Massive Glutamine Cyclization to Pyroglutamic Acid in Human Serum Discovered Using NMR Spectroscopy

Article abstract of DOI:10.1021/ac504435b

Glutamine is one of the most abundant metabolites in blood and is a precursor as well as end product central to numerous important metabolic pathways. A number of surprising and unexpected roles for glutamine, including cancer cell glutamine addiction discovered recently, stress the importance of accurate analysis of glutamine concentrations for understanding its role in health and numerous diseases. Utilizing a recently developed NMR approach that offers access to an unprecedented number of quantifiable blood metabolites, we have identified a surprising glutamine cyclization to pyroglutamic acid that occurs during protein removal. Intact, ultrafiltered and protein precipitated samples from the same pool of human serum were comprehensively investigated using ¹H NMR spectroscopy at 800 MHz to detect and quantitatively evaluate the phenomenon. Interestingly, although glutamine cyclization occurs in both ultrafiltered and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further, virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and biological interpretations. (Graph Presented)

Full text of DOI:10.1021/ac504435b

Article
pubs.acs.org/ac
Massive Glutamine Cyclization to Pyroglutamic Acid in Human
Serum Discovered Using NMR Spectroscopy
,
†
†
,†,‡,§
G. A. Nagana Gowda,* Yashas N. Gowda, and Daniel Raftery*
†
‡
Northwest Metabolomics Research Center, Anesthesiology and Pain Medicine, and Department of Chemistry, University of
Washington, Seattle, Washington 98109, United States
§
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, United States
S Supporting Information
*
ABSTRACT: Glutamine is one of the most abundant metabolites in blood
and is a precursor as well as end product central to numerous important
metabolic pathways. A number of surprising and unexpected roles for
glutamine, including cancer cell glutamine addiction discovered recently,
stress the importance of accurate analysis of glutamine concentrations for
understanding its role in health and numerous diseases. Utilizing a recently
developed NMR approach that offers access to an unprecedented number of
quantifiable blood metabolites, we have identified a surprising glutamine
cyclization to pyroglutamic acid that occurs during protein removal. Intact,
ultrafiltered and protein precipitated samples from the same pool of human
1
serum were comprehensively investigated using H NMR spectroscopy at
8
00 MHz to detect and quantitatively evaluate the phenomenon.
Interestingly, although glutamine cyclization occurs in both ultrafiltered
and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent
serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further,
virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same
pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for
all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic
acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and
biological interpretations.
lutamine is one of the most highly concentrated
metabolites in human blood; compared to all the free
and quantitation of a large number of blood metabolites
8
,9
G
(including glutamine) on a routine basis.
However,
amino acids, glutamine is typically 20% higher in concen-
numerous investigations using standard compounds have
shown that under abnormal conditions, the glutamine molecule
is not stable. Under extreme acidic, alkaline or high-temper-
ature conditions, glutamine undergoes spontaneous cyclization
1
,2
tration. Glutamine is a precursor as well as a metabolic end-
product and thus central to many important metabolic
pathways. In fact, glutamine participates in virtually all core
metabolic functions including bioenergetics, the support of cell
defenses against oxidative stress and as a complement to
glucose metabolism in the production of energy and macro-
molecules. Moreover, due to the somewhat surprising roles i3t
plays in proliferating tumor cells driven by the oncogene Myc,
10−14
to form pyroglutamic acid with the loss of ammonia.
At
pH < 2 and pH > 13, both glutamine and pyroglutamic acid
interconvert reversibly; on heating, glutamine converts into
15
glutamic acid, which then converts into pyroglutamic acid.
Interestingly, more recent investigations show that cyclization
of glutamine to pyroglutamic acid also occurs in situ during
1
,2
glutamine is considered as a “super nutrient” in cancer.
Recent studies have uncovered unexpected roles for glutamine
in signaling pathways that contribute to tumor growth. Such a
vast number of roles played by glutamine highlights the
importance of its accurate analysis in blood for biomarker
discovery as well as mechanistic studies and for understanding
disease pathogenesis.
In the field of metabolomics, metabolite profiling of human
serum/plasma has attracted significant attention, particularly
due to its potentially broad range of applications with clinical
1
0,16
mass spectrometric analysis.
These studies, based on liquid
4
chromatography-mass spectrometry (LC-MS) methods, have
shown that the magnitude of glutamine cyclization to
pyroglutamic acid is significant, which highlights the magnitude
of error introduced in the analysis of these metabolites if not
16
accounted for appropriately. In fact, to avoid the errors in LC-
MS analysis, one of the studies proposed making corrections to
Received: November 27, 2014
Accepted: March 7, 2015
5
−7
relevance.
Nuclear magnetic resonance (NMR) spectrosco-
py and mass spectrometry (MS) enable reliable identification
©
XXXX American Chemical Society
A
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Article
apparent concentrations of glutamine and pyroglutamic acid by
combining suitable chromatographic conditions, isotopic stand-
ards and optimized fragmentor voltage. To date, however,
glutamine has been considered as a relatively stable metabolite
under the mild conditions widely used in the analysis of human
blood serum, for example in NMR analysis.
Ultrafiltration. Ten centrifugal filters (3 kDa cutoff,
Amicon Microcon; YM-3, Sigma-Aldrich) were washed with
water and centrifuged with 350 μL of water at 13400 rcf for 20
min thrice. Serum (350 μL each) was then transferred to each
filter tube and centrifuged for 20 min at 13400 rcf. The filtrates
were dried using an Eppendorf Vacufuge-Plus vacuum
concentrator and the residue mixed with a 100 μL solution
16
In the present study, we question this assumption, focusing
on the stability of glutamine and its proclivity to form
pyroglutamic acid. Intact, ultrafiltered and protein precipitated
samples from the same pool of human serum were
of phosphate buffer (100 mM) in D O containing 66.17 μM
2
TSP; the volume was increased to 600 μL with 100 mM
phosphate buffer in D O and then transferred to 5 mm NMR
2
1
comprehensively investigated using H NMR spectroscopy at
tubes. Separately, five portions of serum (350 μL each) were
subjected to ultrafiltration; the filtrates were mixed with a 15 μL
800 MHz to detect and quantitatively evaluate blood glutamine
levels. The obtained results demonstrate the discovery of a
surprising and unexpected phenomenon, a massive cyclization
of glutamine to pyroglutamic acid in processed human blood
serum. Interestingly, although glutamine apparently does not
cyclize in intact serum, the cyclization occurs upon ultra-
filtration or protein precipitation, with glutamine levels
dropping by up to 75% and a proportionate increase in the
pyroglutamic acid levels. The new findings stress the need to
account for the cyclization reaction to enable accurate analysis
of glutamine in blood.
solution of phosphate buffer (100 mM) in D O containing 0.1
2
mM TSP, and 200 μL was transferred to 3 mm NMR tubes.
Solutions of Intact Serum. Ten intact serum samples (575
μL each) were mixed with 25 μL of phosphate buffer (100
mM) in D O and transferred to 5 mm NMR tubes for analysis.
2
Solutions for Spiking Experiments. Stock solutions (1
mL, 5 mM) of glutamine and pyroglutamic acid were prepared,
separately, in D O by diluting their 50 mM stock solutions,
2
which were first prepared by weighing the standard
compounds. Serum samples were spiked with a standard
solution of glutamine or pyroglutamic acid and used for NMR
analysis either with proteins intact, or after protein removal by
precipitation or ultrafiltration. Separately, four serum samples
were spiked with a 0.25 mM standard solution of glutamine
after protein precipitation using methanol or ultrafiltration.
Four standard glutamine solutions in deionized water (0.25
mM) (without serum) were also subjected to protein
precipitation or ultrafiltration protocols and NMR analysis.
NMR Spectroscopy. All NMR experiments were per-
formed at 298 K on a Bruker Avance III 800 MHz spectrometer
equipped with a cryogenically cooled probe and Z-gradients
suitable for automated gradient shimming, as described
MATERIALS AND METHODS
■
(
Glutamine, pyroglutamic acid, methanol, deuterated methanol
CD OD), sodium phosphate monobasic (NaH PO ), sodium
3
2
4
phosphate dibasic (Na HPO ) and 3-(trimethylsilyl)propionic
2
4
acid-2,2,3,3-d sodium salt (TSP) were obtained from Sigma-
4
Aldrich (St. Louis, MO). Deuterium oxide (D O) was obtained
2
from Cambridge Isotope laboratories, Inc. (Andover, MA).
Pooled human serum (100 mL) was procured from Innovative
Research, Inc. (Novi, MI) and shipped to Seattle under dry ice,
where it was subsequently thawed and aliquoted into 2 mL
Eppendorf tubes and frozen at −80 °C until use for NMR
investigations. Deionized (DI) water was purified using an in-
house Synergy Ultrapure Water System from Millipore
8
previously. The 1D NOESY and CPMG (Carr−Purcell−
Meiboom−Gill) pulse sequences with water suppression using
1
presaturation were used for H 1D NMR experiments.
(
Billerica, MA). All chemicals were used without further
Additional CPMG experiments for intact serum were
performed after spiking with standard pyroglutamic acid.
Spectral width, time domain points, and number of transients
of 9600 Hz, 32 768 and 128, respectively, were used. The
resulting data were Fourier transformed using a spectrum size
of 32 768 points after multiplying by an exponential window
function with a line broadening of 0.5 Hz. Bruker Topspin
versions 3.1 or 3.2 software packages were used for NMR data
acquisition and processing.
purification.
Sample Preparation. Deuterated buffer solution was
prepared by dissolving 928.6 mg of anhydrous NaH PO with
2
4
320.9 mg of anhydrous Na HPO in 100 g of D O and used
2 4 2
without any further pH correction. Eight of the frozen, 2 mL
serum vials (all originally from the same 100 mL “parent”
pooled sample) were thawed at room temperature, repooled
together and used for analysis directly or after removal of serum
proteins using ultrafiltration or protein precipitation. A
summary of the different sample preparations is provided in
Table S1 of the Supporting Information.
Serum Protein Precipitation Using Methanol. Twenty
portions of serum (350 μL each) were mixed with methanol in
a 1:2 ratio (v/v), vortexed and incubated at −20 °C for 20 min.
The mixtures were centrifuged at 13400 rcf for 30 min to pellet
proteins. Supernatants were decanted to fresh vials and dried
using an Eppendorf Vacufuge-Plus vacuum concentrator. The
dried samples were mixed with 100 μL of phosphate buffer
Peak Assignments and Metabolite Quantitation.
Metabolite assignments including identification of peaks from
glutamine and pyroglutamic acid were made based on the
recently published literature on unknown metabolite identi-
fication in human blood serum, which has enabled
identification and quantitation of nearly 70 blood metabolites
8
,17
using 1D NMR.
The assignment of glutamine and
pyroglutamic acid peaks was further confirmed by spiking
with standard compounds. Chenomx NMR Suite Professional
Software package (version 5.1; Chenomx Inc., Edmonton,
Alberta, Canada) was used for absolute quantitation of
glutamine and pyroglutamic acid. The Chenomx software
allows fitting spectral lines using the standard metabolite library
(
100 mM) in D O containing 66.17 μM TSP; the volumes
2
were increased to 600 μL with 100 mM phosphate buffer in
D O and then transferred to 5 mm NMR tubes. Separately, two
2
portions of serum (350 μL each) were subjected to protein
precipitation using 1:2 deuterated methanol (CD OD) as
1
for 800 MHz H NMR spectra. Peak fitting with reference to
3
described above; 600 μL of supernatants was transferred to 5
mm NMR tubes.
the internal TSP signal enabled the determination of absolute
metabolite concentrations.
B
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RESULTS
exemplified in Figures 2 and 3 for different portions of serum
from the same pool obtained after protein precipitation and
■
Intact, ultrafiltered as well as protein precipitated serum
provided highly resolved H NMR spectra. In the intact
1
serum spectrum, however, a large number of metabolites were
either not detected or significantly attenuated. The ultrafiltered
serum, which was completely devoid of proteins, provided rich
spectra in terms of the number of metabolites detected, far
superior to intact serum spectra; however, peak intensities for a
number of metabolites were attenuated significantly. On the
other hand, serum after protein precipitation using methanol
provided the most optimal NMR spectra in terms of both the
number of metabolites detected as well as their peak intensities.
A close evaluation of the spectra showed that although
glutamine did not cyclize in intact serum, a very significant
cyclization of glutamine to pyroglutamic acid occurred in
1
ultrafiltered and protein precipitated serum. Typical H NMR
spectra highlighting the cyclization of glutamine to pyrogluta-
mic acid in both ultrafiltered serum and protein precipitated
serum are shown in Figure 1. It may be striking to note that,
due to cyclization, serum glutamine level drops by up to 75%
and, concomitantly, pyroglutamic acid level increases propor-
tionately.
Interestingly, under virtually identical conditions, the
magnitude of glutamine cyclization varied drastically from
one portion of the same serum sample to another and the
magnitude of cyclization was generally higher for protein
precipitated serum than the ultrafiltered serum. This is
1
Figure 2. (a−c) Regions of 800 MHz H NMR spectra of different
sample portions from the same pool of human serum obtained under
virtually identical conditions after protein precipitation using methanol
(
1:2 v/v). A different degree of cyclization of glutamine to
pyroglutamic acid is observed in different samples.
1
1
Figure 1. Portions of 800 MHz H spectra of the same pooled human
Figure 3. (a−c) Regions of 800 MHz H NMR spectra of different
serum highlighting varied levels of cyclization of glutamine to
pyroglutamic acid. (a) Intact serum spectrum; (b) serum spectrum
obtained after ultrafiltration using a 3 kDa cutoff filter; (c) serum
spectrum obtained after protein precipitation using methanol (1:2 v/
v). Note that the pyroglutamic acid peaks are missing in intact serum,
whereas they are present in both ultrafiltered and protein precipitated
serum spectra.
sample portions from the same pool of human serum obtained after
ultrafiltration, under virtually identical conditions, using 3 kDa
molecular weight cutoff filters. A different degree of cyclization of
glutamine to pyroglutamic acid is again observed in different samples.
C
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ultrafiltration, respectively. Further, it is evident from the
figures that the decrease in peak intensity for glutamine due to
cyclization is proportional to the increase in peak intensities for
pyroglutamic acid. Even when the samples were not dried, they
showed some glutamine cyclization (Figure S1 of the
Supporting Information). Spiking experiments with standard
glutamine followed by protein precipitation showed that
virtually all the added glutamine was converted to pyroglutamic
acid (see Figure 4), whereas no such conversion was detected
acid. However, NMR analysis of standard glutamine solutions
after subjecting to either protein precipitation or ultrafiltration
showed no conversion to pyroglutamic acid.
Further investigations focused on the quantitative evaluation
of cyclization showed that, whereas the magnitude of
cyclization of glutamine to pyroglutamic acid varied drastically
in different portions of samples from the same pool of human
serum, the sum of glutamine and pyroglutamic acid
concentrations in each sample was the same in all portions of
the serum (CV < 3%). This is exemplified in Figure 5 and
Figure S2 (Supporting Information) based on absolute
concentrations of glutamine and pyroglutamic acid from
different sample portions from the same pool of human
serum. The glutamine level in intact serum (213 ± 2 μM) was
virtually equal to the sum of apparent glutamine and
pyroglutamic acid levels for ultrafiltered and protein precipi-
tated samples that exhibit varied degrees of glutamine
cyclization from the same pool of serum (see Figure S3 of
the Supporting Information).
1
H NMR experiments on intact serum samples performed
after spiking with standard pyroglutamic acid showed that the
pyroglutamic acid peaks did not appear in the spectra until its
concentration reached well beyond 170 μM. Further, when the
1
H NMR peaks did appear at higher added concentrations of
pyroglutamic acid, they were unusually broad. Figure 6 shows
typical intact serum spectra before and after spiking with
different amounts of pyroglutamic acid.
DISCUSSION
■
1
Previous studies using pure compounds have shown that
glutamine undergoes spontaneous cyclization to form pyroglu-
tamic acid under extreme conditions of pH and temper-
Figure 4. Regions of 800 MHz H NMR spectra of a pooled human
serum with or without spiking with a standard solution of glutamine
obtained after protein precipitation using methanol (1:2 v/v): (a)
without spiking with glutamine; (b) spiked with 21 μL of 5 mM
glutamine solution. Note, in panel b, virtually all added glutamine is
converted to pyroglutamic acid. Glutamine spiked after methanol
precipitation also showed similar conversion of the spiked glutamine
to pyroglutamic acid (see text), which indicates that the conversion
does not depend on the order of spiking.
10−14
ature;
however, identification of significant cyclization of
glutamine to pyroglutamic acid in human blood serum under
mild conditions typically used in the metabolomics of human
serum/plasma is new. This discovery is significant due to the
importance of the accurate quantitation of blood metabolites,
especially, the growing need to understand the key roles of
glutamine metabolism in health and numerous diseases.
Interestingly, although such glutamine cyclization can delete-
riously affect reliable analysis of blood glutamine levels, the
finding that the sum of the apparent glutamine and
for ultrafiltered serum. The order of spiking, however, did not
matter; glutamine spiked after methanol precipitation still
showed conversion of the spiked glutamine to pyroglutamic
1
Figure 5. Plot of typical concentrations of glutamine, pyroglutamic acid and their sum as determined by 800 MHz H NMR spectroscopy in different
portions of samples from the same pooled human serum obtained after protein precipitation using methanol (1:2 v/v). Although glutamine
massively cyclizes to pyroglutamic acid, the sum of glutamine and pyroglutamic acid is the same in all the portions of serum samples (CV < 3%).
D
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intact serum was essentially equal to the sum of the glutamine
and pyroglutamic acid concentrations in the ultrafiltered and
protein precipitated samples. To further explore the presence
1
and amount of pyroglutamic acid, additional H NMR
experiments on intact serum samples were performed before
and after spiking with different amounts of standard
pyroglutamic acid. Pyroglutamic acid peaks did not appear in
1
the H NMR spectra even after the addition of a large amount
(
∼170 μM) of the standard compound, which indicates a
strong binding of pyroglutamic acid to serum proteins.
Although the binding of pyroglutamic acid to serum proteins
has not been previously reported, it is well-known that many
serum/plasma metabolites bind to proteins, which makes such
metabolites either invisible or significantly attenuated in
intensity in the intact NMR spectra of human serum/
17−20
plasma.
In fact, recognizing the deleterious effects of
metabolite binding to proteins, we recently showed that protein
precipitation by methanol is a far superior approach for the
8,17
quantitative analysis of serum/plasma by NMR.
Because of
the protein binding of pyroglutamic acid, the nonobservance of
its NMR peaks neither confirms its absence in serum nor the
absence of glutamine cyclization. However, the close
association of the glutamine levels in intact serum with the
same value for the sum of apparent glutamine and pyroglutamic
acid levels for ultrafiltered and protein precipitated samples
support the near absence of glutamine cyclization in intact
serum. Based on the similar concentration values of glutamine
in the intact serum (213 ± 2 μM) to the sum of glutamine and
pyroglutamic acid in the protein precipitated serum (219 ± 3
μM) and ultrafiltered serum (220 ± 2 μM), we estimate the
pyroglutamic acid level to be less than 10 μM in intact serum.
In conclusion, investigations of human serum based on the
recently expanded pool of NMR detectable blood metabolites
have led to the discovery of a significant glutamine cyclization
to pyroglutamic acid in the widely used approaches of protein
precipitation and ultrafiltration of human serum for metabolite
profiling. The cyclization causes a decrease in the apparent
blood glutamine level by up to 75% and a proportionate
increase in the levels of pyroglutamic acid. The magnitude of
cyclization varies drastically for different samples from the same
pool of human serum; however, the sum of the apparent
glutamine and pyroglutamic acid concentrations in each sample
remains the same suggesting that virtually all pyroglutamic acid
is derived from glutamine cyclization. Therefore, while
performing serum metabolite profiling, it is important to
consider the sum of the apparent glutamine and pyroglutamic
acid levels as the actual blood glutamine level for biomarker
discovery and biological interpretations. Such an approach can
have adverse effects if pyroglutamic acid, unconnected with
glutamine cyclization, is already present in the sample.
However, our experiments indicate that pyroglutamic acid
levels are likely to be low in intact serum. Currently, the
mechanism for the conversion of glutamine to pyroglutamic
acid is unknown and its investigation is beyond the scope of the
current study. Further, as observed for human serum in this
study, glutamine cyclization may also occur during the analysis
of other biological specimens as well, and hence caution is
needed in the analysis of this biologically important metabolite.
1
Figure 6. Regions of 800 MHz H NMR spectra of the same pooled
intact human serum: (a) serum before spiking with pyroglutamic acid
pGlu); (b−d) serum spiked with standard solution of pyroglutamic
acid so that the added concentration is 86, 172 and 1032 μM,
respectively. Virtually no pyroglutamic acid peaks were seen even after
the addition of up to 172 μM pyroglutamic acid solution (see spectra
in panels b and c); only at very high concentration are broad peaks for
pyroglutamic acid seen (spectrum in panel d).
(
pyroglutamic acid level does not change due to cyclization
provides an avenue for obtaining reliable blood glutamine
concentrations (Figure 5). Therefore, it is important that blood
metabolite profiling using contemporary analytical methods
make corrections appropriately to obtain the correct blood
glutamine level for biomarker discovery and biological
interpretations.
A newly identified factor that contributes to additional
glutamine cyclization to pyroglutamic acid is the electrospray
16
ionization (ESI) source during LC-MS analysis. It may be
striking to note that the magnitude of the cyclization in the ESI
source ranges from 33 to 100% and depends on fragmentor
voltage. MS is a major analytical method in the growing
metabolomics field and ESI is the most widely used source of
ionization. Therefore, the massive in situ cyclization of
glutamine within the ESI source represents an additional
major challenge for the analysis of blood glutamine using MS.
Corrections to such in situ cyclization can be made using a
combination of proper chromatographic conditions, isotopic
internal standards and optimized fragmentor voltage. On the
other hand, NMR spectroscopy, which does not encounter the
phenomenon of in situ cyclization, enables accurate analysis of
blood glutamine and hence potentially represents a reliable
analysis method for blood glutamine.
In this study, evidence to support the observation that
glutamine does not cyclize in intact serum was drawn from the
virtual absence of pyroglutamic acid peaks and undiminished
glutamine peak observed in the NMR spectra of intact serum
samples (see Figure S4 of the Supporting Information). In fact,
the concentration of glutamine (213 ± 2 μM) observed in the
16
ASSOCIATED CONTENT
■
*
S
Supporting Information
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
E
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Article
AUTHOR INFORMATION
Corresponding Authors
G. A. Nagana Gowda. E-mail: ngowda@uw.edu.
Daniel Raftery. E-mail: draftery@uw.edu.
■
*
*
Notes
The authors declare the following competing financial
interest(s): Daniel Raftery reports holding equity and an
executive position at Matrix Bio, Inc.
ACKNOWLEDGMENTS
■
We acknowledge financial support from the NIH (National
Institute of General Medical Sciences 2R01GM085291). We
thank the reviewers for their insightful comments and
suggestions on this work.
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