Article

pubs.acs.org/JAFC

Increase in the Protein-Bound Form of Glutathione in Human Blood

after the Oral Administration of Glutathione
Eun Young Park,† Nami Shimura,† Toru Konishi,‡ Yusuke Sauchi,‡ Sayori Wada,† Wataru Aoi,†
Yasushi Nakamura,† and Kenji Sato*,†
†
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho,
Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan
‡
KOHJIN Life Sciences Company, Ltd., 1-3 Yurakucho 1-chome, Chiyoda-ku, Tokyo 100-0006, Japan

ABSTRACT: The present study examined the impact of the supplementation of glutathione (GSH), γ-L-glutamyl-L-cysteinyl-
glycine, on human blood GSH levels. Healthy human volunteers were orally supplemented with GSH (50 mg/kg body weight).
Venous blood was collected from the cubital vein before and after ingestion. Plasma was mixed with 3 volumes of ethanol. The
supernatant and precipitate were used for the deproteinized and protein fractions of plasma, respectively. Blood cell and plasma
fractions were pretreated with 5% trichloroacetic acid−2% 2-mercaptoethanol to reduce the oxidized form of GSH and liberate
protein-bound GSH. The 2-mercaptoethanol-pretreated GSH was determined by precolumn derivatization with 6-
aminoquinolyl-N-hydroxy succinimidyl carbamate and liquid chromatography−tandem mass spectrometry. There was no
significant difference in GSH contents in the deproteinized fraction of plasma and blood cell fraction after GSH ingestion.
However, the GSH contents in the protein-bound fraction of plasma significantly (P < 0.01) increased from 60 to 120 min after
GSH supplementation.
KEYWORDS: glutathione, GSH, blood, supplementation, oral administration, human trial

■ INTRODUCTION
Glutathione (GSH), γ-L-glutamyl-L-cysteinyl-glycine, is a
used for medical applications. Aside from medical applications,
GSH has also been implicated in skin whitening. In vitro
ubiquitous compound found in humans, animals, plants, and studies have revealed that GSH inhibits melanin synthesis in
microorganisms.1 Within cells, GSH exists in both reduced and the reaction of tyrosinase and L-3,4-dihydroxyphenylalanine in a
oxidized states. In healthy cells, the majority of GSH is present dose-dependent manner.13,14 On the basis of these findings,
in the reduced form.2 Most cells in the human body are capable GSH has been used as a supplement for skin whitening and
of synthesizing GSH from glutamate, cysteine, and glycine. improvement of liver function.
First, γ-glutamyl-cysteine is synthesized from L-glutamate and L- GSH supplementation has been prevalent worldwide.
cysteine by γ-glutamyl-cysteine synthetase. Second, glycine is However, bioavailability and the metabolic fate of orally
conjugated to the C-terminaus of γ-glutamyl-cysteine by GSH administered GSH have not been fully understood. It has
synthetase.1,3 been proposed that GSH is first degraded by γ-glutamyl
GSH is a cofactor or substrate for GSH peroxidase and GSH transferase into γ-glutamyl-X (X = other amino acids) and
S-transferase (GST), which are involved in very important cysteinyl-glycine. The cysteinyl-glycine is degraded into
antioxidant and detoxification systems, respectively.4 The main cysteine and glycine by peptidases.6,15 Orally administered
function of GSH peroxidase is to reduce hydrogen peroxide to GSH could be degraded in this system. In addition, some
water and to reduce lipid hydroperoxides to their correspond- human studies have demonstrated no significant increases in
ing alcohols.3,5 The reduced form of GSH, which is involved in blood GSH levels in human volunteers after the oral
these reactions as a cofactor, is converted to the oxidized form. administration of a relatively large dose of GSH.16,17 These
Thus, the ratio of the oxidized form to the reduced form of findings suggest that GSH supplementation has no impact on
GSH has been thought to reflect oxidative status in cells, blood and organ GSH levels. However, GSH can potentially
organs, and individuals.6 However, oxidized GSH is reduced by bind to proteins by disulfide bonds. Therefore, it is possible
GSH reductase. The reduced form of nicotinamide adenine that orally administered GSH may be present and transported
dinucleotide phosphate, which is a cofactor for GSH reductase, as a protein-bound form in the blood, and this has not been
is supplied by the pentose phosphate pathway.7 GST catalyzes reported in previous studies.
the conjugation of reduced GSH to a wide variety of exogenous The objective of the present study was to elucidate the
chemicals through a sulfhydryl group to make the compounds impact of GSH supplementation on human blood GSH levels
more soluble and to facilitate urinary excretion.8,9 in the free and protein-bound forms.
Decreased GSH levels in cells have been demonstrated to
increase the risks of diseases and poisoning.1,10 GSH has Received: March 18, 2014
therefore been used to treat chronic liver diseases and Revised: May 17, 2014
poisoning through intravenous injections.11,12 In addition to Accepted: May 30, 2014
intravenous injections of GSH, oral administration has been Published: May 30, 2014

© 2014 American Chemical Society 6183 dx.doi.org/10.1021/jf501338z | J. Agric. Food Chem. 2014, 62, 6183−6189
Journal of Agricultural and Food Chemistry Article

Figure 1. Elution profiles of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ) derivatives of 2-mercaptoethanol-pretreated or intact
glutathione (GSH). Pretreated and intact GSH was dried in the presence of triethylamine (TEA) before derivatization with AccQ. Peaks marked A
and B show GSH derivatives.

■ MATERIALS AND METHODS

Chemicals. The reduced form of L-glutathione was produced by
dried under vacuum and added to 100 μL of 5% TCA containing 2%
2-mercaptoethanol, and the mixture was allowed to stand for 30 min at
room temperature and used as the deproteinized plasma fraction in the
fermentation with Torula yeast by KOHJIN Life Sciences, a subsidiary following experiments. The ethanol precipitate of the plasma was
of Mitsubishi Corp. (Tokyo, Japan). This product has been qualified washed with 200 μL of 75% (v/v) ethanol and then mixed with 160
for Generally Recognized As Safe status by the U.S. Food and Drug μL of 10% (w/v) TCA and stirred vigorously. To this suspension were
Administration (U.S. FDA GRAS notified GRN000293). γ-L- added another 320 μL of 5% (w/v) TCA and 10 μL of 10% (v/v) 2-
Glutamyl-L-cysteine (γGlu-Cys) and L-cysteinyl-glycine (Cys-Gly) mercaptoethanol. The mixture was allowed to stand for 30 min at
were obtained from Sigma-Aldrich (St. Louis, MO, USA). An amino room temperature and then centrifuged at 14800g for 5 min. The
acid labeling kit (AccQ Tag) that consisted of 6-aminoquinolyl-N- supernatant was collected and used as the protein-bound plasma
hydroxysuccinimidyl carbamate reagent (AccQ), acetonitrile, and 0.2 fraction. The blood cell from 1 mL of blood was mixed with 500 μL of
mM sodium borate buffer, pH 8.8, was purchased from Waters 10% TCA and 1 mL of 5% TCA and stirred for 3 min. The
(Milford, MA, USA). Acetonitrile [high-performance liquid chroma- supernatant was collected after centrifugation at 14800g for 5 min.
tography (HPLC) grade], triethylamine (TEA), formic acid, trichloro- Fifty microliters of the 5% TCA extract of the blood cell was mixed
acetic acid (TCA), and 2-mercaptoethanol were obtained from Wako with 450 μL of 5% TCA containing 2% 2-mercaptoethanol and
Pure Chemicals (Osaka, Japan). All other reagents were of analytical allowed to stand for 30 min at room temperature and used as the
grade or higher. blood cell fraction.
Clinical Study Design. The experimental protocol was submitted The weights of the blood cell and plasma ethanol precipitate were
to and approved by the experimental ethical committees of Kyoto determined before the addition of TCA solutions. The samples were
Prefectural University (KPU no. 63) and KOHJIN Life Sciences. The stored at −80 °C until use.
human studies were performed according to the Helsinki Declaration Derivatization of GSH with the AccQ Reagent. The reduced
and were under the control of medical doctors. The potential risks of form of GSH was dissolved in 10 mM HCl to result in 2.5 μmol/mL
the ingestion of GSH and the objective of the present study were and used as a stock solution. Suitable amounts of the standard stock
revealed to the volunteers. After careful reading of a consent form, all solution were dried under vacuum in a glass tube (50 mm × 6 mm
subjects signed it. Seven healthy male subjects (mean age ± standard i.d.) with a KLV-CC-105 centrifugal concentrator (Tomy, Tokyo,
deviation 39.8 ± 15.1 years) were fasted for 12 h before the Japan). Fifty microliters of 5% TCA containing 2% 2-mercaptoethanol
experiment and given the reduced form of L-glutathione at 50 mg/kg was added to the tube and allowed to stand for 30 min, and the
body weight and 100 mL of water, which was the same dose as used in solution was then dried under vacuum in a hydrolysis/derivatization
the previous study.17 Approximately 10 mL of venous blood was vial with a resealable valve (Waters). Twenty microliters of an alkaline
collected in the presence of heparin from the cubital vein before and solution, which consisted of methanol, water, and TEA at a ratio of
15, 30, 60, 90, and 120 min after ingestion. 7:1:2 (v/v), was added to the tube, and the mixture was then dried
Preparation of Blood Samples. One milliliter of the blood was under vacuum to increase the pH for derivatization with AccQ. In
centrifuged at 3700g for 5 min to prepare the plasma and blood cell some cases, this treatment was deleted from the protocol. The dried
fractions. Four hundred microliters of the plasma was mixed with 1.2 standards were resolved in 20 μL of 20 mM HCl, and 20 μL of AccQ
mL of ethanol and then centrifuged at 3700g for 5 min. Then, the reagent and 60 μL of borate buffer were added in the AccQ Tag Kit
supernatant was collected. Aliquots of the supernatant (300 μL) were and reacted at 50 °C for 10 min. The resultant AccQ derivatives were

6184 dx.doi.org/10.1021/jf501338z | J. Agric. Food Chem. 2014, 62, 6183−6189

Journal of Agricultural and Food Chemistry Article

diluted with 5 mM sodium phosphate buffer, pH 7.4, containing 10%

(v/v) acetonitrile. The solutions were filtered through a 0.45 μm filter
(column guard LCR4; Millipore, Billerica, MA, USA).
Fifty microliters of the plasma deproteinized and protein-bound
fractions and blood cell fraction were also dried under vacuum in the
presence of TEA and then derivatized with AccQ, as described above.
Isolation of the AccQ Derivatives. The AccQ derivatives of
GSH with and without 5% TCA−2% 2-mercaptoethanol and TEA
pretreatments were isolated by reversed phase HPLC (RP-HPLC)
with an Inertsil ODS-3 (4.6 mm i.d. × 250 mm, GL Sciences, Tokyo,
Japan). Elution was performed with a binary gradient system with 0.1%
formic acid (solvent A) and 60% (v/v) acetonitrile (solvent B). The
gradient profile was as follows: 0−1 min, 5−10% B; 1−10 min, 10−
20% B; 10−25 min, 20−40% B; 25−30 min, 40−45% B; 30−35 min,
45−50% B; 35−40 min, 50−100% B; 40−45 min, 100% B; 45−45.1
min, 100−5% B; and 45.1−55 min, 5% B. The column was
equilibrated with 5% B at 1 mL/min. The column was maintained
at 45 °C, and elution was monitored by fluorescence at 395 nm that
was excited at 250 nm.
Mass Spectrometry Analysis. The isolated AccQ derivatives of
GSH were analyzed by electrospray ionization ion trap mass
spectrometry (ESI-MS) with an LCQ (Thermo Fisher Scientific,
Waltham, MA, USA). The sample was delivered to the ion source by a
syringe pump at 3 μL/min. Detection was conducted in positive mode
and optimized and processed by Xcalibur version 2.07 (Thermo Fisher
Scientific).
Determination of GSH by Liquid Chromatography−Tandem
Mass Spectrometry (LC-MS/MS). The concentrations of GSH,
γGlu-Cys, and Cys-Gly in the blood samples were determined by
precolumn derivatization with AccQ that was followed by an LC-MS/
MS analysis with a Q-TRAP 3200 (AB SCIEX, Framingham, MA,
USA). The AccQ derivative was prepared as described above and
clarified by passing through a filter (0.45 μm column guard; Millipore).
The samples (10 μL) were injected onto an Inertsil ODS-3 column
(2.1 mm i.d. × 250 mm; GL Sciences), and a binary gradient elution
was performed with 0.1% formic acid (solvent A) and 60% acetonitrile
(solvent B) at a flow rate of 0.2 mL/min. The column was equilibrated
with 100% solvent A, and the gradient profile was as follows: 0−15
min, 0−50% B; 15−20 min, 50−100% B; 20−24 min, 100% B; 24−
24.1 min, 100−0% B; and 24.1−30 min, 0% B. The column was
maintained at 40 °C throughout. The multireaction monitoring
(MRM) condition was optimized in positive mode with Analyst
version 4.2 (AB SCIEX) in autoselect mode.
Statistics. Differences between the means were evaluated by
analyses of variance (ANOVA) that were followed by one-way
ANOVAs with StatView 4.11 (Abacus Concepts, Berkeley, CA, USA).
Significant differences between the groups were evaluated by Scheffe’s
post hoc test.

■ RESULTS
Structures of the AccQ Derivatives. The AccQ
Figure 2. Mass spectrometry analyses of the GSH derivative that had
been prepared without (A) and with (B) 2-mercaptoethanol
pretreatment. Peaks marked A and B in Figure 1 were collected and
derivatives of GSH, which had been pretreated with or without analyzed by electrospray ionization (ESI) ion trap MS. The upper and
the 5% TCA−2% 2-mercaptoethanol solution, were resolved by lower panels represent full and zoom scan spectra, respectively.
RP-HPLC. In both cases, the GSH was pretreated by TEA
before derivatization with AccQ. As shown in Figure 1, several
peaks of fluorescence were observed. When different amounts number was 2. Assuming that this derivative had two protons,
of GSH were derivatized, only the peaks marked A and B its molecular weight (x1) can be calculated with the following
changed in a dose-dependent manner, which indicated that formula: (x1 + 2)/2 = 477, x1 = 952. This value coincided with
these peaks corresponded to AccQ−GSH derivatives and that the molecular weight of the oxidized form of GSH that was
the other ones were reagent peaks. The derivative that had been coupled with two AccQ molecules through two amino groups,
pretreated without 2-mercaptoethanol (A) showed a similar but as shown in Figure 3A. However, the AccQ derivative with 2-
slightly different retention time and a smaller peak area mercaptoethanol pretreatment showed a main peak with a m/z
compared to the counterpart (B). These peaks were collected of 554 (Figure 2B). The difference in m/z between the adjacent
and subjected to an ESI-MS analysis. As shown in Figure 2A, isotopes was 1.0. Then, its molecular weight (x2) can be
the AccQ derivative without 2-mercaptoethanol pretreatment calculated as follows: (x2 + 1)/1 = 554, x2 = 553. This value did
showed a main peak with a m/z of 477 (full scan). A zoom scan not coincide with the reduced form of AccQ-GSH that was
analysis revealed that the difference in m/z between the conjugated through an amino group, but it corresponded to the
adjacent isotopes was 0.5, which indicated that the charge AccQ−GSH−2-mercaptoethanol conjugate through an amino
6185 dx.doi.org/10.1021/jf501338z | J. Agric. Food Chem. 2014, 62, 6183−6189
Journal of Agricultural and Food Chemistry Article

followed by the drying treatment with TEA before derivatiza-

tion with AccQ for the determination of GSH by LC-MS/MS
in the MRM mode. The fragment ions with the first and second
high intensities (m/z 554.1 → 171.3 and m/z 554.1 → 145.2)
were automatically selected.
Blood GSH Levels. Figure 5a shows the GSH contents in
the deproteinized fraction of plasma before and after the
ingestion of GSH. There was no significant difference in the
GSH contents after the ingestion of GSH in this fraction. Much
higher contents of GSH were contained in the blood cell
fraction than in the deproteinized fraction of the plasma
(Figure 5b). However, no significant difference was observed in
the GSH contents in the blood cell fraction during the
experimental period. However, the contents of GSH, γGlu-Cys,
and Cys-Gly in the protein-bound fraction of plasma
significantly increased 60−120 min after the supplementation
of GSH (Figure 6). The protein-bound GSH and related
compounds in plasma did not return to the initial level 2 h after
supplementation.

■ DISCUSSION
GSH is present in high concentrations in liver and blood
cells.3,18 Generally, GSH concentrations have been determined
in such samples with Ellman’s reagent, which contains 5,5′-
dithiobis(2-nitrobenzoic acid). To increase sensitivity, an
enzyme recycling reaction using GSH reductase has been
developed.19,20 The GSH content in the blood cells, hepatic
tissues, and other tissues has been determined with these
methods. Allen et al. have reported that oral GSH
supplementation did not improve blood cell GSH levels.16
However, plasma GSH levels are lower (low μM levels) than
Figure 3. Estimated chemical structures of GSH derivatives that were
prepared without (A) and with 2-mercaptoethanol pretreatment (B). those in blood cells. The plasma GSH levels have been
determined by RP-HPLC with the precolumn derivatization of
the sulfhydryl group or the enzyme recycling reaction method.
group and a sulfhydryl group, respectively, as shown in Figure Witschi et al. have observed no increase in plasma GSH levels
3B. The other peaks in Figure 1 could not be assigned to GSH after a single oral supplementation of GSH to healthy human
derivatives, such as the AccQ−GSH conjugate with a free volunteers at 0.15 mmol/kg body weight.17 The present study
sulfhydryl group and the AccQ−GSH−AccQ conjugate confirmed these results (Figure 5). On the basis of these
through an amino and a sulfhydryl group. For preparation of results, it has been suggested that the oral supplementation of
these derivatives, the 5% TCA−2% 2-mercaptoethanol solution GSH does not affect blood GSH levels.
was removed by vacuum in the presence of TEA before It has been demonstrated that plasma proteins, including
reaction with the AccQ reagent. When this drying treatment albumin, can bind to low molecular weight thiol compounds
with TEA was deleted, a similar chromatogram was obtained through a disulfide bond.21,22 Therefore, there is the possibility
(Figure 4, upper) with a larger peak of the derivative (C) that supplemented GSH may be transported as a conjugate of
compared to the derivative with the drying treatment (A). The protein in the blood, and this has not been examined. In the
ESI-MS analysis of the derivative without the drying treatment present study, the effects of the supplementation of GSH on
with TEA (peak C) showed no significant peak of m/z 554, plasma protein-bound GSH levels were examined. To liberate
which was the protonated ion of the AccQ−GSH−2- GSH from protein conjugate, a reducing reagent is necessary.
mercaptoethanol conjugate (Figures 2B and 3B). However, For this purpose, dithiothreitol has been frequently used,23,24
two main peaks with m/z 171 and 249 were observed, and but it is incompatible with the enzyme recycling reaction
these corresponded to the AccQ fragment ion and the AccQ− method. The dithiothreitol also reacts with labeling reagents for
2-mercaptoethanol protonated ion, respectively. These results sulfhydryl and amino groups, which can interfere with the
indicated that the amino group of GSH was dissociated to the precolumn labeling reaction. In the present study, 2-
−NH3+ ion in the presence of a strong acid (TCA) and that it mercaptoethanol was used to liberate GSH from the
then could not react with the AccQ reagent. However, an conjugates. The excess amount of 2-mercaptoethanol can be
excess amount of 2-mercaptoethanol reacted with AccQ removed by vacuum in the presence of TEA. This drying
through the sulfhydryl group under this condition. Therefore, treatment is, therefore, indispensable in the reaction between
the drying treatment with TEA was critical for the AccQ and an amino group of GSH. The final GSH derivative
derivatization of GSH when the sample was pretreated with was double conjugated with AccQ and 2-mercaptoethanol
5% TCA−2% 2-mercaptoethanol to liberate the protein-bound through amino and sulfhydryl groups, respectively. As the final
GSH. GSH derivative had no free sulfhydryl groups, it could not be
On the basis of these findings, all of the blood samples were oxidized to form a disulfide bond. A single GSH derivative was
pretreated with 5% TCA−2% 2-mercaptoethanol, which was obtained, as shown by the MS analysis of the derivative peaks
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Figure 4. Effects of the drying pretreatment with TEA on the derivatization of 2-mercaptoethanol-pretreated GSH with AccQ: elution profiles of the
derivatives with and without the TEA treatment (upper); ESI-MS analysis of the derivative without TEA treatment (lower).

(Figure 1). The GSH derivative could be resolved by RP-HPLC by the oral supplementation of GSH. The protein-bound form
and detected by fluorescence. Other derivatives of thiol GSH level in plasma after supplementation of GSH is much
compounds, such as γGlu-Cy and Cys-Gly, however, show higher (>1000 times) than that of other food-derived peptides
similar retention times (data not shown). These peaks may such as Val-Tyr25 and Ile-Pro-Pro26 but less than that of the
interfere with the determination of GSH by fluorescence food-derived collagen peptides in human blood.27
detection. It has been thought that orally administered GSH is
Therefore, the MRM was used for the specific detection of successively degraded to cysteinyl-glycine, cysteine, and glycine
the GSH and its fragment peptides (γGlu-Cys, Cys-Gly) by by γ-glutamyl-transferase and peptidase.17,28,29 Cysteine could
monitoring m/z 554 → 171.3 and m/z 554 → 145.2 (GSH), be used for GSH synthesis in cells. Increased levels of protein-
m/z 497 → 171.3 and m/z 497 → 145.2 (γGlu-Cys), and m/z bound GSH might be derived from the newly synthesized
425 → 171.2 and m/z 425 → 116.1 (Cys-Gly), respectively, in GSH. The present study also detected fragment peptide (Cys-
the present study. By using this method, protein-bound GSH Gly) and precursor peptide (γGlu-Cys) as protein-bound forms
and its fragment peptides at a concentration of <1 μM in the in human blood, which suggests some GSH is synthesized from
plasma could be successfully determined. degradation products of GSH.
Ikegaya et al. have demonstrated that cysteine and cysteinyl- However, an early study by Kubo that used 35S-labeled GSH
glycine bind to human serum albumins through disulfide bonds, and paper electrophoresis has suggested that GSH could be
whereas no significant amount of GSH is bound to serum directly absorbed from the small intestine into rat portal
albumin that is obtained from human blood.22 The present blood.30 Therefore, there is a possibility that supplemented
study also demonstrated that only a negligible amount of GSH GSH is directly absorbed into human blood and bound to
was bound to plasma protein before the supplementation of plasma protein. To solve these problems, further studies on the
GSH. However, the protein-bound GSH significantly (P < metabolic fate of supplemented GSH that use 13C-labeled GSH
0.01) increased from 60 to 120 min after the oral are in progress. The procedure that was used in the present
supplementation of GSH. This is the first report to study for the determination of protein-bound GSH that was
demonstrate an increase in GSH in the human blood fraction based on LC-MS/MS and precolumn label techniques can
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Journal of Agricultural and Food Chemistry Article

Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
We express our appreciation to the Kyoto Integrated Science
and Technology Bio-Analysis Center for the use of their LC-
MS/MS.

■ ABBREVIATIONS USED
GSH, glutathione; γGlu-Cys-Gly, γ-L-glutamyl-L-cysteinyl-gly-
cine; Cys-Gly, L-cysteinyl-glycine; γGlu-Cys, γ-L-glutamyl-L-
cysteine; AccQ, 6-aminoquinolyl-N-hydroxysuccinimidyl carba-
mate; TEA, triethylamine; TCA, trichloroacetic acid; RP-
HPLC, reversed phase high-performance liquid chromatog-
raphy; ESI-MS, electrospray ionization mass spectrometry; LC-
MS/MS, liquid chromatography−tandem mass spectrometry;
ESI-MS/MS, electrospray ionization tandem mass spectrome-
try

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