Journal Pmed 1002482

RESEARCH ARTICLE
Brain and blood metabolite signatures of

pathology and progression in Alzheimer
disease: A targeted metabolomics study
Vijay R. Varma1, Anup M. Oommen2, Sudhir Varma3, Ramon Casanova4, Yang An1, Ryan
M. Andrews5, Richard O’Brien6, Olga Pletnikova7, Juan C. Troncoso7, Jon Toledo8,
Rebecca Baillie9, Matthias Arnold10,11, Gabi Kastenmueller10,11, Kwangsik Nho12,
P. Murali Doraiswamy13,14, Andrew J. Saykin12, Rima Kaddurah-Daouk13,14,
Cristina Legido-Quigley15, Madhav Thambisetty1*
a1111111111
1 Clinical and Translational Neuroscience Unit, Laboratory of Behavioral Neuroscience, National Institute on
a1111111111 Aging (NIA), National Institutes of Health (NIH), Baltimore, Maryland, United States of America, 2 Consilience
a1111111111 Research Advisors LLP, Bengaluru, Karnataka, India, 3 HiThru Analytics, Laurel, Maryland, United States of
a1111111111 America, 4 Department of Biostatistical Science, Wake Forest School of Medicine, Winston-Salem, North
a1111111111 Carolina, United States of America, 5 Department of Mental Health, Johns Hopkins Bloomberg School of
Public Health, Baltimore, Maryland, United States of America, 6 Department of Neurology, Duke University
School of Medicine, Durham, North Carolina, United States of America, 7 Department of Pathology, Johns
Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 8 Department of
Neurology, Houston Methodist Hospital, Houston, Texas, United States of America, 9 Rosa & Co LLC, San
Carlos, California, United States of America, 10 Institute of Bioinformatics and Systems Biology, Helmholtz
OPEN ACCESS Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany, 11 German
Center for Diabetes Research (DZD), Neuherberg, Germany, 12 Department of Radiology and Imaging
Citation: Varma VR, Oommen AM, Varma S,
Sciences and the Indiana Alzheimer Disease Center, Indiana University School of Medicine, Indianapolis,
Casanova R, An Y, Andrews RM, et al. (2018) Brain
Indiana, United States of America, 13 Department of Psychiatry and Behavioral Sciences, Duke University,
and blood metabolite signatures of pathology and Durham, North Carolina, United States of America, 14 Department of Medicine, Duke University, Durham,
progression in Alzheimer disease: A targeted North Carolina, United States of America, 15 IPS, Faculty of Life Sciences and Medicine, King’s College
metabolomics study. PLoS Med 15(1): e1002482. London, London, United Kingdom
https://doi.org/10.1371/journal.pmed.1002482
* [email protected]
Academic Editor: Carol Brayne, University of
Cambridge, UNITED KINGDOM
Received: April 21, 2017 Abstract

Accepted: November 27, 2017
Background
Published: January 25, 2018
The metabolic basis of Alzheimer disease (AD) is poorly understood, and the relationships
Copyright: This is an open access article, free of all
between systemic abnormalities in metabolism and AD pathogenesis are unclear. Under-
copyright, and may be freely reproduced,
distributed, transmitted, modified, built upon, or standing how global perturbations in metabolism are related to severity of AD neuropathology
otherwise used by anyone for any lawful purpose. and the eventual expression of AD symptoms in at-risk individuals is critical to developing
The work is made available under the Creative effective disease-modifying treatments. In this study, we undertook parallel metabolomics
Commons CC0 public domain dedication.
analyses in both the brain and blood to identify systemic correlates of neuropathology and
Data Availability Statement: Data from the their associations with prodromal and preclinical measures of AD progression.
Baltimore Longitudinal Study of Aging (BLSA) are
available to researchers and can be requested at
https://www.blsa.nih.gov/researchers. Data from
Methods and findings
the Alzheimer’s Disease Neuroimaging Initiative Quantitative and targeted metabolomics (Biocrates AbsoluteIDQ [identification and quantifi-
(ADNI) database are available to researchers at
cation] p180) assays were performed on brain tissue samples from the autopsy cohort of
http://adni.loni.usc.edu/.
the Baltimore Longitudinal Study of Aging (BLSA) (N = 44, mean age = 81.33, % female =
Funding: This research was supported in part by
36.36) from AD (N = 15), control (CN; N = 14), and “asymptomatic Alzheimer’s disease”
the Intramural Research Program of the NIH,
National Institute on Aging. Data collection and (ASYMAD, i.e., individuals with significant AD pathology but no cognitive impairment during
sharing for this project was funded by the life; N = 15) participants. Using machine-learning methods, we identified a panel of 26
PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 1 / 31

Brain and blood metabolite signatures of Alzheimer disease
Alzheimer’s Disease Neuroimaging Initiative (ADNI) metabolites from two main classes—sphingolipids and glycerophospholipids—that discrimi-
(National Institutes of Health Grant U01 nated AD and CN samples with accuracy, sensitivity, and specificity of 83.33%, 86.67%,
AG024904) and DOD ADNI (Department of
Defense award number W81XWH-12-2-0012).
and 80%, respectively. We then assayed these 26 metabolites in serum samples from two
ADNI is funded by the National Institute on Aging, well-characterized longitudinal cohorts representing prodromal (Alzheimer’s Disease Neu-
the National Institute of Biomedical Imaging and roimaging Initiative [ADNI], N = 767, mean age = 75.19, % female = 42.63) and preclinical
Bioengineering, and through generous
(BLSA) (N = 207, mean age = 78.68, % female = 42.63) AD, in which we tested their associ-
contributions from the following: AbbVie;
Alzheimer’s Association; Alzheimer’s Drug ations with magnetic resonance imaging (MRI) measures of AD-related brain atrophy, cere-
Discovery Foundation; Araclon Biotech; BioClinica, brospinal fluid (CSF) biomarkers of AD pathology, risk of conversion to incident AD, and
Inc.; Biogen; Bristol-Myers Squibb Company; trajectories of cognitive performance. We developed an integrated blood and brain endo-
CereSpir, Inc.; Cogstate; Eisai, Inc.; Elan
phenotype score that summarized the relative importance of each metabolite to severity of
Pharmaceuticals, Inc.; Eli Lilly and Company;
EuroImmun; F. Hoffmann-La Roche Ltd and its AD pathology and disease progression (Endophenotype Association Score in Early Alzhei-
affiliated company, Genentech, Inc.; Fujirebio; GE mer’s Disease [EASE-AD]). Finally, we mapped the main metabolite classes emerging from
Healthcare; IXICO Ltd.; Janssen Alzheimer
our analyses to key biological pathways implicated in AD pathogenesis. We found that distinct
Immunotherapy Research & Development, LLC.;
Johnson & Johnson Pharmaceutical Research & sphingolipid species including sphingomyelin (SM) with acyl residue sums C16:0, C18:1, and
Development, LLC.; Lumosity; Lundbeck; Merck & C16:1 (SM C16:0, SM C18:1, SM C16:1) and hydroxysphingomyelin with acyl residue sum
Co., Inc.; Meso Scale Diagnostics, LLC.; NeuroRx C14:1 (SM (OH) C14:1) were consistently associated with severity of AD pathology at autopsy
Research; Neurotrack Technologies; Novartis
and AD progression across prodromal and preclinical stages. Higher log-transformed blood
Pharmaceuticals Corporation; Pfizer, Inc.; Piramal
Imaging; Servier; Takeda Pharmaceutical concentrations of all four sphingolipids in cognitively normal individuals were significantly asso-
Company; and Transition Therapeutics. The ciated with increased risk of future conversion to incident AD: SM C16:0 (hazard ratio [HR] =
Canadian Institutes of Health Research is providing 4.430, 95% confidence interval [CI] = 1.703–11.520, p = 0.002), SM C16:1 (HR = 3.455, 95%
funds to support ADNI clinical sites in Canada.
CI = 1.516–7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373–9.122, p = 0.009),
Private sector contributions are facilitated by the
Foundation for the National Institutes of Health and SM C18:1 (HR = 2.255, 95% CI = 1.047–4.855, p = 0.038). The sphingolipid species iden-
(www.fnih.org). The grantee organization is the tified map to several biologically relevant pathways implicated in AD, including tau phosphory-
Northern California Institute for Research and lation, amyloid-β (Aβ) metabolism, calcium homeostasis, acetylcholine biosynthesis, and
Education, and the study is coordinated by the
Alzheimer’s Therapeutic Research Institute at the
apoptosis. Our study has limitations: the relatively small number of brain tissue samples may
University of Southern California. ADNI data are have limited our power to detect significant associations, control for heterogeneity between
disseminated by the Laboratory for Neuro Imaging groups, and replicate our findings in independent, autopsy-derived brain samples.
at the University of Southern California. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Conclusions
the manuscript. We present a novel framework to identify biologically relevant brain and blood metabolites
Competing interests: I have read the journal’s associated with disease pathology and progression during the prodromal and preclinical
policy and the authors of this manuscript have the stages of AD. Our results show that perturbations in sphingolipid metabolism are consis-
following competing interests: PMD has received
tently associated with endophenotypes across preclinical and prodromal AD, as well as with
research grants from Lilly, Avanir, and Alzheimer’s
Drug Discovery Foundation in the past 12 months AD pathology at autopsy. Sphingolipids may be biologically relevant biomarkers for the
and speaking or advisory fees from Anthrotronix, early detection of AD, and correcting perturbations in sphingolipid metabolism may be a
Cognoptix, Genomind, Neurocog Trials, NeuroPro, plausible and novel therapeutic strategy in AD.
T3D Therapeutics, MindLink, and Global
Alzheimer’s Platform and owns shares in Maxwell
Health, Muses Labs, Anthrotronix, Evidation
Health, Turtle Shell Technologies, and Advera
Health Analytics. PMD is a coinventor on patent(s) Author summary
relating to metabolomics or dementia biomarkers
that are unlicensed. RB is an employee of Rosa &
Co. Rosa & Co. has had no input on and will Why was this study done?
receive no financial benefit from the design of the
study or publication of the results. All other authors • Metabolomics, which measures the biochemical products of cell processes, can be used
have declared that no competing interests exist. to measure alterations in biochemical pathways related to AD.

Abbreviations: ρ, correlation coefficient; ω-3,

omega-3; aa, diacyl; Aβ1–42, amyloid beta 1–42; Aβ, • Several recent studies have applied metabolomics to explore potential blood biomarkers
amyloid-β; ACSL, acyl-CoA synthetase; AD, for Alzheimer disease (AD).
Alzheimer disease; ADMC, Alzheimer Disease
Metabolomics Consortium; ADNI, Alzheimer’s • Prior blood biomarker studies have not linked signals in the blood to those in the brain
Disease Neuroimaging Initiative; ae, acyl-alkyl; and have relied mainly on discriminating between AD/mild cognitive impairment
AKT, protein kinase B; APOE e4, apolipoprotein E (MCI) and control samples.
epsilon 4 allele; APP, amyloid precursor protein;
ARIC, Atherosclerosis Risk in Communities; • These study designs ignore the long preclinical prodrome of AD and do not provide
ASYMAD, asymptomatic Alzheimer’s disease; biological insights into the evolution of AD pathology in the brain and eventual devel-
BACE1, β-secretase; BAD, BCL2 associated agonist
opment of clinical symptoms.
of cell death; BBB, blood-brain barrier; BIMC,
Blessed Information, Memory, and Concentration; • Our study was designed to link alterations in metabolite signals in the brain to those in
BLSA, Baltimore Longitudinal Study of Aging; the blood, explore how those alterations were associated with distinct endophenotypes
CAMKII, calmodulin kinase; CART, classification
of AD, and identify the key biological pathways implicated.
and regression trees methodology; CBL,
cerebellum; CDR, Clinical Dementia Rating;
CERAD, Consortium to Establish a Registry for
What did the research do and find?
Alzheimer’s Disease; CI, confidence interval; CN,
control; CSF, cerebrospinal fluid; CVLT, California
• We used quantitative and targeted metabolomics assays on brain tissue samples (N =
Verbal Learning Test; C3, propionylcarnitine; DAG,
diacylglycerol; DHA, docosahexaenoic acid; DSM,
44) and machine-learning methods to identify a brain metabolite signature of AD, i.e., a
Diagnostic and Statistical Manual; EASE-AD, 26-metabolite panel that discriminated AD and control samples with accuracy, sensitiv-
Endophenotype Association Score in Early ity, and specificity of 83.33%, 86.67%, and 80%, respectively.
Alzheimer’s Disease; ECF, extracellular fluid; ER,
endoplasmic reticulum; FABP, fatty acid binding • We then assayed the same 26 metabolites in blood from two longitudinal cohorts that
protein; FAT, fatty acid transporter; FATP, fatty acid represent prodromal (Alzheimer’s Disease Neuroimaging Initiative [ADNI], N = 767)
transport protein; FIA-MS/MS, flow injection and preclinical (Baltimore Longitudinal Study of Aging [BLSA], N = 207) AD and tested
analysis-tandem mass spectrometry; HPLC-MS/ their associations with MRI measures, CSF biomarkers, risk of conversion to incident
MS, liquid chromatography tandem-mass
AD, and cognitive performance.
spectrometry; HR, hazard ratio; h1, hexose; IDQ,
identification and quantification; ITG, inferior • We found that higher blood concentrations of sphingolipid species were consistently
temporal gyrus; LCFA, long-chain fatty acid; LOD, associated with severity of AD pathology at autopsy and AD progression across prodro-
limit of detection; LOO, leave-one-out; LysoPC,
mal and preclinical stages.
lysophosphatidylcholine; MCI, mild cognitive
impairment; Met-So, methionine sulfoxide; MFG, • These metabolites map to several biologically relevant pathways in AD, including tau
middle frontal gyrus; MRI, magnetic resonance
phosphorylation, Aβ metabolism, calcium homeostasis, acetylcholine biosynthesis, and
imaging; n, number of samples; NIEHS, National
Institute of Environmental Health Science;
apoptosis.
NINCDS-ADRDA, National Institute of Neurological
and Communication Disorders and Stroke–
Alzheimer’s Disease and Related Disorders
What do these findings mean?
Association; p, number of features; p-tau,
• Our study design represents a novel approach for identifying markers of disease pro-
phosphorylated tau; PC, phosphatidylcholine; PET,
positron emission tomography; PHF, paired helical gression in AD and potential avenues for therapeutic intervention.
filaments; PLA2G2A, phospholipase A2 group IIA;
• Perturbations in sphingolipid metabolism are consistently associated with preclinical
PLA2G4A, phospholipase A2 Group IVA; PP2A,
protein phosphatase; PUFA, polyunsaturated fatty
and prodromal AD, as well as with AD pathology at autopsy, providing compelling evi-
acid; QC, quality control; rCBF, resting state dence for their significant role in AD pathogenesis.
cerebral blood flow; RF, random forest; SD,
standard deviation; SGMS, sphingomyelin
synthase; SLC1A5, solute carrier family 1 member
5; SLC5A7, solute carrier family 5 member 7; SM,
sphingomyelin; SML, sphingomyelinase; SPARE-
AD, Spatial Patterns of Abnormality for Recognition Introduction
of Early Alzheimer’s disease; SVM, support vector
The relationships between systemic abnormalities in metabolism and the pathogenesis of
Alzheimer disease (AD) are poorly understood. It is unclear how global perturbations in

machine; S1P, sphingosine 1-phosphate; t-tau, metabolism are related to severity of AD pathology and the eventual expression of AD symp-
total tau. toms in at-risk individuals. Understanding the metabolic basis of AD and its impact on disease
progression during the early, preclinical, and prodromal stages is likely to provide insights into
novel disease-modifying treatments for this irreversible, progressive neurodegenerative
disorder.
Metabolomics, which measures the biochemical products of cell processes downstream of
genomic, transcriptomic, and proteomic systems, has generated excitement because of its
potential to capture snapshots of the complex and multifactorial biochemical pathways that
may be altered in AD [1,2]. These include changes across multiple physiological pathways
driven by the complex interactions between behavioral, genetic, and environmental risk fac-
tors. Recent studies have applied metabolomics to examine alterations in blood metabolite
profiles in AD; such studies have the potential to both discover peripheral biomarkers as well
as identify key metabolic pathways intrinsic to AD pathogenesis [3–7]. However, one of the
key challenges in these metabolomics studies is the inability to link alterations in metabolite
signals in the blood to those in the brain. It is therefore difficult to assess whether a peripheral
signal associated with disease status is also reflected in the brain, where accumulation of dis-
tinct pathological features in specific regions is believed to trigger symptom onset. As is com-
mon with late-onset and gradually progressive diseases, there are many alterations in cell
processes due to chronic comorbid medical conditions that may be reflected in peripheral
blood metabolite concentrations. Additionally, traditional blood biomarker studies have relied
mainly on the binary discrimination of established AD/mild cognitive impairment (MCI)
from control (CN) samples. This study design ignores the long preclinical prodrome of AD,
when brain pathology is accumulating but has not yet triggered the onset of cognitive
impairment and functional decline in individuals eventually diagnosed with AD. As we have
proposed previously [8], alternative study designs in biomarker analyses, in which the primary
end points are well-established endophenotypes of AD pathology rather than binary discrimi-
nation of case versus control, offer the potential to identify biologically relevant blood bio-
markers for AD.
Here, we describe a four-step approach to the discovery of brain and blood metabolites
associated with pathology and progression of AD (Fig 1). (1) Identifying a brain metabolite sig-
nature of AD: in this phase of the study, we first used quantitative and targeted metabolomics
to identify a panel of metabolites that accurately differentiated brain tissue samples from neu-
ropathologically confirmed AD and CN subjects. (2) Testing blood metabolite associations
with AD endophenotypes: we then tested whether serum concentrations of the same metabo-
lites in two independent samples representing preclinical AD and prodromal AD were associ-
ated with distinct clinical, cognitive, neuroimaging, and cerebrospinal fluid (CSF)
endophenotypes of AD. (3) Summarizing results: we developed an integrated blood and brain
endophenotype score (Endophenotype Association Score in Early Alzheimer’s Disease
[EASE-AD]) summarizing the relative importance of specific brain and blood metabolites to
severity of AD pathology and disease progression. (4) Mapping biological pathways: we finally
mapped the main metabolite classes emerging from these analyses to key biological pathways
implicated in AD pathogenesis to understand the potential roles of these molecules and their
interactions in triggering symptom onset and progression of AD.
Methods
Participants
The Baltimore Longitudinal Study of Aging (BLSA) is a prospective cohort study of commu-
nity-dwelling participants that began in 1958 [9,10]. Detailed clinical and cognitive


Fig 1. Schematic representation of study design. In Step 1, we used a quantitative and targeted metabolomics approach followed by
two machine-learning methods to identify a panel of metabolites—a “brain metabolite signature of AD”—that accurately differentiated
brain tissue samples from neuropathologically confirmed AD and CN subjects. In Step 2, using that same metabolite panel, we explored
whether blood concentrations of metabolites in two independent samples representing prodromal AD (ADNI) and preclinical AD
(BLSA) were associated with distinct clinical, cognitive, neuroimaging, and CSF endophenotypes of AD. In Step 3, we summarized
results by developing an integrated blood and brain endophenotype score capturing the relative importance of specific brain and blood
metabolites to severity of AD pathology and disease progression. In Step 4, we mapped the main metabolite classes (emerging from Step
3) to key biological pathways implicated in AD pathogenesis. Aβ1–42, amyloid beta 1–42; AD, Alzheimer disease; ADNI, Alzheimer’s
Disease Neuroimaging Initiative; ASYMAD, asymptomatic Alzheimer’s disease; BLSA, Baltimore Longitudinal Study of Aging;
CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; CN, control; CSF, cerebrospinal fluid; EASE-AD, Endophenotype
Association Score in Early Alzheimer’s disease; IDQ, Identification and Quantification; MCI, mild cognitive impairment; MRI,
magnetic resonance imaging; p-tau, phosphorylated tau; SPARE-AD, Spatial Patterns of Abnormality for Recognition of Early
Alzheimer’s disease; t-tau, total tau.
https://doi.org/10.1371/journal.pmed.1002482.g001
evaluations, including neurological, laboratory, and radiological evaluations, were conducted

every 2 years. Since 2003, participants older than 80 years received yearly assessments. The
autopsy subsample used in Step 1 (i.e., Identifying a brain metabolite signature) to generate
the brain metabolite signature of AD included 44 participants (N = 15 AD; N = 14 CN; N = 15
asymptomatic Alzheimer’s disease [ASYMAD], described below). For Step 2 (i.e., Testing
blood metabolite associations with AD endophenotypes), metabolomic analyses in serum sam-
ples were performed on 207 BLSA (exclusion criteria described below) participants divided
into “converters” and “non-converters.” Converters were defined as participants who were
cognitively normal at the initial blood draw and developed incident AD based on consensus
clinical diagnosis (described below) during follow-up approximately 5 years later. These par-
ticipants were age and sex matched to non-converters and defined as participants who
remained cognitively normal over a similar follow-up interval. Initial serum samples were col-
lected while both groups were cognitively normal; we therefore characterize the converters in
this sample as representing “preclinical AD.” Demographic characteristics of the autopsy sam-
ple and blood study sample in the BLSA are included in Table 1. Written informed consent
was obtained at each visit; the BLSA study protocol has ongoing approval with the institutional
review board of the National Institute of Environmental Health Science (NIEHS), National
Institutes of Health.
As described below, in addition to identifying a brain metabolite signature of AD in Step 1,
the BLSA sample was used in Step 2 to test associations between blood metabolite concentra-
tions and the following AD endophenotypes: (1) differences by diagnoses (i.e., converters ver-
sus non-converters), (2) risk of conversion to incident AD, and (3) trajectories of cognitive
performance prior to onset of AD symptoms.
The Alzheimer’s Disease Neuroimaging Initiative (ADNI) sample was also used in Step 2
(i.e., Testing blood metabolite associations with AD endophenotypes). ADNI is an ongoing,
longitudinal study launched in 2003 as a public–private partnership, led by principal investiga-
tor Michael W. Weiner, MD. The primary goal of ADNI has been to test whether serial mag-
netic resonance imaging (MRI), positron emission tomography (PET), other biological
markers, and clinical and neuropsychological assessments can be combined to measure the
progression of mild cognitive impairment (MCI) and early AD. Details on study design, par-
ticipant recruitment, study approval, and informed consent procedures have been published
previously [11]. The study was approved by the institutional review boards of all of the partici-
pating institutions/study sites. The full list of participating institutions is included in S1 Table.
Informed written consent was obtained from all participants at each site. Metabolomics data
for ADNI samples were generated by the Alzheimer Disease Metabolomics Consortium and
(ADMC) deposited to LONI. The mission of the ADMC is to create a comprehensive metabo-
lomics database for AD. ADNI data used in the preparation of this article were also obtained

Table 1. Demographic characteristics of study samples.

BLSA: brain (autopsy) study sample
Total Sample, N = 44 CN, N = 14 ASYMAD, N = 15 AD, N = 15
Age, mean (SD) 81.33 (10.19) 80.42 (10.98) 85.19 (8.72) 78.25 (10.26)
Sex, n (% female) 16 (36.36) 3 (21.43) 5 (33.33) 8 (53.33)
Race, n (% white) 43 (97.73) 13 (92.86) 15 (100) 15 (100)
APOE e4 carrier, n (%) 7 (17.95) 1 (7.69) 3 (21.43) 3 (25.00)
Postmortem interval (hours), mean (SD) 14.93 (6.86) 15.82 (7.03) 14.79 (8.08) 14.4 (5.87)
BLSA: blood study sample
Total Sample, N = 207 Non-converters, N = 115 Converters, N = 92
Age (mean, SD) 78.68 (7.23) 77.58 (7.08) 80.05 (7.23)
Sex, n (% female) 107 (51.69) 55 (47.83) 52 (56.52)
Race, n (% white) 172 (83.09) 89 (77.39) 83 (90.22)
APOE e4-carrier, n (%) 55 (28.65) 24 (22.64) 31 (36.05)
Storage time in years (mean, SD) 15.30 (6.58) 13.28 (5.98) 17.84 (6.45)
ADNI: blood study sample
Total Sample, N = 767 Normal, N = 216 MCI, N = 366 AD, N = 185
Age, mean (SD) 75.19 (6.82) 75.98 (5.05) 74.69 (7.35)# 75.26 (7.46)
Sex, n (% female) 327 (42.63) 105 (48.61) 132 (36.07)# 90 (48.65)
Race, n (% white) 713 (92.96) 199 (92.13) 341 (93.17) 173 (93.51)
APOE e4-carrier, n (%) 381 (49.67) 58 (26.85) 200 (54.64)# 123 (66.49)#
Storage time in years (mean, SD) 8.89 (0.37) 8.69 (0.42) 8.72 (0.40)

p < 0.05 comparing non-converters and converters at baseline (both samples had normal cognition at baseline)
# p < 0.05 comparing MCI or AD to control group.
Abbreviations: AD, Alzheimer disease; ADNI, Alzheimer’s Disease Neuroimaging Initiative; APOE e4, apolipoprotein E epsilon 4 allele; ASYMAD, asymptomatic
Alzheimer’s disease; BLSA, Baltimore Longitudinal Study of Aging; CN, control; MCI, mild cognitive impairment; SD, standard deviation.
https://doi.org/10.1371/journal.pmed.1002482.t001
from the ADNI-1 database (adni.loni.usc.edu) and included baseline blood serum metabolite
concentrations (with concurrent structural MRI data) on 767 participants and concurrent CSF
AD biomarker data on 403 participants. ADNI was enriched with participants with MCI and
therefore represents “prodromal AD” (participants with MCI at baseline who subsequently
converted back to normal cognition were excluded). Demographic characteristics of the ADNI
sample are included in Table 1.
As described below, the ADNI sample was used in Step 2 to test associations between blood
metabolite concentrations and the following AD endophenotypes: (1) MRI measures of AD-related
brain atrophy, (2) CSF measures of AD pathology, and (3) risk of conversion to incident AD.
Neuropathological studies: Brain tissue samples in BLSA (Step 1)

The autopsy program of the BLSA was initiated in 1986 and has been described previously
[12]. The autopsy subsample is not significantly different from the BLSA cohort as a whole in
terms of the rates of dementia and clinical stroke [13]. Postmortem brain examinations were
performed by an experienced neuropathologist. Assessment of neuritic plaques and neurofi-
brillary tangles using Consortium to Establish a Registry for Alzheimer’s Disease (CERAD)
[14] and Braak criteria [15], respectively, have been described previously [16]. We have previ-
ously described the clinico-pathological features of BLSA participants categorized as ASYMAD
after neuropathological assessment at death [17]. Briefly, these individuals had significant AD
neuropathology at autopsy but were found to be cognitively intact, as assessed by longitudinal
neuropsychological assessments, within 1 year prior to death.

Determining cognitive status in BLSA and ADNI (Step 2)

In BLSA, cognitive status was considered at consensus diagnosis conferences after each
assessment/visit, using established procedures described previously [18]. The consensus
conferences included neurologists, neuropsychologists, and neuroimaging scientists. At
each assessment, participants underwent a battery of neuropsychological testing. Clinical
and neuropsychological data were reviewed at multidisciplinary consensus case conferences
if they made four or more errors on the Blessed Information, Memory, and Concentration
(BIMC) test, if their Clinical Dementia Rating (CDR) score was equal to or greater than 0.5,
or if concerns were raised about their cognitive status by a reliable informant. In addition,
all participants were evaluated by case conference on death or withdrawal. It is also impor-
tant to note that longitudinal data reviewed during consensus case conferences include
(besides detailed cognitive assessments) medication history, self-reported diagnoses of
comorbid medical conditions, neuroimaging data, as well as laboratory evaluation for
reversible causes of cognitive impairment such as serum TSH and B12 levels. The diagnoses
of dementia and AD were based on the Diagnostic and Statistical Manual (DSM)-III-R [19]
and the National Institute of Neurological and Communication Disorders and Stroke–Alz-
heimer’s Disease and Related Disorders Association (NINCDS-ADRDA) criteria, respec-
tively [20].
For individuals diagnosed with AD, age at onset of initial symptoms of AD was estimated at
consensus case conferences using longitudinal cognitive performance data as well as infor-
mant-based history.
In ADNI, dementia diagnosis was determined based on NINCDS-ADRDA criteria for
probable AD. MCI participants met criteria for amnestic MCI [21], and CN participants were
cognitively normal. Additional details on the ADNI protocol are available at http://www.adni-
info.org.
Blood samples in BLSA and ADNI (Step 2)

Blood serum samples were collected from BLSA participants at the NIA Clinical Research
Unit in Harbor Hospital, Baltimore. Details on collection and processing have been pub-
lished previously [7]. Briefly, venous blood samples were collected between 6 and 7 AM fol-
lowing an overnight fast. Serum samples were aliquoted into 0.5-mL volume in Nunc
cryogenic tubes and stored at −80˚C until further use. Samples were not subject to any
freeze–thaw cycles prior to metabolomic assays. Additional details on sample selection have
been published previously [7]. The average storage time of serum samples in BLSA partici-
pants was 17.84 years (SD: 6.45) in converters and 13.28 years (SD: 5.98) in non-converters
(Table 1). In order to minimize potential effects of long storage times on serum sample sta-
bility and metabolite concentrations, we excluded all samples (N = 9 non-converters; N = 34
converters) with methionine sulfoxide (Met-So) concentrations greater than 5 μM (3 SD
above average) [22,23]. The original sample included 250 participants; after excluding sam-
ples with high Met-So concentration, the final sample included 207 participants (N = 115
non-converters; N = 92 converters).
Details on collection and processing of ADNI blood serum samples have been published
previously (http://adni.loni.usc.edu/wp-content/uploads/2010/11/BC_Plasma_Proteomics_
Data_Primer.pdf). Briefly, blood was collected at 8 AM prior to CSF collection after an over-
night fast, immediately placed on dry ice, and shipped on the same day to the ADNI Bio-
marker Core at the University of Pennsylvania for processing. The final sample included 767
participants (N = 216 normal; N = 366 MCI; N = 185 AD). All samples had Met-So concentra-
tions below 5 μM and no samples were excluded.

Brain and blood metabolomics in BLSA and ADNI (Steps 1 and 2)

Quantitative metabolomics was performed on BLSA brain and BLSA and ADNI blood sam-
ples on the Biocrates AbsoluteIDQ p180 platform. This commercially available platform
allows for the quantification of amino acids, acylcarnitines, sphingomyelins (SMs), phos-
phatidylcholines (PCs), hexoses (h1s), and biogenic amines. Details on the assays have been
published previously [24]. Briefly, the validated assay uses two different mass spectrometric
methods with isotope labeled and other internal standards for absolute quantification of
metabolites. The acylcarnitines, lipids, and h1s are analyzed by flow injection analysis-tan-
dem mass spectrometry (FIA-MS/MS). The amino acids and biogenic amines are deriva-
tized using phenylisothiocyanate and analyzed by liquid chromatography tandem-mass
spectrometry (HPLC-MS/MS) using an AB SCIEX 4000 QTrap mass spectrometer (AB
SCIEX, Darmstadt, Germany) with electrospray ionization. Concentration of each metabo-
lite was measured in μM.
For brain tissue metabolomics, regions were selected a priori in the middle frontal gyrus
(MFG), inferior temporal gyrus (ITG), and cerebellum (CBL). The MFG and ITG were sam-
pled to represent brain regions vulnerable to amyloid β and tau deposition, respectively; the
CBL was sampled to represent a brain region resistant to classical AD pathology. A sterile
4-mm-diameter tissue punch was extracted from the cortical surface of the brain tissue
regions, which were stored at −80˚C. To extract metabolites, samples were homogenized
using Precellys with ethanol phosphate buffer; samples were then centrifuged, and the
supernatant was used for analysis. Metabolite concentrations in brain tissue samples indi-
cated as less than the limit of detection (LOD) were imputed as the highest value below the
LOD. This method removed all differences below the LOD but still allowed machine-learn-
ing classifiers to pick up any differences in metabolite concentrations between those above
the LOD and those below.
For blood metabolomics, in BLSA, converter and non-converter samples were randomly
divided into 6 batches. Each batch was processed in separate runs with technicians blinded to
diagnostic status. Additional data processing and checking steps, including reproducibility
and testing for equality of coefficient of variance across metabolites, has been described in
detail previously [25]. BLSA serum samples indicated as less than LOD were not imputed due
to minimal missingness; 25/26 metabolites had 0 < LOD values. ADNI data processing has
been described in detail previously and included imputing values indicated as less than the
LOD as the metabolite LOD/2, as determined by the ADMC [25]. Metabolite concentrations
from participants with duplicate measurements were averaged in all analyses.
Batch effects were controlled for using a set of CN samples. Standardized quality control
(QC) material, i.e., commercially available pooled human plasma spiked with a defined set of
metabolites, was used across all batches to control and adjust for batch effects by applying
MetIDQ software-implemented normalization procedures.
Cognitive assessments in BLSA (Step 2)

Cognitive performance was analyzed from assessments administered to BLSA participants
every two years. Memory was assessed using the California Verbal Learning Test (CVLT),
including learning (total recall over 5 learning trials), immediate free recall, and long delay free
recall. Attention was assessed using the Trails Making Test Part A and the WAIS-R Digits For-
ward test. Executive function was measured using the Trails Making Test Part B and the
WAIS-R Digit Backward test. Language was measured using letter fluency and semantic flu-
ency tests. Visuo-spatial ability was measured using the Clock Drawing Test and the Card
Rotation Test.

Structural MRI measures in ADNI (Step 2)

MRI protocol, including scanner specifications, image acquisition, and image processing, are
described in detail at www.adni-info.org. Briefly, protocol specifications included T1 weighted
MR images, including sagittal volumetric 3D MPRAGE with 1.25 × 1.25-mm in-plane spatial
resolution, 1.2-mm thick sagittal slices, 8˚ flip angle, and target TR of about 8.9 mm and TE of
about 3.9 ms [26]. We utilized the Spatial Patterns of Abnormality for Recognition of Early
Alzheimer’s disease (SPARE-AD) index [27] as a neuroimaging measure of “AD-like” brain
atrophy patterns [28]; this measure was calculated for baseline visits of ADNI-1 participants.
CSF measures of AD pathology in ADNI (Step 2)

Participants underwent lumbar puncture in the morning following overnight fasting and
blood draws. Samples were immediately placed on dry ice and shipped to the ADNI Biomarker
Core for processing. Total tau (t-tau), phosphorylated tau (p-tau), and amyloid beta 1–42
(Aβ1–42) were measured using the multiplex xMAP Luminex platform (Luminex Corp, Austin,
TX) with Innogenetics (INNO-BIA AlzBio3, Ghent, Belgium) immunoassay kit-based
reagents. See http://adni.loni.usc.edu/wp-content/uploads/2012/01/2011Dec28-Biomarkers-
Consortium-Data-Primer-FINAL1.pdf for additional details on sample collection and process-
ing, including reproducibility and data quality checks. CSF samples were available in 395 par-
ticipants (N = 109 normal; N = 186 MCI; N = 100 AD).
Statistical analysis
The first two stages of the analytic plan used for BLSA, including Step 1, Identifying a brain
metabolite signature of AD, and Step 2, Testing blood metabolite associations with AD endo-
phenotypes, were developed conceptually in May 2016 prior to any data analyses. There were
no subsequent data-driven alterations to this conceptual analytic plan; final data visualization
for Step 3, Summarizing results, was based on various iterations during analyses. The inclusion
of ADNI data occurred in fall 2016 after our data request was approved by the ADMC. Step 4,
Mapping biological pathways, occurred after we identified the principal classes of metabolites
emerging from Steps 1–3. Sensitivity analyses testing blood metabolite associations with AD
endophenotypes in BLSA (indicated below in Step 2) were conducted at the request of
reviewers.
Step 1: Identifying a brain metabolite signature of AD. Absolute brain tissue concentra-
tions of 187 targeted metabolites were generated using the quantitative metabolomic methods
described previously on the MFG, ITG, and CBL in the autopsy subsample of the BLSA. We
used two machine-learning methods, support vector machine (SVM) and random forest (RF),
to generate average values of classification accuracy, sensitivity, and specificity for discriminat-
ing between postmortem AD and CN samples in each of the three brain regions examined.
Both machine-learning methods are based on different principles (described below) and were
used in combination to avoid bias towards a particular methodology when selecting relevant
metabolites. The prediction models use the selected metabolites in combination with each
other, effectively modeling interactions between them. The primary aim of the machine-learn-
ing analyses was to discriminate between AD and CN samples, and therefore the ASYMAD
group was not included in defining the brain metabolite signature of AD.
Briefly, SVM is a classification method that attempts to find a separating surface between
two classes with maximum margin [29]. If there is no separating surface in the original feature
space, SVM uses a kernel to implicitly map the features into a higher dimensional space, in
which a separating surface can be found. The performance of an SVM classifier on test data
has rigorous theoretical bounds [29], and it is possible to limit the “complexity” of the

prediction model to match the amount of data available. It has been shown that complex classi-
fier models that have many parameters that can be tuned to match the training data perform
poorly on test data due to overfitting. The restriction on the complexity of the SVM classifier
has been found to generalize well to test data, particularly when the number of features (p) is
much greater than the number of samples (n) [30].
Since its inception in 2001, RF has become popular in the machine-learning and bioinfor-
matics communities [31]. RF is one of the so-called ensemble methods for classification,
because a set of classifiers (instead of one) is generated and each one casts a vote for the pre-
dicted label of a given instance provided to the model. Each classifier is a tree built using the
classification and regression trees (CART) methodology [32]. RF often requires little tuning of
the parameters. RF is nonlinear, multivariate, and can deal with high-dimensional data, even
in small sample size situations. RF contains built-in metrics of variable importance, which
allow evaluating the relative relevance of each variable in a RF model. In the present report, we
used the permutation index of variable importance, which quantifies decreases in accuracy of
the estimated RF model due to random permutation of a given variable. Additional details on
using SVM and RF methodologies to discriminate between diseased and non-diseased individ-
uals in AD have been published previously [33]. To estimate metrics of performance (accuracy,
sensitivity, specificity) we used leave-one-out (LOO) cross validation.
The SVM and RF methods generated a ranked list of the top metabolites that contributed to
discriminating between AD and CN samples. The ranking for SVM was based on the number
of cross-validation iterations that each metabolite was selected in (i.e., higher numbers indicat-
ing higher rank), while that for RF was based on the mean decrease in accuracy when a partic-
ular metabolite was excluded from the prediction model. Because both methods rely on
different analytic principles and differences in feature selection, we expected that the top
metabolites from each method would not necessarily be identical; using both in combination
therefore avoided bias when defining the brain metabolite signature.
The ITG samples had the highest accuracy and sensitivity/specificity in discriminating AD
from CN samples. The top 20 ranked metabolites from each machine-learning classifier (SVM
and RF) in this region (ITG) were therefore used to define the brain metabolite signature of
AD.
Step 1: Identifying a brain metabolite signature of AD: Differences by group and associ-
ations with AD pathology. We next explored differences in concentration of each brain tis-
sue metabolite across 3 groups—AD, CN, and ASYMAD—in the ITG. Importantly, these
analyses included the ASYMAD group, which was not utilized in the development of the brain
metabolite signature of AD through the machine-learning analyses. Concentrations of brain
tissue metabolites were natural log transformed. Proportional odds ordinal logistic models, a
generalization of the Wilcoxon and Kruskal-Wallis test, were used to test for differences across
groups (i.e., AD, ASYMAD, CN) using brain tissue metabolite concentration as the outcome,
group as the nominal predictor, and age at death and sex as covariates. We then explored asso-
ciations between brain tissue metabolite concentrations and severity of AD pathology, specifi-
cally CERAD and Braak scores, again using all three groups, including ASYMAD. Spearman’s
rank correlation tests, adjusting for age at death and sex, were used to test these associations.
Step 2: Testing blood metabolite associations with AD endophenotypes: Risk of conver-
sion to incident AD in cognitively normal older adults (BLSA). In the BLSA sample, we
explored whether the natural log-transformed blood concentration of each metabolite identi-
fied in the brain metabolite signature of AD was associated with risk of conversion to incident
AD. We used Cox regression models, a class of survival models, to explore whether the initial
concentration of each metabolite (i.e., while all participants were cognitively normal) was asso-
ciated with the time to onset of conversion to AD. We included the covariates, age at initial

blood draw and sex, in the model; individuals who remained normal (non-converters) at fol-
low-up were censored at their last visit. Hazard ratios (HRs) indicate the relative increase in
the hazard rate associated with 1 log-unit increase in concentration of the log-transformed
metabolite. An HR greater than 1.0 indicates that higher log-transformed concentration of the
metabolite is associated with increased risk, while an HR less than 1.0 indicates that lower con-
centration of the log-transformed metabolite is associated with increased risk.
Step 2: Testing blood metabolite associations with AD endophenotypes: Associations
with cognitive performance (BLSA). Using the metabolites identified in the brain metabo-
lite signature of AD, we next explored whether the natural log-transformed blood concentra-
tion of each metabolite was associated with longitudinal trajectories of cognitive performance
in cognitively normal individuals who developed incident AD. We first generated domain-spe-
cific composite scores within the following domains: memory, attention, executive function,
language, and visuospatial ability using methods described previously [24]. These methods are
also described in detail in S1 Appendix. Briefly, composite scores were calculated by summing
and averaging the standardized scores from multiple tests within each cognitive domain. Lin-
ear mixed effects regression models were used to test whether the concentration of each
metabolite was associated with longitudinal changes in domain-specific cognitive performance
in cognitively normal individuals converting to incident AD. All models included the follow-
ing predictors: natural log-transformed metabolite concentration, age at initial blood draw,
sex, time (in days between follow-up visit and baseline; baseline indicated as time = 0), and the
two-way interaction of each predictor with time. The main predictor of interest was the inter-
action of metabolite concentration with time, which indicates an increase or decrease in the
annualized rate of change in domain-specific cognitive performance associated with an
increase in metabolite concentration. As our main goal in these analyses was to examine asso-
ciations between blood metabolite concentrations and progression of AD during the early pre-
clinical stage of disease, we excluded all cognitive performance data after the onset of AD
symptoms.
Step 2: Testing blood metabolite associations with AD endophenotypes in BLSA (sensi-
tivity analyses). Due to differences in serum sample storage time among converters and
non-converters in the BLSA cohort and a greater number of converter samples excluded by
the Met-So cutoff, we performed additional sensitivity analyses within a subsample of convert-
ers and non-converters. After excluding all samples with Met-So concentration >5 μM, we
matched converter to non-converter serum samples on the duration of sample storage at
−80˚C within a range of ±2 years. This produced a matched sample of 74 converters (storage
time: 16.15 [SD: 5.83]) and 74 non-converters (storage time: 15.89 [SD: 5.90]). In these sensi-
tivity analyses, we tested whether significant associations observed in the original dataset
between serum metabolites and (i) risk of conversion to incident AD and (ii) cognitive perfor-
mance remained significant after matching on storage time.
with AD-like brain atrophy patterns and CSF biomarkers of AD pathology (ADNI).
Using the metabolites identified in the brain metabolite signature of AD, we next explored
cross-sectional associations between natural log-transformed blood metabolite concentrations
and the SPARE-AD index, a measure of AD-related brain atrophy derived from MRI scans
[27]. We used multivariate linear regression, including the following predictors: natural log-
transformed baseline metabolite concentration, baseline age, and sex; the outcome was the
SPARE-AD index (higher scores represent more “AD-like” brain atrophy patterns).
In the ADNI sample, we additionally examined cross-sectional associations between natural
log-transformed baseline blood metabolite concentrations and natural log-transformed CSF
t-tau, p-tau, and Aβ1–42 concentrations. Similar to the model for the SPARE-AD analysis,

multivariate linear regression models included the following predictors: natural log-trans-
formed baseline metabolite concentration, baseline age, and sex; the outcomes were natural
log-transformed CSF t-tau, p-tau, and Aβ1–42 concentrations.
with risk of conversion to incident AD (ADNI). In the ADNI sample, we explored whether
the natural log-transformed blood concentration of each metabolite identified in the brain
metabolite signature of AD was associated with risk of conversion from MCI to incident AD.
Similar to survival models used in BLSA, Cox regression models were used to explore whether
initial metabolite concentrations in MCI participants were associated with the time to onset of
conversion to AD. We included covariates, age at baseline blood draw, and sex in the model;
individuals who remained MCI at follow-up were censored at their last visit. Similar to survival
models used in BLSA, the HR indicates the relative increase in the hazard rate associated with
1 log-unit increase in concentration of the log-transformed metabolite. An HR greater than
1.0 indicates that a higher log-transformed concentration of the metabolite is associated with
increased risk, while an HR less than 1.0 indicates that a lower concentration of the log-trans-
formed metabolite is associated with increased risk.
Step 3: Summarizing results: Calculating the EASE-AD score. In order to visually sum-
marize results from all analyses of metabolites comprising the brain metabolite signature of
AD and to explore whether metabolites clustered by class in their associations with distinct
AD-related endophenotypes, we generated a heat map indicating statistically significant asso-
ciations between AD metabolites (y-axis) and the specific brain and blood endophenotypes (x-
axis) described above. Significant associations (p < 0.05) are highlighted in red or green indi-
cating that increased or decreased metabolite concentration, respectively, is associated with
the various AD-related endophenotypes. Nonsignificant associations are indicated in gray.
The 26-metabolite panel (brain metabolite signature of AD) was determined specifically based
on metabolite rankings from the machine-learning classifiers following rigorous cross valida-
tion and thus represent a priori hypotheses in subsequent analyses. Additionally, these second-
ary, exploratory analyses were all focused on testing the associations of these metabolites with
distinct measures of AD progression in order to identify consistent trends across two indepen-
dent cohorts [34]. For these reasons, we elected not to use a p-value correction in the second-
ary analyses.
In order to enhance ease of interpretation of the summary heat map, we collapsed all longi-
tudinal domain-specific cognitive performance tests into one category indicating significant
longitudinal associations in any domain. We included the following brain endophenotype cat-
egories: (1) Differences in brain metabolite concentrations in the ITG by diagnosis (i.e., AD,
CN, and ASYMAD) and correlations of metabolite concentrations in the ITG with (2)
CERAD and (3) Braak scores. We included the following blood metabolite versus preclinical
AD endophenotype categories (BLSA): associations of blood metabolite concentrations with
(4) risk of progression from normal to incident AD and (5) longitudinal trajectories of cogni-
tive performance. We included the following blood metabolite versus prodromal AD endophe-
notype categories (ADNI): associations of blood metabolite concentrations with (6) AD-like
brain atrophy patterns on MRI (i.e., SPARE-AD score), (7) CSF Aβ1–42, (8) CSF t-tau, (9) CSF
p-tau, and (10) risk of progression from MCI to incident AD. We then calculated a summary
EASE-AD score indicating the number of significant associations for each AD metabolite with
brain and blood endophenotypes (max score: 10). This score is included as the last column in
the heat map; visualized metabolites are sorted based on this score in order to explore cluster-
ing of metabolite species within main classes.

We present detailed results for representative metabolites that showed significant associa-
tions with multiple AD phenotypes. Detailed results for all 26 AD metabolites are included in
Supporting information tables (S3 Table–S11 Table).
Step 4: Mapping biological pathways. In order to interpret our results within the biologi-
cal context of the metabolic pathways implicated, we mapped the principal metabolite classes
emerging from our analyses to their known primary biosynthetic and catabolic pathways as
well as their known interactions through various enzymatically regulated intermediary reac-
tions (“Metabolic pathway”). We also mapped the main metabolite classes implicated to key
signaling mechanisms related to AD pathogenesis (“Signaling pathway”).
Results
Participants: Demographic characteristics
The demographic characteristics of BLSA participants in the autopsy cohort whose brain tissue
samples were used in the metabolomics assays are included in Table 1. The mean age at death
in the autopsy sample was 81.33 years (SD: 10.19), and the mean interval between last evalua-
tion and death (postmortem interval) was 14.93 h (SD: 6.86). Participants in the three groups
—CN, ASYMAD, and AD—did not significantly vary by age at death, sex, or postmortem
interval. The demographic characteristics of BLSA participants who provided blood data are
included in Table 1. Participants were aged 78.47 years (SD: 6.96) at initial blood draw and
51.69% were female. Converter and non-converter groups did not vary by age or sex. Serum
samples in the converter group were, on average, stored for four years longer than non-con-
verter samples (17.84 years [SD: 6.45] versus 13.28 years [SD: 5.98]; p < 0.05).
The demographic characteristics of ADNI participants are included in Table 1. Participants
were aged 75.19 years (SD: 6.82) at baseline, and 42.63% were female. MCI participants were
significantly younger (74.69 years [SD: 7.35]) and had fewer females (36.07%). Samples did not
vary by storage time.
Step 1: Identifying a brain metabolite signature of AD. Accuracy, sensitivity, and speci-
ficity of the machine-learning classifiers in discriminating between AD and CN samples for
the MFG, ITG, and CBL brain regions are included in Table 2. The SVM algorithm identified
a panel of metabolites that discriminated samples in the ITG with an accuracy of 83.33% and a
Table 2. Machine-learning methods to discriminate between AD and CN samples.

RF SVM
ITG
Accuracy 70.00% Accuracy 83.33%
Sensitivity 66.70% Sensitivity 86.67%
Specificity 73.30% Specificity 80.00%
MFG
CBL
Abbreviations: AD, Alzheimer disease; CBL, cerebellum; CN, control; ITG, inferior temporal gyrus; MFG, middle
frontal gyrus; RF, random forest; SVM, support vector machine.

sensitivity/specificity of 86.67%/80.00%. The RF algorithm derived metabolites that discrimi-

nated samples in the ITG with an accuracy of 70.00% and a sensitivity/specificity of 66.70%/
73.30%. The performance metrics of metabolite concentrations in discriminating samples in
the MFG and CBL were comparatively lower.
Based on the performance of our machine-learning algorithms in the ITG, we chose the top
20 ranked metabolites from the SVM and RF algorithms from this region to define the brain
metabolite signature of AD. Out of 187 metabolites assayed, these top metabolites thus con-
tributed the most to discriminating between pathology-confirmed AD cases and CN in the
ITG. We found that 13 “consensus” metabolites were shared in the top 20 of both SVM and
RF algorithms, and 7 metabolites in each were unique to each algorithm (i.e., a total of 27
metabolites—13 consensus metabolites and 14 unique metabolites). We excluded one metabo-
lite, h1, that was not available in the ADNI dataset from subsequent analyses. Table 3 presents
Table 3. Top metabolites based on SVM and RF algorithms in the ITG.

Abbreviation Full Metabolite Name
Amino Acid
Arg Arginine
Acylcarnitine
C3 Propionylcarnitine
Glycerophospholipids
lysoPC a C17:0 Lysophosphatidylcholine with acyl residue C17:0
lysoPC a C18:0 Lysophosphatidylcholine with acyl residue C18:0
PC aa C38:4 Phosphatidylcholine with diacyl residue sum C38:4
PC ae C34:0 Phosphatidylcholine with acyl-alkyl residue sum C34:0
Biogenic Amines
Serotonin Serotonin
Spermidine Spermidine
Sphingolipids
SM C16:0 Sphingomyelin with acyl residue sum C16:0
SM (OH) C14:1 Hydroxysphingomyelin with acyl residue sum C14:1


Indicates consensus metabolites common to both SVM and RF algorithms.
Abbreviations: ITG, inferior temporal gyrus; RF, random forest; SVM, support vector machine.

the top 27 metabolites, including the 13 consensus metabolites; full ranked lists from both
SVM and RF algorithms from the ITG are included in S2 Table.
Step 1: Identifying a brain metabolite signature of AD: Differences by group and associ-
ations with AD pathology. A total of 16 metabolites showed brain tissue concentrations in
the ITG that differed significantly across clinical groups, i.e., CN, ASYMAD, and AD. The
majority of these were sphingolipids (8 out of 16) and glycerophospholipids (5 out of 16). The
AD group generally showed the highest or lowest metabolite concentrations, while the CN
group showed the opposite. The ASYMAD group generally showed intermediate metabolite
concentrations between the AD and CN samples. In Fig 2A, we show group differences and
global p-values for significance across clinical groups for brain tissue concentrations of 3 repre-
sentative sphingolipids: SM C16:0 (p = 0.005), SM C16:1 (p = 0.017), and SM (OH) C14:1
(p = 0.009) and three representative glycerophospholipids: PC ae C36:0 (p = 0.005), PC ae
C40:1 (p = 0.006), and PC aa C40:4 (p = 0.004). A summary of results across all metabolites is
included in S3 Table.
Brain tissue concentrations of 17 metabolites were significantly associated with severity of
neuritic plaque burden, as reflected in the CERAD scores. The majority (a total of 14 out of 17)
of these were sphingolipids (7 out of 17) and glycerophospholipids (7 out of 17). Brain tissue
concentrations of five metabolites were significantly associated with neurofibrillary pathology, as
assessed by Braak scores: three glycerophospholipids, one sphingolipid, and the amino acid, argi-
nine. Increased concentration of sphingolipids was consistently associated with greater CERAD
and Braak scores. In Fig 2B and 2C, we show correlation coefficient (ρ) and p-value results from
adjusted Spearman rank correlation tests (CERAD and Braak, respectively) for three representa-
tive sphingolipids: SM C16:0 (CERAD: ρ = 0.042, 95% CI = 0.0130–0.070, p = 0.006; Braak: ρ =
0.037, 95% CI = −0.001–0.076, p = 0.057), SM C16:1 (CERAD: ρ = 0.038, 95% CI = 0.011–0.065,
p = 0.008; Braak: ρ = 0.036, 95% CI = 0.000–0.073, p = 0.050), SM (OH) C14:1 (CERAD: ρ =
0.035, 95% CI = 0.005–0.064, p = 0.022; Braak: ρ = 0.025, 95% CI = −0.013–0.065, p = 0.191); and
three representative glycerophospholipids: PC ae C36:0 (CERAD: ρ = −0.037, 95% CI = −0.066–
−0.008, p = 0.014; Braak: ρ = −0.053, 95% CI = −0.089–−0.016, p = 0.006), PC ae C40:1 (CERAD:
ρ = −0.036, 95% CI = −0.064–−0.008, p = 0.012; Braak: ρ = −0.051, 95% CI = −0.086–−0.016,
p = 0.006), and PC aa C40:4 (CERAD: ρ = −0.039, 95% CI = −0.067–−0.011, p = 0.007; Braak:
ρ = −0.049, 95% CI = −0.085–−0.014, p = 0.008). A summary of results across all metabolites is
Step 2: Testing blood metabolite associations with AD endophenotypes: Risk of conver-
sion to incident AD in cognitively normal older adults (BLSA). The mean interval between
initial blood sampling to the onset of AD (for converters) or follow-up (for non-converters) was
4.27 years (SD = 1.33 years). Higher blood concentrations of four sphingolipids were associated
with a significantly greater risk of future conversion to incident AD in cognitively normal older
individuals. These included SM C16:0 (HR = 4.430, 95% CI = 1.704–11.520, p = 0.002), SM C16:1
(HR = 3.455, 95% CI = 1.516–7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373–
9.122, p = 0.009) and SM C18:1 (HR = 2.255, 95% CI = 1.047–4.855, p = 0.038). Lower and higher
baseline blood concentrations of two glycerophospholipids, PC aa 38:4 (HR = 0.253, 95%
CI = 0.102–0.630, p = 0.003) and PC ae C34:2 (HR = 3.055, 95% CI = 1.211–7.705, p = 0.018),
respectively, were also associated with a significantly greater risk of conversion to incident AD. A
summary of results across all metabolites is included in S5 Table.
All metabolites significantly associated with greater risk of future conversion to incident
AD (i.e., 6 out of 6 metabolites) remained significant in sensitivity analyses conducted in the
subsample matched on storage time. A summary of the results from the sensitivity analyses is


Fig 2. Associations between brain tissue metabolite concentration and clinical groups, CERAD scores, and Braak
scores. Please note that vertical axes scales differ across graphs in panels A and B. (A) Group differences and global p-
values for significance across clinical groups for brain tissue concentration of three representative sphingolipids and
three representative glycerophospholipids in the ITG. (B) ρs and p-values showing associations between three
representative sphingolipids and three representative glycerophospholipids and severity of neuritic plaque burden
(CERAD scores). (C) ρs and p-values showing associations between three representative sphingolipids and three
representative glycerophospholipids and severity of neurofibrillary pathology (Braak scores). ρ, correlation coefficient;
AD, Alzheimer disease; ASYMAD, asymptomatic Alzheimer’s disease; CERAD, Consortium to Establish a Registry for
Alzheimer’s Disease; CN, control; ITG, inferior temporal gyrus; OH, hydroxy; PC, phosphatidylcholine; SM,
sphingomyelin.
Step 2: Exploring associations in blood with AD endophenotypes: Associations with

cognitive performance (BLSA). We found that initial blood concentrations of six metabo-
lites predicted longitudinal trajectories of domain-specific cognitive performance prior to the
onset of cognitive impairment in cognitively normal individuals converting to incident AD.
Higher baseline blood concentrations of the sphingolipids were broadly associated with greater
declines in cognition. Specifically, higher baseline blood concentration of SM C18:1 and SM
C26:1 were predictive of greater declines in attention (SM C18:1: β = −0.172, 95% CI =
−0.306–0.038, p = 0.012) and language (β = −0.533, 95% CI = −1.061–−0.005, p = 0.050),
respectively. Similarly, higher blood concentrations of the glycerophospholipids PC aa C40:6
and PC ae C40:1 were also predictive of greater declines in attention (PC aa C40:6: β = −0.122,
95% CI = −0.215–−0.030, p = 0.010) and language (PC ae C40:1: β = −0.251, 95% CI = −1.061–
−0.005, p = 0.041). Higher blood concentration of lysoPC a C18:0 were predictive of greater
declines in language (lysoPC a C18:0: β = −0.1530, 95% CI = −0.266–−0.0330, p = 0.012) and
lower blood concentration of the polyamine, spermidine, was associated with greater declines
in visuospatial ability (β = 1.220, 95% CI = 0.094–2.347, p = 0.034), and higher and lower
blood concentration of arginine were associated with greater declines in language (β = −0.142,
95% CI = −0.270–−0.013, p = 0.031) and visuospatial ability (β = 0.198, 95% CI = 0.007–0.390,
p = 0.043), respectively. Due to conflicting direction of associations between serum arginine
concentrations and cognitive performance, we arbitrarily chose to indicate the association
between arginine and language only, in the summary heat map. Significant associations are
summarized in S7 Table.
The majority of metabolites (i.e., 6 out of 8) remained significant in sensitivity analyses con-
ducted in the subsample matched on storage time. A summary of the results from the sensitiv-
ity analyses is included in S8 Table.
Step 2: Testing blood metabolite associations with AD endophenotypes:

Associations with AD-like brain atrophy patterns and CSF biomarkers of
AD pathology (ADNI)
Higher blood concentrations of sphingolipids were broadly associated with greater AD-like
brain atrophy patterns and more AD-like CSF levels of pathology. Specifically, two sphingoli-
pids, SM C16:0 (β = 0.593, 95% CI = 0.147–1.040, p = 0.009) and SM C18:1 (β = 0.466, 95%
CI = 0.0687–0.863, p = 0.022), were associated with more AD-like patterns of brain atrophy on
MRI scans measured by the SPARE-AD index. Lower blood concentration of the glyceropho-
spholipid, PC aa C40:6 (β = −0.323, 95% CI = −0.617–−0.029, p = 0.032), was associated with a
more “AD-like” pattern of brain atrophy. Fig 3A shows all significant cross-sectional associa-
tions between blood concentrations of metabolites described above and the SPARE-AD index.
A summary of results across all metabolites is included in S9 Table.
Higher blood concentrations of eight metabolites were associated with greater CSF levels of
t-tau, while higher concentrations of 10 metabolites were associated with greater CSF levels of

p-tau. All significant associations were among either sphingolipids (t-tau: 6 out of 8; p-tau: 8
out of 10) or glycerophospholipids (t-tau: 2 out of 8; p-tau: 2 out of 10). Higher blood concen-
trations of two of these sphingolipids (SM C16:0 and SM [OH] C14:1) were also associated
with lower CSF levels of Aβ1–42. Lower blood concentrations of C3 and serotonin were also
associated with lower CSF levels of Aβ1–42. Fig 3B shows associations between blood concen-
trations of SM C16:0 (t-tau: β = 0.347, 95% CI = 0.103–0.592, p = 0.006; p-tau: β = 0.331, 95%
CI = 0.086–0.575, p = 0.008; Aβ1–42: β = −0.169, 95% CI = −0.328–−0.011, p = 0.036) and
SM [OH] C14:1 (t-tau: β = 0.346, 95% CI = 0.109–0.583, p = 0.004; p-tau: β = 0.416, 95%
CI = 0.179–0.653, p = 0.001; Aβ1–42: β = −0.179, 95% CI = −0.333–−0.025, p = 0.023), with all
three CSF biomarkers. We additionally show associations between blood concentrations of PC
aa C38:4 (t-tau: β = 0.279, 95% CI = 0.035–0.522, p = 0.025; p-tau: β = 0.251, 95% CI = 0.008–
0.494, p = 0.043) and PC ae C34:0 (t-tau: β = 0.358, 95% CI = 0.053–0.663, p = 0.022; p-tau: β =
0.423, 95% CI = 0.118–0.728, p = 0.007) and CSF t-tau and p-tau. A summary of significant
results across all metabolites is included in S10 Table.
Step 2: Testing blood metabolite associations with AD endophenotypes:

Associations with risk of conversion to incident AD (ADNI)
The mean interval between baseline blood sampling to the onset of AD or follow-up (for indi-
viduals who remained MCI) was 2.97 years (SD = 2.33 years). Higher blood concentration of
one sphingolipid, SM C18:1 (HR = 2.351, 95% CI = 1.268–4.360, p = 0.007), was associated
with a significantly greater risk of conversion to incident AD among individuals with MCI.
This sphingolipid was also associated with greater risk of conversion to incident AD among
cognitively normal individuals (described above in BLSA results). Higher blood concentration
of one glycerophospholipid, PC aa 38:4 (HR = 2.375, 95% CI = 1.189–4.745, p = 0.014), was
also associated with a significantly greater risk of conversion to incident AD. A summary of
results across all metabolites is included in S11 Table.
Step 3: Summarizing results: Calculating the EASE-AD score

Fig 4 shows the heat map summarizing statistically significant associations between the metab-
olites and brain and blood-specific AD endophenotypes. Metabolites are ranked (in decreasing
order) based on their EASE-AD score. P-values from each cell in Fig 4 are included in S12
Table.
Step 4: Mapping biological pathways: Exploring metabolite interactions

and their impact on AD pathology
Fig 5 summarizes the main biosynthetic and catabolic reactions (“Metabolic pathway”) of the
major metabolite classes and their interactions as well as their roles in signaling cascades (“sig-
naling pathway”) relevant to AD pathogenesis and evolution of the principal pathological fea-
tures of the disease.
Gene symbol and name. SLC5A7, solute carrier family 5 member 7; SLC1A5, solute car-
rier family 1 member 5; SLC7A5, solute carrier family 7 member 5; FATP1, fatty acid trans-
port protein-1; FATP4, fatty acid transport protein-4; FABP5, fatty acid binding protein 5;
CD36, CD36 molecule; SPTLC, serine palmitoyltransferase long chain base; KDSR, 3-ketodi-
hydrosphingosine reductase; CERS, ceramide synthase; DEGS, delta 4-desaturase, sphingoli-
pid; SGMS, sphingomyelin synthase; SML, sphingomyelinase; ACSL, acyl-CoA synthetase
long-chain family member; ELOVL, elongation of very long chain fatty acids; FADS, fatty acid
desaturase; GNPAT, glyceronephosphate O-acyltransferase; AGPS, alkylglycerone phosphate


Fig 3. Associations between blood metabolite concentration and SPARE-AD index, CSF concentrations of Aβ1–42,
t-tau, and p-tau. Please note that vertical axes scales differ across graphs in panels A and B. (A) ρs and p-values showing
associations between representative metabolites and AD-like patterns of brain atrophy on MRI scans (SPARE-AD index).
(B) ρs and p-values showing associations between representative metabolites and CSF markers of AD: Aβ1–42, t-tau, and
p-tau. ρ, correlation coefficient; Aβ1–42, amyloid beta 1–42; AD, Alzheimer disease; CSF, cerebrospinal fluid; MRI,
magnetic resonance imaging; OH, hydroxyl; p-tau, phosphorylated tau; PC, phosphatidylcholine; SM, sphingomyelin;
SPARE-AD, Spatial Patterns of Abnormality for Recognition of Early Alzheimer’s disease; t-tau, total tau.
synthase; PLPP, phospholipid phosphatase; GPAT, Glycerol-3-phosphate acyltransferases;

AGPAT, 1-acylglycerol-3-phosphate O-acyltransferase; PPAP, phospholipid phosphatase;
PLD, phospholipase D; CHK, choline kinase; PCYT1, phosphate cytidylyltransferase 1, cho-
line; CEPT, choline/ethanolamine phosphotransferase; PLA2G2A, phospholipase A2 group II
A; PLA2G4A, phospholipase A2 group IV A; LPCAT, lysophosphatidylcholine acyltransfer-
ase; GPR132 (G2A), G protein-coupled receptor 132; GNAq, G protein subunit alpha q; PLC,
phospholipase C; CALM, calmodulin; CAMKII, calmodulin kinase II; GSK3B, glycogen
synthase kinase 3 beta; TAU, microtubule-associated protein tau; PPP2, serine/threonine pro-
tein phosphatase II; AKT, protein kinase B (PKB); BAD, BCL2 associated agonist of cell death;
BIMEL, BCL2 interacting mediator of cell death (BIM)-extra long; Aβ40–42, amyloid beta (40–
42); BACE1, beta-secretase 1; APP, amyloid protein precursor; sAPPβ, soluble amyloid pro-
tein precursor beta.
Discussion
To the best of our knowledge, this is the first study to apply quantitative and targeted metabo-
lomic analyses of both brain and blood tissue to identify metabolites associated with the sever-
ity of AD pathology as well as measures of AD progression. Our results indicate that distinct
metabolites belonging to the sphingolipid and glycerophospholipid classes are related to the
severity of AD pathology in the brain and that their concentrations in blood are associated
with preclinical disease progression. Furthermore, we were able to identify these specific
metabolites through a data-driven process that first used machine-learning methods to gener-
ate an AD-specific brain metabolite signature, and then clustered these metabolites based on
the EASE-AD summary score representing cumulative associations of each metabolite, with
outcome measures related to AD pathology and progression.
Sphingolipids and AD
This process identified sphingolipids as a class of metabolites that are consistently associated
with preclinical and prodromal AD, as well as with AD pathology at autopsy. Additionally, for
all sphingolipid species—across all endophenotypes in brain, prodromal, and preclinical blood
samples—increased concentration was associated with a more “AD-like” phenotype. Our
results add substantially to a growing body of literature suggesting that perturbations in sphin-
golipid metabolism are related to key aspects of AD pathogenesis [43,44]. SMs are a subclass of
sphingolipids that are enriched in the central nervous system as important constituents of
lipid rafts [38] and play a critical role in neuronal cell signaling [45,46]. In the brain, sphingoli-
pids mediate a diverse array of biological functions that are relevant to critical molecular
mechanisms in AD, including amyloidogeneic processing of the amyloid precursor protein
(APP) within SM-rich lipid rafts [47] and regulation of hippocampal neuronal excitability
[48]. While previous studies in postmortem human brain tissue have demonstrated altered lev-
els of total SM content in AD relative to CN [49,50], few have quantified absolute concentra-
tions of distinct SM species within brain regions differentially vulnerable to AD pathology.

Fig 4. Heat map summarizing associations between metabolites and AD endophenotypes. Meanings of column headings: AD-ASY-CN, association between brain
tissue metabolite concentration and clinical diagnosis of AD; CERAD, association between brain tissue metabolite concentration and plaques measured by CERAD
score; Braak, association between brain tissue metabolite concentration and neurofibrillary tangle burden measured by Braak score; SPARE-AD, association between
blood tissue metabolite concentration in ADNI and SPARE-AD score; A Beta, association between blood tissue metabolite concentration in ADNI and CSF Aβ1–42; t-
tau, association between blood tissue metabolite concentration in ADNI and CSF (t-tau); p-tau, association between blood tissue metabolite concentration in ADNI and
CSF (p-tau); Cog perfor, association between blood tissue metabolite concentration and cognitive performance prior to AD onset; EASE-AD, sum of significant
associations across AD-related endophenotypes. ADNI Cox: association between blood tissue metabolite concentration and risk of incident AD in ADNI among MCI
individuals. BLSA Cox: association between blood tissue metabolite concentration and risk of incident AD/MCI in BLSA among cognitively normal individuals.
Aβ1–42, amyloid beta 1–42; AD, Alzheimer disease; ADNI, Alzheimer’s Disease Neuroimaging Initiative; ASY, asymptomatic Alzheimer’s disease; BLSA, Baltimore
Longitudinal Study of Aging; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease; CN, control; CSF, cerebrospinal fluid; EASE-AD, Endophenotype
Association Score in Early Alzheimer’s disease; MCI, mild cognitive impairment; OH, hydroxyl; p-tau, phosphorylated tau; PC, phosphatidylcholine; SM,
sphingomyelin; SPARE-AD, Spatial Pattern of Abnormality for Recognition of Early Alzheimer’s disease; t-tau, total tau.

Fig 5. Metabolic pathways and signaling cascades involving glycerophospholipids and sphingolipids: relevance to AD pathogenesis. Schematic articulation
of the core metabolic and signaling pathways in neurons, highlighting links between glycerophospholipid and sphingolipid classes of lipid species identified in the
current study to be associated with the severity of AD pathology in the brain. Nutrient transporters (SLC5A7, SLC1A5, CD36, FATPs) present both at the BBB as
well as the neuronal cell membrane mediate the uptake of amino acids, long chain fatty acids, and vitamin precursors into neurons necessary for the de novo
synthesis of glycerophospholipid and SM lipid species [35,36]. The “metabolic pathway” section of the diagram represents the core metabolic pathways involved
in the synthesis and recycling of glycerophospholipid and sphingolipid species. The “signaling pathway” section connects these lipid species to the core
representative signaling cascades implicated in mediating multiple aspects of AD pathology in the brain, such as formation of neuritic plaques, neurofibrillary
tangles, and AD-like brain atrophy. In a condition-dependent manner, incoming free fatty acids are incorporated into glycerolipids or ceramides in the
endoplasmic reticulum. Similarly, LCFAs are processed in peroxisomal organelles to generate ether lipids. Coupling with the Kennedy pathway, glycerolipids and

ether lipids are converted to either aa or ae PC species [37]. PCs are metabolized by the phospholipase or SML enzymes to recycle back phosphatidic acid or DAG
or to generate SM, respectively. These lipid species are critical in the formation of lipid rafts, which represent essential structural and functional domains for
maintaining neuronal function [38]. In AD, remodeling of lipid rafts, especially with enhanced activity of SMLs, results in an increased ceramide to SM ratio,
which facilitates Aβ production by posttranslational stabilization of BACE1 enzyme. This leads to further generation of oligomeric Aβ due to a feed forward
regulatory loop between Aβ and the SML enzymes [39]. Similarly, PC with saturated and unsaturated long-chain fatty acyl groups positively influence activity of
the Ƴ-secretase enzyme by modulating cell membrane thickness and the lipid microenvironment of the enzyme [40]. Meanwhile, generation of
lysophosphatidylcholine from membrane PC by both cytosolic PLA2G4A in Land’s cycle [41] as well as the secretory soluble PLA2G2A can lead to dysregulation
of intracellular calcium signaling in a G-protein receptor (GPR132, G2A) coupled manner. Dysregulated Ca2+ signaling can result in enhanced activity of
CAMKII, which, in coordination with the ceramide–PP2A–GSK3β pathway, results in tau hyperphosphorylation, leading to the generation of PHF and enhanced
neurofibrillary tangle formation [42]. Furthermore, altered ceramide signaling by down-regulation of AKT kinase activity via PP2A can trigger neuronal
apoptosis by augmenting activity of the pro-apoptotic proteins, BAD and BIMEL. aa, diacyl; Aβ, amyloid-β; AD, Alzheimer disease; ae, acyl-alkyl; AKT, protein
kinase B; BACE1, β-secretase; BAD, BCL2 associated agonist of cell death; BBB, blood-brain barrier; BIMEL, BCL2 interacting mediator of cell death-extra long;
CAMKII, calmodulin kinase; CD36, CD36 molecule; DAG, diacylglycerol; ECF, extracellular fluid; ER, endoplasmic reticulum FATP, fatty acid transport protein;
LCFA, long-chain fatty acid; LysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; PHF, paired helical filaments; PLA2G2A, phospholipase A2 group IIA;
PLA2G4A, phospholipase A2 Group IVA; PP2A, protein phosphatase; SLC1A5, solute carrier family 1 member 5; SLC5A7, solute carrier family 5 member 7; SM,
sphingomyelin; SML, sphingomyelinase.
Our findings are broadly consistent with those of Chan and colleagues, who demonstrated
higher levels of the SM species SM d18:1/22:1 and d18:1/26:1 in the prefrontal and entorhinal
cortices of AD patients, relative to CN [51].
Most previous studies reporting on altered blood sphingolipid levels in AD have used an
untargeted lipidomics approach (e.g., [52,53]). Some recent studies have used the p180 tar-
geted metabolomics platform to assay absolute concentrations of metabolites associated with
AD. An important distinction in the design of these previous studies and our current report is
in our use of a brain-derived AD metabolite signature to guide focused analyses of these
metabolites in blood as well as a comprehensive exploration of their associations within both
preclinical and prodromal AD samples. Two studies [25,54] have recently reported on p180
metabolite data within blood samples in the ADNI and the Atherosclerosis Risk in Communi-
ties (ARIC) cohorts. While there is minimal overlap between these results and our current
report, it is striking to note that two sphingolipids we observe to be increased in the temporal
cortex of AD patients and identified in our brain metabolite signature of AD (SM C16:0 and
SM [OH] C14:1) were associated with brain atrophy, cognitive decline, and risk of conversion
from MCI to AD in ADNI [25]. Similarly, blood concentrations of SM C16:0 and SM C26:1
were also associated with a diagnosis of MCI and dementia, respectively, in the predominantly
African-American ARIC cohort [54].
Our findings that blood concentration of sphingolipids represented in the brain metabolite
signature of AD are also associated with progression during preclinical and prodromal AD
suggest that these are biologically relevant, early signals of disease progression. Equally impor-
tantly, correcting perturbations in sphingolipid metabolism may represent a plausible novel
strategy for therapeutic intervention in AD. In this context, the emerging roles of sphingosine
1-phosphate (S1P)-metabolizing enzymes and S1P analogs in ameliorating Aβ-induced neu-
roinflammation in AD [55,56] are especially promising.
Glycerophospholipids and AD
The second major class of metabolites we observed to be related to measures of AD pathology
were glycerophospholipids (i.e., PCs and lysophosphatidylcholines [LysoPCs]). The majority
of associations between these metabolites were in the brain tissue samples: generally, lower
concentrations of glycerophospholipids were associated with greater severity of both amyloid
and neurofibrillary pathology; associations between glycerophospholipids and preclinical and
prodromal AD endophenotypes were sparse. In previous studies using untargeted and semi-
quantitative metabolomics, we demonstrated that AD patients show lower plasma

concentrations of distinct phosphatidylcholines (PC aa C36:5, PC aa C38:6, and PC aa C40:6),

relative to CN [57]. We recently extended these findings to show that reduced plasma concen-
tration of these phosphatidylcholines is also related to lower levels of cognitive performance in
non-demented older individuals and reflects resting state cerebral blood flow (rCBF), a marker
of neuronal activity, in several brain regions related to higher order cognitive processing [58].
Taken together, these prior findings and our current results add further evidence for a role of
altered phosphatidylcholine metabolism in AD pathogenesis.
Network biology: Metabolic pathway alterations in AD

In order to develop an integrated understanding of central–peripheral lipid metabolite fluxes
as well as interactions between the major metabolite classes observed in this study, we applied
a network biology approach. Fig 5 summarizes these networks, based on prior knowledge of
transport mechanisms related to these metabolites and their precursors as well as their known
biosynthetic pathways and catabolic fates. Long-chain fatty acid (LCFA) precursors for glycer-
ophospholipid and sphingolipid biosynthesis are transported both across the blood-brain bar-
rier (BBB) and through plasma membranes within the brain through protein-mediated active
transport by fatty acid transport proteins (FATPs), long-chain acyl-CoA synthetases (ACSLs),
fatty acid binding proteins (FABPs), and the fatty acid transporter (FAT)/CD36 [35,36]. In the
context of neurodegenerative diseases in general and AD in particular, transport of the omega-
3 (ω-3) polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA; 22:6n-3), into the
brain is especially important [59,60]. In a recent untargeted lipidomic analysis in brain tissue
samples from the BLSA, we showed that dysregulation of fatty acid metabolism is associated
with severity of AD pathology [24]. Fig 5 also shows key enzymatically regulated steps in the
biosynthesis of phosphatidylcholines through the Kennedy pathway [37] and their reversible
conversion to LysoPCs through Land’s cycle [41]. The transfer of phosphocholine headgroups
to ceramides by the enzyme phosphatidylcholine transferase (sphingomyelin synthase
[SGMS]) is a key intermediary step in sphingolipid biosynthesis [43] and is a potentially criti-
cal link between glycerophospholipid and sphingolipid metabolism observed in our current
report.
By performing our initial discovery analyses in brain tissue samples at autopsy and subse-
quent validation in preclinical (i.e., BLSA) and prodromal (i.e., ADNI) serum samples, we
were able to ask whether metabolic changes associated with markers of AD neuropathology in
established disease are similar to blood metabolite changes in early AD pathogenesis. Broadly,
our results indicate that there are shared pathways between metabolite changes in brain and
blood, with the prodromal serum samples (i.e., ADNI) sharing more metabolites with brain
samples than the preclinical (i.e., BLSA) serum samples (see Fig 4). A plausible explanation for
these findings is that blood metabolite changes associated with later stages of AD progression
prior to symptom onset are more similar to metabolic correlates of AD pathology in estab-
lished disease. In independent analyses, we have also used the BLSA and ADNI serum samples
as discovery datasets to ask whether principal metabolites associated with preclinical and pro-
dromal AD-related endophenotypes in blood are also represented among the brain metabo-
lites (i.e., in “established disease”) reported in the current study. Among serum metabolites
previously shown to be associated with AD progression in BLSA, we find that propionylcarni-
tine (C3) concentration in serum discriminates between converter and non-converter samples
[7], and its concentration in the ITG is related to the severity of neuritic plaque pathology in
our current study (Fig 4). Among serum metabolites previously shown to be associated with
AD endophenotypes in ADNI, we find that SMC (OH) C14:1 and SM 16:0 concentrations in
serum are associated with CSF Aβ, concentration, brain atrophy, cognitive decline, and risk of

MCI progression [25], and their concentrations in the ITG (i.e., our current report) are both
related to the severity of neuritic plaque pathology and differ across the three groups studied
(Fig 4). Taken together, while these findings suggest that there are metabolic pathways com-
mon to both AD-related neuropathology and blood-related disease progression, there are also
those that are specific to disease stage and tissue compartment. Establishing the relative impor-
tance of common and distinct metabolic pathways across tissue types and disease stages will
require subsequent studies in larger datasets.
Limitations
Our study has limitations. First, the relatively small number of brain tissue samples in our pri-
mary analyses may have limited our power to detect significant associations with other metab-
olites assayed and precluded the use of a discovery and validation dataset. The small number
reflects the challenges of assembling brain tissue samples from well-characterized, longitudi-
nally followed participants who also undergo detailed neuropathological assessment at death;
future studies in larger brain samples are needed to validate our findings. Second, while the
Biocrates AbsoluteIDQ platform is a standardized platform for multiplexed quantitative analy-
sis of 187 different metabolites, these metabolites represent only a small proportion of the
brain and blood metabolomes. Future analyses will expand our study framework across addi-
tional classes of metabolites. Third, it must be noted that we based our primary analyses on
metabolites associated with AD pathology in brain tissue samples. In future studies, it would
be important to perform similar analyses in cognitively normal individuals using primary out-
comes derived from neuroimaging/CSF-based measures of early AD pathology in prodromal/
preclinical AD. Fourth, testing of pre-analytical variables in the BLSA serum samples indicated
a potential selection bias: converter samples were subject to longer storage time at −80˚C, com-
pared to non-converter samples (approximately 17 years versus 13 years, respectively;
Table 1). Additionally, the converter group compared to the non-converter group had more
samples above the cutoff values for Met-So concentration used as an indicator of sample qual-
ity. Therefore, we performed sensitivity analyses within a subsample of converters and non-
converters matched on storage time. In these sensitivity analyses, we confirmed that 10 of the
12 metabolites associated with AD-related outcomes in the BLSA serum samples (Fig 4)
remained significant. We therefore interpret these sensitivity analyses to suggest that our
observed results on serum metabolite concentrations in BLSA are not driven primarily by
group differences in sample storage time or quality. Finally, it is important to note that the
BLSA is a predominantly Caucasian sample of highly educated and relatively healthy older
individuals. Our findings therefore merit confirmation in other cohorts with higher prevalence
of cardiovascular and cerebrovascular disease.
Conclusions
In summary, we have applied quantitative and targeted metabolomics to identify a panel of
sphingolipids, the concentrations of which, in brain tissue, are associated with severity of AD
neuropathology and, in blood, with measures of progression during preclinical and prodromal
AD. We propose that perturbations in sphingolipid metabolism may be integral to the evolu-
tion of AD neuropathology as well as to the eventual expression of AD symptoms in cogni-
tively normal older individuals. Our study design, which takes a machine-learning and data-
driven approach to identify blood metabolites associated with AD progression and explores
how those metabolites are integrated within biologically relevant pathways, suggests a novel
framework for identifying markers for early detection and potential avenues for effective ther-
apeutic intervention in AD.

Supporting information
S1 STROBE Checklist. Checklist of items that should be included in reports of observa-
tional studies.
(DOCX)
S1 Appendix. Description of methods used to generate cognitive domain-specific compos-
ite scores.
(DOCX)
S1 Table. ADNI participating institutions/study sites. ADNI, Alzheimer’s Disease Neuroim-
aging Initiative.
(DOCX)
S2 Table. Full ranking from SVM and RF algorithms from the ITG. ITG, inferior temporal
gyrus; RF, random forest; SVM, support vector machine.
(DOCX)
S3 Table. Brain endophenotype associations: Differences by group.
(DOCX)
S4 Table. Brain endophenotype associations: Associations with AD pathology. AD, Alzhei-
mer disease.
(DOCX)
S5 Table. Blood endophenotype associations: Risk of progression to incident AD in cogni-
tively normal older individuals (BLSA). AD, Alzheimer disease; BLSA, Baltimore Longitudi-
nal Study of Aging.
(DOCX)
S6 Table. Sensitivity analyses in subsample matched on storage time. Blood endophenotype
associations: risk of progression to incident AD in cognitively normal older individuals
(BLSA). AD, Alzheimer disease; BLSA, Baltimore Longitudinal Study of Aging.
(DOCX)
S7 Table. Blood endophenotype associations: Cognitive performance (BLSA). BLSA, Balti-
more Longitudinal Study of Aging.
(DOCX)
S8 Table. Sensitivity analyses in subsample matched on storage time: Blood endopheno-
type associations: Cognitive performance (BLSA). BLSA, Baltimore Longitudinal Study of
Aging.
(DOCX)
S9 Table. Blood endophenotype associations: AD-like brain atrophy patterns and CSF bio-
markers of AD pathology (ADNI). AD, Alzheimer disease; ADNI, Alzheimer’s Disease Neu-
roimaging Initiative; CSF, cerebrospinal fluid.
(DOCX)
S10 Table. Blood endophenotype associations: AD-like brain atrophy patterns and CSF
biomarkers of AD pathology (ADNI). AD, Alzheimer disease; ADNI, Alzheimer’s Disease
Neuroimaging Initiative; CSF, cerebrospinal fluid.
(DOCX)
S11 Table. Blood endophenotype associations: Risk of progression to incident AD in MCI
individuals (ADNI). AD, Alzheimer disease; ADNI, Alzheimer’s Disease Neuroimaging

Initiative; MCI, mild cognitive impairment.

(DOCX)
S12 Table. P-values for all brain- and blood-specific AD endophenotypes included in Fig 4.
AD, Alzheimer disease.
(DOCX)
Acknowledgments
We are grateful to participants in the BLSA for their invaluable contribution. We are thankful
to Dr. Luigi Ferrucci for his guidance throughout the development of the study design and
manuscript. We are additionally thankful for support from the BLSA, the Alzheimer’s Disease
Metabolomics Consortium, and the ADNI studies. Please note: data used in the preparation of
this article were obtained from the ADNI database (adni.loni.usc.edu). As such, the investiga-
tors within the ADNI contributed to the design and implementation of ADNI and/or provided
data but did not participate in the analysis or writing of this report. A complete listing of
ADNI investigators can be found at:
http://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgment_List.
pdf
Author Contributions
Conceptualization: Vijay R. Varma, Madhav Thambisetty.
Data curation: Vijay R. Varma, Sudhir Varma, Ramon Casanova.
Formal analysis: Vijay R. Varma, Sudhir Varma, Ramon Casanova, Yang An.
Investigation: Richard O’Brien, Jon Toledo, Rebecca Baillie, Matthias Arnold, Gabi Kasten-
mueller, Kwangsik Nho, P. Murali Doraiswamy, Andrew J. Saykin, Rima Kaddurah-Daouk,
Cristina Legido-Quigley.
Methodology: Vijay R. Varma, Anup M. Oommen, Sudhir Varma, Ramon Casanova, Yang
An, Olga Pletnikova, Juan C. Troncoso, Cristina Legido-Quigley, Madhav Thambisetty.
Supervision: Madhav Thambisetty.
Visualization: Vijay R. Varma, Anup M. Oommen, Ryan M. Andrews.
Writing – original draft: Vijay R. Varma, Madhav Thambisetty.
Writing – review & editing: Vijay R. Varma, Anup M. Oommen, Sudhir Varma, Ramon
Casanova, Yang An, Ryan M. Andrews, Richard O’Brien, Olga Pletnikova, Juan C. Tron-
coso, Jon Toledo, Rebecca Baillie, Matthias Arnold, Gabi Kastenmueller, Kwangsik Nho,
P. Murali Doraiswamy, Andrew J. Saykin, Rima Kaddurah-Daouk, Cristina Legido-Quig-
ley, Madhav Thambisetty.
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RESEARCH ARTICLE

Brain and blood metabolite signatures of

Received: April 21, 2017 Abstract

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 1 / 31

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 2 / 31

Abbreviations: ρ, correlation coefficient; ω-3,

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evaluations, including neurological, laboratory, and radiological evaluations, were conducted

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Table 1. Demographic characteristics of study samples.

Neuropathological studies: Brain tissue samples in BLSA (Step 1)

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 7 / 31

Determining cognitive status in BLSA and ADNI (Step 2)

Blood samples in BLSA and ADNI (Step 2)

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Brain and blood metabolomics in BLSA and ADNI (Steps 1 and 2)

Cognitive assessments in BLSA (Step 2)

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Structural MRI measures in ADNI (Step 2)

CSF measures of AD pathology in ADNI (Step 2)

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Table 2. Machine-learning methods to discriminate between AD and CN samples.

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 14 / 31

sensitivity/specificity of 86.67%/80.00%. The RF algorithm derived metabolites that discrimi-

Table 3. Top metabolites based on SVM and RF algorithms in the ITG.

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 15 / 31

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Step 2: Exploring associations in blood with AD endophenotypes: Associations with

Step 2: Testing blood metabolite associations with AD endophenotypes:

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 18 / 31

Step 2: Testing blood metabolite associations with AD endophenotypes:

Step 3: Summarizing results: Calculating the EASE-AD score

Step 4: Mapping biological pathways: Exploring metabolite interactions

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 19 / 31

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 20 / 31

synthase; PLPP, phospholipid phosphatase; GPAT, Glycerol-3-phosphate acyltransferases;

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concentrations of distinct phosphatidylcholines (PC aa C36:5, PC aa C38:6, and PC aa C40:6),

Network biology: Metabolic pathway alterations in AD

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PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 26 / 31

PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 27 / 31

Initiative; MCI, mild cognitive impairment.

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PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 29 / 31

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PLOS Medicine | https://doi.org/10.1371/journal.pmed.1002482 January 25, 2018 31 / 31

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