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Enzymes and Protein Markers |
1
HyTest LTD, Tykistokatu 6A, FIN-20520 Turku, Finland.
2
Departments of Biochemistry and Bioorganic Chemistry,
School of Biology, Moscow State University, 119899 Moscow, Russia.
3
Department of Biotechnology, Institute of Medical
Ecology, Simpheropolskiy bull. 8, 113149 Moscow, Russia.
4
Department of Biotechnology, University of Turku,
Tykistokatu 6A, FIN-20520 Turku, Finland.
5
Center for Atherosclerosis, 29th Moscow City Hospital,
119828 Moscow, Russia.
6
Turku University Hospital, Central Laboratory,
Kiinanmyllynkaty 4–8, FIN-20520 Turku, Finland.
a Author for correspondence. Fax 358-2-3338070; e-mail hytest{at}utu.fi.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differences in the measured cTnI concentration using various assays can be explained by the lack of a common calibration standard and/or by the differing cross-reactivities of antibodies with the different cTnI forms (7). Recently it was demonstrated that the major fraction of cTnI in the circulation of AMI patients is complexed with troponin C (TnC) (7)(9). Monoclonal antibodies (MAbs) differ in their ability to recognize free cTnI and cTnI complexed with TnC, and this fact can be the source of disagreement between assays (7)(9).
Necrosis of cardiac tissue caused by infarction is accompanied by liberation of proteolytic enzymes from lysosomes. Because cTnI is highly susceptible to proteolysis (10), necrosis can be expected to induce substantial degradation of cTnI. This means that, depending on conditions (e.g., time after onset of AMI, size of infarction zone, and rate of reperfusion), the blood of AMI patients will contain variable quantities of intact cTnI and its proteolytic fragments, which can circulate in free form or in complexes with the two other troponin components.
In the present study, we followed the process of proteolytic degradation of cTnI in necrotic tissue and in serum by two immunological methods: sandwich immunoassay and Western blots utilizing MAbs with different epitope specificity. We demonstrated that during incubation in necrotic cardiac tissue as well as in serum cTnI undergoes rapid proteolytic cleavage and that different fragments of the cTnI molecule have different stabilities. The region most resistant to proteolysis is the region located between amino acid residues 30 and 110.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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one-step sandwich immunofluorometric assay
The protocol of the assay was similar to that described by
Katrukha et al. (9) with minor modifications. Briefly,
capture MAbs were biotinylated, and detection MAbs were labeled with
the stable chelate of Eu3+. The mixture of antigen (AMI
serum samples or reconstituted in normal human serum) with capture and
detection MAbs was incubated for 30 min at room temperature in
streptavidin-coated plates. After the plates were washed, LANFIA
enhancement solution (0.2 mL/well) was added, and incubation was
continued for 3 min at room temperature with gentle shaking. The
fluorescence (cps) was measured with the 1234 DELFIA Plate Fluorometer.
The fluorescence of the control probe, which did not contain coating
MAbs and which reflected the nonspecific binding of cTnI, was
subtracted from the total fluorescence. The method described has a
detection limit below 0.1 µg/L, and the linearity range is 0.3–100
µg/L. This method provides reliable detection of both free cTnI and
cTnI in the whole ternary complex. In the latter case, the incubation
mixture contained 5 mmol/L EDTA to reduce interaction between troponin
components (9).
serum samples
Serial blood samples from AMI patients were collected at the
University Hospital of Turku (Turku, Finland) and at the 29th Moscow
City Hospital (Moscow, Russia). Blood samples were incubated 5 min at
room temperature and centrifuged, and the serum samples were stored at
-70 °C until use. In all cases, the diagnosis of AMI was confirmed
by electrocardiogram and serial measurements of creatine kinase, its MB
isoenzyme, and lactate dehydrogenase isoenzyme 1. All samples were
collected in accordance with the Helsinki Declaration of 1975 as
revised in 1983.
in vitro necrosis of human cardiac tissue
To simulate the process of necrosis induced by AMI in vitro, we
incubated small pieces (2–3 g) of human cardiac left ventricular
tissue, obtained from four different donors 5–8 h postmortem, at
37 °C. Tissue samples were wetted with 0.15 mol/L sodium chloride
containing 2 g/L sodium azide as a preservative (1 mL of sodium
chloride solution per 3 g of tissue) and incubated with gentle
shaking for 2, 5, 8, and 20 h. After incubation the tissue samples
were frozen and stored at -70 °C.
For protein extraction, each sample was frozen with liquid nitrogen and crushed to powder. Soluble proteins were extracted with 25 mmol/L Tris-HCl buffer (pH 7.5) containing protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L pepstatin A, and 10 mmol/L benzamidine chloride) at a ratio of 9 mL of buffer per gram of tissue powder. After incubation for 5 min on ice with gentle shaking, the sample was centrifuged for 2 min at 5000g, and the supernatant was collected. The pellet was washed with the Tris-HCl buffer, and after centrifugation the supernatant was removed carefully. Supernatants were combined, and 6 mol/L solid urea was added.
The pellet after the second centrifugation was subjected to three cycles of extraction by nine volumes of 50 mmol/L Tris-HCl (pH 7.5) containing 6 mol/L urea, 5 mmol/L EDTA, 10 mmol/L 2-mercaptoethanol, and protease inhibitors (0.1 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L pepstatin A, and 10 mmol/L benzamidine chloride). After centrifugation for 2 min at 5000g, the supernatants were collected. All supernatants (containing soluble and contractile proteins) were combined and stored at -70 °C until use. Less than 2% of the initial cTnI remained in the pellet after extraction (determined by Western blot, data not shown).
Immediately after the samples were thawed, the concentration of cTnI in each sample was measured in sandwich immunoassays after addition of 1 volume of extract to 3000 volumes of normal human serum. The concentration of urea after such dilution was low and did not affect the assay results (9). Protease inhibitors at this dilution also did not affect the assay (data not shown).
n-terminal sequencing of cTnI PROTEOLYTIC PEPTIDE 2
Left ventricular tissue was incubated at 37 °C for 8 h,
and proteins were extracted as described earlier in this section. The
extract was diluted by nine volumes of 20 mmol/L Tris-HCl (pH 8.0)
buffer containing 0.6 mol/L LiCl, 5 mmol/L EDTA, and 15 mmol/L
2-mercaptoethanol. The protein solution was centrifuged for 5 min at
5000g, and the supernatant was collected and loaded on
an affinity column containing anti-cTnI MAb C5 immobilized on Sepharose
[for more details see Katrukha et al. (13)]. Proteins
bound to the affinity matrix were eluted by 0.1 mol/L glycine buffer
(pH 2.0) and dialyzed extensively against 10 mmol/L HCl. After
lyophilization, the protein mixture was subjected to electrophoresis on
a 10–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel and
transferred onto a Immobilon PVDFTM membrane. Sequencing of
N-terminal amino acid residues of peptide 2 was conducted on a Knauer
Protein Sequencer (Knauer), as recommended by the manufacturer.
western blot analysis
Protein samples were separated by electrophoresis on a 10–20%
gradient SDS gel (13) and transferred onto the
nitrocellulose membrane as described previously (14). To
block nonspecific binding of the proteins, the nitrocellulose membrane
was incubated for 15 min at room temperature in phosphate-buffered
saline containing 1 g/L Tween 20 and 5 g/L casein. The membrane was
then incubated for 1 h in the same buffer containing 0.02 g/L
anti-cTnI MAb and 1 g/L casein. After the nitrocellulose membrane was
washed with phosphate-buffered saline containing 1 g/L Tween 20, it was
incubated for 2 h with horseradish peroxidase-labeled anti-mouse
antibodies, and the binding of MAbs was visualized by incubation with
diaminobenzidine.
interaction of cTnI AND ITS PROTEOLYTIC FRAGMENTS WITHTnC
Left ventricular tissue was incubated at 37 °C for 8 h,
and proteins were extracted as described earlier in this section, with
omission of EDTA from the extraction buffer. The extract was loaded on
a column containing TnC immobilized on Sepharose (5 g of TnC per liter
of Sepharose). The column was equilibrated with buffer A (50 mmol/L
Tris-HCl, pH 8.0, 7 mol/L urea, 2 mmol/L CaCl2,
15 mmol/L 2-mercaptoethanol). Unbound proteins were removed by washing
with the same buffer, and the proteins bound to TnC were eluted with
buffer B (50 mmol/L Tris-HCl, pH 8.0, 7 mol/L urea, 10 mmol/L EGTA, 15
mmol/L 2-mercaptoethanol). In the control experiment the same volume of
urea extract was loaded on a Sepharose column without immobilized TnC.
This column was evaluated in the same manner. The proteins bound to the
affinity and control matrices were subjected to 10–20% gradient
SDS-polyacrylamide gel electrophoresis, transferred onto the
nitrocellulose membrane, and detected by MAbs.
stability of cTnI IN SERUM
Intact human cardiac troponin complex (final concentration of
cTnI, 30 µg/L) was dissolved in the pooled normal human serum
containing 1 g/L sodium azide to prevent bacterial growth. Serum
samples from AMI patients were diluted fourfold with the same pooled
serum containing 1 g/L sodium azide. Antigen solutions were aliquoted
and incubated at 23 °C for different periods of time (up to 5 days).
After incubation, samples were frozen and stored at -70 °C until
use.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A).
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cTnI demonstrated different degrees of stability when measured in
different assays. When epitopes of the capture MAbs were located in the
central region of cTnI (19C7-biot–16A11-Eu and 18H7-biot–16A11-Eu),
the rate of decline in cTnI immunoreactivity detected by the assays was
rather slow. Even after a 20-h incubation period, the measured
fluorescence was ~50% of the initial value. cTnI immunoreactivity
was significantly less stable when the epitope of the capture MAb was
located at the extreme N-terminal (14G5) or C-terminal (7F4) ends of
cTnI. When the capture and detection MAbs were located on different
ends of the molecule (14G5-biot–7F4-Eu), incubation at 37 °C
dramatically decreased the measured fluorescence. After 20 h of
incubation, hardly any antigen was detected by this assay (Fig. 1B
).
We hypothesized that during the incubation of necrotic tissue deep
proteolytic degradation of cTnI occurs and that different regions of
the cTnI molecule might have different stabilities. To understand which
region of the molecule is the most stable, we followed the process of
cTnI degradation more closely. Proteins from the tissue extract
incubated at 37 °C for 8 h were subjected to SDS-gel
electrophoresis and subsequent Western blotting. cTnI and its
proteolytic fragments were visualized by MAbs with different epitope
specificities (Fig. 2
). We were able to differentiate seven main peptides with
molecular masses ranging from 14 kDa up to 27–28 kDa. MAbs with
epitopes located in the N- and C-terminal regions of the molecule
recognized only limited numbers of peptides (Fig. 2
, lanes 1, 2 and
8–10), suggesting that the N- and C-terminal peptides of cTnI are
easily removed during proteolysis. On the contrary, MAbs 10F4, 19C7,
414, 16A11, and 18H7, which recognize residues 34–37, 41–49, 56–61,
87–91, and 102–108 of cTnI, respectively, stain the largest number of
peptides, and the peptide pattern stained by these MAbs is very similar
(Fig. 2
, lanes 3–7).
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Continuing our investigation of cTnI degradation, we analyzed the time
course of proteolysis in necrotic tissue. Tissue samples were incubated
for 0, 2, 5, 10, and 20 h at 37 °C, and the proteins were
extracted as described in Materials and Methods. The urea
extracts were subjected to SDS-gel electrophoresis and Western
blotting. cTnI peptides were visualized by MAb 19C7. This MAb was used
because it recognizes the largest number of cTnI peptides (Fig. 2
, lane
4). A tissue sample was obtained from a donor 8 h postmortem.
Therefore, without any additional incubation at 37 °C, this sample
already contained several proteolytic fragments of cTnI (Fig. 3
, lane 1). Incubation of this sample for 2 and 5 h caused
accumulation of peptides 2 and 3, with a substantial decrease in the
intensity of the intact cTnI band (Fig. 3
, lanes 2 and 3). Prolonged
incubation for 8 and 20 h was accompanied by accumulation of short
cTnI peptides (peptides 5, 6, and 7) with molecular masses of 17, 16,
and 14 kDa, respectively, and by further decreases in the intensities
of the intact cTnI band and its two large peptides (peptides 2 and 3).
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Because peptide 2 is the main cTnI peptide detected in tissue
homogenate after incubation in necrotic tissue (Fig. 3
), we tried to
identify the site of protease cleavage. As evident from Fig. 2
, peptide
2, with an apparent molecular mass of 25 kDa, was stained by all MAbs
except 10B11 and 4G6; 10B11 and 4G6 recognize epitopes located in the
extreme N-terminal end of cTnI. Taking into account the size of peptide
2, one may assume that peptide 2 lacks 24–28 amino acid residues from
the N-terminal region of the cTnI molecule. To verify this suggestion
we transferred peptide 2 to Immobilon PVDF and sequenced it on the
Knauer protein sequencer. The peptide 2 fraction was not homogeneous;
we were able to determine two N-terminal sequences–AYAT and
YATE–corresponding to the primary structure of cTnI starting from
residues 27 and 28, respectively.
The rate of cTnI proteolysis may depend on different factors, such as
the size of the infarction zone, the rate of reperfusion, and some
individual biochemical features of the donors. We compared the rate of
cTnI proteolysis in tissue samples obtained from two different donors
(Fig. 4
). Tissue samples were incubated as described in Materials
and Methods for 2–20 h, and the quantity of intact cTnI was
determined by the assay utilizing biotinylated MAb 14G5 as the capture
antibody and 7F4 as the detecting MAb. Because the epitopes of MAbs
utilized in this assay are located at the extreme N- and C-terminal
ends of the cTnI molecule, the assay can be used only for the detection
of the intact cTnI, not its proteolytic fragments. The rate of cTnI
degradation in the two samples tested was different. Twenty hours after
the beginning of incubation, we detected only 1.7% of the initial cTnI
in the tissue sample from the first donor, whereas after the same time
of incubation we detected ~15% of the initial cTnI in the sample
obtained from the second donor (Fig. 4
).
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If our in vitro model correctly reflects the real process of cTnI
proteolytic degradation in vivo, we may expect that the apparent
kinetics of cTnI released into the bloodstream and the sensitivity of
its detection in serum will depend strongly on the specificity of the
MAbs used for the sandwich immunodetection. To test this assumption, we
measured the concentration of cTnI in serial samples obtained from AMI
patients by two different immunoassays. The first assay (assay A)
utilizes 19C7 as the coating MAb and 8E10 as the Eu-labeled detection
MAb. These MAbs recognize epitopes located close to each other in the
central region of cTnI, which is relatively resistant to proteolysis
(Fig. 2
). Therefore assay A is suitable for the detection of both
intact cTnI and its proteolytic fragments. The second assay (assay B)
utilizes 14G5 as the coating biotinylated MAb and 7F4 as the Eu-labeled
detecting MAb. The epitopes of these two MAbs are located at opposite
ends of the cTnI molecule. Because both the N- and C-terminal ends of
cTnI are highly susceptible to proteolytic degradation, the second
assay detects intact cTnI but does not detect short cTnI peptides. We
used both assays to monitor the cTnI concentration in the serum of AMI
patients (Fig. 5
). During the first few hours after the onset of chest pain,
both assays give similar results. Twenty hours after the onset of the
chest pain, however, the cTnI concentration measured by the first assay
was much higher than that measured by the second assay. The ratio of
the concentrations was close to 1–1.5 during the first 10–15 h; 3
days later, however, this ratio increased to 4. In some serum samples
collected 5 days after the onset of chest pain, we were unable to
detect any increase of cTnI using assay B, whereas at the same time we
detected substantial quantities of cTnI, using assay A (data not
shown).
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To study the process of cTnI degradation in calibrators or AMI serum
samples after they were collected, we incubated the troponin complex in
normal human serum and serum from AMI patients (see Materials and
Methods) for different time periods at room temperature
(23 °C). The changes in cTnI concentration were determined by three
differently configured sandwich fluoroimmunoassays. The assays were
constructed using MAb 8E10, which recognizes the epitope located in the
central region of the molecule, as the capture MAb. The MAbs used for
detection recognize the N-terminal (7F4), C-terminal (14G5), or the
central (19C7) regions of the cTnI sequence (Fig. 1A
). Incubation of
the ternary complex in the pooled serum as well as cTnI from AMI serum
samples for 20–120 h caused dramatic decreases in the cTnI
concentration measured by assays based on 8E10–7F4 or 8E10–14G5 (Fig. 6
). At the same time, only a small decrease in cTnI
immunoreactivity was observed even after 120 h of incubation when
measured by the assay using 19C7–8E10 (Fig. 6
).
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We believe that the increased stability of the central region of cTnI
can be explained in part by its interaction with TnC, which protects
this region of the cTnI molecule from proteolysis. If this is correct,
we may expect that the fragments of the cTnI molecule resistant to
endogenous proteolysis will retain their ability to interact with TnC.
To test this hypothesis, the proteins extracted from the tissue sample
incubated at 37 °C for 8 h were treated as described in
Materials and Methods. cTnI fragments bound to TnC-Sepharose
were subjected to SDS-gel electrophoresis and Western blotting. cTnI
and its peptides were visualized by staining with MAb 19C7 (Fig. 7
). As seen in Fig. 7
, intact cTnI and most of the endogenous
proteolytic fragments were bound to immobilized TnC (Fig. 7
). This
interaction is Ca2+-dependent and is highly specific
because cTnI peptides were not retarded on the Sepharose column without
immobilized TnC (Fig. 7
, lane 3). Although the relative intensities of
the peptide bands in lanes 1 and 2 of Fig. 7
are slightly different, we
believe that most of the cTnI peptides were retarded on TnC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have shown that cTnI in necrotic cardiac
tissue is cleaved rapidly by proteases and that this process leads to
the disappearance of the intact molecule and accumulation of short
fragments of cTnI. Depending on tissue samples, <10% of cTnI remained
in the intact form after 20 h of incubation at 37 °C (Figs. 1B
and 3
). We found that the N- and C-terminal regions of cTnI were
extremely unstable and were easily removed when the necrotic tissue was
incubated for short periods of times at 37 °C. We predicted that if
the epitopes of the MAbs used in sandwich immunoassays are well
separated from each other, then the probability that the polypeptide
chain between these epitopes will be cleaved is rather high. In
complete agreement with this prediction, we found that when the
epitopes for MAbs were located at the extreme N-terminal (14G5) and
C-terminal (7F4) ends of cTnI, very small quantities of cTnI were
detected by sandwich immunoassay even after a short (8 h) incubation of
tissue at 37 °C (Fig. 1B
). Our observation is in agreement with
recent data indicating that the N-terminal part of cTnI is very
flexible (17) and is more easily exposed to the action of
proteases.
The central region of cTnI, located between residues 30 and 110, is
more stable and is not cleaved even after extended incubation times
(Fig. 2
). It is well known that in the troponin complex, cTnI tightly
interacts with TnC. This interaction is Ca2+-dependent
(18)(19). The central region of cTnI containing
the inhibitory peptide interacts with the central helix and C-terminal
domain of TnC (19)(20). Because most of the cTnI
proteolytic fragments retain the ability to interact with TnC (Fig. 7
),
we believe that the higher resistance to proteolysis of the central
region of cTnI can at least partly be explained by its interaction with
TnC, which seems to protect it from proteolytic degradation.
The data obtained from our model of necrosis indicate that 20–40 h after AMI onset, the predominant fraction of cTnI in the bloodstream is not intact cTnI but a heterogeneous mixture of proteolytic fragments and their complexes with TnC. Therefore, the sandwich immunoassays for cTnI should meet several important requirements. The antibodies used in these assays should recognize not only intact free cTnI and its complexes with other troponin components (7)(9), but also the proteolytic fragments of cTnI and their complexes.
cTnI can undergo proteolytic degradation not only in the necrotic cardiomyocytes but also in the bloodstream of patients or in collected blood/serum samples. The rate of proteolytic degradation in serum depends on many different factors. For example, the stability of free cTnI is much lower than the stability of cTnI inside intact ternary or binary complexes with TnC (16). Here we demonstrated that the detected stability of cTnI in serum also depends on the assay used for the cTnI measurement. This observation can be of interest to manufacturers who produce cTnI assays and prepare cTnI controls or standards from intact troponin complex, using pooled human serum as a matrix, and for clinicians, providing them with information on the length and conditions of serum storage.
For a long time the N-terminal region of cTnI (the first 32 residues)
was considered to be the best site for antibody production because this
sequence of amino acids is absent in the skeletal isoform of troponin I
(15)(21). Indeed, this region of the molecule is
highly immunogenic and absolutely specific for the cardiac isoform of
troponin I (11)(12). However, our results
indicate that this region of the molecule as well as the extreme
C-terminal end are rapidly cleaved during endogenous proteolysis of
cTnI (
Figs. 1–3
). Therefore, sandwich immunoassays utilizing one
antibody specific to the extreme N-terminal end of cTnI and another
antibody recognizing residues in the central or C-terminal regions of
cTnI will have lower sensitivity, especially in cases of late AMI
diagnosis, compared with assays based on antibodies recognizing the
stable region of the protein.
In addition to superior specificity, cTnI has one important advantage
over the majority of other markers widely used in clinical practice.
cTnI can be detected in the bloodstream of patients 5–7 days after the
onset of the first symptoms of AMI, thus enabling late diagnosis, which
is impossible with some other markers. This advantage turns into a
drawback when reinfarction occurs several days after the first episode.
In that case, the concentration of cTnI can be still increased because
of the initial AMI, and only serial measurements of cTnI can confirm or
reject the new diagnosis. We propose that utilization of two different
cTnI assays specific to both intact cTnI and its proteolytic fragments
(assay A, 19C7–8E10) and to only intact cTnI (assay B, 7F4–14G5) will
help to discriminate the first AMI from the second infarction. If
10–15 h after the onset of symptoms (for the second time, several days
after first AMI), the ratio of the concentrations measured by assays A
and B is high, then one can conclude that it is the first infarction
that leads to the gradual liberation of proteolytic fragments of cTnI
(Fig. 5B
). If the ratio of fluorescence measured by assays A and B at
10–15 h after the appearance of the first symptoms is low (close to
1), then one can conclude that additional intact cTnI has been
liberated into the bloodstream, which may be explained by a recurrent
AMI. The same idea can be used more conveniently in a double-label
assay, with a capture MAb recognizing the epitope localized in the
stable region of the molecule and two detection MAbs, one recognizing
stable regions and the other recognizing unstable regions of the
molecule labeled by different labels (Eu3+ and
Tb3+ or Fluorescein and Texas Red) that can be clearly
detected independently of each other. Currently, such markers as
myoglobin and the creatine kinase MB isoenzyme are used for the
diagnosis of recurrent AMI; however, the much higher specificity of
cTnI can be more valuable in this case.
We conclude that because of high susceptibility to proteolysis, cTnI undergoes rapid degradation after necrosis induced by AMI. A complex mixture of intact cTnI, its proteolytic fragments, and their complexes with other troponin components is liberated into the bloodstream after AMI. The differences in cutoff values and measured cTnI concentrations among the cTnI assays described in the literature can be partially explained by the use in some assays of antibodies that react with unstable epitopes that are rapidly deleted or damaged during proteolysis of cTnI in necrotic cardiac cells or in serum. Therefore, for sensitive and reproducible detection of cTnI in serum, we suggest using antibodies specific to the stable region of the cTnI molecule.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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  D. C Gaze and P. O Collinson Multiple molecular forms of circulating cardiac troponin: analytical and clinical significance Ann Clin Biochem, July 1, 2008; 45(4): 349 - 355. [Abstract] [Full Text] [PDF]
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 R. H. Christenson, S. H. Duh, F. S. Apple, G. S. Bodor, D. M. Bunk, M. Panteghini, M. J. Welch, A. H.B. Wu, S. E. Kahn, and for the American Association for Clinical Chemistr Toward Standardization of Cardiac Troponin I Measurements Part II: Assessing Commutability of Candidate Reference Materials and Harmonization of Cardiac Troponin I Assays Clin. Chem., September 1, 2006; 52(9): 1685 - 1692. [Abstract] [Full Text] [PDF]
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M. N. Fahie-Wilson, D. J. Carmichael, M. P. Delaney, P. E. Stevens, E. M. Hall, and E. J. Lamb Cardiac Troponin T Circulates in the Free, Intact Form in Patients with Kidney Failure Clin. Chem., March 1, 2006; 52(3): 414 - 420. [Abstract] [Full Text] [PDF]
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M. Panteghini Standardization of Cardiac Troponin I Measurements: The Way Forward? Clin. Chem., September 1, 2005; 51(9): 1594 - 1597. [Full Text] [PDF]
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S. Eriksson, T. Ilva, C. Becker, J. Lund, P. Porela, K. Pulkki, L.-M. Voipio-Pulkki, and K. Pettersson Comparison of Cardiac Troponin I Immunoassays Variably Affected by Circulating Autoantibodies Clin. Chem., May 1, 2005; 51(5): 848 - 855. [Abstract] [Full Text] [PDF]
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S. Eriksson, H. Halenius, K. Pulkki, J. Hellman, and K. Pettersson Negative Interference in Cardiac Troponin I Immunoassays by Circulating Troponin Autoantibodies Clin. Chem., May 1, 2005; 51(5): 839 - 847. [Abstract] [Full Text] [PDF]
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 M. Kemp, J. Donovan, H. Higham, and J. Hooper Biochemical markers of myocardial injury Br. J. Anaesth., July 1, 2004; 93(1): 63 - 73. [Abstract] [Full Text] [PDF]
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 J. A. Simpson, J. Van Eyk, and S. Iscoe Respiratory muscle injury, fatigue and serum skeletal troponin I in rat J. Physiol., February 1, 2004; 554(3): 891 - 903. [Abstract] [Full Text] [PDF]
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 S. B. Marston and C. S. Redwood Modulation of Thin Filament Activation by Breakdown or Isoform Switching of Thin Filament Proteins: Physiological and Pathological Implications Circ. Res., December 12, 2003; 93(12): 1170 - 1178. [Abstract] [Full Text] [PDF]
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S. Eriksson, M. Junikka, P. Laitinen, K. Majamaa-Voltti, H. Alfthan, and K. Pettersson Negative Interference in Cardiac Troponin I Immunoassays from a Frequently Occurring Serum and Plasma Component Clin. Chem., July 1, 2003; 49(7): 1095 - 1104. [Abstract] [Full Text] [PDF]
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R. Labugger, J. A. Simpson, M. Quick, H. A. Brown, C. E. Collier, I. Neverova, and J. E. Van Eyk Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry Clin. Chem., June 1, 2003; 49(6): 873 - 879. [Abstract] [Full Text] [PDF]
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A. Lavoinne, B. Cauliez, H. Eltchaninoff, C. Tron, and A. Cribier Release of Macromolecular Cardiac Troponin I Complex after Successful Percutaneous Transluminal Coronary Angioplasty in Acute Myocardial Infarction Clin. Chem., March 1, 2003; 49(3): 505 - 507. [Full Text] [PDF]
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 A. van der Laarse Hypothesis: troponin degradation is one of the factors responsible for deterioration of left ventricular function in heart failure Cardiovasc Res, October 1, 2002; 56(1): 8 - 14. [Abstract] [Full Text] [PDF]
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D. Uettwiller-Geiger, A. H.B. Wu, F. S. Apple, A. W. Jevans, P. Venge, M. D. Olson, C. Darte, D. L. Woodrum, S. Roberts, and S. Chan Multicenter Evaluation of an Automated Assay for Troponin I Clin. Chem., June 1, 2002; 48(6): 869 - 876. [Abstract] [Full Text] [PDF]
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 A. S. Jaffe Testing the wrong hypothesis: the failure to recognize the limitations of troponin assays J. Am. Coll. Cardiol., October 1, 2001; 38(4): 999 - 1001. [Full Text] [PDF]
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 R. C. Payne, B. I. Bluestein, D. L. Morris, R. Labugger, L. Organ, C. Collier, J. E. Van Eyk, and D. Atar Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Response Circulation, July 31, 2001; 104 (5): e26 - e27. [Full Text] [PDF]
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P. Venge, B. Lindahl, and L. Wallentin New Generation Cardiac Troponin I Assay for the Access Immunoassay System Clin. Chem., May 1, 2001; 47(5): 959 - 961. [Full Text] [PDF]
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A. S. Jaffe, J. Ravkilde, R. Roberts, U. Naslund, F. S. Apple, M. Galvani, and H. Katus It's Time for a Change to a Troponin Standard Circulation, September 12, 2000; 102(11): 1216 - 1220. [Full Text] [PDF]
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R. Labugger, L. Organ, C. Collier, D. Atar, and J. E. Van Eyk Extensive Troponin I and T Modification Detected in Serum From Patients With Acute Myocardial Infarction Circulation, September 12, 2000; 102(11): 1221 - 1226. [Abstract] [Full Text] [PDF]
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S. Altinier, M. Mion, A. Cappelletti, M. Zaninotto, and M. Plebani Rapid Measurement of Cardiac Markers on Stratus CS Clin. Chem., July 1, 2000; 46(7): 991 - 993. [Full Text] [PDF]
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J. Ravkilde Risk Stratification in Acute Coronary Syndrome Using Cardiac Troponin I Clin. Chem., April 1, 2000; 46(4): 443 - 444. [Full Text] [PDF]
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D. A. Morrow, N. Rifai, M. J. Tanasijevic, D. R. Wybenga, J. A. de Lemos, and E. M. Antman Clinical Efficacy of Three Assays for Cardiac Troponin I for Risk Stratification in Acute Coronary Syndromes: A Thrombolysis In Myocardial Infarction (TIMI) 11B Substudy Clin. Chem., April 1, 2000; 46(4): 453 - 460. [Abstract] [Full Text] [PDF]
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