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212 Part 2: Biotechnology and EnzymologyMRP-rich diets. Similar results were found in a crossover study
carried out by Wittmann et al. (2001) on 21 healthy volunteers,
where those on the AGE-rich diet gained more weight. In this
study, 21 healthy volunteers were fed on either an AGE-rich or
AGE-poor diet of heated or unheated high protein (3 g/kg/day).
However, to date, the majority of knowledge on the effects of
AGE-rich diets is mainly based on experimental studies of ro-
dents rather than human studies.
In contrast, Ames (2007) supports the idea that dietary AGEs
are not a risk to human health and provides evidence to support
this claim based on information obtained from both animal and
human volunteer studies on the bioavailability and metabolic
fate of dietary AGEs. The majority of the literature to date on
the bioavailability and metabolic fate of AGEs is centred on the
compounds CML and pyrraline, due to their abundance in food
systems. Various studies have investigated the metabolic fate of
dietary CML. One such study involved feeding rats on a diet of
glycated casein (either 1.8 g of CML/kg or 6.2 g of CML/kg)
against the control of native casein (Faist et al. 2000). The results
found that approximately 50% of the initial CML consumed was
excreted through urine and faeces, with a small portion being
deposited on the kidney and liver. Although the majority of
the CML consumed was readily excreted, further investigations
would need to be carried out to determine the metabolic fate
of the rest of the CML, with suggestions that it could have
been degraded by the colonic microflora or metabolised post-
absorption. Another study looked at distribution and elimination
of AGEs, including CML, CEL (Ne-carboxyethyllysine) and
lysine (Bergmann et al. 2001). These AGEs were studied via
fluorine-18-labelled analogues by [18F]-fluorobenzoylation of
theα-amino group of the AGE and then intravenously injected
into the tail vein of rats, which were subsequently sacrificed
within 30 minutes of the injection. The results obtained showed
all three compounds (CML, CEL and lysine) to be distributed
swiftly through the body and be rapidly excreted via the kidneys
with>87% of the radioactivity being excreted within 2 hours of
injection (Bergmann et al. 2001).
Many studies have been carried out investigating the distri-
bution and elimination of the other abundant AGE, pyrraline,
which is excreted via the kidneys within 48 hours (Forster and ̈
Henle 2003). Other studies on excretion of pentosidine, another
MRP, in the body, as well pyrraline and fructoselysine (Miyata
et al. 1998, Forster et al. 2005) have found significant differences ̈
in the excretion rate of the individual MRPs, according to indi-
vidual metabolic fates and whether the MRPs are free or protein
bound. However, the weight of evidence appears to suggest that
the majority of the ARPs and AGEs that are absorbed by the body
are subsequently, rapidly excreted and are therefore considered
not to be harmful to the body. Seiquer et al. (2008) carried out
experiments on healthy adolescent males by feeding some of
the participants a MRP-rich diet and the others a low MRP diet
over a two-week period. Surprisingly, they found there was no
obvious damage to the oxidative status or antioxidant defence
in the MRP-rich diet participants. Lindenmeier et al. (2002) and
Somoza et al. (2005) have suggested beneficial effects of AGEs.
Definitive conclusions await detailed research on the bioactivity
of dietary AGEs (Lopez-Garcia et al. 2004).Ames (2009) suggests there needs to be a more holistic ap-
proach to the research of MRPs. The balance of evidence, based
on many centuries of eating cooked food, is that MRPs/AGEs are
not harmful, but further studies should be done to eliminate the
possibility and provide reassurance to food manufacturers and
consumers. An interdisciplinary approach incorporating food
science, biology, chemistry and medicine is required for a bet-
ter understanding into the biological consequences of thermally
processed foods and more carefully defined human trials should
resolve this. Food manufacturers wishing to harness the Mail-
lard reaction for protein cross-linking or other uses are advised
to keep a watching brief on this research.MELANOIDINS
Very advanced glycation end-products that form as a result of
food processing are dubbed melanoidins. This is a structurally di-
verse class of compounds, which until recently were very poorly
characterised. However, a subset of food melanoidins undoubt-
edly includes those that cross-link proteins.
Melanoidins can have molecular weights of up to 100,000
Da (Hofmann 1998a). Because of their sheer chemical com-
plexity, it has been difficult to isolate and characterise these
molecules (Fayle and Gerrard 2002, Miller and Gerrard, 2005).
However, some recent work in this area has lead to some new
hypotheses. Hofmann proposed that proteins may play an im-
portant role in the formation of these complex, high-molecular
weight melanoidins (Hofmann 1998b). Thus, it was proposed
that a low-molecular weight carbohydrate-derived colourant re-
acts with protein-bound lysine and/or arginine, forming a pro-
tein cross-link. Formation of colour following polymerisation of
protein has indeed been observed following incubation with car-
bohydrates (Cho et al. 1984, 1986a, 1986b, Okitani et al. 1984).
Hofmann tested this hypothesis by the reaction of casein with a
pentose-derived intermediate furan-2-carboxaldehyde and sub-
sequent isolation of a melanoidin-type colourant compound from
this reaction mixture (Figure 10.3). Although non-cross-linking
in nature, the results encouraged further studies to isolate protein
cross-links that are melanoidins, and possible deconvolution of
the chemistry of melanoidin from these data. Further studies
by Hofmann (1998a) proposed a cross-link structure BISARG,
which was formed on reaction ofN-protected arginine with
glyoxal and furan-2-carboxaldehyde (Figure 10.3). This cross-
link was proposed as plausible in food systems due to the large
amount of protein, furan-2-carboxaldehyde and glyoxal that may
be present in food (Hofmann 1998a).
A lysine–lysine radical cation cross-link, CROSSPY (Fig-
ure 10.3C), has been isolated from a model protein cross-link
system involving bovine serum albumin and glycoladehyde, fol-
lowed by thermal treatment (Hofmann et al. 1999). CROSSPY
was also shown to form from glyoxal, but only in the presence
of ascorbic acid, supporting the suggestion that reductones are
able to initiate radical cation mechanisms resulting in cross-links
(Hofmann et al. 1999). EPR spectroscopy of dark-coloured bread
crust revealed results that suggested CROSSPY was most likely
associated with the browning bread crust (Hofmann et al. 1999).