Inherited disorders affecting mitochondrial function
are associated with glutathione deficiency
Kondala R. Atkuria,1, Tina M. Cowanb, Tony Kwanc, Angelina Ngc, Leonard A. Herzenberga, Leonore A. Herzenberga,
and Gregory M. Ennsd,1
aDepartment of Genetics, Stanford University, Stanford, CA 94305;bDepartment of Pathology, Stanford University, Stanford, CA 94304;cStanford University
Medical Center, Stanford, CA 94304; anddDepartment of Pediatrics, Division of Medical Genetics, Stanford University, Stanford, CA 94305-5208
Contributed by Leonard A. Herzenberg, January 2, 2009 (sent for review December 23, 2008)
Disorders affecting mitochondria, including those that directly
branched amino acid metabolism (organic acidemias), have been
shown to be associated with impaired redox balance. Almost all of
the evidence underlying this conclusion has been obtained from
studies on patient biopsies or animal models. Since the glutathione
(iGSH) system provides the main protection against oxidative
damage, we hypothesized that untreated oxidative stress in indi-
viduals with mitochondrial dysfunction would result in chronic
iGSH deficiency. We confirm this hypothesis here in studies using
high-dimensional flow cytometry (Hi-D FACS) and biochemical
analysis of freshly obtained blood samples from patients with
mitochondrial disorders or organic acidemias. T lymphocyte sub-
sets, monocytes and neutrophils from organic acidemia and mito-
chondrial patients who were not on antioxidant supplements
showed low iGSH levels, whereas similar subjects on antioxidant
supplements showed normal iGSH. Measures of iROS levels in
blood were insufficient to reveal the chronic oxidative stress in
untreated patients. Patients with organic acidemias showed ele-
vated plasma protein carbonyls, while plasma samples from all
patients tested showed hypocitrullinemia. These findings indicate
that measurements of iGSH in leukocytes may be a particularly
useful biomarker to detect redox imbalance in mitochondrial
disorders and organic acidemias, thus providing a relatively non-
invasive means to monitor disease status and response to thera-
pies. Furthermore, studies here suggest that antioxidant therapy
may be useful for relieving the chronic oxidative stress that
otherwise occurs in patients with mitochondrial dysfunction.
organic acidemia ? mitochondrial disorders
relatively common inborn errors of metabolism (1). These
conditions may affect any organ system, either in isolation or in
any combination, resulting in significant morbidity and mortal-
ATP production, as well as increases generation of intracellular
reactive oxygen species (iROS) and reactive nitrogen species
(iRNS), which are also byproducts of mitochondrial oxidative
phosphorylation (OXPHOS) under normal conditions (2). Re-
spiratory chain abnormalities have been documented in organic
acidemia patients, such as methylmalonic acidemia (MMA) and
propionic acidemia (PA), a knockout mouse model of MMA,
and other animal models exposed to acids typically produced in
excess in organic acidemias (3–5). The precise mechanism of
respiratory chain impairment in organic acidemias is unknown,
although impaired OXPHOS, generation of free radicals, and
decreased iGSH are likely contributors to disease pathogenesis
(3, 4, 6).
Intracellular reduced glutathione (iGSH) protects against
oxidative damage, but is transformed in the process to its
oxidized form (GSSG) (7). Because individuals with mitochon-
itochondrial disorders in aggregates have an incidence in
the adult population of ?1/8,500 and, therefore, are
drial disease and organic acidemias generate an increased
amount of iROS, it is likely that the GSH system in such
instances is stressed to a higher degree than in individuals with
normal mitochondrial function, resulting in deficiency of GSH
and possibly its precursor cysteine. In support of this theory,
GSH deficiency has been detected in a heterozygous manganese
superoxide dismutase (MnSOD) knockout mouse model and
more recently in a mut MMA mouse model (4, 8). Conversely,
?-glutamyltranspeptidase-deficient knockout mice, which are
characterized by chronic GSH deficiency, have impaired mito-
chondrial respiratory chain function (9). In times of metabolic
crisis, iROS production is likely increased, which could lead to
rapid depletion of iGSH stores and subsequently diminished
cellular capacity to detoxify these intermediates. Such a situation
may explain why individuals with genetic disorders that affect
mitochondrial function or iGSH homeostasis rapidly worsen in
times of intercurrent catabolic illness that may result in over-
production of oxidants.
Although the association of mitochondrial dysfunction with
oxidative stress has been clearly established (2), surprisingly few
reports have examined this relationship directly in blood samples
from patients with mitochondrial disease or other disorders
associated with impaired respiratory chain function such as
organic acidemias (10–12). Despite the growing list of identified
mitochondrial disorders, as well as an increasing appreciation of
the role mitochondrial dysfunction plays in the pathogenesis of
diseases associated with advancing age (such as type 2 diabetes,
cancer, and neurodegenerative disorders), relatively few diag-
nostic and therapeutic monitoring tools are available to physi-
cians caring for individuals who have mitochondrial disease.
Furthermore, the assessment of respiratory chain function after
muscle biopsy, a commonly used but invasive diagnostic proce-
dure, is often insensitive and unreliable (13). With these con-
siderations in mind, we used high-dimensional flow cytometry
(Hi-D FACS) to analyze leukocyte subsets from blood obtained
from individuals with mitochondrial disorders and organic aci-
demias, hypothesizing that increased iROS generation in these
conditions would result in low iGSH levels. We found that in
patients with disorders that affect mitochondrial respiratory
chain function iGSH levels were indeed low in T lymphocyte
subsets, monocytes, and neutrophils, but not B lymphocytes.
Such measurements may serve as potential biomarkers for
mitochondrial disorders and organic acidemias, allowing for
Author contributions: K.R.A., T.M.C., and G.M.E. designed research; K.R.A., T.K., and A.N.
performed research; K.R.A. contributed new reagents/analytic tools; K.R.A., T.M.C., Leo-
Leonard A. Herzenberg, Leonore A. Herzenberg, and G.M.E. wrote the paper.
The authors declare no conflict of interest.
firstname.lastname@example.org, or email@example.com.
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March 10, 2009 ?
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relatively non-invasive monitoring of disease status and response
Mitochondrial Disorders and Organic Acidemias Are Associated with
Glutathione Deficiency. To assess the redox (1) status of patients
with disorders affecting mitochondria we measured levels of
iGSH and iROS in peripheral blood leukocytes; and 2) plasma
protein carbonyl levels. Our results show that mitochondrial
disorders and organic acidemias result in iGSH deficiency and a
significant increase in plasma carbonyl content (Figs. 1–3).
iGSH Levels in Mitochondrial Disorders. Our study population con-
sisted of 20 patients with either definite or probable mitochon-
drial disorders classified according to the criteria described in
at the time of assay, while 11 were supplemented with 1 or more
antioxidants such as vitamin C, vitamin E, and coenzyme Q10
antioxidant supplements after the first draw. For data analysis
we divided the patient cohort into 2 groups based on antioxidant
Levels of iGSH in CD4 T cells (P ? 0.014), CD8 T cells (P ?
0.005), monocytes (P ? 0.016), and neutrophils (P ? 0.044) were
significantly lower in patients with mitochondrial disorders who
were not taking antioxidants compared to healthy controls
(Fig. 1 and Fig. S1). Subjects on antioxidant supplements were
not significantly different in their iGSH levels compared to
iGSH in Organic Acidemias. The organic acidemia cohort included
patients with MMA, PA, and isovaleric acidemia (IVA). Of the
13 blood measurements in this cohort, 6 were obtained during
routine outpatient clinic visits, while the patients were clinically
well, and 7 were obtained during hospitalization for an acute
metabolic crisis (see Table S1). One subject was taking vitamin
C at the time of sample collection. For data analysis we divided
the patients into 2 groups, inpatients (n ? 7) and outpatients
(n ? 6). iGSH levels in CD4 T cells (P ? 0.008), CD8 T cells (P ?
0.003), monocytes (P ? 0.0008), and neutrophils (P ? 0.0006)
are significantly lower in inpatients with organic acidemias as
compared to healthy controls (Fig. 2 and Fig. S1). Lower GSH
levels were detected only in CD4 T cells (P ? 0.040) and CD8
T cells (P ? 0.045) in outpatients. No significant reduction in
iGSH levels was detected in B cells.
iROS Levels Are Not Elevated in Blood Cells in Diseases Affecting
Mitochondria. We did not detect significant overall differences in
the basal levels of iROS between patient cohorts (mitochondrial
1 18-year-old female patient with thymidine kinase 2 deficiency
Plasma Protein Carbonyl Content Is Elevated in Organic Acidemias.
Protein carbonyl levels in plasma, another marker for oxidative
damage, were measured. Because of restrictions in the availabil-
ity of plasma, only select samples were assayed for protein
carbonyl levels (controls, n ? 10; mitochondrial disorders, n ?
12; organic acidemias, n ? 8). Plasma from organic acidemia
patients showed significantly higher levels of protein carbonyls
(P ? 0.014) as compared to healthy controls (Fig. 3). Plasma
from patients with mitochondrial disorders, as a whole, did not
show significantly higher levels of protein carbonyls, although 4
out of 10 samples showed elevated plasma carbonyl levels (Fig. 3).
Mitochondrial Disorders and Organic Acidemias Are Associated with
Lower Citrulline Levels in Plasma.Forty standard and non-standard
amino acids and their derivatives in the plasma of subjects with
mitochondrial disorders and organic acidemias were assayed.
Significantly lower citrulline levels were found in plasma in
patients with mitochondrial disorders and organic acidemias
(P ? 0.009) (Fig. 4). Essential amino acids, particularly the
branched chain amino acids valine, isoleucine, and leucine, were
not significantly different in the patient cohorts as compared to
healthy controls indicating no overall nutritional deficiency.
levels were measured by the MCB assay on whole blood and analyzed by Hi-D
FACS within 3 h of staining (see Materials and Methods). iGSH values are
normalized to iGSH levels of a standard PBMC preparation stained and ana-
bottom panel, iGSH levels in monocytes. Statistical significance was deter-
mined by Wilcoxon/Kruskal Wallis non-parametric test. Each point represents
a single sample. Adult controls (solid circles, n ? 21); subjects not on antiox-
idant supplements (solid circles, n ? 10); and subjects on antioxidant supple-
ments (open circles, n ? 11).
iGSH levels are lower in patients with mitochondrial disorders. iGSH
were measured by the MCB assay on whole blood and analyzed by Hi-D FACS
to iGSH levels of a standard PBMC preparation stained and analyzed at the
same time as patient samples. Top panel, iGSH levels in CD4 T cells; bottom
panel, iGSH levels in monocytes. Statistical significance was determined by
Wilcoxon/Kruskal Wallis non-parametric test. Each point represents a sample.
Two subjects had 2 repeat measurements, and 1 subject had 3 repeat mea-
(n ? 7); and outpatient subjects while clinically stable (n ? 6).
iGSH levels are lower in patients with organic acidemias. iGSH levels
www.pnas.org?cgi?doi?10.1073?pnas.0813409106Atkuri et al.
Studies here demonstrate low iGSH levels in blood cells from
patients with disorders affecting mitochondrial function caused
either by direct inhibition of the respiratory chain or by aberrant
metabolism of branched chain amino acids. The low levels of this
key intracellular antioxidant clearly indicate that these patients
suffer from systemic oxidative stress, even during times of
relatively good health. Our studies also demonstrate that iGSH
levels are normal in mitochondrial patients taking antioxidants,
suggesting that such supplementation may ameliorate some of
the effects of impaired redox balance caused by disorders that
affect mitochondrial respiratory chain function.
As we have shown, iGSH levels in CD4 and CD8 T lympho-
cytes, neutrophils and monocytes are decreased in individuals
with mitochondrial disorders or organic acidemias who are not
on antioxidant supplements. Interestingly, iGSH levels mea-
sured for patients and controls in B lymphocytes are equivalent,
irrespective of the antioxidant status of the patients. The reasons
underlying the difference between B cells and other blood cells
in this respect are unclear.
Although we detected significantly decreased cellular iGSH
levels, we did not detect a concomitant increase in iROS levels
in blood samples. This could be due to the extremely transient
nature of iROS, making their detection difficult in clinical
settings. However, iROS production could be inferred from the
observed decrease in iGSH, thus making iGSH measurement a
more stable index of cellular redox status (14). Measurements of
plasma amino acid levels did not reveal any significant changes
in branched-chain amino acid levels in these patients, suggesting
that nutritional insufficiency is less likely to be a major contrib-
uting factor for low iGSH levels. Since GSH is the main
antioxidant in mammalian cells, a decrease in its intracellular
levels, regardless of the mechanism, indicates chronic oxidative
stress in patients with mitochondrial dysfunction.
There is strong theoretical rationale and previous experimen-
tal evidence suggesting that redox imbalance plays a major role
in the pathogenic effects seen in patients with mitochondrial
disease (2). The most widely accepted mechanism of chronic
oxidative stress pathogenesis involves generation of oxidative
metabolites (iROS, iRNS, and other free radicals) that deplete
cellular antioxidant stores, leading to protein, lipid, and DNA
damage. Numerous in vitro studies have shown that inhibition of
respiratory chain complexes results in elevated levels of ROS
within the mitochondrial matrix, ultimately leading to oxidative
stress (15). These reports are supported by studies documenting
increased production of ROS, decreased GSH, a compensatory
increase in antioxidant enzymes, and elevated lipid hydroper-
oxide levels in blood and biopsy samples from a variety of
mitochondrial disorders (10, 16, 17). Two reports on chronic
progressive external ophthalmoplegia (CPEO) demonstrated
and a compensatory increase in antioxidant enzyme in muscle
fibroblasts (10, 17).
Histochemical and immunohistochemical studies on muscle
biopsies have shown that mitochondrial disorders caused by
point mutations or deletions in mtDNA lead to an induction of
antioxidant enzymes, possibly to counter chronic oxidative stress
(16). Our study further supports the hypotheses that (i) mito-
chondrial diseases are associated with chronic oxidative stress
and (ii) systemic levels of oxidative stress are reflected in
peripheral blood GSH levels, making such measurements a
potentially useful and non-invasive assay to routinely monitor
Patients taking antioxidant supplements did not show de-
creased iGSH levels. This important observation lends support
to the relatively common practice of treating mitochondrial
disorders using a variety of antioxidants (1). Oxidative stress
(iGSH depletion) further inhibits respiratory chain function,
thus initiating a vicious cycle that ultimately increases the
chances of accumulation of new mutations in mtDNA (8, 18).
Further clinical studies are needed to determine whether the
observed improvement of cellular iGSH levels in patients taking
antioxidant supplementation is a general phenomenon, or ap-
plies to only a subset of mitochondrial disease patients. This
study does not address which of the antioxidants or combination
of antioxidants may be most active, nor what dose is optimal to
achieve the observed effects. However, these results lay the
foundation for a prospective study, with blinded and cross-over
design, to address such questions.
Our results indicate that MMA, PA, and IVA patients have
decreased iGSH. This observation supports a previous report of
blood total glutathione deficiency in a 7-year-old boy with MMA
during a metabolic crisis. This child responded favorably to high
dose ascorbate supplementation, which the authors suggested
replaced the antioxidant activity of glutathione (12). Other
organic acidemias have not been studied in this manner. Nev-
ertheless, studies in animal models have demonstrated a clear
link between organic acid metabolites and oxidative stress-
induced mitochondrial dysfunction (3, 4, 19). Our findings also
lend support to evidence of increased ROS production and
mitochondrial impairment found in animal studies and fibro-
blasts or liver samples obtained from organic acidemia patients
(5, 6, 19).
Consistent with the idea of increased oxidative damage in
organic acidemias, we have detected high levels of protein
carbonyls in plasma from these patients. Protein carbonyls are
acidemias. Plasma carbonyl levels were measured in 100 ?L of platelet-free
plasma as described in Materials and Methods. Statistical significance was
determined by Wilcoxon/Kruskal Wallis non-parametric test for ranked sums
using JMP software. Each point represents a single subject. Solid circles rep-
resent subjects not on antioxidants and open circles represent subjects on
antioxidants. Adult controls (n ? 10): subjects with mitochondrial disorders
(n ? 12); and subjects with organic acidemias (n ? 8).
Plasma protein carbonyl levels are increased in subjects with organic
hypocitrullinemia. Citrulline levels were measured in platelet-free plasma as
described in Materials and Methods. Statistical significance was determined
by Wilcoxon/Kruskal Wallis non-parametric test for ranked sums using JMP
software. Each point represents a single subject. Solid circles represent sub-
jects not on antioxidants and open circles represent subjects on antioxidants.
Adults controls (n ? 5): subjects with mitochondrial disorders n ? 8; subjects
with organic acidemias (n ? 6).
Mitochondrial disorders and organic acidemias are associated with
Atkuri et al. PNAS ?
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well-established biomarkers of oxidative protein damage in vivo
in various diseases (20). Although protein carbonyls are primar-
ily caused by ROS-mediated protein damage, high protein
carbonyl content in patients with organic acidemias may also be
secondary to elevated levels of reactive aldehyde intermediates
of organic acids in the blood.
We detected hypocitrullenimia in plasma from mitochondrial
and organic acidemia subjects. Hypocitrullinemia has also been
reported in some individuals with Reye syndrome, MELAS, and
NARP (21–23). Low levels of plasma citrulline are a classic
biochemical hallmark of proximal urea cycle disorders. In ad-
dressing the relationship between citrulline levels and mitochon-
drial function, it has been postulated that primary deficiencies in
OXPHOS result in decreased citrulline synthesis via secondary
impairment of carbamyl phosphate synthetase I, an early urea
cycle enzyme that plays a key role in citrulline synthesis (23), or
by inhibiting production of the citrulline precursor ?-1-pyrroline
carboxylate through inhibition of proline oxidase (22).
There is increasing interest in identifying biomarkers of oxi-
dative stress in human disease. ROS generation due to mito-
chondrial dysfunction likely plays a role in multiple disorders,
including diabetes, atherosclerosis, neurodegenerative diseases,
hypoxic-ischemic encephalopathy, autism, retinopathy of pre-
maturity, and cancer (20, 24). Our results indicate that iGSH
degree of mitochondrial impairment and even response to
therapy in mitochondrial and organic acidemia patients.
At present, antioxidant supplements are often given to mito-
chondrial patients without the ability to monitor therapeutic
response. However, antioxidant supplementation has not been
widely used for the management of organic acidemia patients.
Given the significantly low iGSH levels detected in patients with
organic acidemias, especially during acute illness, there may be
a role for such supplementation in these patients as well. Even
with optimal metabolic control, organic acidemia patients often
demonstrate significant mental retardation. Some patients have
structures, but many without obvious brain imaging abnormal-
ities also display cognitive impairment (25). Because iGSH
deficiency likely plays a role in pathogenesis of neurodegenera-
tive diseases, it is also reasonable to speculate that therapies that
improve redox imbalance may be beneficial for cognitive and
neurologic outcome in organic acidemias and mitochondrial
In this observational study, iGSH deficiency was demon-
strated in patients with disorders characterized by mitochondrial
dysfunction who were not taking antioxidants, despite the het-
erogeneous nature of the subject population. However, subjects
on antioxidants did not show detectable iGSH deficiency. Al-
though these findings need to be confirmed in a larger cohort of
patients, results presented here show that iGSH measurement
represents an important initial step toward a rational assessment
of therapeutic response, and even the development of individ-
ualized treatment regimens, in disorders that affect mitochon-
drial function. Moreover, these findings provide the foundation
to embark on further studies focusing on the relationship of
factors such as age, specific diagnosis, disease severity, clinical
status, and treatment to iGSH deficiency in mitochondrial
Materials. All monoclonal antibodies (either purified or preconjugated to
fluorochromes) were procured from Becton Dickinson Biosciences (BDB). PE
and Allophycocyanin were obtained from Prozyme. Monochlorobimane
(MCB), Dihydrorhodamine 123 (DHR123), and RPMI medium 1640 were ob-
tained from Invitrogen. Probenecid and other fine chemicals were obtained
from Sigma Aldrich. Protein Carbonyl assay kit was procured from Cayman
Human Subjects. Twenty-nine subjects were included in the study, including 9
with organic acidemias (6 MMA mut0, 2 IVA, 1 PA) and 20 with mitochondrial
2 combined complex I/III deficiency, 1 combined complex I/IV deficiency
(tRNALeu3243A?T), 1 combined complex II/III deficiency, 1 complex III defi-
ciency, 1 mtDNA deletion syndrome, 1 mtDNA depletion syndrome (TK2
deficiency), and 3 with undefined disease but with clinical features including
Leigh syndrome or multiorgan system involvement, and biochemical findings
consistent with mitochondrial disease]. Organic acidemia diagnoses were
established by urine organic acid analysis; MMA patients further underwent
complementation studies on cultured skin fibroblasts (Dr. David Rosenblatt,
McGill University, Montreal, Canada). The organic acidemia cohort was fur-
ther classified by clinical status as either inpatient (acutely ill) or outpatient
and symptoms, as well as standard biochemical and molecular analyses (e.g.,
nuclear DNA sequencing). Subjects were classified as having definite (n ? 11)
by Bernier et al. (26). For data analysis the mitochondrial disease cohort was
classified according to whether or not subjects were taking pharmacologic
doses of supplements with antioxidant activity (e.g., ascorbate, vitamin E,
?-lipoic acid, coenzyme Q10) at the time of sample collection. Antioxidant
supplementation was not standardized. All controls were adults and not age
matched. Samples were collected after informed consent. The Stanford Uni-
versity Institutional Review Board approved all study protocols.
Sample Collection and Preparation. Peripheral blood (1–5 mL) from patients
(Vacutainer BDB). Blood was processed as previously described (27). In brief,
blood was centrifuged at 400 ? g and the plasma fraction was collected for
further processing. The remaining cellular fraction was washed with DPBS-
EDTA (DPBS containing 2.5 mM EDTA) and finally resuspended in bimane
platelets and the resulting platelet-free plasma was aliquoted and stored at
-80 °C for amino acid and protein carbonyl assays.
FACS Assays for Intracellular Redox Status. The intracellular redox state of
according to Atkuri et al. (28). In brief, separate aliquots of cells were stained
with 40 ?M monochlorobimane (for iGSH) or 1 ?M DHR123 (for iROS) for 20
min in staining media (RPMI medium 1640, 4% FCS and 2.5 mM probenecid)
at room temperature. The reaction was quenched with excess chilled staining
further processing for Hi-D FACS. iGSH levels were expressed as median
fluorescence intensity (MFI) of intracellular GS-bimane adducts.
High-Dimensional (Hi-D) FACS Analysis. Fifty microliters of washed cellular
fraction was stained with different cocktails of fluorochrome-conjugated
antibodies [CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD235 (glycophorin)]
was performed as described (29, 30). Hi-D FACS data were collected on a
(BD). Flowjo (Treestar) software was used for fluorescence compensation and
analysis. See Fig. S2 for cell gating scheme.
iGSH Levels Normalization to Correct for Experimental Variation. iGSH levels
were expressed relative to iGSH levels measured in the same experiment for a
standard PBMC preparation. The standard PBMC preparation was isolated by
Ficoll gradient centrifugation from a 500-mL blood sample from a healthy
individual, aliquoted, and maintained in liquid nitrogen until immediately
before use. Aliquots of the same standard were used for all normalizations
carried out in this study.
Amino Acid Analysis of Plasma. Plasma amino acids were measured by ninhy-
drin derivatization followed by spectrometric detection with S-aminoethyl-
cystine as an internal standard as described by Spackman et al. (31).
Statistical Analysis. Analyses of FACS data, was performed using FlowJo
software (Treestar). Statistical analyses were performed with the JMP statis-
tical software package (SAS Institute).
www.pnas.org?cgi?doi?10.1073?pnas.0813409106Atkuri et al.
ACKNOWLEDGMENTS. We thank the following members of the Herzen- Download full-text
berg Laboratory (Genetics Department, Stanford University School of Med-
icine): Bahram Aram and Glenn Smith for excellent and devoted technical
support; and John J. Mantovani for administrative help, including the
preparation of the manuscript. We thank Takeshi Fukuhara (VA Hospital)
for help with measurements with protein carbonyls. We thank Vicki Sweet,
RN, PNP; Andrea Kwan, MS; Elizabeth Hadley, RN; and Daphne Nayyar, RN
for coordinating the patient sample collection. This work was generously
supported by our community, the Lucile Packard Foundation For Children’s
Health, and by grants from the United Mitochondrial Disease Foundation,
the Lucile Packard Children’s Fund, and the Arline and Pete Harman
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