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Editorial
Hepatic clearance of advanced glycation end products(AGEs)—myth or truth?
Dmitri Svistounov, Bard Smedsrød*
Department of Experimental Pathology, Institute of Medical Biology, University of Tromsø, N-9037 Tromsø, Norway
0168-8278/$30.00 q 2004 European Association for the
doi:10.1016/j.jhep.2004.10.004
* Corresponding author.
E-mail address: baards@fagmed.uit.no (B. Smedsrød
See Article, pages 913-919
The liver sequesters a number of circulating macromol-
ecular soluble and particular waste products. This blood
clearance function is carried out by the cells that line the
sinusoidal wall: (i) the resident liver macrophages, or the
Kupffer cells (KC), and (ii) the liver sinusoidal endothelial
cells (LSEC). The KC is tuned to phagocytic uptake of large
particles and aggregates, whereas LSEC are specialized on
clathrin mediated endocytosis of soluble macromolecules
and colloids. Until now four major endocytosis receptors
have been observed to mediate waste endocytosis in LSEC.
The mannose receptor, the scavenger receptor (several types
are expressed by LSEC; the hyaluronan/scavenger receptor,
or Stabilin-2 [1] is uniquely expressed and functionally
active in LSEC), the collagen a chain receptor, and the Fc-greceptor. The waste macromolecules that are cleared by
these receptors in LSEC include (i) most types of connective
tissue molecules that are constantly released to the
circulation as a consequence of normal turnover processes
throughout the body [2]; (ii) extracellular enzymes and
products of platelet-mediated coagulation [3]; (iii) intra-
cellular macromolecules (for instance lysosomal enzymes
[4]); (iv) soluble IgG-immune complexes [5]; (v) native
macromolecules that have been modified non-enzymatically
by for instance oxidation (oxidized low density lipoprotein
[6]) or glycation; (vi) foreign molecules (i.e. LPS [7]).
The capacity of endocytosis in LSEC is impressive: some
of the waste macromolecules are turned over in quantities of
several grams per day in a normal adult human individual.
The process of endocytosis in these cells is the most efficient
known: endocytic receptors recycle between the plasma
membrane and early endosomes with a half-life of a few
seconds (most other cell types perform endocytosis with
Study of the Liver. Pub
).
a receptor recycling half-life of minutes). Most of the waste
substances that are destined for uptake via receptor-
mediated endocytosis in LSEC exist for a very short time
in the circulation (less than 1 min in rats).
During the past three decades several groups have
attempted to design experiments to study the fate of
circulating advanced glycation end products (AGEs).
Macromolecules modified in this way are present in normal
individuals throughout life, and are found in higher
concentrations in older people as well as in certain diseases,
most typically diabetes. Available data suggest that AGEs
are eliminated from the blood mainly by scavenger receptor
mediated uptake in KC and LSEC [8]. Alternative
hypotheses hold that the uptake is not necessarily in the
liver, but in the kidneys. One line of research (hereafter
referred to as ‘the in vitro approach’) makes use of in vitro
generated AGEs, that can be labelled and chased after
administration in vivo. Another approach (hereafter referred
to as ‘the in vivo approach’) is based on the notion that
chemical analysis of blood samples, with no prior
administration of in vitro generated AGEs, represents the
key to solve the problem [9,10]. According to the in vivo
approach it would be sufficient to perform sensitive
chemical analyses to check if and to what extent AGEs
are removed by any given tissue. As discussed below these
approaches have distinct strong and weak sides; neither of
them represents a perfect approach to determine the
anatomical site of uptake of circulating AGEs. In vitro
generation of AGEs involves long time (weeks or months)
incubation of protein with glucose or other AGEs precursors
under aseptic conditions. The in vitro approach offers the
possibility of labelling AGEs with high specific radioac-
tivity, enabling very low amounts of AGEs to be chased in
vivo after i.v. administration. The weak side of the in vitro
approach is that we do not know to what extent AGEs
prepared in vitro represent the ‘native’ in vivo generated
Journal of Hepatology 41 (2004) 1038–1040
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D. Svistounov, B. Smedsrød / Journal of Hepatology 41 (2004) 1038–1040 1039
AGEs. One knows for certain that the AGE adducts that are
present in in vitro generated AGEs are also detected on
AGEs formed in vivo. But it is unlikely that the extent of
AGE modification is as high in the in vivo formed specimen
as in the in vitro generated molecules. Using the in vitro
approach it was found that i.v. administered AGEs are very
rapidly taken up in KC and LSEC [8].
The advantage of the in vivo approach is that only native
AGEs are measured. However, using only the in vivo
approach one will not be able to detect the most interesting
AGEs, namely those that are rapidly cleared from the
circulation. Even the most sensitive analytical tools
presently available will not be able to show significant
differences in the concentration of AGEs in blood samples
taken from the portal and hepatic veins, simply because the
speed and efficiency of uptake greatly exceed the rate of
AGEs formation. The speed of blood circulation must also
be considered: in average the recycling time for blood
through liver in humans is 3.6 min, meaning that all the
blood contents are monitored by the liver scavenger
receptors every few minutes. This would make it impossible
for AGEs modified to a ‘high physiological degree’ to
accumulate to a detectable level above the background in
the blood. Moreover, the natural formation of AGEs in the
blood is certainly slower than a few minutes. One of the
authors (P.J.T.) of the presently discussed article previously
used a 24 h incubation schedule to prepare CML-albumin
and methylglyoxal-derived hydroimidazolone-proteins (two
AGEs) with minimal degree of modification [11,12].
Understanding this dynamics is crucial for the appreciation
that AGEs modified to a ‘high physiological degree’ escape
detection due to (i) their very slow formation, (ii) their very
rapid uptake, and (iii) the very efficient blood recycling.
Using the in vivo approach, the high resolution analytical
tool LC-MS/MS has been used to determine the presence of
specified AGEs in peripheral and hepatic venous blood
(control human subjects), or portal venous and hepatic
venous blood (cirrhotic subjects) [13]. With this state-of-
the-art methodology the authors observed no or only minute
differences in the level and type of AGEs that enter and
leave the liver. From this, the authors conclude that liver
does not contribute to extraction of in vivo formed AGEs
from the blood. They also put forward a hypothesis that the
kidneys represent the major site of elimination of AGEs
from the blood. At first glance, this may seem like a
plausible interpretation. However, a closer look at the
premises makes it clear that more solid data is required for a
shift of the current paradigm of hepatic elimination of
AGEs.
The authors state that in vivo formation of proteins
highly modified by AGEs is unlikely considering the
kinetics of albumin glycation under physiological con-
ditions. If ‘highly modified’ means AGE-modification to the
same extent as in vitro modified AGEs prepared by
traditional methods [14], this statement by the authors
would be agreed upon by most AGE-researchers. There is a
general concensus that highly modified AGE-albumin
prepared in vitro is not a perfect model for studies of
AGEs turnover in vivo. Nevertheless, one has to ask the
following question: do in vivo formed AGEs have to be
modified to the same extent as the commonly used in vitro
highly modified AGE albumin in order to be recognized by
receptors for endocytic uptake? In fact, one of the authors
(P.J.T.) of the presently discussed article previously
reported that HSA minimally modified by methylglyoxal
(MGmin-HSA) (1.4–2.4 modified arginine residues per
molecule) is taken up by receptor-mediated endocytosis
and degraded by the monocytic cell line THP-1 [11]. The
degree of AGE-modification was indeed much lower in this
MGmin-HSA (1–2 arginines per albumin molecule) com-
pared with conventionally used in vitro formed highly
modified AGE-BSA (in a typical batch of highly modified
AGE-BSA 37 of 59 lysines and 10 of 23 arginines are
modified). Furthermore, it is logical to assume that LSEC,
which exhibit a higher endocytic activity and express more
scavenger receptors compared to monocytes, would rep-
resent a more efficient site of uptake of MGmin-HSA. The
authors reported previously that approximately 2% of total
HSA contains MG-derived imidazolon (MG-H1) in normal
control subjects [9]. Assuming a total content of 250 g of
albumin in the blood of a normal adult individual, 2% would
correspond to 5 g of MG-H1-albumin, which will be present
in the circulation at any time. There is another important
consideration to take into account when comparing AGE-
modification of protein in vitro and in vivo: due to the fact
that the same individual protein molecules are present
during the in vitro generation of AGE-albumin, each of the
albumin molecules present will bind AGE-adducts with the
same probability. In contrast, since the probability of AGE-
modification increases with increasing life time of any
individual protein in vivo, the generation of AGEs in vivo
will result in differently modified proteins, spanning from
‘young’, newly synthesized proteins containing no or very
few AGE-modifications, to ‘old’ proteins containing most
of the modifications. On this basis, one would come closer
to reality by assuming that only albumin molecules that
have existed for more than one half-life will carry all the
MG-H1 groups. Using this assumption, along with the result
published previously by P.J.T. [11], it follows that 4% of the
albumin molecules older than one half-life carry MG-H1.
Moreover, applying combinatorics analysis it can be
calculated that the probability for any one of these albumin
molecules to carry more than 1 MG-H1 is 5.9%. This means
that at any time 300 mg of albumin molecules in the
circulation of normal humans will have more than 1 MG-H1
residues. According to the paper by P.J.T. cited above [11],
this degree of modification is sufficient to bring about
receptor-mediated uptake in scavenger cells. Of note, this
calculation most likely represents an underestimation, since
it was based on only one type of AGE-modification. In
fact, 12 different AGE-species are presently known, and it
is known that formation of AGEs can results in products
D. Svistounov, B. Smedsrød / Journal of Hepatology 41 (2004) 1038–10401040
that contain several AGE-adducts on the same protein
molecule. On this basis one can safely assume that the
amount of protein sufficiently modified to bring about
endocytic uptake and degradation will be significantly
higher than that calculated on the basis of only MG-H1-
mofication. It should be noted that this calculation applies
to healthy humans. In the diabetic state, for example, the
protein modification will be much higher than in normals
due to the increased levels of glucose, along with increased
serum concentrations of many types of AGE-precursors,
such as glyoxal, methylglyoxal and 3-deoxyglucosone,
that increase 1.2, 3.4 and 3.1 times, respectively [15].
Conceivably, these circumstances when taken together,
generate AGEs with a high enough degree of modification
for scavenger receptor-mediated uptake in liver. The
likelihood for this to happen in the diabetic patient is
even greater. But alas, concluding from the considerations
discussed above it is practically impossible to detect these
AGE-modified proteins in the blood because they will
disappear almost immediately after reaching the modifi-
cation threshold for uptake in the liver RES.
From the above considerations it is conceivable that
more solid evidence is needed if the current paradigm of
elimination of AGEs in liver scavenger cells be exchanged
with a new paradigm stating that AGEs are eliminated
mainly in extrahepatic tissues.
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