Biomarker profiling of ageing and neurodegenerative disease

BIOMARKER PROFILING OF AGING AND NEURODEGENERATIVE DISEASE ZEPING HU (M.Sc., National Institute for the Control of Pharmaceutical & Biological Products, P.R. China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor Assistant Professor Eric Chan Chun Yong and co-supervisor Associate Professor Paul Ho Chi Lui, who have guided me throughout all the phases of my research project. I would like to extend my sincere appreciation to them for their important inputs and invaluable instructions on my thesis. I would like to give my special appreciation to the Associate Professor Chan Sui Yung, Head of Department of Pharmacy, for her continuous support and help. I would like to thank the Department Graduate Committee members for their assistance in both academic and non-academic matters. I would also like to acknowledge the assistance given by Ms. New Lee Sun, Ms. Ng Sek Eng and all the other laboratory officers in our department. I am grateful for the scholarship provided by National University of Singapore (NUS) and the generous support of the NUS Academic Research Fund R-148-000100-112 for my project. Special appreciation should be given to Drs. Paul Chapman and Edward Browne (NGI CEDD R&D Center for Cognitive & Neurodegenerative Disorders, GSK Singapore), Dr. Sashi Kesavapany (Department of Biochemistry, NUS) for their collaboration and assistance in the sample collection, Ms. Cynthia Lahey (Shimadzu Singapore), Mr. Pasikanti Kishore Kumar and Mr. Sudipta Saha for their valuable advice and discussion on my research. Finally, I want to make a special acknowledgement to my family for their love and great moral support. ii DISCLOSURE/DATA CONFIDENTIALITY This project is a joint-program with our collaborator GSK where we have signed a confidentiality agreement. As the data in this thesis have not been published thus far, the reviewers and administrators are advised to keep the data in this thesis confidential. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS . II DISCLOSURE/DATA CONFIDENTIALITY . III TABLE OF CONTENTS IV SUMMARY . IX LIST OF ABBREVIATIONS . XI LIST OF TABLES . XV LIST OF FIGURES XVIII CHAPTER GENERAL INTRODUCTION . 1.1 AGING AND NEURODEGENERATIVE DISEASES . 1.2 AD . 1.2.1 Prevalence of AD 1.2.2 Etiology and pathology of AD 1.2.2.1 Amyloid protein hypothesis 1.2.2.2 NFT hypothesis . 1.2.2.3 Risk factors of AD 1.2.3 Diagnosis of AD . 10 1.2.3.1 1.2.3.2 1.2.3.3 1.2.3.4 Cognitive test 11 Neuroimaging . 12 Biological markers of AD . 13 Combinations of different biomarkers 20 1.3 METABONOMICS 22 1.3.1 Biomarker and “-omics” . 25 1.3.2 Metabonomic platforms 26 1.3.2.1 NMR . 28 1.3.2.2 MS 29 1.3.3 Applications of metabonomics . 33 1.3.3.1 1.3.3.2 1.3.3.3 1.3.3.4 1.3.3.5 Disease diagnosis 33 Preclinical drug candidate safety assessment . 34 Clinical pharmaceutical R&D 34 Plant and microbial sciences . 35 Other applications . 35 1.4 STEROIDS . 36 1.4.1 Physiological roles of endogenous steroids 37 1.4.2 Clinical significances of endogenous steroids 39 iv 1.4.3 Quantitative analysis of endogenous steroids . 40 1.4.3.1 RIA and EIA . 41 1.4.3.2 GC/MS 42 1.4.3.3 LC/MS 43 1.5 RATIONALES AND OBJECTIVES OF THE THESIS 46 1.5.1 Metabonomic profiling in aging and AD models . 46 1.5.2 Steroidal biomarker profiling in aging and AD models 48 1.5.3 Development and comparison of MS-based analytical techniques for bioanalysis of endogenous steroids 48 CHAPTER GC/MS METABOLIC PROFILING OF AGING MODELS 49 2.1 INTRODUCTION 49 2.2 MATERIALS AND METHODS 56 2.2.1 Chemicals and reagents . 56 2.2.2 Animals and grouping schedules 57 2.2.2.1 LH rats 57 2.2.2.2 C57BL/6J mice . 58 2.2.3 Cortical neuron cell culture . 58 2.2.4 Sample collection and preparation 58 2.2.4.1 Urine . 59 2.2.4.2 Plasma . 60 2.2.4.3 Whole brain 61 2.2.4.4 Cell culture and medium . 62 2.2.5 GC/MS analysis 63 2.2.6 Data pre-processing 65 2.2.7 Multivariate and univariate data analyses . 67 2.3 RESULTS . 69 2.3.1 C57BL/6J mice . 69 2.3.2 LH rats 73 2.3.3 LH rats treated with LA 79 2.3.4 Cell culture media and lyses . 84 2.4 DISCUSSION . 90 2.4.1 Metabolite extraction 91 2.4.2 Derivatization 92 2.4.3 Data pre-processing 93 v 2.4.4 Metabolic changes associated with aging . 96 CHAPTER GC/MS METABOLIC PROFILING OF AD MODELS 105 3.1 INTRODUCTION 105 3.2 MATERIALS AND METHODS 110 3.2.1 Chemicals and reagents . 110 3.2.2 Animals and grouping schedules 110 3.2.2.1 TASTPM and wild type mice . 110 3.2.2.2 Human p25 over-expressing transgenic mice . 110 3.2.3 Sample collection and preparation 112 3.2.3.1 Brain, plasma and urine 112 3.2.3.2 Cell culture and medium . 112 3.2.4 GC/MS analysis 113 3.2.5 Chromatogram acquisition and data pre-processing . 113 3.2.6 Multivariate and univariate data analyses . 113 3.3 RESULTS . 113 3.3.1 TASTPM mice 113 3.3.2 Human p25 over-expressing mice 116 3.3.3 Glutamate treated cortical neuron cells . 121 3.4 DISCUSSION . 124 3.4.1 Animal and cell models for AD 124 3.4.2 Metabonomics of AD 128 CHAPTER DETERMINATION OF ENDOGENOUS STEROIDS IN AGING AND AD ANIMAL MODELS . 132 4.1 INTRODUCTION 132 4.2 MATERIALS AND METHODS 134 4.2.1 Materials and Chemicals . 134 4.2.2 Animals and grouping schedules 135 4.2.3 Sample collection and preparation 135 4.2.4 EIA 136 4.2.5 Data processing and statistical analysis 136 4.3 RESULTS . 137 vi 4.3.1 Aging study . 137 4.3.2 AD study . 143 4.4 DISCUSSION . 145 4.4.1 Aging study . 145 4.4.2 AD study . 148 CHAPTER MS-BASED TECHNIQUES FOR THE BIOANLAYSIS OF ENDOGENOUS STEROIDS IN BIOLOGICAL SAMPLES . 150 5.1 INTRODUCTION 150 5.2 MATERIALS AND METHODS 154 5.2.1 Materials and chemicals 154 5.2.2 Preparation of anhydrous solvents 156 5.2.3 Preparation of stock and working solutions 157 5.2.4 Preparation of calibrators and quality controls . 157 5.2.5 Biological samples collection and preparation . 158 5.2.5.1 Extraction of steroids 158 5.2.5.2 Plasma sample preparation . 160 5.2.5.3 Brain sample preparation 160 5.2.6 UPLC/ESI/MS and UPLC/APPI/MS analysis of intact steroids . 162 5.2.6.1 UPLC/ESI/MS instrumentation and optimization 162 5.2.6.2 UPLC/APPI/MS instrumentation and optimization . 164 5.2.7 UPLC/ESI/MS analysis of steroid-HA derivatives . 165 5.2.7.1 Derivatization of steroids with HA . 165 5.2.7.2 UPLC/MS instrumentation and optimization . 166 5.2.8 UPLC/ESI/MS analysis of steroid-SBA derivatives . 167 5.2.8.1 Derivatization of steroids with SBA . 167 5.2.8.2 UPLC/MS instrumentation and conditions 168 5.2.8.3 Calibration curves . 169 5.2.9 GC/EI/MS analysis of steroid-HFBA derivatives . 169 5.2.9.1 Derivatization of steroids with HFBA 169 5.2.9.2 GC/MS 170 5.2.9.3 Linearity, LOD and LOQ, carry-over effect and precision . 172 5.2.10 Data collection, processing and statistical analysis 173 5.3 RESULTS . 173 vii 5.3.1 Extraction recovery of steroids from biological samples by liquid extraction (LE) and SPE 174 5.3.2 UPLC/ESI/MS and UPLC/APPI/MS analysis of intact nonderivatized steroids 176 5.3.3 UPLC/APPI/MS vs UPLC/ESI/MS 178 5.3.4 UPLC/ESI/MS analysis of steroid-HA derivatives . 179 5.3.4.1 Derivatization of steroids with HA . 179 5.3.4.2 UPLC/MS analysis of steroid-HA derivatives . 180 5.3.5 UPLC/ESI/MS analysis of steroid-SBA derivatives . 182 5.3.5.1 Derivatization of steroid with SBA 182 5.3.5.2 UPLC/MS analysis of steroid-SBA derivatives . 185 5.3.6 GC/EI/MS analysis of steroid-HFBA derivatives . 187 5.3.6.1 Derivatization of steroids with HFBA and GC/MS analysis of derivatives 187 5.3.6.2 Quantitation of steroids 190 5.3.6.3 Linearity, LOD and LOQ, carry-over effect and precision . 190 5.4 DISCUSSION . 191 5.4.1 Extraction of steroids 191 5.4.2 MS-based analysis of steroids . 193 CHAPTER CONCLUSIONS AND FUTURE DIRECTIONS 198 6.1 ACHIEVEMENTS AND CONCLUSIONS . 198 6.2 FUTURE DIRECTIONS 200 BIBLIOGRAPHY 203 viii SUMMARY With the increasing life expectancy, the global aging population is expanding rapidly in the last few decades. In some cases, aging is accompanied by neurodegenerative diseases, such as Alzheimer’s Disease (AD) and dementia. Early clinical intervention is crucial for the management and prognosis of AD patients as there is no cure for the disease thus far. However, the lack of specific and accurate biomarkers for its accurate diagnosis hinders the early clinical diagnosis and intervention of AD. In addition, the discovery and development of novel effective therapies for AD necessitate the use of reliable efficacy biomarker to facilitate and accelerate the clinical studies. Mass spectrometry (MS)-based metabonomics provides an important platform for the metabolic profiling of complex biological samples, which may lead to the discovery of biomarkers for the diagnosis of diseases. In this thesis, both non-targeted metabolic and targeted steroidal biomarker profiling of aging and AD were investigated using in vivo and in vitro models. The first objective was to characterize the global metabolic fluxes associated with aging and AD using GC/MS-based metabonomics and multivariate data analysis. Lister Hooded (LH) rat (2-month vs 2-year) and C57BL/6J mouse (1-month vs 5-month) models were analyzed using GC/MS to identify the small molecule metabolite markers of aging. In addition, both young and aged LH rats were treated with αlipoic acid, and their metabolic profiles were investigated and compared. To identify the metabolite markers of AD, the metabolic profiles of two transgenic AD mouse models, TASTPM and p25-induced mice, were examined and compared to those of wild type C57BL/6J mice. Moreover, an in vitro cortical neuron cell ix model was involved to investigate the changes in metabolic profiles associated with aging and glutamate-treatment, which may be implicated in the pathogenesis of AD. As endogenous steroid levels were found to be associated with aging and AD, the brain and plasma samples obtained from our animal models were further investigated for endogenous steroids using enzyme immunoassay. In addition, MSbased analytical techniques for the profiling of endogenous steroids in biological samples were investigated and compared. Our study demonstrated clear differences in metabolic profiles associated with aging and AD in both in vivo and in vitro models. A number of metabolites were found to be associated with the aging process and AD, with different markers being observed between different aging and AD animal models. In addition, the levels of endogenous steroids in brain and plasma of the animal models were found to be modified with aging and AD. While interesting and pertinent data were generated based on the MS-based assays for steroid analysis, none of the investigated methods was found to be suitable for the endogenous steroid profiling in complex biological samples. Our results suggested collectively that small molecule metabolites and steroids are promising biomarkers for the future study of aging and diagnosis of AD. x 402. Ebner M.J., Corol D.I., Havlikova H., Honour J.W., and Fry J.P. Identification of neuroactive steroids and their precursors and metabolites in adult male rat brain. Endocrinology. 147:179-190 (2006). 403. Meffre D., Pianos A., Liere P., Eychenne B., Cambourg A., Schumacher M., Stein D.G., and Guennoun R. Steroid profiling in brain and plasma of male and pseudopregnant female rats after traumatic brain injury: Analysis by gas chromatography/mass spectrometry. Endocrinology (2007). 404. Baulieu E.E. Neurosteroids: a novel function of the brain. Psychoneuroendocrinology. 23:963-987 (1998). 405. Corpechot C., Robel P., Axelson M., Sjovall J., and Baulieu E.E. Characterization and measurement of dehydroepiandrosterone sulfate in rat brain. Proc Natl Acad Sci U S A. 78:4704-4707 (1981). 406. Corpechot C., Synguelakis M., Talha S., Axelson M., Sjovall J., Vihko R., Baulieu E.E., and Robel P. Pregnenolone and its sulfate ester in the rat brain. Brain Res. 270:119-125 (1983). 407. Liere P., Pianos A., Eychenne B., Cambourg A., Liu S., Griffiths W., Schumacher M., Sjovall J., and Baulieu E.E. Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of their sulfated counterparts in rodent brain. J Lipid Res. 45:2287-2302 (2004). 408. Chatman K., Hollenbeck T., Hagey L., Vallee M., Purdy R., Weiss F., and Siuzdak G. Nanoelectrospray mass spectrometry and precursor ion monitoring for quantitative steroid analysis and attomole sensitivity. Anal Chem. 71:2358-2363 (1999). 409. Kauppila T.J., Nikkola T., Ketola R.A., and Kostiainen R. Atmospheric pressure photoionization-mass spectrometry and atmospheric pressure chemical ionization-mass spectrometry of neurotransmitters. J Mass Spectrom. 41:781-789 (2006). 410. Blasco C., Van Poucke C., and Van Peteghem C. Analysis of meat samples for anabolic steroids residues by liquid chromatography/tandem mass spectrometry. J Chromatogr A (2007). 411. Leinonen A., Kuuranne T., and Kostiainen R. Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization. J Mass Spectrom. 37:693-698 (2002). 412. Hanold K.A., Fischer S.M., Cormia P.H., Miller C.E., and Syage J.A. Atmospheric pressure photoionization. 1. General properties for LC/MS. Anal Chem. 76:2842-2851 (2004). 413. Yamamoto A., Kakutani N., Yamamoto K., Kamiura T., and Miyakoda H. Steroid hormone profiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionization and atmospheric pressure photoionization sources. Environ Sci Technol. 40:4132-4137 (2006). 414. Shimada K. and Mukai Y. Studies on neurosteroids. VII. Determination of pregnenolone and its 3-stearate in rat brains using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. J Chromatogr B Biomed Sci Appl. 714:153-160 (1998). 231 415. Mitamura K., Ogasawara C., Shiozawa A., Terayama E., and Shimada K. Determination method for steroid 5alpha-reductase activity using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. Anal Sci. 21:1241-1244 (2005). 416. Higashi T., Nagahama A., Otomi N., and Shimada K. Studies on neurosteroids XIX. Development and validation of liquid chromatography-tandem mass spectrometric method for determination of 5alpha-reduced pregnane-type neurosteroids in rat brain and serum. J Chromatogr B Analyt Technol Biomed Life Sci. 848:188-199 (2007). 417. K. Mitamura, M. Yatera, and Shimada K. Quantitative determination of pregnenolone-3sulfate in rat brains using liquid chromatography/electrospray ionization-mass spectrometry. Anal Sci. 15:951-955 (1999). 418. Higashi T., Daifu Y., Ikeshima T., Yagi T., and Shimada K. Studies on neurosteroids XV. Development of enzyme-linked immunosorbent assay for examining whether pregnenolone sulfate is a veritable neurosteroid. J Pharm Biomed Anal. 30:1907-1917 (2003). 419. Liu S., Sjovall J., and Griffiths W.J. Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes. Rapid Commun Mass Spectrom. 14:390-400 (2000). 420. Singh G., Gutierrez A., Xu K., and Blair I.A. Liquid chromatography/electron capture atmospheric pressure chemical ionization/mass spectrometry: analysis of pentafluorobenzyl derivatives of biomolecules and drugs in the attomole range. Anal Chem. 72:3007-3013 (2000). 421. Kalhorn T.F., Page S.T., Howald W.N., Mostaghel E.A., and Nelson P.S. Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 21:3200-3206 (2007). 422. Higashi T., Takido N., and Shimada K. Studies on neurosteroids XVII. Analysis of stressinduced changes in neurosteroid levels in rat brains using liquid chromatography-electron capture atmospheric pressure chemical ionization-mass spectrometry. Steroids. 70:1-11 (2005). 423. Higashi T., Yamauchi A., and Shimada K. 2-hydrazino-1-methylpyridine: a highly sensitive derivatization reagent for oxosteroids in liquid chromatography-electrospray ionization-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 825:214222 (2005). 424. Wang Y., Hornshaw M., Alvelius G., Bodin K., Liu S., Sjovall J., and Griffiths W.J. Matrix-assisted laser desorption/ionization high-energy collision-induced dissociation of steroids: analysis of oxysterols in rat brain. Anal Chem. 78:164-173 (2006). 425. Griffiths W.J., Liu S., Alvelius G., and Sjovall J. Derivatisation for the characterisation of neutral oxosteroids by electrospray and matrix-assisted laser desorption/ionisation tandem mass spectrometry: the Girard P derivative. Rapid Commun Mass Spectrom. 17:924-935 (2003). 426. Schnackenberg L.K., Sun J., Espandiari P., Holland R.D., Hanig J., and Beger R.D. Metabonomics evaluations of age-related changes in the urinary compositions of male Sprague Dawley rats and effects of data normalization methods on statistical and quantitative analysis. BMC bioinformatics. Suppl 7:S3 (2007). 427. Dobson J.G., Jr., Fray J., Leonard J.L., and Pratt R.E. Molecular mechanisms of reduced beta-adrenergic signaling in the aged heart as revealed by genomic profiling. Physiol Genomics. 15:142-147 (2003). 232 428. Kayo T., Allison D.B., Weindruch R., and Prolla T.A. Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci U S A. 98:5093-5098 (2001). 429. Zou S., Meadows S., Sharp L., Jan L.Y., and Jan Y.N. Genome-wide study of aging and oxidative stress response in Drosophila melanogaster. Proc Natl Acad Sci U S A. 97:13726-13731 (2000). 430. D'Ascenzo M., Relkin N.R., and Lee K.H. Alzheimer's disease cerebrospinal fluid biomarker discovery: a proteomics approach. Curr Opin Mol Ther. 7:557-564 (2005). 431. Selle H., Lamerz J., Buerger K., Dessauer A., Hager K., Hampel H., Karl J., Kellmann M., Lannfelt L., Louhija J., Riepe M., Rollinger W., Tumani H., Schrader M., and Zucht H.D. Identification of novel biomarker candidates by differential peptidomics analysis of cerebrospinal fluid in Alzheimer's disease. Comb Chem High Throughput Screen. 8:801806 (2005). 432. Davidsson P. and Sjogren M. The use of proteomics in biomarker discovery in neurodegenerative diseases. Dis Markers. 21:81-92 (2005). 433. Zhang J., Goodlett D.R., and Montine T.J. Proteomic biomarker discovery in cerebrospinal fluid for neurodegenerative diseases. J Alzheimers Dis. 8:377-386 (2005). 434. Curtis C., Landis G.N., Folk D., Wehr N.B., Hoe N., Waskar M., Abdueva D., Skvortsov D., Ford D., Luu A., Badrinath A., Levine R.L., Bradley T.J., Tavare S., and Tower J. Transcriptional profiling of MnSOD-mediated lifespan extension in Drosophila reveals a species-general network of aging and metabolic genes. Genome Biol. 8:R262 (2007). 435. Kuzdzal S., Lopez M., Mikulskis A., Golenko E., DiCesare J., Denoyer E., Patton W., Ediger R., Sapp L., Ziegert T., Ackloo S., Wall M.R., Mannion D.P., della Cioppa G., Wolfe G., Bennett D., and Melov S. Biomarker discovery and analysis platform: application to Alzheimer's disease. Biotechniques. 39:606-607 (2005). 436. Pears M.R., Cooper J.D., Mitchison H.M., Mortishire-Smith R.J., Pearce D.A., and Griffin J.L. High resolution 1H NMR-based metabolomics indicates a neurotransmitter cycling deficit in cerebral tissue from a mouse model of Batten disease. J Biol Chem. 280:4250842514 (2005). 437. Inagaki S., Noda T., Min J.Z., and Toyo'oka T. Metabolic profiling of rat hair and screening biomarkers using ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry. J Chromatogr A. 1176:94-99 (2007). 438. Lawler D.F., Larson B.T., Ballam J.M., Smith G.K., Biery D.N., Evans R.H., Greeley E.H., Segre M., Stowe H.D., and Kealy R.D. Diet restriction and ageing in the dog: major observations over two decades. Br J Nutr:1-13 (2007). 439. Salek R.M., Colebrooke R.E., Macintosh R., Lynch P.J., Sweatman B.C., Emson P.C., and Griffin J.L. A Metabolomic Study of Brain Tissues from Aged Mice with Low Expression of the Vesicular Monoamine Transporter (VMAT2) Gene. Neurochem Res. 33:292-300 (2008). 440. Wu B., Yan S., Lin Z., Wang Q., Yang Y., Yang G., Shen Z., and Zhang W. Metabonomic study on ageing: NMR-based investigation into rat urinary metabolites and the effect of the total flavone of Epimedium. Mol Biosyst. 4:855-861 (2008). 441. Lu Y., A J., Wang G., Hao H., Huang Q., Yan B., Zha W., Gu S., Ren H., Zhang Y., Fan X., Zhang M., and Hao K. Gas chromatography/time-of-flight mass spectrometry based 233 metabonomic approach to differentiating hypertension- and age-related metabolic variation in spontaneously hypertensive rats. Rapid Commun Mass Spectrom. 22:2882-2888 (2008). 442. Jiang N., Yan X., Zhou W., Zhang Q., Chen H., Zhang Y., and Zhang X. NMR-Based Metabonomic Investigations into the Metabolic Profile of the Senescence-Accelerated Mouse. J Proteome Res. 7:3678-3686 (2008). 443. Finn D.P., Jhaveri M.D., Beckett S.R., Madjd A., Kendall D.A., Marsden C.A., and Chapman V. Behavioral, central monoaminergic and hypothalamo-pituitary-adrenal axis correlates of fear-conditioned analgesia in rats. Neuroscience. 138:1309-1317 (2006). 444. Dowd E., Monville C., Torres E.M., Wong L.F., Azzouz M., Mazarakis N.D., and Dunnett S.B. Lentivector-mediated delivery of GDNF protects complex motor functions relevant to human Parkinsonism in a rat lesion model. Eur J Neurosci. 22:2587-2595 (2005). 445. Gengler S., Gault V.A., Harriott P., and Holscher C. Impairments of hippocampal synaptic plasticity induced by aggregated beta-amyloid (25-35) are dependent on stimulationprotocol and genetic background. Exp Brain Res (2006). 446. Hagen T.M., Ingersoll R.T., Lykkesfeldt J., Liu J., Wehr C.M., Vinarsky V., Bartholomew J.C., and Ames A.B. (R)-alpha-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased metabolic rate. Faseb J. 13:411-418 (1999). 447. Liu J., Head E., Gharib A.M., Yuan W., Ingersoll R.T., Hagen T.M., Cotman C.W., and Ames B.N. Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: partial reversal by feeding acetyl-L-carnitine and/or R-alpha -lipoic acid. Proc Natl Acad Sci U S A. 99:2356-2361 (2002). 448. Liu J., Killilea D.W., and Ames B.N. Age-associated mitochondrial oxidative decay: improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-L- carnitine and/or R-alpha -lipoic acid. Proc Natl Acad Sci U S A. 99:1876-1881 (2002). 449. Haugaard N. and Levin R.M. Activation of choline acetyl transferase by dihydrolipoic acid. Mol Cell Biochem. 229:103-106 (2002). 450. Farr S.A., Poon H.F., Dogrukol-Ak D., Drake J., Banks W.A., Eyerman E., Butterfield D.A., and Morley J.E. The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice. J Neurochem. 84:1173-1183 (2003). 451. Quinn J.F., Bussiere J.R., Hammond R.S., Montine T.J., Henson E., Jones R.E., and Stackman R.W., Jr. Chronic dietary alpha-lipoic acid reduces deficits in hippocampal memory of aged Tg2576 mice. Neurobiol Aging. 28:213-225 (2007). 452. Ziegler D., Nowak H., Kempler P., Vargha P., and Low P.A. Treatment of symptomatic diabetic polyneuropathy with the antioxidant alpha-lipoic acid: a meta-analysis. Diabet Med. 21:114-121 (2004). 453. Jonsson P., Bruce S.J., Moritz T., Trygg J., Sjostrom M., Plumb R., Granger J., Maibaum E., Nicholson J.K., Holmes E., and Antti H. Extraction, interpretation and validation of information for comparing samples in metabolic LC/MS data sets. Analyst. 130:701-707 (2005). 234 454. Serkova N.J. and Christians U. Biomarkers for toxicodynamic monitoring of immunosuppressants: NMR-based quantitative metabonomics of the blood. Ther Drug Monit. 27:733-737 (2005). 455. Schauer N., Steinhauser D., Strelkov S., Schomburg D., Allison G., Moritz T., Lundgren K., Roessner-Tunali U., Forbes M.G., Willmitzer L., Fernie A.R., and Kopka J. GC-MS libraries for the rapid identification of metabolites in complex biological samples. FEBS Lett. 579:1332-1337 (2005). 456. Jonsson P., Johansson E.S., Wuolikainen A., Lindberg J., Schuppe-Koistinen I., Kusano M., Sjostrom M., Trygg J., Moritz T., and Antti H. Predictive metabolite profiling applying hierarchical multivariate curve resolution to GC-MS data--a potential tool for multi-parametric diagnosis. J Proteome Res. 5:1407-1414 (2006). 457. Coen M., Lenz E.M., Nicholson J.K., Wilson I.D., Pognan F., and Lindon J.C. An integrated metabonomic investigation of acetaminophen toxicity in the mouse using NMR spectroscopy. Chem Res Toxicol. 16:295-303 (2003). 458. Stentiford G.D., Viant M.R., Ward D.G., Johnson P.J., Martin A., Wenbin W., Cooper H.J., Lyons B.P., and Feist S.W. Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study. Omics. 9:281-299 (2005). 459. Bligh E.G. and Dyer W.J. A rapid method of total lipid extraction and purification. Canadian journal of biochemistry and physiology. 37:911-917 (1959). 460. Want E.J., O'Maille G., Smith C.A., Brandon T.R., Uritboonthai W., Qin C., Trauger S.A., and Siuzdak G. Solvent-dependent metabolite distribution, clustering, and protein extraction for serum profiling with mass spectrometry. Anal Chem. 78:743-752 (2006). 461. Kimball E. and Rabinowitz J.D. Identifying decomposition products in extracts of cellular metabolites. Anal Biochem. 358:273-280 (2006). 462. Villas-Boas S.G., Hojer-Pedersen J., Akesson M., Smedsgaard J., and Nielsen J. Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast. 22:11551169 (2005). 463. Lu W., Kimball E., and Rabinowitz J.D. A high-performance liquid chromatographytandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom. 17:37-50 (2006). 464. Bajad S.U., Lu W., Kimball E.H., Yuan J., Peterson C., and Rabinowitz J.D. Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A. 1125:76-88 (2006). 465. Miura D., Tanaka H., and Wariishi H. Metabolomic differential display analysis of the white-rot basidiomycete Phanerochaete chrysosporium grown under air and 100% oxygen. FEMS Microbiol Lett. 234:111-116 (2004). 466. Maharjan R.P. and Ferenci T. Global metabolite analysis: the influence of extraction methodology on metabolome profiles of Escherichia coli. Anal Biochem. 313:145-154 (2003). 467. Gullberg J., Jonsson P., Nordstrom A., Sjostrom M., and Moritz T. Design of experiments: an efficient strategy to identify factors influencing extraction and derivatization of Arabidopsis thaliana samples in metabolomic studies with gas chromatography/mass spectrometry. Anal Biochem. 331:283-295 (2004). 235 468. Weckwerth W., Wenzel K., and Fiehn O. Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their coregulation in biochemical networks. Proteomics. 4:78-83 (2004). 469. A J., Huang Q., Wang G., Zha W., Yan B., Ren H., Gu S., Zhang Y., Zhang Q., Shao F., Sheng L., and Sun J. Global analysis of metabolites in rat and human urine based on gas chromatography/time-of-flight mass spectrometry. Anal Biochem. 379:20-26 (2008). 470. Smith C.A., Want E.J., O'Maille G., Abagyan R., and Siuzdak G. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem. 78:779-787 (2006). 471. Tikunov Y., Lommen A., de Vos C.H., Verhoeven H.A., Bino R.J., Hall R.D., and Bovy A.G. A novel approach for nontargeted data analysis for metabolomics. Large-scale profiling of tomato fruit volatiles. Plant Physiol. 139:1125-1137 (2005). 472. Katajamaa M., Miettinen J., and Oresic M. MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics. 22:634636 (2006). 473. Gao P., Lu C., Zhang F., Sang P., Yang D., Li X., Kong H., Yin P., Tian J., Lu X., Lu A., and Xu G. Integrated GC-MS and LC-MS plasma metabonomics analysis of ankylosing spondylitis. Analyst. 133:1214-1220 (2008). 474. Chen J., Zhao X., Fritsche J., Yin P., Schmitt-Kopplin P., Wang W., Lu X., Haring H.U., Schleicher E.D., Lehmann R., and Xu G. Practical Approach for the Identification and Isomer Elucidation of Biomarkers Detected in a Metabonomic Study for the Discovery of Individuals at Risk for Diabetes by Integrating the Chromatographic and Mass Spectrometric Information. Anal Chem. 80:1280-1289 (2008). 475. Brand A., Richter-Landsberg C., and Leibfritz D. Multinuclear NMR studies on the energy metabolism of glial and neuronal cells. Dev Neurosci. 15:289-298 (1993). 476. Isaacks R.E., Bender A.S., Kim C.Y., Prieto N.M., and Norenberg M.D. Osmotic regulation of myo-inositol uptake in primary astrocyte cultures. Neurochem Res. 19:331338 (1994). 477. Saraf-Lavi E., Bowen B.C., Pattany P.M., Sklar E.M., Murdoch J.B., and Petito C.K. Proton MR spectroscopy of gliomatosis cerebri: case report of elevated myoinositol with normal choline levels. AJNR Am J Neuroradiol. 24:946-951 (2003). 478. Benedetti M.S., Russo A., Marrari P., and Dostert P. Effects of ageing on the content in sulfur-containing amino acids in rat brain. J Neural Transm Gen Sect. 86:191-203 (1991). 479. Moini H., Packer L., and Saris N.E. Antioxidant and prooxidant activities of alpha-lipoic acid and dihydrolipoic acid. Toxicol Appl Pharmacol. 182:84-90 (2002). 480. Biewenga G.P., Haenen G.R., and Bast A. The pharmacology of the antioxidant lipoic acid. Gen Pharmacol. 29:315-331 (1997). 481. Bilska A. and Wlodek L. Lipoic acid - the drug of the future? Pharmacol Rep. 57:570-577 (2005). 482. Jones W., Li X., Qu Z.C., Perriott L., Whitesell R.R., and May J.M. Uptake, recycling, and antioxidant actions of alpha-lipoic acid in endothelial cells. Free Radic Biol Med. 33:83-93 (2002). 236 483. Haugaard N. and Levin R.M. Regulation of the activity of choline acetyl transferase by lipoic acid. Mol Cell Biochem. 213:61-63 (2000). 484. Suh J.H., Moreau R., Heath S.H., and Hagen T.M. Dietary supplementation with (R)alpha-lipoic acid reverses the age-related accumulation of iron and depletion of antioxidants in the rat cerebral cortex. Redox Rep. 10:52-60 (2005). 485. Wong A., Dukic-Stefanovic S., Gasic-Milenkovic J., Schinzel R., Wiesinger H., Riederer P., and Munch G. Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxide synthase mediated by advanced glycation endproducts in murine microglia. Eur J Neurosci. 14:1961-1967 (2001). 486. Suh J.H., Shenvi S.V., Dixon B.M., Liu H., Jaiswal A.K., Liu R.M., and Hagen T.M. Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid. Proc Natl Acad Sci U S A. 101:3381-3386 (2004). 487. Suh J.H., Wang H., Liu R.M., Liu J., and Hagen T.M. (R)-alpha-lipoic acid reverses the age-related loss in GSH redox status in post-mitotic tissues: evidence for increased cysteine requirement for GSH synthesis. Arch Biochem Biophys. 423:126-135 (2004). 488. Arivazhagan P., Ramanathan K., and Panneerselvam C. Effect of DL-alpha-lipoic acid on mitochondrial enzymes in aged rats. Chem Biol Interact. 138:189-198 (2001). 489. Bitar M.S., Wahid S., Pilcher C.W., Al-Saleh E., and Al-Mulla F. Alpha-lipoic acid mitigates insulin resistance in Goto-Kakizaki rats. Horm Metab Res. 36:542-549 (2004). 490. Thirunavukkarasu V., Anitha Nandhini A.T., and Anuradha C.V. Lipoic acid attenuates hypertension and improves insulin sensitivity, kallikrein activity and nitrite levels in high fructose-fed rats. J Comp Physiol [B]. 174:587-592 (2004). 491. Lee W.J., Song K.H., Koh E.H., Won J.C., Kim H.S., Park H.S., Kim M.S., Kim S.W., Lee K.U., and Park J.Y. Alpha-lipoic acid increases insulin sensitivity by activating AMPK in skeletal muscle. Biochem Biophys Res Commun. 332:885-891 (2005). 492. Visioli F., Smith A., Zhang W., Keaney J.F., Jr., Hagen T., and Frei B. Lipoic acid and vitamin C potentiate nitric oxide synthesis in human aortic endothelial cells independently of cellular glutathione status. Redox Rep. 7:223-227 (2002). 493. Zhang L., Xing G.Q., Barker J.L., Chang Y., Maric D., Ma W., Li B.S., and Rubinow D.R. Alpha-lipoic acid protects rat cortical neurons against cell death induced by amyloid and hydrogen peroxide through the Akt signalling pathway. Neurosci Lett. 312:125-128 (2001). 494. Lovell M.A., Xie C., Xiong S., and Markesbery W.R. Protection against amyloid beta peptide and iron/hydrogen peroxide toxicity by alpha lipoic acid. J Alzheimers Dis. 5:229239 (2003). 495. Muller U. and Krieglstein J. Prolonged pretreatment with alpha-lipoic acid protects cultured neurons against hypoxic, glutamate-, or iron-induced injury. J Cereb Blood Flow Metab. 15:624-630 (1995). 496. Kelly P.H., Bondolfi L., Hunziker D., Schlecht H.P., Carver K., Maguire E., Abramowski D., Wiederhold K.H., Sturchler-Pierrat C., Jucker M., Bergmann R., Staufenbiel M., and Sommer B. Progressive age-related impairment of cognitive behavior in APP23 transgenic mice. Neurobiol Aging. 24:365-378 (2003). 237 497. Ziegler D., Hanefeld M., Ruhnau K.J., Hasche H., Lobisch M., Schutte K., Kerum G., and Malessa R. Treatment of symptomatic diabetic polyneuropathy with the antioxidant alphalipoic acid: a 7-month multicenter randomized controlled trial (ALADIN III Study). ALADIN III Study Group. Alpha-Lipoic Acid in Diabetic Neuropathy. Diabetes Care. 22:1296-1301 (1999). 498. Packer L., Kraemer K., and Rimbach G. Molecular aspects of lipoic acid in the prevention of diabetes complications. Nutrition. 17:888-895 (2001). 499. Boushey C.J., Beresford S.A., Omenn G.S., and Motulsky A.G. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA. 274:1049-1057 (1995). 500. Seshadri S., Beiser A., Selhub J., Jacques P.F., Rosenberg I.H., D'Agostino R.B., Wilson P.W., and Wolf P.A. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. N Engl J Med. 346:476-483 (2002). 501. Nilsson K., Gustafson L., and Hultberg B. Plasma homocysteine concentration relates to the severity but not to the duration of Alzheimer's disease. Int J Geriatr Psychiatry. 19:666-672 (2004). 502. Braak H. and Braak E. Frequency of stages of Alzheimer-related lesions in different age categories. Neurobiol Aging. 18:351-357 (1997). 503. Pericak-Vance M.A., Bass M.P., Yamaoka L.H., Gaskell P.C., Scott W.K., Terwedow H.A., Menold M.M., Conneally P.M., Small G.W., Vance J.M., Saunders A.M., Roses A.D., and Haines J.L. Complete genomic screen in late-onset familial Alzheimer disease. Evidence for a new locus on chromosome 12. Jama. 278:1237-1241 (1997). 504. Rogaev E.I., Sherrington R., Wu C., Levesque G., Liang Y., Rogaeva E.A., Ikeda M., Holman K., Lin C., Lukiw W.J., de Jong P.J., Fraser P.E., Rommens J.M., and St GeorgeHyslop P. Analysis of the 5' sequence, genomic structure, and alternative splicing of the presenilin-1 gene (PSEN1) associated with early onset Alzheimer disease. Genomics. 40:415-424 (1997). 505. Maes O.C., Xu S., Yu B., Chertkow H.M., Wang E., and Schipper H.M. Transcriptional profiling of Alzheimer blood mononuclear cells by microarray. Neurobiol Aging. 28:17951809 (2007). 506. Unger T., Korade Z., Lazarov O., Terrano D., Schor N.F., Sisodia S.S., and Mirnics K. Transcriptome differences between the frontal cortex and hippocampus of wild-type and humanized presenilin-1 transgenic mice. Am J Geriatr Psychiatry. 13:1041-1051 (2005). 507. Zhang J. and Montine T.J. Proteomic discovery of CSF biomarkers for Alzheimer's disease. Ann Neurol. 61:497; author reply 497-498 (2007). 508. Tsuji T. and Shimohama S. Analysis of the proteomic profiling of brain tissue in Alzheimer's disease. Dis Markers. 17:247-257 (2001). 509. Lovestone S., Guntert A., Hye A., Lynham S., Thambisetty M., and Ward M. Proteomics of Alzheimer's disease: understanding mechanisms and seeking biomarkers. Expert Rev Proteomics. 4:227-238 (2007). 510. Soreghan B.A., Lu B.W., Thomas S.N., Duff K., Rakhmatulin E.A., Nikolskaya T., Chen T., and Yang A.J. Using proteomics and network analysis to elucidate the consequences of synaptic protein oxidation in a PS1 + AbetaPP mouse model of Alzheimer's disease. J Alzheimers Dis. 8:227-241 (2005). 238 511. Davidsson P. and Sjogren M. Proteome studies of CSF in AD patients. Mech Ageing Dev. 127:133-137 (2006). 512. Papassotiropoulos A., Fountoulakis M., Dunckley T., Stephan D.A., and Reiman E.M. Genetics, transcriptomics, and proteomics of Alzheimer's disease. J Clin Psychiatry. 67:652-670 (2006). 513. Chishti M.A., Yang D.S., Janus C., Phinney A.L., Horne P., Pearson J., Strome R., Zuker N., Loukides J., French J., Turner S., Lozza G., Grilli M., Kunicki S., Morissette C., Paquette J., Gervais F., Bergeron C., Fraser P.E., Carlson G.A., George-Hyslop P.S., and Westaway D. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J Biol Chem. 276:21562-21570 (2001). 514. Games D., Adams D., Alessandrini R., Barbour R., Berthelette P., Blackwell C., Carr T., Clemens J., Donaldson T., Gillespie F., and et al. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 373:523527 (1995). 515. Sturchler-Pierrat C., Abramowski D., Duke M., Wiederhold K.H., Mistl C., Rothacher S., Ledermann B., Burki K., Frey P., Paganetti P.A., Waridel C., Calhoun M.E., Jucker M., Probst A., Staufenbiel M., and Sommer B. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc Natl Acad Sci U S A. 94:13287-13292 (1997). 516. Duff K., Eckman C., Zehr C., Yu X., Prada C.M., Perez-tur J., Hutton M., Buee L., Harigaya Y., Yager D., Morgan D., Gordon M.N., Holcomb L., Refolo L., Zenk B., Hardy J., and Younkin S. Increased amyloid-beta42(43) in brains of mice expressing mutant presenilin 1. Nature. 383:710-713 (1996). 517. Holcomb L., Gordon M.N., McGowan E., Yu X., Benkovic S., Jantzen P., Wright K., Saad I., Mueller R., Morgan D., Sanders S., Zehr C., O'Campo K., Hardy J., Prada C.M., Eckman C., Younkin S., Hsiao K., and Duff K. Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin transgenes. Nat Med. 4:97-100 (1998). 518. Richards J.G., Higgins G.A., Ouagazzal A.M., Ozmen L., Kew J.N., Bohrmann B., Malherbe P., Brockhaus M., Loetscher H., Czech C., Huber G., Bluethmann H., Jacobsen H., and Kemp J.A. PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation. J Neurosci. 23:8989-9003 (2003). 519. Kantarci K., Xu Y., Shiung M.M., O'Brien P.C., Cha R.H., Smith G.E., Ivnik R.J., Boeve B.F., Edland S.D., Kokmen E., Tangalos E.G., Petersen R.C., and Jack C.R., Jr. Comparative diagnostic utility of different MR modalities in mild cognitive impairment and Alzheimer's disease. Dement Geriatr Cogn Disord. 14:198-207 (2002). 520. Zakzanis K.K., Graham S.J., and Campbell Z. A meta-analysis of structural and functional brain imaging in dementia of the Alzheimer's type: a neuroimaging profile. Neuropsychol Rev. 13:1-18 (2003). 521. Krishnan K.R., Charles H.C., Doraiswamy P.M., Mintzer J., Weisler R., Yu X., Perdomo C., Ieni J.R., and Rogers S. Randomized, placebo-controlled trial of the effects of donepezil on neuronal markers and hippocampal volumes in Alzheimer's disease. Am J Psychiatry. 160:2003-2011 (2003). 239 522. Miller B.L., Moats R.A., Shonk T., Ernst T., Woolley S., and Ross B.D. Alzheimer disease: depiction of increased cerebral myo-inositol with proton MR spectroscopy. Radiology. 187:433-437 (1993). 523. Dedeoglu A., Choi J.K., Cormier K., Kowall N.W., and Jenkins B.G. Magnetic resonance spectroscopic analysis of Alzheimer's disease mouse brain that express mutant human APP shows altered neurochemical profile. Brain Res. 1012:60-65 (2004). 524. Marjanska M., Curran G.L., Wengenack T.M., Henry P.G., Bliss R.L., Poduslo J.F., Jack C.R., Jr., Ugurbil K., and Garwood M. Monitoring disease progression in transgenic mouse models of Alzheimer's disease with proton magnetic resonance spectroscopy. Proc Natl Acad Sci U S A. 102:11906-11910 (2005). 525. Kantarci K., Weigand S.D., Petersen R.C., Boeve B.F., Knopman D.S., Gunter J., Reyes D., Shiung M., O'Brien P C., Smith G.E., Ivnik R.J., Tangalos E.G., and Jack C.R., Jr. Longitudinal (1)H MRS changes in mild cognitive impairment and Alzheimer's disease. Neurobiol Aging (2006). 526. von Kienlin M., Kunnecke B., Metzger F., Steiner G., Richards J.G., Ozmen L., Jacobsen H., and Loetscher H. Altered metabolic profile in the frontal cortex of PS2APP transgenic mice, monitored throughout their life span. Neurobiol Dis. 18:32-39 (2005). 527. Tukiainen T., Tynkkynen T., Makinen V.P., Jylanki P., Kangas A., Hokkanen J., Vehtari A., Grohn O., Hallikainen M., Soininen H., Kivipelto M., Groop P.H., Kaski K., Laatikainen R., Soininen P., Pirttila T., and Ala-Korpela M. A multi-metabolite analysis of serum by (1)H NMR spectroscopy: Early systemic signs of Alzheimer's disease. Biochem Biophys Res Commun. 375:356-361 (2008). 528. Holmes E., Tsang T.M., Huang J.T., Leweke F.M., Koethe D., Gerth C.W., Nolden B.M., Gross S., Schreiber D., Nicholson J.K., and Bahn S. Metabolic profiling of CSF: evidence that early intervention may impact on disease progression and outcome in schizophrenia. PLoS Med. 3:e327 (2006). 529. Crowther D.C., Kinghorn K.J., Page R., and Lomas D.A. Therapeutic targets from a Drosophila model of Alzheimer's disease. Curr Opin Pharmacol. 4:513-516 (2004). 530. Crowther D.C., Page R., Chandraratna D., and Lomas D.A. A Drosophila model of Alzheimer's disease. Methods Enzymol. 412:234-255 (2006). 531. Bayer T.A., Schafer S., Simons A., Kemmling A., Kamer T., Tepest R., Eckert A., Schussel K., Eikenberg O., Sturchler-Pierrat C., Abramowski D., Staufenbiel M., and Multhaup G. Dietary Cu stabilizes brain superoxide dismutase activity and reduces amyloid Abeta production in APP23 transgenic mice. Proc Natl Acad Sci U S A. 100:14187-14192 (2003). 532. Bornemann K.D., Wiederhold K.H., Pauli C., Ermini F., Stalder M., Schnell L., Sommer B., Jucker M., and Staufenbiel M. Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice. Am J Pathol. 158:63-73 (2001). 533. Boncristiano S., Calhoun M.E., Kelly P.H., Pfeifer M., Bondolfi L., Stalder M., Phinney A.L., Abramowski D., Sturchler-Pierrat C., Enz A., Sommer B., Staufenbiel M., and Jucker M. Cholinergic changes in the APP23 transgenic mouse model of cerebral amyloidosis. J Neurosci. 22:3234-3243 (2002). 534. van Dooren T., Dewachter I., Borghgraef P., and van Leuven F. Transgenic mouse models for APP processing and Alzheimer's disease: early and late defects. Subcell Biochem. 38:45-63 (2005). 240 535. Pugh P.L., Richardson J.C., Bate S.T., Upton N., and Sunter D. Non-cognitive behaviours in an APP/PS1 transgenic model of Alzheimer's disease. Behav Brain Res. 178:18-28 (2007). 536. Howlett D.R., Richardson J.C., Austin A., Parsons A.A., Bate S.T., Davies D.C., and Gonzalez M.I. Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes. Brain Res. 1017:130-136 (2004). 537. Howlett D.R., Bowler K., Soden P.E., Riddell D., Davis J.B., Richardson J.C., Burbidge S.A., Gonzalez M.I., Irving E.A., Lawman A., Miglio G., Dawson E.L., Howlett E.R., and Hussain I. Abeta deposition and related pathology in an APP x PS1 transgenic mouse model of Alzheimer's disease. Histol Histopathol. 23:67-76 (2008). 538. Pugh P.L., Vidgeon-Hart M.P., Ashmeade T., Culbert A.A., Seymour Z., Perren M.J., Joyce F., Bate S.T., Babin A., Virley D.J., Richardson J.C., Upton N., and Sunter D. Repeated administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2bromobenzylamine (DSP-4) modulates neuroinflammation and amyloid plaque load in mice bearing amyloid precursor protein and presenilin-1 mutant transgenes. J Neuroinflammation. 4:8 (2007). 539. Pardon M.C., Sarmad S., Rattray I., Bates T.E., Scullion G.A., Marsden C.A., Barrett D.A., Lowe J., and Kendall D.A. Repeated novel cage exposure-induced improvement of early Alzheimer's-like cognitive and amyloid changes in TASTPM mice is unrelated to changes in brain endocannabinoids levels. Neurobiol Aging (2007). 540. Sankaranarayanan S. Genetically modified mice models for Alzheimer's disease. Curr Top Med Chem. 6:609-627 (2006). 541. Richardson J.C., Kendal C.E., Anderson R., Priest F., Gower E., Soden P., Gray R., Topps S., Howlett D.R., Lavender D., Clarke N.J., Barnes J.C., Haworth R., Stewart M.G., and Rupniak H.T. Ultrastructural and behavioural changes precede amyloid deposition in a transgenic model of Alzheimer's disease. Neuroscience. 122:213-228 (2003). 542. Chui D.H., Tanahashi H., Ozawa K., Ikeda S., Checler F., Ueda O., Suzuki H., Araki W., Inoue H., Shirotani K., Takahashi K., Gallyas F., and Tabira T. Transgenic mice with Alzheimer presenilin mutations show accelerated neurodegeneration without amyloid plaque formation. Nat Med. 5:560-564 (1999). 543. Culbert A.A., Skaper S.D., Howlett D.R., Evans N.A., Facci L., Soden P.E., Seymour Z.M., Guillot F., Gaestel M., and Richardson J.C. MAPK-activated protein kinase deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. J Biol Chem. 281:23658-23667 (2006). 544. Cruz J.C. and Tsai L.H. Cdk5 deregulation in the pathogenesis of Alzheimer's disease. Trends Mol Med. 10:452-458 (2004). 545. Patrick G.N., Zukerberg L., Nikolic M., de la Monte S., Dikkes P., and Tsai L.H. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature. 402:615-622 (1999). 546. Smith P.D., Crocker S.J., Jackson-Lewis V., Jordan-Sciutto K.L., Hayley S., Mount M.P., O'Hare M.J., Callaghan S., Slack R.S., Przedborski S., Anisman H., and Park D.S. Cyclindependent kinase is a mediator of dopaminergic neuron loss in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A. 100:13650-13655 (2003). 241 547. Lee M.S., Kwon Y.T., Li M., Peng J., Friedlander R.M., and Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature. 405:360-364 (2000). 548. Giese K.P., Ris L., and Plattner F. Is there a role of the cyclin-dependent kinase activator p25 in Alzheimer's disease? Neuroreport. 16:1725-1730 (2005). 549. Tseng H.C., Zhou Y., Shen Y., and Tsai L.H. A survey of Cdk5 activator p35 and p25 levels in Alzheimer's disease brains. FEBS Lett. 523:58-62 (2002). 550. Esposito G.I., Giovacchini G., Liow J.-S., Bhattacharjee A., Greenstein D., Schapiro M., Hallett M., Herscovith P., and Eckleman W. Imaging neuroinflammation in Alzheimer disease with radiolabeled arachidonic acid and PET. J Nucl Med. 49:1414-1421 (2008). 551. McGeer P.L. and McGeer E.G. NSAIDs and Alzheimer disease: epidemiological, animal model and clinical studies. Neurobiol Aging. 28:639-647 (2007). 552. Hinkelbein J., Feldmann R.E., Jr., Peterka A., Schubert C., Schelshorn D., Maurer M.H., and Kalenka A. Alterations in cerebral metabolomics and proteomic expression during sepsis. Curr Neurovasc Res. 4:280-288 (2007). 553. Thomas C.E. and Ganji G. Integration of genomic and metabonomic data in systems biology--are we 'there' yet? Curr Opin Drug Discov Devel. 9:92-100 (2006). 554. Kitteringham N.R., Jenkins R.E., Lane C.S., Elliott V.L., and Park B.K. Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics. J Chromatogr B Analyt Technol Biomed Life Sci (2008). 555. Lambert J.J., Belelli D., Peden D.R., Vardy A.W., and Peters J.A. Neurosteroid modulation of GABAA receptors. Prog Neurobiol. 71:67-80 (2003). 556. Henderson L.P. Steroid modulation of GABA(A) receptor-mediated transmission in the hypothalamus: Effects on reproductive function. Neuropharmacology. 52:1439-1453 (2007). 557. Monnet F.P. and Maurice T. The sigma1 protein as a target for the non-genomic effects of neuro(active)steroids: molecular, physiological, and behavioral aspects. J Pharmacol Sci. 100:93-118 (2006). 558. Lagrange A. Dancing the Delta Shuffle: Neurosteroids Regulate GABA(A) Receptor Expression. Epilepsy Curr. 6:14-17 (2006). 559. Rasmuson S., Nasman B., Carlstrom K., and Olsson T. Increased levels of adrenocortical and gonadal hormones in mild to moderate Alzheimer's disease. Dement Geriatr Cogn Disord. 13:74-79 (2002). 560. Dorgan J.F., Fears T.R., McMahon R.P., Aronson Friedman L., Patterson B.H., and Greenhut S.F. Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry. Steroids. 67:151-158 (2002). 561. Lo M.J., Kau M.M., Cho W.L., and Wang P.S. Aging effects on the secretion of corticosterone in male rats. J Investig Med. 48:335-342 (2000). 562. DeKosky S.T., Scheff S.W., and Cotman C.W. Elevated corticosterone levels. A possible cause of reduced axon sprouting in aged animals. Neuroendocrinology. 38:33-38 (1984). 563. Oxenkrug G.F., McIntyre I.M., and Gershon S. Effects of pinealectomy and aging on the serum corticosterone circadian rhythm in rats. J Pineal Res. 1:181-185 (1984). 242 564. Lo M.J., Kau M.M., and Wang P.S. Effect of aging on corticosterone secretion in diestrous rats. J Cell Biochem. 97:351-358 (2006). 565. Goya R.G., Naylor P.H., Goldstein A.L., and Meites J. Changes in circulating levels of neuroendocrine and thymic hormones during aging in rats: a correlation study. Exp Gerontol. 25:149-157 (1990). 566. Lupo-di Prisco C. and Dessi-Fulgheri F. Endocrine and behavioral modifications in aging male rats. Horm Res. 12:149-160 (1980). 567. Meldrum D.R., Davidson B.J., Tataryn I.V., and Judd H.L. Changes in circulating steroids with aging in postmenopausal women. Obstetrics and gynecology. 57:624-628 (1981). 568. Maki T. [Age-related changes in secretion of adrenocortical steroid hormones in normal healthy men]. Nippon Naibunpi Gakkai Zasshi. 62:672-682 (1986). 569. Pirke K.M., Sintermann R., and Vogt H.J. Testosterone and testosterone precursors in the spermatic vein and in the testicular tissue of old men. Reduced oxygen supply may explain the relative increase of testicular progesterone and 17 alpha-hydroxyprogesterone content and production in old age. Gerontology. 26:221-230 (1980). 570. Vallee M., Mayo W., Darnaudery M., Corpechot C., Young J., Koehl M., Le Moal M., Baulieu E.E., Robel P., and Simon H. Neurosteroids: deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus. Proc Natl Acad Sci U S A. 94:14865-14870 (1997). 571. Melcangi R.C., Magnaghi V., Cavarretta I., Riva M.A., Piva F., and Martini L. Effects of steroid hormones on gene expression of glial markers in the central and peripheral nervous system: variations induced by aging. Exp Gerontol. 33:827-836 (1998). 572. Popplewell P.Y., Tsubokawa M., Ramachandran J., and Azhar S. Differential effects of aging on adrenocorticotropin receptors, adenosine 3'5'-monophosphate response, and corticosterone secretion in adrenocortical cells from Sprague-Dawley rats. Endocrinology. 119:2206-2213 (1986). 573. Prahalathan C., Selvakumar E., and Varalakshmi P. Lipoic acid modulates adriamycininduced testicular toxicity. Reprod Toxicol. 21:54-59 (2006). 574. Attal-Khemis S., Dalmeyda V., Michot J.L., Roudier M., and Morfin R. Increased total alpha-hydroxy-dehydroepiandrosterone in serum of patients with Alzheimer's disease. J Gerontol A Biol Sci Med Sci. 53:B125-132 (1998). 575. Baulieu E.E., Robel P., and Schumacher M. Neurosteroids: beginning of the story. Int Rev Neurobiol. 46:1-32 (2001). 576. Higashi T., Takido N., Yamauchi A., and Shimada K. Electron-capturing derivatization of neutral steroids for increasing sensitivity in liquid chromatography/negative atmospheric pressure chemical ionization-mass spectrometry. Anal Sci. 18:1301-1307 (2002). 577. Sandhoff R., Brugger B., Jeckel D., Lehmann W.D., and Wieland F.T. Determination of cholesterol at the low picomole level by nano-electrospray ionization tandem mass spectrometry. J Lipid Res. 40:126-132 (1999). 578. Richard I. Robinson, John C. Stephens, Steve M. Worden, Alexander J. Blake, Claire Wilson, and Woodward. S. Sulfonic acid libraries attained through opening of 2sulfobenzoic acid anhydride. Eur J Org Chem. 2004:4586-4605 (2004). 243 579. Ukena T., Satake M., Usami M., Oshima Y., Fujita T., Naoki H., and Yasumoto T. Structural confirmation of ostreocin-D by application of negative-ion fast-atom bombardment collision-induced dissociation tandem mass spectrometric methods. Rapid Commun Mass Spectrom. 16:2387-2393 (2002). 580. Dryrwa R., Droescher P., Ring S., Elger W., Schneider B., Hillisch A., and Reddersen G. Preparation of steroid prodrugs with androgenic activity. PCT Int Appl:57 pp. CODEN: PIXXD52 WO 2005113575 A2005113571 2020051201 CAN 2005113144:2005116967 AN 2005112005:2001260616 (2005). 581. Sjovall J. Fifty years with bile acids and steroids in health and disease. Lipids. 39:703-722 (2004). 582. Griffiths W.J., Karu K., Hornshaw M., Woffendin G., and Wang Y. Metabolomics and metabolite profiling: past heroes and future developments. Eur J Mass Spectrom (Chichester, Eng). 13:45-50 (2007). 583. Wang Y. and Griffiths W.J. Capillary liquid chromatography combined with tandem mass spectrometry for the study of neurosteroids and oxysterols in brain. Neurochem Int. 52:506-521 (2008). 584. New L.S., Saha S., Ong M.M., Boelsterli U.A., and Chan E.C. Pharmacokinetic study of intraperitoneally administered troglitazone in mice using ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 21:982-988 (2007). 585. Kushnir M.M., Rockwood A.L., Roberts W.L., Pattison E.G., Owen W.E., Bunker A.M., and Meikle A.W. Development and performance evaluation of a tandem mass spectrometry assay for adrenal steroids. Clin Chem. 52:1559-1567 (2006). 586. Zimmerman J.J. Moving beyond Babel. Pediatr Crit Care Med. 8:73-75 (2007). 587. Labombarda F., Pianos A., Liere P., Eychenne B., Gonzalez S., Cambourg A., De Nicola A.F., Schumacher M., and Guennoun R. Injury elicited increase in spinal cord neurosteroid content analyzed by gas chromatography mass spectrometry. Endocrinology. 147:18471859 (2006). 588. di Michele F., Caltagirone C., Bonaviri G., Romeo E., and Spalletta G. Plasma dehydroepiandrosterone levels are strongly increased in schizophrenia. J Psychiatr Res. 39:267-273 (2005). 589. Marx C.E., Trost W.T., Shampine L., Behm F.M., Giordano L.A., Massing M.W., and Rose J.E. Neuroactive steroids, negative affect, and nicotine dependence severity in male smokers. Psychopharmacology (Berl). 186:462-472 (2006). 590. Daeseleire E., Vandeputte R., and Peteghem C.V. Validation of multi-residue methods for the detection of anabolic steroids by GC-MS in muscle tissues and urine samples from cattle. Analyst. 123:2595-2598 (1998). 591. Van Poucke C., Van De Velde M., and Van Peteghem C. Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine. J Mass Spectrom. 40:731738 (2005). 592. Folch J., Lees M., and Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 226:497-509 (1957). 244 593. Svoboda J.A., Rees H.H., Thompson M.J., and Hoggard N. Intermediates of stigmasterol metabolism in Spodoptera littoralis. Steroids. 53:329-343 (1989). 594. Shimamoto A., Liu J., Kozawa S., and Fujimiya T. Determination of endogenous testosterone in rat tissues following fetal alcohol exposure using HPLC with UV detection. J Chromatogr B Analyt Technol Biomed Life Sci. 836:69-73 (2006). 595. Yu N.H., Ho E.N., Leung D.K., and Wan T.S. Screening of anabolic steroids in horse urine by liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 37:1031-1038 (2005). 596. Guo T., Chan M., and Soldin S.J. Steroid profiles using liquid chromatography-tandem mass spectrometry with atmospheric pressure photoionization source. Arch Pathol Lab Med. 128:469-475 (2004). 597. Guo T., Taylor R.L., Singh R.J., and Soldin S.J. Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry. Clin Chim Acta. 372:76-82 (2006). 598. Gelpi M.E., Cadenas R.A., Mosettig J., and Zuazo B.N. Nucleosteroids: carbocyclic nucleoside analogs of androst-4-en-17 beta-ol. Steroids. 67:263-267 (2002). 599. Ho E.N., Leung D.K., Wan T.S., and Yu N.H. Comprehensive screening of anabolic steroids, corticosteroids, and acidic drugs in horse urine by solid-phase extraction and liquid chromatography-mass spectrometry. J Chromatogr A. 1120:38-53 (2006). 600. Storbeck K.H., Kolar N.W., Stander M., Swart A.C., Prevoo D., and Swart P. The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids. Anal Biochem. 372:11-20 (2008). 601. Robb D.B., Covey T.R., and Bruins A.P. Atmospheric pressure photoionization: an ionization method for liquid chromatography-mass spectrometry. Anal Chem. 72:36533659 (2000). 602. Syage J.A., Evans M.D., and Hanold K.A. Photoionization Mass Spectrometry. Am Lab. 32:24-29 (2000). 603. Kauppila T.J., Kuuranne T., Meurer E.C., Eberlin M.N., Kotiaho T., and Kostiainen R. Atmospheric pressure photoionization mass spectrometry. Ionization mechanism and the effect of solvent on the ionization of naphthalenes. Anal Chem. 74:5470-5479 (2002). 604. Kauppila T.J., Kostiainen R., and Bruins A.P. Anisole, a new dopant for atmospheric pressure photoionization mass spectrometry of low proton affinity, low ionization energy compounds. Rapid Commun Mass Spectrom. 18:808-815 (2004). 605. Takino M., Daishima S., and Nakahara T. Liquid chromatography/mass spectrometric determination of patulin in apple juice using atmospheric pressure photoionization. Rapid Commun Mass Spectrom. 17:1965-1972 (2003). 606. Nilsson S.L., Andersson C., Sjoberg P.J., Bylund D., Petersson P., Jornten-Karlsson M., and Markides K.E. Phosphate buffers in capillary electrophoresis/mass spectrometry using atmospheric pressure photoionization and electrospray ionization. Rapid Commun Mass Spectrom. 17:2267-2272 (2003). 245 607. Hsieh Y., Merkle K., Wang G., Brisson J.M., and Korfmacher W.A. High-performance liquid chromatography-atmospheric pressure photoionization/tandem mass spectrometric analysis for small molecules in plasma. Anal Chem. 75:3122-3127 (2003). 608. Moriwaki H., Ishitake M., Yoshikawa S., Miyakoda H., and Alary J.F. Determination of polycyclic aromatic hydrocarbons in sediment by liquid chromatography-atmospheric pressure photoionization-mass spectrometry. Anal Sci. 20:375-377 (2004). 609. Cai Y., Kingery D., McConnell O., and Bach A.C., 2nd. Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery. Rapid Commun Mass Spectrom. 19:1717-1724 (2005). 610. Raffaelli A. and Saba A. Atmospheric pressure photoionization mass spectrometry. Mass Spectrom Rev. 22:318-331 (2003). 611. Pereira Ados S., Oliveira L.S., Mendes G.D., Gabbai J.J., and De Nucci G. Quantification of betamethasone in human plasma by liquid chromatography-tandem mass spectrometry using atmospheric pressure photoionization in negative mode. J Chromatogr B Analyt Technol Biomed Life Sci. 828:27-32 (2005). 612. Henley S.M., Bates G.P., and Tabrizi S.J. Biomarkers for neurodegenerative diseases. Curr Opin Neurol. 18:698-705 (2005). 246 [...]... area of intense research, as oxidative stress in brain is emerging as a potential causal factor in aging and ageassociated neurodegenerative diseases [6-11] 1 Neurodegenerative diseases are amongst the most common and most disabling of all diseases Resulted from premature progressive degeneration of specific neurons, neurodegenerative diseases are characterized by progressive dysfunction and death of. .. effective against in vivo oxidation of CSF lipoproteins and brain lipids, and offer new perspectives in the treatment of AD and other neurodegenerative disorders [111] Metal ions Several studies in mice have shown that one of the consequences of normal aging is a rise in the levels of copper and iron in brain tissue [113-115] The brain is an organ that concentrates metal ions and recent evidence suggests... cause of neuronal death in each disease appears to be multifactorial Current therapies provide only symptomatic relief, while none significantly alter the course of disease [12] 1.2 AD Following the general briefing of aging and neurodegenerative diseases, a specific introduction on AD including its prevalence, etiology and pathology, diagnosis and treatment will be presented here In addition, biomarkers... clinical and neuropathological characteristics of the disease in a 51-year-old woman that was subsequently named after him [15] One hundred years later, AD is the most common neurodegenerative disease and cause of dementia in old population It accounts for 2 at least two thirds of all dementias, more than dementia with Lewy bodies and vascular dementia combined [16] 1.2.1 Prevalence of AD The prevalence of. .. chapter, an overview on aging and neurodegenerative diseases is provided Alzheimer’s disease (AD), including its etiology, pathology, diagnosis and clinical interventions are also discussed In addition, the scopes of biomarker discovery, particularly the metabonomic profiling techniques, are presented Finally, the clinical roles of endogenous steroids in neurodegeneration and the associated analytical... Na+ and Ca2+ influx and further exocytosis of glutamate Ca2+ influx leads to delayed necrosis of the neuron, and to a lesser extent activation of apoptotic pathways Continuing release of glutamate leads to a spreading of the process [77], which is termed excitotoxicity Excitotoxicity resulting from excessive activation of N-methyl-Daspartate (NMDA) receptors may enhance the localized vulnerability of. .. cytokines are also able to promote the accumulation of Aβ peptide Altogether, IL-1, IL-6, TNF-α and TGF-β should be considered as key players of a vicious cycle leading to the progression of the disease [90, 93] Cardiovascular diseases Recently, several studies demonstrated the associations between cardiovascular disease and its risk factors and the incidence of AD [94-97] Previous study examined the evidence... several types of heart disease and stroke on the development of AD The evidence suggested that CBH is responsible for protein synthesis defects that later result in 8 the classic AD neurodegenerative lesions such as the formation of excess betaamyloid plaques and NFT [98] In addition, several studies reported that blood pressure is increased in victims of AD decades before the onset of the disease [99102]... progressive dysfunction and death of specific populations of neurons, and manifest as syndromes with varied combinations of cognitive, motor, sensory and autonomic dysfunctions [12] The most common of these age-associated chronic illnesses are AD, Lewy body diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), Prion disease and amyotrophic lateral sclerosis (ALS) [12] Although several... standard clinical criteria that take into account patient age, cognitive testing [such as the Mini-Mental State Exam (MMSE) or Mini-Cog], and functional and behavioral testing (such as the Dementia Severity Rating Scale) The National Institute of Neurological and Communicative Diseases and Stroke/Alzheimer’s Disease and Related Disorders Association criteria use this information to give a diagnosis of . RATIONALES AND OBJECTIVES OF THE THESIS 46 1.5.1 Metabonomic profiling in aging and AD models 46 1.5.2 Steroidal biomarker profiling in aging and AD models 48 1.5.3 Development and comparison of MS-based. metabolic profiling of complex biological samples, which may lead to the discovery of biomarkers for the diagnosis of diseases. In this thesis, both non-targeted metabolic and targeted steroidal biomarker. BIOMARKER PROFILING OF AGING AND NEURODEGENERATIVE DISEASE ZEPING HU (M.Sc., National Institute for the Control of Pharmaceutical & Biological