Blood carotenoids

A concern has been raised that phytosterol doses that are effective for cholesterol reduction may impair the absorption and lower blood concentrations of fat-soluble vitamins and antioxidants. A number of studies showed that phytosterols had no effect on plasma concentrations of vitamin D, retinol, or plasma-lipid-standardized alpha-tocopherol. Moreover, the reports of the effect of phytosterols on concentrations of blood carotenoids (lutein, lycopene, and alpha-carotene) are controversial. There seems to be general agreement that phytosterol doses >1 g/d significantly decrease LDL-C standardized beta-carotene concentrations however, it remains to be determined whether a reported 15-20% reduction in beta-carotene due to phytosterol supplementation is associated with adverse health effects. Noakes et al. found that consumption of one or more carotenoid-rich vegetable or fruit servings a day was sufficient to prevent lowering of plasma carotenoid concentrations in 46 subjects with hypercholesterolemia treated with 2.3 g of either sterol or stanol esters. 

At the other end of the spectrum of serum values, populations with high mean serum carotenoids often are reported to have lower or no different mean retinol levels than found among populations with normal or low carotenoid values. This is illustrated in Table X by comparing data from surveys conducted in Senegal where 98% of the dietary vitamin A activity comes from carotenoids with surveys from the United States where about 50% is from preformed sources. It is difficult to determine if the high blood carotenoids are causally related to a real shift downward in the retinol distribution curve or if this is a methodologic artifact. Several of the colorimetric and fluorometric analytical methods for... 

Observational studies of carotenoids and chronic disease have examined the association between carotenoid intake and disease incidence, or between blood or tissue levels of carotenoids, and disease incidence. One type of observational study is prospective, with the carotenoid intake and/or status measured years before the diagnosis of disease. Another type of observational study is retrospective, wherein individuals with disease and comparable controls are asked to recall usual dietary intake prior to the development of the disease (cases) or prior to interview (controls). Retrospective studies of carotenoids and disease commonly rely on recalled dietary intake rather than blood carotenoid levels to assess exposure, as altered blood carotenoid status could easily be a consequence, rather than a cause, of the disease, particularly for diseases like cancer. 

Carotenoid and tocopherol concentrations in plasma, peripheral blood mononuclear cells and red blood cells after long-term beta-carotene supplementation in men. Am J Clin Nutr 63(4) 553-8. 

With investigations of phytochemicals and functional foods, the outcome measure is generally going to be a biomarker of disease, such as serum cholesterol level as a marker of heart disease risk, or indicators of bone turnover as markers of osteoporosis risk. Alternatively, markers of exposure may also indicate the benefit from a functional food by demonstrating bioavailability, such as increased serum levels of vitamins or carotenoids. Some components will be measurable in both ways. For instance, effects of a folic acid-fortified food could be measured via decrease in plasma homocysteine levels, or increase in red blood cell folate. 

In contrast, the carotenes such as p-carotene and lycopene may position themselves parallel to the membrane surfaces to remain in a more lipophilic environment in the inner cores of the bilayer membranes. To move through an aqueous environment, carotenoids can be incorporated into lipid particles such as mixed micelles in the gut lumen or lipoproteins in the blood circulation and they can also form complexes with proteins with unspecific or specific bindings. 

More than 600 carotenoids have been isolated from natural sources, but only about 60 have been detected in the human diet — about 20 in human blood and tissues. P-Carotene, a-carotene, lycopene, lutein, and P-cryptoxanthin are the five most prominent carotenoids present in the human body. 

In intestinal cells, carotenoids can be incorporated into CMs as intact molecules or metabolized into mainly retinol (or vitamin A), but also in retinoic acid and apoc-arotenals (see below for carotenoid cleavage reactions). These polar metabolites are directly secreted into the blood stream via the portal vein (Figure 3.2.2). Within intestinal cells, retinol can be also esterified into retinyl esters. 

Both intact carotenoids and their apolar metabolites (retinyl esters) are secreted into the lymphatic system associated with CMs. In the blood circulation, CM particles undergo lipolysis, catalyzed by a lipoprotein lipase, resulting in the formation of CM remnants that are quickly taken up by the liver. In the liver, the remnant-associated carotenoid can be either (1) metabolized into vitamin A and other metabolites, (2) stored, (3) secreted with the bile, or (4) repackaged and released with VLDL particles. In the bloodstream, VLDLs are transformed to LDLs, and then HDLs by delipidation and the carotenoids associated with the lipoprotein particles are finally distributed to extrahepatic tissues (Figure 3.2.2). Time-course studies focusing on carotenoid appearances in different lipoprotein fractions after ingestion showed that CM carotenoid levels peak early (4 to 8 hr) whereas LDL and HDL carotenoid levels reach peaks later (16 to 24 hr). 

Kaplan, L.A., Lau, J.M., and Stein, E.A., Carotenoid composition, concentrations, and relationship in various human organs, Clin. Physiol. Biochem., 8, 1, 1990. Parker, R.S., Carotenoids in human blood and tissues, J. Nutr, 119, 101, 1989. Clifford, M.N., Anthocyanins nature, occurrence, and dietary burden, J. Sci. Food Agric., 80, 1063, 2000. 

Experimental evidence in humans is based upon intervention studies with diets enriched in carotenoids or carotenoid-contaiifing foods. Oxidative stress biomarkers are measured in plasma or urine. The inhibition of low density lipoprotein (LDL) oxidation has been posmlated as one mechanism by which antioxidants may prevent the development of atherosclerosis. Since carotenoids are transported mainly via LDL in blood, testing the susceptibility of carotenoid-loaded LDL to oxidation is a common method of evaluating the antioxidant activities of carotenoids in vivo. This type of smdy is more precisely of the ex vivo type because LDLs are extracted from plasma in order to be tested in vitro for oxidative sensitivity after the subjects are given a special diet. 

Jyonouchi, H., Sun, S., and Gross, M., Effect of carotenoids on in vitro immunoglobulin production by human peripheral blood mononuclear cells astaxanthin, a carotenoid without vitamin A activity, enhances in vitro immunoglobulin production in response to a T-dependent stimulant and antigen, Nutr. Cancer, 23, 171, 1994. 

Sterlie, M, Bjerkeng, B., and Liaaen-Jensen, S., Blood appearance and distribution of astaxanthin E/Z isomers among plasma lipoproteins in humans administered a single meal with astaxanthin, m Abstracts of 12th International Carotenoid Symposium, Cairns, Australia, Abstract 2A-13, 1999, 72. 

Lee, H.S. and Castle, W.S., Seasonal changes of carotenoid pigments and color in Hamlin, Earlygold, and Budd blood orange juices, J. Agric. Food Chem., 49, 877, 2001. 

Carotenoids are also present in animals, including humans, where they are selectively absorbed from diet (Furr and Clark 1997). Because of their hydrophobic nature, carotenoids are located either in the lipid bilayer portion of membranes or form complexes with specific proteins, usually associated with membranes. In animals and humans, dietary carotenoids are transported in blood plasma as complexes with lipoproteins (Krinsky et al. 1958, Tso 1981) and accumulate in various organs and tissues (Parker 1989, Kaplan et al. 1990, Tanumihardjo et al. 1990, Schmitz et al. 1991, Khachik et al. 1998, Hata et al. 2000). The highest concentration of carotenoids can be found in the eye retina of primates. In the retina of the human eye, where two dipolar carotenoids, lutein and zeaxan-thin, selectively accumulate from blood plasma, this concentration can reach as high as 0.1-1.0mM (Snodderly et al. 1984, Landrum et al. 1999). It has been shown that in the retina, carotenoids are associated with lipid bilayer membranes (Sommerburg et al. 1999, Rapp et al. 2000) although, some macular carotenoids may be connected to specific membrane-bound proteins (Bernstein et al. 1997, Bhosale et al. 2004). 

Parker, R. S. 1989. Carotenoids in human blood and tissues. J. Nutr. 119 101-104. 

Lutein and zeaxanthin are the dominant carotenoids in nonretinal eye tissue, and lycopene and p-carotene have been found in the ciliary body, which after the retina and the retinal pigment epithelium (RPE) contains the highest quantity of carotenoids (Bernstein et al. 2001). The orbital adipose tissue also contains measurable quantities of lutein and p-carotene, and possibly other carotenoids as minor constituents (Sires et al. 2001). It is also interesting to note that lutein was recently identified in the vitreous body of human fetuses, 15-28 weeks old (Yakovleva et al. 2007). However, these results may have to be considered with caution, because the vitreous bodies were described as substantially being penetrated with hyaloid blood vessels, which could have contaminated the vitreous with blood. 

Carotenoids accumulating in the human body are obtained exclusively from our diet. Out of almost 50 carotenoids present in a typical human diet, about 14 are absorbed into the blood (Khachik et al., 1997), and only two of them—lutein and zeaxanthin (Figure 15.1)—accumulate in the retina (Bernstein et al., 2001 Bone and Landrum, 1992 Bone et al., 1988, 1997 Davies and Morland, 2004 Khachik et al., 1997, 2002). Lutein and zeaxanthin are particularly concentrated in photoreceptor axons and inner plexiform layer in the area including and surrounding... 

Interestingly, carotenoids more abundant in the blood plasma than zeaxanthin, such as lycopene, P-carotene, and P-cryptoxanthin, do not accumulate in the retina. RPE cells express p,p-carotene 15,15 -monooxygenase (BCO), formerly known as P-carotene 15,l5 -dioxygcnase, an enzyme that catalyzes the oxidative cleavage of P-carotene into two molecules of all-trans-retinal (Aleman et al., 2001 Bhatti et al., 2003 Chichili et al., 2005 Leuenberger et al., 2001 Lindqvist and Andersson, 2002). Therefore it may be suggested that p -carotene transported into RPE-cells is efficiently cleaved into retinal molecules. BCO cleaves also P-cryptoxanthin (Lindqvist and Andersson, 2002), and its absence in the retina may also be explained by its efficient cleavage to retinoids. However, lycopene, often the most abundant carotenoid in human plasma, cannot serve as a substrate for BCO, and yet it is not detectable in the neural retina (Khachik et al., 2002). 

Recent data indicate that SR-BI is a nonspecific receptor for many lipophilic molecules (Lorenzi et al., 2008 Reboul et al., 2007b). Apart from HDLs, rodent SR-BI also binds to LDL, VLDL, acetylated LDL, oxidized LDL, and maleylated bovine serum albumin. SR-BII has a similar ligand specificity and function to that of SR-BI (Webb et al., 1998). However, it has been shown that vitamin E (which like carotenoids is carried in the bloodstream mainly by LDL and HDL) is transported more efficiently into the endothelial cells from HDLs than from LDLs (Balazs et al., 2004 Kaempf-Rotzoll et al., 2003 Mardones and Rigotti, 2004). This is in striking contrast to cholesterol, which is taken up much more efficiently from LDLs than HDLs by the RPE to the retina (Tserentsoodol et al., 2006b). It remains to be shown which lipoproteins are the main carriers for carotenoids transported from blood into the RPE. 

The expression of all these apo-lipoproteins by the RPE, and its ability to form lipoprotein particles suggest that these newly formed lipoproteins may be involved in the transport of lipophilic molecules, including carotenoids, from the RPE to the neural retina and/or to the choroidal blood supply. Testing the roles of apolipoproteins and lipoprotein particles in carotenoid secretion from the RPE is another subject awaiting experimental investigation. 

While it may be speculated that in the RPE both lipoprotein and/or scavenger receptors are likely to be involved in carotenoid uptake from the blood, it is not clear what mechanism(s) are responsible for carotenoid transport through the RPE into the neural retina. Also, it is not clear what mechanism(s) are responsible for selective accumulation in the retina of only two carotenoids. 

Apart from SR-BI, SR-BII, CD36, and ABCA1, a microarray analysis of gene expression in human RPE reveals some additional lipid transporters that might potentially be involved in intracellular transport of carotenoids and/or their efflux from the RPE cells into the neural retina or out of the retina into the choroidal blood (van Soest et al., 2007). These include other ABC... 

FIGURE 15.3 Hypothetical pathways responsible for carotenoid uptake, metabolic transformations, tran-scytosis to the neural retin, or secretion to the blood. 

Despite the feasibility of using cultured RPE cells for studies similar to those performed using Caco-2 cells, the role of the RPE in carotenoid uptake and dynamic regulation has only just begun to be investigated. As carotenoids are carried in blood by lipoproteins, lipoprotein-rich serum seems to be the most appropriate vehicle for carotenoid delivery to cultured RPE cells. Indeed, recent studies comparing carotenoid delivery from fetal calf serum and from organic solvents showed that delivery in the presence of serum was superior to tetrahydrofuran (Shafaa et al., 2007). 

So far, we have focused mainly on the potential pathways of carotenoid uptake by the RPE from the choroidal blood supply. As mentioned earlier, POS contain lutein and zeaxanthin, and their distal tips are phagocytosed by the RPE. Therefore POS is another source of xanthophylls in the RPE and this pathway of carotenoid delivery and their further fate can be easily tested in cultured RPE. It has been shown that exposure of RPE cells in vitro to HDL stimulates efflux of phospholipids from phagocytosed POS out of the cell (Ishida et al., 2006). Thus it is of interest to determine whether that transport may potentially include xanthophylls and whether other types of lipoproteins may... 

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