Inherited disorders affecting mitochondrial function are associated with glutathione deficiency and hypocitrullinemia

Article (PDF Available)inProceedings of the National Academy of Sciences 106(10):3941-5 · March 2009with20 Reads
DOI: 10.1073/pnas.0813409106 · Source: PubMed
Abstract
Disorders affecting mitochondria, including those that directly affect the respiratory chain function or result from abnormalities in 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 individuals 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 subsets, monocytes and neutrophils from organic acidemia and mitochondrial 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 elevated 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 therapies. Furthermore, studies here suggest that antioxidant therapy may be useful for relieving the chronic oxidative stress that otherwise occurs in patients with mitochondrial dysfunction.
Inherited disorders affecting mitochondrial function
are associated with glutathione deficiency
and hypocitrullinemia
Kondala R. Atkuri
a,1
, Tina M. Cowan
b
, Tony Kwan
c
, Angelina Ng
c
, Leonard A. Herzenberg
a
, Leonore A. Herzenberg
a
,
and Gregory M. Enns
d,1
a
Department of Genetics, Stanford University, Stanford, CA 94305;
b
Department of Pathology, Stanford University, Stanford, CA 94304;
c
Stanford University
Medical Center, Stanford, CA 94304; and
d
Department 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
affect the respiratory chain function or result from abnormalities in
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
M
itochondrial disorders in aggregates have an incidence in
the adult population of 1/8,500 and, therefore, are
relatively common inborn errors of met abolism (1). These
c onditions may affect any organ system, either in isolation or in
any combination, resulting in significant morbidity and mortal-
it y. Dysfunction of the mitochondrial respiratory chain decreases
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 abnor malities have been documented in organic
acidemia patients, such as methylmalon ic acidemia (MMA) and
propion ic 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 impair ment 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 indiv iduals with mitochon-
drial disease and organic acidemias generate an increased
amount of iROS, it is likely that the GSH system in such
inst ances is stressed to a higher degree than in individuals w ith
nor mal 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 dismut ase (MnSOD) knock out mouse model and
more recently in a mut MMA mouse model (4, 8). Conversely,
-glut amyltranspeptidase-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 inter mediates. 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.
A lthough the association of mitochondrial dysfunction with
oxidative stress has been clearly established (2), surprisingly few
reports have examined this relationship directly in blood samples
f rom patients with mitochondrial disease or other disorders
associated with impaired respiratory chain function such as
organ ic 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.
Further more, the assessment of respiratory chain function af ter
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
f rom individuals with mitochondrial disorders and organic aci-
demias, hypothesizing that increased iROS generation in these
c onditions 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 lymphocy te
subsets, monocy tes, 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-
nard A. Herzenberg, Leonore A. Herzenberg, and G.M.E. analyzed data; and K.R.A., T.M.C.,
Leonard A. Herzenberg, Leonore A. Herzenberg, and G.M.E. wrote the paper.
The authors declare no conflict of interest.
1
To whom correspondence may be addressed. E-mail: greg.enns@stanford.edu,
atkuri@stanford.edu, or lenherz@darwin.stanford.edu.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0813409106/DCSupplemental.
www.pnas.orgcgidoi10.1073pnas.0813409106 PNAS
March 10, 2009
vol. 106
no. 10
3941–3945
MEDICAL SCIENCES
relatively non-invasive monitoring of disease status and response
to therapies.
Results
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 deficienc y and a
sign ificant 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
Mater ials and Methods. Ten subjects were not t aking antioxidants
at the time of assay, while 11 were supplemented with 1 or more
antioxidants such as vitamin C, vitamin E, and coenz yme Q
10
(see Table S1). One subject underwent 2 blood draws and started
antioxidant supplements after the first draw. For data analysis
we divided the patient cohort into 2 groups based on antioxidant
st atus.
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
sign ificantly 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
healthy controls.
iGSH in Organic Acidemias. The organic acidemia cohort included
patients with MM A, 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
met abolic 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 organ ic acidemias as
c ompared to healthy controls (Fig. 2 and Fig. S1). L ower GSH
levels were detected only in CD4 T cells (P 0.040) and CD8
T cells (P 0.045) in outpatients. No sign ificant 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
disorders and organic acidemias) and healthy controls. However,
1 18-year-old female patient with thymidine kinase 2 deficiency
and 1 20-year-old male with MELAS showed high levels of iROS.
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-
it y of plasma, only select samples were assayed for protein
carbonyl levels (controls, n 10; mitochondrial disorders, n
12; organ ic acidemias, n 8). Plasma f rom organic acidemia
patients showed significantly higher levels of protein carbonyls
(P 0.014) as compared to healthy controls (Fig. 3). Plasma
f rom 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 st andard and non-standard
amino acids and their derivatives in the plasma of subjects with
mitochondrial disorders and organic acidemias were assayed.
Sign ificantly 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 sign ificantly different in the patient cohorts as compared to
healthy controls indicating no overall nutritional deficiency.
Fig. 1. iGSH levels are lower in patients with mitochondrial disorders. iGSH
levels were measured by the MCB assay on whole blood and analyzed by Hi-D
FACS within3hofstaining (see Materials and Methods). iGSH values are
normalized to iGSH levels of a standard PBMC preparation stained and ana-
lyzed at the same time as patient samples. Top panel, iGSH levels in CD4 T cells;
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).
Fig. 2. iGSH levels are lower in patients with organic acidemias. iGSH levels
were measured by the MCB assay on whole blood and analyzed by Hi-D FACS
within3hofstaining (see Materials and Methods). iGSH levels are normalized
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-
surements. Adult controls (n 21); inpatient subjects during an acute episode
(n 7); and outpatient subjects while clinically stable (n 6).
3942
www.pnas.orgcgidoi10.1073pnas.0813409106 Atkuri et al.
Discussion
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
met abolism of branched chain amino acids. The low levels of this
key intracellular antioxidant clearly indicate that these patients
suf fer from systemic oxidative stress, even during times of
relatively good health. Our studies also demonstrate that iGSH
levels are normal in mitochondrial patients tak ing antioxidants,
suggesting that such supplementation may ameliorate some of
the effects of impaired redox balance caused by disorders that
af fect mitochondrial respiratory chain function.
As we have shown, iGSH levels in CD4 and CD8 T lympho-
c ytes, neutrophils and monocy tes are decreased in individuals
with mitochondrial disorders or organic acidemias who are not
on antioxidant supplements. Interestingly, iGSH levels mea-
sured for patients and c ontrols 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.
A lthough we detected significantly decreased cellular iGSH
levels, we did not detect a conc omitant increase in iROS levels
in blood samples. This could be due to the extremely transient
nature of iROS, mak ing their detection difficult in clinical
settings. However, iROS production c ould 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 ox idative
stress in patients with mitochondrial dysfunction.
There is strong theoretical rationale and previous experimen-
t al 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
met abolites (iROS, iRNS, and other f ree 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 variet y of
mitochondrial disorders (10, 16, 17). Two reports on chron ic
progressive external ophthalmoplegia (CPEO) demonstrated
low GSH levels in plasma and ery throcytes, higher levels of ROS,
and a compensatory increase in antioxidant enz yme 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 enz ymes, 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 mon itor
redox imbalance.
Patients taking antioxidant supplements did not show de-
creased iGSH levels. This import ant observation lends support
to the relatively common practice of treating mitochondrial
disorders using a variet y of antioxidants (1). Oxidative stress
(iGSH depletion) further inhibits respiratory chain function,
thus in itiating 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 tak ing
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 glut athione (12). Other
organ ic acidemias have not been studied in this manner. Nev-
ertheless, studies in animal models have demonstrated a clear
link between organ ic 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
organ ic acidemias, we have detected high levels of protein
carbonyls in plasma from these patients. Protein carbonyls are
Fig. 3. Plasma protein carbonyl levels are increased in subjects with organic
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).
Fig. 4. Mitochondrial disorders and organic acidemias are associated with
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).
Atkuri et al. PNAS
March 10, 2009
vol. 106
no. 10
3943
MEDICAL SCIENCES
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
sec ondary to elevated levels of reactive aldehyde intermediates
of organic acids in the blood.
We detected hypocitrullen imia in plasma from mitochondrial
and organic acidemia subjects. Hypocitrullinemia has also been
reported in some individuals with Reye syndrome, MELAS, and
NA RP (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 v ia sec ondary
impair ment of carbamyl phosphate synthetase I, an early urea
c ycle 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 identif ying 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-
maturit y, and cancer (20, 24). Our results indicate that iGSH
measured by Hi-D FACS may be a useful biomarker for assessing
degree of mitochondrial impair ment and even response to
therapy in mitochondrial and organic acidemia patients.
At present, antiox idant supplements are of ten given to mito-
chondrial patients without the ability to mon itor 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
organ ic acidemias, especially during acute illness, there may be
a role for such supplementation in these patients as well. Even
with optimal met abolic control, organic acidemia patients often
demonstrate significant mental retardation. Some patients have
had so-called ‘‘metabolic strokes,’’ i.e., injury to deep gray matter
str uctures, but many w ithout obvious brain imaging abnormal-
ities also display cognitive impair ment (25). Because iGSH
deficienc y 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 cogn itive and
neurologic outcome in organic acidemias and mitochondrial
disorders.
In this observational study, iGSH deficiency was demon-
strated in patients with disorders characterized by mitochondrial
dysfunction who were not tak ing antioxidants, despite the het-
erogeneous nature of the subject population. However, subjects
on antioxidants did not show detectable iGSH deficiency. A l-
though these findings need to be confir med in a larger cohort of
patients, results presented here show that iGSH measurement
represents an import ant 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 severit y, clinical
st atus, and treatment to iGSH deficiency in mitochondrial
disease.
Methods
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
Chemicals.
Human Subjects. Twenty-nine subjects were included in the study, including 9
with organic acidemias (6 MMA mut
0
, 2 IVA, 1 PA) and 20 with mitochondrial
disorders [4 tRNA
Leu
3243AG, 4 complex I deficiency, 2 complex IV deficiency,
2 combined complex I/III deficiency, 1 combined complex I/IV deficiency
(tRNA
Leu
3243AT), 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
(clinically stable). Mitochondrial disease was diagnosed based on clinical signs
and symptoms, as well as standard biochemical and molecular analyses (e.g.,
muscle or skin fibroblast respiratory chain activities and mitochondrial DNA or
nuclear DNA sequencing). Subjects were classified as having definite (n 11)
or probable (n 9) mitochondrial disease based on diagnostic criteria defined
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 Q
10
) 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
and healthy volunteers was collected by venipuncture into heparanized tubes
(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
medium in a volume equivalent to the original volume of blood collected. The
plasma fraction was further centrifuged for 10 min at 3,000 g to remove the
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
peripheral blood leukocytes was determined by FACS assays for iGSH and iROS
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
media. The cells were then centrifuged and resuspended in staining media for
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)]
prepared in our laboratory or obtained from BD-PharMingen. Surface staining
was performed as described (29, 30). Hi-D FACS data were collected on a
modified BD FACStar with Moflo electronics (Cytomation, MO) or BD FACSAria
(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.
Plasma Protein Carbonyl Assay. Plasma protein carbonyls were assayed accord-
ing to the protocol provided by the manufacturer (Cayman Chemicals; catalog
# 10005020).
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).
3944
www.pnas.orgcgidoi10.1073pnas.0813409106 Atkuri et al.
ACKNOWLEDGMENTS. We thank the following members of the Herzen-
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
Scholarship.
1. Chinnery PF, Turnbull DM (2001) Epidemiology and treatment of mitochondrial dis-
orders. Am J Med Genet 106:94–101.
2. Wallace DC (1999) Mitochondrial diseases in man and mouse. Science 283:1482–1488.
3. Fontella FU, et al. (2000) Propionic and L-methylmalonic acids induce oxidative stress
in brain of young rats. Neuroreport 11:541–544.
4. Chandler RJ, et al. (2008) Mitochondrial dysfunction in mut methylmalonic acidemia.
FASEB J, in press.
5. Mardach R, Verity MA, Cederbaum SD (2005) Clinical, pathological, and biochemical
studies in a patient with propionic acidemia and fatal cardiomyopathy. Mol Genet
Metab 85:286 –290.
6. Richard E, Alvarez-Barrientos A, Perez B, Desviat LR, Ugarte M (2007) Methylmalonic
acidaemia leads to increased production of reactive oxygen species and induction of
apoptosis through the mitochondrial/caspase pathway. J Pathol 213:453– 461.
7. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the
redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med
30:1191–1212.
8. Williams MD, et al. (1998) Increased oxidative damage is correlated to altered mito-
chondrial function in heterozygous manganese superoxide dismutase knockout mice.
J Biol Chem 273:28510 –28515.
9. Will Y, et al. (2000) gamma-glutamyltranspeptidase-deficient knockout mice as a
model to study the relationship between glutathione status, mitochondrial function,
and cellular function. Hepatology 32:740–749.
10. Piccolo G, et al. (1991) Biological markers of oxidative stress in mitochondrial myo-
pathies with progressive external ophthalmoplegia. J Neurol Sci 105:57–60.
11. Piemonte F, et al. (2001) Glutathione in blood of patients with Friedreich’s ataxia. Eur
J Clin Invest 31:1007–1011.
12. Treacy E, et al. (1996) Glutathione deficiency as a complication of methylmalonic
acidemia: response to high doses of ascorbate. J Pediatr 129:445– 448.
13. Thorburn DR, Smeitink J (2001) Diagnosis of mitochondrial disorders: Clinical and
biochemical approach. J Inherit Metab Dis 24:312–316.
14. Pastore A, Federici G, Bertini E, Piemonte F (2003) Analysis of glutathione: Implication
in redox and detoxification. Clin Chim Acta 333:19 –39.
15. Turrens JF (1997) Superoxide production by the mitochondrial respiratory chain. Biosci
Rep 17:3– 8.
16. Filosto M, et al. (2002) Antioxidant agents have a different expression pattern in muscle
fibers of patients with mitochondrial diseases. Acta Neuropathol (Berl) 103:215–220.
17. Lu CY, Wang EK, Lee HC, Tsay HJ, Wei YH (2003) Increased expression of manganese-
superoxide dismutase in fibroblasts of patients with CPEO syndrome. Mol Genet Metab
80:321–329.
18. Jha N, et al. (2000) Glutathione depletion in PC12 results in selective inhibition of
mitochondrial complex I activity. Implications for Parkinson’s disease. J Biol Chem
275:26096–26101.
19. Hayasaka K, et al. (1982) Comparison of cytosolic and mitochondrial enzyme alter-
ations in the livers of propionic or methylmalonic acidemia: A reduction of cytochrome
oxidase activity. Tohoku J Exp Med 137:329 –334.
20. Dalle-Donne I, Rossi R, Colombo R, Giustarini D, Milzani A (2006) Biomarkers of
oxidative damage in human disease. Clin Chem 52:601– 623.
21. Schiff GM (1976) Reye’s syndrome. Annu Rev Med 27:447–452.
22. Naini A, et al. (2005) Hypocitrullinemia in patients with MELAS: An insight into the
‘‘MELAS paradox.’’ J Neurol Sci 229- 230:187–193.
23. Parfait B, et al. (1999) The neurogenic weakness, ataxia, and retinitis pigmentosa
(NARP) syndrome mtDNA mutation (T8993G) triggers muscle ATPase deficiency and
hypocitrullinaemia. Eur J Pediatr 158:55–58.
24. Enns GM (2003) The contribution of mitochondria to common disorders. Mol Genet
Metab 80:11–26.
25. Heidenreich R, et al. (1988) Acute extrapyramidal syndrome in methylmalonic
acidemia: ‘‘Metabolic stroke’’ involving the globus pallidus. J Pediatr 113:1022–
1027.
26. Bernier FP, et al. (2002) Diagnostic criteria for respiratory chain disorders in adults and
children. Neurology 59:1406 –1411.
27. Tirouvanziam R, et al. (2006) High-dose oral N-acetylcysteine, a glutathione prodrug,
modulates inflammation in cystic fibrosis. Proc Natl Acad Sci USA 103:4628 4633.
28. Atkuri KR, Herzenberg LA, Niemi AK, Cowan T, Herzenberg LA (2007) Importance of
culturing primary lymphocytes at physiological oxygen levels. Proc Natl Acad Sci USA
104:4547–4552.
29. De Rosa SC, Roederer M (2001) Eleven-color flow cytometry. A powerful tool for
elucidation of the complex immune system. Clin Lab Med 21:697–712.
30. Sahaf B, Heydari K, Herzenberg LA (2003) Lymphocyte surface thiol levels. Proc Natl
Acad Sci USA 100:4001– 4005.
31. Spackman DH, Moore S, Stein WH (1958) Automatic recording apparatus for use in the
chromatography of amino acids. Fed Proc 17:1107–1115.
Atkuri et al. PNAS
March 10, 2009
vol. 106
no. 10
3945
MEDICAL SCIENCES
    • "Oxidative stress is strictly associated with the physiophatology of several metabolic disorders including methylmalonic aciduria, phenylketonuria, and maple syrup urine disease [441][442][443]. Atkuri and co-workers reported diminished intracellular glutathione levels in leukocytes of patients with organic acidemias and normal glutathione concentrations in patients receiving antioxidant supplementation [444]. ROS overproduction and increased apoptosis levels have been found in fibroblasts of cblC patients [445] . "
    [Show abstract] [Hide abstract] ABSTRACT: Neurodegenerative diseases are characterized by a gradual and selective loss of neurons. ROS overload has been proved to occur early in this heterogeneous group of disorders, indicating oxidative stress as a primer factor underlying their pathogenesis. Given the importance of a better knowledge of the cause/effect of oxidative stress in the pathogenesis and evolution of neurodegeneration, recent efforts have been focused on the identification and determination of stable markers that may reflect systemic oxidative stress. This review provides an overview of these systemic redox biomarkers and their responsiveness to antioxidant therapies. Redox biomarkers can be classified as molecules that are modified by interactions with ROS in the microenvironment and antioxidant molecules that change in response to increased oxidative stress. DNA, lipids (including phospholipids), proteins and carbohydrates are examples of molecules that can be modified by excessive ROS in vivo. Some modifications have direct effects on molecule functions (e.g. to inhibit enzyme function), but others merely reflect the degree of oxidative stress in the local environment. Testing of redox biomarkers in neurodegenerative diseases has 3 important goals: 1) to confirm the presence or absence of systemic oxidative stress; 2) to identify possible underlying (and potentially reversible) causes of neurodegeneration; and 3) to estimate the severity of the disease and the risk of progression. Reflecting pathological processes occurring in the whole body, redox biomarkers may pinpoint novel therapeutic targets and lead to diagnose diseases before they are clinically evident.
    Article · Jul 2015
    • "Decreased activities of mitochondrial complexes I–IV have also been reported in hippocampus of a murine model of SSADH deficiency [16]. Low glutathione levels have been demonstrated in the blood of patients with primary mitochondrial disease [24] as well as in the blood and liver tissue of patients with organic acidemias such as methylmalonic acidemia [24] [25] [26] indicating secondary mitochondrial dysfunction and redox imbalance organic acidemias. Increased lipid peroxidation [14] [15] [23] and low glutathione levels [10] [14] [16] in murine models of SSADH deficiency as well as dicarboxylic aciduria reported in patients [3] and mitochondrial dysfunction in SSADH deficiency, similar to other organic acidemias. "
    [Show abstract] [Hide abstract] ABSTRACT: The pathophysiology of succinic semialdehyde dehydrogenase (SSADH) deficiency is not completely understood. Oxidative stress, mitochondrial pathology, and low reduced glutathione levels have been demonstrated in mice, but no studies have been reported in humans. We report on a patient with SSADH deficiency in whom we found low levels of blood reduced glutathione (GSH), and elevations of dicarboxylic acids in urine, suggestive of possible redox imbalance and/or mitochondrial dysfunction. Thus, targeting the oxidative stress axis may be a potential therapeutic approach if our findings are confirmed in other patients.
    Full-text · Article · Dec 2014
    • "During oxidative stress, GSH is transformed to the oxidized form (GSSG) to counter-balance increased amounts of cellular ROS (Schafer and Buettner, 2001). GSH is lower in patients with genetically inherited mitochondrial diseases compared with healthy individuals, ostensibly due to the prolonged exposure to oxidative stress caused by mitochondrial dysfunction (Atkuri et al., 2009). However, a specific decrease of GSH was not observed in HL-60 cells treated with known mitochondrial disruptors in our assays. "
    [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial perturbation has been recognized as a contributing factor to various drug-induced organ toxicities. To address this issue, we developed a high-throughput flow cytometry-based mitochondrial signaling assay to systematically investigate mitochondrial/cellular parameters known to be directly impacted by mitochondrial dysfunction: mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (ROS), intracellular reduced glutathione (GSH) level, and cell viability. Modulation of these parameters by a training set of compounds, comprised of established mitochondrial poisons and 60 marketed drugs (30 nM to 1mM), was tested in HL-60 cells (a human pro-myelocytic leukemia cell line) cultured in either glucose-supplemented (GSM) or glucose-free (containing galactose/glutamine; GFM) RPMI-1640 media. Post-hoc bio-informatic analyses of IC50 or EC50 values for all parameters tested revealed that MMP depolarization in HL-60 cells cultured in GSM was the most reliable parameter for determining mitochondrial dysfunction in these cells. Disruptors of mitochondrial function depolarized MMP at concentrations lower than those that caused loss of cell viability, especially in cells cultured in GSM; cellular GSH levels correlated more closely to loss of viability in vitro. Some mitochondrial respiratory chain inhibitors increased mitochondrial ROS generation; however, measuring an increase in ROS alone was not sufficient to identify mitochondrial disruptors. Furthermore, hierarchical cluster analysis of all measured parameters provided confirmation that MMP depletion, without loss of cell viability, was the key signature for identifying mitochondrial disruptors. Subsequent classification of compounds based on ratios of IC50s of cell viability:MMP determined that this parameter is the most critical indicator of mitochondrial health in cells and provides a powerful tool to predict whether novel small molecule entities possess this liability.
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