⫻Tlens⫻TMP⫽Bref⫻Tlens
(1)
Where Bm and Bref are the radiance of the blue light to minimize flicker at the measured locus and reference point. Tlens and TMP are the transmission of the crystalline lens and MP. Because Tlens can be eliminated from both sides of the equation (1), equation (1) becomes Bm⫻TMP⫽Bref
Therefore, TMP transmission of MP ⫽Bref ⁄ Bm. Since MPOD ⫽ log共1/TMP兲, MPOD⫽log共Bm/Bref兲. This psychophysical measurement of MPOD is relatively simple to understand. The repeatability and reliability found in young and elderly subjects are reasonably high.48,49 Spatial distribution of MP may be estimated by increasing the size of the test field from the fixation (foveal center). However, eccentric perception of flicker induces the Troxler effect, which may affect the determination of the endpoint of the measurement. Because the experimental conditions (such as refractive error, head movement, eye movement, and flicker frequency) may affect the accuracy and the values of MPOD measured, standardized protocol of the HFP method has been proposed.46,50
Most common method New method
Motion detection photometer There is a newly modified psychophysical method to measure MP by motion photometry.51 The perceived motion, because of absorption of the MP, is minimized by changing the blue luminance observed. The optical principle and measurement procedure are similar to flicker photometry. The design is based on the simplicity of motion detection compared with flicker detection. A recent study suggests that the measured optical density is comparable with other methods such as the autofluorescence method.52
Fundus reflectance spectroscopy The MPOD may be assessed indirectly by measuring the fundus reflectance in the living eye.53 Several tools such as the reflectometer,54 fundus camera attached to chargecouple device (CCD),55 and scanning laser ophthalmoscope (SLO)56 have been modified to quantify MP. The objective is to measure and compare the reflectance of the retina at a specific wavelength at the macular and peripheral regions. Spectral analysis of the reflected light has also been used to eliminate the reflection caused by other absorbers (e.g., red blood cells, melanin granules, or lipofuscin granules) and stray light. Because of the directional reflectance and scatter light within the eye, great care is needed in the analysis of the reflectance to determine the density of the MP.
Raman spectrometer The resonance Raman spectrometer measures the amount of zeaxanthin and lutein in the retina by directing a low-energy argon laser (488 nm) at the retina.57 The Raman spectrometer detects the Raman shift of the scattered light reflected by zeaxanthin and lutein molecules in the fundus. Gellerman et al.58 noted a reduction of MP (as
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measured by the Raman method) with increasing age. This observation has been challenged by others59 because other methods did not find significant age dependency in MP.60
Autofluorescence spectrometry The autofluorescence of the lipofuscin in the retinal pigment epithelium has provided an innovative approach to assess MP.61 Since the range of excitation (400 nm to 590 nm) of lipofuscin is broader than the absorption range (400 nm to 550 nm) of MP, excitation light of the lipofuscin is partially absorbed by MP. However, the emitted fluorescent light (between 520 nm and 800 nm) of the lipofuscin is not absorbed by MP. Therefore, it allows single-pass measurement of MPOD by detection of the autofluorescent light rather than the usual double pass reflection in other physical methods such as the reflectance method. The procedure starts with the dilation of a subject’s pupil. The autofluorescence images are then taken with excitation at 2 distinct wavelengths, which are absorbed differently by the MP. The subtraction of the signals between these 2 wavelengths creates a MP density map in the central retina.62 The measurement of MPOD in autofluorescence imaging is based on the following equation61: ⌬MPOD f,ref (460) ⫽
冋
1 K(1) ⫺ K(2)
⫻ log
册
Fref (1) Fref (2) . . . equation 共2兲 ⫺ log F f (1) F f (2)
Where ⌬MPOD f,ref (460) is the difference of MPOD between a field location (f) and the reference (ref) location at 460 nm, and K is the extinction coefficient of the MP at a particular wavelength. For this calculation, 2 excited wavelengths (1, 2) are used. F is the fluorescence at the field (f) and reference (ref) locations respectively. Because of the availability of 1 excitation light source (argon laser 488 nm) in current confocal scanning laser ophthalmoscopes (SLO), some investigators used only 1 wavelength to determine the optical density of the MP in autofluorescence imaging.63 A higher percentage of error in MP was created at greater eccentricities because of nonuniform excitation and emission across the fundus. Comparisons of the 1-wavelength and 2-wavelength methods suggested that the 2-wavelength methods provide a better estimation of MP profile in vivo.64 For the 2-wavelength method, however, an additional light source (e.g., 514 nm), as well as software, is needed to incorporate into the current SLO technology.
Limitation of current clinical methods Currently there is no gold standard to measure MP in vivo. Researchers are trying to validate and optimize their meth-
ods and protocols for routine MP assessment. Because HFP was developed with well-recognized optical principles, it is generally accepted by researchers in measuring MP.47 Comparisons are being made between psychophysical methods and others.61,65 With the exception of Raman spectrometry, all other methods (psychophysical methods, autofluorescent method, and reflectance methods) use the spectral properties of zeaxanthin/lutein to quantify the levels of MP.46 HFP is known for its noninvasive and low-cost device. Its measurement is not affected by cataract or intraocular light scatter caused by aging. However, the examiner and subject need to learn the examination skill and procedure before the measurements. For optimal measurement, the flicker frequency needs to be determined for each tested individual. Other objective methods such as AF and reflectance method measure the profile/distribution of the MP within a short period of time. Nevertheless, pupillary dilation is needed, and expensive instrumentation (e.g., SLO) is required. As primary eye care practitioners, optometrists should take a role in the early detection and prevention of degenerative eye diseases such as AMD. Early determination of risk factors of AMD, such as the detection of low levels of MP, may identify which patients are susceptible to the development of AMD. Optometrists should promote healthy living styles to their patients. Recommendations such as smoking cessation and healthy diet intake should be given. When low levels of MPOD are detected, optometrists may prescribe dietary supplements to increase MP and monitor the change of MP after supplementation. Although lutein is commonly available in the marketplace, zeaxanthin, as a major component in MP, should be considered for dietary supplementation. Because MP may serve as a biomarker for ocular health, measurement of MP can provide a unique tool for AMD risk assessment. According to the current advantages and disadvantages of the MP assessment methods, HFP is a fast, safe, and affordable tool to measure MP. Imaging methods (e.g., methods using SLO or Raman spectroscopy) are more difficult to access in the private optometric practice. HFP may become part of optometric practice for the assessment of ocular health in the near future.
Acknowledgments The author gratefully acknowledges funding from ZeaVison LLC and Dr. Dennis Gierhart. The author has no financial or proprietary interest in any of the products or devices described in the article.
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AMD and healthy subjects. Graefes Arch Clin Exp Ophthalmol 2002;240:666-71. Bernstein PS, Zhao DY, Sharifzadeh M, et al. Resonance Raman measurement of macular carotenoids in the living human eye. Arch Biochem Biophys 2004;430:163-9. Gellerman W, Ermakov IV, Ermakov MR, et al. In vivo resonant Raman measurement of macular carotenoid pigments in the young and the aging human retina. J Opt Soc Am A 2002;19:1172-86. Wooten BR, Hammond BR. Assessment of the Raman method of measuring human macular pigment [Letter]. Invest Ophthalmol Vis Sci [serial online]. Available at: http://www.iovs.org/cgi/eletters?lookup⫽ by_date&days⫽9999#73. Last accessed August 15, 2003. Wooten BR, Hammond BR. Assessment of the Raman method of measuring human macular pigment [Letter]. II. Invest Ophthalmol Vis Sci [serial online]. Available at: http://www.iovs.org/cgi/eletters? lookup⫽by_date&days⫽9999#73. Last accessed December 30, 2003. Delori FC, Goger DG, Hammond BR, et al. Macular pigment density measured by autofluorescence spectrometry: comparison with reflectometry and heterochromatic flicker photometry. J Opt Soc Am A 2001;18:1212-30. Delori FC. Autofluorescence method to measure macular pigment optical densities fluorometry and autofluorescence imaging. Arch Biochem Biophys 2004;430:156-62. Trieschmann M, Spital G, Lommatzsch A, et al. Macular pigment: quantitative analysis on autofluorescence images. Graefes Arch Clin Exp Ophthalmol 2003;241:1006-12. Trieschmann M, Heimes B, Hense HW, et al. Macular pigment optical density measurement in autofluorescence imaging: comparison of oneand two-wavelength methods. Graefes Arch Clin Exp Ophthalmol 2006;244:1565-74. Neelam K, O’Gorman N, Nolan J, et al. Measurement of macular pigment: Raman spectroscopy versus heterochromatic flicker photometry. Invest Ophthalmol Vis Sci 2005;46:1023-32.
Macular pigment: New clinical methods of detection and the role of carotenoids in age-related macular degeneration Ivan Y-F. Leung, Ph.D., M.Phil. Wilmer Eye Institute, Johns Hopkins University, Baltimore, Maryland. KEYWORDS Age-related macular degeneration; Macular pigment; Carotenoids; Zeaxanthin; Lutein; Dietary supplementation
Abstract Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over the age of 65. The Age-Related Eye Disease Study (AREDS) suggests antioxidants may delay the advance of age-related macular degeneration. The macular pigments zeaxanthin and lutein may serve as antioxidants as well as blue filter to protect the retina. In this review, the general characteristics of macular pigment are described. The nutritional value of zeaxanthin/lutein and methods to assess macular pigment are discussed. Several emerging instruments to assess macular pigment, including heterochromatic flickering photometer, motion detection photometer, fundus reflectance spectroscope, Raman spectrometer, and autofluorescence spectrometry, are introduced and reviewed. Optometrists should be aware that they may play a role to assess and monitor the risk of AMD. There is an opportunity to incorporate measurement of macular pigment in optometric practice. Optometry 2008;79:266-272
Age-related macular degeneration Age-related macular degeneration (AMD) is one of the leading causes of adult blindness in the United States.1 It is a degenerative eye disease that affects the macula of people over the age of 65. Consequently, central vision is affected, and daily tasks such as reading, writing, driving, and sewing can be impaired. Degenerative changes of AMD occur primarily in the photoreceptor-retinal pigment epithelium (RPE) complex. Because the photoreceptor cells cannot regenerate, current treatment strategies focus on delaying or preventing the advancement of the disease process. Currently, there are several treatment options for AMD, but satisfactory and efficient therapy in the majority of AMD patients is lacking.2,3
Corresponding author: Ivan Y-F Leung, Ph.D., M.Phil., 633 Tower 1, Grand Central Plaza, 138 Shatin Rural Committee Road, Shatin, NT, Hong Kong. E-mail address: [email protected]
By 2030, about 72 million people will be 65 years or older in the United States. This older population will be twice as large as in 2000.4 The number of individuals with AMD is estimated to increase by more than 50% from 1.75 million to 2.95 million between 2000 and 2020.1 Therefore, AMD will have enormous social and economic impact on the health care system.
Anatomic features of the human macula The adult macula is approximately 5.5 mm in diameter and the fovea approximately 1.5 mm in diameter. The numbers of cone cells and RPE cells attain highest densities at the floor of the fovea (foveloa), and the high density of cells in the fovea decrease toward the periphery.5-8 The rod cells, however, are relatively sparse in the foveloa for about 0.2 mm and then gradually increase with eccentricity.6,7 Ganglion cells and the inner retinal layers are displaced away from the foveloa, and this area is avascular. Although the
1529-1839/08/$ -see front matter © 2008 American Optometric Association. All rights reserved. doi:10.1016/j.optm.2007.03.017
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Figure 1
267
Chemical structures of zeaxanthin, meso-zeaxanthin, and lutein.
fovea may be exposed to high oxidative stress and strong light exposure,9-10 the morphologic features allow for maximal sensitivity and resolution of vision in the macular region. The specific morphologic characteristics of the fovea coexist with another unusual feature of the retina—the macular pigment (MP). The existence of the yellow macular pigment has drawn the attention of medical practitioners for hundreds of years.11 The biological functions of the yellow macular pigment, however, are still under investigation. Several lines of evidence suggest that the macular pigment plays a unique role in the central retina.
Macular pigment MP contains a mixture of carotenoids consisting of zeaxanthin, meso-zeaxanthin, and lutein (see Figure 1).12 All 3 of these carotenoids are stereoisomers with the same molecular size but are not distributed randomly in the macula. Both zeaxanthin and lutein decrease in density with increased eccentricity. However, zeaxanthin is higher in amount than lutein in the central fovea. Because zeaxanthin and lutein cannot be synthesized in our body, these molecules must be absorbed from our diet. Meso-zeaxanthin is found only in the central macula but it is neither found in the diet nor in the human blood stream.12 Recently, a biochemical study in monkey retinas found that meso-zeaxanthin is converted
from lutein in the primate retina.13 These specific distributions of the stereoisomers suggest that zeaxanthin and/or meso-zeaxanthin may have a specific function in the center of the macula.
Nutritional role of carotenoids in macular diseases Zeaxanthin and lutein are members of the carotenoid family. Some common foods contain different types of carotenoids in a way shown by their color (see Table 1). One common carotenoid, -carotene, is known for its conversion to vitamin A, whereas zeaxanthin and lutein cannot be converted to vitamin A. Although there are more than 600 carotenoids found in nature, only 13 of them are found in the human body.14,15 Zeaxanthin and lutein are the only 2 transported from serum into the retina. Thus, the retina exhibits a specific predilection for uptake of zeaxanthin and lutein. Because of the potential therapeutic effect of zeaxanthin and lutein in macular degeneration, many dietary supplementation studies have been carried out in different laboratories.16-21 Recently, no adverse effect has been reported or found in both monkey and human supplementation studies,22-25 and so lutein and zeaxanthin are safe for human
268 Table 1
Optometry, Vol 79, No 5, May 2008 Examples of carotenoids in various foods
Food
Major carotenoid
Color appearance
Carrot Maize Tomato Spinach Egg yolk Orange pepper Shrimp Salmon
Beta-carotene Lutein/zeaxanthin Lycopene Lutein Lutein Zeaxanthin Astaxanthin Astaxanthin
Orange Yellow Red Green* Yellow Orange Pink/red Orange/pink
* The yellow color of lutein is covered by the green-colored chlorophylls in the spinach leaves.
large-scale randomized clinical trial to show the protective role of nutritional vitamins and minerals to the eye. The next phase of the study is the AREDS II study. The formulation of the oral supplements has been revised according to current knowledge in eye research.39 The macular carotenoids zeaxanthin and lutein are added with or without omega-3 polyunsaturated fatty acids. The formulations are also modified with/without the addition of -carotene or a lower dosage of zinc. This phase III clinical trial started to recruit subjects in late 2006, and eligible participants will be followed up for 5 years.
Detection of MP in the retina consumption. Some studies found the increase of zeaxanthin and lutein in both serum and retina after dietary supplementation.16,17,19 A few studies also suggested improvement of visual functions in normal subjects after supplementation.26,27 A depletion study of zeaxanthin and lutein in monkeys has also shown the reduction of RPE density in the fovea.28 Although the biological functions of the MP are not fully understood, some evidence has suggested that zeaxanthin and lutein absorb blue light, which is most damaging to the retina.10 In addition, scattering and chromatic aberration of blue light may be minimized by the macular pigment.29 In vitro and in vivo studies have found that these carotenoids are also potent antioxidants.9,10,30,31 Hence, zeaxanthin and lutein may protect the retina against oxidative damage in the macula where free radicals may be generated by lengthy light exposure, high oxygen tension, and high metabolic rate. This hypothesis is supported by observational studies that showed that high dietary intake in zeaxanthin/lutein32 and high serum zeaxanthin33,34 lower the risk of AMD. Several small-size clinical trials suggested that zeaxanthin and/or lutein supplementation improve human visual function or visual performance.27,35,36 However, the biological effects of zeaxanthin/lutein supplementation need to be fully addressed in large clinical trial. In 1992, the National Eye Institute initiated a multicenter, prospective clinical trial known as the Age-Related Eye Disease Study (AREDS).37 One of the objectives of AREDS was to investigate the potential benefit of high levels of vitamin C, vitamin E, -carotene, and zinc in AMD patients. From 1992 to 1998, 4,757 participants age 55 to 80 were recruited and followed up for at least 7 years. During the process of selecting several antioxidants for the AREDS formulation, zeaxanthin and lutein were strong candidates for inclusion in AREDS. However, these macular carotenoids were not commercially available when AREDS started.37 In 2001, AREDS reported the protective role of vitamins C, E, zinc, and -carotene in advanced AMD.38 The AREDS formulation reduced the risk of development of advanced AMD by 25% for certain categories of AMD. The oral supplement also reduced the vision loss by 19% caused by advanced AMD. This study is the first
Because low serum zeaxanthin/lutein and low MP may be associated with high risk of AMD,32,40 assessment of MP may serve as an early screening tool to evaluate the risk of AMD. Although it may take years for the AREDS II study to determine the protective effects of zeaxanthin and lutein for AMD subjects, optometrists should be aware of the new development in this area and prepare for the opportunity to measure MP in their practice. Two methods have been established to measure MP in the human retinal tissues. High-pressure liquid chromatography (HPLC) is the standard method to identify the quantity and components of the MP in the retina.40-42 The second method, microspectrophotometry, measures optical density of the MP in the retinal sections.43,44 Both methods provide the basic data of the distribution of MP in the postmortem human retina. However, these methods cannot be applied to the living eye. Several clinical methods of assessing MP are being developed for patient evaluation, including (1) psychophysical photometry, (2) fundus reflectance spectroscopy, (3) Raman spectroscopy, and (4) autofluorescence spectrometry. In this review, 2 specific methods are emphasized: heterochromatic flickering photometry and autofluorescent spectrometry. These techniques have potential to become clinical procedures to assess MP in the next decades. The advantages and disadvantages of different methods are summarized in Table 2.
Heterochromatic flickering photometer Heterochromatic flickering photometer (HFP) is a well-established method to measure the concentration of MP psychophysically.45-46 Test stimuli from 0.5° to 1° in diameter are routinely used in different laboratories to measure MP at the fovea. The principle of measurement is based on the absorption properties and the distribution of the MP. Two sources of monochromatic light, which are substantially absorbed (e.g., blue light at 460 nm) or not absorbed (e.g., green light at 550 nm) by the MP, are presented to the testing eye at the fovea (point of fixation) alternatively. Because of the MP absorption of the blue light at 460 nm, the blue light becomes dimmer
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269
The advantages and disadvantages of different methods to assess macular pigment Nature of the test
Postmortem/ in vivo
Cost
Pupil dilation
Light scatter
Availability
Reproducibility
Comment
Microspectrophotometry
Objective, optical
Postmortem
Moderate-High
N/A
Mild
Yes
Good
High-pressure liquid chromatography (HPLC) Heterochromatic flicker photometry Motion detection photometry Fundus reflectance spectroscopy Raman spectroscopy
Biochemical separation and optical detection Subjective psychophysical test Subjective psychophysical test Objective optical imaging Objective Raman signal detection Objective optical imaging
Postmortem
Moderate-High
N/A
N/A
Yes
Good
Standard optical method Standard biochemical method
In vivo
Low
No
N/A
Yes
Fair to good
In vivo
Low
No
N/A
No
Fair to good
In vivo
High
Yes
Mild-moderate
Yes
Fair
In vivo
High
Yes
Mild-moderate
Yes
Good
In vivo
High
Yes
Mild
Yes
Good
Autofluorescence spectrometry
when compared to green light at 550 nm. Thus, the subject will perceive flickering of blue and green light. The flicker will be eliminated when the intensity of the blue light is increased to match with that of the green light. This is considered the endpoint of measurement, and the intensity of the blue light is recorded. The procedure will be repeated at a reference point, for example at 5° to 7° from fixation, where MP is optically undetectable. The MP optical density (MPOD) is determined by calculating the ratio of the blue light intensity at the fixation point and the reference point.47 Once the minimal flicker is achieved at both the fixation and reference point, the intensity of the blue light absorbed by the photoreceptors at both the measured locus and the reference point are presumably the same. It can be expressed as: Bm⫻Tlens⫻TMP⫽Bref⫻Tlens
(1)
Where Bm and Bref are the radiance of the blue light to minimize flicker at the measured locus and reference point. Tlens and TMP are the transmission of the crystalline lens and MP. Because Tlens can be eliminated from both sides of the equation (1), equation (1) becomes Bm⫻TMP⫽Bref
Therefore, TMP transmission of MP ⫽Bref ⁄ Bm. Since MPOD ⫽ log共1/TMP兲, MPOD⫽log共Bm/Bref兲. This psychophysical measurement of MPOD is relatively simple to understand. The repeatability and reliability found in young and elderly subjects are reasonably high.48,49 Spatial distribution of MP may be estimated by increasing the size of the test field from the fixation (foveal center). However, eccentric perception of flicker induces the Troxler effect, which may affect the determination of the endpoint of the measurement. Because the experimental conditions (such as refractive error, head movement, eye movement, and flicker frequency) may affect the accuracy and the values of MPOD measured, standardized protocol of the HFP method has been proposed.46,50
Most common method New method
Motion detection photometer There is a newly modified psychophysical method to measure MP by motion photometry.51 The perceived motion, because of absorption of the MP, is minimized by changing the blue luminance observed. The optical principle and measurement procedure are similar to flicker photometry. The design is based on the simplicity of motion detection compared with flicker detection. A recent study suggests that the measured optical density is comparable with other methods such as the autofluorescence method.52
Fundus reflectance spectroscopy The MPOD may be assessed indirectly by measuring the fundus reflectance in the living eye.53 Several tools such as the reflectometer,54 fundus camera attached to chargecouple device (CCD),55 and scanning laser ophthalmoscope (SLO)56 have been modified to quantify MP. The objective is to measure and compare the reflectance of the retina at a specific wavelength at the macular and peripheral regions. Spectral analysis of the reflected light has also been used to eliminate the reflection caused by other absorbers (e.g., red blood cells, melanin granules, or lipofuscin granules) and stray light. Because of the directional reflectance and scatter light within the eye, great care is needed in the analysis of the reflectance to determine the density of the MP.
Raman spectrometer The resonance Raman spectrometer measures the amount of zeaxanthin and lutein in the retina by directing a low-energy argon laser (488 nm) at the retina.57 The Raman spectrometer detects the Raman shift of the scattered light reflected by zeaxanthin and lutein molecules in the fundus. Gellerman et al.58 noted a reduction of MP (as
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measured by the Raman method) with increasing age. This observation has been challenged by others59 because other methods did not find significant age dependency in MP.60
Autofluorescence spectrometry The autofluorescence of the lipofuscin in the retinal pigment epithelium has provided an innovative approach to assess MP.61 Since the range of excitation (400 nm to 590 nm) of lipofuscin is broader than the absorption range (400 nm to 550 nm) of MP, excitation light of the lipofuscin is partially absorbed by MP. However, the emitted fluorescent light (between 520 nm and 800 nm) of the lipofuscin is not absorbed by MP. Therefore, it allows single-pass measurement of MPOD by detection of the autofluorescent light rather than the usual double pass reflection in other physical methods such as the reflectance method. The procedure starts with the dilation of a subject’s pupil. The autofluorescence images are then taken with excitation at 2 distinct wavelengths, which are absorbed differently by the MP. The subtraction of the signals between these 2 wavelengths creates a MP density map in the central retina.62 The measurement of MPOD in autofluorescence imaging is based on the following equation61: ⌬MPOD f,ref (460) ⫽
冋
1 K(1) ⫺ K(2)
⫻ log
册
Fref (1) Fref (2) . . . equation 共2兲 ⫺ log F f (1) F f (2)
Where ⌬MPOD f,ref (460) is the difference of MPOD between a field location (f) and the reference (ref) location at 460 nm, and K is the extinction coefficient of the MP at a particular wavelength. For this calculation, 2 excited wavelengths (1, 2) are used. F is the fluorescence at the field (f) and reference (ref) locations respectively. Because of the availability of 1 excitation light source (argon laser 488 nm) in current confocal scanning laser ophthalmoscopes (SLO), some investigators used only 1 wavelength to determine the optical density of the MP in autofluorescence imaging.63 A higher percentage of error in MP was created at greater eccentricities because of nonuniform excitation and emission across the fundus. Comparisons of the 1-wavelength and 2-wavelength methods suggested that the 2-wavelength methods provide a better estimation of MP profile in vivo.64 For the 2-wavelength method, however, an additional light source (e.g., 514 nm), as well as software, is needed to incorporate into the current SLO technology.
Limitation of current clinical methods Currently there is no gold standard to measure MP in vivo. Researchers are trying to validate and optimize their meth-
ods and protocols for routine MP assessment. Because HFP was developed with well-recognized optical principles, it is generally accepted by researchers in measuring MP.47 Comparisons are being made between psychophysical methods and others.61,65 With the exception of Raman spectrometry, all other methods (psychophysical methods, autofluorescent method, and reflectance methods) use the spectral properties of zeaxanthin/lutein to quantify the levels of MP.46 HFP is known for its noninvasive and low-cost device. Its measurement is not affected by cataract or intraocular light scatter caused by aging. However, the examiner and subject need to learn the examination skill and procedure before the measurements. For optimal measurement, the flicker frequency needs to be determined for each tested individual. Other objective methods such as AF and reflectance method measure the profile/distribution of the MP within a short period of time. Nevertheless, pupillary dilation is needed, and expensive instrumentation (e.g., SLO) is required. As primary eye care practitioners, optometrists should take a role in the early detection and prevention of degenerative eye diseases such as AMD. Early determination of risk factors of AMD, such as the detection of low levels of MP, may identify which patients are susceptible to the development of AMD. Optometrists should promote healthy living styles to their patients. Recommendations such as smoking cessation and healthy diet intake should be given. When low levels of MPOD are detected, optometrists may prescribe dietary supplements to increase MP and monitor the change of MP after supplementation. Although lutein is commonly available in the marketplace, zeaxanthin, as a major component in MP, should be considered for dietary supplementation. Because MP may serve as a biomarker for ocular health, measurement of MP can provide a unique tool for AMD risk assessment. According to the current advantages and disadvantages of the MP assessment methods, HFP is a fast, safe, and affordable tool to measure MP. Imaging methods (e.g., methods using SLO or Raman spectroscopy) are more difficult to access in the private optometric practice. HFP may become part of optometric practice for the assessment of ocular health in the near future.
Acknowledgments The author gratefully acknowledges funding from ZeaVison LLC and Dr. Dennis Gierhart. The author has no financial or proprietary interest in any of the products or devices described in the article.
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