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Liposomal-Glutathione Provides Maintenance of Intracellular Glutathione and Neuroprotection in Mesencephalic Neuronal Cells



A liposomal preparation of glutathione (GSH) was investigated for its ability to replenish intracellular GSH and provide neuroprotection in an in vitro model of Parkinson's disease using paraquat plus maneb (PQMB) in rat mesencephalic cultures. In mixed neuronal/glial cultures depleted of intracellular GSH, repletion to control levels occurred over 4 h with liposomal-GSH or non-liposomal-GSH however, liposomal-GSH was 100-fold more potent; EC(50s) 4.75 μM and 533 μM for liposomal and non-liposomal-GSH, respectively. Liposomal-GSH utilization was also observed in neuronal cultures, but with a higher EC(50) (76.5 μM), suggesting that glia facilitate utilization. Blocking γ-glutamylcysteine synthetase with buthionine sulfoxamine prevented replenishment with liposomal-GSH demonstrating the requirement for catabolism and resynthesis. Repletion was significantly attenuated with endosomal inhibition implicating the endosomal system in utilization. Liposomal-GSH provided dose-dependent protection against PQMB with an EC(50) similar to that found for repletion. PQMB depleted intracellular GSH by 50%. Liposomal-GSH spared endogenous GSH during PQMB exposure, but did not require GSH biosynthesis for protection. No toxicity was observed with the liposomal preparation at 200-fold the EC(50) for repletion. These findings indicate that glutathione supplied in a liposomal formulation holds promise as a potential therapeutic for neuronal maintenance.
Liposomal-Glutathione Provides Maintenance of Intracellular
Glutathione and Neuroprotection in Mesencephalic Neuronal
Gail D. Zeevalk Laura P. Bernard
F. T. Guilford
Accepted: 3 June 2010
ÓSpringer Science+Business Media, LLC 2010
Abstract A liposomal preparation of glutathione (GSH)
was investigated for its ability to replenish intracellular
GSH and provide neuroprotection in an in vitro model of
Parkinson’s disease using paraquat plus maneb (PQMB) in
rat mesencephalic cultures. In mixed neuronal/glial cul-
tures depleted of intracellular GSH, repletion to control
levels occurred over 4 h with liposomal-GSH or non-
liposomal-GSH however, liposomal-GSH was 100-fold
more potent; EC
4.75 lM and 533 lM for liposomal
and non-liposomal-GSH, respectively. Liposomal-GSH
utilization was also observed in neuronal cultures, but
with a higher EC
(76.5 lM), suggesting that glia facili-
tate utilization. Blocking c-glutamylcysteine synthetase
with buthionine sulfoxamine prevented replenishment with
liposomal-GSH demonstrating the requirement for catab-
olism and resynthesis. Repletion was significantly attenu-
ated with endosomal inhibition implicating the endosomal
system in utilization. Liposomal-GSH provided dose-
dependent protection against PQMB with an EC
to that found for repletion. PQMB depleted intracellular
GSH by 50%. Liposomal-GSH spared endogenous GSH
during PQMB exposure, but did not require GSH biosyn-
thesis for protection. No toxicity was observed with the
liposomal preparation at 200-fold the EC
for repletion.
These findings indicate that glutathione supplied in a
liposomal formulation holds promise as a potential thera-
peutic for neuronal maintenance.
Keywords Glutathione Neurodegeneration Autism
Schizophrenia Parkinson’s disease
Oxidative stress and free radical damage to cells is a
consequence of life in an aerobic environment. Many
cellular defenses have evolved to protect cells. Glutathi-
one and its associated enzymes form one of the major
antioxidant defense systems in all cells. A strong anti-
oxidant defense is particularly important in the brain as
this organ relies solely on aerobic metabolism which can
generate significant reactive oxygen species (ROS). In
addition, neurotransmitters such as dopamine generate
ROS during their metabolism and can form reactive
quinones [1] that can conjugate with proteins and alter
protein function. Most neurodegenerative diseases involve
oxidative damage [2,3]. Furthermore a number of
neuropathological and neuropsychiatric conditions have
been shown to specifically present derangements in the
glutathione system. In Parkinson’s disease (PD), there is a
40–50% decrease in total GSH [46]. The decrease in
GSH is found in the brain region most affected in the
disease, i.e., the substantia nigra and is thought to be one
of the earliest dysfunctions in the disease process [5].
GSH levels in the prefrontal cortex of patients with
schizophrenia are decreased by 52% [7]. Evidence indi-
cates that this is due to a polymorphism in the catalytic
subunit of glutamylcysteine ligase, the first enzyme
involved in the synthesis of GSH [8]. Plasma levels of
GSH were recently shown to be decreased in patients with
G. D. Zeevalk (&)L. P. Bernard
Department of Neurology, UMDNJ-Robert Wood Johnson
Medical School, Staged Research Building, 661 Hoes Lane,
Piscataway, NJ 08854, USA
F. T. Guilford
Your Energy Systems, LLC, Palo Alto, CA 94301, USA
Neurochem Res
DOI 10.1007/s11064-010-0217-0
autism spectrum disorders [9,10] and in the blood of
children with Down syndrome [11].
Given the contribution of GSH in defense of cells from
oxidative stress, as well as its more recently delineated role
in protein modification and cell signaling [12,13], main-
tenance of intracellular GSH is of significant import to cell
function and viability. Neurons, like many other cell types,
however, do not have a transport mechanism for uptake of
exogenous GSH [14]. Other strategies have, therefore, been
tried in an effort to elevate or maintain intracellular GSH.
Cysteine is the rate limiting amino acid in GSH biosyn-
thesis and the L-amino acid uptake system aids to transport
cysteine across the blood brain barrier to maintain adequate
supplies to the brain [15]. Cysteine supplementation can
replenish GSH [16], however, elevations in extracellular
cysteine can damage neurons via an excitotoxic process
[17,18]. N-acetyl-cysteine (NAC), a derivative of cysteine
has been shown in animal studies to provide neuroprotec-
tion in an ischemia model and in a 6’OHDA model of
Parkinson’s disease [19,20], but can cause acidification
[21] and neuronal toxicity [19] as well as induce astroglial
and microglial activation [19]. Ethyl ester derivatives of
glutathione readily increase intracellular GSH both in vitro
and in vivo [14]. The ethanol formed following esterase
cleavage of the ethyl ester, however, can cause toxicity to
neurons [14]. Elevations in extracellular GSH per se can
also enhance neuronal toxicity through modulation of
NMDA receptors [22] and enhancement of excitotoxicity
and ischemic damage [23,24].
Here we examine a liposomal formulation of GSH
encapsulated in lipid vesicles made from lecithin and
glycerol as a safe alternative for maintenance of neuronal
GSH. This formulation of GSH was recently shown to have
antioxidant and anti-atherogenic properties [25]. Apolipo-
protein-E deficient mice fed daily for 2 mo with 50 mg/kg/d
liposomal-GSH showed a significant reduction in lipid
peroxides, macrophage cholesterol mass and a reduction in
atherosclerotic lesion development. Using mesencephalic
neuronal cultures, the present study examined the ability of
liposomal-GSH to replenish intracellular GSH following its
depletion and to provide neuroprotection in the paraquat
plus maneb environmental model of Parkinson’s disease
[26]. In addition, studies were conducted to provide a more
mechanistic understanding of the utilization of liposomal-
GSH for neuronal repletion and protection.
Materials and methods
Acivicin was purchased from Enzo Life Sciences Inc.
(Plymouth, PA). Maneb was obtained from ChemService
(West Chester, PA). The purity of maneb is reported to be
[95%. N2 supplement was from Invitrogen (Carlsbad,
CA). Liposomal-glutathione was provided by Your Energy
Systems (Palo Alto, CA). The liposomal preparation of
glutathione consists of reduced glutathione encapsulated in
hydroxylated lecithin (1.5%) and suspended in water con-
taining 15% glycerol and 0.1% potassium sorbate as pre-
servative. Glutathione content per ml of undiluted stock
was 82 mg. Stock solutions of liposomal-GSH and non-
liposomal-GSH were pH adjusted to 6.0 prior to use. All
other chemicals were from Sigma Chemical Co. (St. Louis,
Mixed Mesencephalic Cultures
Timed pregnant Sprague–Dawley rats were obtained from
Charles River (Wilmington, MA). The ventral mesen-
cephalon from embryonic day 15 rat fetuses were dis-
sected, pooled, dissociated and plated as described
previously [27]. Briefly, cells were mechanically dissoci-
ated by trituration and plated at 2.5 910
onto polyornithine and serum coated substrate in 48 well
trays. Cultures were incubated at 37°C in a 95% air/5%
humidified atmosphere. Cultures were grown in
Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 5 mM glucose, 2 mM glutamine, 2.2 g/L
bicarbonate plus 10% fetal bovine and horse serums
(DMEM/serum). 5-Fluoro-2-deoxyuridine (13 lg) plus
uridine 33 lg) was added from days 7–9 in vitro to reduce
glial growth. Under these conditions, cultures contain
approximately 70% neurons and 30% glia. The medium
was supplemented with 5 mM glucose every 72 h until the
conclusion of the studies.
Mesencephalic Neuronal Enriched Cultures
Neuronal enriched cultures were established as described
previously [28]. Mesencephalic cells were isolated from
embryonic day 15 rat brains as described above. Within
4–6 h after plating in DMEM/serum, the medium was
replaced with DMEM supplemented with serum-free N2
(Invitrogen, Eugene, OR) as described by [29]. N2 sup-
plement containing 8.6 lM insulin, 1 mM human trans-
ferrin, 2 lM progesterone, 10 mM putrescine and 3 lM
selenite promotes the growth of post-mitotic neurons, but
not astroglial cells.
Glutathione Measurement
Total glutathione was measured by HPLC as we have
previously described [30]. Briefly, just prior to extraction
of intracellular contents with 0.2 N perchloric acid, cul-
tures were rinsed 3 times with warm bicarbonate buffered
Neurochem Res
Krebs–Ringer (KRB; 119 mM NaCl, 4.8 mM KCl; 25 mM
3-[N-morpholino]propane sulfonic acid; 1.7 mM CaCl
1.2 mM KH
, 1.2 mM MgSO
, 5.5 mM glucose,
23.8 mM sodium bicarbonate, pH 7.4). The acid extract
was neutralized to—pH 5.0 with K
derivatized with
r—phthalaldehyde and separated on a C18 column using a
gradient of sodium acetate and methanol and a Beckman
System Gold HPLC with fluorometric detection. Quantifi-
cation was by comparison with a standard of glutathione.
General Cell Viability Assay
Overall cell viability was determined using the Cell Titer-
Cell Viability Assay kit (Promega, Madison, WI).
Viability is determined by the ability of cells to reduce
resazurin into resorufin. The procedure followed that sup-
plied with the kit using a 1 h incubation. Product formation
was determined in a CytoFluor 4000 microplate reader
(PerSeptive Biosystems) set at an excitation/emission of
530/580. Two vehicles were used to control for nonspecific
antioxidant or neuroprotective effects. Vehicle 1 was a
solution of liposomal constituents, lecithin plus glycerol.
Vehicle 2 was liposomal vesicles made of hydroxylated
lecithin surrounding water. Vehicles 1 and 2 were diluted
to the same concentration as that found in the final con-
centration of liposomal-GSH used for treatment. Neither
vehicle showed any antioxidant or neuroprotective efficacy
and results from toxicity assays report the combined results
from both vehicles.
Viability of Dopamine Neurons
Toxicity to the dopamine neuronal population in the mes-
encephalic cultures was determined by a functional assay
for the high affinity transport of a
H-labeled dopamine as
routinely reported [14,30,31].
H-dopamine (final con-
centration 37.5 nM) uptake was carried out in a HEPES
buffer (25 mM HEPES, 5.6 mM glucose, 125 mM NaCl,
4.8 mM KCl, 1.2 mM KH
, 1.3 mM CaCl
containing 1 mM ascorbate, and 100 lM pargy-
line) for 15 min at 37°C. Following extensive washing,
radioactivity in the cell extract was quantified by scintil-
lation counting. Values were corrected for background
binding determined in samples incubated at 4°C.
c-Glutamyltranspeptidase Assay
c-Glutamyltranspeptidase (c-GT) was determined by a
spectrophotometric assay that measures the release of
p-nitroaniline from c-glutamyl-p-nitroanilide. Cultures
were put into 1 ml of KRB containing 2 mM c-glutamyl-p-
nitroanilide plus 20 mM glycinylglycine and incubated for
1 h in a 5% CO
incubator, 37°C. Some wells contained
the c-GT inhibitor acivicin (1 mM). For competition
studies, 2 mM liposomal-GSH or 2 mM non-liposomal-
GSH was added to the wells just prior to the addition of
c-glutamyl-p-nitroanilide. After incubation, 0.6 ml media
was added to 0.15 ml glacial acetic acid and the absor-
bance read at 410 nm. Quantification was by comparison to
a standard curve derived from p-nitroaniline.
Glutathione S-Transferase Assay
Glutathione S-transferase (GST) activity was determined
via a spectrophotometric assay that measures the conju-
gation of glutathione to chlorodinitrobenzene (CDNB). The
assay was carried out in 0.1 M phosphate buffer to which
was added various concentrations of non-liposomal-GSH
or liposomal-GSH (0.1–1 mM) and CDNB (1 mM). The
reaction rate was monitored at 340 nM for 2 min. The
reaction was initiated by the addition of 50 ll of cytosol
from rat brain homogenate and the reaction monitored for
an additional 2 min. Any background rate was subtracted
from the rate with brain homogenate. Results are reported
as change in absorbance per minute.
Peroxide Assay
Peroxide in solution was determined using the Amplex Red
Hydrogen Peroxide/Peroxidase Assay Kit (Molecular
Probes, Eugene, OR) as we have published on previously
[28]. The Ampled Red reagent (10-acetyl-3,7-dehydroxy-
phenoxazine) in the presence of horseradish peroxidase
(HRP) reacts with peroxides to generate a fluorescent
product. Wells containing 5 lM peroxide in HEPES buffer
were incubated with various concentrations of liposomal or
non-liposomal-GSH or vehicle for 10 min at room tem-
perature. Reaction mix containing Amplex Red plus HRP
was then added and the plate incubated an additional 5 min
at room temperature. The amount of peroxide remaining in
solution was determined in a CytoFluor 4000 multiwell
plate reader at 530/580 ex/em. Quantification was by
comparison to a peroxide standard curve.
Data were analyzed for statistical significance by ANOVA
(GraphPad Instat 3.0) with Tukey post-hoc treatment.
APvalue of\0.05 was considered statistically significant.
were calculated using Prism 2.01 and a sigmoidal
dose–response equation. For each set of experiments where
were derived for intracellular GSH repletion, Top
and bottom values were set as follows: the top value was
the intracellular GSH level in untreated control cultures
incubated in Krebs–Ringer buffer for 4 h; the bottom value
was the intracellular GSH level in DEM treated cultures
Neurochem Res
further incubated in Krebs–Ringer buffer for 4 h without
Exogenous Liposomal-GSH is More Efficacious
than Non-Liposomal GSH for Replenishment
of Intracellular Glutathione Levels
A model for GSH depletion and optimal time for repletion
was determined in mixed mesencephalic cultures. Intra-
cellular levels of GSH were transiently reduced by a
30 min treatment with diethylmaleate (DEM), a compound
that readily chelates with GSH for removal. This treatment
reduced intracellular GSH by approximately 70% (Fig. 1;
intracellular GSH 2.01 ±0.23 and 0.65 ±0.12 lmol/
100 mg protein ±sem for untreated control and DEM
treated cultures, respectively). Optimal repletion was car-
ried out in Krebs–Ringer supplemented with cysteine,
glycine and glutamine. Glutamine was used as the gamma-
glutamyl donor instead of glutamate since glutamate causes
an excitotoxicity in the mesencephalic cultures [32]. Four
hours were required to return GSH levels to those found in
controls not treated with DEM but incubated in Krebs–
Ringer for 4 h. (Fig. 1a). Consistent with cysteine being
the rate limiting amino acid for GSH repletion [16], cys-
teine alone was sufficient to replenish intracellular GSH in
4 h. No repletion occurred in the absence of supplements
(Fig. 1a). A similar lack of repletion was found with sup-
plementation with glutamine or glycine in the absence of
cysteine (data not shown), and is consistent with findings
reported by others [16].
The ability of liposomal-GSH was compared with non-
liposomal-GSH for replenishment of intracellular levels of
the antioxidant following DEM treatment. Both com-
pounds restored intracellular levels by 4 h, however, lipo-
somal-GSH was 100-fold more potent in serving as a
source for intracellular GSH repletion; EC
, 4.75 lM±
1.38 and 533 lM±1.35 (mean ±sem) for liposomal-
GSH and non-liposomal-GSH, respectively (Fig. 1b).
Cultures exposed to 1 mM liposomal-GSH for 24 h
showed no toxicity as assessed by the Cell-Titer Blue
viability assay. At 1 mM liposomal-GSH, viability was not
statistically different from control (100 ±6.5 and 93 ±
5.0 percent of control ±sem, n=3 for control and 1 mM
liposomal-GSH, respectively).
Mixed mesencephalic cultures contain both neurons and
glia. To determine if liposomal-GSH could be utilized by
neurons per se, neuronal enriched cultures were estab-
lished as described previously [28]. Four hours after DEM
treatment, liposomal-GSH supplemented KRB restored
Fig. 1 a Replenishment of intracellular GSH was studied in mesen-
cephalic cultures consisting of neurons and glia. Cultures were
depleted of intracellular GSH with a 30 min treatment with 0.5 mM
diethylmaleate (DEM) on day 8 in vitro. Repletion was followed over
time in a balanced bicarbonate buffered Krebs–Ringer (KRB)
supplemented with either 200 lM cysteine, 200 lM glycine and
1 mM glutamine (Gline/cys/gly); 200 lM cysteine (cys) or no
additives. bMixed mesencephalic cultures or cneuronal enriched
mesencephalic cultures were depleted of GSH with DEM treatment.
Cells were then incubated in KRB supplemented with various
concentrations of liposomal-GSH or non-liposomal-GSH as indicated.
Intracellular GSH levels were measured at 4 h by HPLC as described
in Methods. The nis from (a)3,(b) 3–5 and (c) 5 determinations per
condition with all samples run in duplicate
Neurochem Res
intracellular GSH to 86% of controls. The potency for
repletion of intracellular GSH by liposomal-GSH in neu-
ronal enriched cultures was approximately 15-fold less than
in mixed neuronal/glial cultures; EC
76.5 lM±1.26
(Fig. 1c).
Liposomal-GSH Requires Catabolism and Resynthesis
Prior to Repletion
The mechanism by which liposomal-GSH was utilized by
mesencephalic neuronal cells was investigated. To
accomplish this, replenishment with liposomal-GSH in
mixed mesencephalic cultures was carried out with glu-
tathione synthesis blocked with buthionine sulfoxamine
(BSO, 10 lM), an inhibitor of c-glutamylcysteine syn-
thetase. Liposomal-GSH returned intracellular GSH to
control levels by 4 h (Fig. 2a). In contrast, in the presence
of BSO, repletion was completely blocked indicating that
liposomal-GSH needed to be catabolized and its constit-
uent amino acids utilized for resynthesis of intracellular
Since catabolism and resynthesis of the GSH packaged
in the lipid vesicles was required prior to its utilization for
intracellular replenishment, we investigated whether aci-
vicin, an inhibitor of the ectoenzyme c-GT, thought to be
responsible for the extracellular breakdown of GSH to a
c-glutamyl amino acid plus cysteinylglycine, would prevent
liposomal-GSH utilization in the cultures. A modest, but
statistically significant decrease of 32% in liposomal-GSH
utilization was observed (Fig. 2b) indicating that while
some GSH was processed extracellularly, the majority
(*70%) of the liposomal-GSH was not processed in this
manner. Studies were then conducted to determine whether
liposomal-GSH would serve as substrate for c-GT. This
investigation would also address the integrity of the lipo-
somal preparation with regards to the packaging of GSH
within the lipid vesicle as leakage of GSH from vesicles
might account for extracellular metabolism by c-GT.
Equimolar concentrations of liposomal-GSH or non-lipo-
somal-GSH (2 mM) were tested for their ability to compete
with 2 mM c-glutamyl-p-nitroanilide in a c-GT activity
assay (Fig. 2c). While non-liposomal-GSH effectively
Fig. 2 a Depletion of intracellular GSH with DEM treatment was
carried out in mixed mesencephalic cultures on day 8 in vitro as
described in Methods. Cultures were then incubated in KRB
supplemented with various concentrations of liposomal-GSH
(L-GSH) in the presence or absence of 10 lM buthionine sulfoxamine
(BSO). At 4 h, cell content was extracted and GSH levels were
determined by HPLC. The nis from 3–5 determination per condition
run in duplicate. bCultures were treated as in A to deplete GSH and
then incubated for 4 h with 100 lM liposomal-GSH plus or minus
1 mM acivicin. The nis from 3–4 determinations per condition.
Different from control;
different from DEM;
different from
DEM ?L-GSH. cc-glutamyltranspeptidase (c-GT) activity was
measured in mixed mesencephalic cultures as described in Methods.
Liposomal-GSH (2 mM) and non-liposomal-GSH (2 mM) were used
to compete with the assay substrate, 2 mM c-glutamyl-p-nitroanilide.
The nis from 3 determinations per condition.
Different from control
c-GT activity. dGST activity was measured in rat brain cytosolic
homogenates in the presence of different concentrations of either
GSH or liposomal-GSH as described in Methods. Only GSH could act
as substrate for GST. The nis from 3 determinations per condition
Neurochem Res
competed with c-glutamyl-p-nitroanilide to reduce the rate
of product formed by c-GT by 75%, liposomal-GSH was
without effect demonstrating that the GSH in the liposomal
preparation was not available as a substrate for c-GT. As
expected, acivicin, an inhibitor of c-GT, completely
blocked activity. The lack of extravesicular GSH in the
liposomal preparation was further verified by studies to
examine if liposomal-GSH could directly serve as substrate
for GST. Similar to what was found for c-GT, non-lipo-
somal-GSH, but not liposomal-GSH supported GST
activity (Fig. 2d).
Utilization of Liposomal-GSH in Mesencephalic
The finding with acivicin indicated that most of the lipo-
somal-GSH was not processed extracellularly. Liposomes
can be taken up into cells via an endocytic process [33].
Once inside the cell, endosomes can fuse with lysosomes
for intravesicular degradation and subsequent reutiliza-
tion of constituent components [34]. To determine if
the endosomal/lysosomal system was involved in the
utilization of liposomal-GSH for intracellular repletion,
liposomal-GSH utilization was followed in the presence of
phenylarsine oxide (PAO), an endosomal inhibitor [35], or
concanavalin A (ConA), shown to inhibit endosomal/lyso-
somal fusion [36]. PAO (1 lM) and ConA (1–30 lM) sig-
nificantly attenuated liposomal-GSH utilization (Fig. 3a, b).
PAO can also serve as a metabolic inhibitor [35] and could
cause cell toxicity. Even though cell loss was not evident at
the time of intracellular GSH determination (i.e. following
2 h of exposure), toxicity studies when assayed after a 72 h
recovery indicated severe cell loss (Fig. 3c) bringing to
question if blockage of GSH repletion at 2 h could have
been due to compromised cells. ConA, however, showed
only modest loss of cell viability at the highest dose tested
(32% loss at 30 lg/ml). At 1 and 10 lg/ml, ConA was not
toxic (Fig. 3d), yet produced a 48 and 60% loss, respec-
tively, of liposomal-GSH supported repletion. Repletion of
intracellular GSH by cysteine was not affected by ConA
treatment. Repletion was 92 ±2.8 percent of control with
200 lMcysteineand89±6.2 percent of control with
cysteine in the presence of 10 lg/ml ConA (mean ±sem,
n=3 with duplicate runs). These findings provide evidence
for utilization of liposomal-GSH via the endosomal/lyso-
somal pathway.
Fig. 3 The effect of endosomal inhibition on intracellular GSH
repletion with liposomal-GSH was tested in mixed mesencephalic
cultures transiently depleted of GSH with DEM treatment. Replen-
ishment was carried out for 2 h in KRB containing 100 lM
liposomal-GSH in the presence or absence of a1lM phenylarsine
oxide (PAO) or b1–30 lg/ml concanavalin A (ConA). Intracellular
GSH content was measured by HPLC from cell extracts. PAO and
ConA significantly inhibited liposomal-GSH supported repletion.
Toxicity of c1uM PAO or d1–30 lg/ml ConA was determined by
the Cell Titer Blue Viability Assay in cultures treated for 2 h with the
agents followed by 72 h of recovery in growth medium. This viability
assay measures the ability of cells to reduce resazurin to resorufin.
The nis from 3 separate experiments with each parameter run in
Different from control;
different from DEM;
from DEM plus L-GSH
Neurochem Res
Liposomal-GSH Provides Neuroprotection
in an Environmental Model of Parkinson’s Disease
Perturbation of intracellular glutathione is associated with a
number of disease states including PD and is thought to be
a contributing factor to the loss of midbrain dopamine
neurons in PD [5,37]. Paraquat plus maneb are widely
used pesticides that have been linked to PD [38,39]. In
animal models, paraquat plus maneb causes loss of dopa-
mine neurons [26] via a mechanism that involves oxidative
stress and the GSH system [14,40]. Here we investigated if
liposomal-GSH provided neuroprotection in an in vitro
paraquat plus maneb model. Mixed mesencephalic cultures
were exposed to 45 lM paraquat plus 45 lM maneb for
4 h in KRB in the presence or absence of various con-
centrations of liposomal-GSH. After exposure, cultures
were returned to their growth medium and allowed to
recover for 72 h. Control cultures were incubated in
Krebs–Ringer buffer for 4 h and then returned to growth
medium for a 72 h recovery. General cell viability was
then assessed. Treatment with paraquat plus maneb
reduced overall cell viability by approximately 80%.
Liposomal-GSH provided dose-dependent protection with
an EC
for protection of 14.1 lM±1.75 (Fig. 4a).
Vehicle control was without affect.
Mesencephalic cultures contain a small population of
presumptive midbrain dopamine neurons which are repre-
sentative of the population of neurons that degenerate in
PD. In order to determine if paraquat plus maneb was toxic
to dopamine neurons and if liposomal-GSH protected these
neurons from the toxic insult, viability was assessed in the
dopamine population by a functional assay that monitors
the high affinity transport of dopamine. Our past work has
shown that assessment of toxicity in the dopamine popu-
lation via monitoring of DAT activity parallels that
observed with counts of tyrosine hydroxylase positive cells
when a recovery period of 48–72 h is allowed between
treatment and toxicity assessment [31]. Paraquat plus
maneb caused a 75% loss of dopamine cell viability which
was similar to the extent of loss in the total cell population.
Liposomal-GSH dose-dependently provided protection of
dopamine neurons with an EC
of 10.5 lM±1.08. The
Fig. 4 Neuroprotection by liposomal-GSH was tested in mixed
mesencephalic cultures using an environmental model of Parkinson’s
disease. Cultures were treated on day 8 in vitro with 45 lM paraquat
plus 45 lM maneb plus or minus various concentrations of liposomal-
GSH for 4 h in KRB. Following treatment, cultures were returned to
complete medium and allowed to recover for 72 h. aOverall toxicity
in the cultures was determined by the Cell Title Blue Viability assay.
bToxicity specific to the dopamine neuronal population in the
cultures was determined by a functional assay that measured the high
affinity transport of
H-labeled dopamine. cComparison of protection
from paraquat plus maneb exposure in dopamine neurons by several
agents known to have antioxidant properties was carried out in
mesencephalic cultures on day 8 in vitro. Cultures were treated with
paraquat plus maneb as described in 3A in the presence or absence of
liposomal-GSH, non-liposomal-GSH, a-tocopherol, cysteinylglycine
and glutamylcysteine at 100 lM, ascorbate at 400 lM and combined
liposomal vehicles 1 and 2 at a concentration of empty liposomes or
lecithin plus glycerol equivalent to that found in 100 lM liposomal-
GSH. The nis from 3–5 for aand band for the antioxidant
compounds shown in c. The nis from 6–8 for combined control and
paraquat plus maneb data shown in c.aand b:
different from
paraquat plus maneb; c:
different from control,
different from
paraquat plus maneb
Neurochem Res
vehicle for lipid vesicles, i.e. lecithin plus glycerol pro-
vided no protection (Fig. 4b).
Protection by liposomal-GSH was compared with sev-
eral other compounds with known antioxidant properties.
Liposomal-GSH at 100 lM was compared with equimolar
concentrations of non-liposomal-GSH, alpha-tocopherol,
and the precursor GSH dipeptides cysteinylglycine and
glutamylcysteine. Ascorbate was tested at 400 lMto
approximate reported brain levels. While all the antioxi-
dants provided significant neuroprotection, only the GSH
precursor dipeptides showed a similar potency to liposomal-
GSH to provide complete protection at 100 lM (Fig. 4c).
Liposomal-GSH Spares Endogenous GSH
during Paraquat Plus maneb Exposure but Protection
does not Require GSH Biosynthesis
In order to determine if liposomal-GSH provided protec-
tion by maintaining intracellular GSH levels, mixed mes-
encephalic cultures were treated with paraquat plus maneb
and liposomal-GSH while GSH biosynthesis was blocked
with BSO. As shown in Fig. 5a, paraquat plus maneb
treatment caused a 79% toxicity in the dopamine popula-
tion. Liposomal-GSH provided significant protection.
Inhibition of GSH biosynthesis with BSO during paraquat
plus maneb exposure did not prevent neuroprotection
(Fig. 5a) suggesting that the utilization of liposomal-GSH
for GSH biosynthesis was not required for protection.
Intracellular GSH levels were significantly decreased
following 4 h of treatment with paraquat plus maneb to
approximately 50% of control. The presence of liposomal-
GSH during paraquat plus maneb exposure spared intra-
cellular GSH as levels remained similar to controls
(Fig. 5b). When BSO was added with paraquat plus maneb,
intracellular GSH was further reduced to 25% of control
values (control shown in Fig. 5b), indicating the active
depletion and resynthesis of GSH during paraquat plus
maneb exposure (Fig. 5c). In the presence of liposomal-
GSH with BSO and paraquat plus maneb, intracellular
levels of GSH were depressed as compared with control,
but were significantly higher than with BSO plus paraquat
and maneb in the absence of liposomal-GSH.
Liposomal-GSH has Direct Antioxidant Properties
The finding of protection by liposomal-GSH from paraquat
plus maneb exposure in the presence of BSO suggested that
liposomal-GSH may have direct antioxidant properties. To
Fig. 5 a The effect of inhibition of GSH biosynthesis on neuropro-
tection by liposomal-GSH was studied in mixed mesencephalic
cultures treated with 45 lM each paraquat plus maneb for 4 h in
KRB. Following a 3d recovery in complete medium, viability in the
dopamine population was determined as described in Methods. The n
is from 4 determinations per condition run in duplicate.
from control,
different from paraquat plus maneb. bIntracellular
GSH levels in mesencephalic cultures were determined following
treatment for 4 h with paraquat plus maneb. The nis from 3
determinations run in duplicate.
different from control,
from paraquat plus maneb. cCultures were treated with paraquat plus
maneb for 4 h in the presence of 10 lM BSO plus or minus 100 lM
liposomal-GSH and intracellular GSH content was determined by
HPLC immediately following treatment. The nis from 3 determina-
tions per condition.
different from BSO with paraquat plus maneb.
dLiposomal-GSH was tested for its ability to remove H
from a
cell-free solution using the Amplex Red peroxide detection kit as
described in Methods. The nis from 3 determinations run in duplicate
Neurochem Res
test this, the ability of liposomal-GSH to remove H
from a cell-free solution was tested using an Amplex red
peroxide detection kit (Molecular Probes). Liposomal-GSH
was found to remove H
from solution in a concentra-
tion-dependent manner (Fig. 5d). By comparison, non-
liposomal-GSH, as expected, also removed peroxide,
whereas liposomal vehicle equivalent to the equimolar
concentration of lecithin plus glycerol found in the differ-
ent concentrations of liposomal-GSH was without effect
(Fig. 5d).
Two major findings result from these studies: firstly that
liposomal-GSH can be utilized for repletion and mainte-
nance of intracellular GSH in neuronal cells and secondly,
that liposomal-GSH can provide significant protection to
neurons in a model system relevant to Parkinson’s disease.
Glutathione plays multifunctional and diverse roles in
neuronal cells including peroxide and toxin removal,
maintenance of the redox state of proteins and protein
signaling via glutathionylation [13,37,41]. In animal
studies, disturbances in glutathione can result in damage to
neurons per se [42] or in enhancement of toxicity due to
metabolic or oxidative stress [23,24,43]. In humans, a
number of neurodegenerative and neuropsychiatric condi-
tions are associated with disturbances in glutathione
[3,5,7,9,11,44]. Repletion and maintenance of neuronal
GSH is, therefore, important to cell health and viability and
could provide therapeutic benefit in situations when GSH is
deficient. Repletion of neuronal GSH, however, has met
with difficulty. Neurons, like most other cells, do not
possess transport mechanisms for GSH. In addition, ele-
vation of extracellular GSH may pose potential toxicity
problems that increase neuronal vulnerability during
ischemia [23] or enhance toxicity involving NMDA
receptors [24]. Other approaches such as the use of cys-
teine, NAC and ethyl esters of glutathione while effective
in repleting and in the case of the ethyl ester, elevating
intracellular GSH, have limited usefulness due to potential
toxicities [14,1719,21]. Encapsulation of GSH into lipid
vesicles may avoid the potential toxicity to neurons asso-
ciated with extracellular GSH elevation and may facilitate
drug delivery to cells as has been shown for other liposo-
mal preparations [45,46].
Facilitation of intracellular GSH repletion was greatly
enhanced by liposomal delivery. The concentration needed
for half maximal repletion in mixed mesencephalic cultures
containing approximately 70% neurons and 30% glia
(unpublished observations) was 100-fold less when GSH
was encapsulated into liposomal vesicles (4.75 lM for
liposomal-GSH versus 533 lM for non-liposomal fully
reduced GSH). Neuronal enriched cultures also directly
utilized liposomal-GSH for intracellular repletion, but with
an EC
15-fold higher than in the mixed cultures
(76.5 lM). Glia are known to support GSH utilization by
neurons. In vitro studies using enriched cultures of neurons
and glia have shown that glia can efflux GSH [47,48]
which can then be metabolized by the ectoenzyme c-GT
and dipeptidases to supply neurons with the substrates for
GSH biosynthesis. While the findings demonstrate direct
neuronal utilization of liposomal-GSH, they also suggest
that glia facilitate liposomal-GSH utilization by neurons.
This would be consistent with a significant, but modest
inhibitory effect of acivicin on liposomal-GSH utilization
in the mixed mesencephalic cultures and with liposomal-
GSH not serving as a substrate for c-GT. Since exogenous
liposomal-GSH was found not to be available for catabo-
lism by c-GT (Fig. 2c), only liposomal-GSH taken-up into
cells would be further metabolized and its amino acid
products used for GSH resynthesis. This is in accord with
the finding that BSO, an inhibitor of the first GSH bio-
synthetic reaction completely prevented intracellular GSH
repletion with exogenous liposomal-GSH administration as
well as with the modest effects of acivicin and lack of
metabolism of liposomal-GSH by c-GT.
If liposomal-GSH is not available for extracellular
catabolism by c-GT, how is the liposomal-GSH utilized?
Liposomal-GSH appears to gain access to the cytosol and
its constituent amino acids utilized for GSH resynthesis
via endosomal uptake and lysosomal degradation. Lipo-
somes have been shown to gain entry into cells through
macropinocytosis or phagocytosis [33,45]. Endosomes can
then fuse with lysosomes for digestion of intravesicular
contents [34]. PAO, an inhibitor of endosomal uptake
significantly attenuated liposomal-GSH utilization, how-
ever, PAO was also toxic and it is not clear if non-specific
cell compromise or endosomal inhibition was responsible
for the block of GSH repletion. The lectin, ConA, on the
other hand, has been shown to inhibit endosomal/lysosomal
fusion [36] and can inhibit degradation of endocytosed
molecules [49]. ConA at concentrations that were not toxic
to cells, caused a dose dependent reduction in the ability of
liposomal-GSH to replenish intracellular GSH (Fig. 3b, d).
Cysteine is taken up into neurons predominately via the
glutamate transporter system X
[50,51]. Consistent
with this, ConA had no effect on the ability of cysteine to
replenish intracellular GSH. In total, the data provide evi-
dence for uptake of liposomal-GSH into endosomes and
fusion with lysosomes where GSH is hydrolyzed to its
constituent amino acids. The amino acids can then be used
by the cell for further GSH biosynthesis. Neurons could
accomplish this independent of glia. Glia could also utilize
liposomal-GSH in this manner and in addition, continue to
efflux GSH for extracellular metabolism and utilization of
Neurochem Res
its metabolites by neurons. It is this latter component that
would be inhibited by acivicin (Fig. 2b). A schematic
representing the postulated utilization of liposomal-GSH is
shown in Fig. 6.
The lack of extracellular catabolism of the GSH in the
liposomal preparation is important since catabolism to
constituent amino acids, in particular, glutamate and cys-
teine can overstimulate glutamate receptors on neurons and
lead to an excitotoxicity [17,18]. In addition, there appears
to be little leakage of GSH from the liposomal vesicles as
the GSH in the preparation was neither a substrate for c-GT
nor glutathione S-transferase. This is also a point of interest
as elevations in extracellular GSH may pose a toxicity risk
as well [23,24]. The mesencephalic cultures exposed to a
concentration of the liposomal prepa ration [200-times the
for GSH repletion did not cause toxicity. This finding
attests to the relative safety of the liposomal preparation.
Mesencephalic cultures contain a small population of
presumptive midbrain dopamine neurons. These neurons
are representative of the midbrain neurons that degenerate
in Parkinson’s disease and are a commonly used in vitro
system to model Parkinson’s disease [14,31]. Sporadic
Parkinson’s disease is thought to result from a combination
of genetic susceptibility and environmental factors [52].
Exposure to pesticides is a risk factor for the development
of Parkinson’s disease [38,39,53]. Paraquat plus maneb
are widely used pesticides and their combination has been
shown in animal models to cause selective loss of
substantia nigra pars compacta dopamine neurons [26]. In
vitro, paraquat plus maneb caused loss of dopamine neu-
rons, however, the extent of cell loss observed in the
dopamine population was similar in magnitude to the loss
observed in the general population which consists pre-
dominately of midbrain GABAregic neurons. The differ-
ence in the relative vulnerability of dopamine neurons to
paraquat plus maneb in vivo versus in vitro could be due to
a chronic dosing paradigm in vivo (5–6 weeks) in com-
parison with the acute exposure used in vitro (4 h). Chronic
versus acute exposure in other Parkinson’s disease models
in vitro, i.e. rotenone, can alter the relative vulnerability of
GABergic and dopaminergic mesencephalic neurons [31].
Regardless of differences in neuronal vulnerability,
liposomal-GSH provided complete neuroprotection of
dopamine neurons and the general mesencephalic popula-
tion with EC
in the low lM range (14.1 and 10.5 lM for
the general population and dopamine neurons respec-
tively). Thus liposomal-GSH may be of therapeutic benefit
not only in Parkinson’s disease, but in other CNS condi-
tions where there is perturbation of the glutathione system.
While the EC
for protection were similar to those
observed for intracellular GSH repletion, protection was
not dependent on the utilization of liposomal-GSH for
repletion since the same degree of protection was observed
in the presence or absence of BSO. This suggested that the
liposomal preparation had direct antioxidant properties and
this was confirmed in a cell free assay where liposomal-
Fig. 6 A schematic summary of the findings for liposomal-GSH
(L-GSH) utilization in the mixed mesencephalic cultures is shown.
L-GSH is taken up into neurons and astrocytes via an endosomal
process inhibited by phenylarsine oxide (PAO). Once inside the cell,
endosomes containing L-GSH can fuse with lysosomes (L), where
hydrolysis and release of GSH to its constituent amino acids,
glutamate (Glu), cysteine (Cys) and glycine (Gly) occur. ConA can
inhibit the formation of the phago-lysosomes. Hydrolyzed amino
acids released from the lysosomes can be used for GSH biosynthesis,
the first step of which is inhibited by buthionine sulfoxamine (BSO).
Some efflux of GSH occurs by astrocytes and can be metabolized
extracellularly by the ectoenzyme c-glutamyltranspeptidase (c-GT)
and dipeptidases (DP). Acivicin inhibits c-GT activity. The amino
acids derived from the extracellular breakdown of GSH, and in
particular cysteine, can be taken up by neurons to further supply
substrates for GSH synthesis
Neurochem Res
GSH was demonstrated to remove H
from solution.
This is possible as the readily diffusible H
can traverse
the lipid vesicle to access intravesicular GSH. There was
no contribution of the lecithin and glycerol used for the
liposomal preparation as lecithin plus glycerol were unable
to remove H
. The direct removal of ROS by liposomal-
GSH would account for the sparing of endogenous intra-
cellular GSH during paraquat plus maneb exposure. This
sparing effect has also been noted for other antioxidants
such as ascorbate [54] as some of the burden for ROS
removal by endogenous GSH is shared. The antioxidant
property of liposomal GSH could provide a dual benefit as
a potential therapeutic agent, firstly, in that it can be
metabolized to supply substrates for GSH synthesis par-
ticularly when there is increased oxidative stress and
increased demand for substrates and secondly, liposomal-
GSH can serve to directly remove ROS.
In a limited study in 9 Parkinson’s patients, i.v. admin-
istration of 600 mg GSH twice daily for 30d provided sig-
nificant improvement in disability as assessed using a
modified Columbia University Rating Scale [55]. Due to the
open label design of the study and the limited number of
patients, however, conclusions from this study should be
viewed with caution. A controlled, double-blinded clinical
trial is planned for study of i.v. glutathione administration in
the treatment of Parkinson’s disease [56]. Non-liposomal-
GSH requires i.v. administration to avoid the hydrolysis and
gut absorption that follows oral administration. An advan-
tage of the liposomal preparation over non-liposomal-GSH
is that it can be administered orally [25] thus allowing self-
administered daily application.
One enigmatic issue at present, is whether liposomal-
GSH crosses the blood brain barrier. There is some evi-
dence for non-liposomal-GSH transport across the blood
brain barrier [57], but this is thought to be of low capacity
[58]. Direct transport of non-liposomal-GSH or liposomal-
GSH into brain may not be necessary, however, to support
brain maintenance of GSH. The reasons underlying low
brain GSH levels in neurodegenerative diseases such as
Parkinson’s disease are unknown. In contrast with
Schizophrenia [7,8], synthesizing enzymes for glutathione
function normally in Parkinson’s disease [59]. Cysteine can
serve as an antioxidant and is the rate limiting enzyme in
the production of glutathione [16,60]. High levels of
oxidative stress as well as elevations in cysteine conjugates
such as cysteinyldopamine have been reported in Parkin-
son’s disease brain [61,62]. These findings point towards
an imbalance in supply and demand with low levels of
glutathione due to limiting amounts of cysteine. Peripheral
GSH and/or liposomal-GSH could serve as a source of
peripherally generated cysteine which readily traverses the
blood brain barrier via the L-system carrier [15]. Thus
benefit from peripherally administered GSH could derive
from GSH per se or from a peripherally generated metab-
olite. Further studies on the blood brain barrier perme-
ability of the liposomal preparation and its neuroprotective
potential in vivo are needed.
In summary, the studies presented here show that a
liposomal preparation of GSH is 100-fold more potent than
non-liposomal-GSH in providing substrates for mainte-
nance of intracellular glutathione in neuronal cells and
provides complete protection of neurons in an environ-
mental model of Parkinson’s disease. These findings pro-
vide a rationale for in vivo studies of liposomal-GSH in a
variety of disease models where oxidative stress and/or
perturbations in the glutathione system are thought to be
Acknowledgments This work was supported by a grant from Your
Energy Systems, LLC and from The National Institutes of Health
(NS36157). As a study sponsor, Your Energy Systems, LLC played
no role in the study design, data acquisition, analysis or writing of the
report for publication.
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Neurochem Res
... Some lipophilic derivatives, conjugated codrugs, or analogues of GSH have been examined in experimental studies. Liposomal GSH, which can cross the blood-brain barrier, spared endogenous GSH and protected paraquat plus maneb-induced neuronal death [79]. Pinnen et al. developed codrugs, which are conjugates of L-DOPA and GSH, as potential treatment for PD. ...
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Glutathione (GSH) is the most abundant intrinsic antioxidant in the central nervous system, and its substrate cysteine readily becomes the oxidized dimeric cystine. Since neurons lack a cystine transport system, neuronal GSH synthesis depends on cystine uptake via the cystine/glutamate exchange transporter (xCT), GSH synthesis, and release in/from surrounding astrocytes. Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), a detoxifying master transcription factor, is expressed mainly in astrocytes and activates the gene expression of various phase II drug-metabolizing enzymes or antioxidants including GSH-related molecules and metallothionein by binding to the antioxidant response element (ARE) of these genes. Accumulating evidence has shown the involvement of dysfunction of antioxidative molecules including GSH and its related molecules in the pathogenesis of Parkinson’s disease (PD) or parkinsonian models. Furthermore, we found several agents targeting GSH synthesis in the astrocytes that protect nigrostriatal dopaminergic neuronal loss in PD models. In this article, the neuroprotective effects of supplementation and enhancement of GSH and its related molecules in PD pathology are reviewed, along with introducing new experimental findings, especially targeting of the xCT-GSH synthetic system and Nrf2–ARE pathway in astrocytes.
... If there is not enough Glutathione to generate the Phase two enzymes, toxins will build to dangerous levels in the liver (Koch, 2011). Glutathione is the most important physiological chelator and the glutathione in reduced form protects cells from reactive oxygen species related with heavy metals (Becker & Soliman, 2009;Kaur et al., 2006;Rosenblat et al., 2007); Zeevalk et al., 2010;Flora, 2009). GSH forms metal complexes via non-enzymatic reactions (Ballatori, 1994). ...
... [91] Glutathione encapsulated liposomes were found to be useful for PD treatment, which showed neuroprotection by maintaining intracellular glutathione in neuronal cells. [92] Several studies demonstrated use of gold nanoparticles for the quantitative detection of neurotransmitters such as epinephrine, L-DOPA, α-synuclein, norepinephrine and dopamine. [93] Exosomes, released from CNS and altered during the disease process, were marked as excellent aspirants for carrying biomarkers. ...
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Recent times have witnessed an upsurge in the incidence of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Prion disease, and amyotrophic lateral sclerosis. The treatment of the same remains a daunting challenge due to the limited access of therapeutic moieties across the blood–brain barrier. Engineered nanoparticles with a size less than 100 nm provide multifunctional abilities for solving these biomedical and pharmacological issues due to their unique physico‐chemical properties along with capability to cross the blood–brain barrier. Needless to mention, there is a scarcity of review articles summarizing recent developments of various nanomaterials including liposomes, polymeric nanoparticles, metal nanoparticles, and bio‐nanoparticles toward the therapeutic and theranostics applications for various neurodegenerative disorders. Here, a broad spectrum of nanomedicinal approaches to eradicate neurodegenerative disorders is provided, along with a brief account of neuroprotection and neuronal tissue regeneration, current clinical status, issues related to safety, toxicity, challenges, and future outlook.
... chorobach układu immunologicznego (AIDS), chorobach neurodegeneracyjnych czy cukrzycy. Działania profilaktyczne czy terapeutyczne prowadzące do wzrostu stężenia GSH w organizmie mogą spowalniać i łagodzić przebieg tych schorzeń [20][21][22][23][24][25][26][27][28]. ...
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Glutathione (GSH) is a endogenous , low molecular weight thiol compound. It has a wide spectrum of biological activity in the body. It is an important element of the antioxidant system that protects cells against the effects of oxidative stress. As an antioxidant, GSH inactivates free radicals and reactive forms of oxygen and nitrogen in enzymatic and non-enzymatic reactions, regenerates other antioxidants, e.g. vitamins C and E, maintains - SH groups in proteins in a reduced state, participates in the detoxification of xenobiotics. The decrease in GSH concentration occurs in many diseases and in the aging process. Increasing the level of GSH in the body's cells is possible by consuming dietary components containing GSH or amino acids (especially cysteine) for its endogenous synthesis and supplementation with GSH pharmaceutical preparations. The aim of the study was to characterize dietary supplements available in Poland containing glutathione (GSH). The analysis covered 39 supplements from GSH, which were available in Poland. Their characteristics include: place / country of production; type of preparations (single and multi-component); bioavailability of GSH contained therein - preparations in the liposome formula (lipophilic GSH) and in the non-liposome formula (non-lipophilic GSH); pharmaceutical form in which these preparations were available on the commercial offer and GSH content in supplements. Among 39 GSH supplements, there were 24 single-component and 15 multi-component preparations. The largest number of GSH supplements available in Poland came from the USA and Great Britain. Among GSH supplements, there were 15 liposome (lipophilic) formula preparations, of which 12 were lipophilic one-component and 3 multi-component and 24 non-liposome (non-lipophilic) formula preparations - 12 single and 12 multi-component. Capsules, gel, liquid and tablets were the most common pharmaceutical form among all analyzed GSH supplements. The rarest pharmaceutical form was lozenges, aerosol, powder and ampoule. Analysis of GSH supplements available in Poland showed that in one single dose of the preparation was from 18 mg to 750 mg GSH. The most common dose was 250, 450 and 500 mg GSH in a single dose. One-component supplements most often contained 450 mg, then 250 and 500 mg GSH, while multi-component supplements - 250 mg GSH.
... BCP is usually dissolved in polyoxyethylated castor oil or olive oil, which results in inaccuracy of dosage and inconvenient for experimental operations [10]. As a new type of microparticle delivery system, liposomes have many advantages: prolonging the action time of drugs and exert sustained-release effects, and have thus been widely used in drug delivery systems [36,37]. Studies have confirmed that liposomes can be used in the preparation of various volatile oil delivery systems [16]. ...
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This study was conducted to prepare β-caryophyllene loaded liposomes (BCP-LP) and investigated their effects on neurovascular unit (NVU) damage after subarachnoid hemorrhage (SAH) in rats. A blood injection into the pre-chiasmatic cistern was used to achieve SAH. BCP-LP were prepared, characterized and administrated to rats with SAH. The prepared BCP-LP were spherical with a size distribution of approximately 189.3 nm and Zeta potential of − 13.9 mV. Neurological scoring, the balance beam test, cerebral blood flow monitoring, brain edema and biochemical analyses were applied to evaluate the effects of BCP-LP on rat NVU damage after SAH. The results demonstrated that BCP-LP treatment improved neurological function disorder, balance ability and cerebral blood perfusion in rats. Brain edema detection and blood–brain barrier permeability detection revealed that BCP-LP could reduce brain edema and promote repairment of blood–brain barrier after SAH. Using the western blot experiments, we demonstrated that BCP-LP attenuated the loss of tight junction proteins Occludin and Zonula occludens-1, inhibit the high expression of VEGFR-2 and GFAP, and promote the repair of laminin. These results demonstrate the protective effect BCP-LP exert in the NVU after SAH in rats, and supports the use of BCP-LP for future study and therapy of SAH.
... Definitely, the difference of uptake pathway for free Dox and NC-Dox is another factor. To assess the effect of GSH on Dox release in cells, we applied glutathione ethyl ester (GSHee) as a GSH donor 53,54 and incubated another group of cells with GSHee (2 mM) to elevate intracellular GSH levels before treating the cells with NC-Dox. Accordingly, in comparison to NC-Dox-treated cells, it is seen that GSHee/ NC-Dox-treated cells were more fluorescent in the red channel, and the amount of Dox accumulated in nucleus was also slightly increased ( Figure 7a). ...
The anti-alcoholic drug disulfiram (DSF) has attractive biomedical interests for its anticancer effects, particularly in combination use with copper. CuET, the complex of copper and diethyldithiocarbamate (CuET) that is derived from disulfiram, exhibits high redox stability against glutathione, cysteine and hydrogen peroxide, but appears reactive to ascorbic acid and superoxide ions. By virtue of the copper binding property, apoferritin is developed as a carrier of CuET to alleviate concerns of poor water solubility and nonspecific toxicity, and shows superior stability and protection over human serum albumin in nanocomposite manipulation. CuET and doxorubicin are co-delivered by apoferritin with excellent efficiency (> 97%). The glutathione-responsive system demonstrates enhanced antitumor effect and potentials for combination tumor therapy.
... To supplement with lipoceutical GSH, GSH is first packaged into liposomes, small droplets that fuse with cellular membranes, directly releasing their contents into target cells. Recent studies have underscored the beneficial effects of liposomal GSH on intracellular GSH concentrations [204,205]. For instance, liposomal GSH was given to children (3-13 years old) with autism spectrum disorder, and the treatment significantly increased plasma GSH, cysteine, and taurine levels [29]. ...
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Glutathione (GSH) is a critical endogenous antioxidant found in all eukaryotic cells. Higher GSH concentrations protect against cellular damage, tissue degeneration, and disease progression in various models, so there is considerable interest in developing interventions that augment GSH biosynthesis. Oral GSH supplementation is not the most efficient option due to the enzymatic degradation of ingested GSH within the intestine by γ-glutamyltransferase, but supplementation of its component amino acids—cysteine, glycine, and glutamate—enhances tissue GSH synthesis. Furthermore, supplementation with some non-precursor amino acids and micronutrients appears to influence the redox status of GSH and related antioxidants, such as vitamins C and E, lowering systemic oxidative stress and slowing the rate of tissue deterioration. In this review, the effects of oral supplementation of amino acids and micronutrients on GSH metabolism are evaluated. And since specific dietary patterns and diets are being prescribed as first-line therapeutics for conditions such as hypertension and diabetes, the impact of overall diets on GSH homeostasis is also assessed.
Significance: Glutathione (GSH) represents the most abundant and the main antioxidant in the body with important functions in the brain related to Alzheimer's disease (AD). Recent Advances: Oxidative stress is one of the central mechanisms in AD. We and others have demonstrated the alteration of GSH in AD brain, its important role for the detoxification of advanced glycation end products and of acrolein. Recent in vivo studies found a decrease of GSH in different area of the brain from control, mild cognitive impairment and AD subjects which is correlated with cognitive functions. Critical issues: Several strategies were developed to restore its intracellular level with the L-cysteine prodrugs or the oral administration of γ-glutamylcysteine (γ-GC) to prevent alterations observed in AD. To date, no benefit on GSH levels or oxidation biomarkers have been reported in clinical trials. Thus, it remains uncertain if GSH could be considered as a potential preventive or therapeutic approach or as a biomarker for AD. Future directions: We will address how GSH-coupled nanocarriers represent a promising approach for the functionalization of nanocarriers to overcome the blood brain barriers (BBB) for the delivery of GSH to the brain while avoiding cellular toxicity. It is also important to address the presence of GSH in exosomes for its potential intercellular transfer or to cross the BBB under certain conditions.
Reduced glutathione (GSH) and some food-derived phenolic acids are welcome as natural antioxidant, but their synergistic antioxidant effect is unknown. The purpose of this study was to screen out the combination of GSH and the phenolic acid with synergistic antioxidant effect, improve their coexistence and protect the activity. Antioxidant evaluation systems including ABTS, DPPH, ORAC and reducing power assays were implemented, and dynamic light scattering, transmission electron microscopy analysis was carried out. Caffeic acid (CA) and GSH exhibited significant synergistic effect via electron transfer when the concentration ratio was 1:5. This combination was well embedded into chitosan-coated liposomes, which consist of evenly distributed and uniformly spherical microparticles, and its average particle size was 247.63 nm with polymer dispersity index of 0.29 and zeta potential of 57.73 mV. The encapsulation efficiencies of GSH and CA were 61.32% and 68.92%, respectively. The successfully prepared microparticles showed higher markedly antioxidant activity than the simple mixture and liposomes in 10-week storage. Therefore, encapsulation of GSH and CA using chitosan-coated liposomes is a potential approach to prepare natural potent antioxidant in food manufacture.
The incidence of Parkinson's disease (PD), the second most common neurodegenerative disorder, has increased exponentially as the global population continues to age. Although the etiological factors contributing to PD remain uncertain, its average incidence rate is reported to be 1% of the global population older than 60 years. PD is primarily characterized by the progressive loss of dopaminergic (DAergic) neurons and/or associated neuronal networks and the subsequent depletion of dopamine (DA) levels in the brain. Thus, DA or levodopa (l-dopa), a precursor of DA, represent cardinal targets for both idiopathic and symptomatic PD therapeutics. While several therapeutic strategies have been investigated over the past decade for their abilities to curb the progression of PD, an effective cure for PD is currently unavailable. Even DA replacement therapy, an effective PD therapeutic strategy that provides an exogenous supply of DA or l-dopa, has been hindered by severe challenges, such as a poor capacity to bypass the blood-brain barrier and inadequate bioavailability. Nevertheless, with recent advances in nanotechnology, several drug delivery systems have been developed to bypass the barriers associated with central nervous system therapeutics. In here, we sought to describe the adapted lipid-based nanodrug delivery systems used in the field of PD therapeutics and their recent advances, with a particular focus placed on DA replacement therapies. This work initially explores the background of PD; offers descriptions of the most recent molecular targets; currently available clinical medications/limitations; an overview of several lipid-based PD nanotherapeutics, functionalized nanoparticles, and technical aspects in brain delivery; and, finally, presents future perspectives to enhance the use of nanotherapeutics in PD treatment.
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Context The cause of Parkinson disease (PD) is unknown. Genetic linkages have been identified in families with PD, but whether most PD is inherited has not been determined, Objective To assess genetic inheritance of PD by studying monozygotic (MZ) and dizygotic (DZ) twin pairs. Design Twin study comparing concordance rates of PD in MZ and DZ twin pairs. Setting and Participants A total of 19 842 white male twins enrolled in the National Academy of Sciences/National Research Council World War II Veteran Twins Registry were screened for PD and standard diagnostic criteria for PD were applied. Zygosity was determined by polymerase chain reaction or questionnaire. Main Outcome Measure Parkinson disease concordance in twin pairs, stratified by zygosity and age at diagnosis. Results Of 268 twins with suspected parkinsonism and 250 presumed unaffected twin brothers, 193 twins with PD were identified (concordance-adjusted prevalence, 8.67/1000). In 71 MZ and 90 DZ pairs with complete diagnoses, pairwise concordance was similar (0.129 overall, 0.155 MZ, 0.111 DZ; relative risk, 1.39; 95% confidence interval, 0.63-3.1). In 16 pairs with diagnosis at or before age 50 years in at least 1 twin, MZ concordance was 1.0 (4 pairs), and DZ was 0.167 (relative risk, 6.0; 95% confidence interval, 1.69-21.26). Conclusions The similarity in concordance overall indicates that genetic factors do not play a major role in causing typical PD, No genetic component is evident when the disease begins after age 50 years. However, genetic factors appear to be important when disease begins at or before age 50 years.
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The level of glutathione (GSH) is often reduced in brains that are affected by neurodegeneration. It is not known, however,whether this is a cause or a consequence of the disease. Here we have examined the effects of GSH depletion on the viability of human neurons cultured in either the presence or the absence of astrocytes, both derived from NT2/D1 cells. We established that the endogenous concentration of GSH is 10 times lower in neurons than in astrocytes (1.42 versus 18.9 pmol microg protein(-1)) and that pure neuronal cultures begin to die by apoptosis within 24 h of GSH depletion. By contrast, neurons that are co-cultured with astrocytes remain viable for several days, even with a profoundly decreased GSH content. However, they die rapidly when challenged additionally with nitrative stress. In addition, astrocytes survive for prolonged periods of time (>12 days) under severely reduced GSH concentrations. Our study shows clear differences in the content and sensitivity to depletion of GSH in neurons and astrocytes and establishes the significance of neuronal-glial interactions for the maintenance of neuronal viability under reduced GSH content. However, with chronic GSH depletion, these interactions might not be sufficient to protect neurons from other injurious factors (i.e. reactive oxygen and nitrogen species), which indicates that defective GSH metabolism might facilitate the progression of neurodegeneration.
Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long-time disability. Ischemia/reperfusion to any organ triggers a complex series of biochemical events, which affect the structure and function of every organelle and subcellular system of the affected cells. The purpose of this study was to investigate the therapeutic efficacy of N-acetyl cysteine (NAC), a precursor of glutathione and a potent antioxidant, to attenuate ischemia/reperfusion injury to brain tissue caused by a focal cerebral ischemia model in rats. A total of 27 male Sprague-Dawley rats weighing 250-300 g were used in this study. Focal cerebral ischemia (45 min) was induced in anesthetized rats by occluding the middle cerebral artery (MCA) with an intra-luminal suture through the internal carotid artery. The rats were scored post-reperfusion for neurological deficits. They were then sacrificed after 24 h of reperfusion and infarct volume in the brain was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC). Brain sections were immunostained for tumor necrosis factor (TNF-alpha) and inducible nitric oxide synthase (NOS). Animals treated with NAC showed a 49.7% (S.E.M. = 1.25) reduction in brain infarct volume and 50% (S.E.M. = 0.48) reduction in the neurological evaluation score as compared to the untreated animals. NAC treatment also blocked the ischemia/reperfusion-induced expression of tumor necrosis factor and inducible nitric oxide synthase. The data suggest that pre-administration of NAC attenuates cerebral ischemia and reperfusion injury in this brain ischemia model. This protective effect may be as a result of suppression of TNF-alpha and NOS.
Oxidative stress is now recognized as accountable for redox regulation involving reactive oxygen species (ROS) and reactive nitrogen species (RNS). Its role is pivotal for the modulation of critical cellular functions, notably for neurons astrocytes and microglia, such as apoptosis program activation, and ion transport, calcium mobilization, involved in excitotoxicity. Excitotoxicity and apoptosis are the two main causes of neuronal death. The role of mitochondria in apoptosis is crucial. Multiple apoptotic pathways emanate from the mitochondria. The respiratory chain of mitochondria that by oxidative phosphorylation, is the fount of cellular energy, i.e. ATP synthesis, is responsible for most of ROS and notably the first produced, superoxide anion (O2 ̇−). Mitochondrial dysfunction, i.e. cell energy impairment, apoptosis and overproduction of ROS, is a final common pathogenic mechanism in aging and in neurodegenerative disease such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Nitric oxide (NO ̇), an RNS, which can be produced by three isoforms of NO-synthase in brain, plays a prominent role. The research on the genetics of inherited forms notably ALS, AD, PD, has improved our understanding of the pathobiology of the sporadic forms of neurodegenerative diseases or of aging of the brain. ROS and RNS, i.e. oxidative stress, are not the origin of neuronal death. The cascade of events that leads to neurons, death is complex. In addition to mitochondrial dysfunction (apoptosis), excitotoxicity, oxidative stress (inflammation), the mechanisms from gene to disease involve also protein misfolding leading to aggregates and proteasome dysfunction on ubiquinited material.
We examine the evidence for free radical involvement and oxidative stress in the pathological process underlying Parkinson's disease, from postmortem brain tissue. The concept of free radical involvement is supported by enhanced basal lipid peroxidation in substantia nigra in patients with Parkinson's disease, demonstrated by increased levels of malondialdehyde and lipid hydroperoxides. The activity of many of the protective mechanisms against oxidative stress does not seem to be significantly altered in the nigra in Parkinson's disease. Thus, activities of catalase and glutathione peroxidase are more or less unchanged, as are concentrations of vitamin C and vitamin E. The activity of mitochondrial superoxide dismutase and the levels of the antioxidant ion zinc are, however, increased, which may reflect oxidative stress in substantia nigra. Levels of reduced glutathione are decreased in nigra in Parkinson's disease; this decrease does not occur in other brain areas or in other neurodegenerative illnesses affecting this brain region (i.e., multiple system atrophy, progressive supranuclear palsy). Altered glutathione metabolism may prevent inactivation of hydrogen peroxide and enhance formation of toxic hydroxyl radicals. In brain material from patients with incidental Lewy body disease (presymptomatic Parkinson's disease), there is no evidence for alterations in iron metabolism and no significant change in mitochondrial complex I function. The levels of reduced glutathione in substantia nigra, however, are reduced to the same extent as in advanced Parkinson's disease. These data suggest that changes in glutathione function are an early component of the pathological process of Parkinson's disease. The data presented suggest (1) there is oxidative stress in the substantia nigra at the time of death in advanced Parkinson's disease that manifests in terms of increased lipid peroxidation, superoxide dismutase activity, and zinc levels; (2) there is a major impairment of the glutathione pathway in Parkinson's disease; and (3) alterations in reduced glutathione levels may occur very early in the illness.
Oxidative stress has been implicated in both normal aging and in various neurodegenerative disorders and may be a common mechanism underlying various forms of cell death including necrosis, apoptosis, and excitotoxicity. In this review, we develop the hypothesis that oxidative stress-mediated neuronal loss may be initiated by a decline in the antioxidant molecule glutathione (GSH). GSH plays multiple roles in the nervous system including free radical scavenger, redox modulator of ionotropic receptor activity, and possible neurotransmitter. GSH depletion can enhance oxidative stress and may also increase the levels of excitotoxic molecules; both types of action can initiate cell death in distinct neuronal populations. Evidence for a role of oxidative stress and diminished GSH status is presented for Lou Gehrig's disease (ALS), Parkinson's disease, and Alzheimer's disease. Potential links to the Guamanian variant of these diseases (ALS–PD complex) are discussed. In context to the above, we provide a GSH-depletion model of neurodegenerative disorders, suggest experimental verifications of this model, and propose potential therapeutic approaches for preventing or halting these diseases.
Recent studies indicate that protein glutathionylation is an important regulatory mechanism. The develop-ment of redox proteomics techniques to identify proteins undergoing glutathionylation has a key role in defining the importance of this post-translational modification, although the available methods are not yet comparable to those for the study of other modifications like phosphorylation. We describe here methods that have been successfully employed to identify in vitro glutathionylated proteins.