Enzymes In Cerebrospinal Fluid Research Articles

State of the Art in the Development of Human Serum Carnosinase Inhibitors.

Human serum carnosinase is an enzyme that operates the preferential hydrolysis of dipeptides with a C-terminus histidine. Only higher primates excrete such an enzyme in serum and cerebrospinal fluid. In humans, the serum hydrolytic rate has high interindividual variability owing to gene polymorphism, although age, gender, diet, and also diseases and surgical interventions can modify serum activity. Human genetic diseases with altered carnosinase activity have been identified and associated with neurological disorders and age-related cognitive decline. On the contrary, low peripheral carnosinase activity has been associated with kidney protection, especially in diabetic nephropathy. Therefore, serum carnosinase is a druggable target for the development of selective inhibitors. However, only one molecule (i.e., carnostatine) has been discovered with the purpose of developing serum carnosinase inhibitors. Bestatin is the only inhibitor reported other than carnostatine, although its activity is not selective towards serum carnosinase. Herein, we present a review of the most critical findings on human serum carnosinase, including enzyme expression, localization and substrate selectivity, along with factors affecting the hydrolytic activity, its implication in human diseases and the properties of known inhibitors of the enzyme.

Potential roles of oxidative distress on neurodegeneration in Parkinson's disease with neuropsychiatric symptoms.

Neuropsychiatric symptoms (NPSs) belong to a category of non-motor symptoms of Parkinson's disease (PD), which seriously compromise the quality of life and prognosis of PD. This study focused on the correlations between NPSs, free radicals, neuroinflammatory factors, and neuropathological proteins in cerebrospinal fluid (CSF) in patients with PD, aiming to provide insights into the potential mechanisms and therapeutic target for PD with NPSs (PD-NPSs). In total, 129 patients with PD were enrolled and assessed by the Neuropsychiatric Symptoms Inventory (NPI); they were divided into the PD-NPSs group (75 patients) and PD with no NPSs (PD-nNPSs) group (54 patients). The levels of hydrogen peroxide (H2O2) and nitric oxide (NO), and hydroxyl radical (·OH), anti-oxidative enzyme, neuroinflammatory factors, and neuropathological proteins in CSF from patients with PD were measured. The levels of the above variables were compared between PD-NPSs and PD-nNPSs groups, and correlation analyses among the above variables were conducted. (1) The levels of H2O2 and NO in CSF from the PD-NPSs group were significantly elevated compared with the PD-nNPSs group (p = 0.001), and NPI score positively correlated with the levels of H2O2 and NO (r = 0.283, P = 0.001; r = 0.231, P = 0.008). Reversely, total superoxide dismutase (tSOD) activity in CSF from the PD-NPSs group was significantly reduced compared with the PD-nNPSs group (p = 0.011), and negatively correlated with NPI score (r = -0.185, p = 0.036). (2) The tumor necrosis factor (TNF)-α level in CSF from the PD-NPSs group was significantly decreased compared with the PD-nNPSs group (p = 0.002) and negatively correlated with NPI score (r = -0.211, p = 0.016). (3) The total tau (T-tau) level in CSF from the PD-NPSs group was significantly higher than in the PD-nNPSs group (p = 0.014) and positively correlated with the NPI score (r = 0.167, p = 0.060). (4) The levels of H2O2 and NO positively correlated with the T-tau level in CSF from the PD-NPSs group (r = 0.183, p = 0.039; r = 0.251, P = 0.004), and the levels of TNF-α and T-tau showed a negative correlation (r = -0.163, p = 0.067). Oxidative distress characterized by the elevations of H2O2 and NO levels may closely correlate with the neurodegeneration in brain regions related to PD-NPSs. Thus, therapeutic antioxidants may become an important target for PD-NPSs therapy.

BACE1 and Other Alzheimer's-Related Biomarkers in Cerebrospinal Fluid and Plasma Distinguish Alzheimer's Disease Patients from Cognitively-Impaired Neurosyphilis Patients.

Patients with spirochetal infection, which causes neurosyphilis (NS) and at a later stage general paresis of the insane (GPI), present with brain pathology features of Alzheimer's disease (AD). However, the relationships among these illnesses regarding biomarker levels are still unclear. To explore biomarker levels in NS and GPI compared with those in AD and the relationship between biomarker levels and cognitive function in NS and GPI. Levels of neurogranin (NGRN) and β-amyloid precursor protein cleaving enzyme (BACE1) in cerebrospinal fluid (CSF)/plasma, together with amyloid-β 1-40 (Aβ40), Aβ42, and total tau in the CSF of 23 AD patients, 55 GPI patients, and 13 NS patients were measured. Patients were classified into none-to-mild, moderate, and severe stages of cognitive impairment. Levels of CSF NGRN, BACE1, and tau as well as plasma BACE1 levels were significantly different among groups. In the none-to-mild stage, plasma BACE1 levels correlated with the protein levels in CSF and were significantly increased in AD patients versus GPI patients. The CSF tau levels in AD patients were significantly increased versus GPI patients in the moderate and severe stages. Pooling data from GPI and NS patients, both CSF tau and plasma NGRN levels correlated with cognitive scale scores. GPI and NS patients might have different biomarker level patterns compared to AD patients. While plasma BACE1 could be a promising early biomarker for distinguishing AD from GPI, CSF tau and plasma NGRN levels might be valuable in indications of cognitive function in pooled NS populations.

Evaluation of H2S-producing enzymes in cerebrospinal fluid and its relationship with interleukin-6 and neurologic deficits in subarachnoid hemorrhage

Background and purposeRecent studies have suggested that H2S may be involved in the pathophysiology of subarachnoid hemorrhage (SAH). Endogenous H2S is mainly formed by cystathionine β-synthase (CBS), d-amino-acid oxidase (DAO), and 3-mercaptopyruvate sulfotransferase (3-MST) from the substrate cysteine in the central nervous system. In this study, we assessed the expression of CBS, 3-MST, and DAO in cerebrospinal fluid (CSF) from patients with SAH and rats and the expression in the rat brain. MethodsCSF samples were collected within 48 h of aneurysm rupture in SAH patients. The CBS, DAO and 3-MST levels in CSF were measured using Western blot analyses, and correlations with the inflammatory parameter Interleukin-6 (IL-6) were assessed. Six months after SAH, the clinical outcomes were assessed. ResultsIn human CSF samples, the CBS and DAO protein levels were detected and increased after SAH. However, 3-MST was not detected in the control group CSF but increased after SAH. Strong correlations were observed between the increasing levels of CBS, DAO, and 3-MST and IL-6 2 days after SAH. Furthermore, high CBS, 3-MST and DAO levels in the CSF samples were correlated with poor outcomes at 6 months after SAH onset. We also found that the expression of CBS, DAO and 3-MST in the rat CSF and brain (parietal cortex and hippocampus) increased following SAH. We detected strong correlations between the increases in CBS, 3-MST and IL-6 in the rat CSF and brain samples. ConclusionsThese results indicate that the upregulated expression of CBS, DAO and 3-MST after SAH was closely associated with the inflammatory response and neurological deficits after SAH.

Cardio embolic stroke and blood biomarkers: diagnosis and predictors of short-term outcome

BackgroundThe brain is a productive source of a variety of enzymes and any brain injury like a stroke to brain tissue could similarly result in an increase in these enzymes in cerebrospinal fluid and serum. Evaluation of these enzymes represents a simple method for the ischemic stroke subtype diagnosis and prognosis. Objective: This study aimed to determine the role of brain natriuretic peptide (BNP), d-dimer, creatine–kinase-MB (CK-MB), C-reactive protein (CRP) serum levels, and globulin/albumin ratio in the diagnosis of CES stroke and its ability to predict short-term outcome.MethodsThis study was conducted on 96 patients with acute ischemic stroke, subdivided into two groups: group Ι was 48 patients with cardio-embolic stroke and group ΙΙ was 48 patients with non-cardio-embolic. All patients were subjected to the assessment of serum BNP, d-dimer and CK-MB, and CRP and globulin/albumin ratio within the first 24 h of stroke. In the third week, they were assessed by mRS.ResultsThe mean levels of BNP, d-dimer level, and CK-MB were significantly higher in patients with cardio-embolic stroke than in patients with non-cardio-embolic stroke (P < 0.001) and also were associated with poor short-term outcome.ConclusionElevated plasma levels of BNP, d-dimer levels, and CK-MB can be used as surrogate biomarkers for the diagnosis of cardio-embolic stroke and prediction of poor short-term outcomes.

Role of Serum Lactate Dehydrogenase in Isolated Head Injury

AbstractIn recent years, some encouraging reports have appeared on assessing the degree of brain damage by estimating serum and cerebrospinal fluid enzymes such as lactate dehydrogenase (LDH). In our study, we included 100 patients with isolated head injury arriving at trauma center, PGIMS Rohtak within 24 hours of injury, studied levels of serum LDH on days 1, 3, and 7 of injury, and concluded that levels have diagnostic as well as prognostic significance.

Studying of Oxidative Stress and Activity of Some Enzymes in Cerebrospinal Fluid from Children with Hydrocephalus Disease

This research include studying the oxidative stress state and the activity of some enzymes in the cerebrospinal fluid (CSF) from children with hydrocephalus by the determination of (15) Biochemical parameters which include: Acetylcholinesterase (AChE), Pseudocholinesterase (PChE), Monoamin oxidase A (MAOA), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Creatinine kinase (CK), Lactate dehydrogenase (LDH), Superoxide dismutase (SOD), Uric acid (UA), Vitamin D, Total protein (Tp), Albumin (Alb), Ceruloplasmin (Cp), Malonadialdehyde (MDA), The study was done on (176) child with hydrocephalus (110) were male , (66) female and (67) healthy child as control group (39) were male and (28) female ,the age of children with hydrocephalus and control group ranged from 1-14 month for both sexes. The results showed that there was a significant increase in the activity of AChE, PChE, MAOA, AST, ALT, ALP, CK and LDH in CSF for children with hydrocephalus compared with healthy group for both sexes, these result were indicated that these parameters may be good biochemical markers for this disease . Beside of that, the results showed there was a highly oxidative stress in the patients for both sexes, so there was a significant increase in the activity of SOD, Tp, Alb and oxidant indicator of MDA, with a significant decrease in vit. D, uric acid and Cp levels in CSF patients compared with healthy group for both sexes,

Role of cerebrospinal fluid, creatine kinase and lactate dehydrogenase enzyme levels in diagnostic and prognostic evaluation of tubercular and pyogenic meningitis

Background: Etiological diagnosis for meningitis remains a challenge and often a cerebrospinal fluid (CSF) examination may not give the precise diagnosis. Many enzymes are known to be present in abundance in nervous system. In meningitis, nerve tissue damage leads to release of these enzymes into CSF, which may have important diagnostic and prognostic role. Aim was to evaluate the diagnostic and prognostic significance of creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes in CSF of tubercular and pyogenic meningitis.Methods: A prospective case control study of 50 subjects with pyogenic or tubercular meningitis, admitted in Department of Medicine, S.R.N. Hospital, M.L.N.M.C. Allahabad, Uttar Pradesh, India from September 2015-August 2016 was done. Level of CK and LDH were estimated in CSF and serum on day of admission and 7th day. 50 subjects with non-neurological diseases were taken as control.Results: Out of total 50 patients, 36 were of tubercular meningitis and 14 were of pyogenic meningitis. Mean CSF CK and CSF LDH levels were significantly raised in both conditions with respect to controls (p&lt;0.001 and p&lt;0.05 respectively). Mean CSF CK and CSF LDH level were significantly reduced from day of admission to 7th day in both tubercular and pyogenic meningitis patients.Conclusions: Enzymes level in CSF like CK and LDH are significantly raised in pyogenic and tubercular meningitis. Accessing its value can be an important diagnostic and prognostic marker.

Correlation of secretory phospholipase-A2 activity and fatty acids in cerebrospinal fluid with liver enzymes tests

Introduction: The aim was to determine whether secretory phospholipase-A2 (sPLA2) activity and fatty acids in cerebrospinal fluid (CSF) are correlated with liver enzymes tests. Methods: CSF and serum samples were collected from 49 patients (age 18-65) as part of routine diagnostic testing. Along with serum liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP), the fatty acid composition of CSF was measured by gas liquid chromatography. CSF enzyme activities of sPLA2 were measured using the standard assay with diheptanoyl thio-phosphatidylcholin as substrate. Results: The saturated fatty acids (SFAs) including palmitic acid and stearic acid were positively, and the unsaturated fatty acids including oleic acid and linoleic acid were negatively correlated with liver enzymes tests. In regression analysis with adjustment for body mass index (BMI), the elevated liver enzymes tests were positively associated with activity of sPLA2 (β > 0.31, P 0.38, P < 0.010) and negatively with total monounsaturated fatty acids (MUFAs) (β < -0.40, P < 0.001) contents of CSF. Conclusion: CSF activity of sPLA2 and fatty acids may be linked to peripheral markers of liver function, suggesting an indirect impact of central fatty acids on hepatocytes function and metabolism.

Neurocysticercosis presenting as central diabetes insipidus in a boy

Neurocysticercosis (NCC) is known to have varied presentations ranging from simple psychosis to stroke and death. While NCC may affect any part of brain or spinal cord, endocrinopathies due to NCC involving hypothalamus and pituitary gland are very rare. A 7-year-old child presented with polyuria and polydipsia for 1 year. After ruling out peripheral diabetes insipidus, a magnetic resonance imaging scan of the brain was done which showed a small cystic lesion in the proximal pituitary stalk. Similar cystic lesions were also seen in other areas of the brain. Cerebrospinal fluid enzyme linked immunosorbent assay for cysticercosis was positive. Such a case of NCC involving pituitary stalk and presenting as central diabetes insipidus is, to the best of our knowledge, the first report of both the location and clinical presentation of NCC in children.

Factors influencing the measurement of lysosomal enzymes activity in human cerebrospinal fluid.

Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders.

Disease stage determines the efficacy of treatment of a paediatric neurodegenerative disease.

Lysosomal storage disorders are a large group of inherited metabolic conditions resulting from the deficiency of proteins involved in lysosomal catabolism, with resulting accumulation of substrates inside the cell. Two-thirds of these disorders are associated with a neurodegenerative phenotype and, although few therapeutic options are available to patients at present, clinical trials of several treatments including lysosomal enzyme replacement are underway. Although animal studies indicate the efficacy of presymptomatic treatment, it is largely unknown whether symptomatic disease-related pathology and functional deficits are reversible. To begin to address this, we used a naturally-occurring mouse model with Sanfilippo syndrome (mucopolysaccharidosis type IIIA) to examine the effectiveness of intracisternal cerebrospinal fluid enzyme replacement in early, mid- and symptomatic disease stage mice. We observed a disease-stage-dependent treatment effect, with the most significant reductions in primary and secondary substrate accumulation, astrogliosis and protein aggregate accumulation seen in mucopolysaccharidosis type IIIA mice treated very early in the disease course. Affected mice treated at a symptomatic age exhibited little change in these neuropathological markers in the time-frame of the study. Microgliosis was refractory to treatment regardless of the age at which treatment was instigated. Although longer-term studies are warranted, these findings indicate the importance of early intervention in this condition.

Role of CSF CK, LDH, GGTP Enzyme Levels in Diagnostic and Prognostic Evaluation of Meningitis.

Aetiological diagnosis for meningitis remains a challenge and often a thorough Cerebrospinal fluid (CSF) examination may not give a precise diagnosis as at times CSF findings may simulate meningitis like picture as in carcinomatous meningitis, chemical meningitis, early stages of Guillan bare syndrome, subarachnoid hemorrhage, ICSOL, brain abscess, intracranial extension of epidural or subdural infection. Lesions of tissue rich in enzymes may cause enzymatic release on tissue destruction, so also in meningitis and many enzymes are known to be present in abundance in nervous system. To evaluate the diagnostic and prognostic significance of Creatine Kinase (CK), Lactate Dehydrogenase (LDH) and Gamma Glutamyl Transpeptidase (GGTP) enzymes in CSF of meningitis. A prospective cases control study of pyogenic and tubercular meningitis patients of 44 cases admitted in department of medicine from September 2011-September 2012 was done. Enzymes like CK, LDH and GGTP were estimated on day 0 (day of admission) and 7th day in the CSF and in serum. CSF and serum CK, LDH, GGTP was estimated on 0th day 0 only in cases of controls. Out of 44 patients, total 32 were tubercular meningitis and 12 were pyogenic meningitis. Mean CSF CK, LDH levels were significantly raised in both conditions with respect to controls in day 0 with p < 0.0001 and p < 0.05 respectively. Mean CSF CK, LDH level were also significantly reduced from 0 to 7th day in both tubercular, pyogenic meningitis, while CSF GGTP was not significantly raised with respect to control in day 0. Enzymes level in CSF like CK, LDH are significantly raised in the meningitis and accessing its value can be an important diagnostic and prognostic marker. But more studies are required to establish the usefulness of estimating these enzymes.

Use of cerebrospinal fluid enzyme immunoassay for diagnosis of neurosyphilis

Neurosyphilis is a difficult clinical stage of syphilis as there is no ideal method for diagnosis and workup requires lumbar puncture which may sometimes provide ambiguous results especially in HIV co-infected patients. Enzyme immunoassay is a widely used screening test for syphilis in serum, but its test performance was not well studied in cerebrospinal fluid. To examine the diagnostic performance of cerebrospinal fluid-enzyme immunoassay (CSF-EIA) in neurosyphilis, we conducted a prospective study for two years. All consecutive patients admitted for workup of neurosyphilis under the Social Hygiene Service, in Hong Kong, were included. Laboratory tests on CSF included several serological tests, CSF cell count, and protein. Forty-five patients were prospectively recruited, of which 29 people were living with HIV / AIDS. Using diagnostic case definition standard stipulated in the IUSTI 2008 guidelines, 17 patients satisfied the diagnosis of neurosyphilis. The CSF-EIA test provided 100% in both sensitivity and negative predictive value; its specificity was 46.4% (13/28, 95% CI 31.8-61%). Specificity improved to 80.8% (95% CI: 68.4-93.2%) with optical density cut-off value at 1.4 for cases with CSF red cell counts <600/mm(3) This is the first study on use of CSF-EIA in neurosyphilis. CSF-EIA showed high sensitivity and high negative predictive value in the study population and the presence of CSF red cells < 600/mm(3)might not affect its accuracy.

Changes in behaviour and cytokine expression upon a peripheral immune challenge

Depression is frequently associated with inflammation. Animal studies have shown that peripheral inflammation induces depressive-like behaviour, but the underlying mechanisms remain unclear. A distinction between sickness- and depressive-like behaviour has been proposed. We hypothesize that the behavioural distinction is due to changes in the central production of immune mediators.As a model of peripheral inflammation, we administered lipopolysaccharide (LPS) intraperitoneally daily for 4 days in rats. The effect of LPS on sickness- and depressive-like behaviour was assessed. We examined protein levels and mRNA expression of cytokines and cyclooxygenase (COX) enzymes in serum, cerebrospinal fluid (CSF) and specific brain regions.Two hours post-LPS, the rats displayed sickness behaviour and cytokine levels were elevated in both serum and CSF. This was paralleled by specific alterations of mRNA transcription of IL-1β, IL-6 and TNF-α in frontal cortex, hippocampus and striatum. Twenty-four hours post-LPS the rats showed depressive-like behaviour and peripheral cytokine levels were back close to baseline. In contrast, the central transcription of IL-1β mRNA had increased even further, as well as IL-1β CSF levels. IL-6 and TNF-α transcription was unaltered compared to controls. COX enzymes were downregulated in the hippocampus during sickness behaviour and unaltered during depressive-like behaviour.Our results show for the first time that a peripheral immune challenge induces a region specific transcription of cytokines and COX-enzymes in the brain, at time-points corresponding to behavioural sickness and depression. When the peripheral inflammation and sickness behaviour had ceased, a production of proinflammatory cytokines remained within the brain parenchyma.

Cerebrospinal fluid phospholipase C activity increases in migraine

Adrenaline, serotonin, cannabinoid and estrogen receptors are involved in migraine pathophysiology. The signaling of these receptors change phosphatidylcholine-specific phospholipase C (PC-PLC) activity, but there have been no reported PC-PLC studies in migraine. We identified PC-PLC activity in blood and cerebrospinal fluid (CSF), and quantified it in samples from ictal and interictal migraineurs without aura and healthy controls. Pre-incubation with a specific PC-PLC inhibitor, D609, inhibited enzyme activity (p < .0001) and confirms its presence in CSF. PC-PLC activity was higher in the CSF from ictal migraineurs compared to controls (mean relative fluorescence unit [RFU]/µg/min [standard deviation, SD] 13.1 [3.07] vs. 9.3 [1.97]; p = .002) and, in a paired analysis, in migraineurs during ictal compared to interictal states (11.7 [1.6] vs. 7.9 [1.5]; p = .02). CSF PC-PLC activity in the ictal state correlated negatively with migraine frequency (r = -0.82). Plasma PC-PLC activity was 250-300 times less than in CSF and did not increase in migraine, implicating the brain as the source of the CSF enzyme changes. This is the first report of PC-PLC activity in CSF and of its alteration in migraine. We propose that these PC-PLC changes in CSF reflect the overall receptor fluctuations in migraine.

Cerebrospinal fluid enzymes in neurological diseases.

The activities of adenylate kinase, creatine kinase and lactate dehydrogenase were measured in cerebrospinal fluid and serum from 127 patients admitted to the Department of Neurology. All cerebrospinal fluid samples with hemoglobin greater than 1 mg/l were excluded. Upper reference limits for the enzyme determinations were established, using samples from patients without objective criteria of organic involvement of the nervous system. High enzyme activities did not correlate to any particular group of diseases and were also found in patients without organic brain diseases. We conclude that determination of the three enzymes in cerebrospinal fluid is of limited value in the diagnosis of neurological diseases.

Surface plasmon resonance biosensors for detection of Alzheimer disease biomarker

This paper describes direct label-free detection of 17beta-hydroxysteroid dehydrogenase type 10 (17β-HSD10) using a surface plasmon resonance (SPR) biosensor. This multifunctional mitochondrial enzyme is involved in pathogenesis of Alzheimer disease (AD) and represents a potential target for AD diagnostics. A multichannel SPR sensor with spectral modulation is functionalized with amyloid beta or polyclonal antibody against 17β-HSD10 peptide using a self-assembled monolayer of alkylthiolates and amino coupling chemistry. Detection of a synthetic peptide of 14 amino acids from the 17β-HSD10 molecule (residues 133–146aa) and whole enzyme 17β-HSD10 is performed. Detection of 17β-HSD10 enzyme in artificial cerebrospinal fluid (ACSF) buffer is also carried out. It is demonstrated that the reported SPR biosensor is capable of detecting 17β-HSD10 enzyme at ng/ml levels.

Decrease in age-adjusted cerebrospinal fluid β-secretase activity in Alzheimer's subjects

Objectives To develop a novel cerebrospinal fluid (CSF) β-secretase-1 activity assay and evaluate β-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease. Methods The assay consisted of an enzymatic reaction of CSF samples with an optimized β-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody. Results The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to < 1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~ 1.0 pM/year, p < 0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p = 0.02). Conclusion We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.