Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

Fluoxetine administration modulates the cytoskeletal microtubular system in the rat hippocampus.

A number of studies suggest that stressful conditions can induce structural alterations in the hippocampus and that antidepressant drugs may prevent such deficits. In particular, the selective serotonin reuptake inhibitor (SSRI) fluoxetine was more effective in modulating different neuronal plasticity phenomena and related molecules in rat hippocampus. Cytoskeletal microtubule dynamics are fundamental to dendrites and axons remodeling, leading to the hypothesis that fluoxetine may affect the microtubular system. However, despite reports of stress-induced alterations in microtubule dynamics by different stressors, only few studies investigated the in vivo effects of antidepressants on microtubules in specific rat brain regions. The present study investigated the dose-related (1, 5, or 10 mg/kg i.p.) effects of acute and chronic (21 days) treatments with fluoxetine on the ratio of hippocampal alpha-tubulin isoforms which is thought to reflect microtubule dynamics. Western Blot analysis was used to quantify alpha-tubulin isoforms, high-performance liquid chromatography and fluorescence detection was used to measure ex vivo monoamine metabolism. The results showed that acute fluoxetine increased the stable forms acetylated and detyrosinated alpha-tubulin. Conversely, chronic fluoxetine decreased acetylated alpha-tubulin, indicative of increased microtubule dynamics. The neuron-specific Delta2-Tubulin was increased by chronic fluoxetine indicating neuronal involvement in the observed cytoskeletal changes. Although acute and chronic fluoxetine similarly altered serotonin metabolism by inhibition of serotonin reuptake, this showed no apparent correlation to the cytoskeletal perturbations. Our findings demonstrate that fluoxetine administration modulates microtubule dynamics in rat hippocampus. The cytoskeletal effect exerted by fluoxetine may eventually culminate in promoting events of structural neuronal remodeling.

Development and application of a liquid chromatography/tandem mass spectrometric assay for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate in brain slice superfusates and tissue extracts.

A liquid chromatography-tandem mass spectrometric method has been developed for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate. The analytes were separated within 5 min using an anion exchange/reverse phase column. The lower limit of quantification for Glu, NAA and NAAG was found to be 5, 50 and 6 nM, respectively, with a signal-to-noise ratio of 5:1. Using this methodology the basal levels of Glu, NAA and NAAG could be measured consistently in in vitro superfusion samples from rat hippocampus. The assay was also used for measurement of the distribution of Glu, NAA and NAAG in different regions of the rat brain.

SB-399885 is a potent, selective 5-HT6 receptor antagonist with cognitive enhancing properties in aged rat water maze and novel object recognition models.

SB-399885 (N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide) has high affinity for human recombinant and native 5-HT(6) receptors, with pK(i) values 9.11+/-0.03 and 9.02+/-0.05, respectively and is a potent competitive antagonist (pA(2) 7.85+/-0.04). It displays over 200-fold selectivity for the 5-HT(6) receptor over all other receptors, ion channels and enzymes tested to date. SB-399885 inhibited ex vivo [(125)I]SB-258585 (4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-benzenesulfonamide) binding with an ED(50) of 2.0+/-0.24 mg/kg p.o. in rats. It had a minimum effective dose of 1 mg/kg p.o. in a rat maximal electroshock seizure threshold test and a long duration of action, overall demonstrating an excellent pharmacokinetic-pharmacodynamic correlation. Repeated administration of this agent (10 mg/kg p.o., b.i.d. for 7 days) significantly reversed a scopolamine-induced deficit (0.5 mg/kg i.p.) in a rat novel object recognition paradigm. Moreover, in aged rats (22 months old) SB-399885 (10 mg/kg p.o., b.i.d. for 7 days) fully reversed the age-dependent deficit in water maze spatial learning compared to vehicle-treated age-matched controls and significantly improved recall of the task measured by increases in the searching of the target quadrant on post-training days 1, 3 and 7. In vivo microdialysis in the rat medial prefrontal cortex demonstrated that acute SB-399885 (10 mg/kg p.o.) significantly increased extracellular acetylcholine levels. These data demonstrate that SB-399885 is a potent, selective, brain penetrant, orally active 5-HT(6) receptor antagonist with cognitive enhancing properties that are likely to be mediated by enhancements of cholinergic function. These studies provide further support for the potential therapeutic utility of 5-HT(6) receptor antagonists in disorders characterised by cognitive deficits such as Alzheimer's disease and schizophrenia.

Postsynaptic 5-HT1B receptors modulate electroshock-induced generalised seizures in rats.

1. Although an important regulatory role for serotonin (5-HT) in seizure activation and propagation is well established, relatively little is known of the function of specific 5-HT receptor subtypes on seizure modulation. 2. The aim of the present study was to investigate the role of 5-HT(1A, 1B and 1D) receptors in modulating generalised seizures in the rat maximal electroshock seizure threshold (MEST) test. 3. The mixed 5-HT receptor agonists SKF 99101 (5-20 mg kg(-1) i.p.) and RU 24969 (1-5 mg kg(-1) i.p.), 0.5 h pretest, both produced marked dose-related increases in seizure threshold. These agents share high affinity for 5-HT(1A, 1B and 1D) receptors. 4. Antiseizure effects induced by submaximal doses of these agonists were maintained following p-chlorophenylalanine (150 mg kg(-1) i.p. x 3 days)-induced 5-HT depletion. 5. The anticonvulsant action of both SKF 99101 (15 mg kg(-1) i.p.) and RU 24969 (2.5 mg kg(-1) i.p.) was dose-dependently abolished by the selective 5-HT1B receptor antagonist SB-224289 (0.1-3 mg kg(-1) p.o., 3 h pretest) but was unaffected by the selective 5-HT1A receptor antagonist WAY 100635 (0.01-0.3 mg kg(-1) s.c., 1 h pretest). This indicates that 5-HT1B receptors are primarily involved in mediating the anticonvulsant properties of these agents. 6. In addition, the ability of the 5-HT(1B/1D) receptor antagonist GR 127935 (0.3-3 mg kg(-1) s.c., 60 min pretest) to dose-dependently inhibit SKF 99101-induced elevation of seizure threshold also suggests possible downstream involvement of 5-HT1D receptors in the action of this agonist, although confirmation awaits the identification of a selective 5-HT1D receptor antagonist. 7. Overall, these data demonstrate that stimulation of postsynaptic 5-HT1B receptors inhibits electroshock-induced seizure spread in rats.

Localisation of NMU1R and NMU2R in human and rat central nervous system and effects of neuromedin-U following central administration in rats.

RATIONALE: Neuromedin-U (NmU) is an agonist at NMU1R and NMU2R. The brain distribution of NmU and its receptors, in particular NMU2R, suggests widespread central roles for NmU. In agreement, centrally administered NmU affects feeding behaviour, energy expenditure and pituitary output. Further central nervous system (CNS) roles for NmU warrant investigation. OBJECTIVES: To investigate the CNS role of NmU by mapping NMU1R and NMU2R mRNA and measuring the behavioural, endocrine, neurochemical and c-fos response to intracerebroventricular (i.c.v.) NmU. METHODS: Binding affinity and functional potency of rat NmU was determined at human NMU1R and NMU2R. Expression of NMU1R and NMU2R mRNA in rat and human tissue was determined using semi-quantitative reverse-transcription polymerase chain reaction. In in-vivo studies, NmU was administered i.c.v. to male Sprague-Dawley rats, and changes in grooming, motor activity and pre-pulse inhibition (PPI) were assessed. In further studies, plasma endocrine hormones, [DOPAC + HVA]/[dopamine] and [5-HIAA]/[5-HT] ratios and levels of Fos-like immunoreactivity (FLI) were measured 20 min post-NmU (i.c.v.). RESULTS: NmU bound to NMU1R ( K(I), 0.11+/-0.02 nM) and NMU2R ( K(I), 0.21+/-0.05 nM) with equal affinity and was equally active at NMU1R (EC(50), 1.25+/-0.05 nM) and NMU2R (EC(50), 1.10+/-0.20 nM) in a functional assay. NMU2R mRNA expression was found at the highest levels in the CNS regions of both rat and human tissues. NMU1R mRNA expression was restricted to the periphery of both species with the exception of the rat amygdala. NmU caused a marked increase in grooming and motor activity but did not affect PPI. Further, NmU decreased plasma prolactin but did not affect levels of corticosterone, luteinising hormone or thyroid stimulating hormone. NmU elevated levels of 5-HT in the frontal cortex and hypothalamus, with decreased levels of its metabolites in the hippocampus and hypothalamus, but did not affect dopamine function. NmU markedly increased FLI in the nucleus accumbens, frontal cortex and central amygdala. CONCLUSIONS: These data provide further evidence for widespread roles for NmU and its receptors in the brain.

A selected reaction monitoring (SRM)-based method for absolute quantification of Aß38, Aß40, and Aß42 in cerebrospinal fluid of Alzheimer's disease patients and healthy controls.

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-ß (Aß) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aß (Aß42), together with Aß40 and Aß38 in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aß peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aß42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aß42 similar to that obtained by ELISA and even better separation was obtained using the Aß42/Aß40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aß42, Aß40, and Aß38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aß peptide quantification in human CSF valuable for clinical research and trials.

Endogenous purification reveals GREB1 as a key estrogen receptor regulatory factor.

Estrogen receptor-α (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.

Chronic fluoxetine differentially modulates the hippocampal microtubular and serotonergic system in grouped and isolation reared rats.

Social isolation from weaning in rats produces behavioural and hippocampal structural changes at adulthood. Here, rats were group or isolation reared for eight-weeks. Following the initial four-week period of rearing, fluoxetine (10 mg/kg i.p.) was administered for 28 days. Changes in recognition memory, hippocampal monoamines, and cytoskeletal microtubules were investigated. Isolation-rearing for four- or eight-weeks produced recognition memory deficits that were not reversed by fluoxetine. Eight-weeks of isolation decreased alpha-tubulin acetylation (Acet-Tub) and the tyrosinated/detyrosinated alpha-tubulin ratio (Tyr/Glu-Tub), suggesting major alterations in microtubule dynamics and neuronal plasticity. In grouped rats, fluoxetine decreased Acet-Tub without changes in Tyr/Glu-Tub. In isolates, fluoxetine did not affect Acet-Tub but increased Tyr/Glu-Tub. Finally, fluoxetine altered serotonin metabolism in grouped, but not in isolated animals. Therefore, isolation-rearing changes the hippocampal responses of the serotonergic and microtubular system to fluoxetine. These findings show that early-life experience induces behavioural changes paralleled by alterations in cytoskeletal and neurochemical functions.

GSK189254, a novel H3 receptor antagonist that binds to histamine H3 receptors in Alzheimer's disease brain and improves cognitive performance in preclinical models.

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H(3) receptor antagonist with high affinity for human (pK(i) = 9.59 -9.90) and rat (pK(i) = 8.51-9.17) H(3) receptors. GSK189254 is >10,000-fold selective for human H(3) receptors versus other targets tested, and it exhibited potent functional antagonism (pA(2) = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC(50) = 8.20 versus basal guanosine 5'-O-(3-[(35)S]thio)triphosphate binding] at the human recombinant H(3) receptor. In vitro autoradiography demonstrated specific [(3)H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H(3) binding was detected in medial temporal cortex samples from severe cases of Alzheimer's disease, suggesting for the first time that H(3) receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(-)-alpha-methyl[imidazole-2,5(n)-(3)H]histamine dihydrochloride ([(3)H]R-alpha-methylhistamine) binding (ED(50) = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3-3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50) = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimer's disease and other cognitive disorders.