Serine ether glycerophospholipids: Decrements in the frontal cortex associated with mild cognitive impairment and Alzheimer's disease.

Ether glycerophospholipids (GPL) are involved in membrane fluidity and fusion. Vinyl-ether GPL are also conjectured to provide antioxidant capacity in the brain. The roles of these lipids in the processes involved in the development of dementia are not understood but choline and ethanolamine vinyl-ether GPL (i.e., plasmalogens) are decreased in the brains of subjects with dementia. In contrast, serine ether and vinyl-ether GPL have not been investigated in human brain. We therefore undertook an evaluation of these lipids, utilizing high-resolution mass spectrometry (HR-MS), in tissues from control and dementia subjects that we had previously characterized in-depth. We can report for the first time that a number of serine ether GPL and a more limited number of serine plasmalogens are present in human frontal cortex. In addition, we found that some of these frontal cortex lipids are decreased in Mild Cognitive Impairment (MCI), early-onset Alzheimer's disease (EOAD), and late-onset AD (LOAD). In contrast no alterations in serine ether GPL were monitored in the frontal cortex of donors with schizophrenia, demonstrating disease specificity. These data suggest that further studies of the roles of ether GPL, including serine ether GPL, in brain function are worthy of undertaking.

The future of paediatric and adolescent gynaecology in Europe.

Paediatric and Adolescent Gynaecology (PAG) is a subspecialty under the umbrella of Obstetrics and Gynaecology but linked to other branches of medicine including Paediatrics, Surgery, Endocrinology and Urology. Therefore future developments in clinical care and education requires a multidisciplinary approach combining aspects of all the above medical specialties, and also with inputs from Public Health, Genetics, Radiology and Psychology. A multidisciplinary collaboration among different specialists is as important as the establishment and adoption of standards in education, training and management. PAG in Europe has evolved from its first steps and it is still growing with the aim of providing increasing protection of the gynaecological and reproductive health of female children and adolescents. In fact, without proper advice and care, inappropriate management of gynaecological issues in childhood and adolescence can be expected to have significant repercussions throughout later years, and into adulthood. The aim of this third paper in this mini-symposium is to explore how PAG should develop in Europe in the near future.

Reduced Plasma Levels of Very-Long-Chain Dicarboxylic Acid 28:4 in Italian and Brazilian Colorectal Cancer Patient Cohorts.

BACKGROUND: There are currently no blood-based biomarkers for early diagnosis of colorectal cancer. Previous research has suggested that very-long-chain dicarboxylic acid (VLCDCA) 28:4 might be such a biomarker. METHODS: Using high-resolution mass spectrometry, we analyzed VLCDCA 28:4 in the plasma of colorectal cancer patients in Italian [n = 62] and Brazilian [n = 52] cohorts. Additionally, we investigated individuals diagnosed with familial adenomatous polyposis (FAP; n = 27), one of the most important clinical forms of inherited susceptibility to colorectal cancer. Results: Decrements in plasma levels of VLCDCA 28:4 were monitored in colorectal cancer patients. These decreases were independent of the stage of tumor development and the individual's age. However, no decrements in VLCDCA 28:4 were monitored in FAP patients. CONCLUSIONS: The plasma levels of VLCDCA 28:4 represent a potential biomarker of sporadic colorectal cancer. In addition, it is possible that resupply of this anti-inflammatory lipid may represent a new therapeutic strategy for CRC and inflammatory disorders.

Endogenous Anti-Inflammatory Very-Long-Chain Dicarboxylic Acids: Potential Chemopreventive Lipids.

In a paradigm shift, cancer research efforts are being dedicated to the discovery of chemopreventive agents. The goal of this approach is to delay or prevent the progression of augmented cell division to established cancer. Research has focused on dietary supplements, drugs, and endogenous lipids that possess anti-inflammatory properties. We undertook a lipidomics analysis of potential endogenous anti-inflammatory/anti-proliferative lipids in human plasma. We performed high-resolution mass spectrometric lipidomics analyses of plasma samples from controls and patients with colorectal, kidney, pancreatic, glioblastoma, and breast cancers. We present evidence that endogenous very-long-chain dicarboxylic acids (VLCDCA) are anti-inflammatory lipids that possess chemopreventative properties. In a family of VLCDCAs, we characterized VLCDCA 28:4, which is decreased in the plasma of patients with colorectal, kidney, and pancreatic cancers. The structure of this biomarker was validated by derivatization strategies, synthesis of the analytical standard, and tandem mass spectrometry. Our data suggest that VLCDCA 28:4 may be a useful blood biomarker for a number of cancers and that resupplying this lipid, via a prodrug for example, may offer a new anti-inflammatory therapeutic strategy for delaying or preventing the progression of cancer and other inflammatory diseases.

Cosmetic genital surgery in children and adolescents.

Clinicians are faced with increasing requests for female cosmetic genital surgery and prominent amongst these is labiaplasty. The implications of labiaplasty in adolescence are explored with emphasis on what is known about normal genital appearances, pubertal development, anatomy and physiology and the options for surgical intervention including risks and implications. Faced with what is known to date on female cosmetic genital surgery then such interventions should be avoided in adolescence in the absence of defined medical indications until at least the age of 18 years.

Cosmetic Labiaplasty in an Adolescent Population.

Labiaplasty (defined as the surgical reduction of the labia minora) is the most common procedure under the umbrella of female genital cosmetic surgery with the prevalence increasing over the past 10-15 years. However, the concept of labial hypertrophy holds an arbitrary definition, with no research into labial size undertaken within the pediatric and adolescent populations. Under the tenets of medical ethics there is acceptance of the need to avoid harm and so, for reasons to be outlined, performance of labiaplasty in children and adolescents should be avoided. This Mini-Review does not extend to pathological conditions that affect the labia minora.

ESI-MS/MS and MALDI-IMS Localization Reveal Alterations in Phosphatidic Acid, Diacylglycerol, and DHA in Glioma Stem Cell Xenografts.

Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism toward these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N = 9) with those that do not attract (N = 9) to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6), were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.

Gynaecological issues affecting the obese adolescent.

The implications of obesity in childhood and adolescence resonate into adulthood and have implications at different levels that include psychosocial and health issues that impact beyond reproductive performance. This chapter explores the various facets and consequences on gynaecological issues of increased Body Mass Index in childhood, including the link with puberty, pubertal menorrhagia (also affecting children with complex needs) and the all too common problems surrounding hyperandrogenism, insulin resistance and the polycystic ovarian syndrome in particular which need to be seen in the specific context of the adolescent years. The wider ramifications of obesity on the psychosocial welfare of adolescents merits special attention. Finally management strategies are considered in the context of the needs of adolescents.

Mass spectrometry strategies for clinical metabolomics and lipidomics in psychiatry, neurology, and neuro-oncology.

Metabolomics research has the potential to provide biomarkers for the detection of disease, for subtyping complex disease populations, for monitoring disease progression and therapy, and for defining new molecular targets for therapeutic intervention. These potentials are far from being realized because of a number of technical, conceptual, financial, and bioinformatics issues. Mass spectrometry provides analytical platforms that address the technical barriers to success in metabolomics research; however, the limited commercial availability of analytical and stable isotope standards has created a bottleneck for the absolute quantitation of a number of metabolites. Conceptual and financial factors contribute to the generation of statistically under-powered clinical studies, whereas bioinformatics issues result in the publication of a large number of unidentified metabolites. The path forward in this field involves targeted metabolomics analyses of large control and patient populations to define both the normal range of a defined metabolite and the potential heterogeneity (eg, bimodal) in complex patient populations. This approach requires that metabolomics research groups, in addition to developing a number of analytical platforms, build sufficient chemistry resources to supply the analytical standards required for absolute metabolite quantitation. Examples of metabolomics evaluations of sulfur amino-acid metabolism in psychiatry, neurology, and neuro-oncology and of lipidomics in neurology will be reviewed.

Oral bioavailability of the ether lipid plasmalogen precursor, PPI-1011, in the rabbit: a new therapeutic strategy for Alzheimer's disease.

INTRODUCTION: Docosahexaenoic acid (DHA) and DHA-containing ethanolamine plasmalogens (PlsEtn) are decreased in the brain, liver and the circulation in Alzheimer's disease. Decreased supply of plasmalogen precursors to the brain by the liver, as a result of peroxisomal deficits is a process that probably starts early in the AD disease process. To overcome this metabolic compromise, we have designed an orally bioavailable DHA-containing ether lipid precursor of plasmalogens. PPI-1011 is an alkyl-diacyl plasmalogen precursor with palmitic acid at sn-1, DHA at sn-2 and lipoic acid at sn-3. This study outlines the oral pharmacokinetics of this precursor and its conversion to PlsEtn and phosphatidylethanolamines (PtdEtn). METHODS: Rabbits were dosed orally with PPI-1011 in hard gelatin capsules for time-course and dose response studies. Incorporation into PlsEtn and PtdEtn was monitored by LC-MS/MS. Metabolism of released lipoic acid was monitored by GC-MS. To monitor the metabolic fate of different components of PPI-1011, we labeled the sn-1 palmitic acid, sn-2 DHA and glycerol backbone with (13)C and monitored their metabolic fates by LC-MS/MS. RESULTS: PPI-1011 was not detected in plasma suggesting rapid release of sn-3 lipoic acid via gut lipases. This conclusion was supported by peak levels of lipoic acid metabolites in the plasma 3 hours after dosing. While PPI-1011 did not gain access to the plasma, it increased circulating levels of DHA-containing PlsEtn and PtdEtn. Labeling experiments demonstrated that the PtdEtn increases resulted from increased availability of DHA released via remodeling at sn-2 of phospholipids derived from PPI-1011. This release of DHA peaked at 6 hrs while increases in phospholipids peaked at 12 hr. Increases in circulating PlsEtn were more complex. Labeling experiments demonstrated that increases in the target PlsEtn, 16:0/22:6, consisted of 2 pools. In one pool, the intact precursor received a sn-3 phosphoethanolamine group and desaturation at sn-1 to generate the target plasmalogen. The second pool, like the PtdEtn, resulted from increased availability of DHA released during remodeling of sn-2. In the case of sn-1 18:0 and 18:1 plasmalogens with [(13)C(3)]DHA at sn-2, labeling was the result of increased availability of [(13)C(3)]DHA from lipid remodeling. Isotope and repeated dosing (2 weeks) experiments also demonstrated that plasmalogens and/or plasmalogen precursors derived from PPI-1011 are able to cross both the blood-retinal and blood-brain barriers. CONCLUSIONS: Our data demonstrate that PPI-1011, an ether lipid precursor of plasmalogens is orally bioavailable in the rabbit, augmenting the circulating levels of unesterified DHA and DHA-containing PlsEtn and PtdEtn. Other ethanolamine plasmalogens were generated from the precursor via lipid remodeling (de-acylation/re-acylation reactions at sn-2) and phosphatidylethanolamines were generated via de-alkylation/re-acylation reactions at sn-1. Repeated oral dosing for 2 weeks with PPI-1011 resulted in dose-dependent increases in circulating DHA and DHA-containing plasmalogens. These products and/or precursors were also able to cross the blood-retinal and blood-brain barriers.

Cellular diamine levels in cancer chemoprevention: modulation by ibuprofen and membrane plasmalogens.

BACKGROUND: To develop effective strategies in cancer chemoprevention, an increased understanding of endogenous biochemical mediators that block metastatic processes is critically needed. Dietary lipids and non-steroidal anti-inflammatory drugs (NSAIDs) have a published track record of providing protection against gastrointestinal malignancies. In this regard, we examined the effects of membrane plasmalogens and ibuprofen on regulation of cellular levels of diamines, polyamine mediators that are augmented in cancer cells. For these studies we utilized Chinese hamster ovary (CHO) cells and NRel-4 cells, a CHO cell line with defective plasmalogen synthesis. RESULTS: NRel-4 cells, which possess cellular plasmalogen levels that are 10% of control CHO cells, demonstrated 2- to 3-fold increases in cellular diamine levels. These diamine levels were normalized by plasmalogen replacement and significantly reduced by ibuprofen. In both cases the mechanism of action appears to mainly involve increased diamine efflux via the diamine exporter. The actions of ibuprofen were not stereospecific, supporting previous studies that cyclooxygenase (COX) inhibition is unlikely to be involved in the ability of NSAIDs to reduce intracellular diamine levels. CONCLUSIONS: Our data demonstrate that ibuprofen, a drug known to reduce the risk of colorectal cancer, reduces cellular diamine levels via augmentation of diamine efflux. Similarly, augmentation of membrane plasmalogens can increase diamine export from control and plasmalogen-deficient cells. These data support the concept that membrane transporter function may be a therapeutic point of intervention for dietary and pharmacological approaches to cancer chemoprevention.

In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor.

BACKGROUND: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency. RESULTS: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver. CONCLUSIONS: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.

Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines.

Despite the fact that N-acetyl methionine (NAM) supplementation has long been reported as a bioavailable source of methionine in humans, and known to reduce liver toxicity after acetaminophen overdose, its cellular endogenous presence has never been investigated. We demonstrate for the first time that NAM is present in both human and mouse tissues and cells in culture. A wide variety of cultured cells, including a number of brain derived cell types, as well as mouse and human brain tissue all have clearly detectable levels of NAM. Methionine is rapidly acetylated to form NAM in cultured human oligodendroglioma cells with an initial rate of 0.44 ± 0.064 atom percent excess per minute. The presence of measurable quantities of NAM in brain cells in combination with its rapid formation point to a potential physiological role for N-acetylated methionine in the brain. Aminoacylase 1 is responsible for metabolism of NAM to methionine and acetate. Deficiencies in aminoacylase 1 have been linked to a variety of neurological disorders; however, it is unclear whether and how the brain is affected by this defect. The reported presence of NAM in the human brain may provide an invaluable key to discovering the link between aminoacylase 1 mutations and neurological problems.

Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study.

BACKGROUND: Disrupted cholesterol regulation leading to increased circulating and membrane cholesterol levels is implicated in many age-related chronic diseases such as cardiovascular disease (CVD), Alzheimer's disease (AD), and cancer. In vitro and ex vivo cellular plasmalogen deficiency models have been shown to exhibit impaired intra- and extra-cellular processing of cholesterol. Furthermore, depleted brain plasmalogens have been implicated in AD and serum plasmalogen deficiencies have been linked to AD, CVD, and cancer. RESULTS: Using plasmalogen deficient (NRel-4) and plasmalogen sufficient (HEK293) cells we investigated the effect of species-dependent plasmalogen restoration/augmentation on membrane cholesterol processing. The results of these studies indicate that the esterification of cholesterol is dependent upon the amount of polyunsaturated fatty acid (PUFA)-containing ethanolamine plasmalogen (PlsEtn) present in the membrane. We further elucidate that the concentration-dependent increase in esterified cholesterol observed with PUFA-PlsEtn was due to a concentration-dependent increase in sterol-O-acyltransferase-1 (SOAT1) levels, an observation not reproduced by 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibition. CONCLUSION: The present study describes a novel mechanism of cholesterol regulation that is consistent with clinical and epidemiological studies of cholesterol, aging and disease. Specifically, the present study describes how selective membrane PUFA-PlsEtn enhancement can be achieved using 1-alkyl-2-PUFA glycerols and through this action reduce levels of total and free cholesterol in cells.

Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection.

BACKGROUND: There are currently no accurate serum markers for detecting early risk of colorectal cancer (CRC). We therefore developed a non-targeted metabolomics technology to analyse the serum of pre-treatment CRC patients in order to discover putative metabolic markers associated with CRC. Using tandem-mass spectrometry (MS/MS) high throughput MS technology we evaluated the utility of selected markers and this technology for discriminating between CRC and healthy subjects. METHODS: Biomarker discovery was performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Comprehensive metabolic profiles of CRC patients and controls from three independent populations from different continents (USA and Japan; total n = 222) were obtained and the best inter-study biomarkers determined. The structural characterization of these and related markers was performed using liquid chromatography (LC) MS/MS and nuclear magnetic resonance technologies. Clinical utility evaluations were performed using a targeted high-throughput triple-quadrupole multiple reaction monitoring (TQ-MRM) method for three biomarkers in two further independent populations from the USA and Japan (total n = 220). RESULTS: Comprehensive metabolomic analyses revealed significantly reduced levels of 28-36 carbon-containing hydroxylated polyunsaturated ultra long-chain fatty-acids in all three independent cohorts of CRC patient samples relative to controls. Structure elucidation studies on the C28 molecules revealed two families harbouring specifically two or three hydroxyl substitutions and varying degrees of unsaturation. The TQ-MRM method successfully validated the FTICR-MS results in two further independent studies. In total, biomarkers in five independent populations across two continental regions were evaluated (three populations by FTICR-MS and two by TQ-MRM). The resultant receiver-operator characteristic curve AUCs ranged from 0.85 to 0.98 (average = 0.91 +/- 0.04). CONCLUSIONS: A novel comprehensive metabolomics technology was used to identify a systemic metabolic dysregulation comprising previously unknown hydroxylated polyunsaturated ultra-long chain fatty acid metabolites in CRC patients. These metabolites are easily measurable in serum and a decrease in their concentration appears to be highly sensitive and specific for the presence of CRC, regardless of ethnic or geographic background. The measurement of these metabolites may represent an additional tool for the early detection and screening of CRC.

Novel plasma phospholipid biomarkers of autism: mitochondrial dysfunction as a putative causative mechanism.

Autism is a neurological disorder that manifests as noticeable behavioral and developmental abnormalities predominantly in males between the ages of 2 and 10. Although the genetics, biochemistry and neuropathology of this disease have been extensively studied, underlying causal factors to this disease have remained elusive. Using a longitudinal trial design in which three plasma samples were collected from 15 autistic and 12 non-autistic age-matched controls over the course of 1 year, universal and unambiguous alterations in lipid metabolism were observed. Biomarkers of fatty acid elongation and desaturation (poly-unsaturated long chain fatty acids (PUFA) and/or saturated very long chain fatty acids (VLCFA)-containing ethanolamine phospholipids) were statistically elevated in all autistic subjects. In all 8 of the affected/non-affected sibling pairs, the affected sibling had higher levels of these biomarkers than the unaffected sibling. Exposure of neurons, astrocytes and hepatocytes in vitro to elevated extracellular glutamate levels resulted in lipid biomarker changes indistinguishable from those observed in autistic subjects. Glutamate stress also resulted in in vitro decreased levels of reduced glutathione (GSH), methionine and cysteine, in a similar way to the decreases we observed in autism plasma. Impaired mitochondrial fatty acid oxidation, elevated plasma VLCFAs, and glutamate toxicity as putative causal factors in the biochemistry, neuropathology, and gender bias in autism are discussed.

Cellular thiol pools are responsible for sequestration of cytotoxic reactive aldehydes: central role of free cysteine and cysteamine.

Cellular thiol pools have been shown to be important in the regulation of the redox status of cells, providing a large antioxidant pool consisting of free thiols, thiols bound in the disulfide form and thiols bound to proteins. However, experimental studies with the thiol cysteamine and its disulfide cystamine have demonstrated dramatic cytoprotection in experimental models where antioxidants provide only minor protection. These data suggest that an alternate action of thiols is important in their cytoprotective actions. A common feature of the in vitro and in vivo models, where these thiol agents demonstrate cytoprotection, is the generation of cytotoxic aldehydes. We therefore studied the actions of cystamine, cysteamine and several reference thiol agents as cytoprotectants against cell death induced by increased "aldehyde load". We found that all the thiol agents examined provided dramatic protection against aldehyde-induced cell death in SN56 cholinergic neurons, under conditions in which acrolein induced 100% cell death. With regard to mechanism of action, the reference thiols cysteine, N-acetylcysteine, 2-mercaptoethanesulfonic acid, mercapto-propionyglycine, and cysteamine can directly sequester aldehydes. In addition, these thiols were all found to augment intracellular cysteine levels via disulfide interchange reactions. Cysteamine and cystamine also augmented basal intracellular cysteamine levels. Our data, for the first time, demonstrate the importance of intracellular thiols in sequestering toxic reactive aldehyde products of lipid peroxidation and polyamine metabolism. In addition it appears that pharmacological manipulation of intracellular thiol pools might offer a new approach in the design of neuroprotective drug candidates.

Neurochemical analysis of amino acids, polyamines and carboxylic acids: GC-MS quantitation of tBDMS derivatives using ammonia positive chemical ionization.

The GC-MS quantitation of a large number of neurochemicals utilizing a single derivatization step is not common but is provided by the reagent N-(tert-butyldimethylsilyl)-N-methyltrifluro-acetamide (MTBSTFA). Previous workers have utilized this derivative for GC-MS analyses of amino acids, carboxylic acids and urea with electron impact (EI) and with positive chemical ionization (PCI; methane as reagent gas). However, these conditions yield significant fragmentation, decreasing sensitivity and in some cases reducing specificity for quantitation with selected ion monitoring (SIM). Additionally, the majority of studies have used a single internal standard to quantitate many compounds. In this study we demonstrate that using isotopic dilution combined with ammonia as the reagent gas for PCI analyses, results in high precision and sensitivity in analyzing complex neurochemical mixes. We also demonstrate for the first time the utility of this derivative for the analysis of brain polyamines and the dipeptide cysteinyl glycine. In the case of ammonia as the reagent gas, all amino acids, polyamines and urea yielded strong [MH](+) ions with little or no fragmentation. In the case of carboxylic acids, [M+18](+) ions predominated but [MH](+) ions were also noted. This approach was used to analyze superfusates from hippocampal brain slices and brain tissue extracts from brain lesion studies. The advantages of this methodology include: (i) simple sample preparation; (ii) a single derivatization step; (iii) direct GC-MS analysis of the reaction mix; (iv) high precision as a result of isotopic dilution analyses; (v) high sensitivity and specificity as a result of strong [MH](+) ions with ammonia reagent gas; (vi) no hydrolysis of glutamine to glutamate or asparagine to aspartate; and (vii) applicability to a wide range of neurochemicals.