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Quantitation of glutathione by multiple label SIDA / J. Reinbold et al. / Anal. Biochem. 445 (2014) 41–48
cðAÞ ¼ ½m ꢁ ðAðAÞ=AðSÞÞ þ tꢂ ꢁ cðSÞ;
distinguished from the label resulting from the oxidized labeled
GSH standard. In contrast to this, the other strategy for a SIDA re-
cently was reported by Haberhauer-Troyer et al. [15], who used the
same label for GSH and GSSG, but calculated the degree of oxidized
GSH by monitoring the mixed disulfide formed from unlabeled
GSH and the labeled GSH internal standard. However, this ap-
proach would not allow one to quantify simultaneously GSH and
GSSG and to differentiate oxidation of GSH from reduction of GSSG.
Taking these considerations into account, a differently sixfold-
labeled GSSG generated from [13C2,15N]GSH labeled in the glycine
moiety was chosen. Taking into account possible oxidation as well
as reduction or thiol/disulfide exchange of the GSH isotopologues,
for reduced GSH the occurrence of three isotopologues (unlabeled
GSH M+0; [13C2,15N]GSH M+3; [13C3,15N]GSH M+4) and for GSSG
the occurrence of six isotopologues (unlabeled GSSG M+0;
where c(A) is the concentration of analyte in erythrocytes, c(S) is the
concentration of internal standard in erythrocytes, A(A) is the area
of analyte m/z 1083 ? 1008, A(S) is the area of internal standard
m/z 1085 ? 1007, m is the slope of response curve for Didans-GSSG
M+6 (Table 1), and t is the y intercept of response curve for Didans-
GSSG M+6 (Table 1).
A statistical distribution of GSSG isotopologues formed from
GSH and GSH M+4 was assumed to calculate their abundances P
from the following equations:
PðGSHÞ þ PðGSH M þ 4Þ ¼ 100% results in GSSG distributions :
2
PðGSSGÞ ¼ PðGSHÞ ;
PðGSSG M þ 4Þ ¼ 2 ꢁ PðGSHÞ ꢁ PðGSH M þ 4Þ;
[
[
13C2,15N]GSSG M+3; [13C3,15N]GSSG M+4; [13C4,15N2]GSSG M+6;
2
13C5,15N2]GSSG M+7; 13C6,15N2]GSSG M+8) was conceivable.
[
PðGSSG M þ 8Þ ¼ PðGSH M þ 4Þ :
Therefore, we tested in preliminary experiments whether the
isotopologues could be differentiated by LC–MS/MS. For GSH, the
fragmentation of the carboxymethyl-dansyl-GSH resulted mainly
in two products upon loss (1) of 2-aminoacetic acid and (2) of
2-carbonylaminoacetic acid, both from the C-terminus (Fig. 1).
The isotopologues shown in Fig. 1 reveal a mass difference of at
least 3 u either in the precursor or in the product ions.
For didansylated GSSG, MS/MS fragmentation revealed the most
intense fragment by loss of one glycine as depicted in Fig. 2. How-
ever, differentiation of the isotopologues is more difficult than for
GSH as the mass differences between the possible isotopologues
are smaller. Moreover, owing to the higher number of atoms
present in the dansylated GSSG molecule, natural isotopes of car-
bon, sulfur, oxygen, and nitrogen are likely to cause interferences
between the single isotopologues.
Hence, a factor, ft, for the theoretical distribution of GSSGformed
GSSG M+4 was calculated from the areas of GSH and GSH M+4 in
the characteristic mass traces according to the following equation:
/
ft ¼ GSSGformed=GSSG M þ 4 ¼ AðGSHÞ=2 ꢁ AðGSH M þ 4Þ:
Glutathione disulfide formed during analysis (GSSGformed) was
calculated then by multiplication of the GSSG M+4 concentration
with the factor ft of the theoretical distribution of GSSG/GSSG M+4.
Method validation
As there was no material available that was similar to the eryth-
rocyte matrix and did not contain the analyte GSH, the limit of
detection (LOD) was derived from the LOD calculated for cereal
flour [19] considering the different sample dilutions.
Intraday precision was evaluated by measuring GSH, GSSG, and
GSO3H in the erythrocytes of one volunteer in triplicate.
For measuring the recovery, erythrocytes were spiked (each in
triplicate) with three different amounts of GSH and analyzed by
SIDA.
When reacting the single GSH isotopologues GSH M+0,
[
13C2,15N]GSH M+3, and [13C3,15N]GSH M+4 in model experiments
separately to mixed GSSG isotopologues, in particular significant
interferences for the GSSG isotopologues [13C3,15N]GSSG M+4,
[
13C5,15N2]GSSG M+7, and [13C6,15N2]GSSG M+8 were observed. As
shown in Table 3, the natural isotopologues with an additional
mass of 1 u of [13C2,15N]GSSG M+3+1, of [13C4,15N2]GSSG M+6+1,
and of [13C5,15N2]GSSG M+7+1 interfered with [13C3,15N]GSSG
M+4, [13C5,15N2]GSSG M+7, and [13C6,15N2]GSSG M+8, respectively,
each by giving signal abundances of about 50% of the respective
major isotopologue.
Results and discussion
Quantitation of glutathione by stable-isotope dilution assays
The glutathione present in erythrocytes is susceptible to degra-
dation and oxidation because of the occurrence of hemoglobin,
iron, and various enzymes. Therefore, even careful sample prepara-
tion under exclusion of oxygen is not likely to prevent oxidation of
GSH. For similarly challenging analytes such as folates [21] or
thiol-containing odorants [22], SIDAs have proven their superiority
over other alternatives. Therefore, we decided also to use this
methodology for quantitation of GSH in erythrocytes by applica-
tion of [13C3,15N]GSH labeled in the cysteine moiety. This standard
already has been used for quantitation of total GSH in cereals after
derivatization with IAA and dansylation [19]. In that study we re-
ported that IAA was preferred as the thiol protection agent over
NEM as the former does not react with amino groups. Nevertheless,
in a recent study the suitability of NEM for analyzing GSH in blood
was shown [23]. In our SIDA for cereals we dansylated the analytes
to enhance retention in HPLC and sensitivity in LC–MS. However,
This could be particularly crucial for monitoring the GSH
oxidation during analysis, as this is reflected by the formation of
[
13C3,15N]GSSG M+4 and [13C6,15N2]GSSG M+8 generated by reac-
tion of the labeled standard [13C3,15N]GSH M+4 with itself or with
unlabeled GSH. Therefore, the respective overlap with natural iso-
topologues of GSSG M+3 and GSSG M+7 has to be considered.
Synthesis and mass spectrometry of glutathione sulfonic acid
GSO3H has been described as an oxidation product from GSH
and GSSG when reacted with oxidants such as hydroperoxide
[20]. Therefore, blood was also analyzed for this possible oxidation
product of GSH.
13C3,15N-labeled glutathione sulfonic acid was prepared by oxi-
dation of [13C3,15N]GSH with performic acid according to Henschen
[24]. For quantitation by LC–MS/MS, GSO3H was dansylated using
the same conditions as for GSH and GSSG. As selective transition,
the simultaneous loss of glycine and cysteine was chosen. Thus,
for the labeled GSO3H isotopologues, all labels were lost and the
product ion at m/z 363 is the same for all labeled and unlabeled
GSO3H isotopologues (Fig. 3). However, the labeled compounds
still could be distinguished from the unlabeled compound by their
different precursor ion masses.
[
13C3,15N]GSH would not allow for simultaneous quantitation of
GSSG as the amount of GSH isotopologues oxidized to GSSG would
be unknown. Moreover, we also observed reduction of GSSG to
GSH upon some extraction conditions, which would not be recog-
nized when using identically labeled GSH and GSSG as applied in
the studies of Haberhauer-Troyer et al. [15] and Harwood et al.
[14]. Therefore, we decided to use a label for GSSG that can be
Reinbold, Julia
