From the Biomedical Proteomics Research Group, Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland, and
Department of Neurology, Geneva University Hospital, Geneva, Switzerland
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ABSTRACT |
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Ideally, a biomarker for the diagnosis, monitoring and prognosis of stroke should include at least the following characteristics: 1) it should be brain specific; 2) because of obvious difficulties to obtain CSF samples in patients with acute stroke, detection in serum is highly desirable; 3) it should appear very early, i.e. hours at the most, after the insult; 4) its peak level, alternatively the area under the curve of sequential concentrations, should reflect the extension of brain damage; 5) it should possibly distinguish between transient and established stroke, between hemorrhage and ischemia and perhaps between the necrotic and penumbra aspect of the brain lesion; finally, 6) it should be indicative of functional outcome. While such an ideal stroke biomarker does not exist yet, we investigate a novel stroke biomarker and compare it with S100 and NSE, the two molecules, which have been most extensively assessed for this purpose.
In this pilot study, we describe how fatty acid binding protein (FABP) has been identified as a novel diagnostic biomarker for stroke using a proteomics-based analysis of CSF from deceased patients as a model of massive brain damage. And we report on results obtained after serum FABP levels have been sequentially determined using an enzyme-linked immunosorbant assay (ELISA) in patients with acute stroke, as compared with S100 and NSE.
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EXPERIMENTAL PROCEDURES |
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The population used for the assessment in serum of the various markers detailed below included a total of 64 prospectively studied patients (Table I) equally distributed into three groups: 1) a control group including 14 men and 8 women aged 65 years (ranges: 3486 years) with no known peripheral or central nervous system condition; 2) a group of patients with acute myocardial infarction (AMI group) including 14 men and 6 women aged 65 years (ranges: 2990 years); the diagnosis of AMI was established in all cases by typical electrocardiography modifications and elevated levels of creatine kinase-MB (CK-MB; above a cut-off value of 9.3 ng/ml) and of troponin-I (above a cut-off value of 2 ng/ml); 3) a group of patients with acute stroke (stroke group) including 14 men and 8 women aged 65 years (ranges: 3087 years); the diagnosis of stroke was established by a trained neurologist and was based on the sudden appearance of a focal neurological deficit and the subsequent delineation of a lesion consistent with the symptoms on brain computed tomography (CT) or magnetic resonance imaging (MRI) images, with the exception of transient ischemic attacks (TIAs), where a visible lesion was not required for the diagnosis. The stroke group was separated according to the type of stroke (ischemia (16 patients) or hemorrhage (6 patients)), the location of the lesion (brainstem (4 patients) or hemisphere (18 patients)), and the clinical evolution over time (TIA when complete recovery occurred within 24 h (5 patients) or established stroke when the neurological deficit was still present after 24 h (11 patients)). The diagnostic of TIA was further supported by the absence of any recent lesion on the CT or MRI scans in all five TIA patients, whereas all 11 patients with established ischemic stroke had a definite and recent lesion on neuroimaging exams. The size of the lesion varied from lacune to large infarct.
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CSF Two-dimensional Gel Electrophoresis (2-DE)
All reagents and apparatus used have been described in detail elsewhere (9). Two hundred fifty microliters of CSF was mixed with 500 µl of ice-cold acetone (-20 °C) and centrifuged at 10,000 x g at 4 °C for 10 min. The pellet was mixed with 10 µl of a solution containing 10% SDS (w/v) and 2.3% dithioerythritol (DTE) (w/v). The sample was heated to 95 °C for 5 min and then diluted to 60 µl with a solution containing 8 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) (w/v), 40 mM Tris, 65 mM DTE, and a trace of bromphenol blue. The whole final diluted CSF sample corresponding to 45 µg was loaded in a cup at the cathodic end of the immobilized pH gradient (IPG) strips. 2-DE was performed as described previously (10). In brief, the first dimensional protein separation was performed using a commercial 18-cm nonlinear IPG going from pH 3.5 to 10 from Amersham Biosciences (Uppsala, Sweden). The second dimensional separation was performed onto in-house manufactured vertical gradient slab gels (916% T, 2.6% C). Analytical gels were then stained with ammoniacal silver staining (11). Gels were scanned using a laser densitometer (Amersham Biosciences). 2-DE computer image analysis was carried out with the MELANIE 3 software package (12). Spots were detected and quantified automatically. The optical density (OD), the area, and the volume were computed and directly related to protein concentration. The relative OD and relative volume were also calculated in order to correct for differences in gel staining. Differential analysis (>200%) and Student t test (p < 0.05) using the relative volume of each spot (>0.05%) allowed the detection of significantly overexpressed polypeptides with a minimum ratio of 2.
Mass Spectrometry Identification
Differentially expressed spots were found through the comparison of analytical gels of deceased versus healthy CSF (n = 4). Spots of interest were analyzed by peptide mass fingerprinting using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (Voyager STR MALDI-TOF-MS; PerSeptive Biosystems, Framingham, MA) (10) and identified through database using the PeptIdent tool (www.expasy.ch/sprot/peptident.html).
FABP Measurement
Heart-FABP (H-FABP) levels were measured in plasma by a sandwich ELISA. Specificity of anti H-FABP antibodies was tested by Western 2-DE blots. They specifically detected the H-FABP spot and not the other forms (data not shown). A 96-well polystyrene microplate (Nunc, Polylabo, Switzerland) was coated with 100 µl/well polyclonal goat anti-human muscle FABP (Spectral Diagnosis HC, Ontario, Canada), 20.4 µg/ml in carbonate buffer 0.1 M, pH 9.6, overnight at 4 °C. The plate was automatically washed with phosphate-buffered saline (PBS; 15 mM Na2PO4-120 mM NaCl-2.7 mM KCl, pH 7.4; Sigma, St. Louis, MO) on a BioRad NovapathTM washer (Hercules, CA). Every washing step was performed with fresh PBS. Nonspecific binding sites were blocked with 200 µl/well 2% casein (w/v) in carbonate buffer for 2 h at 37 °C. After the washing step, the crude samples were pipetted in duplicate at 100 µl/well. The plate was incubated 2 h at 37 °C. After the washing step, 100 µl/well of mouse anti-human H-FABP (clone 66E2; HyCult Biotechnology b.v., Uden, Netherlands), 0.3 µg/ml in PBS-1% BSA (w/v), were incubated for 1 h at room temperature with shaking. After the washing step, 100 µl/well of phosphatase-labeled anti-mouse immunoglobulins (Dako, Glostrub, Denmark), 15 µg/ml in PBS, were incubated 1.5 h at room temperature with shaking. After the washing step, 50 µl/well of phosphatase substrate, 1.5 mg/ml paranitrophenylphosphate in diethanolamine, were incubated 30 min. Reaction was stopped with 100 µl/well NaOH 1 M. Color development was assessed with a microplate reader, MileniaTM kinetic analyzer (Diagnostic Products Corporation, Los Angeles, CA), at a wavelength of 405 nm.
CK-MB and Troponin-I Measurement
Plasma samples were centrifuged at 1500 x g for 15 min, and aliquots were stored at -20 °C. Serum CK-MB and troponin-I levels were determined using a fluorescent microparticle enzyme immunoassay with an automated chemical analyser AxSYMTM system (Abbott Laboratories, Abbott Park, IL). The formation rate of fluorescent products was directly proportional to the amount of troponin-I in the sample. The detection limit for troponin-I was 0.3 µg/liter. CK-MB measurement is proportional to the amount of fluorescent probes, and the detection limit was 0.7 µg/liter.
NSE and S100 Measurement
Similar to H-FABP measurements, NSE and S100B were assayed in the four serial plasma samples of the stroke group. The SMART S100 and SMART-NSE ELISA kits were used and commercialized both by Skye PharmaTech Inc. (Ontario, Canada). The detection limits for NSE and S100B were 1 µg/liter and 0.01 µg/liter, respectively.
Statistical Analysis
H-FABP levels were expressed in OD values as mean ± SD. Because recombinant H-FABP was not available, external calibration and limit of sensitivity could not be performed to express results as concentration units (ng/ml). Troponin-I and CK-MB levels were expressed in ng/ml. Because plasma H-FABP, troponin-I, and CK-MB concentrations did not fulfill the criteria for a Gaussian distribution in neither of the normal, stroke, and AMI populations according to the Kolmogorov-Smirnov test, comparisons between the three groups was carried out using the nonparametric Kruskall-Wallis test with the post hoc Dunns procedure. Comparisons between the stroke subgroups defined above were made by means of the Mann-Whitney U test, and longitudinal assessment of H-FABP concentrations over time were analyzed using the repeated measures analysis of variance. Reference limits for H-FABP aiming at distinguishing stroke versus normal patients were delineated using receiver operating characteristic (ROC) curves (Analyze-ItTM software for Microsoft ExcelTM; Microsoft, Redmond, WA) (13). Statistical significance was set at p < 0.05.
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RESULTS |
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In the stroke group, seven false negative results were found with H-FABP levels below the cut-off value of OD 0.531 at any time point following the neurological event. Of these seven patients, two had a negative MRI and a rapid and complete recovery of their neurological deficits within 24 h consistent with a TIA, and two have had a lacunar stroke on MRI images, one located in the brainstem. While TIA and lacunar stroke may explain false negative results in a majority of patients, no explanation was consistently found for the three remaining stroke patients with low H-FABP levels.
Sequential determinations of H-FABP level after stroke showed that 10 out of 15 (67%) H-FABP-positive stroke patients had a very early increase of H-FABP levels (<12 h). Moreover, as shown in Fig. 3, when all stroke patients were considered, the mean H-FABP concentrations decreased steadily after the insult, the highest value being found before 12 h. The differences between the initial measurement and the <48 h and afterward measurements were significant (analysis of variance, p < 0.05). When H-FABP levels were compared between the different subgroups of the stroke group, no statistically significant differences were found. H-FABP levels were similar for ischemia (0.955 + 0.668, n = 15) versus hemorrhage (1.346 + 1.139, n = 7) strokes, and for hemispheric (0.987 + 0.783, n = 18) versus brainstem (1.493 + 1.080) strokes, but the statistical power of the analyses was limited by the small size of the samples to be compared. However, when comparing established strokes versus TIAs, the former (1.2002 + 0.892) showed nearly twice as high H-FABP levels as the latter (0.652 + 0.499), although this difference failed to reach significance (Mann-Whitney U test, p = 0.24). Interestingly, H-FABP levels were found moderately increased in three of the five TIA patients, despite the absence of any detectable lesion on the CT or MRI scans, suggesting that the marker may be sensitive to extremely short periods of ischemia.
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DISCUSSION |
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In a first step, identification of H-FABP was carried out using an original approach based on proteomics analysis of CSF samples obtained at autopsy from deceased patients, as a model of massive brain damage, which were compared with CSF from healthy living subjects. Comparisons of 2-DE maps showed two distinct spots highly expressed in the CSF of deceased patients, both of which being definitely identified by various MS techniques as two forms of FABP. The FABP family encompasses at least eight different proteins (14) functionally involved in intracellular transport systems, oxidation of fatty acids, and membrane lipids trafficking (15, 16). Brain-FABP (B-FABP) is localized exclusively in neuronal and glial cells (17), whereas H-FABP has a more widespread distribution including, in addition to brain, myocardial muscle, endothelial cells, lung, and kidney (18). B-FABP and H-FABP have a 69% sequence homology. In fact, several forms of FABP with variable degree of sequence homology are present in both the brain and the heart, but they seem to have distinct distribution and different functions.
The second step of the study was to develop and to apply a quantitative FABP detection method in the serum for the diagnosis of stroke. The study was designed to discriminate stroke patients not only from normal controls but also from patients with AMI, because H-FABP has been recently demonstrated to increase in this condition. A sandwich ELISA was therefore developed using a human anti-H-FABP (clone 66E2) (19) monoclonal antibody in combination with a polyclonal anti-human FABP antibody. In another study, the H-FABP assay has been shown to allow adequate detection of FABP in the serum after heart injury (20). Ideally, anti-B-FABP antibodies would have been preferred, but those antibodies are currently not commercially available. When applied to two groups of clinically well-characterized patients, including patients with stroke and subjects for whom a blood draw was performed for unrelated conditions, the H-FABP demonstrated a specificity of 22 patients out of 22 (cut-off at OD 0.531), a sensitivity for the diagnosis of stroke of 15 patients out of 22, when controls were taken as the comparative variable. H-FABP was compared with the two other well-established markers for stroke in the same samples of patients, NSE and S100B proteins.
S100 is an acidic, calcium-binding protein present in the brain, striated muscle, heart, and kidney. The isoform S100B is a major cytosolic constituent of glial and Schwann cells. S100 protein has been reported as a useful marker for stroke, head injury, brain metastases, and central nervous system complications of cardiac surgery (2123). NSE is a dimeric glycolytic enzyme that catalyzes the interconversion of phosphoglycerate and phosphoenolpyruvate. It is found primarily in the cytoplasm of neurons, as -NSE, and neuroendocrine cells (24). NSE has been reported as a useful marker not only for stroke and head injury but also for the prognosis of neuroblastoma (25), neuroendocrine tumors (26), and lung carcinoma (27). Several studies have shown that S100 and NSE are elevated not only in the CSF but also in the blood of stroke patients. Moreover, in a recent comparative study (28), Fassbender and colleagues have demonstrated that S100 and NSE significantly increased in the serum of stroke patients as early as 8 h following the cerebrovascular event, though only in a small fraction of cases. Unlike NSE, S100 levels correlated with the size of the infarct, as estimated by CT-scan-based volumetry, and with clinical outcome. In contrast, an increased level of S100 within 24 h was found only in 14 out of 22 patients yielding a sensitivity of 64% at day 1 post-stroke. Overall, in another study (29), sensitivity and specificity for stroke have been found to range from 44 to 67% for S100.
In comparison to NSE and S100, all validity measures turned out to be much higher for H-FABP with the exception of the specificity of S100B, which was found almost as high as that of H-FABP. The kinetics of FABP release in the blood, as determined by serial blood sampling after stroke, showed that FABP increases very early after the cerebrovascular event, even within a few hours in some patients. This early rise seems consistent because elevated levels of H-FABP were detected within 24 h in 80% of the H-FABP-positive stroke patients and a delayed H-FABP peak after 24 h was observed in only a few cases. Although this aspect was not fully explored in the present study, NSE and S100B having been measured only at admission, it appears that the very early, that is within 12 h, elevation of H-FABP has been found by others in only a minority of patients with S100B and in none with NSE (28, 30). In that respect, H-FABP has clearly the potential of being not only a valid but also a very early marker of stroke.
Unsurprisingly, the H-FABP levels did not correlate with either the type of stroke (hemorrhage or ischemia) or its location (hemisphere or brainstem), but it is noteworthy that the number of patients was very small in some categories. In contrast, although the size of the lesion was not quantitatively assessed, we found that more than half of the H-FABP falsely negative stroke patients have suffered from either TIAs or small lacunar lesions. There was a trend for TIAs (n = 5) to have lower H-FABP levels than established strokes (n = 11). Taken together, these preliminary results prompt further studies looking at potential correlation between H-FABP levels and lesion size to be undertaken.
Because AMI patients have also elevated levels of H-FABP (20), the assessment of stroke patients must exclude a concomitant acute heart condition, which may contribute to the net value of H-FABP and yield erroneous clinical conclusions. Thus, the H-FABP assay has to be performed along with a dosage of troponin-I (31) or CK-MB. Because the latter is not heart-specific and may increase in response to any muscle damage (32), we propose to combine the H-FABP assay with the troponin-I assay. In this study, we demonstrated that this combination could clearly separate the AMI from the stroke population with almost no overlap, except in rare instances where the two conditions may develop simultaneously.
We believe that the H-FABP assay in its present form still needs further refinements before a more widespread use for the diagnosis of stroke. First, the ELISA was performed in 8 h, which is slower than the S100B assay (29). The use of a rapid microparticle-enhanced turbidimeric immunoassay, already developed for the assessment of H-FABP levels in AMI (33), should drastically shorten the availability of the results at bedside. The full automation in a widely used clinical chemistry analyzer like the COBAS MIRA Plus system from Roche (Rotkreuz, Switzerland) or the AxSYM system from Abbott Laboratories should be possible and applied for routine testing. Second, the additional need of the troponin-I assay might become unnecessary provided an ELISA using antibodies directed toward the B-FABP form be manufactured in the future. Finally, further clinical studies are still warranted to definitely confirm and validate the preliminary data presented here. These studies will have to include a large and well-categorized cohort of patients, to establish the sensitivity of the test using MRI-based volumetry of stroke lesion and perhaps to approach the early prognosis of any vascular brain insult.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, October 26, 2003, DOI 10.1074/mcp.M300066-MCP20
1 The abbreviations used are: CSF, cerebrospinal fluid; AMI, acute myocardial infarction; CK-MB, creatine kinase-MB; FABP, fatty acid binding protein; H-FABP, heart-FABP; NSE, neuron-specific enolase; ELISA, enzyme-linked immunosorbant assay; CT, computed tomography; MRI, magnetic resonance imaging; TIA, transient ischemic attack; 2-DE, two-dimensional gel electrophoresis; DTE, dithioerythritol; IPG, immobilized pH gradient; PBS, phosphate-buffered saline; OD, optical density; ROC, receiver operating characteristic; MS, mass spectrometry; B-FABP, brain-FABP.
* This work was supported in part by Proteome Science (U.K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Biomedical Proteomics Research Group, Laboratoire Central de Chimie Clinique, Hôpital Universitaire de Genève, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. Tel.: +41-22-372-73-74; Fax: +41-22-372-73-99; E-mail: sanchez{at}dim.hcuge.ch
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REFERENCES |
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