Bioavailability of lutein from a lutein-enriched egg-yolk-beverage and its dried re-suspended versions.

Age-Related Macular Degeneration (AMD, both wet and dry form) is a degenerative
eye disease and the leading cause of irreversible visual loss in the elderly (>
60years) in the Western world. This disease of the elderly robs them of central
vision in one or both eyes. People who are affected by AMD have problems
reading, driving and performing activities that require clear central vision
(Bernstein Paul, Bhosale Prakash et al. 2008). Recent studies have estimated
the current prevalence of AMD from around 1.47% (Friedman, O'Colmain et al.
2004) in the United States, to as high as 3.5% in United Kingdom (Wang,
Rochtchina et al. 2007). More than half of the registered blindness (54.5%) is
due to degeneration at the macula.

Macular pigment is composed primarily of the xanthophylls lutein and
zeaxanthin, members of the carotenoid family. The current believe is that
lutein and zeaxanthin can help in the prevention of AMD by absorbing blue light
and protecting the retina from oxidative stress by neutralizing free radicals
(Landrum, Bone et al. 1997; Bernstein, Khachik et al. 2001; Landrum and Bone
2001; O'Connell, Neelam et al. 2006). Epidemiological evidence indicates that a
higher intake of lutein and zeaxanthin is associated with a lower risk on AMD
(Moeller, Parekh et al. 2006).

Eggs are among other foods an important source of lutein. Moreover, it is
possible to increase the natural lutein concentration in egg yolk
(lutein-enriched eggs =>macular eggs) (Thielen Wilhelmus Johannes, Berendschot
Toussaint Theresia et al.), which creates opportunities to further increase
lutein intake when needed, as has been suggested for subjects suffering from
age-related macula degeneration (AMD).

In earlier studies, it has been proven that the lipid matrix of the egg yolk is
a vehicle for the efficient absorption of dietary lutein (Granado, Olmedilla et
al. 2003; Chung, Rasmussen et al. 2004; Leeson and Caston 2004; Calvo 2005) and
that it is possible to increase serum/ plasma levels of lutein and macula
lutein levels (Bone, Landrum et al. 1997).
Plat et al. showed in a pilot study (ClinicalTrials.gov Identifier:
NCT00527553) with healthy subjects that the consumption of lutein-enriched eggs
indeed increased plasma and macular levels of lutein when compared with
consumption of regular eggs (not yet published data, NWO-2010, voedings-dagen
and personal conversation). In that study they also evaluated the changes in
plasma and macular lutein concentrations when the egg was eaten boiled or when
the egg yolk was incorporated into a beverage, based on buttermilk.

However, this fresh lutein-enriched egg yolk beverage has a quite limited shelf
life of about two to three weeks (NIZO, product report E2010/089). Drying this
fresh beverage can extent shelf life, however, this should be performed without
loosing the functional properties concerning lutein bioavailability. The effect
of pasteurization, spray- and freeze-drying egg-yolks with respect to
xanthophyll stability has recently been investigated (Wenzel, Seuss-Baum et al.
2010). It was shown that freeze-drying as well as spray-drying (to 98.9% of dry
matter) resulted in a increased concentration per gram egg-yolk compared to
either fresh or pasteurized egg-yolks. Authors suggest this to happen, possibly
by an irreversible denaturation of the egg-yolk lipo-proteins, resulting in an
increased release of xanthophylls that had not been accessible for extraction
previously (Wenzel, Seuss-Baum et al. 2010).

The question remaining is, whether lutein bioavailability (increase in lutein
serum concentration) is affected by the different drying procedures.

The primary objective of this study is to investigate whether consumption of a
dried lutein-enriched egg-yolk containing beverage significantly increases
serum lutein concentration in healthy volunteers.
A secondary objective is to investigate whether the bioavailability of lutein
from a lutein-enriched egg-yolk containing beverage is comparable to its dried
version(s).

Research questions
Specific research questions for this proposal are:
1)Does the consumption of a dried lutein-enriched egg-yolk containing beverage
significantly increase serum lutein concentration in healthy volunteers?
2)Is the increase in lutein serum concentration from the dried re-suspended
lutein-enriched egg-yolk containing beverages comparable to the fresh beverage?
3)Is the consumption of the dried lutein-enriched egg-yolk containing beverage
well-accepted in healthy volunteers?

To answer the research questions the study will have a parallel design with
four groups of 25 subjects, consuming either the *plain* control beverage, the
fresh lutein-enriched egg-yolk beverage or two differently dried
lutein-enriched egg-yolk beverages for 6 weeks. (see also appendix 1)

All participants will start with a three weeks run-in period on a diet low in
lutein, as usually is done when studying lutein and other carotenoid serum
concentrations. During the whole study (run-in and intervention), subjects will
be asked to avoid lutein-rich vegetables and fruits (see Table 2). Furthermore,
no vitamin capsules are allowed during the whole study period.

After the run-in period, subjects will randomly assigned to either one of the
four groups, stratified for age and gender, for an additional 6 weeks. Blood
will be sampled at in total 6 different time points during the study, before
and after the run-in period, and after 6 weeks of interventions, at two
different in duplicate. The technician will be blinded for the lutein analysis.

The study will start with a three weeks run-in period for all participants, as
usually has been done when studying lutein and other carotenoid
bioavailability. During the whole study, subjects will be asked to avoid
lutein-rich vegetables and fruits such as spinach, broccoli, peas (see table 1
for the top 10 contributors to lutein intake in the Netherlands). No vitamin
capsules are allowed during the whole study period for the whole study period
of 9 weeks. After three weeks of run-in, subjects are randomly assigned to one
of the four groups
N Treatment
Control 25 *plain* beverage/ placebo
Treatment 1 25 fresh lutein-enriched egg-yolk beverage
Treatment 2 25 Dried-1 lutein-enriched egg-yolk beverage
Treatment 3 25 Dried-2 lutein-enriched egg-yolk beverage
.
Subjects are asked to consume either 1 bottle of 80 ml each day or an
equivalent dried version which they need to re-suspended in tapwater, in the
morning between 7 and 9am. Bottles or sachets will be provided every two weeks,
and subjects will be asked to return empty bottles or sachets to evaluate the
compliance of the volunteers.

There are no risks for the participants during the intervention as the
consumption of the beverage is not expected to have any associated with any
risk. Venapunctures can occasionally cause a local haematoma or bruise and some
participants may report pain or discomfort.
There is no direct (health-related) benefit for the participant.