Detection of Localized Hepatocellular Amino Acid Kinetics by using Mass Spectrometry Imaging of Stable Isotopes

Article abstract of DOI:10.1002/anie.201702669

Mass spectrometry imaging (MSI) simultaneously detects and identifies the spatial distribution of numerous molecules throughout tissues. Currently, MSI is limited to providing a static and ex vivo snapshot of highly dynamic systems in which molecules are constantly synthesized and consumed. Herein, we demonstrate an innovative MSI methodology to study dynamic molecular changes of amino acids within biological tissues by measuring the dilution and conversion of stable isotopes in a mouse model. We evaluate the method specifically on hepatocellular metabolism of the essential amino acid l-phenylalanine, associated with liver diseases. Crucially, the method reveals the localized dynamics of l-phenylalanine metabolism, including its in vivo hydroxylation to l-tyrosine and co-localization with other liver metabolites in a time course of samples from different animals. This method thus enables the dynamics of localized biochemical synthesis to be studied directly from biological tissues.

Full text of DOI:10.1002/anie.201702669

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Communications
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International Edition: DOI: 10.1002/anie.201702669
German Edition: DOI: 10.1002/ange.201702669
Isotopic Labeling
Detection of Localized Hepatocellular Amino Acid Kinetics by using
Mass Spectrometry Imaging of Stable Isotopes
Martijn Arts+, Zita Soons+,* Shane R. Ellis, Keely A. Pierzchalski, Benjamin Balluff,
Gert B. Eijkel, Ludwig J. Dubois, Natasja G. Lieuwes, Stijn M. Agten, Tilman M. Hackeng,
Luc J. C. van Loon, Ron M. A. Heeren, and Steven W. M. Olde Damink
Abstract: Mass spectrometry imaging (MSI) simultaneously
detects and identifies the spatial distribution of numerous
molecules throughout tissues. Currently, MSI is limited to
providing a static and ex vivo snapshot of highly dynamic
systems in which molecules are constantly synthesized and
consumed. Herein, we demonstrate an innovative MSI meth-
odology to study dynamic molecular changes of amino acids
within biological tissues by measuring the dilution and
conversion of stable isotopes in a mouse model. We evaluate
the method specifically on hepatocellular metabolism of the
essential amino acid l-phenylalanine, associated with liver
diseases. Crucially, the method reveals the localized dynamics
of l-phenylalanine metabolism, including its in vivo hydrox-
ylation to l-tyrosine and co-localization with other liver
metabolites in a time course of samples from different animals.
This method thus enables the dynamics of localized biochem-
ical synthesis to be studied directly from biological tissues.
systems and cannot elucidate dynamic chemical conversions
within the tissue. As MSI evolves to study localized biochem-
ical pathways and their alteration in disease, spatially resolved
dynamics of molecular synthesis and turnover becomes
critical. Incorporation of stable isotopes into tissues, and
thus their biosynthetic processes, provides a unique oppor-
tunity to study molecular kinetics in complex biological
systems. Conventional MS modalities with the capability to
analyze metabolic fluxes generally use stable-isotope-labeled
precursor molecules.[2] To date, such approaches have been
widely employed using tissue/cell homogenates but are not
able to detect spatial changes in heterogeneous tissues.[3]
Recent work has extended this approach to MSI to reveal
localized incorporation throughout tissues. For example,
nanoscale secondary ion mass spectrometry (SIMS) is
employed to study localized turnover and isotopic incorpo-
ration in a variety of systems with subcellular resolution (ca.
50–100 nm).[4] However, this approach suffers from the
extensive fragmentation induced under dynamic SIMS con-
ditions. Typically, such experiments monitor fragment signals
of 13CH and 15N, making it difficult to assign incorporation to
a specific biochemical precursor, especially if endogenous
metabolism of the initially labeled compounds occurs. Louie
et al. have developed similar approaches to study localized
phospholipid synthesis following D2O administration using
softer matrix-assisted laser desorption ionization (MALDI).[5]
It was demonstrated that rates of lipid synthesis are altered in
different tumor cell subpopulations, thus highlighting the
need to study localized synthesis kinetics in tumor metabo-
lism. In addition, MALDI-MSI in combination with stable
isotopes has been applied to study the formation of athero-
sclerotic plaques[6] and the nitrogen cycle within plants.[7]
In this paper, we present a new method based on stable
isotope-labeling, which enables detection of localized amino
acid metabolism over time for the first time. Stable isotopes of
l-phenylalanine (Phe) are commonly used in clinical studies
to measure protein turnover.[8] In liver, Phe is enzymatically
hydroxylated into l-tyrosine (Tyr), and altered levels are
related to nonalcoholic steatohepatitis, nonalcoholic fatty
M
ass spectrometry imaging (MSI) enables the study of
molecular distributions and processes throughout biological
tissues.[1] Typically performed ex vivo on frozen sections, it
provides only a static snapshot of inherently highly dynamic
[*] M. Arts,[+] Dr. Z. Soons,[+] Prof. Dr. S. W. M. Olde Damink
Department of General Surgery (NUTRIM), Maastricht University
Postbus 616, 6200 MD, Maastricht (The Netherlands)
E-mail: zita.soons@maastrichtuniversity.nl
Prof. Dr. S. W. M. Olde Damink
Department of General, Visceral and Transplantation Surgery,
University Hospital RWTH Aachen
52075 Aachen (Germany)
Dr. S. R. Ellis, Dr. K. A. Pierzchalski, Dr. B. Balluff, G. B. Eijkel,
Prof. Dr. R. M. A. Heeren
Maastricht MultiModal Imaging Institute (M4I), Division of Imaging
Mass Spectrometry, Maastricht University
Postbus 616, 6200 MD, Maastricht (The Netherlands)
Dr. S. M. Agten, Prof. Dr. T. M. Hackeng
Department of Biochemistry (CARIM), Maastricht University
Postbus 616, 6200 MD, Maastricht (The Netherlands)
Prof. Dr. L. J. C. van Loon
Department of Human Biology and Movement Sciences (NUTRIM),
Maastricht University
liver disease, cirrhosis, and several cancer phenotypes.[9]
A
bolus of ring-13C6-labeled l-phenylalanine (13C6-Phe) was
injected into 11 mice and healthy liver tissue was collected at
three time points (10, 30, and 60 min) after injection to track
the incorporation of 13C6-Phe into tissue using MALDI. As
a control, four mice were injected with saline solution and
sacrificed at 10 min (sham). All measurements were per-
formed using four biological and two technical replicates.
Postbus 616, 6200 MD, Maastricht (The Netherlands)
Dr. L. J. Dubois, N. G. Lieuwes
Department of Radiation Oncology (MAASTRO, GROW)
Maastricht University
Postbus 616, 6200 MD, Maastricht (The Netherlands)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
https://doi.org/10.1002/anie.201702669.
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To address the low ionization efficiency of many amino
acids with MALDI, we performed on-tissue derivatization
using the reagent p-N,N,N-trimethylammonioanilyl N’-
hydroxysuccinimidyl carbamate iodide (TAHS).[10] This ena-
bled the sensitive detection of a variety of free amino acids
and their labeled analogues as well as a suite of amine-
containing metabolites due to the addition of a quaternary
nitrogen group. Tissues were imaged using MALDI-Fourier
transform ion cyclotron resonance (FTICR) mass spectrom-
etry following on-tissue derivatization. The experimental
workflow is summarized in the Supporting Information,
Figures S1 and S2. High resolution (25 mm) MSI data is
provided in Figure S3 to demonstrate the preservation of
tissue distributions during the sample preparation steps. Phe,
Tyr, 13C6-Phe, and 13C6-Tyr could not be detected in non-
derivatized tissue 10 minutes after tracer injection. In con-
trast, the derivatization enabled the detection throughout the
tissues (Figure 1). Representative spectra for labeled and
unlabeled Phe are shown in the Supporting Information,
Figures S4A and S3B. In addition, TAHS derivatization
enabled the simultaneous detection of approximately
60 amino metabolites in liver tissue (Supporting Information,
Table S1 and S2). Using an accurate mass tolerance of
< 1 ppm, each m/z value could be assigned to a unique
metabolite. Structural assignments of TAHS-derivatized 13C6-
Phe and 13C6-Tyr were also supported by tandem mass
spectrometry, which revealed a characteristic TAHS fragment
for derivatized metabolites (Figure S4). The detection of 13C6-
Tyr (Figure 1B) indicated that 13C6-labeled Phe was metab-
olized to 13C6-Tyr through active hydroxylation in liver tissue.
Localized enrichment of 13C6-Phe and 13C6-Tyr in tissue
was determined by calculating the ratio of labeled to total
(labeled + unlabeled) peaks, also referred to as the molar
percentage excess (MPE; Figure 1C and Supporting Infor-
mation). MPE was applied to measure the incorporation of
13C6-Phe into liver and adjacent muscle and pancreas tissue.
Computed for each location in 150-mm increments, MPE
values varied throughout the tissue (Figure 1C). Although
ionization efficiency and matrix effects can distort the
absolute detection and quantification of metabolites (Sup-
porting Information, Figures S5A and S5B),[11] MPE values
represent a robust measure (Figure S5C), since the tracer and
target have the same chemical properties and are ionized in
the same way.[12] We obtained the same average MPE values
for each biological replicate (n = 4), measured in duplicate
and in two randomized batches (standard error < 1.2%)
(Supporting Information, Figure S6). Such reproducible and
quantitative measurements are important to determine the
Figure 1. Representative MALDI-FTICR-MS images with (+TAHS) and without (ÀTAHS) derivatization showing the direct localization of the
in vivo metabolism of Phe (left) and Tyr (right) in liver sections at 10 min after injection with 13C6-Phe. (Top) l-Phe is metabolized to l-Tyr in the
liver by phenylalanine hydroxylase. Consecutive sections with identical intersections, rotated 1808, are denoted by similarly colored asterisks.
A) Molecular ion [M+TAHS]+ image of derivatized Phe and Tyr; B) molecular ion [M+TAHS]+ image of derivatized 13C6-Phe and de novo
synthesized 13C6-Tyr; C) MPE of 13C6-Phe and 13C6-Tyr shows localized enrichment; D) H&E image of a representative tissue section.
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kinetics of biologically altered amino acids, which are
Spectra were generated from homogenized liver tissue
implicated in impairments in liver function.[13]
using gas chromatography mass spectrometry (GC-MS; Fig-
ure S6) and confirmed that 13C6-Phe was successfully incor-
porated into tissue. Importantly, GC-MS measurements gave
similar enrichment trends across the time-series as the MSI
data, validating the accuracy of the MSI approach. These
average enrichments may reflect the presence of other cells
(muscle, pancreas, inflammatory cells) as the measurements
were performed with homogenized tissues. This explains the
slight differences in absolute enrichment between the meth-
ods.
The enrichment precision for MSI data was evaluated by
comparing the detected natural enrichments of Phe and Tyr
with their theoretical isotopic enrichments in tissues (M + 1
vs. M + 0, M + 2 vs. M + 0, and M + 3 vs. M + 0). In particular,
the highly abundant Phe allowed more accurate reconstruc-
tion of low-abundance natural 13C isotopes, whereas lower
abundant isotopes (for example, Tyr M + 3) may not be
detected in all pixels due to a detection limit (Supporting
Information, Table S3). Overall, an isotope ratio of more than
2% is required to facilitate precise detection.
We measured the fraction of molecules isotopically
labeled with 13C6 at 10, 30, and 60 min after bolus injection
and compared these with control tissues to determine the
kinetics of 13C6-Phe. The MPE values for 13C6-Phe and 13C6-
Tyr showed clear abundance throughout the liver at 10 min,
indicating the transport and conversion of these labeled
amino acids (Figure 2B,C). At 30 min, 13C6-Tyr levels were
diminished, and 13C6-Phe levels were cleared in liver tissue. By
60 min, both 13C6-Phe and 13C6-Tyr were cleared from liver
tissue, indicating their predominant metabolism and incorpo-
ration into liver proteins (assessed by GC-C-IRMS in the
Supporting Information, Figure S7). De novo hydroxylation
was determined by the MPE ratio 13C6-Tyr:13C6-Phe to be
approximately 1 (Figure 2D). In addition, we measured
enrichments in adjacent muscle and pancreas tissue (Fig-
ure S8). The MPE was lower for 13C6-Tyr compared to 13C6-
Phe in the adjacent tissue at 10 min, which might be due to the
absence of enzymes hydroxylating 13C6-Phe into 13C6-Tyr in
non-hepatic tissue.[14] At 30 min, 13C6-Phe and 13C6-Tyr levels
were elevated in adjacent pancreatic tissue (*, Figure S8A).
These data suggest 13C6-Phe and 13C6-Tyr may have different
uptake and diffusion mechanisms in pancreas compared to
liver tissue.[15]
Principal component analysis combined with linear dis-
criminant analysis (PCA-LDA) was applied to MSI data on
a pixel-by-pixel basis to identify the main metabolic differ-
ences across time points associated with Phe metabolism. The
first discriminant function
captures
differences
between matrix and tissue
(data not shown). The
second discriminant func-
tion (Supporting Informa-
tion, Figure S9) demon-
strated that the greatest
metabolic differences were
observed
10 min
after
tracer infusion, followed
by 30 min, and almost
reverted back to normal
after 60 min, which is in
agreement with the enrich-
ments by MPE (Figure 2).
The loadings of the nega-
tive discriminant (from the
10- and 30-min samples)
comprise the expected
metabolites Phe, 13C6-Phe,
and Tyr, metabolites gener-
ated from Tyr, and poly-
amine synthesis (Table S4).
The positive function (from
the sham-control and 60-
min samples) predomi-
nantly comprises other
amino acids, pathways
related to oxidative stress
(both the substrates and
product of glutathione syn-
thase), and the urea cycle.
We applied PCA to each
Figure 2. Detection of localized Phe and Tyr kinetics in liver tissue at three time points at a 150-mm spatial
resolution. Sections taken from mice 10 min after injection with saline (sham) are shown as a control.
A) H&E stains; B–D) dynamic MS images for 13C6-Phe MPE, 13C6-Tyr MPE, and hepatic hydroxylation
(Hydrox), respectively.
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pixel of each sample to gain further insight into local
Keywords: amino acids · dynamics · isotopic labeling ·
mass spectrometry imaging · metabolism
metabolic changes. As an example, the first principal compo-
nent in the 30-min sample correlates with different anatom-
ical structures, liver tissue in the positive component and
pancreatic tissue in the negative component (Supporting
Information, Figure S10). This principal component is asso-
ciated with taurine and glutamic acid in liver and Phe and
13C6-Phe in pancreatic tissue. These findings may be explained
by substrate competition for transporters of Phe with
branched chain amino acids.[16] Cellular import of aromatic
amino acids is mainly mediated by l-type amino acid
transporters (LAT), which are found in most tissues. We
found increased aromatic amino acid levels and diminished
BCAA levels in liver sections after tracer infusion (Fig-
ure S9). Clinically, increased LAT transporter expression was
shown to be of diagnostic and prognostic value.[17]
In conclusion, we present a novel and reproducible MSI
method to explore the dynamic chemical changes of complex
biological samples, allowing for the first time, the detection of
co-localization of hepatocellular 13C6-Phe and amino acid
kinetics. The method enabled the broad detection of over
60 endogenous metabolites, thus providing a visual map of
liver function. This map enables translation of molecular
kinetics to morphology for a wide variety of biological tissues.
This allows differential kinetics between healthy and diseased
tissue or altered amino acid metabolism in tumor cells and
their microenvironment to be intimately followed.
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A bolus injection of 13C6-Phe (n = 11) or saline solution (n = 4)
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Acknowledgements
The authors thank Hang Nguyen for revising the manuscript,
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supported by the Dutch Province of Limburg as part of the
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Manuscript received: March 14, 2017
Revised: April 21, 2017
Final Article published: && &&, &&&&
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Communications
Isotopic Labeling
Imaging isotopes: A mass spectrometry
imaging method using isotope labeling to
study dynamic molecular changes of l-
phenylalanine (Phe) in mouse liver tissue
was developed. Using this method, hy-
droxylation of Phe to l-tyrosine (Tyr), as
well as its co-localization with other
amino acids could be monitored. This
study demonstrates the potential to spa-
tially detect local molecular kinetics of
amino acids in research and clinical
applications.
M. Arts, Z. Soons,* S. R. Ellis,
K. A. Pierzchalski, B. Balluff, G. B. Eijkel,
L. J. Dubois, N. G. Lieuwes, S. M. Agten,
T. M. Hackeng, L. J. C. van Loon,
R. M. A. Heeren,
S. W. M. Olde Damink
&&&& — &&&&
Detection of Localized Hepatocellular
Amino Acid Kinetics by using Mass
Spectrometry Imaging of Stable Isotopes
6
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