Carotenes do not contain electron withdrawing groups such as hydroxyl (OH) and carbonyl (C=O) groups which can amplify sterically induced restrictions and as a result can more readily exist in a cis-form. This is even more true for aliphatic carotenoids, where the cis isomer is more easily formed due to the lack of cyclic end groups. Di-cis and poly-cis configurations are possible but are not energetically favored and are much less common in nature.

Cis and trans isomers have different physical properties and can be separated based on these differences. The melting and boiling points of these compounds are just two of the ways these isomers can differ. Usually cis configuration assignment can be carried out using UV–VIS spectra and high performance liquid chromatography (HPLC) using known standards of cis configurations for comparison. NMR is also a powerful tool in carotenoid isomer identification.

30.5Terminal Groups

Carotenoids are identified by their end groups, referred to as terminal groups (or rings), which can have many distinguishing features. These end groups in most cases are cyclic in structure and can be substituted with hydroxyl groups (OH) and/ or carbonyl groups (C=O). The orientation of these substituent groups in space is also a critical feature of the carotenoids, not just in compound identification and characterization, but also in the functionality of the carotenoid (see below). MZ is an isomer of Z and L. MZ differs from Z only in the orientation of one hydroxyl group, whereas it differs from L by the location of a double bond in the terminal ring (see Fig. 30.1) [6]. These variations in structure result in a large difference in the distribution of these compounds in the eye, i.e., MZ is the dominant carotenoid at the foveal center, whereas L is the dominant carotenoid at the parafovea, suggesting that each compound has a different prioritized protective effect (this is discussed below under function of the macular carotenoids).

The functional groups are also predominantly responsible for the degree of polarity, the solubility and the chemical behavior of the carotenoids, parameters that vary between carotenoids based on the type and number of substituent groups. This allows accurate identification and characterization of these compounds, which is an advantage in practical work, i.e., allowing these compounds to be separated from blood serum samples, or synthesized in a laboratory environment.

The terminal rings on which the functional substituents reside are identified as beta (b) or epsilon (e) rings due to the different placement of the only double bond in the ring structure. Beta indicates a double bond between C5 and C6 on the ring, while epsilon indicates a double bond between C4 and C5. Z has two b-rings whereas L has one b-ring and one e-ring. Z has a high level of symmetry due to the two symmetrical b-rings and therefore only has two chiral centers, C3 and C3¢.

As previously stated the orientation of the substituent groups in space has an effect on functionality. This is the case for the substituent groups on the ring end structures. R (clockwise), S (counterclockwise) isomerization can occur giving rise

to various isomers of the compound. For example, L, which has three chiral centers, C3, C3¢, and C6¢, has a total of eight isomers, or four pairs of enantiomers (nonidentical mirror images). These do not exist in equal ratios, as factors such as steric hindrance and level of conjugation present in the backbone of the compound can cause the isomers that are more thermodynamically or sterically favored to occur predominantly, i.e., in L, the most abundant naturally occurring isomer is described as 3R,3¢R,6¢R L. The other isomers of L that can be found are usually seen as metabolites in animal tissues.

The terminal end groups can also affect the physical properties of the compound by using other reactive pathways. For example, modification of dietary carotenoids such as L by animals can vary the observed coloration of the compound. The C4 location on the epsilon ring can be ketonized, allowing the double bond formed to resonate with the already conjugated structure of the backbone, resulting in increased conjugation of the compound. This changes the observed coloration from a soft yellow to red, denoting a shift in absorption known as bathochromic shift.

30.5.1Source of Macular Carotenoids

An average western diet contains 1.3–3 mg/day of L and Z combined [51], with significantly more L than Z (represented by an estimated ratio of circa 7:1). It has been reported that approximately 78% of dietary L and Z is sourced from vegetables, with L found in highest concentrations in dark green leafy vegetables (including spinach, broccoli, kale, and collard greens) [52]. However, as most current dietary databases report intakes of L and Z combined, it has been difficult to assess the relative intakes and respective roles of the individual macular carotenoids at the macula. Recently, however, a study by Perry et al. did report concentrations of L and Z separately within the major food sources, as determined by the National Health and Nutrition Examination Survey (NHANES). In their study, they confirmed that green leafy vegetables were the richest source of L (e.g., cooked spinach and kale), whereas corn and corn products were confirmed as being a major source of Z [53]. Eggs are also a good source of L and Z, especially given their reported high bioavailability of L and Z when consumed in a normal diet [53, 54].

It appears that humans ingest relatively low levels of MZ (if any); however, research is ongoing in this area given the recent interest in this centrally located macular carotenoid. To date, there has been no exhaustive assessment of the amounts of MZ in a normal diet. However, eggs from hens fed MZ are known to be a rich human dietary source [55]. Also, a study by Maoka et al. in 1986 reported that MZ and Z are present in 21 species of edible fish, shrimp, and sea turtles [56]. The presence of MZ in the serum of unsupplemented individuals has never been demonstrated unambiguously. Efforts to extract and quantify MZ in human blood have demonstrated that if it is present, the concentrations are low [57]. Interestingly, in spite of its absence or low concentrations in a normal diet, MZ accounts for about one third of total MP at the macula, consistent with the hypothesis that retinal MZ is produced primarily by isomerization of retinal L at the macula [6, 58].

30.5.2Macular Carotenoids: The Origins of Macular Pigment

The macula lutea (yellow spot) was first identified more than two centuries ago. In 1792, Buzzi first described it in the human eye [59], and later in 1795 Soemmering independently discovered the foramine centrali limbo luteo (the central yellow-edged hole) [60]. The first review on “macular yellow” was published by Home [61], which began an era of investigation into the composition, and function, of what has become known as macular MP [62], a term first coined in 1933 by Walls [63]. The hypothesis that this pigment provides protection against the damaging effects of short-wavelength visible light was first proposed by Schultze [64], and its function was further discussed in a series of studies in the early twentieth century [63, 65–67].

In 1945, Wald demonstrated the spectral sensitivity of MP (using a spectral adaptometer), indicating that it had a characteristic carotenoid absorption spectrum and belonged to a family of xanthophylls found in green leaves [68]. However, it was not until as recent as 1985 that Bone and Landrum first proposed that the pigment was composed of the carotenoids, L and Z [7], and this was later confirmed in 1988 by Handelman et al. [69]. MZ was later identified as being the third carotenoid present in the central retina, where it is the dominant carotenoid at the epicenter of the macula [70]. Bone et al. proposed that MZ was primarily formed at the macula following conversion from retinal L [6], and this has subsequently been confirmed [58, 71, 72].

30.5.3The Functions of the Macular Carotenoids as Macular Pigment for AMD

The putative protective role of MP for AMD rests on at least one of the two following properties of this pigment. First, its absorbance spectrum (peak absorption of this pigment is 460 nm), and therefore its ability to filter (damaging) short-wavelength light. Second, the ability of the macular carotenoids to quench ROIs, referred to as antioxidant capacity (Fig. 30.2).

30.5.4Short-Wavelength Light Filtration

Although almost all UV-B (290–320 nm) and UV-A (320–400 nm) light is absorbed by the cornea and lens, blue light of slightly longer wavelength (400–520 nm) passes through the anterior media, and irradiates the macula [73]. Given that the peak absorption of MP is at 460 nm [68], it has the ideal light filtration properties to screen short-wavelength light prereceptorally. This allows MP to attenuate the amount of blue light incident upon the central retina.

L is reported to be a superior filter of blue light when compared to Z, due to its orientation with respect to the plane of the phospholipid bilayer of the cell membrane [74], which is both parallel and perpendicular. In contrast, Z and MZ only exhibit perpendicular orientation to this layer. However, it is important to note that the

Fig. 30.2 Illustration showing (a) a macula without macular pigment and at increased risk of oxidative stress with increased number of free radicals and a consequential damaged photoreceptor layer, and (b) a macula with macular pigment and therefore less free radicals produced as a result of blue light irradiation at the macula. Also, the free radicals produced directly from oxidative stress are neutralized by the macular carotenoids because of their antioxidant activity