**8. Association of carotenoids with plasma lipoproteins**

The majority of plasma carotenoids are transported on LDL, with 55% of total carotenoids associated with this lipoprotein, whereas HDL is associated with 33%, and VLDL is associated with 10-19%, of the total carotenoids.[55] However, in the case of the hydroxycarotenoids, L and Z, some studies have reported that they are relatively equally distributed between LDL and HDL molecules, but other studies have reported that HDL is the preferential carrier of the MP carotenoids in plasma.[56;57]

MP is inversely related to percentage body fat.[58] Interestingly, Viroonudomphol *et al.* have demonstrated lower levels of HDL in overweight and obese subjects, consistent with the possibility that a relative lack of HDL may impair transport and/or retinal capture of the carotenoids.[59] Furthermore, Seddon and co-workers have demonstrated a significantly increased risk of AMD in association with obesity.[33] These findings have prompted the suggestion that an individual's lipoprotein, and apolipoprotein, profile may influence the transport and delivery of these carotenoids to the retina, with a consequential impact on MP.

A recent study, designed to investigate the respective relationships between lipoprotein profile, MP optical density and serum concentrations of L and Z, was conducted in 302 healthy adult subjects.[60] This study found that there was a statistically significant inverse association between serum triglyceride concentration and MP optical density, and an inverse association between serum triglyceride concentration and serum L concentration in subjects with a positive family history of AMD. There have been no previous reports on the association between serum triglyceride concentration and either MP optical density or serum concentrations of L and/or Z. Elevated serum triglyceride concentration is an element of an undesirable lipoprotein profile and represents risk for cardiovascular disease.[61;62] Since there is an inverse association between serum triglyceride concentration and serum HDL concentration,[62] one could expect an inverse association between serum triglyceride concentration and serum L, since HDL appears to be the most important lipoprotein involved in the transport of L in serum. This expected inverse association was observed in subjects with a positive family history of AMD. In this study sample there was a positive and significant association between serum HDL concentration (and serum cholesterol concentration) and serum L and Z concentrations. Of note, there was no significant association observed between MP optical density and either serum cholesterol concentration or serum HDL concentration. There was also no association between serum LDL concentration and MP optical density (or serum concentrations of its constituent carotenoids). These findings suggest that a desirable lipoprotein profile (higher serum HDL, lower serum LDL and lower serum triglyceride concentrations) is associated with greater serum L concentration. However, the impact of lipoprotein profile on the capture and/or stabilization of these carotenoids at the macula, where they comprise MP, is less clear from this data.

364 Lipoproteins – Role in Health and Diseases

Subsequently, the liver synthesises a second class of triglyceride-rich lipoprotein, referred to as VLDL, which, upon secretion, functions as a transporter of lipids and cholesterol. In the bloodstream, VLDL undergoes progressive removal of triglycerides from its core by lipoprotein lipase, in a similar way to chylomicrons. The VLDL particles thus become increasingly smaller, leading to the formation of IDL, and LDL. LDL are the final metabolic products of VLDL and are responsible for most of the cholesterol transport in serum.[53]

HDL are the smallest lipoproteins, arising from several sources including the intestine and liver. HDL are involved in a process known as 'reverse cholesterol transport', whereby HDL acquire cholesterol from cells and deliver it to the liver.[53] This is a particularly important mechanism in humans, as the quantities of cholesterol transported out of the gut and liver far exceed the quantities converted to steroid hormones, or those lost through the skin in sebum. Thus, unless the requirement for cell membrane repair or synthesis is high, excess

The majority of plasma carotenoids are transported on LDL, with 55% of total carotenoids associated with this lipoprotein, whereas HDL is associated with 33%, and VLDL is associated with 10-19%, of the total carotenoids.[55] However, in the case of the hydroxycarotenoids, L and Z, some studies have reported that they are relatively equally distributed between LDL and HDL molecules, but other studies have reported that HDL is the

MP is inversely related to percentage body fat.[58] Interestingly, Viroonudomphol *et al.* have demonstrated lower levels of HDL in overweight and obese subjects, consistent with the possibility that a relative lack of HDL may impair transport and/or retinal capture of the carotenoids.[59] Furthermore, Seddon and co-workers have demonstrated a significantly increased risk of AMD in association with obesity.[33] These findings have prompted the suggestion that an individual's lipoprotein, and apolipoprotein, profile may influence the transport and delivery of these carotenoids to the retina, with a

A recent study, designed to investigate the respective relationships between lipoprotein profile, MP optical density and serum concentrations of L and Z, was conducted in 302 healthy adult subjects.[60] This study found that there was a statistically significant inverse association between serum triglyceride concentration and MP optical density, and an inverse association between serum triglyceride concentration and serum L concentration in subjects with a positive family history of AMD. There have been no previous reports on the association between serum triglyceride concentration and either MP optical density or serum concentrations of L and/or Z. Elevated serum triglyceride concentration is an element of an undesirable lipoprotein profile and represents risk for cardiovascular disease.[61;62] Since there is an inverse association between serum triglyceride concentration and serum HDL concentration,[62] one could expect an inverse association between serum triglyceride concentration and serum L, since HDL appears to be the most important lipoprotein

cholesterol must be returned to the liver for excretion.[54]

preferential carrier of the MP carotenoids in plasma.[56;57]

consequential impact on MP.

**8. Association of carotenoids with plasma lipoproteins** 

In this study, the lipoprotein particle-concentration of L and/or Z in serum was not directly measured, nor were lipoprotein subspecies measured, as performed by Goulinet *et al*.[57] In their study, they fractionated HDL and LDL subspecies on the basis of their hydrated density by gradient ultracentrifugation, and they found that serum L and Z (combined) were relatively equally distributed between HDL and LDL; but more importantly, they found that there was a progressive decrease in the concentration of these carotenoids with increasing density (and decreasing lipoprotein particle size) from light to dense LDL. They also found that the majority of macular carotenoid transport by LDL was accounted for by the most abundant subspecies, LDL3 (intermediate LDL) and LDL4 (dense LDL). This is highly relevant to the transport of L and Z in serum, as LDL3 and LDL4, despite being the most abundant subspecies of LDL in that study, had reduced particle-concentrations of these carotenoids compared to less dense LDL subspecies, making them more vulnerable to oxidation.[63] LDL is the primary component of total cholesterol,[62] and has previously been reported in various studies to transport between 22-44% of L and Z in serum.[55;57;64- 66] Of note, it has been shown that there is no significant difference in the transport of L and Z by lipoproteins between subjects with and without AMD.[65]

The findings of Goulinet *et al* in relation to HDL were similar to that of LDL, in that there was a progressive and marked decrease in HDL particle concentration of L and Z, with maximal carotenoid concentration evident in the lightest, largest HDL subspecies (HDL2-1), and minimal concentration in the densest HDL. Certainly, the findings of Goulinet *et al* with respect to HDL, in concert with our findings, are consistent with the view that HDL plays an important role in the transport of L and Z in human serum, and are provocative given that AMD and cardiovascular disease share certain antecedants.[32;57;60;67-70] Furthermore, and again consistent with a shared pathogenesis between AMD and cardiovascular disease, the finding of an inverse association between serum triglyceride concentration and MP optical density (and between serum triglyceride concentration and serum L concentration) in subjects with a positive family history of AMD, is noteworthy.[60] Since AMD has been shown to be associated with low serum concentrations of L,[71] and given that risk factors for AMD are associated with a relative lack of MP,[31] our observations are yet another example of how AMD and cardiovascular disease share risk factors.[32;60-62;67-70]

In 2007, Connor *et al* reported on the role that HDL plays in the transport of L and Z in serum in a study involving WHAM chicks.[64] WHAM chickens have a recessive sex-linked mutation in the *ABCA1* transporter gene that results in very low circulating HDL concentration, with normal, or increased, concentrations of other plasma lipoproteins, particularly LDL. The analogous mutation in humans results in Tangier disease, which is characterized by a similar deficiency in circulating HDL concentration.[72] In their study, involving 24 WHAM chicks and 24 control chicks, Connor *et al* found that one-day old WHAM chicks had only 9% of the L concentration in plasma when compared with control chicks, and only 6% of the retinal concentration of controls (the corresponding concentrations of Z were 6% and 9%, respectively). Following a high-L diet for 28 days, there was a significant increase in the plasma and retinal concentrations of L in WHAM chicks and controls, but the increases were still greatly inferior in the WHAM chicks when compared with control chicks and, furthermore, still did not reach the concentrations observed in the one-day old control chicks. The observations of Connor *et al* suggest an important role for HDL in the transport of L and Z in serum and/or their incorporation into the retina, and are consistent with our findings.[60;64]

Interestingly, although all subjects in our study were healthy volunteers with no evidence of ocular pathology, it is notable that, on average, subjects with a positive family history of AMD had a higher serum concentration of L than subjects with a negative family history of AMD, yet MP optical density levels in both groups were comparable, as were serum concentrations of HDL.[60] As was shown in this study, and as has previously been documented,[73] serum concentrations of L and Z generally correlate positively with MP optical density. Therefore, it is plausible to suggest that in the subjects in this study with a positive family history of AMD, the delivery to, and/or uptake by, the retina of the macular carotenoids is defective when compared to subjects without such a family history.[60] Indeed, although MP optical density levels were comparable between subjects with and without a family history of AMD, subjects with a positive family history of this disease also had higher serum L concentrations. This is consistent with the observations of Nolan *et al*, where a relative lack of MP was seen in association with a positive family history of AMD in 828 healthy subjects, but where dietary and serum concentrations of L and Z were comparable for subjects with and without a family history of this condition, suggesting defective retinal capture of circulating L and/or Z in persons who are genetically predisposed to AMD.[31] Mechanisms governing the retinal capture and/or stabilization of L and/or Z may be subject to influence by HDL subspecies profile, by affecting receptormediated uptake of these carotenoids from serum. Indeed, apolipoprotein profile is probably a determinant of retinal uptake of the macular carotenoids from serum, reflected in our recently reported finding that individuals with at least one Apo ε4 allele exhibit significantly higher MP optical density than individuals without this protective allele, despite statistically comparable serum concentrations of L and Z.[74] Interestingly, the lack of an association between MP optical density and either serum cholesterol concentration or serum HDL concentration in our study would suggest that our observations are more likely due to impaired uptake and/or stabilization of circulating L and/or Z by the macula than due to any impact the HDL subspecies profile may have on the transport of the macular carotenoids in serum.

Another recent study has shown somewhat conflicting evidence regarding the association between circulating lipoprotein levels and MP levels in serum and in the macula.[75] These differences may be attributable to differences in the methods used to measure serum lipoproteins, although it should be noted that this study also found a positive association between serum L and serum HDL levels, underscoring the importance of HDL as a transporter of L in serum. However, it should be emphasised that a notable paucity of data still remains regarding the mechanism(s) whereby L and Z accumulate in the liver, are repackaged into lipoproteins, and transported via the circulatory system to specific target tissues such as the retina.
