Heterochromatic flicker photometry (HFP) is the most commonly used method to measure macular pigment in clinical research.
17 18 24 26 27 39 40 A small spot of light projected onto the fovea rapidly alternates between blue, which is well absorbed by the macular carotenoids, and green, which is not. The subject varies the relative intensities of the two lights until the perception of flicker is minimized or eliminated, and the average energy of the blue light (
B fov) at minimal flicker from multiple tests is recorded. Because subjects vary markedly in the ratio of L and M cones that mediate color perception in HFP, the test must be repeated with an eccentric reference fixation point. To determine
B ref, macular pigment optical density (MPOD) is assumed to be zero. Typical reference points are at 4° to 9° eccentricity (1.2–2.6 mm at the retinal surface). The MPOD is then calculated with the equation MPOD = log (
B fov /B ref). The zero MPOD assumption at approximately 7° is likely to be reasonable in an unsupplemented primate eye based on microspectrophotometry using absorbance and resonance Raman techniques in cadaver eyes,
32 33 34 but our data in this study raised questions as to whether this assumption remains valid in the supplemented state. In some subjects, substantial increases in carotenoid content (up to 10-fold) are exhibited in the equatorial region, very far beyond the traditional reference point of approximately 7°. We calculated that the average optical density attributable to xanthophyll carotenoids in the equatorial retina in one of these donors would be up to 0.01 at 460 nm. This would introduce a 3% underestimate of foveal MPOD for a subject with a typical foveal MPOD of 0.3, which is not particularly problematic if the equatorial periphery were used for
B ref. In HFP, however,
B ref is less than 2.6 mm from the center of the fovea. It is not unreasonable to predict that MPOD at approximately 7° might be in the 0.1 range or even higher in a supplemented subject because of a steep gradient of xanthophyll concentration from the periphery to the fovea, as evidenced in a previous human retinal HPLC microdissection study demonstrating that macular carotenoid levels at this eccentricity are typically more than 10 times higher than at the equatorial periphery.
11 This level of MPOD at
B ref would introduce a very large and potentially variable error in the foveal MPOD calculation (up to a 30% underestimate for a typical foveal MPOD of 0.3 or a 10% underestimate for a very high foveal MPOD of 1.0). The best way to avoid this problem would be to place
B ref as far as possible in the periphery. Unfortunately, reliable psychophysical performance of HFP much beyond 7° (i.e., >14°) is extraordinarily difficult.