Carotenoids in Lipid Membranes - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 23

lipids or cholesterol, resulted in decreases in the incorporation rate of both carotenoids (Socaciu
et al., 2000). Similar differences in the incorporation rate have been observed in the lipid mixture
based on SBPC and cholesterol: 80% incorporation in the case of zeaxanthin and 64% in the case of
β -carotene (Grolier et al., 1992). The lower initial pigment concentration (2.5 mol% with respect to

DPPC) resulted in higher incorporation rate in the case of zeaxanthin (ca. 92%) but lower in the case
of

-carotene (ca. 28%) (Socaciu et al., 1999). Moreover, the similar nonlinear relationship between
the initial concentration and the incorporated carotenoid fraction has been shown for zeaxanthin
in the liposomes formed with EYPC (Lazrak et al., 1987). Interestingly the incorporation rate of
zeaxanthin was found to be higher in the case of the liposomes formed with DMPC as compared to
DPPC but the opposite effect has been observed in a longer zeaxanthin homologue, decaprenozeax-
anthin (Lazrak et al., 1987). Such a i nding clearly indicates the importance of the match, between
the distance separating the polar groups of the carotenoid and the thickness of the hydrophobic core
of the bilayer, in incorporation of polar carotenoids into the lipid membranes.

β

2.2.4 S OLUBILITY
The miscibility of carotenoids and lipids within the membrane represents a kind of two-dimensional
solubility of pigment molecules within the lipid bilayer. The lateral diffusion of the pigment mol-
ecules incorporated can cause an aggregation. Owing to the fact that the carotenoid backbone is a
polyene chain, characterized by the conjugated double bond system, the molecules readily polarize
and bind to each other by van der Waals interactions. Carotenoid aggregation in the lipid phase
restricts their effect with respect to the membrane. It appears that even at the low molar fractions of
the pigments with respect to lipid, despite the efi cient incorporation rate, carotenoids form molecu-
lar aggregates in the membranes. Pigment aggregation is associated with dipole-dipole interactions
responsible for the excitonic splitting of the electronic energy levels (Kasha et al., 1965; Parkash
et al., 1998). The splitting results in the hypsochromic or/and the bathochromic spectral shift(s),
depend on the actual structure formed. The analysis of the shifts in the electronic absorption spectra
has been applied to investigate the process of carotenoid aggregate formation, both in the water envi-
ronment and in the lipid membranes (Gruszecki et al., 1999; Kolev and Kafalieva, 1986; Mendelsohn
and Van Holten, 1979; Sujak et al., 2000, 2005). Figure 2.3 presents the temperature dependency
of the absorption spectrum of zeaxanthin incorporated into the liposomes formed with DPPC. As
can be seen, even at relatively low pigment concentration (the initial concentration used for the lipo-
some preparation 5 mol%) the zeaxanthin absorption spectrum is different from the characteristic
absorption spectrum of the monomeric form and is elevated in the short-wavelength spectral region.
Lowering of the temperature results in abrupt spectral changes that accompany the L α → phase

transition of the membranes formed with DPPC (~41°C). According to the absorption spectrum,
below the transition temperature the carotenoid exists entirely in the aggregated form within the
membrane, despite relatively low concentration. This is demonstrated by the hypsochromic shift of
the main absorption maximum to 385 nm and by the loss of the i ne vibrational substructure (Sujak
et al., 2000, 2002). The miscibility threshold of the same system (zeaxanthin in DPPC liposomes)
has been determined as 29 and 6 mol% in the l uid phase and the crystalline phase of the mem-
brane, respectively, by differential scanning calorimetric experiments (Kolev and Kafalieva, 1986).
The comparison of these i ndings with the spectral analysis shows that the pigment aggregates can
have similar effects on the membrane properties to those of monomers and therefore one has to be
very cautious in concluding a carotenoid miscibility on the basis of different experimental tech-
niques. A monomolecular layer approach seems to provide a good system to study carotenoid-lipid
miscibility because the analysis of molecular area in a monolayer, in terms of the additivity rule,
is very sensitive to the phenomenon of perfect miscibility, poor miscibility, and phase separation
(Gruszecki et al., 1999; Milanowska et al., 2003; N'soukpoe-Kossi et al., 1988; Sujak and Gruszecki
2000; Sujak et al., 2007a). The comparative monomolecular layer study of the organization of
DPPC membranes containing the xanthophyll pigments, zeaxanthin and lutein, shows pronounced

P β ′