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Identification of a new plasma biomarker of Alzheimer's disease using metabolomics technology

2011; Elsevier BV; Volume: 53; Issue: 3 Linguagem: Inglês

10.1194/jlr.m022376

1539-7262

Yoshiaki Sato, Ikumi Suzuki, Tatsuji Nakamura, François P. Bernier, Ken Aoshima, Yoshiya Oda,

Fatty Acid Research and Health

We performed unbiased analysis of steroid-related compounds to identify novel Alzheimer's disease (AD) plasma biomarkers using liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy. The analysis revealed that desmosterol was found to be decreased in AD plasma versus controls. To precisely quantify variations in desmosterol, we established an analytical method to measure desmosterol and cholesterol. Using this LC-based method, we discovered that desmosterol and the desmosterol/cholesterol ratio are significantly decreased in AD. Finally, the validation of this assay using 109 clinical samples confirmed the decrease of desmosterol in AD as well as a change in the desmosterol/cholesterol ratio in AD. Interestingly, we could also observe a difference between mild cognitive impairment and control. In addition, the decrease of desmosterol was somewhat more significant in females. Receiver operating characteristic (ROC) analysis between controls and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power for this marker in the two reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. Further analysis showed a significant correlation of plasma desmosterol with Mini-Mental State Examination scores. Although larger sample populations will be needed to confirm this diagnostic marker sensitivity, our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest that plasma desmosterol could become a powerful new specific biomarker for early and easy AD diagnosis. We performed unbiased analysis of steroid-related compounds to identify novel Alzheimer's disease (AD) plasma biomarkers using liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy. The analysis revealed that desmosterol was found to be decreased in AD plasma versus controls. To precisely quantify variations in desmosterol, we established an analytical method to measure desmosterol and cholesterol. Using this LC-based method, we discovered that desmosterol and the desmosterol/cholesterol ratio are significantly decreased in AD. Finally, the validation of this assay using 109 clinical samples confirmed the decrease of desmosterol in AD as well as a change in the desmosterol/cholesterol ratio in AD. Interestingly, we could also observe a difference between mild cognitive impairment and control. In addition, the decrease of desmosterol was somewhat more significant in females. Receiver operating characteristic (ROC) analysis between controls and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power for this marker in the two reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. Further analysis showed a significant correlation of plasma desmosterol with Mini-Mental State Examination scores. Although larger sample populations will be needed to confirm this diagnostic marker sensitivity, our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest that plasma desmosterol could become a powerful new specific biomarker for early and easy AD diagnosis. Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system characterized by a progressive loss of short-term memory accompanied by a gradual loss of cognitive functions (1Ross C.A. Poirier M.A. Protein aggregation and neurodegenerative disease.Nat. Med. 2004; 10 (Suppl.): 10-17Crossref PubMed Scopus (2468) Google Scholar). AD pathology is characterized by brain atrophy reflecting neuronal and synaptic loss and by the presence of amyloid plaques and neurofibrillary tangles. AD pathogenic mechanisms contributing to neuronal loss and brain dysfunction are still unclear. Biomarkers are very useful for diagnosing and monitoring disease progression (2Ward M. Biomarkers for Alzheimer's disease.Expert Rev. Mol. Diagn. 2007; 7: 635-646Crossref PubMed Scopus (26) Google Scholar) and are important for patient selection, monitoring side-effects, aiding selection of appropriate patient treatment, and helping new drug discovery. For the clinical studies of AD therapeutics, there is an increasing need for diagnostic markers to ensure that therapies are targeted at the right patient population, to initiate early treatment when disease-modifying drugs will be available, and to monitor disease progression (3Hye A. Lynham S. Thambisetty M. Causevic M. Campbell J. Byers H.L. Hooper C. Rijsdijk F. Tabrizi S.J. Banner S. et al.Proteome-based plasma biomarkers for Alzheimer's disease.Brain. 2006; 129: 3042-3050Crossref PubMed Scopus (391) Google Scholar). Several studies have investigated AD biochemical biomarkers in various tissues including blood (4Blennow K. Hampel H. Weiner M. Zetterberg H. Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.Nat. Rev. Neurol. 2010; 6: 131-144Crossref PubMed Scopus (1390) Google Scholar, 5Song F. Poljak A. Smythe G.A. Sachdev P. Plasma biomarkers for mild cognitive impairment and Alzheimer's disease.Brain Res. 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Aging. 2009; 30: 1895-1901Crossref PubMed Scopus (111) Google Scholar), although they cannot predict conversion from mild cognitive impairment (MCI) to AD accurately and are not useful for guiding drug treatment. In addition, analyzing those markers requires performing delicate CSF collection from patients. Hence, access to less-invasive biomarkers found in easy-to-acquire fluids such as plasma would accelerate and reduce the cost of AD diagnosis and offer windows of opportunity for selecting and treating patients with disease-modifying drugs once they are available. Metabolomics has particular relevance to drug discovery and development, because metabolites often mirror the end result of genomic and protein perturbations in disease and are most closely associated with phenotypic changes. Current metabolomics research involves the identification and quantification of hundreds to thousands of small-molecular-mass metabolites (<1,500 Daltons) in cells, tissues, or biological fluids. The aims of such studies are typically to understand new diagnosis biomarkers, to understand the mechanism of action of therapeutic compounds, and to uncover the pharmacodynamics and kinetic markers of drugs in patients and in preclinical in vivo and in vitro models (8Wilcoxen K.M. Uehara T. Myint T.T. Sato Y. Oda Y. Practical metabolomics in drug discovery.Expert Opinion on Drug Discovery. 2010; 5: 249-263Crossref PubMed Scopus (21) Google Scholar). We had previously established in our laboratory a metabolomics platform focused on hydrophilic cationic compounds (9Myint K.T. Aoshima K. Tanaka S. Nakamura T. Oda Y. Quantitative profiling of polar cationic metabolites in human cerebrospinal fluid by reversed-phase nanoliquid chromatography/mass spectrometry.Anal. Chem. 2009; 81: 1121-1129Crossref PubMed Scopus (65) Google Scholar) and could identify 55 hydrophilic AD biomarker candidates in human AD CSF using nano-LC/MS. 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Investigating lipid biochemistry using a lipidomics approach will not only provide insights into the specific roles of lipid molecular species in healthy individuals and patients but will also assist in identifying potential biomarkers for establishing preventive or therapeutic approaches for human health (10Hu C. van der Heijden R. Wang M. van der Greef J. Hankemeier T. Xu G. Analytical strategies in lipidomics and applications in disease biomarker discovery.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2836-2846Crossref PubMed Scopus (168) Google Scholar, 13Wenk M.R. The emerging field of lipidomics.Nat. Rev. Drug Discov. 2005; 4: 594-610Crossref PubMed Scopus (986) Google Scholar, 14Rosenson R.S. New technologies personalize diagnostics and therapeutics.Curr. Atheroscler. Rep. 2010; 12: 184-186Crossref PubMed Scopus (15) Google Scholar–15Kochanek P.M. Berger R.P. Bayir H. Wagner A.K. Jenkins L.W. Clark R.S. Biomarkers of primary and evolving damage in traumatic and ischemic brain injury: diagnosis, prognosis, probing mechanisms, and therapeutic decision making.Curr. Opin. Crit. Care. 2008; 14: 135-141Crossref PubMed Scopus (195) Google Scholar). Lipidomics has recently captured attention, owing to the well-recognized roles of lipids in numerous human diseases such as diabetes, obesity, atherosclerosis, and Alzheimer's disease (13Wenk M.R. The emerging field of lipidomics.Nat. Rev. Drug Discov. 2005; 4: 594-610Crossref PubMed Scopus (986) Google Scholar, 16Watson A.D. Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: a global approach to lipid analysis in biological systems.J. Lipid Res. 2006; 47: 2101-2111Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 17Steinberg D. Thematic review series: the pathogenesis of atherosclerosis. An interpretive history of the cholesterol controversy: part II: the early evidence linking hypercholesterolemia to coronary disease in humans.J. Lipid Res. 2005; 46: 179-190Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar–18Sato Y. Nakamura T. Aoshima K. Oda Y. Quantitative and wide-ranging profiling of phospholipids in human plasma by two-dimensional liquid chromatography/mass spectrometry.Anal. Chem. 2010; 82: 9858-9864Crossref PubMed Scopus (67) Google Scholar). In support of the hypothesis that lipid dysfunction plays an important role in AD pathogenesis, our laboratory also previously established a lipidomics method for comprehensive phospholipids evaluation that identified 31 phospholipids as AD biomarker candidates in human plasma using LC/MS (18Sato Y. Nakamura T. Aoshima K. Oda Y. Quantitative and wide-ranging profiling of phospholipids in human plasma by two-dimensional liquid chromatography/mass spectrometry.Anal. 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Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.J. Am. Med. Assoc. 1997; 278: 1349-1356Crossref PubMed Google Scholar–22Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.Proc. Natl. Acad. Sci. USA. 1993; 90: 1977-1981Crossref PubMed Scopus (3703) Google Scholar). In this study, we applied lipidomics technologies we developed in-house that focus on sterols, to find new AD plasma biomarkers. The reliable quantification method we established allows the detection of selected lipids and validates the change of the candidate in AD. The specificity of candidate was also confirmed using unrelated neurological disorders samples to clarify the specificity of the biomarker candidate. Most of the reagents used in the experiments were of analytical grade and were purchased from Wako Pure Chemicals Co. (Osaka, Japan). All other chemicals and solvents were analytical reagent grade. Zymosterol was purchased from Steraloids, Inc. (Newport, RI). Desmosterol and cholesterol were purchased from Sigma Aldrich (Poole; Dorset, UK). 5α-Cholesta-7,24-dien-3β-ol, cholesta-5,7-dien-3β-ol, and cholesta-5,8-dien-3β-ol were purchased from Sumika Technoservice Corporation. Cholesterol-25,26,26,26,27,27,27-D7 (D7-cholesterol) was purchased from Kanto Chemicals Co., Inc. (Tokyo, Japan). Desmosterol-26,26,26,27,27,27-D6 (D6-desmosterol) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Plasma and CSF samples of elderly controls and subjects diagnosed with AD, MCI, schizophrenia, and Parkinson's disease were purchased from PrecisionMed, Inc. (San Diego, CA) and stored at −80°C until use. For circadian rhythm studies, plasma samples of healthy volunteers were collected at various time points during the day (9:30, 11:00, 13:00, 15:00, and 17:00) and stored at −80°C until use. The deep-frozen plasma samples were thawed on ice, and 25 μl of plasma was spiked with 50 μl of 5 μg/ml D7-cholesterol in ethanol as internal standard. One hundred microliters of 50% potassium hydroxide (w/v) was then added to the solution, mixed thoroughly, and incubated at 70°C for 60 min. Following the incubation, 2 ml hexane and 0.5 ml PBS (pH 6.8) were added and mixed well. The solution was centrifuged for 10 min at 2,000 g, and the upper organic phase was transferred to a new tube. The lower layer was extracted with an additional 1 ml hexane, which was also added to the organic phase extract. The solvents were evaporated to dryness under a nitrogen gas stream at 40°C, the pellet was reconstituted in 100 μl ethanol, and the solution was subjected to liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy (LC/APCI-MS) analysis. The omics study of neutral lipids was performed using a Shimadzu 20 AD system with an SIL-20AC auto-sampler, a CTO-20A column oven, and an LTQ Orbitrap mass spectrometer (ThermoFisher; San Jose, CA) with an APCI probe. For each run, we injected a total of 20 μl sample onto a 3.0 inner diameter (ID) × 100 mm Shim-Pak XR-ODS column (Shimadzu Corporation; Kyoto, Japan) at a flow rate of 0.8 ml/min, with a total run time of 60 min. The gradient used consisted of solvent A (water-methanol, 50:50) and solvent B (methanol) starting at 40% B, ramping to 90% B over 20 min, holding at 90% B for 10 min, ramping to 100% B over 10 min, holding at 100% B for 10 min, back to 40% B in 0.1 min, and then holding to 10 min. The mass spectrometer was operated in the positive-ion mode. The spray voltage was set at 2.5 kV. A cycle of one full fourier transform (FT) scan mass spectrum (200–1,000 m/z, resolution of 30,000) followed by three data-dependent MS/MSs acquired in the linear ion trap with normalized collision energy (setting of 35%) was repeated continuously. Application of mass spectrometer scan functions and HPLC solvent gradients were controlled by the Xcalibur data system. The mass spectrometric data were acquired using Xcalibur, and the initial metabolomics profiling was performed using in-house-developed Mass ++ data analysis software (http://groups.google.com/group/massplusplus) to obtain a peak list and align retention times and peak area, which is normalized with internal standard (D7-cholesterol). One-way variance analysis (ANOVA) was applied to the data. Finally, some peaks, whose ANOVA values were lower than 0.05 and fold change more than 2 or less than half, were picked up for further identification. Individual primary methanolic stock solutions of desmosterol, zymosterol, 5α-cholesta-7,24-dien-3β-ol, cholesta-5,7-dien-3β-ol, and cholesta-5,8-dien-3β-ol were prepared at concentrations high enough that when combined, a secondary dilution of each in a 10 ml volume of methanol resulted in concentrations of 20–30 μg/ml for each compound. This solution was subjected to LC/MS analysis to compare retention times. The identification study of unknown peak A was formed using a Shimadzu 20AD system with a SIL-20AC auto-sampler, a CTO-20AC column oven, and an LCMS-2010EV high-performance single-quadrupole mass analyzer (Shimadzu) equipped with an APCI probe. For each run, we injected a total of 20 μl sample onto a 4.6 mm ID × 250 mm YMC-Pack Pro C18 RS column (YMC, Inc.; Wilmington, NC) at a flow rate of 1 ml/min, with a total run time of 70 min for desmosterol analysis or 20 min for cholesterol analysis. Column temperature was maintained at 50°C. For desmosterol analysis, the gradient used consisted of solvent A (water-methanol, 50:50) and solvent B (methanol) starting at 85% B for 45 min, ramping to 100% B over 0.1 min, holding at 100% B for 10 min, back to 85% B in 0.1 min, and then holding to 15 min. For the cholesterol analysis, the mobile phase B flow rate was 1 ml/min isocratically. The mass spectrometer was operated in the positive-ion mode. MS conditions were as follows: resolution, ±0.15 Da; capillary temperature, 250°C; APCI vaporizer temperature, 400°C; ionization voltage, 4.5 kV; sheath gas flow, 2.5 l/min; and drying gas pressure, 0.02 MPa. To increase sensitivity, selected-ion monitoring (SIM) was used in this study. Programmed SIM was used, in which specific ions were monitored for compound throughout the chromatographic run. Ions monitored for identification and quantification of desmosterol and its isomers were m/z 367.3 and 369.3 for cholesterol. The deep-frozen CSF samples were thawed on ice, and 100 μl of CSF was spiked with 100 μl of 1 μg/ml D7-cholesterol in ethanol and 100 μl of 100 ng/ml of D6-desmosterol in ethanol as internal standard. One hundred microliters of 50% potassium hydroxide (w/v) was then added to the solution, mixed thoroughly, and incubated at 70°C for 60 min. Following the incubation, 2 ml hexane and 0.5 ml PBS (pH 6.8) were added and mixed well. The solution was centrifuged for 10 min at 2,000 g, and the upper organic phase was transferred to a new tube. The lower layer was extracted with an additional 1 ml hexane, which was also added to the organic-phase extract. The solvents were evaporated to dryness under a nitrogen gas stream at 40°C, the residue was dissolved in 100 μl of ethanol, 20 μl of this solution was injected into the LC/APCI-MS system in desmosterol analysis, and 2 μl of this solution was injected in cholesterol analysis of CSF. For preparation of standard stock solutions, desmosterol and cholesterol were dissolved in ethanol at a concentration of 0.1 mg/ml. Samples were diluted to concentrations of 10, 30, 100, 300, 1,000, 3,000, 10,000, and 30,000 ng/ml using ethanol for desmosterol and 0.1, 0.2, 0.5, 1, 2, 5, and 10 mg/ml using ethanol for cholesterol for the calibration curve in the plasma analysis. And samples were diluted to concentrations of 1, 2, 5, 10, 20, 50, 100, and 200 ng/ml using ethanol for desmosterol and 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 μg/ml using ethanol for cholesterol for the calibration curve in CSF analysis. Internal standard stock solutions containing 500 ng/ml or 100 ng/ml of D6-desmosterol and 200 μg/ml or 1 μg/ml of D7-cholesterol in ethanol were also prepared in ethanol for plasma or CSF analysis, respectively. In the calibration study, a 25 μl or 100 μl aliquot of each standard solution was mixed with 100 μl of each internal standard solution in ethanol for plasma or CSF analysis and evaporated under a nitrogen gas stream at room temperature. The residue was dissolved in 100 μl ethanol, 20 μl of this solution was injected into the LC/APCI-MS system in desmosterol analysis, and 2 μl of this solution was injected in cholesterol analysis of CSF. The residual 10 μl was diluted with 1990 μl ethanol for analysis of cholesterol, and 2 μl of this solution was injected into the LC/APCI-MS system in cholesterol analysis of plasma. The LC/MS is LCMS-2010EV high-performance single quadrupole mass analyzer. SIM was used in this study. Programmed SIM was also used in these studies. Ions monitored for quantification of desmosterol were m/z 367.3 and 373.3 (D6-incorporated standard); cholesterol ions were m/z 369.3 and 376.3 (D7-incorporated standard). The calibration curves were constructed by 1/x2 weighted least-squares linear regression of the peak area ratio of the analyte to the internal standard, against the concentration of each compound. For the accuracy and precision studies using human plasma, the interassay precision, expressed as relative standard deviation (RSD), was determined by the extraction and analysis of the four different samples. Briefly a 25 μl aliquot of human plasma was added to 25 μl aliquots of 0, 30, 300, or 10,000 ng/ml stock solution of desmosterol, or 25 μl aliquots of 0, 0.2, 1, or 5 mg/ml stock solution of cholesterol were separately spiked into the plasma. Then the concentrations of desmosterol and cholesterol were analyzed with LC/APCI-MS on the same day (n = 3) and on a different day (n = 3). The relative error was calculated as [(observed concentration−theoretical concentration)/spiked concentration] × 100 (%), and the precision was obtained in terms of the RSD. Samples were aliquoted and frozen at −80°C until assayed. Plasma concentration of desmosterol was measured by a modified method using combined GC-MS as described previously (23Kölsch H. Heun R. Jessen F. Popp J. Hentschel F. Maier W. Lutjohann D. Alterations of cholesterol precursor levels in Alzheimer's disease.Biochim. Biophys. Acta. 2010; 1801: 945-950Crossref PubMed Scopus (51) Google Scholar). In brief, 1 μg D6-desmosterol (100 μl from a stock solution of D7-desmosterol in ethanol; 10 μg/ml) was added to 100 μl plasma. One hundred microliters of 50% potassium hydroxide was then added to the solution, mixed thoroughly, and incubated at 70°C for 60 min. Following the incubation, 2 ml hexane and 0.5 ml PBS (pH 6.8) were added and mixed well. The solution was centrifuged for 10 min at 2,000 g, and the upper organic phase was transferred to a new tube. The lower layer was extracted with an additional 1 ml hexane, which was also added to the organic-phase extract. The organic solvents were evaporated, and the residual sterols were derivatized to TMS ethers by adding 1 ml TMS reagent (pyridine-hexamethylsisilazane-trimethylchlorosilane, 9:3:1[Supelco; Bellefonte, PA]) and incubation for 1 h at 64°C. The derivatization reagent was evaporated under nitrogen, and the silyl sterol ethers from plasma were dissolved in 160 μl n-decane. Eighty microliters of the solution was transferred into micro-vials for GC-MS SIM, and an aliquot of 2 μl was injected in a splitless mode at 280°C by an automated sampler and injector. GC-MS SIM was performed on a GC-2010 (Shimadzu) combined with a GCMS-QP2010 plus (Shimadzu) equipped with a DB-XLB 122-1232 fused silica capillary column (J&W Scientific, Inc.; Folsom, CA) (30 m × 0.25 mm, i.e., ×0.25 μm film thickness) in the splitless mode using helium (1 ml/min) as the carrier gas as described previously (23Kölsch H. Heun R. Jessen F. Popp J. Hentschel F. Maier W. Lutjohann D. Alterations of cholesterol precursor levels in Alzheimer's disease.Biochim. Biophys. Acta. 2010; 1801: 945-950Crossref PubMed Scopus (51) Google Scholar, 24Teunissen C.E. Lutjohann D. von Bergmann K. Verhey F. Vreeling F. Wauters A. Bosmans E. Bosma H. van Boxtel M.P. Maes M. et al.Combination of serum markers related to several mechanisms in Alzheimer's disease.Neurobiol. Aging. 2003; 24: 893-902Crossref PubMed Scopus (82) Google Scholar). The temperature program was as follows: 150°C for 1 min, followed by 20°C/min up to 260°C, and 10°C/min up to 280°C (for 15 min). 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