A role for vessel-associated extracellular matrix proteins in multiple sclerosis pathology.

Multiple sclerosis (MS) is unsurpassed for its clinical and pathological hetherogeneity, but the biological determinants of this variability are unknown. HLA-DRB1*15, the main genetic risk factor for MS, influences the severity and distribution of MS pathology. This study set out to unravel the molecular determinants of the heterogeneity of MS pathology in relation to HLA-DRB1*15 status. Shotgun proteomics from a discovery cohort of MS spinal cord samples segregated by HLA-DRB*15 status revealed overexpression of the extracellular matrix (ECM) proteins, biglycan, decorin, and prolargin in HLA-DRB*15-positive cases, adding to established literature on a role of ECM proteins in MS pathology that has heretofore lacked systematic pathological validation. These findings informed a neuropathological characterisation of these proteins in a large autopsy cohort of 41 MS cases (18 HLA-DRB1*15-positive and 23 HLA-DRB1*15-negative), and seven non-neurological controls on motor cortical, cervical and lumbar spinal cord tissue. Biglycan and decorin demonstrate a striking perivascular expression pattern in controls that is reduced in MS (-36.5%, p = 0.036 and - 24.7%, p = 0.039; respectively) in lesional and non-lesional areas. A concomitant increase in diffuse parenchymal accumulation of biglycan and decorin is seen in MS (p = 0.015 and p = 0.001, respectively), particularly in HLA-DRB1*15-positive cases (p = 0.007 and p = 0.046, respectively). Prolargin shows a faint parenchymal pattern in controls that is markedly increased in MS cases where a perivascular deposition pattern is observed (motor cortex +97.5%, p = 0.001; cervical cord +49.1%, p = 0.016). Our findings point to ECM proteins and the vascular interface playing a central role in MS pathology within and outside the plaque area. As ECM proteins are known potent pro-inflammatory molecules, their parenchymal accumulation may contribute to disease severity. This study brings to light novel factors that may contribute to the heterogeneity of the topographical variation of MS pathology.

Regional contribution of vascular dysfunction in white matter dementia: clinical and neuropathological insights.

The maintenance of adequate blood supply and vascular integrity is fundamental to ensure cerebral function. A wide range of studies report vascular dysfunction in white matter dementias, a group of cerebral disorders characterized by substantial white matter damage in the brain leading to cognitive impairment. Despite recent advances in imaging, the contribution of vascular-specific regional alterations in white matter dementia has been not extensively reviewed. First, we present an overview of the main components of the vascular system involved in the maintenance of brain function, modulation of cerebral blood flow and integrity of the blood-brain barrier in the healthy brain and during aging. Second, we review the regional contribution of cerebral blood flow and blood-brain barrier disturbances in the pathogenesis of three distinct conditions: the archetypal white matter predominant neurocognitive dementia that is vascular dementia, a neuroinflammatory predominant disease (multiple sclerosis) and a neurodegenerative predominant disease (Alzheimer's). Finally, we then examine the shared landscape of vascular dysfunction in white matter dementia. By emphasizing the involvement of vascular dysfunction in the white matter, we put forward a hypothetical map of vascular dysfunction during disease-specific progression to guide future research aimed to improve diagnostics and facilitate the development of tailored therapies.

Brain biopsy in neurological disease of unknown etiology: A single-center 12-year retrospective analysis.

There are no international guidelines for brain biopsy in neurological disease of unknown etiology, yet most practicing neurologists will encounter difficult cases in which biopsy is considered. This patient cohort is heterogenous, and it is unclear in which circumstances biopsy is most useful. We performed an audit of brain biopsies reviewed in our neuropathology department from 2010 to 2021. Of 9,488 biopsies, 331 biopsies undertaken for an undiagnosed neurological disease were identified. Where documented, the commonest symptoms were hemorrhage, encephalopathy, and dementia. 29% of biopsies were non-diagnostic. The most common clinically relevant findings on biopsy were infection, cerebral amyloid angiopathy with or without angiitis, and demyelination. Rarer conditions included CNS vasculitis, non-infectious encephalitis, and Creutzfeldt Jakob Disease. We highlight the value of brain biopsy in the workup of cryptogenic neurological disease despite recent advances in less invasive diagnostics.

BK polyomavirus associated progressive multifocal leukoencephalopathy in a person living with HIV.

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the white matter central nervous system occurring in immunocompromised patients particularly those with T cell deficiency such as in HIV, haematological and solid organ malignancies and those taking immunomodulatory medications. PML is caused by JC virus however in rare cases BK virus has been isolated in the cerebral spinal fluid of patients presenting with PML. In this case we describe a 49 year old man who presented to the emergency department with a 2 week history of progressive right sided weakness and dysarthria. His background history included HIV diagnosed in 2005, he had not engaged with care in the past 2 years and had not been taking anti-retroviral therapy (ART). Other past medical history included untreated hepatitis C. His CD4 count was 90 (11%) cells/mm3 on admission and his HIV viral load VL) was 141,000 copies/ml. Magnetic resonance imaging(MRI) showed a hypointense lesion on T1, hyperintense on T2 and FLAIR without diffusion restriction and without mass effect. A lumbar puncture was performed which confirmed JC virus was positive (PCR <50 copies/ml) and also revealed BK virus was positive (PCR 46,511 copies/ml). The patient was commenced on tenofovir alafenamide fumarate/emtricitabine/darunavir/cobicistat in combination with dolutegravir 50mg twice daily. On day 40 post commencement of ART the patient was readmitted with worsening of his right arm weakness and dysarthria. A repeat MRI was performed which showed the hyperdense lesion on T2 and FLAIR appeared slightly larger with some slight enhancement with gadolinium contrast but no other features suggesting PML immune reconstitution inflammatory syndrome (IRIS). The CD4 count had increased to 141(17%) and HIV VL had decreased to 85 copies/ml. A clinical diagnosis of PML IRIS was made and the patient was commenced on prednisolone 30mg BD which lead to an initial improvement in symptoms. Interestingly in this case, both JC virus and BK virus were detected in the CSF of this patient with the level of JC virus being too low to quantify. BK virus was not detectable on peripheral serum sampling suggesting that BK virus is replicating in the CNS independent of other body sites. There have been 5 case reports in the literature of BK virus as the cause of PML. Testing for BK virus should be considered in patients presenting with signs and symptoms of PML and encephalitis particularly when no other cause is found.

Paraneoplastic neurologic syndromes with multiple neural autoantibodies: A report of two cases.

We present two patients who presented with classical paraneoplastic syndromes with multiple central nervous system (CNS) autoantibodies in each case. The presence of multiple antibodies made the detection of a malignancy more likely and both patients were subsequently diagnosed with small cell lung carcinoma (SCLC). We highlight that the presence of multiple CNS autoantibodies increases the likelihood of detecting a malignancy but that the clinical presentation and response to treatment can vary despite similar antibody profiles. Clinicians should be alert to the need to search for occult malignancy in patients with multiple CNS autoantibodies.

Rash developing after cessation of Daclizumab for relapsing remitting MS; a case series.

Daclizumab, a monoclonal antibody directed against CD25, a subunit of the high-affinity IL-2 receptor, was licensed as a disease modifying therapy (DMT) for relapsing remitting multiple sclerosis in 2017. Interference with IL-2 signalling is hypothesised to modulate T cell function. For example it results in a preferential shift of innate lymphoid cell (ILC) into CD56bright natural killer cells and a decrease in regulatory T Cells. We present three patients who developed urticarial papulovesicular rashes at a median of 3 months after discontinuation of Daclizumab. We propose an unexpected T cell mediated immune reaction as the cause.

New versus old: Implications of evolving diagnostic criteria for relapsing-remitting multiple sclerosis.

The International Panel on Diagnosis of Multiple Sclerosis (MS) recently revised the 2010 McDonald criteria and made recommendations for revision, allowing for the earliest possible, accurate diagnosis of MS. For relapsing-remitting MS, positive, unmatched cerebrospinal fluid oligoclonal bands may substitute for dissemination in time. Symptomatic lesions, including brainstem and spinal cord, may demonstrate dissemination in space or in time if enhancing (with the exception of the optic nerve). Cortical and juxtacortical lesions are equivalent. In this retrospective analysis, we applied revised criteria to 250 patients previously diagnosed with relapsing-remitting MS according to 2010 criteria and assessed for change in diagnostic times. There was a significant improvement in time to diagnosis between 2010 and 2017 groups ( p < 0.01). Median time to diagnosis according to McDonald 2010 was 7.4 months, compared with 2.3 months for McDonald 2017. Use of cerebrospinal fluid results most frequently resulted in a reduction in time to diagnosis. Symptomatic gadolinium-enhancing lesions led to earliest diagnostic times.

Mechanisms of vascular disease in dementia: what does industry want to know?

Despite recent advances in basic and clinical science, dementia remains an area of high unmet medical need. The role of cerebrovascular mechanisms in the pathogenesis and progression of cognitive and functional impairment in dementia is being revived. In order to facilitate the development of therapeutic approaches, it is critical that a number of fundamental elements are integrated into research strategies investigating cerebrovascular pathologies as these will maximize the opportunity of bringing medicines to patients in a timely manner.

Investigation of the Brain Biodistribution of the Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitor [18F]GSK2647544 in Healthy Male Subjects.

PURPOSE: GSK2647544 is a potent and specific inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), which was in development as a potential treatment for Alzheimer's disease (AD). In order to refine therapeutic dose predictions and confirm brain penetration, a radiolabelled form of the inhibitor, [18F]GSK2647544, was manufactured for use in a positron emission tomography (PET) biodistribution study. PROCEDURES: [18F]GSK2647544 was produced using a novel, copper iodide (Cu(I)) mediated, [18F]trifluoromethylation methodology. Healthy male subjects (n = 4, age range 34-42) received an oral dose of unlabelled GSK2647544 (100 mg) and after 2 h an intravenous (iv) injection of [18F]GSK2647544 (average injected activity and mass were 106 ± 47 MBq and 179 ± 55 µg, respectively) followed by dynamic PET scans for 120 min. Defined regions of interest (ROI) throughout the brain were used to obtain regional time-activity curves (TACs) and compartmental modelling analysis used to estimate the primary outcome measure, whole brain volume of distribution (VT). Secondary PK and safety endpoints were also recorded. RESULTS: PET dynamic data were successfully obtained from all four subjects and there were no clinically significant variations of the safety endpoints. Inspection of the TACs indicated a relatively homogenous uptake of [18F]GSK2647544 across all the ROIs examined. The mean whole brain VT was 0.56 (95 % CI, 0.41-0.72). Secondary PK parameters, Cmax (geometric mean) and Tmax (median), were 354 ng/ml and 1.4 h, respectively. Metabolism of GSK2647544 was relatively consistent across subjects, with 20-40 % of the parent compound [18F]GSK2647544 present after 120 min. CONCLUSIONS: The study provides evidence that GSK2647544 is able to cross the blood brain barrier in healthy male subjects leading to a measurable brain exposure. The administered doses of GSK2647544 were well tolerated. Exploratory modelling suggested that a twice-daily dose of 102 mg, at steady state, would provide ~80 % trough inhibition of brain Lp-PLA2 activity. TRIAL REGISTRATION: Clintrials.gov: NCT01924858.

Evaluation of the safety, pharmacokinetics, pharmacodynamics, and drug-drug interaction potential of a selective Lp-PLA2 inhibitor (GSK2647544) in healthy volunteers
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OBJECTIVE: To evaluate in healthy volunteers the safety, pharmacokinetics (PK), pharmacodynamics (PD), and drug-drug interaction (DDI) potential of GSK2647544, (a selective lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibitor). METHODS: Study 1 was a single-blind, randomized, placebo-controlled, crossover study with healthy male volunteers randomized to receive single escalating oral doses (0.5 - 750 mg) of GSK2647544. Study 2 was a single-blind, randomized, placebo-controlled study with healthy volunteers randomized to receive repeat doses (80 mg) of GSK2647544. The drug-drug interaction of GSK2647544 with simvastatin was also evaluated in study 2. RESULTS: Across both studies GSK2647544 doses were generally well tolerated with no GSK2647544-related clinically significant findings. GSK2647544 was readily absorbed and its plasma concentration declined bi-exponentially with a terminal half-life ranging from 8 to 16 hours. Plasma exposure of GSK2647544 increased approximately dose-proportionally. There was GSK2647544 dose-dependent inhibition of plasma Lp-PLA2 activity, with a trough inhibition (12 hours after dose) of 85.6% after 7-day twice daily dosing. The administration of simvastatin concomitantly with GSK2647544 increased the overall exposure (area under the plasma concentration-time curve and maximum plasma concentration) of simvastatin and simvastatin acid by 3.6- to 4.3-fold and 1.5- to 3.1-fold, respectively. CONCLUSIONS: GSK2647544 was generally well tolerated and had a reasonable PK-PD profile. The clinically significant drug-drug interaction led to an early termination of study 2.
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A 24-week study to evaluate the effect of rilapladib on cognition and cerebrospinal fluid biomarkers of Alzheimer's disease.

BACKGROUND: The lipoprotein-associated phospholipase A2 inhibitor (Lp-PLA2), rilapladib (SB659032), is being evaluated as a potential treatment to slow the progression of Alzheimer's disease (AD). METHODS: One hundred twenty-four subjects with possible mild AD and with neuroimaging evidence of cerebrovascular disease were randomized to placebo or 250-mg rilapladib once daily, for 24 weeks, in addition to stable background acetylcholinesterase inhibitor and/or memantine. The study assessed the safety and tolerability of rilapladib and its effects on cognition, mechanistic, and disease-related biomarkers. Although the overall intent behind the study was to take a broad exploratory view of the data, two primary end points of interest (cerebrospinal fluid [CSF] amyloid beta peptide 1-42 [Aß1-42] and CogState executive function/working memory [EF/WM] composite score at week 24) were prespecified in the analysis plan for inferential statistical analysis. RESULTS: Rilapladib was well tolerated with no significant safety concerns. A significant difference from placebo was observed for rilapladib on change from baseline in EF/WM (effect size, 0.45; P = .026). There was no significant difference between groups on the change from baseline in CSF Aß1-42 (P = .133). Preliminary evidence of effects was detected on other mechanistic (albumin quotient) and disease-related biomarkers (tau/P-tau and neurofilament light chain). CONCLUSION: These data provide initial evidence supporting Lp-PLA2 inhibition as a novel treatment for dementia. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT01428453.

Imaging as a biomarker in drug discovery for Alzheimer's disease: is MRI a suitable technology?

This review provides perspectives on the utility of magnetic resonance imaging (MRI) as a neuroimaging approach in the development of novel treatments for Alzheimer's disease. These considerations were generated in a roundtable at a recent Wellcome Trust meeting that included experts from academia and industry. It was agreed that MRI, either structural or functional, could be used as a diagnostic, for assessing worsening of disease status, for monitoring vascular pathology, and for stratifying clinical trial populations. It was agreed also that MRI implementation is in its infancy, requiring more evidence of association with the disease states, test-retest data, better standardization across multiple clinical sites, and application in multimodal approaches which include other imaging technologies, such as positron emission tomography, electroencephalography, and magnetoencephalography.

Cholestenoic acids regulate motor neuron survival via liver X receptors.

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3ß,7α-dihydroxycholest-5-en-26-oic acid (3ß,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3ß-hydroxy-7-oxocholest-5-en-26-oic acid (3ßH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3ß,7α-diHCA and 3ßH,7O-CA, 3ß-hydroxycholest-5-en-26-oic acid (3ß-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3ß-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3ß,7α-diHCA. Moreover, 3ß,7α-diHCA prevented the loss of motor neurons induced by 3ß-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Plasma lipoprotein-associated phospholipase A2 activity in Alzheimer's disease, amnestic mild cognitive impairment, and cognitively healthy elderly subjects: a cross-sectional study.

INTRODUCTION: Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a circulating enzyme with pro-inflammatory and oxidative activities associated with cardiovascular disease and ischemic stroke. While high plasma Lp-PLA2 activity was reported as a risk factor for dementia in the Rotterdam study, no association between Lp-PLA2 mass and dementia or Alzheimer's disease (AD) was detected in the Framingham study. The objectives of the current study were to explore the relationship of plasma Lp-PLA2 activity with cognitive diagnoses (AD, amnestic mild cognitive impairment (aMCI), and cognitively healthy subjects), cardiovascular markers, cerebrospinal fluid (CSF) markers of AD, and apolipoprotein E (APOE) genotype. METHODS: Subjects with mild AD (n = 78) and aMCI (n = 59) were recruited from the Memory Clinic, University Hospital, Basel, Switzerland; cognitively healthy subjects (n = 66) were recruited from the community. Subjects underwent standardised medical, neurological, neuropsychological, imaging, genetic, blood and CSF evaluation. Differences in Lp-PLA2 activity between the cognitive diagnosis groups were tested with ANOVA and in multiple linear regression models with adjustment for covariates. Associations between Lp-PLA2 and markers of cardiovascular disease and AD were explored with Spearman's correlation coefficients. RESULTS: There was no significant difference in plasma Lp-PLA2 activity between AD (197.1 (standard deviation, SD 38.4) nmol/min/ml) and controls (195.4 (SD 41.9)). Gender, statin use and low-density lipoprotein cholesterol (LDL) were independently associated with Lp-PLA2 activity in multiple regression models. Lp-PLA2 activity was correlated with LDL and inversely correlated with high-density lipoprotein (HDL). AD subjects with APOE-ε4 had higher Lp-PLA2 activity (207.9 (SD 41.2)) than AD subjects lacking APOE-ε4 (181.6 (SD 26.0), P = 0.003) although this was attenuated by adjustment for LDL (P = 0.09). No strong correlations were detected for Lp-PLA2 activity and CSF markers of AD. CONCLUSION: Plasma Lp-PLA2 was not associated with a diagnosis of AD or aMCI in this cross-sectional study. The main clinical correlates of Lp-PLA2 activity in AD, aMCI and cognitively healthy subjects were variables associated with lipid metabolism.

Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid ß42.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in clinical settings, research and drug trials. However, their broad-scale use on different technology platforms is hampered by the lack of standardization at the level of sample handling, determination of concentrations of analytes and the absence of well-defined performance criteria for in vitro diagnostic or companion diagnostic assays, which influences the apparent concentration of the analytes measured and the subsequent interpretation of the data. There is a need for harmonization of CSF AD biomarker assays that can reliably, across centers, quantitate CSF biomarkers with high analytical precision, selectivity and stability over long time periods. In this position paper, we discuss reference procedures for the measurement of CSF AD biomarkers, especially amyloid ß42 and tau. We describe possible technical approaches, focusing on a selected reaction monitoring mass spectrometry assay as a candidate reference method for quantification of CSF amyloid ß42.

Cerebrospinal fluid steroidomics: are bioactive bile acids present in brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C(27) and C(24) intermediates of the bile acid biosynthetic pathways with structures corresponding to 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 +/- 2.826 ng/ml, mean +/- S.D., six subjects), 3beta-hydroxycholest-5-en-26-oic acid (0.416 +/- 0.193 ng/ml), 7alpha,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 +/- 0.543 ng/ml), and 7alpha-hydroxy-3-oxochol-4-en-24-oic acid (0.172 +/- 0.085 ng/ml), and the C(26) sterol 7alpha-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 +/- 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3beta-hydroxycholest-5-en-26-oic acid and 3beta,7alpha-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7alpha-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7Alpha-hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3beta,7alpha-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3beta-hydroxy-Delta(5)-C(27)-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239-248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.

Interaction of the amyloid imaging tracer FDDNP with hallmark Alzheimer's disease pathologies.

The distinctive cortical uptake of the tracer (18)F-FDDNP (2-(1-{6-[(2-fluoroethyl(methyl)amino]-2-naphthyl}ethylidene)malononitrile) in Alzheimer's disease (AD) is believed to be because of its binding to both neurofibrillary tangles (NFTs) and highly fibrillar senile plaques. We therefore investigated the binding of a tracer concentration of (3)H-FDDNP to brain sections containing AD hallmark pathologies. Semi-adjacent sections were labelled with (3)H-PIB (Pittsburgh compound-B, 2-[4'-(methylamino)phenyl]-6-hydroxybenzothiazole) and (14)C-SB13 (4-N-methylamino-4'-hydroxystilbene) for comparison. Neocortical sections containing widespread senile plaques and cerebrovascular amyloid angiopathy, produced a sparse and weak labelling following incubation with (3)H-FDDNP. Furthermore, in sections containing NFTs, there was no overt labelling of the pathology by (3)H-FDDNP. In contrast, sections labelled with (3)H-PIB displayed extensive labelling of diffuse plaques, classical plaques, cerebrovascular amyloid angiopathy and NFTs. (14)C-SB13 produced a broadly similar binding pattern to PIB. Radioligand binding assays employing in vitro generated amyloid-beta peptide fibrils demonstrated a approximately 10-fold reduced affinity for (3)H-FDDNP (85.0 +/- 2.0 nM) compared with (3)H-PIB (8.5 +/- 1.3 nM). These data provide an alternative mechanistic explanation for the observed low cortical uptake of (18)F-FDDNP in AD; in that the ligand is only weakly retained by the hallmark neuropathology because of its low affinity for amyloid structures.

Monitoring the amyloid beta-peptide in vivo--caveat emptor.

As a wave of 'disease modifying' (DM) therapies for Alzheimer's disease (AD) progresses towards the later stages of clinical development, an evaluation of our ability to measure relevant pharmacodynamic effects of such therapies is warranted. Reducing accumulation of amyloid beta (Abeta)-peptide in the brain parenchyma is the primary objective of most current DM approaches. Although a number of methods are available to measure Abeta in blood, cerebrospinal fluid (CSF) and the cerebrum, putative DM-induced changes in the levels of the peptides may not be fully captured, and the reasons for any such changes are not fully understood. Additional candidate biofluid (tau and isoprostanes) and imaging (MRI, FDG-PET) measures may provide alternative supporting evidence of drug activity and subsequent clinical efficacy in patient populations.

Discovering oxysterols in plasma: a window on the metabolome.

While the proteome defines the expressed gene products, the metabolome results from reactions controlled by such gene products. Plasma represents an accessible "window" to the metabolome both in regard of availability and content. The wide range of the plasma metabolome, in terms of molecular diversity and abundance, makes its comprehensive analysis challenging. Here we demonstrate an analytical method designed to target one region of the metabolome, that is, oxysterols. Since the discovery of their biological activity as ligands to nuclear receptors there has been a reawakening of interest in oxysterols and their analysis. In addition, the oxysterols, 24S- and 27-hydroxycholesterol, are currently under investigation as potential biomarkers associated with neurodegenerative disorders such as Alzheimer's disease and multiple sclerosis; widespread analysis of these lipids in clinical studies will require the development of robust, sensitive and rapid analytical techniques. In this communication we present results of an investigation of the oxysterols content of human plasma using a newly developed high-performance liquid chromatography-mass spectrometry (HPLC-MS) method incorporating charge-tagging and high-resolution MS. The method has allowed the identification in plasma of monohydroxylated cholesterol molecules, 7alpha-, 24S-, and 27-hydroxycholesterol; the cholestenetriol 7alpha,27-dihydroxycholesterol; and 3beta-hydroxycholest-5-en-27-oic acid and its metabolite 3beta,7alpha-dihydroxycholest-5-en-27-oic acid. The methodology described is also applicable for the analysis of other sterols in plasma, that is, cholesterol, 7-dehydrocholesterol, and desmosterol, as well as cholesterol 5,6- seco-sterols and steroid hormones. Although involving derivatization, sample preparation is straightforward and chromatographic analysis rapid (17 min), while the MS method offers high sensitivity (ng/mL of sterol in plasma, or pg on-column) and specificity. The methodology is suitable for targeted metabolomic analysis of sterols, oxysterols, and steroid hormones opening a "window" to view this region of the metabolome.