Biomarker Insights 2007: 2 299–306 299
ORIGINAL RESEARCH
Alteration of Transthyretin Microheterogeneity in Serum
of Multiple Trauma Patients
Beate Gericke1, Jens Raila1, Maria Deja2, Sascha Rohn3, Bernd Donaubauer2, Britta
Nagl1, Sophie Haebel4, Florian J. Schweigert1 and Udo Kaisers2,
1Department of Physiology and Pathophysiology, Institute of Nutritional Science, University of
Potsdam. 2Department of Anaesthesiology and Intensive Care Medicine, Charité, Campus Virchow-
Klinikum, Universitätsmedizin Berlin. 3Department of Food Analysis, Institute of Food Technology and
Food Chemistry, Technical University of Berlin. 4Interdisciplinary Center for Mass Spectrometry of
Biopolymers, University of Potsdam.
Abstract: Transthyretin (TTR) which exists in various isoforms, is a valid marker for acute phase response and subclinical
malnutrition. The aim of the study was to investigate the relationship between infl ammation, oxidative stress and the
occurrence of changes in microheterogeneity of TTR.
A prospective, observational study at a level-I trauma center of a large urban medical university was performed. Patients
were severely injured (n = 18; injury severity score (ISS): 34–66), and were observed within the fi rst 24 hours of admit-
tance and over the following days until day 20 after injury. 20 healthy subjects, matched by age and sex, were used as
controls.
TTR was enriched by immunoprecipitation. Microheterogeneity of TTR was determined by linear matrix assisted laser
desorption/ionization-time of fl ight-mass spectrometry (MALDI-TOF-MS). Four major mass signals were observed for
TTR representing native, S-cysteinylated, S-cysteinglycinylated and S-glutathionylated TTR. In the course of their ICU
stay, 14 of the 18 patients showed a transient change in microheterogeneity in favour of the S-cysteinglycinylated form of
TTR (p < 0.05 vs. controls). The occurrence of this variant was not associated with the severity of trauma or the intensity
of the acute-phase response, but was associated with oxidative stress as evidenced by Trolox.
Our results demonstrate that changes in microheterogeneity of TTR occur in a substantial number of ICU trauma patients.
The diagnostic values of these changes remains to be elucidated. It is speculated that TTR modifi cation may well be the
mechanism underlying the morphological manifestation of amyloidose or Alzheimer’s diseases in patients surviving multiple
trauma.
Keywords: polytrauma, modifi cation, microheterogeneity, TTR
Introduction
Transthyretin (TTR), formerly called prealbumin, belongs to the group of proteins including thyroxine-
binding globulin and albumin, which bind to and transport thyroid hormones in the blood. TTR is also
involved in the metabolism of vitamin A as it binds retinol-binding protein (RBP), the specifi c serum
transport protein for retinol. First identifi ed in 1942 by Kabat et al. in serum and cerebrospinal fl uid, TTR
has been described as a so-called visceral protein that is synthesized in the liver in response to nutritional
supply. Therefore, TTR serum levels can be used as a sensitive biochemical parameter of subclinical
malnutrition, since both the synthesis of proteins as well as energy intake are refl ected in its serum levels.
Serum levels of TTR are however also affected by acute and chronic diseases associated with an acute-
phase response. Under these conditions, liver activity is concentrated on the synthesis of acute-phase
response proteins, resulting in a drop in visceral proteins despite adequate nutritional supply (Ingenbleek
and Young, 1994; Lasztity et al. 2002; Power et al. 2000; Abraham et al. 2003). Mutations in the TTR
gene are the most common cause of autosomal dominant systemic amyloidosis such as the familial
amyloid polyneuropathy (Quintas et al. 1999; Merlini and Bellotti, 2003). In these amyloidoses, the
pathological deposits are characterized by the high abundance of TTR-β-structured-fi brils (Merlini and
Bellotti, 2003). TTR has a single cysteine residue in position 10 that can exist in the SH form or as a
mixed disulfi de with the amino acid cysteine as well as with the peptides cysteinylglycine and glutathione
Correspondence: Florian J. Schweigert, Department of Physiology and Pathophysiology, Institute of
Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, D-14558 Potsdam-Rehbrücke,
Germany. Tel: +49 33200 88527/528; Fax: +49 33200 88573; Email: [email protected]
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm
300
Gericke et al
Biomarker Insights 2007: 2
(Bernstein and Ingenbleek, 2002; Schweigert et al.
2004). Additionally, Cys10 adducts of the
S-homocysteine of TTR have been detected in
serum of humans with hyperhomocysteinemia
(Sass et al. 2003). It has recently been discussed
that post-translational modifi cations of TTR repre-
sent a potential risk factor for the development of
senile systemic amyloidosis (Zhang and Kelly,
2003). In-vitro TTR modifi ed at the cys10 is found
to be more susceptible to the formation of fi brils as
precursors of amyloid deposits (Zhang and Kelly,
2003). Modifi cations of the TTR molecule might
not only result in changes in its function but may
also serve, for instance, as a biochemical marker
for oxidative stress.
Free radical-derived reactive oxygen species
(ROS) are constantly generated in living tissues,
potentially damaging DNA, proteins and lipids.
“Oxidative stress” occurs if ROS reaches abnor-
mally high concentrations. Several pathological
conditions such as atherosclerosis, carcinogenesis,
neurodegenerative diseases, rheumatoid arthritis,
and cataracts, as well as aging and cell death, have
been linked to oxidative damage in various cell
components (Berger, 2005). Antioxidative
compounds, such as glutathione, play a protective
role in minimizing the deleterious consequences
of oxygen activation processes. Essentially, their
benefi cial effect occurs due to their ability to
reduce disulfi des or to oxidize themselves to disul-
fi des. Once formed, thiol groups can also generate
mixed disulfi de bonds with protein thiol residues.
Thus, besides the potential protection of protein-
cysteine(Cys)-residues from irreversible oxidative
modifi cations, thiol/disulfi des can recruit protein
thiol residues for the antioxidative buffering
system (Tobaben et al. 2003).
Procedures which occur in connection with
polytrauma induce a host defense response. This
is characterized by local and systemic release of
pro-inflammatory cytokines, arachidonic acid
metabolites, contact phase and coagulation
proteins, complement factors and acute phase
proteins, as well as hormonal mediators. It is
defined as systemic inflammatory response
syndrome and is determined according to clinical
parameters. However in parallel, anti-infl amma-
tory mediators are produced (compensatory
anti-infl ammatory response syndrome) (Keel and
Trentz, 2005). The production of free radicals is
supported by several stress factors. Enzymatic
protecting systems react to oxidative stress by
positive adaptation. The non-enzymatic antioxida-
tive systems (α-tocopherol, ascorbic acid, selen)
are diminished, indicating an increased require-
ment (Kreinhoff et al. 1990).
Due to the fact that TTR acts as a negative
acute-phase protein and based on the assumed
interaction between changes in its mircrohetero-
geneity through the exchange at the Cys on position
10 and oxidative stress, we assumed a role for TTR
modifi cations in acute phase reaction and oxidative
stress in multiple trauma patients. Therefore, we
investigated the microheterogeneity of TTR and
its relation to systemic infl ammation in severely
trauma patients.
Methods
Patients
A total of 18 severely injured intensive care unit
(ICU) patients were randomly selected and
assessed within the fi rst 24 hours after trauma
and over the following days until day 20. The
study protocol was approved by the ethic
committee of the hospital and informed consent
was obtained from legal substitutes. 20 healthy
(based on physical and clinical-chemical param-
eters) subjects, matched by age and sex, were
used as controls. The Injury Severity Score (ISS)
is an anatomical scoring system whereby the
score from the 3 most severely injured body
regions (Head, Face, Chest, Abdomen, Extrem-
ities (including Pelvis), External) is squared and
added together to produce the ISS score (ISS:
45 ± 7.5, mean ± SD). Blood samples were taken
within the fi rst 24 hours and at day 20 after
injury.
Immunoprecipitation of TTR
and MALDI-TOF-MS
15 µl of serum was treated with equal amounts
of a polyclonal rabbit anti human TTR or RBP
(DakoCytomation, Denmark) and with 15 µl
Sephadex G-15, 1 mg/ml phosphate buffer saline
(PBS), (Pharmacia Fine Chemicals, Sweden). The
mixture was incubated for two hours at 37 °C and
then centrifuged at 15.000 × g for 15 min at room
temperature. The supernatant was removed and
the immunoprecipitated complex of TTR was
extensively washed three times with PBS and then
after fi nal centrifugation, it was dissolved in 5 µl
301
Alteration of transthyretin microheterogeneity in serum
Biomarker Insights 2007: 2
PBS. This was followed by a two step preparation
procedure: Firstly, 1 µl immunoprecipitated
sample was deposited on the target and dried.
Secondly, 0.5 µl matrix (a saturated solution of
sinapinic acid in acetonitrile plus water (1:2; v/v)
containing 0.1% trifl uoroacetic acid) was placed
on the serum drop and also dried. This step was
repeated.
MALDI mass spectra of the immunoprecipi-
tated TTR was obtained using a Refl ex II MALDI-
TOF mass spectrometer (Bruker-Daltonik,
Bremen, Germany). MALDI-TOF MS was
performed in linear mode at 20k acceleration
voltage. For ionisation, a nitrogen laser (VFL-
337ND, LSI, MA, Newton, USA; 337 nm, 3 ns
pulse width, 3 Hz) was used. The spectra were
calculated by using external calibration with ions
produced from horse cytochrome c (m/z 12,360.08)
and horse myoglobin (m/z 16,951.46). After
external calibration, this mass spectrometer was
capable of achieving ~0.1% mass accuracy in the
linear mode.
The samples were prepared in a two step proce-
dure as follows. To determine the disulfi de linkage
of TTR adducts, the immunoprecipitated TTR was
treated with dithiothreitol (DTT). DTT solution
(100 mM) in buffer (100 mM NH4CO3, pH 8.8)
was added to the solution at a ratio of 1:1 v/v (DTT
solution volume/TTR solution volume). The
mixture was incubated for 2 h at room temperature.
Thereafter samples were immunoprecipitated as
described and subsequently subjected to MALDI-
TOF-MS.
Quantitative analysis of C-reactive
protein (CRP) and TTR
Serum TTR was quantitatively determined by an
ELISA method using a polyclonal rabbit anti-
human antibody (DakoCytomation, Hamburg,
Germany). The TTR was measured by use of an
ELISA technique adapted from the RBP proce-
dure (Schweigert et al. 2003). In detail, wells of
microtiter plates were coated through the addition
to each well of rabbit anti-human TTR IgG
(DakoCytomation, Hamburg, Germany) diluted
1:2000 in 50 µl of 50 mM carbonate buffer
(pH 9.6) and then incubated for 2 hours at 37 °C.
Plates were washed 4 times with PBS-Tween
(pH 7.4), shaken dry, wrapped in plastic, and
stored overnight at 4 °C. For analysis, nonspecifi c
binding was blocked by the addition of 1% BSA
diluted in PBS and incubated for 2 hours at 37 °C.
After 4 additional washings, 50 µl of TTR
standard (N Protein Standard/Standard SL OQIM
13, Dade Behring GmbH, Marburg, Germany)
or serum sample diluted with PBS-Tween
(pH 7.4) was placed in triplicate wells and incu-
bated for 2 hours at 37 °C with constant shaking.
After rinsing the wells with PBS-Tween (pH 7.4),
50 µl of peroxidase-conjugated sheep anti-human
TTR IgG (Biotrend, Cologne, Germany), diluted
1:2000 in PBS-Tween with 1% BSA was added
to each well, and plates were incubated for 1 hour
at 37 °C. After 4 fi nal washings, color was devel-
oped by the addition of o-phenylenediamine
dihydrochloride solution (OPD, Sigma, Deisen-
hofen, Germany) and incubated for 20 minutes
at 25 °C. OPD solution (100 µl/well) consisted
of 3.7 mM solution in 50 mM disodium
phosphate-25 mM citric acid buffer (pH 5.2)
containing 0.012% H2O2. The reaction was
stopped by the addition of 1 M H2SO4 (50 µl/well)
and absorbance was measured at 490 nm by use
of a spectrophotometer (Microplate Reader, Bio-
Rad, Munich, Germany). The intra-assay
coeffi cient of variation was 8.8%, and interassay
coeffi cient of variation was 8.1%.
CRP levels in serum were measured with a
high sensitivity latex turbimetric immunoassay
using a latex-coupled monoclonal mouse anti-
human antibody (Olympus AU 600, Biomed,
Germany). The sensitivity of this assay was
0.005 mg/dl. The 90th percentile of normal CRP
distribution was 0.3 mg/dl.
Trolox equivalent antioxidant capacity
(TEAC) assay
To measure the antioxidant capacity, the TEAC
assay, described by Re et al. (Re et al. 1999), was
used with minor modifi cations. This method is
based on the reaction of the blue/green stable
ABTS radical (ABTS*), which is formed through
the reaction of ABTS and K2O8S2, with antioxi-
dants. In this reaction, the blue/green color
disappears because the ABTS* reacts with the
antioxidant. This decolorization is determined
spectrophotometrically at 734 nm after 6 min.
The reduction in absorbance is related to that of
Trolox, a synthetic, hydrophilic vitamin E
analogue, which gives the TEAC value. The
TEAC is calculated as millimoles of Trolox
equivalents per litre.
302
Gericke et al
Biomarker Insights 2007: 2
Analysis of Carotenoids
and α-Tocopherol
For separation and quantifi cation of carotenoids
(α-carotene, β-carotene, lutein, zeaxanthin,
canthaxanthin, β-cryptoxanthin, lycopene) and
α-tocopherol, a gradient reversed-phase HPLC-
system was used (Waters, Eschborn, Germany) as
described in the literature. (Schweigert et al. 2003).
Results of α-tocopherol and β-carotene were
compared with the standard reference material 968a
[National Institute of Standards Technology (NIST),
Gaithersburg, MD, USA]. Carotenoid standards of
lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin
and lycopene were a generous gift from Hoffman-
La Roche, Switzerland and the standards for
α-carotene, β-carotene and α-tocopherol were from
Sigma (Deisenhofen, Germany).
Statistical procedures
All data are given as a mean value ± SD. Statistical
analysis was performed using nonparametric
procedures. The Mann-Withney U-rank test was
used to test for signifi cant differences between
patients and controls. The Wilcoxon test was used
for the comparison between results of the fi rst 24
hours and day 20 after injury. Values of p < 0.05
were considered signifi cant.
Results
Patients studied were predominantly male (age
32 ± 11 yrs) who had a mean Injury Severity Score
(ISS) of 45.2 ± 7.5 (Table 1). Levels of TTR did
not differ within the fi rst 24 hours up to day 20
after trauma (4.0 ± 2.6 µmol/L vs. 4.1 ± 3.0 µmol/L;
mean ± SD; n.s.). The CRP values tended to be
lower on day 20 after injury (13.3 ± 8.4, 6.1 ± 4.6;
mean ± SD; Table 2).
Four dominant peaks were detected after
immunoprecipitation by MALDI-TOF-MS
(external calibration) in the range where TTR
and its conjugated forms should normally appear
(m/z 13,700–14,100), with a dominant peak at
13,878 ± 7 m/z (controls). The molecular mass
of 13,757 ± 6 Da (controls) corresponded to the
native, unmodifi ed TTR. The other ion peaks
showed molecular masses of 121 ± 3, 179 ± 3
and 298 ± 5 Da larger than native TTR,
representing Cys10 adducts for S-cysteine
(TTR-Cys10-S-S-Cys, mass = 13,877 ± 7 Da),
S-cysteinylglycine (TTR-Cys10-S-S-CysGly,
mass = 13,934 ± 6) and S-glutathione (TTR- Cys10-
S-S-SG, mass = 14,056 ± 15) for controls.
Figure 1 compares a representative mass spec-
trum obtained from a trauma patient within the
fi rst 24 hours after injury with the mass spectrum
of a healthy individual. The treatment with DTT
of the immunoprecipitated TTR from the same
patient resulted in a loss of microheterogeneity
and a shift towards the native form of TTR.
No differences in molecular masses of the indi-
vidual signals were detected between controls and
patients at different time points after injury, or
between groups with or without modifi cation.
However a transient change in the microheteroge-
neity was observed. The extent of modifi cation was
defi ned on the basis of the relationship between
peak height of the dominant cysTTR and the other
variants as has been done previously (24, 26). In
healthy persons the relation between cysTTR
(100%) and cysglycTTR is 40%. A substantial
change was thus defi ned if the extent of modifi ca-
tion of cysglyc reached values twice as high as in
healthy controls. We therefore used 80% as a cut-
off. In 14 out of 18 multiple trauma patients a more
dominant peak for the cysglycTTR (>80%) was
present; this was again normalized at day 20 after
hospitalization (Fig. 1). At this time point the TTR
microheterogeneity pattern was comparable to
healthy controls. Seven patients developed infec-
tion during their ICU stay and all of them showed
an increased cysglycTTR modifi cation, whilst none
of those without a high cysglycTTR actually devel-
oped infection. The peak height ratio in patients
Table 1. General characteristics of ICU patients (n = 18)
at day of admission.
Age (years) 32.2 ± 11.6
Sex 3 female/15 male
Height (cm) 179.3 ± 11.6
Weight (kg) 85.0 ± 10.7
Injury Severity Score (ISS) 45.2 ± 7.5
Abbreviated Injury Scale (AIS) 13.1 ± 2.1
Acute Physiology And Chronic 16.5 ± 8.2
Health Evaluation (APACHE II)
Simplifi ed Acute Physiology 27.1 ± 9.5
Score (SAPS II) fi rst 24 hours
SAPS II day 20 13.9 ± 7.9
Sepsis-related Organ Failure 7.9 ± 3.1
Assessment (SOFA) fi rst
24 hours
SOFA day 20 1.7 ± 2.1
303
Alteration of transthyretin microheterogeneity in serum
Biomarker Insights 2007: 2
within the fi rst 24 hours after injury was signifi cantly
different to controls (Fig. 1). On day 20, no signif-
icant difference in peak height ratio was measured
between patients and controls.
Trolox values were signifi cantly lower in patients
with the modifi cation in TTR molecules in favor of
the cysglyc form (3.1 mmol/L ± 0.4 versus 2.9 ± 0.3,
p < 0.05); Trolox values were signifi cantly higher
on day 20 after injury. Serum α-tocopherol levels
were signifi cantly increased (p < 0.05) whilst for
serum levels of carotenoids in total as well as for
the individual carotenoids, except for β-carotene
there was no difference between day 20 and the
initial values at admission (Table 2).
Discussion
The detection and characterization of variants and
modifi ed structures of proteins are clinically impor-
tant for diagnosis purposes and for the elucidation of
the pathogenesis of various diseases. Modern mass
spectrometry methods have largely replaced analyses
by conventional protein chemistry. MALDI-TOF-MS
after immunoprecipitation allows the measurement
of the molecular weight of intact TTR and the eluci-
dation of the modifi ed structures. Post-translational
modifi cations of TTR have enormous diagnostic
value. Some variant proteins cause diseases, and some
diseases result in increase of proteins with abnormally
modifi ed structures. The protein TTR occurs in over
100 variants and most of them cause amyloidosis
(6, 7). A recently published study reported on differ-
ential post-translational modifi cations of TTR in
Alzheimer’s disease (26). The results of our study
demonstrate the link between polytrauma and a clear
change in microheterogeneity of TTR. Although the
exact consequence on the stability of TTR and the
long term outcome of polytrauma patients remains
to be elucidated, modifi cations may infl uence the
interaction of TTR with other proteins (RBP,
Thyroxin), as well as having an effect on many
aspects of the metabolism of the protein such as
receptor binding, tissue uptake, degradation and
excretion.
A negative correlation between levels of CRP
and TTR in serum has already been shown
Table 2. The most important parameters used to characterize the antioxidative status and the post trauma
response of ICU patients.
First 24 hours Day 20 after Modifi cation on Without changes
injury cysGlysTTR in modifi cation
Trolox 2.8 ± 0.4a 3.1 ± 0.4 3.1 ± 0.4 2.9 ± 0.3
(mmol/L)
α-tocopherol 17.4 ± 5.5b 22.3 ± 6.7 19.5 ± 7.3 19.3 ± 6.9
(µmol/L)
α-carotene 0.04 ± 0.02 0.04 ± 0.03 0.03 ± 0.02 0.04 ± 0.03
(µmol/L)
β-carotene 0.22 ± 0.10 0.23 ± 0.20 0.21 ± 0.14 0.20 ± 0.11
(µmol/L)
Lutein 0.06 ± 0.02 0.04 ± 0.03 0.05 ± 0.03 0.04 ± 0.02
(µmol/L)
Zeaxanthin 0.006 ± 0.002 0.004 ± 0.004 0.005 ± 0.003 0.004 ± 0.002
(µmol/L)
Canthaxanthin 0.013 ± 0.005 0.006 ± 0.003 0.011 ± 0.006 0.010 ± 0.005
(µmol/L)
β-cryptoxanthin 0.06 ± 0.03 0.04 ± 0.05 0.05 ± 0.03 0.04 ± 0.02
(µmol/L)
Lycopene 0.06 ± 0.03 0.04 ± 0.02 0.05 ± 0.03 0.05 ± 0.03
(µmol/L)
CRP 13.3 ± 8.4 6.1 ± 4.6 13.3 ± 9.0 12.3 ± 7.0
(mg/dL)
Total carotenoids 0.5 ± 0.2 0.4 ± 0.3 0.4 ± 0.2 0.4 ± 0.2
(µmol/L)
TTR 4.0 ± 2.6 4.1 ± 3.0 4.7 ± 3.4 3.5 ± 2.7
(µmol/L)
aTrolox is signifi cantly different from patients with or without modifi cation (p < 0.05) and between both time points (p < 0.05).
bα-tocopherol tends to be different between patient within the fi rst 24 hours and on day 20 after injury.
Loading more pages...