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J Proteome Res. Author manuscript; available in PMC 2016 March 01.
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Published in final edited form as:

J Proteome Res. 2016 February 5; 15(2): 659–666. doi:10.1021/acs.jproteome.5b01111.

A Urinary Metabolic Signature for Multiple Sclerosis and

Neuromyelitis Optica
Teklab Gebregiworgis1, Helle H. Nielsen2, Chandirasegaran Massilamany3, Arunakumar
Gangaplara3, Jay Reddy3, Zsolt Illes2, and Robert Powers1
1Department of Chemistry, University of Nebraska–Lincoln, Lincoln, Nebraska 68588-0304,
United States
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2Department of Neurology, Odense University Hospital, Institute of Clinical Research, University

of Southern Denmark, Odense, Denmark
3School of Veterinary Medicine and Biomedical Sciences, University of Nebraska–Lincoln,
Lincoln, Nebraska 68583-0905, United States

Abstract
Urine is a metabolite-rich biofluid that reflects the body’s effort to maintain chemical and osmotic
homeostasis. Clinical diagnosis routinely relies on urine samples because the collection process is
easy and noninvasive. Despite these advantages, urine is an under-investigated source of
biomarkers for multiple sclerosis (MS). Nuclear magnetic resonance spectroscopy (NMR) has
become a common approach for analyzing urinary metabolites for disease diagnosis and
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biomarker discovery. For illustration of the potential of urinary metabolites for diagnosing and
treating MS patients, and for differentiating between MS and other illnesses, 38 urine samples
were collected from healthy controls, MS patients, and neuromyelitis optica-spectrum disorder
(NMO-SD) patients and analyzed with NMR, multivariate statistics, one-way ANOVA, and
univariate statistics. Urine from MS patients exhibited a statistically distinct metabolic signature
from healthy and NMO-SD controls. A total of 27 metabolites were differentially altered in the
urine from MS and NMO-SD patients and were associated with synthesis and degradation of
ketone bodies, amino acids, propionate and pyruvate metabolism, tricarboxylic acid cycle, and
glycolysis. Metabolites altered in urine from MS patients were shown to be related to known
pathogenic processes relevant to MS, including alterations in energy and fatty acid metabolism,
mitochondrial activity, and the gut microbiota.
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Graphical Abstract

Corresponding authors: Jay Reddy, School of Veterinary Medicine and Biomedical Sciences, Room 202, VBS, East Campus,
University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA; [email protected] Phone (402) 472 8541 Fax (402) 472 9690. Zsolt
Illes, University of Southern Denmark Institute of Clinical Research, Odense University Hospital, Department of Neurology Odense,
Denmark; [email protected] Phone 45 6541-5332. Robert Powers, University of Nebraska-Lincoln, Department of Chemistry, 722
Hamilton Hall Lincoln, NE 68588-0304, USA; [email protected] Phone (402) 472-3039 Fax (402) 472-9402.
Present address: Arunakumar Gangaplara, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, MD 20892–1892, USA
The authors declare no competing financial interest.
 Gebregiworgis et al. Page 2
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Keywords
urine biomarkers; multiple sclerosis; neuromyelitis optica-spectrum disorder; NMR;
metabolomics; multivariate statistics
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Introduction
Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) with both
neurodegenerative and inflammatory demyelinating components.1 The disease has a
heterogeneous clinical presentation and is characterized by clinical symptoms that involve
different parts of the CNS. Especially in the early stages, MS shares features with other
demyelinating diseases like neuromyelitis optica-spectrum disorders (NMO-SD).2 Despite
recently updated classification criteria,3,4 differentiation between the two diseases can be
difficult but nevertheless vital, because misclassification can lead to increased disease
activity due to incorrect treatment.5 Thus, the identification of metabolite biomarkers for MS
may help improve MS diagnostic protocols and help better understand the pathogenesis of
the disease.
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Metabolite biomarkers are small chemical entities (<1500 Da) found in biofluids where their
presence or concentration has a correlation with either the prognosis, existence, or
progression of a disease or the therapeutic response to a medication or treatment.6
Metabolites are the end products of enzymatic reactions or protein activity that are readily
modulated by genetic alterations, environmental stress, toxins, or drugs.7 Thus, all
phenotypic alteration caused by a disease or a medical treatment is expected to exhibit a
unique metabolic profile or fingerprint.8 The ability to accurately and efficiently detect these
metabolic alterations presents a potential avenue for personalized medicine and disease
diagnosis through the identification of metabolite biomarkers.9 Unlike a single gene or
protein routinely used as a medical biomarker, a metabolomics biomarker is commonly
comprised of a dozen or more metabolites and provides a highly unique signature that may
increase the likelihood of a correct diagnosis.10
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The analysis of urine to obtain a metabolic profile has a number of well-known advantages
that includes ready availability; a rapid, easy, inexpensive, and noninvasive sample
collection procedure; the ability to collect multiple, large samples over a range of time-
points; and well-established protocols for storing, handling, and examining urine samples.11
The investigation of urine metabolites using nuclear magnetic resonance spectroscopy
(NMR) is experiencing a rapid growth of interest, where NMR metabolomics is routinely

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 Gebregiworgis et al. Page 3

being used for drug and biomarker discovery.12 NMR is an attractive technique because it
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requires minimal sample preparations and is able to simultaneously detect and quantify a
variety of compounds from a complex mixture without separation. NMR is commonly
combined with multivariate statistics to efficiently identify and statistically validate the
metabolomics profile.13

To date, investigations into MS metabolite biomarkers has primarily focused on the analysis
of cerebrospinal fluid (CSF) and serum samples from MS patients.14,15 Little attention has
been given to the analysis of urine16 despite the fact that metabolites excreted into urine are
readily accessible and are easily detected compared to CSF or blood samples.16 We
previously reported an NMR metabolomics analysis of urinary markers of MS using the
animal model experimental autoimmune encephalomyelitis (EAE).17 The results of our prior
EAE animal study demonstrated the potential of using urine as a source of metabolite
biomarkers for MS. Herein, we report an NMR metabolomics analysis of human urine
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samples collected from healthy controls, MS patients, and NMO-SD patients. Our results
demonstrate a statistically significant difference in the urinary metabolites observed between
MS patients and healthy controls and between MS and NMO-SD patients.

Methods and Materials

Patient Information and Clinical Manifestation
To ensure as much homogeneity as possible within the groups, we selected definite
aquaporin-4 (AQP4)-seropositive NMO-SD patients with high antibody titers in the serum
directed against the water channel AQP4, and ensured that all were diagnosed according to
the criteria proposed by Wingerchuk 2006.4 Seven of the patients had definite seropositive
NMO, whereas 2 had seropositive NMO-SD in the form of optic neuritis (ON) and
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longitudinally extensive transverse myelitis (LETM) (Table 1 and Table S1). The mean age
was 39.3 with a female predominance as expected,18 and all received immunosuppressive
therapy in the form of azathioprine.

Similarly, all MS patients were diagnosed with relapsing-remitting MS (RR-MS) according

to the McDonald’s 2010 criteria19 and accordingly received immunomodulatory therapy.
Among this group, 5 patients received first-line therapy (glatiramer acetate and interferon-
β), 1 patient received second-line therapy (natalizumab), and 2 patients received no
treatment (Table 1 and Table S1). The mean age was 44.6 years with a female
predominance. On average, the age at disease onset is 34 years; however, none of these
patients were newly diagnosed. All healthy subjects were healthy volunteers with no known
neurological or autoimmune diseases. Neither MS nor NMO/NMO-SD patients had
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experienced a relapse within 30 days of the sample collection. The study was conducted in
accordance with both the Hungarian and Danish National Ethics Committee
(38.93.316-12464/KK4/2010, 42341-2/2013/EKU, S-20120066).

Urine Collection
Using the NMO-SD and MS database of the University of Pecs in Hungary, we collected
urine samples from 9 patients with NMOSD seropositive for antibodies against AQP4, 8

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patients with RRMS, and 7 healthy subjects. Treatment, age, and gender of the study
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populations are shown in Table 1 and Table S1. Urine samples were collected as spot urine
in the morning before breakfast and administration of drug treatment and processed within 2
h of collection. The time intervals between replicate urine sample collections were variable.
All patients were on chronic treatment as indicated in Table 1 and Table S1. Azathioprine
and glatiramer acetate were administered daily, interferon β-1b every other day, and
natalizumab was administered once a month. In cases of interferon β-1b treatment, urine was
collected the day after administration. Urine was collected the day before the monthly
natalizumab infusion in the single patient treated with natalizumab. Samples were
centrifuged at 20,000g for 20 min at room temperature (RT) to pellet cell debris, and the
supernatants were stored at −80 °C until use. Except for one MS patient, two analytical
replicate urine samples were obtained from each MS patient and each healthy subject.
Conversely, only one urine sample was collected from each NMO-SD patient.
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NMR Sample Preparation

The urine samples were thawed and then centrifuged at 13000 rpm for 5 min at RT to
remove any precipitate. Then, 100 μL of each urine sample was transferred into a new
Eppendorf tube and mixed with 500 μL of 50 mM phosphate buffer in 99.8% D2O (Isotec,
St. Louis, MO) at pH 7.2 (uncorrected); 50 μM of 3-(trimethylsilyl) propionic acid-2,2,3,3-
d4 (TMSP-d4) was added to each sample as a chemical shift reference. The urine samples
were then transferred to a 5 mm NMR tube for NMR data acquisition.

NMR Data, Collection, and Processing and Multivariate Statistical Analysis

The one-dimensional (1D) 1H NMR experiments were collected and processed as described
previously.17 The NMR data processing and multivariate statistical analysis were
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accomplished using our MVAPACK software suite (http://bionmr.unl.edu/mvapack.php).20

The 1D 1H NMR spectra were aligned with the icoshift algorithm when the full-resolution
spectra were modeled using orthogonal projections to latent structure discriminant analysis
(OPLS-DA). Alternatively, the 1D 1H NMR spectra were binned using an intelligent
adaptive binning algorithm when S-plots and a shared and unique structure (SUSplot) plot
were generated from the OPLS-DA model. The data were normalized with a probabilistic
quotient normalization function and Pareto scaled prior to multivariate statistical analysis.
Fractions of explained variation (R2 X and R2 Y) were computed during OPLS-DA model
training. OPLS-DA models were internally cross-validated using 7-fold Monte Carlo cross
validation to compute Q2 values, which were compared to a distribution of null model Q2
values in 1000 rounds of response permutation testing. Model results were further validated
using CV-ANOVA significance testing.
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Metabolite Identification
An SUS plot was generated from the OPLS-DA models using MVAPACK to compare the
MS and NMO-SD group against healthy controls. The plot visualizes the correlation
between predictive components of each model and was used to identify metabolite changes
unique to either the MS or the NMO-SD group. The chemical shift information from the
loadings and SUS plots were assigned to metabolites using Chenomx NMR suite 7.0

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(Chenomx Inc., Edmonton, Alberta, Canada) and the Human Urine Metabolome database
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(http://www.urinemetabolome.ca/). A 1H chemical shift error of 0.08 ppm was used to

match the experimental chemical shifts with database values. Metabolite pathway analysis
was accomplished using the Metabolomics Pathway Analysis (MetPA) Web server (http://
metpa.metabolomics.ca/MetPA).

One-Way ANOVA and Univariate Statistical Analysis

One-way ANOVA and univariate calculations were conducted using the R statistical
package version 3.2.0.21 One-way ANOVA was used to determine the statistical
significance of individual metabolite differences between healthy, MS, and NMO-SD
patients. Metabolites with a p-value ≤ 0.05 were then subjected to Tukey’s multiple
comparison of means test to identify the set of metabolites that are statistically different
between the paired groups.22 A Student’s t test was also applied to determine the statistical
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significance of metabolite differences between the two groups. The p-values from the
Student’s t test were further adjusted using the Benjamini–Hochberg multiple hypothesis
method.23

Results
Urine Metabolomics Signature for MS Patients
A 1D 1H NMR spectrum was acquired for each of the 29 urine samples collected from seven
healthy individuals and eight patients previously diagnosed with MS. The 1D 1HNMR
spectra capture a “snapshot” of the state of the urinary metabolome and provides a direct
means of determining if the metabolic profiles differ between healthy and MS patients. The
NMR data set was modeled by OPLS-DA, and the resulting scores plot (Figure 1a) shows a
clear separation between the healthy controls and MS patients. Importantly, all of the
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biological replicates were assigned to the correct class in the OPLS-DA scores plot. The
leave-n-out cross-validation metrics of R2Y = 0.77 and Q2 = 0.39 indicates an acceptable
level of fit and predictive ability. A reliable model is also indicated by a p-value of 7.8 ×
10−4 from the CV-ANOVA test and a p-value of 8.0 × 10−3 from the response permutation
test (Figure 1b).

A back-scaled loadings plot was generated from the OPLS-DA model to identify the spectral
regions (metabolites) that primarily contribute to the observed class separation in the scores
plot (Figure 2a). Twenty-six metabolites are differentially altered in the urine samples
collected from healthy individuals and MS patients. The identified metabolites are from
metabolic pathways associated with energy metabolism, fatty acid synthesis, and gut
microflora, which include amino acid derivatives and amino acid degradation products
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(Table S2). An S-plot was then used to identify the major contributors to these observed
class differences in the OPLS-DA scores plot (Figures S1 and 2b). The S-plot identified
creatinine, hippurate, 3-hydroxybutyrate, malonate, oxaloacetate, and trimethylamine N-
oxide as having the highest covariance and correlation (from the 26 metabolites) with the
OPLS-DA model from the healthy and MS data sets. A one-way ANOVA analysis was
performed for each metabolite identified from the multivariate statistical analysis to
determine if a statistically significant difference exists between the healthy and MS groups.

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Metabolites with a p-value ≤ 0.05 were selected (Table S3) and further subjected to Tukey’s
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multiple comparisons of means test.22 This analysis indicated that the set of metabolites,
including creatinine (p-value = 4.2 × 10−4), 3-hydroxyisovalerae (p-value = 3.2 × 10−02),
and oxaloacetate (p-value = 5.0 × 10−02) discriminate between healthy controls and MS
patients (Figure S2). A Student’s t test followed by the Benjamini–Hochberg multiple
hypothesis test (Figure S3) identified acetate and creatinine as statistically different between
healthy controls and MS patients.23 The Benjamini–Hochberg adjusted p-value, one-way
ANOVA, and multivariate analyses are fundamentally distinct techniques that emphasize
different aspects of the data and have no expectations of producing identical results. Thus, a
subset of the eight metabolites corresponding to acetate, creatinine, hippurate, 3-
hydroxybutyrate, 3-hydroxyisovalerae, malonate, oxaloacetate, and trimethylamine N-oxide
have the potential of differentiating between healthy controls and MS patients.

Urine Metabolomics Signature for NMO-SD Patients

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Because NMO-SD is an inflammatory demyelinating disease of the CNS similar to MS, but
antibodies play a major role in the pathogenesis in contrast to the supposedly heterogeneous
pathogenesis in MS, we considered NMO-SD as a valuable negative control for identifying
biomarkers specific to MS.2 A 1D 1H NMR spectrum was acquired for each of the 23 urine
samples collected from seven healthy individuals and nine patients previously diagnosed
with NMO-SD. The NMR data set was modeled by OPLS-DA, and the resulting scores plot
(Figure 3a) shows a clear separation between the healthy controls and NMOSD patients. All
of the biological replicates were assigned to the correct class in the OPLS-DA scores plot.
The leave-n-out cross-validation metrics of R2Y = 0.93 and Q2 = 0.68 indicates a reasonable
level of fit and predictive ability. A reliable model is also indicated by a p-value of 7.3 ×
10−3 from the CV-ANOVA test and a p-value of zero from the response permutation test
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(Figure 3b). A back-scaled loadings plot (Figure 4a) identified 20 metabolites differentially
altered in the urine samples collected from healthy individuals and NMO-SD patients. The
identified metabolites are amino acids and amino acid derivatives, tricarboxylic acid (TCA)
cycle intermediates, choline-containing compounds, and metabolites from the gut microflora
(Table S2). An S-plot was then used to identify the major contributors to this observed class
differences in the OPLS-DA scores plot (Figures S4 and 4b). The S-plot identified acetate,
creatinine, 3-hydroxybutyrate, methylmalonate, oxaloacetate, and succinate as having the
highest covariance and correlation (from the 20 metabolites) with the OPLS-DA model from
the healthy and NMO-SD data sets. A one-way ANOVA analysis was performed for each
metabolite identified from the multivariate statistical analysis to determine if a statistically
significant difference exists between the healthy and NMO-SD groups. Metabolites with a p
value ≤ 0.05 were selected (Table S3) and further subjected to Tukey’s multiple
comparisons of means test.22 This analysis indicated that the set of metabolites, including
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creatinine (p value 4.0 × 10−07), 3-hydroxybutyrate (p-value 2.9 × 10−02), oxaloacetate (p-
value 2.7 × 10−02), and methylmalonate (p-value 2.6 × 10−06) discriminates between healthy
controls and NMOSD patients (Figure S2). A Student’s t test followed by the Benjamini–
Hochberg multiple hypothesis test (Figure S3) also identified the same set of metabolites as
statistically different between healthy controls and NMO-SD patients. Similar to our
comparison between healthy controls and MS patients, we identified a subset of the six
metabolites corresponding to acetate, creatinine, 3-hydroxybutyrate, methylmalonate,

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oxaloacetate, and succinate that have the potential of differentiating between healthy
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controls and NMO-SD patients.

Metabolite Pathway Analysis

The complete list of 27 metabolites (Tables S2 and S4) differentially altered in urine
samples collected from MS and NMO-SD patients relative to healthy controls were
uploaded to the MetPA Web server. MetPA used the Homo sapiens pathway library, the
hypergeometric test for the over-representation analysis, and the out-degree centrality for the
pathway topology analysis. MetPA estimates a metabolite’s relative importance, provides a
global overview of the metabolic changes, and assists in identifying important pathways
associated with the disease phenotype. The synthesis and degradation of ketone bodies,
amino acid metabolism, propionate metabolism, pyruvate metabolism, the TCA cycle, and
glycolysis were identified as high impact pathways in MS and NMO-SD patients (Figure 5
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and Table S5).

Urine NMR Metabolomics Signatures That Differentiate MS and NMO-SD Patients

The two back-scaled loadings plots and the two S-plots were directly compared to identify
metabolites that were distinctly altered in the urine samples from either MS or NMO-SD
patients relative to healthy controls (Figures 2 and 4 and Figure S5). Eight metabolites,
alanine, hippurate, 2-hydroxyisovalerate, 3-hydroxybutyrate, isovalerate, malonate,
oxaloacetate, and trimethylamine N-oxide were identified as being uniquely altered in urine
samples from MS patients relative to NMO-SD patients. Eight other metabolites, acetate,
acetone, citrate, creatine, creatinine, lactate, methylmalonate, and succinate were identified
as being uniquely altered in urine samples from NMO-SD patients relative to MS patients.

For further refinement of a metabolic signature unique to MS patients, an SUS plot (Figure
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5a) was generated from the MS vs healthy and the NMO-SD vs healthy OPLS-DA models
using binned NMR data (Figures S1 and S4). The two sets of metabolites identified from the
overlaid back-scaled loading plots were then assigned to appropriate bins in the SUS plot. In
this manner, only metabolites that clearly fell within the MS specific or NMO-SD specific
regions of the SUS plot were identified as statistically significant and retained. This analysis
reduced the number of urine metabolites specific to MS (2-hydroxyisovalerate, alanine,
hippurate, isovalerate, trimethylamine N-oxide) or to NMO-SD (acetate, creatine, lactate,
methylmalonate, and citrate) to five each.

The results of the multivariate statistical analysis was further supported by a follow-up
univariate analysis.24 A one-way ANOVA analysis was performed for each metabolite
identified from the multivariate statistical analysis to determine if a statistically significance
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difference exists between the healthy, MS, and NMO-SD groups. Metabolites with a p-value
≤ 0.05 were selected (Table S3) and further subjected to Tukey’s multiple comparisons of
means test.22 This analysis indicated that the set of metabolites, including creatinine (p-
value = 1.5 × 10−02), 3-hydroxybutyrate (p-value = 8.0 × 10−3), 3-hydroxyisovalerate (p-
value = 8.4 × 10−05), and methylmalonate (p-value = 1.1 × 10−4), discriminate between MS
and NMO-SD patients (Figure S2). An identical set of metabolites was also obtained using
the Student’s t test followed by the Benjamini–Hochberg multiple hypothesis test (Figure S–

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3).23 It is important to note that, in addition to being identified by the univariate analysis, 3-
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hydroxybutyrate was also identified in both S-plots but was absent in the SUS plot. 3-
Hydroxybutyrate was likely missing in the SUS plot due to a serendipitous cancellation
because it was located in opposite regions in the two S-plots (Figures 2 and 4).

Discussion
A wide range of chemicals from food, medication, environmental contaminants, normal
biological processes, and disease conditions are routinely excreted into the urine to maintain
chemical homeostasis. Consequently, urinalysis has been used to diagnose diseases and
evaluate a patient’s well-being for years. More recently, there has been a growing interest in
identifying urinary metabolite biomarkers to monitor the prognosis, existence, or
progression of various cancers, cardiovascular diseases, and neurological diseases.25 Despite
the high-rate of misdiagnosis, little attention has been paid toward the analysis of urinary
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metabolites for diagnosing MS.26 To address this oversight, we previously demonstrated

that urinary metabolites can differentiate between EAE mice (prototypic disease model for
MS) from healthy and fingolimod (MS drug)-treated EAE mice.17 We extended this initial
animal study by using NMR, multivariate statistics, one-way ANOVA, and univariate
statistics to analyze changes in urine samples collected from MS and NMO-SD patients.
Although the sample size was limited, we were still able to observe a potentially unique
metabolic signature in urine samples collected from MS patients. Importantly, the MS
metabolic signature was likely to be distinct from the urinary metabolites identified for
NMO-SD patients. Our results are also consistent with a recent study by Moussallieh et al.15
that showed an increase in serum acetate levels in NMO-SD patients relative to MS patients.
However, the limited sample size requires us to describe our statistical model and the set of
potential urinary metabolite biomarkers as only a working hypothesis. A major revision may
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occur as the number of patients per group increases significantly. Nevertheless, we are
encouraged by the fact that we were able to link all of the metabolites and metabolic
pathways identified from the urine of MS patients to known pathologic processes associated
with MS.

Glycolysis and the synthesis and degradation of ketone bodies were identified as two
potentially high-impact pathways from our NMR metabolomics analysis of urine samples
from MS patients. Specifically, we observed significant concentration changes in the
glycolysis intermediates lactate and acetate and the ketone bodies 3-hydroxybutyrate and
acetoacetate. This observation is consistent with basic brain chemistry that is expected to be
altered by MS. The brain has a high energy need and uses 25% of the total available glucose.
Interestingly, the brain also uses ketone bodies as an alternative energy source.27 Thus,
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alteration in glycolysis and the synthesis and degradation of ketone bodies is consistent with
an alteration in energy generation. Besides energy generation, ketone bodies are also
associated with fatty acid metabolism. This is pertinent because prior analysis of CSF and
serum samples from MS patients indicated altered energy and fatty acid metabolism.28

The observed alterations in energy generation have led to the speculation that MS might be
associated with mitochondrial defects.29 In fact, Witte et al. demonstrated an occurrence of
severe mitochondrial defects in MS lesions.30 Because the mitochondria are the primary

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source of energy in axons, the observed defects would be expected to alter energy
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generation. In the mitochondria, ATP is produced via the TCA cycle, the electron transport
chain (respiratory chain), and oxidative phosphorylation. From our NMR metabolomics
analysis, we identified the TCA cycle as another possible high-impact pathway that was
altered in the urine samples from MS patients. Intermediates of the TCA cycle, such as
citrate, oxaloacetate, and succinate were altered in the urine from MS patients. Of particular
note, pyruvate and amino acid metabolism (alanine, aspartate, and glutamate) pathways,
which are directly coupled to the TCA cycle, were also identified as potential high-impact
pathways. Defects in the amino acid metabolism pathway are known to cause a range of
neurological issues.31

We also observed an increase in creatine and a decrease in creatinine in the urine from MS
patients. The creatine/phosphocreatine/creatine kinase system is critical for maintaining
energy levels in the brain and as a high-energy phosphate shuttle from the mitochondria to
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the cytoplasm.32

Our NMR metabolomics analysis also identified propionate metabolism as another likely
high-impact pathway altered in the urine of MS patients. Propionate (short-chain fatty acid)
is primarily derived from the catabolism of lipids (fatty acid metabolism) or proteins, where
its accumulation is toxic and inhibits TCA cycle enzymes and cell growth.33 Propionate and
propionyl-CoA are detoxified by the mitochondria through the methyl-malonyl-CoA
pathway.34 Genetic flaws in the propionate metabolism pathway cause faulty amino acid
and fatty acid metabolism that lead to various neurological problems.34 Thus, the observed
alteration in propionate metabolism appears to be consistent with a recent view that MS may
also be associated with dysfunction in lipid metabolism.35
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Gut microbiota are known to establish a symbiotic relationship that provides essential health
benefits to the host.36 A similar correlation between gut microbiota and MS has been
previously observed.36 In fact, Cantarel et al. described a difference in specific operational
taxonomic units of gut microbiota in MS patients. Their finding also indicated that
immunomodulatory medications cause alterations in the gut microbiota of MS patients.37
Herein, we observed hippurate, a mammalian microbial cometabolite that was altered in the
urine of MS patients. Propionate (described above) is also a metabolite of the gut
microbiota. Thus, differences in urinary hippurate and propionate levels may be a result of a
change in the gut microbiota or alterations in the relevant metabolic pathway.38 Taken
together, these findings suggest a potential role of gut microbiota in the pathogenesis and
treatment of MS.
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Conclusions
Although CNS and serum metabolites have been previously considered as a source of MS
and NMO-SD biomarkers, we have demonstrated that the urine metabolome shows
significant promise for investigating and diagnosing MS and NMO-SD. We observed a set
of eight potential urinary metabolites (acetate, creatinine, hippurate, 3-hydroxybutyrate, 3-
hydroxyisovalerae, malonate, oxaloacetate, and trimethylamine N-oxide) associated with
MS that may be prospective biomarkers. Similarly, we observed a set of eight potential

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urinary metabolites (acetate, creatinine, 3-hydroxybutyrate, 3-hydroxyisovalerae,

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methylmalonate, oxaloacetate, and succinate) associated with NMO-SD. Critically, we

observed a distinct set of urinary metabolites (creatinine, 3-hydroxybutyrate, 3-
hydroxyisovalerate, methylmalonate) that possibly differentiates MS from NMO-SD
patients. This is despite the fact that NMO-SD is also an inflammatory immune-mediated
disease of the CNS that was once considered a variant of MS. The observation that all of the
metabolites and metabolic pathways identified from the urine of MS patients are linked to
known pathologic processes associated with MS supports the potential reliability of our
study, although the sample size was small. Specifically, metabolite changes are associated
with alterations in energy and fatty acid metabolism, mitochondrial activity, and the gut
microbiota. Although our results highlight the promise of urinary biomarkers as a tool to
diagnose MS and NMO-SD, the limited sample size necessitates interpreting our results as
only a proof-of-principle that requires further validation. It is possible that an increase in the
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number of patients per group may lead to a change in the observed set of urinary metabolites
that differentiates between MS patients, NMO-SD patients, and healthy controls.
Nevertheless, as evident by a number of other recent MS and NMO-SD studies where
practical considerations limit sample size,39–43 a conservative interpretation does not negate
the inherent scientific value of our observations. Instead, given the ease and ready access of
urine samples from MS and NMO-SD patients, our results establish the urine metabolome as
a potentially valuable resource for investigating the pathology of MS and NMO-SD for
obtaining a rapid and reliable diagnosis and for monitoring a patient’s response to treatment.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
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Acknowledgments
This manuscript was supported in part by funds from the National Institutes of Health (NIH), USA (P30
GM103335, R.P., J.R.; HL114669, J.R.), NIH National Center for Research Resources (P20 RR-17675, J.R.),
University of Nebraska Research Council (J.R., R.P.), Scleroseforeningen (A-19412, Denmark, Z.I.),
Lundbeckfonden (R118-A11472, Denmark, Z.I.), and a grant from Odense University Hospital (Z.I.). The research
was performed in facilities renovated with support from the National Institutes of Health (RR015468-01).

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Figure 1.
(a) OPLS-DA scores resulting from modeling of the 1D 1H NMR data matrix from human
urine samples collected from MS patients (cyan) and healthy controls (red). A statistically
significant degree of separation is observed between the two experimental classes. The
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leave-n-out cross-validation metrics are R2Y = 0.77 and Q2 = 0.39, and the CV-ANOVA and
a response permutation test p-values are 7.8 × 10−4 and 8.0 × 10−3, respectively. Ellipses
enclose the 95% confidence intervals estimated by the sample means and covariances of
each class. (b) Response permutation testing results for OPLS-DA scores after 1000 random
permutations of the group membership information (Y). The model significance is inferred
from the degree of vertical separation between the null distribution (leftmost) and the true
R2Y and Q2 values (rightmost). The apparent discretization along the correlation axis is a
result of using binary class labels in Y.
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 Gebregiworgis et al. Page 14
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Figure 2.
(a) Back-scaled OPLS-DA loadings plot resulting from modeling of the 1D 1H NMR data
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matrix from human urine samples collected from MS patients and healthy controls. (b) S-
plot from the OPLS-DA model generated from binned 1D 1H NMR spectra from the MS
and healthy controls data sets (Figure S1). The x- and y-axis of the S-plot measures the
covariance and correlation, respectively. The green and red triangles identify metabolites
with a relative increase or decrease in concentration in urine samples from MS patients
compared to healthy controls, respectively. The blue triangles correspond to unknown
metabolites. The black triangles correspond to all other bins or metabolites. The metabolites
are labeled as follows: 1, 2-hydroxyisovalerate; 2, isovalerate; 3, 3-hydroxyisobutyrate; 4,
propylene glycol; 5, 3-hydroxybutyrate; 6, methylmalonate; 7, 3-hydroxyisovalerate; 8,
lactate; 9, alanine; 10, acetate; 11, N-acetylglutamine; 12, acetone; 13, acetoacetate; 14,
oxaloacetate; 15, succinate; 16, citrate; 17, creatine; 18, creatinine; 19, malonate; 20,
choline-containing compounds; 21, trimethylamine N-oxide; 22, glycine; 23, phenylalanine;
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24, phenylacetylglycine; 25, hippurate; and 26, xanthine.

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Figure 3.
(a) OPLS-DA scores resulting from modeling of the 1D 1H NMR data matrix from human
urine samples collected from NMO-SD patients (cyan) and healthy controls (red). A
statistically significant degree of separation is observed between the two experimental
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classes. The leave-n-out cross-validation metrics are R2Y = 0.93 and Q2 = 0.68, and the CV-
ANOVA and a response permutation test p-values are 7.3 × 10−3 and 0, respectively.
Ellipses enclose the 95% confidence intervals estimated by the sample means and
covariances of each class. (b) Response permutation testing results for OPLSDA scores after
1000 random permutations of the group membership information (Y). The model
significance is inferred from the degree of vertical separation between the null distribution
(leftmost) and the true R2Y and Q2 values (rightmost). The apparent discretization along the
correlation axis is a result of using binary class labels in Y.
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 Gebregiworgis et al. Page 16
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Figure 4.
(a) Back-scaled OPLS-DA loadings plot resulting from modeling of the 1D 1H NMR data
matrix from human urine samples collected from NMO-SD patients and healthy controls. (b)
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S-plot from the OPLS-DA model generated from binned 1D 1H NMR spectra from NMO-
SD and healthy controls data sets (Figure S2). The x- and y-axis of the S-plot measures the
covariance and correlation, respectively. The green and red triangles identify metabolites
with a relative increase or decrease in concentration in urine samples from NMO-SD
patients compared to healthy controls. The blue triangles correspond to unknown
metabolites. The black triangles correspond to all other bins or metabolites. The metabolites
are labeled as follows: 6, methylmalonate; 8, lactate; 9, alanine; 10, acetate; 11, N-
acetylglutamine; 12, acetone; 13, acetoacetate; 14, oxaloacetate; 15, succinate; 16, citrate;
17, creatine; 18, creatinine; 19, malonate; 20, choline-containing compounds; 21,
trimethylamine N-oxide; 22, glycine; 23, phenylalanine; 24, phenylacetylglycine; 25,
hippurate; and 27, timethylamine.
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 Gebregiworgis et al. Page 17
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Figure 5.
(a) SUS plot generated from the OPLS-DA models compare the MS and NMO-SD groups
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against healthy controls. NMR bins within the MS specific or NMO-SD specific regions of
the SUS plot are labeled with red or blue triangles, respectively. The resulting chemical shift
information from the SUS plot was assigned to metabolites using ChenomxNMRsuite 7.0.
The metabolites are numbered accordingly: 1, 2-hydroxyisovalerate/isovalerate; 6,
methylmalonate; 8, lactate; 9, alanine; 10, acetate; 16, citrate; 17, creatine; 21,
trimethylamine N-oxide; and 25, hippurate. (b) Overview of the pathway topology analysis
produced by MetPa (http://metpa.metabolomics.ca/MetPA). The highest ranked pathways
are numbered accordingly: 1, synthesis and degradation of ketone bodies; 2, propionate
metabolism; 3, pyruvate metabolism; 4, TCA cycle; 5, alanine, aspartate, and glutamate
metabolism; and 6, glycolysis or gluconeogenesis.
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Table 1

Demographic Data of Hungarian Cohort*

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HS AQP4-NMO/NMO-SD MS
n=7 n=9 n=8
Disease Subtype
NMO-SD 0 7 0
ON 0 1 0
LETM 0 1 0
RR-MS 0 0 8
Sex
female 4 6 6
male 3 3 2
mean age (range) 26.7 (25–30) 39.3 (22–55) 44.6 (31–70)
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Treatment
azathioprine 0 9 0
natalizumab 0 0 1
interferon β-1b 0 0 3
glatiramer acetate 0 0 2
none of the above 7 0 2

*
HS, healthy subjects; AQP4, aquaporin 4; NMO, neuromyelitis optica; NMO-SD, neuromyelitis optica-spectrum disorder; MS, multiple sclerosis;
ON, optic neuritis; LETM, longitudinally extensive transverse myelitis; RR-MS, relapsing-remitting multiple sclerosis
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