Kallikrein-related peptidases 6 and 10 are elevated in cerebrospinal fluid of patients with Alzheimer's disease and associated with CSF-TAU and FDG-PET.

BACKGROUND: Alterations in the expression of human kallikrein-related peptidases (KLKs) have been described in patients with Alzheimer's disease (AD). We elucidated the suitability of KLK6, KLK8 and KLK10 to distinguish AD from NC and explored associations with established AD biomarkers. METHODS: KLK levels in cerebrospinal fluid (CSF), as determined by ELISA, were compared between 32 AD patients stratified to A/T/(N) system with evidence for amyloid pathology and of 23 normal controls with normal AD biomarkers. Associations between KLK levels and clinical severity, CSF and positron emission tomography (PET) based AD biomarkers were tested for. RESULTS: Levels of KLK6 and KLK10 were significantly increased in AD. KLK6 differed significantly between AD A+/T+/N+ and AD A+/T-/N+ or NC with an AUC of 0.922. CSF pTau and tTau levels were significantly associated with KLK6 in AD. CONCLUSIONS: KLK6 deserves further investigations as a potential biomarker of Tau pathology in AD.

Neuronal pentraxin receptor-1 is a new cerebrospinal fluid biomarker of Alzheimer's disease progression.

Background: Alzheimer's disease (AD) is the most common type of dementia, with progressive onset of clinical symptoms. The main pathological hallmarks are brain deposits of extracellular amyloid beta plaques and intracellular neurofibrillary tangles (NFT). Cerebrospinal fluid reflects pathological changes in the brain; amyloid beta 1-42 is a marker of amyloid plaques, while total and phosphorylated tau are markers of NFT formation. Additional biomarkers associated with disease pathogenesis are needed, for better prognosis, more specific diagnosis, prediction of disease severity and progression and for improved patient classification in clinical trials. The aim of the present study was to evaluate brain-specific proteins as potential biomarkers of progression of AD. Methods: Overall, 30 candidate proteins were quantified in cerebrospinal fluid (CSF) samples from patients with mild cognitive impairment (MCI) and mild, moderate and severe AD dementia (n=101) using mass spectrometry-based selected reaction monitoring assays. ELISA was used for neuronal pentraxin receptor-1 (NPTXR) confirmation. Results: The best discrimination between MCI and more advanced AD stages (moderate and severe dementia) was observed for protein NPTXR (area under the curve, AUC=0.799). A statistically different abundance of this protein was observed between the two groups, with severe AD patients having progressively lower levels (p<0.05). ELISA confirmed lower levels in AD, in a separate cohort that included controls, MCI and AD patients. Conclusions: We conclude that NPTXR protein in CSF is a novel potential biomarker of AD progression and could have important utility in assessing treatment success in clinical trials.

Targeted Mass Spectrometry-Based Assays for Relative Quantification of 30 Brain-Related Proteins and Their Clinical Applications.

Cerebrospinal fluid (CSF) is a promising clinical sample for identification of novel biomarkers for various neurological disorders. Considering its direct contact with brain tissue, CSF represents a valuable source of brain-related and brain-specific proteins. Multiple sclerosis is an inflammatory, demyelinating neurological disease affecting the central nervous system, and so far there are no diagnostic or prognostic disease specific biomarkers available in the clinic. The primary aim of the present study was to develop a targeted mass spectrometry assay for simultaneous quantification of 30 brain-related proteins in CSF and subsequently to demonstrate assay feasibility in neurological samples derived from multiple sclerosis patients. Our multiplex selected reaction monitoring assay had wide dynamic range (median fold range across peptides = 8.16 × 103) and high assay reproducibility (median across peptides CV = 4%). Candidate biomarkers were quantified in CSF samples from neurologically healthy individuals (n = 9) and patients diagnosed with clinically isolated syndrome (n = 29) or early multiple sclerosis (n = 15).

Brain-related proteins as potential CSF biomarkers of Alzheimer's disease: A targeted mass spectrometry approach.

Alzheimer's disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline. The main disease hallmarks include amyloid beta aggregates and neurofibrillary tangles. Brain pathology is reflected in cerebrospinal fluid (CSF); the core biomarkers amyloid beta 1-42, total and phosphorylated tau protein levels are changed, relative to cognitively normal elderly. Still, there is a need for additional biomarkers which could identify disease more accurately and at an earlier stage, predict severity and be used in research settings. Here we evaluated 30 brain-related proteins as candidate biomarkers of AD. Proteins were quantified in CSF samples from cognitively healthy individuals (n = 23) and patients with mild cognitive impairment (MCI) due to AD (n = 20) or dementia due to AD (n = 10) using selected reaction monitoring mass spectrometry assays. APLP1 protein was increased in MCI relative to control (p < 0.001). The best discrimination between MCI vs. controls was observed with a model combining APLP1 and SPP1 proteins (area under the curve, AUC = 0.84). The strongest associations between protein abundance and disease severity were found for APLP1, CNTN2 and SPP1 proteins, which had a significant correlation with MMSE and CDR tests (p < 0.05). This study identifies new proteins with biomarker potential at various stages of AD severity. SIGNIFICANCE: The current study evaluated 30 brain-related, highly specific proteins as candidate biomarkers of AD diagnosis. Protein APLP1 showed promise as early AD biomarker; protein panel APLP1 and SPP1 had the best diagnostic potential in discriminating MCI from control group, while proteins APLP1, SPP1 and CNTN2 may be indicators of disease progression, demonstrating weak to moderate correlation with cognitive tests. This study therefore identifies new proteins with biomarker potential at early AD stage. If the performance of proposed biomarkers is further confirmed, these proteins may add value in the clinic or clinical trial settings as diagnostic biomarkers (alone or in combination with the existing biomarkers) of the prodromal AD stage, and in monitoring disease progression.

Assessment of kallikrein 6 as a cross-sectional and longitudinal biomarker for Alzheimer's disease.

BACKGROUND: Kallikrein 6 (KLK6) is known to be an age-related protease expressed at high levels in the central nervous system. It was previously shown to be involved in proteolysis of extracellular proteins implicated in neurodegenerative diseases such as Alzheimer's disease (AD), prompting validation of KLK6 as a potential biomarker of disease. However, analyses of both plasma and cerebrospinal fluid (CSF) levels of KLK6 in patients with AD have been inconclusive. We present a detailed analysis of KLK6 in plasma and CSF in two separate cohorts in a cross-sectional and a longitudinal clinical setting. METHODS: The cross-sectional cohort included control subjects without dementia and patients with AD, and the longitudinal cohort included patients with MCI and patients with AD followed over a 2-year period. Plasma and CSF levels of KLK6 were quantified by use of a previously developed and validated enzyme-linked immunosorbent assay. Statistical analyses were performed to compare KLK6 levels between diagnostic groups and to identify potential associations between KLK6 level, age, apolipoprotein E (APOE) genotype, total apoE level and the classical CSF AD biomarkers. RESULTS: In the cross-sectional setting, KLK6 levels in plasma but not in CSF were significantly higher in the AD group than in control subjects. CSF but not plasma KLK6 levels were positively correlated with age in both the cross-sectional and longitudinal settings. In both cohorts, the CSF KLK6 levels were significantly and positively correlated with the CSF levels of core AD biomarkers. Total plasma and CSF apoE levels were positively associated with KLK6 in the cross-sectional study. Finally, during the 2-year monitoring period of the longitudinal cohort, CSF KLK6 levels increased with disease progression over time in the investigated patient groups. CONCLUSIONS: In two separate cohorts we have confirmed the previously reported correlation between age and CSF levels of KLK6. Increased plasma KLK6 levels in patients with AD with a more advanced disease stage suggest KLK6 as a potential biomarker in patients with AD with more severe dementia. Significant correlations between KLK6 levels and core CSF AD biomarkers suggest molecular links between KLK6 and AD-related pathological processes.

Identification of brain-enriched proteins in the cerebrospinal fluid proteome by LC-MS/MS profiling and mining of the Human Protein Atlas.

BACKGROUND: Cerebrospinal fluid (CSF) is a proximal fluid which communicates closely with brain tissue, contains numerous brain-derived proteins and thus represents a promising fluid for discovery of biomarkers of central nervous system (CNS) diseases. The main purpose of this study was to generate an extensive CSF proteome and define brain-related proteins identified in CSF, suitable for development of diagnostic assays. METHODS: Six non-pathological CSF samples from three female and three male individuals were selected for CSF analysis. Samples were first subjected to strong cation exchange chromatography, followed by LC-MS/MS analysis. Secreted and membrane-bound proteins enriched in the brain tissues were retrieved from the Human Protein Atlas. RESULTS: In total, 2615 proteins were identified in the CSF. The number of proteins identified per individual sample ranged from 1109 to 1421, with inter-individual variability between six samples of 21 %. Based on the Human Protein Atlas, 78 brain-specific proteins found in CSF samples were proposed as a signature of brain-enriched proteins in CSF. CONCLUSION: A combination of Human Protein Atlas database and experimental search of proteins in specific body fluid can be applied as an initial step in search for disease biomarkers specific for a particular tissue. This signature may be of significant interest for development of novel diagnostics of CNS diseases and identification of drug targets.

The role of human kallikrein 6, clusterin and adiponectin as potential blood biomarkers of dementia.

OBJECTIVES: Progressive degenerative syndromes which affect brain, altering memory, behavior, cognition and emotion, are commonly defined as dementia. It was suggested that serum human kallikrein 6 (KLK6), clusterin (CLU) and adiponectin (ADPN) in combination with inflammation markers, neuroimaging and neuropsychological testing could assist in discriminating dementia patients from control individuals. Our aim was therefore to compare serum concentrations of KLK6, CLU and ADPN and inflammatory marker, interleukin-6 (IL-6), in patients suffering from Alzheimer's disease (AD), patients with vascular dementia (VAD), cognitively healthy participants (CHP) and those with mild cognitive impairment (MCI). DESIGN AND METHODS: Serum samples were collected from AD, VAD and MCI patients admitted to the University Department of Neurology (Zagreb, Croatia) for regular follow-up. All patients underwent standard neuroimaging procedures including brain CT, neurosonological assessment with intima-media thickness (IMT) and breath holding index (BHI) calculations. Cognitive abilities were tested using standard Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA). Concentrations of KLK6, CLU, ADPN and IL-6 were determined in all serum samples. RESULTS: We have recruited a total of 235 participants, divided in 4 groups: AD (N=70), VAD (N=67), MCI (N=48) and CHP (N=50). Serum concentrations of KLK6 (P=0.137), CLU (P=0.178) and ADPN (P=0.268) did not differ between AD, VAD, MCI and cognitively healthy control group of participants, whereas IL-6 was significantly higher in VAD patients than in AD, MCI and CHP individuals (P=0.014). There was no association between investigated biomarkers and clinical patient parameters. CONCLUSIONS: Serum concentrations of KLK6, CLU and ADPN do not differ between AD, VAD and controls with and without mild cognitive impairment. Higher IL-6 levels in VAD group point to the inflammatory component in the development of vascular dementia. Investigated biomarkers are not associated with neuroimaging findings and neuropsychological patient data.

Assessment of peptide chemical modifications on the development of an accurate and precise multiplex selected reaction monitoring assay for apolipoprotein e isoforms.

Apolipoprotein E (ApoE) is a polymorphic protein that plays a major role in lipid metabolism in the central nervous system and periphery. It has three common allelic isoforms, ApoE2, ApoE3, and ApoE4, that differ in only one or two amino acids. ApoE isoforms have been associated with the occurrence and progression of several pathological conditions, such as coronary atherosclerosis and Alzheimer's disease. The aim of this study was to develop a mass spectrometry (MS)-based assay for absolute quantification of ApoE isoforms in cerebrospinal fluid and plasma samples using isotope-labeled peptides. The assay included five tryptic peptides: CLAVYQAGAR (ApoE2), LGADMEDVCGR (ApoE2 and 3), LAVYQAGAR (ApoE3 and 4), LGADMEDVR (ApoE4), and LGPLVEQGR (total ApoE). Both cerebrospinal fluid and plasma samples were assayed to validate the method. The digestion yield and the extension of chemical modifications in selected amino acid residues (methionine oxidation, glutamine deamidation, and cyclization of N-terminus carbamidomethylcysteine) were also studied. The ApoE phenotype was successfully assigned to all samples analyzed in a blinded manner. The method showed good linearity (R(2) > 0.99) and reproducibility (within laboratory imprecision <13%). The comparison of the MS-based assay with an ELISA for total ApoE concentration showed a moderate correlation (R(2) = 0.59). This MS-based assay can serve as an important tool in clinical studies aiming to elucidate the association between ApoE genotype, total ApoE, and ApoE isoform concentrations in various disorders related to ApoE polymorphisms.

Identification of novel biomarkers of brain damage in patients with hemorrhagic stroke by integrating bioinformatics and mass spectrometry-based proteomics.

Hemorrhagic stroke (HS) is a significant cause of mortality that requires rapid diagnosis and prompt medical attention. A time-efficient diagnostic test to assist in the early classification of patients with stroke would be of great value. The aims here were to (a) select "brain-specific" proteins using a bioinformatics approach, (b) develop selected reaction monitoring (SRM) assays for candidate proteins, and (c) quantify these proteins in cerebrospinal fluid (CSF). "The Human Protein Atlas" and the "Peptide Atlas" were used to select proteins specifically and abundantly expressed in brain tissue, excluding high-abundance plasma proteins. Protein extracts from brain tissue were used for SRM assay development of proteins of interest. The levels of 68 "brain-specific" proteins were measured by SRM in 36 age-matched patients, including individuals with HS (n = 15), ischemic stroke (n = 11), and controls (n = 10). Additionally, S100B was measured using an electrochemoluminometric immunoassay. CSF levels of S100B and eight of the "brain-specific" proteins (NSE, GFAP, α-Inx, MBP, MT3, NFM, ß-Syn, and γ-Syn) were increased in a subset of samples from HS patients, especially in those individuals with intraventricular hemorrhage and poor outcome. Seven of these proteins (S100B, NSE, GFAP, α-Inx, MBP, NFM, and ß-Syn) showed significant differences between patients with and without brain hemorrhage. Novel biomarkers of brain injury (α-Inx, NFM, and ß-Syn) were identified in the CSF of patients with HS. Investigating the role of these proteins in blood with more sensitive methods is warranted.

Can cranberry extract and vitamin C + Zn supplements affect the in vivo activity of paraoxonase 1, antioxidant potential, and lipid status?

BACKGROUND: The modern way of life exposes us to substantial oxidative stress, putting the focus on the research of antioxidant effects of dietary supplements. Recent studies have shown that the effectiveness of particular vitamins and herbal preparations might have an effect on paraoxonase activity. Paraoxonase 1 is an HDL associated enzyme which prevents the oxidation of LDL. Several studies have shown the beneficial effect of some dietary components to the activity of paraoxonase. The aim of this study was to analyze the effects of cranberry extract and vitamin C and zinc preparations (vitamin C + Zn) on serum paraoxonase 1 activity, antioxidant status, and glucose and lipid concentration. METHODS: The study included 31 healthy volunteers (median age 24 years). They were divided into 3 groups according to the intervention type and smoking status and exposed to commercially available preparations of the cranberry extract (2 g/day) and vitamin C + Zn (300 mg/day) during 4 weeks. RESULTS: The results have shown that there is a significant increase in the activity of the paraoxonase 1 in nonsmokers after the intervention with the cranberry extract as well as with vitamin C + Zn preparations. Also, total antioxidant status increased in the non-smokers subgroup after intervention with vitamin C + Zn. However, the lipid profile did not change significantly in response to antioxidant preparations. CONCLUSIONS: Our results show that antioxidant supplements can increase the antioxidant potential of an organism as well as paraoxonase 1 activity. This observation is pointing to the potential complementary role of dietary supplements in the primary prevention of atherosclerosis.

Semiquantitative proteomic analysis of human hippocampal tissues from Alzheimer's disease and age-matched control brains.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid ß plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Aß fragment 1-42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD. RESULTS: In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with cerebrospinal fluid (CSF) literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples. CONCLUSIONS: Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.

Reference intervals for reproductive hormones in prepubertal children on the automated Roche cobas e 411 analyzer.

OBJECTIVES: To establish reference intervals for luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), progesterone (P), total and free testosterone (T) and sex-hormone binding globulin (SHBG) in prepubertal children and to assess age- and gender-related differences. DESIGN AND METHODS: A total of 948 subjects, 480 girls and 468 boys, between 1 and 11 years of age, were included in this study. All assays were performed on a Roche cobas e 411 immunoassay analyzer. Reference intervals have been evaluated according to the most recent CLSI guidelines. RESULTS: Median values of LH, FSH and T were significantly higher in subgroups ranging from ≥ 8 to < 11 years, for both genders. In girls of that age, reference values of E2 were significantly higher than in younger ones, and in boys of the corresponding age. CONCLUSION: Established reference intervals are applicable to other laboratories that use the same instrumentation.