| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Lipids, Lipoproteins, and Cardiovascular Risk Factors |
Department of Pathology and Immunology, Division of Laboratory Medicine, Washington University School of Medicine, St. Louis, MO.
aAddress correspondence to this author at: Department of Pathology and Immunology, Division of Laboratory Medicine, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8118, St. Louis, MO 63110. Fax 314-454-5208; e-mail ladenson{at}labmed.wustl.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: We performed gene array analyses on a mouse model to look for biomarkers that are both preferentially and abundantly produced in the brain. Via bioinformatics databases, we identified the human homologs of genes that appeared abundant in brain but not in other tissues. We then confirmed protein production of the genes via Western blot of various tissue homogenates and assayed for one of the markers, visinin-like protein 1 (VLP-1), in plasma from patients after ischemic stroke.
Results: Twenty-nine genes that were preferentially and abundantly expressed in the mouse brain were identified; of these 29 genes, 26 had human homologs. We focused on 17 of these genes and their protein products on the basis of their molecular characteristics, novelty, and/or availability of antibodies. Western blot showed strong signals in brain homogenates for 13 of these proteins. Tissue specificity was tested by Western blot on a human tissue array, and a sensitive and quantitative sandwich immunoassay was developed for the most abundant gene product observed in our search, VLP-1. VLP-1 was detected in plasma of patients after stroke and in cerebrospinal fluid of a rat model of stroke.
Conclusions: The use of relative mRNA production appears to be a valid method of identifying possible biomarkers of tissue injury. The tissue specificity suggested by gene expression was confirmed by Western blot. One of the biomarkers identified, VLP-1, was increased in a rat model of stroke and in plasma of patients after stroke. More extensive, prospective studies of the candidate biomarkers identified appear warranted.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
700 000 patients in the US have a new or recurrent stroke each year(1). The clinical diagnosis of stroke by specialists is reasonably accurate. On clinical grounds but in general practice, up to 20% of the patients suspected of stroke have a different diagnosis(2). The evaluation of patients with suspected stroke, an acute brain injury, would be greatly aided by the availability of a readily measured biomarker such as is available for acute coronary syndrome and myocardial infarction, e.g., troponin I or troponin T(3). Such a brain biomarker for brain injury might also be useful in chronic neurodegenerative diseases.
There have been several studies of possible biomarkers of acute brain damage such as stroke, with S-100B, neuron-specific enolase (NSE),1 glial fibrillary associated protein (GFAP), and myelin basic protein (MBP) generating the most interest.
S-100B(4) is found at high concentrations in glial and Schwann cells. Numerous reports(4) show that S-100B is increased in blood and cerebrospinal fluid (CSF) after traumatic brain damage, stroke, and a variety of neurodegenerative diseases. The increase of S-100B in serum after acute ischemic stroke peaks at 2–3 days, and it can be observed at 12 h(5). S-100B does not appear to be specific for brain injury; high amounts of S-100B are released in response to trauma of muscle, fat, and bone, and values in trauma without head injury are also increased(6)(8).
NSE(9) has been found to increase after brain injury, such as stroke(10)(11), but it is also increased in cancers of neuroendocrine origin, such as small-cell lung cancer, neurobastoma, carcinoid tumors, and melanoma(12). In addition, hemolysis has been reported to cause positive interference in at least one of the commercially available procedures for its measurement(13).
GFAP is a monomeric intermediate filament protein thought to be produced almost exclusively in astrocytes. It has been reported to be increased after stroke, with peak values around 3 days(14). Recently, promising results for fatty acid binding proteins after ischemic stroke have been reported as well(15).
Other efforts to identify stroke have included the monitoring of hemostatic function. D-dimer appears to be more sensitive than other hemostatic markers, such as fibrinopeptide A and B-thromboglobulin, particularly for cardioembotic stroke(16)(17).
Inflammatory markers have been studied as well, including various cytokines(18) and matrix metalloproteinases (MMPs), particularly MMP-9 (gelatinase B)(18)(19)(20); MMP-9 is associated with hemorrhagic transformation(20).
Recently, several studies combining various biomarkers have been reported. McGirth et al.(21) reported that a combination of serum von Willebrand factor, MMP-9, and vascular endothelial growth factor could predict the onset of cerebral vasospasm and subarachoid hemorrhage. Another study reported that S-100B, B-type neurotrophic growth factor, von Willebrand factor, MMP-9, and monocyte chemotactic protein-1 could be effectively used. The authors stated that if any 3 of the 5 biomarkers exceeded their reference range, then the sensitivity was 92% and specificity was 93% for ischemic stroke for samples taken within 6 h from symptom onset(22). Another study screened 26 biomarkers and found that 4 were highly correlated with stroke: S100-B, MMP-9, vascular cell adhesion molecule, and von Willebrand factor(23).
Proteomic approaches have been used with only moderate success to identify potential brain biomarkers(24)(25)(26)(27). A consortium has been formed, The Human Brain Proteome Project, to facilitate the identification of brain proteins that may be involved in disease (http://www.hupo.org).
Despite a large amount of effort, there have been few successful systematic approaches to the discovery of new brain biomarkers for disease such as stroke.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Brain, liver, spleen, kidney, skeletal muscle, lung, pancreas, heart, and small intestine from 3 (2 male and 1 female) C57BL/6 mice (Jackson Laboratory), ages 4–6 weeks, were obtained by careful dissection. The organ samples were snap frozen in liquid nitrogen and processed as described(28) to isolate RNA. The quality of the RNA was confirmed by (a) spectrophotometry of RNA with an A260/A280 ratio >1.9, and (b) a 28S/18S ratio of extracted RNA >1.4, as observed by RNA LabChip (Agilent 2100 Bioanalyzer RNA 6000 LabChip reagent set).
Ten micrograms of the total RNA was converted to double-stranded cDNA with an oligo(dT) primer containing the T7 promoter and used to prepare biotinylated cRNA with the Bioarray HighYield reagent set (Enzo) according to the manufacturer’s directions. The biotinylated cRNA probes were fragmented and applied as described(29)(30) to Mouse MU74A (Version 1) Genechip® arrays (Affymetrix). The overall fluorescence intensity across each chip was scaled to 1500 with Affymetrix analysis software Microarray Suite. The data were transferred to a Microsoft Excel worksheet. One selection criterion was gene expression, with mean difference values >10 000 in the brain (i.e., signal intensity of each mRNA as computed by Affymetrix software that calculates the difference between the perfect match and mismatch oligonucleotides for each nucleotide sequence represented on the gene chip array). The mean difference values as described above reflect an expression signal well above "background" and suggest sufficient abundance of the expression products to provide a suitable marker. The second criterion was the preferential expression of the gene in the brain. In this case, we considered the gene to be preferentially expressed in the brain if its RNA expression value was 10-fold higher than spleen, kidney, skeletal muscle, lung, pancreas, heart, and small intestine. For the genes that met these 2 criteria, we then perused their human homologs in the bioinformatics databases Entrez Gene (http://ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene), Online Mendelian Inheritance in Man (OMIM; http://ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM), and Unigene (http://ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene).
preparation of antibodies
Nucleotide sequences encoding putative marker proteins were inserted into pGEX or pET vectors for production of protein in Escherichia coli. Proteins from pET vectors were purified with Qiagen Ni-NTA according to the manufacturer’s protocol (The Qiaexpressionist 06/2003; Qiagen). Those from the pGEX vector were purified with immobilized glutathione from Pierce following the manufacturer’s protocol. In some cases, plasmids were sent to GenWay Biotech, Inc., for large-scale production of protein from E. coli. These included pET 100 GAD67, pET 102 GAD67, pET 28 Zygin, and Zygin I. Quality control of the produced protein included analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, N-terminal Edman sequencing, and mass spectrometry as appropriate(31). Protein concentration was estimated by absorbance at 280 nm with calculated extinction coefficients from the protein sequence plus construct obtained at the Swiss-Prot web site (http://www.expasy.org/sprot) and agreed with visual inspection of protein-stained bands in sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Antipeptide immunogens were prepared as described by Li et al.(31). Rabbits were immunized at Harlan Bioproducts for Science, Inc. Rabbit serum was immunopurified over an affinity column containing the cognate peptide or protein antigen.
Mice were immunized with 25 µg protein/mouse immunogen in MPL-TDM adjuvant (Sigma-Aldrich) followed by at least 2 boosts in adjuvant and 1 boost in phosphate-buffered saline (150 nm NaCl, 50 nm sodium phosphate pH 7.2) 3 days before fusion. Armenian hamsters were immunized with 100 µg protein/hamster in complete Freund’s adjuvant followed by boosts in incomplete Freund’s adjuvant and a final boost in phosphate-buffered saline. All fusions were performed at the Washington University School of Medicine Hybridoma Center. Monoclonal antibodies were purified from culture media on protein A-agarose or goat antimouse IgG-agarose or produced in ascites by Maine Biotechnology. All purified antibodies were dialyzed vs phosphate-buffered saline/azide, pH 7.2, and protein concentration was estimated from absorbance at 280 nm with an absorptivity of 1.4 (L · g–1 · cm–1). Subclass determination for monoclonal antibodies was performed with the IsoStrip reagent set from Roche. Minimum epitope assignment was based on immunostaining of ABIMED spot peptide arrays prepared at the MIT Biopolymers Facility. Each spot comprised a 10-mer contiguous peptide, and depending on the number of residues in the antigen of interest, either a 1-, 2-, or 3-residue offset was used to cover the entire antigen sequence. For example, for a 1-residue offset, spot 1 contains sequence 1–10, spot 2 contains sequence 2–11, spot 3 contains sequence 3–12, etc. Epitope mapping was attempted in some cases without success (noted as not established), and in others, it was not experimentally determined (noted as not determined).
Monoclonal antibodies were raised against visinin-like protein 1 (VLP-1) with a combination of DNA and protein injections. The vector VR1012 (Vical Inc.) and certain sequences from CTLA4Ig were used for the DNA injections. VR1012 has been optimized for protein production in mouse skeletal muscle, whereas sequences contained in CTLA4Ig were previously shown to greatly increase the antibody response in mice(32).
Standard procedures used at the Hybridoma Center at Washington University in St. Louis for the creation and maintenance of the fusions were followed.
Antibodies against some of the candidate biomarkers, used to further characterize the utility of the protein products of candidate genes in the detection of brain injury, were purchased through commercial sources (Chemicon, Santa Cruz Biotechnology, Synaptic Systems, or Abnova Corp.).
western blot analysis
To confirm the presence of the proteins encoded by the candidate genes, we performed Western blots on unaffected human brain homogenate (Geno Technology, Inc.). Likewise, to assess the specificity of the coded protein for brain, we used a human tissue array (Geno Technology, Inc.; 50 µg per tissue) and followed the manufacturer’s specifications for the Western blot. The human tissue array included liver, brain, lung, kidney, spleen, testis, ovary, heart, pancreas, uterus, breast, cervix, rectum, prostate, thyroid, laryngopharynx, stomach, and skin. For some of the Western blots, different antibodies were mixed 1:1 before use so that more epitopes would be detected.
rat stroke model
A rat stroke model was used to assess the appearance of VLP-1 in CSF as a function of time. Ischemia was induced in femoral vein–cannulated male Sprague–Dawley rats (Charles-River) by the previously described permanent (p) or transient (t) middle cerebral artery occlusion (MCAO) intraluminal filament method with some modifications(33). Both pMCAO and tMCAO methods were used so that different degrees of injury could be obtained. As controls, some animals were subjected to sham operations (no filament placed). In brief, a midline incision was performed, and the right common, internal, and external carotid arteries were exposed. The external carotid and occipital arteries were ligated. The common carotid artery was ligated, and the internal carotid artery was temporarily closed. A small incision was made in the common carotid artery and a filament (3.0 Ethilon; heat-blunted tip) was inserted into the internal carotid artery through the common carotid artery. The filament was advanced 17.5 mm to occlude the origin of the middle cerebral artery, either permanently or transiently (60–90 min). The filament was secured in place in the right common carotid artery with a surgical nylon suture. After surgery, anesthesia (isoflurane) was discontinued, and the animals were allowed to recover.
CSF was collected by cisternal puncture when the animals were killed at either 2 h [sham (n = 4), pMCAO (n = 5), tMCAO (n = 3)] or 24 h [sham (n = 5), pMCAO (n = 4), tMCAO (n= 3)] postsurgery. Groups were compared by an unpaired t-test (GraphPad Prism 4 software) at a significance of P = 0.05. Blood (EDTA plasma) was collected, when possible, at 0, 2, 4, 8, and 24 h postsurgery (sham- and MCAO-operated).
quantitative assays
Sandwich immunoassays were developed for VLP-1 (monoclonal antibody 3A8–1 as a capture antibody and rabbit antibody R3471 for detection), GAD67 (rabbit antibody Rb4043 as capture and Rb4610 for detection), and zygin (mouse monoclonal IG4.4 as capture and hamster monoclonal 4G3.1 as detection) with ruthenium-labeled detection antibodies and electrochemiluminescent detection (Meso Scale Discovery). The lower limit of detection for the human plasma VLP-1 assay was set to be the first point on the calibration curve (0.04 µg/L), which was at least 2 SDs above the background. In addition, 3 quality control samples were prepared by adding to heparinized plasma various amounts of postmortem CSF (high concentration of VLP-1), and were measured in every run. The interassay CVs for these samples were 
15% (n = 10).
Human heparinized plasma samples were retrospectively collected and frozen from excess clinical laboratory specimens that were obtained for routine care from a group of patients who presented to Barnes-Jewish Hospital with an acute neurological deficit between March and November of 2002. The medical histories of these patients were reviewed by one of us (J.-M.L.) without knowledge of the VLP-1 results, and patients with a discharge diagnosis of ischemic stroke and a clear time of onset of stroke were identified. Eighteen patients met this criterion, and their samples were analyzed.
Human heparinized samples were also obtained from healthy males and females and used as controls. These samples were not age- or sex-matched to the stroke patients.
Excess plasma samples from the Clincial Chemistry Laboratory were obtained with the approval of the Washington University IRB committee. Human CSF postmortem samples were obtained from Analytical Biological Services.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Mr 70 000 cutoff was selected because albumin, a protein abundant in the plasma, is known to enter the brain after injury because of damage to the blood–brain barrier(34)(35)(36), suggesting that this cutoff value for the egress of proteins from the brain might also be similar. The 20 genes and their gene products are listed in Table 1
in order of the mRNA production of their mouse homologs in brain.
|
Antibodies for the gene products of SLC32A1, VMP, and GABRG2 are not commercially available, nor had we generated antibodies as of the time of preparation of this report, and they could not be evaluated further. Thus, 17 possible brain-specific biomarkers were selected for further investigation. A summary of the antibodies used to further assess the candidate biomarker is shown in Table 2
(antibodies prepared internally) and Table 3
(antibodies purchased commercially). The immunogen was in the protein form unless otherwise indicated.
|
|
western blot analysis of gene products
Of the 17 gene products for which antibodies were available, Western blots showed strong signals from brain extracts for 13 of them. We did not find any signal for the gene products of the genes family member 1, proteolipid protein 1, neuronatin, or olfactomedin 1 (ZIC1, PLP1, NNAT, or OLFM1) with the antibodies described in Table 3
. We then tested the reactivity of the 13 gene products found in brain in different tissues by Western blot. The tissue Western blot for VLP-1 is shown in Fig. 1
. We scored the intensity of the Western blots of the tissue arrays for the 13 candidate gene products as follows: 5, very strong; 4, strong; 3, moderate; 2, weak; 1, very weak. Results are summarized in Table 4
. Western blots were also performed on human postmortem CSF at various dilutions, and the intensity of the bands compared visually with standards of the respective proteins. Zygin, neuroserpin, and GAD67 were below the limit of detection of this method (<2.5 µg/L), and the intensity of the VLP-1 band was between the 25 and 250 µg/L VLP-1 standards. This range was in agreement with the quantitative assay determination of 17 µg/L for VLP-1. The quantitative assays detected GAD67 at 0.3 µg/L and zygin at 0.6 µg/L in the postmortem spinal fluid. No quantitative assay for neuroserpin was available at the time of these experiments.
|
|
rat stroke model
VLP-1 was not detected in the CSF of sham-operated rats, whereas VLP-1 was found in high abundance in the CSF of MCAO-operated rats 24 h post injury (Fig. 2
), suggesting neuronal damage. The concentration [mean (SE)] of VLP-1 in the CSF of pMCAO rats was higher [16.8 (6.5 µg/L)] than in tMCAO [6.0 (2.4 µg/L)], although the difference was not statistically different (P = 0.23, unpaired t-test). No VLP-1 was detected in the CSF of sham- or tMCAO-operated rats at 2 h postinjury. Interestingly, no VLP-1 were detected in EDTA plasma of either sham- or MCAO-operated rats at any of the time points tested up to 24 h postinjury.
|
determination of vlp-1 in stroke patients
Thirty-nine plasma samples from unaffected donors were analyzed, and 37 of them were below the limit of detection of 0.04 µg/L; the others had low values of 0.08 and 0.11 µg/L. Of the 18 patients with confirmed stroke, only 2 had no samples with detectable VLP-1; most were considerably higher. Shown in Fig. 3
are the results at various times after stroke onset for 18 patients.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Through our analysis, we were able to identify genes that are preferentially and abundantly expressed in the mouse brain, and through the use of bioinformatics, were able to confirm the presence of the homologs of these genes in the human brain and gained further insight into their molecular characteristics. Western blot analyses showed expression in the brain with reasonable specificity for 10 of 13 of the gene products.
Furthermore, we developed sandwich immunoassays to detect the presence of some of these proteins in postmortem CSF and the CSF and sera of rats that had undergone stroke. Postmortem CSF was tested because it should reflect a condition in which the brain has been deprived of oxygen for a period of time long enough to cause extensive cell death. We put special emphasis on the development of a sensitive assay for VLP-1, given its ideal molecular characteristics (cytoplasmic protein of low molecular weight) and its high abundance in brain (most abundant mRNA in our search). Also, VLP-1, a calcium-sensor protein found in mammalian CNS neurons, has been found to have a widespread distribution in the brain and to be abundant in all brain areas except the caudate–putamen by in situ mRNA hybdridization studies(37). We found similar intensity for Western blots of various human brain regions for VLP-1 (TB57 brain tissue region blot; Geno Technology) (data not shown). We were able to demonstrate that VLP-1 was present in the CSF of an animal model of stroke and in the blood of stroke patients, as well as in CSF obtained postmortem. It was interesting that we were not able to detect VLP-1 in the serum of a rat model of stroke. This could be caused by several factors; we observed a decrease in the detection limit of our electrochemiluminescence assay with rat EDTA plasma compared with human heparinized plasma. Also, 24 h postinjury may not be long enough to allow VLP-1 to dissipate from the CSF into the bloodstream of the injured rats. These rats showed no increase in VLP-1 in CSF 2 h postinjury but did at 24 h. Similarly, 24 h may not be sufficient time for VLP-1 to appear in the blood. It would be interesting to have blood measurements in these animals beyond 24 h, but it will be difficult because there are complications in keeping these animals alive beyond 24 h postinjury. One study supports the idea of delayed appearance of neuronal injury biomarkers into the CSF, as has been reported for other biomarkers(38). This study looked at CSF markers after acute stroke and saw no increase of total Tau in CSF at day 0–1 after acute ischemic stroke, but we did see increase at days 2–3, peaking after 1 week and returning to unaffected levels after 3–5 months. Our studies of VLP-1 samples from patients after stroke are promising, but they were from samples obtained retrospectively from excess samples obtained for their routine clinical care and therefore were not systematic as regards time after stroke.
Our results demonstrate that the identification of possible cell damage biomarkers via a genomic approach is a viable one and likely generally applicable for other cell damage markers. This approach has led to the identification of several potential biomarkers of brain injury. We are currently developing sensitive assays for these markers and further systematically assessing their ability to assess brain damage in human disease.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
4 Current affiliation: Merck & Co., Inc., Rahway, NJ 07065-0900.
2 Human genes: VSNL1, visinin-like 1; SNAP25, synaptosomal-associated protein, Mr 25; GAD1, glutamate decarboxylase 1 (brain, Mr 67); MOBP, myelin-associated oligodendrocyte basic protein; SYT1, synaptotagmin I; TUBB4, tubulin ß4; FEZ1, fasciculation and elongation protein 
1 (zygin I); GLRB, glycine receptor ß; VMP, ;Vesicular membrane protein p24; OLFM1, olfactomedin 1; ZIC1, zic family member 1 (odd-paired homolog, Drosophila); PACSIN1, protein kinase C and casein kinase substrate in neurons 1; PLP1, proteolipid protein 1 (Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated); INA, internexin neuronal intermediate filament protein 
; SLC32A1, solute carrier family 32 (
-aminobutyric acid vesicular transporter), member 1; SERPINI1, serine (or cysteine) proteinase inhibitor, clade I (neuroserpin), member 1; NNAT, neuronatin; GABRG2, -aminobutyric acid A receptor 2; VAMP2, vesicle-associated membrane protein 2 (synaptobrevin 2); NRGN, neurogranin (protein kinase C substrate, RC3).
1 Nonstandard abbreviations: NSE, neuron-specific enolase; GFAP, glial fibrillary-associated protein; MBP, myelin basic protein; CSF, cerebrospinal fluid; MMP, matrix metalloproteinase; MCAO, middle cerebral artery occlusion.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  O. F. Laterza, L. Lim, P. W. Garrett-Engele, K. Vlasakova, N. Muniappa, W. K. Tanaka, J. M. Johnson, J. F. Sina, T. L. Fare, F. D. Sistare, et al. Plasma MicroRNAs as Sensitive and Specific Biomarkers of Tissue Injury Clin. Chem., November 1, 2009; 55(11): 1977 - 1983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Foerch, J. Montaner, K. L. Furie, M. M. Ning, and E. H. Lo Invited Article: Searching for oracles?: Blood biomarkers in acute stroke Neurology, August 4, 2009; 73(5): 393 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. S. Weiss, K. K.W. Wang, J. G. Allen, M. E. Blue, L. U. Nwakanma, M. C. Liu, M. S. Lange, J. Berrong, M. A. Wilson, V. L. Gott, et al. Alpha II-Spectrin Breakdown Products Serve as Novel Markers of Brain Injury Severity in a Canine Model of Hypothermic Circulatory Arrest Ann. Thorac. Surg., August 1, 2009; 88(2): 543 - 550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-G. Bernstein and K.-H. Braunewell Some Notes on Visinin-Like Protein 1 and Alzheimer Disease Clin. Chem., May 1, 2009; 55(5): 1041 - 1043. [Full Text] [PDF]
|
|
|
|
|
|
M. M. Verbeek and M. G.M. Olde Rikkert Cerebrospinal Fluid Biomarkers in the Evaluation of Alzheimer Disease Clin. Chem., October 1, 2008; 54(10): 1589 - 1591. [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Lee, K. Blennow, N. Andreasen, O. Laterza, V. Modur, J. Olander, F. Gao, M. Ohlendorf, and J. H. Ladenson The Brain Injury Biomarker VLP-1 Is Increased in the Cerebrospinal Fluid of Alzheimer Disease Patients Clin. Chem., October 1, 2008; 54(10): 1617 - 1623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Whiteley, M.-C. Tseng, and P. Sandercock Blood Biomarkers in the Diagnosis of Ischemic Stroke: A Systematic Review Stroke, October 1, 2008; 39(10): 2902 - 2909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Jeyaseelan, K. Y. Lim, and A. Armugam MicroRNA Expression in the Blood and Brain of Rats Subjected to Transient Focal Ischemia by Middle Cerebral Artery Occlusion Stroke, March 1, 2008; 39(3): 959 - 966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Rifai and R. E. Gerszten Biomarker Discovery and Validation Clin. Chem., September 1, 2006; 52(9): 1635 - 1637. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
0.04 µg/L. VLP-1 was undetected in 36 of 39 samples from unaffected donors.