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\begin{document}

\title{Metabolomic Identification of Predictive and Early Biomarkers of
Cisplatin-induced Acute Kidney Injury in Adult Head and Neck Cancer
Patients}



\author[1]{Yong Jin Lim}%
\author[1]{Steven Xiu}%
\author[1]{M Kuruvilla}%
\author[1]{Eric Winquist}%
\author[1]{Stephen Welch}%
\author[1]{Morgan Black}%
\author[1]{Lauren  Faught}%
\author[2]{Jasmine Lee}%
\author[3]{Michael (Guest Editor) Rieder}%
\author[4]{Tom Blydt-Hansen}%
\author[2]{Michael Zappitelli}%
\author[1]{Brad Urquhart}%
\affil[1]{Western University Schulich School of Medicine \& Dentistry}%
\affil[2]{SickKids Research Institute}%
\affil[3]{University of Western Ontario}%
\affil[4]{British Columbia Children's Hospital}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aim: Cisplatin causes acute kidney injury (AKI) in approximately
one-third of patients. Serum creatinine and urinary output are poor
markers of cisplatin-induced (AKI). Metabolomics was utilized to
identify predictive or early diagnostic biomarkers of cisplatin-induced
AKI. Methods: Thirty-one adult head and neck cancer patients receiving
cisplatin (dose [?] 70 mg m2 -1) were recruited for metabolomics analysis.
Urine and serum samples were collected prior to cisplatin (pre), 24-48
hours after cisplatin (24-48h), and 5-14 days (post) after cisplatin.
Based on serum creatinine concentrations measured at the post timepoint,
11/31 patients were classified with clinical AKI. Untargeted
metabolomics was performed using liquid chromatography-mass
spectrometry. Results: Metabolic discrimination was observed between
``AKI'' patients and ``no AKI'' patients at all timepoints. Urinary
glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid, and suberate
were significantly different between AKI patients and no AKI patients
prior to cisplatin infusion. Urinary glycine and hippuric acid sulfate
were lower (-2.22-fold and -8.85-fold), whereas 3-hydroxydecanedioc acid
and suberate were higher (3.62-fold and 1.91-fold) in AKI patients
relative to no AKI patients. Several urine and serum metabolites were
found to be altered 24-48 hours following cisplatin infusion,
particularly metabolites involved with mitochondrial energetics.
Conclusion: We propose glycine, hippuric acid sulfate,
3-hydroxydecanedioc acid, and suberate as predictive biomarkers of
predisposition to cisplatin-induced AKI. Metabolites indicative of
mitochondrial dysfunction may serve as early markers of subclinical AKI.%
\end{abstract}%



\sloppy


\textbf{TITLE:} Metabolomic Identification of Predictive and Early
Biomarkers of Cisplatin-induced Acute Kidney Injury in Adult Head and
Neck Cancer Patients

\textbf{RUNNING TITLE:} Metabolomics of cisplatin-induced AKI

\textbf{AUTHORS}

Yong Jin Lim\textsuperscript{1}(ylim33@uwo.ca)

Steven G. Xiu\textsuperscript{1}(sxiu3@uwo.ca)

M. Sara Kuruvilla\textsuperscript{2}(sara.kuruvilla@lhsc.on.ca)

Eric Winquist\textsuperscript{2}(eric.winquist@lhsc.on.ca)

Stephen Welch\textsuperscript{2}(stephen.welch@lhsc.on.ca)

Morgan Black\textsuperscript{2}(morgan.black@lhsc.on.ca)

Lauren N. Faught\textsuperscript{1}(lauren.faught@icloud.com)

Jasmine Lee\textsuperscript{3} (jasmine.lee@sickkids.ca)

Michael J. Rieder\textsuperscript{1,4,5}(mrieder@uwo.ca)

Tom D. Blydt-Hansen\textsuperscript{6}(tom.blydthansen@cw.bc.ca)

Michael Zappitelli\textsuperscript{3}(michael.zappitelli@sickkids.ca)

Bradley L. Urquhart\textsuperscript{1,7}(brad.urquhart@schulich.uwo.ca)

\textbf{AFFILIATIONS}

\textsuperscript{1}Department of Physiology and Pharmacology, Schulich
School of Medicine and Dentistry, Western University, London, ON, Canada

\textsuperscript{2}Division of Medical Oncology, Department of Oncology,
Schulich School of Medicine and Dentistry, Western University, London,
ON, Canada

\textsuperscript{3}Division of Nephrology, Department of Pediatrics,
Hospital for Sick Children, Toronto, ON, Canada

\textsuperscript{4}Department of Paediatrics, Schulich School of
Medicine and Dentistry, Western University, London, ON, Canada,

\textsuperscript{5}Division of Clinical Pharmacology, Department of
Medicine, Schulich School of Medicine and Dentistry, Western University,
Canada

\textsuperscript{6}Division of Nephrology, Department of Pediatrics,
British Columbia Children's Hospital, Vancouver, BC, Canada

\textsuperscript{7}Division of Nephrology, Department of Medicine,
Schulich School of Medicine \& Dentistry, Western University, London,
ON, Canada

\emph{The authors confirm that Bradley L. Urquhart is the PI for this
paper. M. Sara Kuruvilla and Eric Winquist had direct clinical
responsibility for patients in this study.}

\textbf{CORRESPONDENCE}

Bradley L. Urquhart, PhD

1151 Richmond Street

Medical Sciences Building Room 216

London, ON, N6A 5C1

E-mail: Brad.Urquhart@schulich.uwo.ca

Phone: (519) 661-3756

Fax: (519) 661-3827

\textbf{KEYWORDS:} Metabolomics, biomarker, cisplatin, acute kidney
injury

\textbf{Word count} : 3985 (Introduction, Methods, Results, Discussion)

\textbf{Table count} : 2

\textbf{Figure count} : 6

\textbf{What is already known about this subject:}

Nephrotoxicity is a dose-limiting complication of cisplatin treatment,
resulting in acute kidney injury (AKI) in approximately a third of
patients receiving cisplatin

Serum creatinine and urinary output are poor markers of kidney damage,
only detectable after severe functional impairment of kidneys

\textbf{What this study adds:}

\begin{itemize}
\tightlist
\item
  Patients who develop clinical AKI following cisplatin infusion exhibit
  metabolic differences compared to patients who do not
\item
  We reveal glycine, hippuric acid sulfate, 3-hydroxydecanedioc acid,
  and suberate as potential predictive markers of predisposition to
  cisplatin-induced AKI
\item
  Identification of several urine and serum metabolites exhibiting early
  alterations after cisplatin infusion are potential markers of
  subclinical nephrotoxicity
\end{itemize}

\textbf{ABSTRACT}

\textbf{Aim:} Cisplatin causes acute kidney injury (AKI) in
approximately one-third of patients. Serum creatinine and urinary output
are poor markers of cisplatin-induced (AKI). Metabolomics was utilized
to identify predictive or early diagnostic biomarkers of
cisplatin-induced AKI.

\textbf{Methods:} Thirty-one adult head and neck cancer patients
receiving cisplatin (dose [?] 70 mg m\textsuperscript{2 -1}) were
recruited for metabolomics analysis. Urine and serum samples were
collected prior to cisplatin (pre), 24-48 hours after cisplatin
(24-48h), and 5-14 days (post) after cisplatin. Based on serum
creatinine concentrations measured at the post timepoint, 11/31 patients
were classified with clinical AKI. Untargeted metabolomics was performed
using liquid chromatography-mass spectrometry.

\textbf{Results:} Metabolic discrimination was observed between ``AKI''
patients and ``no AKI'' patients at all timepoints. Urinary glycine,
hippuric acid sulfate, 3-hydroxydecanedioc acid, and suberate were
significantly different between AKI patients and no AKI patients prior
to cisplatin infusion. Urinary glycine and hippuric acid sulfate were
lower (-2.22-fold and -8.85-fold), whereas 3-hydroxydecanedioc acid and
suberate were higher (3.62-fold and 1.91-fold) in AKI patients relative
to no AKI patients. Several urine and serum metabolites were found to be
altered 24-48 hours following cisplatin infusion, particularly
metabolites involved with mitochondrial energetics.

\textbf{Conclusion:} We propose glycine, hippuric acid sulfate,
3-hydroxydecanedioc acid, and suberate as predictive biomarkers of
predisposition to cisplatin-induced AKI. Metabolites indicative of
mitochondrial dysfunction may serve as early markers of subclinical AKI.



\textbf{1. INTRODUCTION}

Cisplatin is an effective chemotherapeutic agent widely used for the
treatment of a variety of malignancies, including head and neck,
testicular, ovarian, cervical, and bladder cancers. Cisplatin is
primarily eliminated by the kidneys through tubular secretion and
glomerular filtration, and consequently accumulates in the kidneys to
cause kidney injury. Cisplatin-induced nephrotoxicity presents as acute
kidney injury (AKI) in approximately one-third of patients receiving
cisplatin. AKI is characterized as a rapid decline in kidney function
and has been associated with increased risk for chronic kidney disease,
major cardiovascular events, and mortality. Clinical diagnosis of AKI is
based on increases in serum creatinine (SCr) concentrations or a
decrease in urine output. However, serum creatinine and decreased urine
output are markers of functional impairment, only manifesting after
significant kidney injury and impairment of glomerular filtration.
Biomarkers for earlier detection or prediction of cisplatin-induced
nephrotoxicity are needed to guide cisplatin therapy, improve AKI
prognosis, and allow for development of nephroprotective interventions.

Novel markers for the early detection of AKI are currently under
investigation, including neutrophil gelatinase-associated lipocalin,
kidney injury molecule-1, cystatin C, tissue inhibitor of
metalloproteinase 2, and insulin-like growth factor binding protein 7.
However, these markers are not necessarily specific to AKI, do not allow
for discrimination of AKI etiology, and do not predict a patient's
predisposition to developing cisplatin-induced nephrotoxicity. There is
consensus that a combination of kidney function or damage markers should
be utilized to not only diagnose AKI, but to also discriminate AKI
etiology, assess severity, and evaluate the prognosis of AKI.

In this study, we utilized untargeted metabolomics to analyze urine and
serum samples from a cohort of adult head and neck cancer patients. We
aimed to identify both early diagnostic markers of cisplatin-induced
AKI, as well as predictive markers of patient predisposition to
cisplatin-induced AKI. Although untargeted metabolomics has been used in
rodent models of cisplatin-induced AKI, to our knowledge, our study is
the first to use untargeted metabolomics in a cohort of patients
receiving cisplatin therapy.

\textbf{2. METHODS}

\textbf{2.1 Study participants and sample collection}

Participants were recruited as part of a pilot for the ACCENT
(\textbf{A} \textbf{C} anadian study of \textbf{C} isplatin m\textbf{E}
tabolomics and \textbf{N} ephro\textbf{T} oxicity) study, an ongoing
Canada-wide, multi-center initiative to identify serum and urine
metabolites for the prediction or early diagnosis of cisplatin-induced
nephrotoxicity. The ACCENT study is being conducted in accordance with
the Medical Association Declaration of Helsinki. After receiving written
informed consent, 31 adult patients were recruited at the London
Regional Cancer Program (Victoria Hospital, London, ON, Canada) between
2018-2020. Ethics approval for this study was obtained from the Health
Sciences Research Ethics Board at the University of Western Ontario. The
cohort included adult patients (\textgreater{}18 years of age)
initiating cisplatin treatment (dose [?] 70 mg m\textsuperscript{2 -1})
for head and neck cancer. Exclusion criteria were chronic kidney disease
(GFR \textless{} 60 ml min\textsuperscript{-1}) at baseline, previous
exposure to cisplatin/other nephrotoxic drugs in the 2 weeks leading up
to cisplatin treatment, radiotherapy within 1 month prior to the study,
or previous hematopoietic stem cell transplant. Patient demographic
information (\textbf{Table 1} ) was recorded upon enrollment. Urine and
blood samples were collected from patients prior to (``pre''), 24--48
hours after (``24-48h''), and 5-14 days following (``post'') each
cisplatin infusion, and laboratory results/patient data were collected
for each sample collection timepoint. Samples were stored at -80\selectlanguage{ngerman}°C prior
to analysis. A detailed outline of the ACCENT study has been previously
published.

\textbf{2.2 AKI classification}

Patients were classified as ``no AKI'' or ``AKI'' based on the Kidney
Disease Improving Global Outcomes (KDIGO) guidelines, which defines AKI
as a \selectlanguage{english}[?] 1.5 times increase in serum creatinine (SCr) versus baseline or
as an increase in SCr \selectlanguage{english}[?] 26.5 \selectlanguage{greek}µ\selectlanguage{english}mol L\textsuperscript{-1}. Serum
creatinine concentrations measured at the post timepoint were compared
to those measured at the pre timepoint to classify AKI.

\textbf{2.3 Sample preparation for untargeted metabolomics}

For untargeted metabolomics analysis, serum and urine samples were
thawed on ice. Ice-cold acetonitrile (ACN) containing internal standards
chlorpropamide (5 \selectlanguage{greek}µ\selectlanguage{english}M), atenolol-d7 (1.8 \selectlanguage{greek}µ\selectlanguage{english}M), was used for protein
precipitation of urine and serum samples as described previously.

\textbf{2.4 Phenylisothiocyanate and 3-nitrophenylhydrazine
derivatization of serum and urine samples}

Derivatization was employed to increase the coverage of the metabolites
captured by metabolomics analysis. Phenylisothiocyanate (PITC) was used
to derivatize amino acids, amino acid derivatives, biogenic amines,
glucose/hexose, lipids, and acylcarnitines, whereas
3-nitrophenylhydrazine (3-NPH) was used to derivatize keto- and
carboxyl-containing compounds. The protocols for PITC and 3-NPH
derivatization were adapted from previously published work by Zheng et.
al.

\textbf{2.5 Chromatography and mass spectrometry}

Metabolites were separated by reverse-phase liquid chromatography using
a Waters ACQUITY UPLC HSS T3 column (1.8 \selectlanguage{greek}µ\selectlanguage{english}m particle size, 2.1 mm x 100
mm) and subsequent by time-of-flight mass spectrometry on a Waters
Xevo-G2S QTof mass spectrometer. Parameters for liquid chromatography
and mass spectrometer sample acquisition can be found in the
supplementary information (\textbf{Table S1, S2} ). LC-MS sample
injection order was randomized, and pooled samples were injected every
six sample injections.

\textbf{2.6 Data Processing}

Waters raw data files generated from the metabolomics analysis were
converted to mzData files using the convert.waters.raw R package. Using
the isotopologue parameter optimization (IPO) R package, the quality
control pooled sample injections were used to find the optimal peak
processing parameters, retention time corrections and grouping
parameters. Parameters generated from IPO were used for XCMS processing
of metabolomics data. The CAMERA package was applied to XCMS processed
features to annotate possible isotopes and adducts. The resulting data
was subsequently normalized to internal standards, and features with
\textgreater{}30\% relative standard deviation (rsd) within quality
control injections were excluded from analysis. Urine features were
normalized to their corresponding urinary creatinine and internal
standard signals, to account for differences in urine concentration.
Using the CAMERA package, features were grouped based on Pearson
correlation coefficients and retention time into ``pcgroups''. Within
each pcgroup, only the feature with the highest mean raw intensity was
kept for further data analysis. Duplicate features found in both the
untargeted and derivatized experiments were removed from the derivatized
dataset before analysis. The raw intensity values of all features were
log transformed using MetaboAnalyst 5.0, to remove heteroscedasticity
and correct for skewed data distribution. Any 0 values during log
transformation were treated as 1/5 of the minimum intensity values of
each feature. Log transformed feature intensity values were used for all
analyses unless stated otherwise.

\textbf{2.7 Statistical Analysis}

The EZInfo 3.0 software (Umetrics, Ume\selectlanguage{ngerman}å, Sweden) was used to perform
multivariate analysis on the metabolomics dataset. Data was centered,
pareto scaled and subsequently analyzed by principal component analysis
(PCA), an unsupervised approach to visualize the metabolic differences
between no AKI and AKI patients at each timepoint. Orthogonal partial
least squares discriminant analysis (OPLS-DA), a supervised
discriminatory analysis, was used for the pairwise discrimination of no
AKI and AKI patients at each timepoint. For each OPLS-DA, metabolites
were ranked by their variable importance in projection (VIP) values and
features with VIP values \selectlanguage{english}[?] 1 were considered to have discriminatory
value in discriminating between no AKI and AKI. This VIP filtering was
repeated until OPLS-DA model statistics (R2 and Q2 values) were
maximized to select for the most important features to annotate. The
final optimized OPLS-DA model was used to generate a list of features to
identify, using a VIP value threshold of \selectlanguage{english}[?] 1 and correlation (p(corr))
values less than -0.4 and greater than 0.4.

Features determined to have discriminatory value were analyzed by
two-way ANOVA with Benjamini-Hochberg false discovery rate (FDR)
correction. Individual features that were found to be significantly
different by AKI classification following two-way ANOVA and FDR
correction were further analyzed by pairwise t-tests comparing no AKI
and AKI patients at each timepoint, with p-values adjusted for multiple
comparisons using Bonferroni correction. To find metabolites altered
over time, serum and urine features were analyzed by one-way ANOVA with
FDR correction, followed by Tukey's test for metabolites significant by
one-way ANOVA after FDR correction. p\textless{}0.05 was considered as
significantly significant for all univariate data analysis.

Receiver operating characteristic (ROC) curves were generated and area
under the ROC (AUROC) values were calculated using MetaboAnalyst 5.0.

\textbf{2.8 Putative Metabolite Annotation and Identification}

The \emph{m/z} and fragmentation spectra of features were cross
referenced with online metabolite databases Human Metabolome Database
(HMDB), METLIN, or MassBank for putative metabolite annotation.
Analytical standards were purchased where available and analyzed in
parallel with biological samples to achieve a level 1 identification
based on the Chemical Analysis Working Group (CAWG) guidelines on
metabolomics reporting standards.

Derivatized features were identified by cross-referencing \emph{m/z} and
retention time with a panel of derivatized metabolites previously
generated in-house by the derivatization of the mass spectrometry
library of standards (MSMLS) kit, purchased from IROA technologies (Sea
Girt, NJ, USA).

\textbf{3. RESULTS}

\textbf{3.1 Patient demographics}

Thirty-one patients were recruited with the majority male (29/31) and
all patients were Caucasian. Twenty patients (64.5\%) were classified as
no AKI and 11 patients (35.5\%) were classified as AKI based on serum
creatinine concentrations measured at the post timepoint (\textbf{Table
1} ). Baseline demographics including age, BMI, baseline SCr/eGFR, and
cigarette packs/day were nearly identical between no AKI and AKI
patients (\textbf{Table 1} ). Out of the 11 patients in the AKI group,
seven patients had stage 2 AKI (2.0-2.9 times increase in SCr compared
to baseline) and 4 patients had stage 1 AKI (1.5-1.9 times increase in
SCr compared to baseline).

\textbf{3.2 Metabolomic profiling of no AKI vs. AKI patients}

Following XCMS processing of chromatographic data collected from
untargeted metabolomics, exclusion of features with high variability in
pooled quality control samples, and removal of potential
adducts/isotopes, 758 serum features and 484 urine features were
included for subsequent multivariate analysis. The derivatized
metabolomics dataset was similarly processed, without the removal of
potential adducts or isotopes. Additionally, any duplicate features
detected in both the derivatized and untargeted datasets were removed.
Ultimately, a total of 975 serum features and 2355 urine features
remained after processing of derivatized data. After cross referencing
the derivatized features with a library of 263 derivatized small
molecule standards, 35 serum and 117 urine features were matched
by\emph{m/z} and retention time with the library of standards.

To select the features most important in discriminating between no AKI
and AKI patients at each timepoint, OPLS-DA models were sequentially
generated, each time excluding features with VIP values \textless{} 1.
This sequential exclusion of features was repeated until model
statistics of OPLS-DA models were maximized. With the remaining
features, PCA score plots were generated to visualize the metabolic
differences between no AKI and AKI patients at each timepoint. Score
plots of urine samples showed moderate separation at the pre
(\textbf{Figure 1A} ) and 24-48h timepoints (\textbf{Figure 1B} ), and
clear separation at the post timepoint (\textbf{Figure 1C} ). In serum,
strong separation was observed in both the pre (\textbf{Figure 2A} ) and
post (\textbf{Figure 2C} ) timepoints, with moderate separation observed
at the 24-48h timepoint (\textbf{Figure 2B} ). Corresponding OPLS-DA
models comparing no AKI and AKI patients at each timepoint mirrored the
visual separation observed in the PCA score plots, with a high degree of
fit (R2Y) and predictive ability (Q2Y) at the post timepoint for both
urine and serum (\textbf{Figure 1F, 2F} ), as well as the pre timepoint
for serum (\textbf{Figure 2D} ). Moderate model statistics were observed
for the 24-48h timepoint for both urine and serum (\textbf{Figure 1E,
2E} ), as well as the pre timepoint for urine (\textbf{Figure 1D} ).
Features with 0.4 \textless{} p(corr) \textless{} -0.4 and VIP
\textgreater{} 1 in the OPLS-DA models were considered as important
discriminators of AKI and were thus followed up for identification.
Identified metabolites that were significantly different between no AKI
and AKI groups by two-way ANOVA are summarized in \textbf{Table 2} .

\textbf{3.3 Predictive and early urinary markers of cisplatin-induced
acute kidney injury}

In urine, glycine, 3-hydroxydecanedioc acid, hippuric acid sulfate, and
suberate were found to be significantly different between no AKI and AKI
patients prior to cisplatin infusion (\textbf{Figure 3} ). Levels of
hippuric acid sulfate were 8.85-fold and 9.08-fold lower in the AKI
group compared to the no AKI group at the pre and 24-48h timepoints,
respectively (\textbf{Table 2} , \textbf{Figure 3C, 3E} ).
3-hydroxydecanedioc acid and suberate were significantly higher in AKI
patients compared to no AKI patients at the pre timepoint (3.62-fold and
1.91-fold, respectively) and trended towards being higher at the 24-48h
timepoint (1.98-fold and 1.82-fold, respectively), though the difference
was not significant (\textbf{Table 2} , \textbf{Figure 3B, 3D} ).
Finally, glycine levels were 2.22-fold and 2.55-fold lower in AKI
patients relative to no AKI patients at the pre and post timepoints,
respectively (\textbf{Table 2, Figure 3A} ). Diagnostic performance of
the markers was assessed by calculation of AUROC. AUROC values \selectlanguage{english}[?] 0.7 are
generally considered to be acceptable discrimination. Though serum
creatinine was an excellent discriminatory marker of no AKI vs. AKI at
the post timepoint (AUROC of 0.947), it performed poorly (AUROC
\textless{} 0.7) at the pre and 24-48h timepoints (\textbf{Figure 3F} ).
Glycine, 3-hydroxydecanedioc acid, hippuric acid sulfate, and suberate
all had AUROC \textgreater{} 0.7 at the pre timepoint, with suberate
exhibiting the strongest performance (AUROC \textgreater{} 0.8). These
metabolites, with the exception of glycine, also had AUROC
\textgreater{} 0.7 at the 24-48h timepoint, with hippuric acid sulfate
and suberate possessing AUROC \textgreater{} 0.8.

\textbf{3.4 Late urinary markers of cisplatin-induced acute kidney
injury}

Aminoisobutanoate (\selectlanguage{english}|4.69-fold), betaine (\selectlanguage{english}|2.67-fold), glutamine
(\selectlanguage{english}|1.98-fold), glycolate (\selectlanguage{english}|2.71-fold), histidine (\selectlanguage{english}|2.63-fold),
indole-3-acetate (\selectlanguage{english}|4.03-fold), serine (\selectlanguage{english}|2.80-fold), taurine
(\selectlanguage{english}|2.56-fold), threonine (\selectlanguage{english}|2.90), and tyramine (\selectlanguage{english}|2.87-fold) were all
significantly lower at the post timepoint in the AKI group relative to
the no AKI group (\textbf{Table 2} , \textbf{Figure 4} ). With the
exception of aminoisobutanoate, all of the aforementioned metabolites
exhibited AUROC \selectlanguage{english}[?] 0.8 at the post timepoint, with AUROC of glycolate and
tyramine being greater than 0.9 (\textbf{Figure 4H).} Though not
statistically significant, betaine, indole-3-acetate, and threonine
trended lower in AKI patients compared to no AKI patients at the pre
timepoint with AUROC values \selectlanguage{english}[?] 0.7 (\textbf{Figure 4B, 4F, 4I} ).
Similarly, urine levels of aminoisobutanoate, indole-3-acetate, and
tyramine trended lower in AKI patients at the 24-48h timepoint with
AUROC \selectlanguage{english}[?] 0.7, though the difference was not statistically significant
(\textbf{Figure 4A, 4F, 4J} ).

\textbf{3.5 Late serum markers of cisplatin-induced acute kidney injury}

Serum metabolites that were significantly different between AKI and no
AKI were predominantly elevated in the AKI group (\textbf{Figure 5} ),
with the exception of glutamine, which was 1.55-fold lower in AKI
patients at the post timepoint (\textbf{Table 2} , \textbf{Figure 5B} ).
Serum levels of creatinine (\selectlanguage{english}|1.81-fold), linolenic acid (\selectlanguage{english}|1.97-fold),
octenoylcarnitine (\selectlanguage{english}|2.09-fold), octanoylcarnitine (\selectlanguage{english}|2.46-fold), indoxyl
sulfate (\selectlanguage{english}|2.87-fold), and N,N,N-trimethyl-L-alanyl-L-proline betaine
(TMAP, \selectlanguage{english}|1.87-fold) were significantly higher in AKI patients at the post
timepoint compared to no AKI patients (\textbf{Table 2, Figure 5A,
5C-5G} ). AUROC values for all serum metabolites at the post timepoint
were greater than 0.7, with octenoylcarnitine and TMAP having AUROC
\textgreater{} 0.8, and creatinine, octanoylcarnitine, and indoxyl
sulfate AUROC values \textgreater{} 0.9 (\textbf{Figure 5H} ).
Additionally, the AUROC values of ocanoylcarnitine at the pre timepoint
and indoxyl sulfate and TMAP at the 24-48h timepoint were greater than
0.7, though the differences were not statistically significant
(\textbf{Figure 5H} ).

\textbf{3.6 Alterations of urine and serum metabolites over time in
patients without clinical acute kidney injury}

Urine and serum samples from the no AKI group were analyzed via one-way
ANOVA to investigate the effects of cisplatin treatment independent of
AKI. Following cisplatin infusion, metabolites primarily followed one of
two trends: 1) an initial increase or decrease 24-48 hours after
cisplatin, and a subsequent return to baseline at the post timepoint; 2)
an initial increase or decrease at the 24-48h timepoint that was
sustained at the post timepoint. In urine, L-acetylcarnitine
(\selectlanguage{english}|3.64-fold), L-carnitine (\selectlanguage{english}|8.91-fold), 3-hydroxydecanedioc acid
(\selectlanguage{english}|2.42-fold), malate (\selectlanguage{english}|3.56-fold), pyruvate (\selectlanguage{english}|4.87-fold), and
valerylcarnitine (\selectlanguage{english}|4.76-fold), followed the first trend, where
metabolite levels were significantly increased 24-48h following
cisplatin treatment and returned to baseline levels by the post
timepoint (\textbf{Figure 6A, 6B, 6E, 6F, 6H} ). Serum levels of
L-arginine (\selectlanguage{english}|1.96-fold), L-carnitine (\selectlanguage{english}|1.40-fold), proline (\selectlanguage{english}|1.63-fold),
TMAP (\selectlanguage{english}|4.14-fold), and valerylcarnitine (\selectlanguage{english}|1.79-fold) followed the same
pattern, with a significant increase at 24-48h, and a return to baseline
at the post timepoint (\textbf{Figure 6I, 6K, 6N, 6O, 6P} ). Conversely,
levels of urine indole-3-acetate (\selectlanguage{english}|1.82-fold) and serum cortisol
(\selectlanguage{english}|5.18-fold) were significantly lower 24-48h following cisplatin
treatment, with a subsequent return to baseline by the post timepoint
(\textbf{Figure 6D, 6J} ). Urinary succinate and serum 4-hydroxycinammic
acid and phenylalanine followed the second trend, where metabolite
levels were significantly altered 24-48h following cisplatin infusion
(\selectlanguage{english}|1.88-fold for succinate; \selectlanguage{english}|1.56-fold and \selectlanguage{english}|1.43-fold for
4-hydroxycinammic acid and phenylalanine, respectively), with the
alterations being sustained at the post timepoint (\textbf{Figure 6G,
6L, 6M} ). Similar metabolite alterations were observed over time for
patients in the AKI group (\textbf{Figure S1} ).

\textbf{4. DISCUSSION}

Untargeted metabolomics was employed in urine and serum samples of adult
head and neck cancer patients receiving cisplatin for the identification
of predictive or early biomarkers of cisplatin-induced AKI. This allowed
for the identification of important metabolites that are early or
predictive biomarkers of cisplatin AKI. Future metabolomics studies in
larger adult and pediatric cohorts recruited as part of the ACCENT study
will employ fully quantitative metabolite analysis. Metabolomics has
been used to investigate cisplatin-induced acute kidney injury in the
past but has predominantly been utilized in rodent models. To our
knowledge, this study is the first metabolomic investigation of
cisplatin-induced AKI in human patients, providing insight into the
metabolic differences present between patients who present with clinical
AKI upon cisplatin infusion and those who do not, in addition to
highlighting the early metabolic alterations induced by cisplatin.

Four urinary biomarkers were identified as predictive markers of
clinical AKI: glycine, 3-hydroxydecanedioc acid, hippuric acid sulfate,
and suberate. All four metabolites were significantly different between
the no AKI and AKI groups at the pre timepoint with fold changes of
-2.2-fold, 3.62-fold, 8.85-fold, and 1.91-fold, respectively, in AKI
patients relative to no AKI patients (\textbf{Table 2} ).

Glycine is an amino acid component of the potent antioxidant molecule
glutathione and has been associated with beneficial effects in reducing
oxidative stress. Alterations in glycine levels have been observed
previously in a mouse model of ischemia-reperfusion AKI, where glycine
levels were decreased in kidney and heart tissues following ischemic
AKI. A metabolomic investigation of urine samples from combat casualties
also revealed that lower levels of glycine were associated with need for
renal replacement therapy, and glycine levels were higher in patients
with moderate to severe AKI compared to mild AKI. In both cases, it was
suggested that decreases in glycine levels were associated with
upregulation of glutathione production under oxidative stress.
Furthermore, glycine was shown to protect against cisplatin
nephrotoxicity and ischemia reperfusion renal injury \emph{in vivo} when
administered to rats before cisplatin treatment or ischemic insult.
Cisplatin is well documented to cause mitochondrial dysfunction and
oxidative stress, and availability of glycine may be an important factor
in antioxidant defense against cisplatin-induced oxidative stress.

3-hydroxydecanedioc acid and suberate are dicarboxylic acids that have
been associated with fatty acid \selectlanguage{greek}β-\selectlanguage{english}oxidation disorders. Increased urinary
excretion of 3-hydroxydecanedioc acid and suberate have been used to
diagnose medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and
indicates a block in fatty acid oxidation. Dysfunctional mitochondrial
fatty acid oxidation is believed to be a crucial mechanism in
cisplatin-induced AKI. Cisplatin has previously been shown to inhibit
mitochondrial fatty acid \selectlanguage{greek}β-\selectlanguage{english}oxidation by deactivating PPAR-\selectlanguage{greek}α, \selectlanguage{english}a crucial
nuclear receptor in the regulation \selectlanguage{greek}β-\selectlanguage{english}oxidation. An accumulation of
intracellular acyl-CoAs due to disorders of fatty acid \selectlanguage{greek}β-\selectlanguage{english}oxidation is
associated with lipotoxicity and detrimental to mitochondrial function.
Additionally, serum levels of acylcarnitines octanoylcarnitine and
octenoylcarnitine were significantly higher at the post timepoint in AKI
patients relative to the no AKI group and showed increased trends in the
pre and 24-48h timepoints, though the differences were not significant
(\textbf{Figure 5D, 5E} ). Elevation of serum acylcarnitines is also a
marker of dysfunction in fatty acid \selectlanguage{greek}β-\selectlanguage{english}oxidation. Taken together, the
elevation of urinary 3-hydroxydecanedioc acid, urinary suberate, and
serum acylcarnitines in AKI patients suggest a lower capacity for fatty
acid oxidation in patients who develop clinical AKI following cisplatin
therapy. An underlying diminished capability for fatty acid oxidation
may leave these patients more susceptible towards cisplatin-induced
mitochondrial dysfunction and accumulation of toxic lipid compounds.

Hippuric acid sulfate was identified to potentially be both a predictive
and early diagnostic marker of cisplatin-induced AKI. Hippuric acid
sulfate is not well studied, and very few articles have been published
regarding this metabolite. Hippuric acid sulfate is a sulfated
derivative of hippuric acid, a uremic toxin that accumulates in chronic
kidney disease (CKD). Hippuric acid is derived from the conversion of
dietary polyphenols into benzoic acid by the gut microbiome, followed by
conjugation with glycine by hepatic or renal glycine-N-acyltransferase.
Though hippuric acid has been implicated in both CKD and AKI, hippuric
acid sulfate has yet to be implicated with kidney disease.

Cisplatin is well known to be nephrotoxic, manifesting as AKI in
approximately one third of patients. It is likely that patients who
don't develop AKI are able to withstand the nephrotoxic insult mediated
by cisplatin. To evaluate the metabolic response to cisplatin in
patients that don't progress to AKI, we evaluated metabolic alterations
in no AKI patients over the three timepoints of this study. This
analysis revealed cisplatin induces early metabolic changes in both the
urine and serum even in patients who don't progress to AKI
(\textbf{Figure 6} ). Many of the metabolites found to be altered at the
24-48h were intermediates of the citric acid cycle or associated with
fatty acid oxidation, further emphasizing the central role of
mitochondrial dysfunction in cisplatin-induced nephrotoxicity. Of
special interest was TMAP, a dipeptide biomarker of reduced kidney
function in CKD, which was elevated in both no AKI and AKI patients at
the 24-48h timepoint but only remained elevated at the post timepoint in
patients with clinical AKI (\textbf{Figure 5G} ). These findings are in
accordance with the concept of subclinical AKI induced by cisplatin
whereby there is an increase in AKI biomarkers without presentation of
clinical AKI. In other words, subclinical AKI is kidney damage without
substantial loss of function. Though there has been some work
highlighting potential prognostic benefits of using markers of
subclinical AKI, the clinical relevance of subclinical AKI is unclear.
Further investigation of these early subclinical markers of AKI may
provide further insight into the mechanisms of cisplatin nephrotoxicity.

One strength of our study was the high degree of similarity in baseline
patient demographics such as age, BMI, ethnicity, and baseline SCr/eGFR
between the AKI and no AKI groups, minimizing interindividual
variability that could potentially confound metabolic profiling
(\textbf{Table 1} ). Furthermore, the collection of three separate
timepoints allowed for comprehensive metabolic profiling of patients
prior to and shortly after cisplatin infusion, as well as upon
establishment of clinical AKI (or lack thereof).

There were some limitations to this study. Firstly, the sample size for
our study was relatively small, ranging from 11 in the AKI group and 20
in the no AKI group. Despite this small sample size, a number of key
metabolic alterations were characterized. Future metabolomics studies
with larger discovery cohorts may help extract more distinct and robust
differences between AKI and no AKI patients. Furthermore, the incidence
of head and neck cancer is 2-4-fold higher in men compared to women, and
this disparity was reflected in our cohort. As only 2 out of 31 patients
were female, sex differences could not be investigated in our analysis.
Similarly, all 31 patients in this study were Caucasian which limits the
generalizability of our findings to other ethnicities.

Though serum creatinine remains the principal biomarker in AKI
diagnosis, cisplatin nephrotoxicity occurs prior to the detection of
elevated serum creatinine. Accordingly, there is a need for biomarkers
capable of early diagnosis of AKI or prediction of AKI onset prior to
cisplatin therapy. In this study, we identified glycine,
3-hydroxydecanedioc acid, hippuric acid sulfate, and suberate as
potential predictive markers of clinical cisplatin-induced AKI.
Additionally, we provided insight into early metabolic alterations
following cisplatin infusion. Further investigations are necessary to
validate the applicability and clinical utility of these proposed
biomarkers. Future metabolomics studies are planned in large discovery
and validation cohorts to further investigate the metabolic effects of
cisplatin and elucidate the underlying metabolic differences between
patients who present with clinical AKI and patients who do not.

\textbf{AUTHOR CONTRIBUTIONS}

B.L.U., M.Z., and T.D.B. designed the study. S.K., E.W., S.W., M.B.,
J.L., M.J.R. and L.N.F. were responsible for research coordination and
sample collection. Y.J.L. and S.G.X. performed the research and data
analysis. Y.J.L. drafted the manuscript. B.L.U. edited the manuscript
for final submission. All authors have reviewed and approved the
submitted manuscript.

\textbf{ACKNOWLEDGEMENTS}

The authors are grateful to the support from the nurses and staff at the
London Regional Cancer Program for support of this study.

\textbf{FUNDING INFORMATION}

This study was funded by a project grant from the Canadian Institutes of
Health Research and an Accelerator grant from Western University.

\textbf{CONFLICTS OF INTEREST}

The authors declare no conflicts of interest.

\textbf{DATA AVAILABILITY}

Data collected in this study are available upon request from the
corresponding author Bradley L. Urquhart.

\textbf{ORCID}

Bradley L. Urquhart: 0000-0001-8324-1472

\textbf{REFERENCES}

\textbf{TABLES}

\textbf{Table 1.} Baseline demographics of study population. Patient
serum creatinine (SCr) levels at the post timepoint were used to
classify patients as no AKI (n=20) or AKI (n=11) according to KDIGO
guidelines. Patient sex is presented as a ratio of males to females
(M/F). Ethnicity is given as a ratio of Caucasian, Black, Asian,
Indigenous, and Other (C/B/A/I/O). Demographic characteristics of age,
body mass index (BMI), baseline SCr, baseline eGFR, and smoking are
presented as mean \selectlanguage{ngerman}± standard deviation. Statistical differences between
no AKI and AKI groups were determined using unpaired t-tests for each
characteristic, with *p\textless{}0.05. Groups were not statistically
different from each other for any of the baseline characteristics.\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Sex (M/F)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Age (years)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Body mass index (kg/m\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Ethnicity (C/B/A/I/O)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Baseline SCr (\selectlanguage{greek}µ\selectlanguage{english}mol/L)\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Baseline eGFR (mL/min/1.73m\textsuperscript{2})\strut
\end{minipage} & \begin{minipage}[b]{0.10\columnwidth}\raggedright\strut
Smoking (packs/day)\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{No AKI} (n=20)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
19/2\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
59.5 (7.1)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26.7 (5.2)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
21/0/0/0/0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
69.2 (13.4)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
101.9 (9.0)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.8 (0.6)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{AKI} (n=11)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10/0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
61.7 (3.8)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
26.7 (6.0)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10/0/0/0/0\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
63.2 (10.4)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
104.3 (6.8)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0.7 (0.6)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Summary of metabolite alterations in AKI patients
compared to no AKI patients. Fold change was calculated using mean raw
intensity values for each metabolite. All observed \emph{m/z} values
were \textless{}5 ppm mass error relative to the theoretical \emph{m/z}
.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllllllll@{}}
\toprule
\begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Metabolite Identity}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{ID Level}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Chemical Formula}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Observed \emph{m/z}}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{t\textsubscript{R}} \textbf{(min)}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Sample Type}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage} & \begin{minipage}[b]{0.06\columnwidth}\raggedright\strut
\textbf{Change in AKI Patients vs. No AKI Patients}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Pre}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Pre}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Pre}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{24-48 hours}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{24-48 hours}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{24-48 hours}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Post}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Post}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Post}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Direction}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Fold}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Direction}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Fold}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Direction}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{Fold}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\textbf{p}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Aminoisobutanoate (NPH)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C4H9NO2 + C6H5N3O {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
237.0987\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.44\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.08\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.7735\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
3.58\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1717\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4.69\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0115\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Betaine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C5H11NO2 {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
118.0863\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.62\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.08\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.22\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1569\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.12\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.7524\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.21\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.5552\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.79\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1653\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.67\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Creatinine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C4H7N3O {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
114.0664\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.61\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.14\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.5215\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.16\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.4374\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.81\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4.16E-06\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Glutamine (fragment)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C5H7NO3 {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
130.0499\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.56\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.32\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.4544\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.13\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.55\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0179\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.38\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.6298\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.17\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3192\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.98\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0231\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Glycine (PITC)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C2H5NO2 + C7H5NS {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
211.0542\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.94\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.09\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.45\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.4264\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.51\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.22\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0412\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.99\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1871\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.55\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0369\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Glyocolate (NPH)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C2H4O3 + C6H5N3O {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
210.0514\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.74\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.04\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.23\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.7735\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.47\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3539\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.59\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0829\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.27\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.2142\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.71\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4.12E-05\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Hippuric acid sulfate\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C9H9NO6S {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
258.0072\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.36\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
8.85\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0437\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
9.08\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0071\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.82\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.9553\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Histidine\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C6H9N3O2 {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
154.0616\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.53\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.44\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.2450\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.48\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0418\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.63\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0089\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
3-Hydroxydecan edioc acid\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C10H18O5 + C6H5N3O {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
217.1075\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.76\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
3.62\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0305\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.98\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0984\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.90\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3837\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Indole-3-acetate (NPH)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C10H9NO2 + C6H5N3O {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
309.0987\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.55\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.77\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0549\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.91\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1226\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4.03\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0067\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Indoxyl sulfate\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C8H7NO4S {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
212.0017\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.87\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.16\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.68\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.3071\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2.87\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
9.71E-05\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.75\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3584\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.54\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.5244\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.11\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3951\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Linolenic acid\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C18H30O2 {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
279.2319\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
4.36\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.60\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.3398\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.40\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.97\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0428\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
L-Octanoylcarni tine\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C15H29NO4 {[}M+Na{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
310.2015\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2.00\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.29\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.5105\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.14\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.5053\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2.46\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
4.87E-05\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.31\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.2154\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.08\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.79\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0979\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2-Octenoylcarni tine\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C15H27NO4 {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
286.2016\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.88\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.40\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.4940\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1.18\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.6974\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
2.09\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.0080\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.10\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.9346\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.07\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.47\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0250\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serine (PITC)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C3H7NO3 + C7H5NS {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
241.0647\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.86\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.39\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.2089\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.37\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.6135\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.80\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0176\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Suberate (NPH)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C8H14O4 + C6H5N3O {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
443.1676\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.59\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.91\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0395\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.82\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0673\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.05\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Taurine\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
C2H7NO3S {[}M-H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
124.006\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
0.57\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.06\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.16\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.07\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.5806\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.56\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0023\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Threonine (PITC)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C4H9NO3 + C7H5NS {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
255.0803\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.86\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.03\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.2927\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.35\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.7733\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.90\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0201\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
TMAP\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C11H20N2O3 {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
229.1554\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.15\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.58\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.30\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.1353\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.87\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0195\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.23\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.9216\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.11\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.27\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.4621\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Tyramine (PITC)\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
C8H11NO + C7H5NS {[}M+H{]}\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
273.1061\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.52\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Serum\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
-\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
Urine\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.05\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.0000\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
1.45\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
0.0521\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
|\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.87\strut
\end{minipage} & \begin{minipage}[t]{0.04\columnwidth}\raggedright\strut
2.38E-07\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{FIGURE LEGENDS}

\textbf{Figure 1} . Principle component analysis (A-C) and orthogonal
parti al least squares discriminant analysis (D-F) scores plots
comparing urine samples from no AKI (black) and AKI (red) patients at
each timepoint: (A, D) pre, (B, E) 24-48h, and (C, F) post. Model
statistics for OPLS-DA were as follows: (D) pre: R2Y = 0.784, Q2Y =
0.389 (E) 24-48h: R2Y = 0.752, Q2Y = 0.384 (F) post: R2Y = 0.969, Q2Y =
0.700. n=18-20 for no AKI patients and n=10-11 for AKI patients.

\textbf{Figure 2.} Principle component analysis (A-C) and orthogonal
partial least squares discriminant analysis (D-F) scores plots comparing
serum samples from no AKI (black) and AKI (red) patients at each
timepoint: (A, D) pre, (B, E) 24-48h, and (C, F) post. Model statistics
for OPLS-DA were as follows: (D) pre: R2Y = 0.991, Q2Y = 0.668 (E)
24-48h: R2Y = 0.776, Q2Y = 0.317 (F) post: R2Y = 0.995, Q2Y = 0.813.
n=18-20 for no AKI patients and n=10-11 for AKI patients.

\textbf{Figure 3} . (\textbf{A-D} ) Log transformed relative intensity
of urine features significantly different between no AKI and AKI
patients at the pre timepoint. Data are presented as box and whisker
plots, where boxes represent the median and interquartile range (IQR),
and whiskers represent 1.5x IQR. \textbf{(F)} Diagnostic performance of
urine metabolites as assessed by the area under the receiver operating
curve. Statistical analysis was performed using two-way ANOVA,
*p\textless{}0.05, **p\textless{}0.01 compared to no AKI patients at
each timepoint. n=18-20 for no AKI patients and n=10-11 for AKI
patients.

\textbf{Figure 4.} (\textbf{A-J} ) Log transformed relative intensity of
urine features significantly different between no AKI and AKI patients
at the post timepoint. Data are presented as box and whisker plots,
where boxes represent the median and interquartile range (IQR), and
whiskers represent 1.5x IQR. \textbf{(K)} Diagnostic performance of
urine metabolites as assessed by the area under the receiver operating
curve. Statistical analysis was performed using two-way ANOVA,
*p\textless{}0.05, **p\textless{}0.01, ***p\textless{}0.001 compared to
no AKI patients at each timepoint. n=18-20 for no AKI patients and
n=10-11 for AKI patients.

\textbf{Figure 5.} (\textbf{A-G} ) Log transformed relative intensity of
serum features significantly different between no AKI and AKI patients
at the post timepoint. Data are presented as box and whisker plots,
where boxes represent the median and interquartile range (IQR), and
whiskers represent 1.5x IQR. \textbf{(H)} Diagnostic performance of
urine metabolites as assessed by the area under the receiver operating
curve. Statistical analysis was performed using two-way ANOVA,
*p\textless{}0.05, **p\textless{}0.01, ***p\textless{}0.001 compared to
no AKI patients at each timepoint. n=18-20 for no AKI patients and
n=10-11 for AKI patients.

\textbf{Figure 6.} Log transformed relative intensity of urine and serum
features significantly altered over time in no AKI patients. Data are
presented as box and whisker plots, where boxes represent the median and
interquartile range (IQR), and whiskers represent 1.5x IQR. Statistical
analysis was performed using one-way ANOVA, *p\textless{}0.05,
**p\textless{}0.01, ***p\textless{}0.001 compared to saline control at
each timepoint, n=18-20.\selectlanguage{english}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-4/Figure-4}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

\end{center}
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\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

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\end{document}

