Arachidonic acid pathway alterations in cerebrospinal fluid of dogs with naturally occurring spinal cord injury.

BACKGROUND: Canine intervertebral disc πherniation causes a naturally-occurring spinal cord injury (SCI) that bears critical similarities to human SCI with respect to both injury pathomechanisms and treatment. As such, it has tremendous potential to enhance our understanding of injury biology and the preclinical evaluation of novel therapies. Currently, there is limited understanding of the role of arachidonic acid metabolites in canine SCI. RESULTS: The CSF concentrations of PLA2 and PGE2 were higher in SCI dogs compared to control dogs (p = 0.0370 and 0.0273, respectively), but CSF LCT4 concentration in SCI dogs was significantly lower than that in control dogs (p < 0.0001). Prostaglandin E2 concentration in the CSF was significantly and positively associated with increased severity of SCI at the time of sampling (p = 0.041) and recovery 42 days post-injury (p = 0.006), as measured by ordinal behavioral scores. CONCLUSION: Arachidonic acid metabolism is altered in dogs with SCI, and these data suggest that these AA metabolites reflect injury severity and recovery, paralleling data from other model systems.

Hydrolysis of a lipid membrane by single enzyme molecules: accurate determination of kinetic parameters.

The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis-Menten parameters with an accuracy better than 20% without variation of the initial substrate concentration. The combined single-liposome and single-enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature.

Alterations in cerebrospinal fluid glycerophospholipids and phospholipase A2 activity in Alzheimer's disease.

Our aim is to study selected cerebrospinal fluid (CSF) glycerophospholipids (GP) that are important in brain pathophysiology. We recruited cognitively healthy (CH), minimally cognitively impaired (MCI), and late onset Alzheimer's disease (LOAD) study participants and collected their CSF. After fractionation into nanometer particles (NP) and supernatant fluids (SF), we studied the lipid composition of these compartments. LC-MS/MS studies reveal that both CSF fractions from CH subjects have N-acyl phosphatidylethanolamine, 1-radyl-2-acyl-sn-glycerophosphoethanolamine (PE), 1-radyl-2-acyl-sn-glycerophosphocholine (PC), 1,2-diacyl-sn-glycerophosphoserine (PS), platelet-activating factor-like lipids, and lysophosphatidylcholine (LPC). In the NP fraction, GPs are enriched with a mixture of saturated, monounsaturated, and polyunsaturated fatty acid species, while PE and PS in the SF fractions are enriched with PUFA-containing molecular species. PC, PE, and PS levels in CSF fractions decrease progressively in participants from CH to MCI, and then to LOAD. Whereas most PC species decrease equally in LOAD, plasmalogen species account for most of the decrease in PE. A significant increase in the LPC-to-PC ratio and PLA2 activity accompanies the GP decrease in LOAD. These studies reveal that CSF supernatant fluid and nanometer particles have different GP composition, and that PLA2 activity accounts for altered GPs in these fractions as neurodegeneration progresses.

Decreased phospholipase A2 activity in cerebrospinal fluid of patients with dementia.

Phospholipase A2 (PLA2) is involved in important aspects of dementia, for example neurotransmission and memory processing, membrane function, choline availability, and antioxidative defense. Reduced PLA2-activity has been reported so far in blood samples and postmortem neuronal tissue in Alzheimer disease. For the first time, we studied PLA2 in cerebrospinal fluid (CSF) in Alzheimer disease (AD), vascular (VD), and mixed Alzheimer/vascular dementia (MD). Intracellular PLA2 was assessed in CSF of 16 AD, 12 VD, 15 MD patients, and 19 healthy control subjects. A fluorometric assay was applied using the PLA2-specific substrate NBDC6-HPC. Significantly reduced PLA2 activity was not only found in AD, but also in VD and MD. This finding was independent of demographic co-variates and medication. PLA2 results in CSF corroborate previous findings of impaired PLA2 function in Alzheimer's disease and extend these to patients with VD. They are likely to reflect an involvement of PLA2 impairment in a variety of pathomechanisms crucial in different dementia subtypes, in which disruption of cholinergic neurotransmission and disturbance of intact membrane function appear to be the key mechanisms.