Elemental fingerprint as a cerebrospinal fluid biomarker for the diagnosis of Parkinson's disease

The diagnosis of Parkinson's disease (PD) still lacks objective diagnostic markers independent of clinical criteria. Cerebrospinal fluid (CSF) samples from 36 PD and 42 age‐matched control patients were subjected to inductively coupled plasma‐sector field mass spectrometry and a total of 28 different elements were quantified. Different machine learning algorithms were applied to the dataset to identify a discriminating set of elements yielding a novel biomarker signature. Using 19 stably detected elements, the extreme gradient tree boosting model showed the best performance in the discrimination of PD and control patients with high specificity and sensitivity (78.6% and 83.3%, respectively), re‐classifying the training data to 100%. The 10 times 10‐fold cross‐validation yielded a good area under the receiver operating characteristic curve of 0.83. Arsenic, magnesium, and selenium all showed significantly higher mean CSF levels in the PD group compared to the control group (p  = 0.01, p  = 0.04, and p  = 0.03). Reducing the number of elements to a discriminating minimum, we identified an elemental cluster (Se, Fe, As, Ni, Mg, Sr), which most importantly contributed to the sample discrimination. Selenium was identified as the element with the highest impact within this cluster directly followed by iron. After prospective validation, this elemental fingerprint in the CSF could have the potential to be used as independent biomarker for the diagnosis of PD. Next to their value as a biomarker, these data also argue for a prominent role of these highly discriminating six elements in the pathogenesis of PD.

     

A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high‐abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC‐MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti‐HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method.     

Depletion of abundant proteins from non-human primate serum for biomarker studies

Non-human primates are an important biomedical research model organism and offer great promise for serum biomarker proteomic studies. However, potential obstacles to these studies include affinity serum depletion methods based on human antigens, depletion methods altering quantitation, and incomplete non-human primate genome sequences for protein identification. In the present study, high-abundance protein removal from monkey serum using a human multiple affinity removal system (MARS) was shown to be specific and did not alter quantitation. Depleted serum also demonstrated greater sensitivity for previously masked, lower-abundance proteins.     

The biochemistry of blister fluid from pediatric burn injuries: proteomics and metabolomics aspects

Burn injury is a prevalent and traumatic event for pediatric patients. At present, the diagnosis of burn injury severity is subjective and lacks a clinically relevant quantitative measure. This is due in part to a lack of knowledge surrounding the biochemistry of burn injuries and that of blister fluid. A more complete understanding of the blister fluid biochemistry may open new avenues for diagnostic and prognostic development. Burn insult induces a highly complex network of signaling processes and numerous changes within various biochemical systems, which can ultimately be examined using proteome and metabolome measurements. This review reports on the current understanding of burn wound biochemistry and outlines a technical approach for ‘omics’ profiling of blister fluid from burn wounds of differing severity.     

Clinical utility of decorin in follicular fluid as a biomarker of oocyte potential

This study investigated the concentration of decorin (DCN) in mature follicular fluid and the existence in the granulosa cells. It also investigated whether DCN is useful as a biomarker for outcomes of assisted reproductive technology (ART). A retrospective cohort study was performed involving 130 oocytes of 88 patients treated with ART because of unexplained infertility. The concentration of DCN in the follicular fluid (F-DCN) was 39.26 ng/ml (median value); it was higher than that in serum. F-DCN of the oocytes fertilized by intracytoplasmic sperm injection (ICSI) was significantly lower than that of oocytes that were not fertilized (33.24 ng/ml vs 40.18 ng/ml; P = 0.043). When a cut-off level of 34.5 ng/ml was set according to the receiver-operating characteristic curve, the fertilization rate of the oocytes from the follicles in which F-DCN was lower than the cut-off level tended to be good compared to that of the oocytes with F-DCN higher than the cut-off level (P = 0.052). DCN is less likely to be produced by the granulosa cells (GCs), because it was not detected in GCs by immunostaining and Western blot analysis. F-DCN has a possibility to be a biomarker indicating the quality of oocytes collected from the corresponding follicle.     

Xenobiotic-induced apoptosis: significance and potential application as a general biomarker of response

The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute and chronic chemical stress, loss of cellular function and structure, and organismal health. Examples are provided of the application of this methodology in studies of health of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes.     

Xenobiotic-induced apoptosis: significance and potential application as a general biomarker of response

The process of apoptosis, often coined programmed cell death, involves cell injury induced by a variety of stimuli including xenobiotics and is morphologically, biochemically, and physiologically distinct from necrosis. Apoptotic death is characterized by cellular changes such as cytoplasm shrinkage, chromatin condensation, and plasma membrane asymmetry. This form of cell suicide is appealing as a general biomarker of response in that it is expressed in multiple cell systems (e.g. immune, neuronal, hepatal, intestinal, dermal, reproductive), is conserved phylogenetically (e.g. fish, rodents, birds, sheep, amphibians, roundworms, plants, humans), is modulated by environmentally relevant levels of chemical contaminants, and indicates a state of stress of the organism. Further, apoptosis is useful as a biomarker as it serves as a molecular control point and hence may provide mechanistic information on xenobiotic stress. Studies reviewed here suggest that apoptosis is a sensitive and early indicator of acute and chronic chemical stress, loss of cellular function and structure, and organismal health. Examples are provided of the application of this methodology in studies of health of lake trout (Salvelinus namaycush) in the Laurentian Great Lakes.     

Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples

Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.     

microRNAs in the blastocoel fluid as accessible indicators of chromosomal normality

MicroRNAs (miRNAs) derived from the pre-implantation blastocoel fluid (BF) have attracted interest as accessible biomarkers indicative of embryonic health in ongoing IVF cycles. Therefore, we investigated expression levels of some aneuploidy-associated miRNAs and implantation-related mRNAs as predictive markers for embryo chromosomal normality. In this study, the BF of 25 blastocysts that had been checked for aneuploidy (aneuploid=17 and euploid=8) was aspirated and the expression of 10 miRNAs (miR-20a, miR-30c, miR‐661, miR-372, miR-142, miR-191, miR‐345, miR‐339, miR‐141, and miR-27b) and four genes (ERBB4, SELL, ITGB3, ITGAV) were evaluated using real time-PCR. Results showed that the levels of miR-661 and miR-20a were significantly higher in the BF of the aneuploid embryos compared to the euploid group (p = 0.0017 and 0.004, respectively). A comparison of the mRNA levels between the aneuploid and euploid groups also demonstrated a significant difference in ITGAV (p = 0.013) and SELL (p = 0.0317) levels. In the euploid group, a negative correlation was found between ITGB3 and miR-30c (r = ?0.71, p = 0.08), and in the aneuploid group, a positive correlation was found between ERBB4 and miR-345 (r = 0.71, p = 0.02). It can be suggested that miR-20a, miR-661, and ITGAV levels of BF could be used as less-invasive biomarkers to evaluate embryonic health. Moreover, aneuploidy-related miRNA levels were associated with levels of genes involved in embryo implantation.     

Preliminary characterizations of a serum biomarker for sarcoidosis by comparative proteomic approach with tandem-mass spectrometry in ethnic Han Chinese patients

Background

The diagnosis of sarcoidosis is still a significant challenge in China because of the need to exclude other diseases including granulomatous infections and malignancies that may be clinically and radiographically similar. The specific aim of the study is to search for serum protein biomarkers of sarcoidosis and to validate their clinical usefulness in differential diagnosis.

Methods

Serum samples were collected from patients with sarcoidosis (n = 37), and compared to those from patients with tuberculosis (n = 20), other pulmonary diseases (n = 20), and healthy volunteers (n = 20) for determination of sarcoidosis-specific or -associated protein expression profiles. The first part of this study focused on proteomic analysis of serum from patients with sarcoidosis to identify a pattern of peptides capable of differentiating the studied populations using the ClinProt profiling technology based on mass spectrometry. Enzyme Linked Immunosorbent Assay (ELISA) was then used to verify corresponding elevation of the serum protein concentration of the potential biomarkers in the same patients sets. Receiver operating characteristic curve (ROC) analyses was performed to determine the optimal cutoff value for diagnosis. Immunohistochemistry was carried out to further confirm the protein expression patterns of the biomarkers in lung tissue.

Results

An unique protein peak of M/Z 3,210 Daltons (Da) was found to be differentially expressed between the sarcoidosis and control groups and was identified as the N-terminal peptide of 29 amino acids (94-122) of serum amyloid A (SAA). ELISA confirmed that the serum SAA level was significantly higher in the sarcoidosis group than that of the other 3 control groups (p < 0.05). The cutoff for serum SAA concentration determined by ROC analysis was 101.98 ng/ml, with the sensitivity and specificity of 96.3% and 52.5%, respectively. Immunohistochemical staining showed that the SAA depositions in lung tissue of the sarcoidosis patients were also significantly more intense than in non-sarcoid lung tissue (p < 0.05).

Conclusion

This is the first study to investigate serum protein markers in Chinese subjects with sarcoidosis. This study shows that the serum SAA expression profiles were different between the sarcoidosis and non-sarcoidosis groups. SAA may be a potential serum biomarker for ruling-out the diagnosis of sarcoidosis in Chinese subjects.     

Circulating endothelium-enriched microRNA-126 as a potential biomarker for coronary artery disease in type 2 diabetes mellitus patients

Circulating microRNAs (miRNAs) have been shown as promising biomarkers for various diseases. We investigated the predictive potential of circulating endothelium-enriched miR-126 in type 2 diabetes patients (T2D) without chronic complications and T2D patients with coronary artery diseases (CAD). The expression levels of circulating miR-126, determined by quantitative real time PCR, were decrease in peripheral blood of T2D patients and T2D with CAD compared with healthy controls. MiR-126 strongly associated with T2D and CAD, negatively correlated with LDL in CAD patients and differentiated between T2D patients, T2D patients with CAD and healthy subjects. Circulating miR-126 may serve as a biomarker for predicting patients with T2D and diabetic CAD.     

Serum protein profiling in mice: identification of Factor XIIIa as a potential biomarker for muscular dystrophy

Protein profiling in blood serum by fractionation and MS analysis has been applied in mice to assess its applicability as a fast, economical alternative to current DNA and RNA analyses for diagnosis of neuromuscular disorders. Mass spectra of peptides and proteins were generated using serum from dystrophin-deficient mdx and control mice by WCX ClinProt bead fractionation, followed by MALDI-MS. Double cross-validatory linear discriminant and logistic regression data analysis methods were compared with a new Bayesian logistic regression method. These were evaluated on their ability to discriminate between healthy and dystrophic samples, and to identify the discriminatory peaks in the mass spectra. All three approaches classified the spectra with comparable misclassification rates (between 18.4 and 20.6%), with much overlap between the differential peaks identified between the methods. The differential peak pattern from the Bayesian method was sparser and easier to interpret than from the other two methods, without compromising classifying strength. One of the two main differentiating peaks at m/z 3908 was identified as an N-terminal peptide of coagulation Factor XIIIa, previously identified in human serum. This work underlines the translational aspect of serum protein profiling in mice and supports a further study with serum from patients with neuromuscular disorders.     

Ascorbylated 4-hydroxy-2-nonenal as a potential biomarker of oxidative stress response

Oxidative stress, resulting from the generation of reactive oxygen species, contributes to the development of a multitude of age-related diseases. Current methods of assessing oxidative stress levels range from the detection of lipid peroxidation products, such as F(2)-isoprostanes and malondialdehyde, to monitoring the redox status of glutathione. While useful, traditional biomarkers of oxidative stress are not without their drawbacks, including low in vitro concentrations and possible artifact formation. In the present study, we utilize liquid chromatography coupled with tandem mass spectrometry for investigation into the use of a novel compound, ascorbylated 4-hydroxy-2-nonenal, as a potential biomarker of oxidative stress.     

The potential use of the neurotensin high affinity receptor 1 as a biomarker for cancer progression and as a component of personalized medicine in selective cancers

A growing challenge in medicine today, is the need to improve the suitability of drug treatments for cancer patients. In this field, biomarkers have become the “flags” to provide additional information in tumor biology. They are a relay between the patient and practitioner and consequently, aid in the diagnosis, providing information for prognosis, or in some cases predicting the response to specific therapies. In addition to being markers, these tumor “flags” can also be major participants in the process of carcinogenesis.Neurotensin receptor 1 (NTSR1) was recently identified as a prognosis marker in breast, lung, and head and neck squamous carcinomas. Neurotensin (NTS) was also shown to exert numerous oncogenic effects involved in tumor growth and metastatic spread. These effects were mostly mediated by NTSR1, making the NTS/NTSR1 complex an actor in cancer progression. In this review, we gather information on the oncogenic effects of the NTS/NTSR1 complex and its associated signaling pathways in order to illuminate its significant role in tumor progression and its potential as a biomarker and a therapeutic target in some tumors.     

8 Hydroxydeoxyguanosine as a biomarker of workplace exposures

To date, the 8-hydroxydeoxyguanosine (8OHdG) DNA adduct has been used as a biomarker in 11 occupational health studies examining the potential for ten different workplace exposures to cause oxidative DNA damage. Exposures examined include asbestos, azo-dyes, benzene, chromium, coal dust, glassworks, rubber manufacturing, styrene, toluene and environmental tobacco smoke (ETS). Experimental designs that applied 8OHdG as biomarker varied dramatically among the studies. For example, one study detected increased urinary excretion in retired workers with a history of exposure to mining dusts, while a study of workers exposed to benzene showed that the pattern of urinary excretion of 8OHdG varied over a 24h period following exposure. All but one study reported increased 8OHdG relative to controls, but in three cases the increases were not statistically significant. Only one study demonstrated a dose-response relationship between a chemical exposure (benzene) in the workplace and elevated 8OHdG. In most cases, exposure data were lacking and the elevated 8OHdG could only be considered to be associated with a generalized job category. Numerous animal and human studies have demonstrated an effect of tobacco smoke on 8OHdG, including a study of ETS in the workplace. In the majority of occupational studies, however, smoking was found not to be a confounding variable. 8OHdG levels tended to be higher in women than men as did the response to an occupational exposure and/or smoking. Two of three studies that stratified workers by age found it to be a confounder for the 8OHdG adduct, but the relationship between age and 8OHdG was non-linear. Only one study considered the impact of dietary supplements on 8OHdG levels in workers despite the fact that diet can have a marked effect on an individual's response to oxidative stress. It is premature to consider 8OHdG as biomarker that can be used for decision making or for regulatory purposes. Nonetheless, these studies demonstrate that with additional characterization of the role 8OHdG plays in the exposure-disease continuum it may well serve as a powerful biomonitoring toolin the future.     

Hydrogen peroxide in urine as a potential biomarker of whole body oxidative stress

The level of hydrogen peroxide (H2O2) in urine has been suggested as a potential biomarker of whole body oxidative stress, but issues of stability, reproducibility and biological variation have not been investigated to date. In this study, we used a refined protocol, which demonstrated improved sensitivity and precision, to determine the stability of H2O2 in urine, and to measure its concentration in apparently healthy subjects. We also investigated intra-individual variation within and between days. Results showed that H2O2 in urine is stable for up to 48 h at 4 degrees C, however, storage of urine at room temperature was associated with up to 50% increase in H2O2 concentration over a few hours. Total H2O2 in freshly voided urine from 55 healthy, fasting subjects ranged from 0.84 to 5.71 microM, or 90-1164 micromol H2O2/mol creatinine. Intra-individual variation was wide. Even when concentration corrected and collected at the same time of day, 2- to 3-fold variation was seen over 4 consecutive days, and over the course of a single day the creatinine-corrected H2O2 also varied significantly. We suggest that this large biological variation limits the usefulness of urine H2O2 as a biomarker of oxidative stress, the exception being when the effects of disease, therapy or diet induce very large changes in its concentration.     

Suction capillaries with U-shaped opening for holding moving cells during intracellular electrical recording

The production of suction capillaries with an U-shaped cross-section is described. The capillary may be used for holding contractile cells during intracellular recording to diminish movement artifacts. In addition microelectrode penetration is facilitated by stretching the surface of the cell.     

Humoral response to cancer as a tool for biomarker discovery

There is an important need to find relevant biomarkers that show high sensitivity and specificity for early diagnosis and prognosis of cancer. An immune response to cancer is elicited in humans, as demonstrated in part by the identification of autoantibodies against a number of tumor-associated antigens in sera from patients with different types of cancer. Identification of tumor-associated antigens and their cognate autoantibodies is a promising strategy for the discovery of relevant biomarkers. During the past few years, proteomic approaches, including SEREX, SERPA and, more recently, protein microarrays, have been the dominant strategies used to identify tumor-associated antigens and their cognate autoantibodies. In this review, we aim to describe advantages, drawbacks, and recent improvements of these approaches for the study of humoral responses.     

Protein phosphorylation studies of cerebral spinal fluid for potential biomarker development

Subarachnoid hemorrhage (SAH) followed by cerebral vasospasm (CV) leads to severe debilitation or death of an estimated one million people worldwide every year. A biomarker that would predict the onset of CV after a SAH would be useful in informing treatment protocols, but has yet to be found. The focus of this study is to explore differences in protein phosphorylation in cerebral spinal fluid (CSF) among healthy patients, SAH patients and SAH-CV patients. A significant difference in phosphorylation among the three sample types could be an important step towards the discovery of a diagnostic marker. The identification and validation of phosphorylated protein differences for study is manifested in the nature of signaling involved in the pathological events seen post SAH. Capillary liquid chromatography (cap-LC) coupled to inductively coupled plasma mass spectrometry (ICPMS) and nano-liquid chromatography-CHIP/ion trap mass spectrometry (nanoLC-CHIP/ITMS) are used to identify and measure protein phosphorylation changes in the CSF of the aforementioned groups. ICPMS represents a suitable method for screening ultra-trace phosphorus levels at the natural isotope, (31)P, while nano-LC-CHIP/ITMS is used to identify phosphoproteins by searching appropriate protein databases.