Comments on the Use of Raman Spectroscopy for the in vivo Measurement of Human Macular Pigment

In a recent article in this journal Ermakov et al. describe the further development of a device for measuring the in vivo concentration of carotenoids within the retina using Raman spectroscopy. Since some readers of this journal may not be familiar with the literature on macular pigment (MP), we thought it would be helpful to identify some of the controversy surrounding some of the points made by Ermakov et al. Rather than be comprehensive, we simply point out a few representative examples and refer readers to the published literature. In the introduction the authors state that ‘‘recent Raman and flicker photometry clinical studies have shown that MP concentrations are on average declining with age in normally aged healthy eyes,’’ citing only two studies (Bernstein et al., 2002, and Neelam et al., 2005). A number of reviews have been published on this issue that have surveyed extensive results obtained from both ex vivo and in vivo methods. They have concluded that MP does not change meaningfully with age. For example, we pointed out to the authors in 2003 that results from eight large studies show ‘‘moderate to small changes in MP density over the lifespan (averaging less than 1% across studies)’’. We also noted that ‘‘most of the other studies have found actual increases in the range of MP with age (averaging about a 23% increase across studies), as opposed to the large restriction in range found with the Raman method (about 86%)’’. More recently, Bernendschot et al. (2005) reviewed 20 different studies on many populations using a variety of methods and concluded that ‘‘Taken together, most studies, including the present one, found no age effect; a few found either a slight decrease or a slight increase. This brings us to our conclusion that the MPOD shows no systematic change with age’’. In contrast, based on their Raman method the authors conclude that both the MP average and MP range drastically decline with age. Whereas this is clearly true of the Raman signal, it is unlikely that this is true of MP. We have argued that methodological problems cause the near disappearance of the Raman signal for nearly all older subjects (as shown in the data of Gellerman et al., 2001, and Zhao et al., 2003). The authors also often describe their technique as ‘‘fast. ’’ The term ‘‘fast’’ is, of course, relative and is only relevant when compared to other methods of measuring MP. Is, for instance, the standard Raman measurement (one MP value referenced to a location on the retina) faster than other methods of measuring MP? Using the standardized protocol in the CAREDs study, for instance, a standard HFP measure takes only about 20 to 30 minutes. Is the Raman method faster? As noted by Ermakov et al., their subjects were first dilated to 9 mm (a very large diameter that is necessary to avoid confounding by the pupil). Afterwards, subjects must recover from this dilation procedure. Typically, five Raman counts (RCs) are recorded, the lowest two are discarded, and the RC is taken as the average of the remaining three. Subjects must be re-aligned after each trial. Neelam et al. notes that when making these five measures, 2–3 minutes are necessary between each trial in order to allow the ‘‘flash after-image to fade. ’’ Until a comparative study of measurement times is done, their assertion that the Raman method is faster is not based on actual evidence. A fundamental issue for all methods that measure posterior structures within the eye is interference due to anterior structures. Most MP methods avoid this problem by using a normalizing method referenced to a parafoveal site where MP is optically undetectable. Since a reliable Raman signal cannot be obtained at a parafoveal reference site, however, their method relies on an absolute signal. The method requires a double-pass through the anterior segment and therefore is confounded by any structure that alters the stimulation beam or the return signal. In this case, the stimulation beam (input) and the signal (output) refer to lights at or around 488 and 527 nm, respectively. We have reviewed a large body of literature showing that the lens both scatters and absorbs these wavelengths. The real issue is determining the magnitude of these confounds for a given individual. Until the authors actually supply empirical data regarding the magnitude of these potential confounds, their assertion that the method has no ‘‘systematic error’’ is unfounded. Ermakov et al.’s study, as well as past studies using the Raman method, supplies only Raman counts as if they were actual MP values. The authors have supplied external calibration curves that they argue can be used to translate RCs into optical density values that represent true variation in MP. Such calibrations, however, can only be accurate to the extent that the model eye is a valid representation of the living human eye. One obvious difference is that the optical properties of the human eye vary, especially in the elderly, and the model eye is static. Real eyes display many attributes that are highly variable, including pupil size, density of the lens, and intraocular diffusion of light. Unless the magnitudes of each of these factors are known for a given individual and are used to correct the Raman signal, the final RC is impossible to interpret. The authors have disagreed, yet acknowledge that calibration of the Raman signal is of central importance to Received 18 November 2005; accepted 15 May 2006. * Author to whom correspondence should be sent. E-mail: bhammond@ egon.psy. uga.edu