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Effects of N-acetylcysteine, oral glutathione (GSH) and a novel sublingual form of GSH on oxidative stress markers: A comparative crossover study

Authors:
  • Laboratoires Le Stum, France, Larmor Plage

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Glutathione (GSH) is critical to fight against oxidative stress. Its very low bioavailability limits the interest of a supplementation. The purpose of this study was to compare the bioavailability, the effect on oxidative stress markers and the safety of a new sublingual form of GSH with two commonly used dietary supplements, N-acetylcysteine (NAC) and oral GSH. The study was a three-week randomized crossover trial. 20 Volunteers with metabolic syndrome were enrolled. GSH levels and several oxidative stress markers were determined at different times during each 21-days period. Compared to oral GSH group, an increase of total and reduced GSH levels in plasma and a higher GSH/GSSG ratio (p=0.003) was observed in sublingual GSH group. After 3 weeks of administration, there was a significant increase of vitamin E level in plasma only in sublingual GSH group (0.83µmol/g; p=0.04). Our results demonstrate the superiority of a new sublingual form of GSH over the oral GSH form and NAC in terms of GSH supplementation. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
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Effects of N-acetylcysteine, oral glutathione (GSH) and a novel
sublingual form of GSH on oxidative stress markers: A comparative
crossover study.
Bernard Schmitt
a
, Morgane Vicenzi
b
, Catherine Garrel
c
, Frédéric M. Denis
b,
n
a
Centre dEnseignement et de Recherche en Nutrition Humaine, Centre Hospitalier de Bretagne Sud, 5 Avenue De Choiseul, BP 12233, 56322 Lorient Cedex,
France
b
Laboratoires Le Stum, 4 impasse de Kerhoas, 56260 Larmor Plage, France
c
Unité de Biochimie Hormonale et Nutritionnelle, Département de Biochimie, Toxicologie et Pharmacologie, Institut de Biologie et de Pathologie, Centre
Hospitalier Universitaire de Grenoble, CS10217, 38043 Grenoble, France
article info
Article history:
Received 16 June 2015
Received in revised form
20 July 2015
Accepted 26 July 2015
Available online 29 July 2015
Keywords:
Glutathione
Oral bioavailability
N-acetyl cysteine
Oxidative stress
Metabolic syndrome
Dietary supplement
Nutraceuticals
Sublingual
abstract
Glutathione (GSH) is critical to ght against oxidative stress. Its very low bioavailability limits the interest
of a supplementation. The purpose of this study was to compare the bioavailability, the effect on oxi-
dative stress markers and the safety of a new sublingual form of GSH with two commonly used dietary
supplements, N-acetylcysteine (NAC) and oral GSH. The study was a three-week randomized crossover
trial. 20 Volunteers with metabolic syndrome were enrolled. GSH levels and several oxidative stress
markers were determined at different times during each 21-days period. Compared to oral GSH group, an
increase of total and reduced GSH levels in plasma and a higher GSH/GSSG ratio (p¼ 0.003) was observed
in sublingual GSH group. After 3 weeks of administration, there was a signicant increase of vitamin E
level in plasma only in sublingual GSH group (0.83 mmol/g; p¼0.04). Our results demonstrate the su-
periority of a new sublingual form of GSH over the oral GSH form and NAC in terms of GSH supple-
mentation.
& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Glutathione (GSH) is an ubiquitous water-soluble molecule
found in millimolar concentration in many tissues and cells. It is
the most abundant intracellular low molecular weight peptide
containing a thiol group. This thiol function is critical for the
biological activity of GSH [1].
GSH is made from three amino acids: glycine, cysteine and
glutamic acid. This tripeptide exists in reduced (GSH) and oxidized
(GSSG) forms. The relative amounts of each form determine the
cellular redox status (GSH/GSSG ratio) which is often used as a
marker of antioxidative capacity of cells [2].
GSH exhibits diverse physiological roles. It is a potent free ra-
dical and reactive oxygen species scavenger [3]. It reacts with
various molecules (metabolites, xenobiotics) to form conjugates
[4]. GSH functions as a thiol buffer for many cellular proteins
(metallothioneins, thioredoxins). GSH is an essential cofactor for
many enzymes and it is involved in several metabolic and sig-
naling pathways [5]. GSH is also critical for the regeneration of
other antioxidants such as tocopherols and ascorbate [6].
There is growing evidence that dysfunctional GSH homeostasis
is implicated in the etiology of several diseases. The most well-
known conditions associated with GSH depletion include neuro-
degenerative diseases [7,8], pulmonary diseases [9], liver diseases
[10], immune disorders [11], cardiovascular diseases [12,13] as
well as the aging process itself [14].
Several studies showed that plasma GSH levels decrease with
age. This deterioration of GSH homeostasis could participate, with
other physiological events, in the ageing process and the appear-
ance of age-related diseases[15].
Thus, dietary supplementation with antioxidants has been
studied extensively as a potential way to prevent these diseases by
countering the negative effects of oxidative stress.
Researchers suggest that GSH is poorly absorbed by oral route
mainly due to the action of an intestinal enzyme, the
γ
-glutamyl
transpeptidase (GGT) which degrades GSH[16]. Several studies
showed that GSH supplementation in animals is effective, with
benets in the enhancement of immune function[17]; protection
against the carcinogenesis process[18]; and improvement of the
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/redox
Redox Biology
http://dx.doi.org/10.1016/j.redox.2015.07.012
2213-2317/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
n
Corresponding author. Fax: þ33 297880155
E-mail addresses: schmitt.lorient@yahoo.fr (B. Schmitt),
innov@labo-lestum.com (M. Vicenzi), CGarrel@chu-grenoble.fr (C. Garrel),
infomed@labo-lestum.com (F.M. Denis).
Redox Biology 6 (2015) 198205
elimination of toxic chemicals[19].
However, in humans, the effectiveness of an oral supple-
mentation with GSH is very controversial. A number of studies
were conducted but using intravenous or nebulized GSH. The re-
sults of studies conducted with oral GSH are mixed [20,21]. This
differential absorption of oral GSH between humans and rats/mice
could be explained by a difference in quantity and activity of the
intestinal GGT enzyme [21].
Although oral GSH appears to be the most convenient and safe
way to take GSH, the lack of efcacy explains why this form is not
often used in clinical trials.
Another strategy to enhance GSH production relies on the use
of N-acetylcysteine (NAC), a cysteine precursor. Indeed, the amino
acid cysteine is the main factor limiting the synthesis of GSH.
Several studies showed that NAC is well absorbed by the intestine
and that a supplementation with NAC is effective for increasing
GSH levels [22]. However, supplementing with NAC relies upon
the body's ability to synthesize glutathione from available raw
materials, an ability that diminishes with age and in the presence
of certain diseases, especially dysfunction of the liver.
Finding a way to safely, conveniently and rapidly improve GSH
levels by directly delivering the whole GSH molecule in its active
reduced form would be medically compelling.
Because oral GSH is enzymatically degraded in the intestine,
one solution could be using a sublingual delivery system for GSH.
Argument for that delivery route is that the GSH tripeptide is very
well absorbed through mucosa. It is also well-known that sub-
lingual route allows to by-pass the effect of hepatic rst-pass
metabolism. In order to address this hypothesis, a food-grade
sublingual dosage form was designed.
The purpose of this study was to compare the use of this novel
sublingual form of GSH with two commonly used dietary sup-
plements, NAC and oral GSH, to determine their respective interest
for raising GSH levels and acting on the GSH/GSSG ratio.
2. Experimental section
This randomized crossover study focused on the efciency of a
sublingual form and a conventional oral form of GSH compared to
effects of a precursor (NAC) taken as a baseline. The evaluation
focused on the bioavailability of both GSH forms, their respective
effect on oxydative stress markers, and their tolerability.
2.1. Participants
The study protocol was reviewed and approved by ethics
committee (CPP Ouest) and the Agence Nationale de la Sécurité du
Médicament (ANSM). The approval code of this study is AEC/
B120599-40. All clinical activities were conducted at the Centre
Hospitalier Bretagne Sud (Lorient, France), in accordance with the
Helsinki Declaration, the French Public Health Code concerning
the biomedical researches and the rules of Clinical Best Practice.
A total of 20 voluntary subjects (5 men and 15 women) was
enrolled. Baseline characteristics of the participants are given in
Table 1 below.
Regarding the inclusion criteria, all subjects had risk factors of
low-grade inammatory state corresponding to metabolic syn-
drome as dened in the guidelines of the National Cholesterol
Education Program ATP III[23]: abdominal obesity associated with
at least two of the following factors: impaired fasting glucose with
glucose levels between 1.10 and 1.27 g/l (Z 6 and o 7 mmol/l),
elevated blood pressure (SBP 4 130 mm Hg and/or DBP 4 85 mm
Hg), hypertriglyceridemia 4 1.45 g/l (Z 1.65 mmol), LDL-C be-
tween 1.6 and 2.2 g/l (4.13 and 5.68 mmol/l), HDL-C o 0.40 g/l
(1 mmol/l) (male) and 0.50 g/l (1.3 mmol/l) (female). They had a
low tobacco consumption (o 5 cigarettes/day) associated with a
sedentary lifestyle.
Regarding the exclusion criteria, the subjects took no anti-in-
ammatory or non-steroidal anti-inammatory drugs. They had
no previous history of cardiovascular disease or symptomatic
chronic inammatory disease. They did not consume antioxidant
supplements or cholesterol-lowering agents. They had a free reg-
ular diet respecting their family habits, but neither limited nor
extensive (average energy: 2500 kcal/day) and a moderate alcohol
intake (o 20 g/day) according to the criteria of the Alcohol Use
Disorder Identication Test. GGT were inferior to 35 IU/l and Car-
bohydrate Decient Transferrin (CDT) inferior to 3%.
20 Participants completed the study and all the data were
analyzed.
2.2. Products tested
2.2.1. Composition of the sublingual form of glutathione
This new and patented sublingual form of GSH (Sublinthion
s
)
was provided by Laboratoires Le Stum (Larmor-Plage, France). One
sublingual tablet contains 150 mg of reduced GSH. The dosage was
one tablet 3 times per day (in the morning, the midday and the
evening), to let melt under the tongue. It represents a daily intake
of 450 mg of GSH.
2.2.2. Composition of the oral form of glutathione
The oral form of reduced GSH was
L
-Glutathione Reduce
s
(Laboratoire Equi-Nutri, Belgium). One capsule contains 150 mg of
GSH. The dosage used in the study was one capsule 3 times per
day providing a total intake of 450 mg of GSH.
Table 1
Baseline characteristics of study participants (mean 7 SD).
Female (n ¼ 15) Male ( n ¼ 5)
Mean Max Min Mean Max Min
AGE (years) 59.737 8.4 67 38 53.67 4.67 59 47
BODY WEIGHT (kg) 75.977 10.17 87.5 52 88.447 5.18 95 84
HEIGHT (cm) 152.30 7 5.34 168 150 175.20 7 5.54 182 169
BMI (kg/m
2
) 28.587 2.81 32.42 22.51 28.807 0.75 30 28
SBP (mm Hg) 1357 15.3 160 110 129.27 11.6 150 130
DBP (mm Hg) 77.8 7 11.2 100 60 80.07 8.4 90 65
TRIGLYCERIDES (g/l) 1.277 0.63 2.61 0.44 1.747 0.70 2.68 0.60
LDL-C (g/l) 1.487 0.29 1.97 0.87 1.307 0.41 1.74 0.60
HDL-C (g/l) 0.687 0.28 1.38 0.41 0.657 0.37 1.39 0.36
GLYCEMIA (g/l) 0.957 0.11 1.19 0.81 1.077 0.11 1.27 0.96
BMI: Body Mass Index; SBP: Systolic Blood Pressure; DBP: Diastolic Blood Pressure
B. Schmitt et al. / Redox Biology 6 (2015) 198205 199
2.2.3. Composition of the precursor NAC taken orally
The NAC drug used for this study was marketed under the
brand name Fluimucil
s
(Laboratoires Zambon, France). A sachet
contains 200 mg of NAC. The dosage was one sachet per day.
Cysteine is the limiting factor for GSH synthesis and its re-
presents 33.6% of the GSH molecule [24]. Providing 200 mg of
cysteine (commercial dosage) would be sufcient for the body to
theoretically synthetize de novo up to 600 mg of GSH.
2.3. Study design
Each product was administered successively during three per-
iods (P1, P2 and P3) of 21 days each. A wash-out period of 14 days
was observed between each product. Six combinations of admin-
istration were possible: [NAC-PO-SL]; [NAC-SL-PO]; [PO-SL-NAC];
[PO-NAC-SL]; [SL-PO-NAC]; [SL-NAC-PO] (PO¼ oral form of GSH;
SL¼ sublingual form). Each combination was randomly assigned
to volunteers at the inclusion, by respecting a minimum of three
subjects per combination. Each period included three visits: V1;
V2 (V1þ 10 7 2 days) and V3 (V2þ 10 7 2 days). In total, each vo-
lunteer received two preliminary visits (pre-inclusion and inclu-
sion) and nine visits during the protocol.
During the pre-inclusion visit, inclusion/exclusion criteria were
explained to subjects, the initial screening was done according to
these criteria and the informed consent of each subject was
collected.
A full medical history from each participant was obtained
during the inclusion visit. Medical examinations were also con-
ducted to check the digestive, cardiovascular and pulmonary sta-
tus of the subjects.
The study was conducted according to the following plan
(Fig. 1).
During the test there was no change of the diet habits or the
lifestyle of the participants (diet, physical activity, smoking, etc.).
Each patient was his own control to avoid a potential bias of a
residual effect of the taking of the previous product on the fol-
lowing product. Table 2 presents the average characteristics for
each group at the time of inclusion in the study.
2.4. Markers and parameters measured
Blood tests were realized at the beginning (visit 1, before taking
treatment), at the middle (visit 2) and at the end (visit 3) of every
period, for all the volunteers. At each visit, the following para-
meters were measured.
2.4.1. Total GSH; GSH (reduced); GSSG (oxidized); ratio GSH/GSSG
Endogenous glutathione was measured according to the fol-
lowing analytical protocol. Briey, arterial blood samples were
collected in lithium heparin vacutainers as an anticoagulant.
400 ml of whole blood were collected in 3.6 ml of metaphosphoric
acid for the determination of total, reduced (GSH) and oxidized
(GSSG) glutathione. After centrifugation (4000g, 10 min, 4 °C) total
and reduced GSH were determined enzymatically in the acidic
protein-free-supernatant. The assay of GSSG was performed after
having masked GSH by adding 2-vinylpyridine to the deprotei-
nized extract and also determined enzymatically[25].
2.4.2. Reduced thiols
The rest of whole blood was centrifuged (4000 g, 10 min, 4 °C)
to separate the plasma for thiols, Plasma was collected in Eppen-
dorff sterile tubes and stored at -80 °C until assayed. The mea-
surement of thiol groups was performed using Ellman's reagent
[26,27]. Briey, 5,5-dithio-bis (2-nitrobenzoic acid) 2.5 mM in
0.2 M phosphate buffer, pH 8.0 was mixed with 500 ml of sample
and 750 ml of 50 mM phosphate buffer 50 mM pH 8.0 and baseline
absorbance recorded at 412 nm. Then, 250 m
l of freshly prepared
Ellman's reagent were added, reaction allowed to proceed for
15 min at room temperature in the dark and nal absorbance
measured. Thiol values were expressed in mmol/g protein using a
molar absorption coefcient of 13,600 l mol
1
cm
1
for thiol 5,5-
dithio-bis (2-nitrobenzoic acid) complex. A calibration curve was
performed by sequential dilution of a 1 mM N-acetyl cysteine
stock solution.
2.4.3. Vitamin E: alpha and gamma tocopherols
Serum concentrations of tocopherols were measured by high
performance liquid chromatography as previously described[28].
2.4.4. Lipid status: total cholesterol (TC), LDL-C, HDL-C, triglycerides
(TG)
Total cholesterol, HDL, and TG were measured by spectro-
photometric methods on a routine chemistry system (Vitros Fu-
sion 5.1, Ortho Clinical Diagnostics, USA). Serum LDL-cholesterol
was calculated using the following Friedewald formula [29]:
gLDLC TC HDLC 0,20 TG expressedin /l
[
]=[ ]−[ ]−
*
[]
()
2.5. Statistical analysis
The individual characteristics (age, sex) and all the parameters
at D0 are described with regard to the mean and the standard
deviation. The conditions of normality are veried in advance by
the ShapiroWilk test, comparability of groups is performed by
Student's t-test. From a value of s estimated on the basis of a
previous pilot study (internal documents), we used a sample size
of 20 volunteers, who were included. To search a carry-over ef-
fect (i.e. a residual effect of a product of a previous period over the
next period), the measures obtained at the end of 3 treatment
periods (P1V3; P2V3; P3V3) were compared by using a mixed
linear regression model for each of three treatments.
The statistical analysis was performed with the SAS software
Fig. 1. Design of the study.
B. Schmitt et al. / Redox Biology 6 (2015) 198205200
version 9.3 (SAS Institute Inc., Cary, NC). Pr 0.05 was taken as the
level of statistical signicance for all procedures.
2.6. Outcomes
The purpose of the study was to compare the bioavailability of
each product, the effects of the different forms of glutathione on
markers of oxidative stress and the safety of each product.
2.6.1. Bioavailability evaluation
The bioavailability of each product was measured by comparing
respective total glutathione (GSHt), reduced glutathione (GSH) and
oxidized glutathione (GSSG) indices. The measurements were
made by comparing these markers between the rst and second
visit (V2V1) and between the rst and last visit (V3V1).
2.6.2. Evaluation of the antioxidant efcacy
The primary outcome was the ratio between reduced glu-
tathione and oxidized glutathione expressed as GSH/GSSG.
The GSH/GSSG ratios were compared at the end of each period
by using a mixed linear regression model. The equation is the
following one:
aaia a
a
Primary outcome GSH/GSSG
0 1 treatment 2 period
3 GSH/GSSH ratio at visit 1
[
()
=(+)+* +*
+*
()
In the equation above, ai (a index i) represents the individual
variability, to which the standard intercept (a0) is added. To
evaluate the effect of each treatment, the model is adjusted to the
value of the primary outcome at the start of each treatment period
(P1V1; P2V1; P3V1). The model is also adjusted to the treat-
ment period (3 periods for each subject corresponding to the
successive intake of the 3 treatments). This adjustment allows to
take into consideration a potential effect of time on the primary
outcome [30].
The absence of Carry-Over effect, whatever the period and
the product considered, allowed us to apply the above equation to
all parameters studied.
The secondary outcomes contributed to estimate the oxidative
stress status of the volunteers: plasma reduced thiols, vitamin E,
Total Cholesterol, HDL-C, LDL-C and plasma triglycerides were also
measured.
2.6.3. Tolerance of the treatments
The tolerance of the treatments was analyzed by evaluating
changes in plasma levels of CRPus (ultra-sensitive C-reactive pro-
tein) and liver function tests (Alanine amino transferase (ALAT),
Aspartate amino transferase (ASAT), alkaline phosphatase (AP) and
GGT between the rst visit (V1) and the last visit (V3) for each
treatment). The values did not have to exceed the superior limit of
the range of normality (N): ALAT (N:3050 IU/l); ASAT (N:15
41 IU/l); AP (N:40150 IU/l); GGT (N:550 IU/l). The percentage
increase of these biological data was analyzed using the Wilcoxon
test of signed rank.
The evaluation of safety also included the monitoring of any
clinical adverse events as well as vital signs (heart rate, blood
pressure, respiratory frequency, body temperature). The general
appearance was checked by a physical examination of each subject
at every visit (V1, V2, V3).
3. Results
3.1. Bioavailability
The comparative analysis of bioavailability between oral GSH
and the sublingual form is summarized in Tables 3 and 4.
The potential problem in crossover design is that carryover
effects may bias the direct effects of the treatment. Regardless of
the period and the treatment, no signicant carryover effect was
observed (p4 0.75). Therefore, the data were pooled for a given
treatment (SL, PO) to analyze the results (n¼ 20 for each
treatment).
3.2. Effects of the treatments
3.2.1. Primary outcome (GSH/GSSG)
The comparison of the effect of each treatment on the GSH/
GSSG ratio was performed after pooling the results as there was no
carryover effect.
The evolution of the GSH/GSSG ratio for the 3 groups (NAC, oral
GSH or sublingual GSH) is reported in Table 5.
A comparative analysis was performed: rst by taking NAC as
reference (comparison NAC vs oral GSH and NAC vs sublingual
GSH) and second by comparing the oral GSH to the sublingual GSH
form.
In the oral GSH group, the GSH/GSSG ratio was low at each time
and signicantly different at V3 (p¼ 0.03) compared to the NAC
group.
In the sublingual GSH group, this ratio tended to be high at
each time and was statistically signicant at V2 (p¼ 0.03) com-
pared to the NAC group.
Compared to the oral GSH group, the sublingual group ex-
hibited a higher GSH/GSSG ratio, in particular at V3 (p¼ 0.02).
Table 2
Characteristics of volunteer groups at inclusion (mean7 SD).
Group
a
Age (years) Height (m) Weight (kg) BMI (kg/m
2
) Systolic pressure (mm Hg) Diastolic pressure (mm Hg)
1 NAC-PO-SL 557 15 1.61 7 0.06 70.97 5.1 27.47 0.8 1327 19 77 76
(n¼ 3)
2 NAC-SL-PO 547 10 1.657 0.06 77.97 7.3 28.67 1.9 138 7 18 80 79
(n¼ 4)
3 PO-SL-NAC 637 5 1.617 0.04 75.17 7.4 28.97 2.0 1357 14 82 74
(n¼ 4)
4 PO-NAC-SL 597 8 1.547 0.06 71.47 17.0 30.07 5.4 1377 12 83 7 15
(n¼ 3)
5 SL-PO-NAC 527 5 1.697 0.09 85.17 10.6 29.67 1.7 138 7 17 84 713
(n¼ 3)
6 SL-NAC-PO 617 7 1.637 0.15 76.67 14.3 28.67 2.0 1257 9777 6
(n
¼ 3)
Comparison groups
b
p¼ 0.26 p¼ 0.23 p¼ 0.28 p¼ 0.59 p¼ 0.80 p¼ 0.72
a
Successive treatment.
b
KruskalWallis test.
B. Schmitt et al. / Redox Biology 6 (2015) 198205 201
3.2.2. Secondary outcomes
3.2.2.1. Reduced thiols. Results are detailed in the Table 6. Reduced
thiols levels are expressed on an albumin gram basis.
An intragroup analysis was performed: in the NAC group, a
signicant increase was observed at V2 compared to baseline
(0.12 mmol/g, p¼ 0.04). In the oral GSH group, the level of reduced
thiols increased signicantly at V2 and V3 compared to baseline
(respectively 0.14 mmol/g; p¼ 0.004 and 0.13 mmol/g, p¼ 0.001). For
sublingual GSH, this level increased only in the rst period
(0.14 mmol/g, p¼ 0.01). No signicant differences were observed
between the 3 groups.
3.2.2.2. Vitamin E. The effects of the 3 treatments on the levels of
vitamin E in plasma were also examined (Table 7).
After 3 weeks of administration, there was a signicant in-
crease of vitamin E level in plasma only in the sublingual GSH
group (0.83 mmol/g; p¼ 0.04). No signicant differences were ob-
served between the 3 groups or for the oral GSH and NAC arms.
3.2.2.3. Lipid status. Results are detailed in the Table 8.
After performing an intragroup analysis, no changes were
observed at any time points or in either groups, whatever the lipid
biomarker monitored (total cholesterol, HDL-C, LDL-C, TG).
When taking the lipid values of the NAC group as baseline, total
cholesterol and LDL-C were slightly decreased at V3 in both oral
and sublingual GSH groups. However, it was not statistically
signicant.
In the meantime, HDL-C level decreased in the oral GSH group
and increased in the sublingual GSH group but these differences
were not signicant. However, compared to the oral GSH group, a
signicant increase of HDL-C level was observed in the sublingual
GSH group (0.0397 0.013, p¼ 0.0043).
3.2.3. Adverse effects
Values of the plasma levels of CRPus and liver function markers
at each visit for each treatment arm were reported in Table 9.
Whatever the marker (hepatic status or ultra-sensitive CRP), no
signicant changes were reported. For all the markers monitored,
values were always within the range of normality.
All the dosage forms were very well tolerated and no adverse
events were reported by the participants, whatever the treatment
used.
4. Discussion
Conducting such a study is always dif
cult, as the supple-
mentation product (GSH) is also produced endogenously by the
body. Furthermore, like most of our antioxidants, it is tightly
regulated.
Whatever the treatment considered, our results sometimes
show high standard deviations. This can be explained by the
heterogeneity of the studied population. Indeed, the main inclu-
sion criterion was the presence of metabolic syndrome. It is not a
strictly dened disease entity, but a convergence of at least 3 risk
factors. As each patient may have a number and/or a combination
of different risk factors while meeting the strict denition of me-
tabolic syndrome, this heterogeneity was expected. Otherwise, it
corresponds to the remaining diversity commonly encountered in
a standard population. Therefore, bias in the recruitment of the
Table 3
Total glutathione, GSH and GSSG levels (mmol/l) (mean7 SD).
Product Dosage V1 V2 V3 ΔV2V1 ΔV3V2 ΔV3V1
NAC (n¼ 20) Total GSH 800.24794.87 825.537 127.62 821.07 124.88 30.357 74.73 4.317 47.6 27.07 77.75
GSSG 17.327 5.24 17.877 4.38 15.60 7 4.83 0.557 4.03 1.66 7 2.91 7.337 11.0
GSH 765.597 91.94 789.797 122.83 789.807 119.40 24.217 63.64 11.147 45.80 30.95 7 71.84
PO (n¼ 20) Total GSH 823.297 90.51 782.69 7 96.89 789.887 133.55 -37.447 72.41 3.197 103.63 -33.417 84.01
GSSG 16.32 7 3,48 16.08 7 4.10 18.60 7 5.29 0.067 3.34 2.337 6.32 2.287 4.62
GSH 790.667 87.99 750.54 7 96.90 752.68
7129.53 37.587 67,78 -1.467 99.74 37.98 7 80.58
SL (n¼ 20) Total GSH 811.127 99.77 846.07 127.88 838.76797.69 34.887 61.52 7.247 50.57 27.657 57.71
GSSG 17.617 4.03 16.547 4.70 15.62 7 3.62 1.0774.17 0.927 4.38 2.017 4.26
GSH 774.717 99.45 812.92 7 122.90 807.537 96.15 38.737 57.96 5.397 48.25 32.417 57.54
Table 4
Evolution of total GSH, GSH and GSSG: oral versus sublingual GSH (mmol/l).
Dosage ΔV2V1 ΔV3V2 ΔV3V1
Comparison Total GSH PO 37.44 3.19 33.41
PO vs SL SL 34.88 7.24 27.65
(n¼ 20) p 0.02 0.37 0.05
GSSG PO 0.06 2.33 2.28
SL 1.07 0.92 2.01
p 0.23 0.12 0.04
GSH PO 37.58 1.46 37.98
SL 38.73 5.39 32.41
p 0.01 0.41 0.03
Compared to the oral GSH group, an increase of total and reduced GSH levels in
plasma was observed in the sublingual GSH group. The GSSG level also decreased
following the supplementation with the sublingual GSH. These differences between
the 2 groups were statistically signicant (pr 0.05), whatever the parameter
considered.
Table 5
GSH/GSSG ratio and their evolution (mean7 SD).
Product V1 V2 V3 ΔV2V1 ΔV3V2 ΔV3V1
NAC (n¼ 20) 50.037 14.02 46.257 7.17 56.447 13.74 3.797 11.39 9.717 10.81 7.387 11.12
Oral GSH (n ¼ 20) 51.687 11.04 51.547 14.28 44.767 14.23 0.917 9.35 6.317 17.41 6.927 15.90
Sublingual (n¼ 20) 47.557 12.50 53.697 13.84 56.977 16.22 6.157 10.41 3.277 14.75 9.427 14.62
Comparison NAC/PO p¼ 0.28 p ¼ 0.20 p¼ 0.03 p¼ 0.29 p¼ 0.06 p¼ 0.01
Comparison NAC/SL p¼ 0.22 p¼ 0.03 p¼ 0.37 p¼
0.02 p¼ 0.22 p¼ 0.20
Comparison PO/SL p¼ 0.11 p¼ 0.20 p ¼ 0.02 p¼ 0.03 p¼ 0.07 p¼ 0.002
B. Schmitt et al. / Redox Biology 6 (2015) 198205202
volunteers can be excluded for this study.
Oral administration of GSH is not considered optimal due to its
very poor bioavailability and rapid oxidation. Other indirect means
have been developed to circumvent this problem. One of them is
the oral delivery of NAC as a source of cysteine. Indeed, after its
intestinal absorption, NAC undergoes rst-pass metabolism in the
liver where it is deacetylated to cysteine. Then, unless there is
hepatic dysfunction, the hepatic tissue synthetizes de novo GSH
from this cysteine. This GSH replenishes the hepatic stock before
being released in the plasma [22,31]. Consequently, we considered
NAC as the reference treatment for this study.
Regarding the bioavailability of oral GSH, our data are con-
sistent with previous results from a study of oral GSH supple-
mentation (1 g/day for 4 weeks) that showed a non-signicant
decrease of total and reduced GSH indices [20] . This results in an
overall decrease of the GSH/GSSG ratio. High levels of GSSG are
indicative of periods of oxidative stress. The ratio GSH/GSSG is an
important marker of redox status. The restoration of a normal
redox equilibrium results in an increase in GSH/GSSG ratio. In our
study, the GSH/GSSG ratios in the NAC and sublingual GSH arms
are signicantly higher than oral GSH one. It seems that the sub-
lingual GSH form is more useful than the oral form to improve this
balance.
One possible explanation of these results is that oral GSH un-
dergoes partial hydrolysis and oxidation during the digestive
process. Therefore, the liver has to synthetize de novo GSH from
precursors.
With the sublingual dosage form, the GSH is directly assimi-
lated through the buccal mucosa and avoid the hepatic rst-pass
effect. Our results suggest that the sublingual GSH form exhibits a
better bioavailability than the oral GSH.
This increase of the GSH/GSSG ratio may suggest a reduction in
oxidative stress resulting from the sublingual GSH supplementa-
tion. Therefore, we tried to determine whether the improved
bioavailability of the sublingual form of GSH resulted in an effect
on oxidative stress markers. The outcome measures were ex-
tended to reduced thiols and vitamin E.
Some evidence suggest that GSH is critical for the recycling of
antioxidants like vitamin C [32,33] and consequently, vitamin E
[34], which is an important inhibitor of the lipid peroxidation. Our
ndings are consistent with these previous observations. We
found that there was a signicant increase in the plasma vitamin E
level following the supplementation with sublingual GSH.
Considering that GSH forms were given at physiological dose to
subjects, obtaining such signicant differences between these two
dosage forms, either on the primary endpoint or on several sec-
ondary endpoints, reinforces the legitimacy of the sublingual form
over the oral GSH.
It would be interesting to conduct the same study on a popu-
lation with greater GSH deciency or high oxidative stress (smo-
kers, type 2 diabetics, HIV-positive subjects). Larger differences
between the two forms of GSH on the GSH/GSSG ratio or on other
markers would likely be expected.
Given the results observed between NAC and sublingual GSH, it
seems useful to advise this new sublingual form for the same in-
dications as those described for the NAC in the literature [35]. The
recommendation of this product should be preferentially based on
clinical observation and inventory of risk factors rather than based
on costly and variable blood tests.
Regarding the duration of treatment necessary to obtain an
antioxidative effect, 21 days of treatment were sufcient to
achieve signicant results, mostly for the NAC and the sublingual
GSH form. During these 3 weeks, no adverse effects were reported.
However, the overall duration of treatment must take into account
the importance and the multiplicity of risk factors.
5. Conclusions
Conducted on a population at risk with metabolic syndrome,
Table 6
Evolution of reduced thiols (mmol/g) for each treatment (mean7 SD).
V1 V2 V3 Intragroup evolution
ΔV2V1 ΔV3V1 ΔV3V2
NAC (n¼ 20) 6.247 0.32 6.367 0.32 6.297 0.44 0.12, p¼ 0.04 0.05, p¼ 0.29 0.07, p¼ 0.17
Oral GSH (n ¼ 20) 6.157 0.28 6.297 0.28 6.287 0.38 0.14, p¼ 0.004 0.13, p¼ 0.001 0.01, p¼ 0.5
Sublingual GSH (n¼ 20) 6.147 0.29 6.287 0.36 6.147 0.33 0.14, p ¼ 0.01 0.00, p¼ 0.46 0.14, p¼ 0.07
Comparison NAC/PO p¼ 0.40 p¼ 0.40 p¼ 0.39
Comparison NAC/SL p¼ 0.21 p¼ 0.20
p¼ 0.15
Table 7
Vitamin E levels (mmol/g) and their evolution (mean7 SD).
Product V1 V2 V3 ΔV2V1 ΔV3V1 ΔV3V2
NAC (n¼ 20) 26.637 6.02 25.88 76.39 27.167 5.56 0.75, p¼ 0.10 0.53, p¼ 0.18 1.28, p¼ 0.10
Oral GSH (n ¼ 20) 26.707 4.94 26.417 5.52 26.237 5.64 0.29, p ¼ 0.24 0.47, p ¼ 0.15 0.18, p¼ 0.31
Sublingual GSH (n¼ 20) 26.59 75.76 26.717 5.92 27.427 6.32 0.12, p¼ 0.44 0.83, p¼ 0.04 0.71, p ¼ 0.25
Comparison NAC/PO p¼ 0.32 p¼ 0.31 p¼ 0.17
Comparison NAC/SL p¼ 0.49 p¼ 0.25
p¼ 0.45
Table 8
Lipid biomarkers levels (g/l) and their evolution (mean7 SD).
Product Dosage V1 V2 V3
NAC (n ¼ 20) TG 1.527 0.59 1.497 0.67 1.597 0.63
TC 2.297 0.39 2.287 0.45 2.287 0.41
HDL-C 0.527 0.11 0.51 7 0.1 0.527 0.1
LDL-C 1.48 70.34 1.477 0.4 1.457 0.39
Oral GSH (n ¼ 20) TG 1.527 0.58 1.627 0.92 1.547 0.72
TC 2.327 0.38 2.297 0.41 2.237 0.36
HDL-C 0.527 0.1 0.527 0.12 0.51 7 0.1
LDL-C 1.49 70.33 1.467 0.35 1.417 0.33
Sublingual GSH (n ¼ 20) TG 1.7 7 0.74 1.47 0.61 1.557 0.63
TC 2.37 0.4 2.297 0.39 2.257 0.43
HDL-C 0.51 7 0.12 0.537 0.12 0.547
0.13
LDL-C 1.45 7 0.38 1.49 7 0.35 1.417 0.4
B. Schmitt et al. / Redox Biology 6 (2015) 198205 203
the objective of this study was to assess the bioavailability, the
effect on biomarkers, and the short-term safety of 2 dosage forms
of glutathione, the cornerstone of antioxidant defenses.
Overall, our results demonstrate the signicant superiority of a
new sublingual form of GSH over the oral form, both in terms of
bioavailability and positive effects on oxidative stress. Compared
to NAC, better effects of the sublingual form of GSH were also
observed.
Metabolic syndrome increases the risk of developing cardio-
vascular diseases and diabetes. Because of the impact of the de-
leterious effects of reactive oxygen species (ROS) in the promotion
and the development of the metabolic syndrome, it is important to
establish a preventive strategy and ght against oxidative stress.
In addition to the usual dietary recommendations, it is reasonable
to propose to concerned people a product whose interest is
demonstrated.
This new sublingual formulation of GSH meets this require-
ment. It should nd its place in the primary and secondary pre-
vention strategies.
Author contributions
Conceived and designed the experiments: Bernard Schmitt,
Morgane Vicenzi and Frederic M. Denis. Performed the experi-
ments: Bernard Schmitt. Analyzed the data: Bernard Schmitt.
Contributed reagents/materials/analysis tools: Catherine Garrel.
Wrote the paper: Bernard Schmitt, Morgane Vicenzi and Frederic
M. Denis. All authors read and approved the nal manuscript.
Conicts of interest
Frederic M. Denis and Morgane Vicenzi are employees of La-
boratoires Le Stum. The other authors declare no con ict of
interest.
Acknowledgments
The study was sponsored by Laboratoires Le Stum. The authors
thank Cheryl Myers for her careful reading of our manuscript.
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GGT (IU/l) 38.287 29.6 39.647 29.68 36.837 24.73
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... GSH has been orally administered in forms such as sublingual [10], orobuccal [11,12], and liposomal [13] for rapid absorption. We note that these forms of GSH are not only not easily available commercially but also sublingual and orobuccal formulations include GSH as one of the (primary) ingredients, which makes it difficult to attribute the effects to GSH alone. ...
... GSH has been orally administered in forms such as sublingual [10], orobuccal [11,12], and liposomal [13] for rapid absorption. We note that these forms of GSH are not only not easily available commercially but also sublingual and orobuccal formulations include GSH as one of the (primary) ingredients, which makes it difficult to attribute the effects to GSH alone. ...
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Complications in type 2 diabetes (T2D) arise from hyperglycemia-induced oxidative stress. Here, we examined the effectiveness of supplementation with the endogenous antioxidant glutathione (GSH) during anti-diabetic treatment. A total of 104 non-diabetic and 250 diabetic individuals on anti-diabetic therapy, of either sex and aged between 30 and 78 years, were recruited. A total of 125 diabetic patients were additionally given 500 mg oral GSH supplementation daily for a period of six months. Fasting and PP glucose, insulin, HbA1c, GSH, oxidized glutathione (GSSG), and 8-hydroxy-2-deoxy guanosine (8-OHdG) were measured upon recruitment and after three and six months of supplementation. Statistical significance and effect size were assessed longitudinally across all arms. Blood GSH increased (Cohen’s d = 1.01) and 8-OHdG decreased (Cohen’s d = −1.07) significantly within three months (p < 0.001) in diabetic individuals. A post hoc sub-group analysis showed that HbA1c (Cohen’s d = −0.41; p < 0.05) and fasting insulin levels (Cohen’s d = 0.56; p < 0.05) changed significantly in diabetic individuals above 55 years. GSH supplementation caused a significant increase in blood GSH and helped maintain the baseline HbA1c overall. These results suggest GSH supplementation is of considerable benefit to patients above 55 years, not only supporting decreased glycated hemoglobin (HbA1c) and 8-OHdG but also increasing fasting insulin. The clinical implication of our study is that the oral administration of GSH potentially complements anti-diabetic therapy in achieving better glycemic targets, especially in the elderly population.
... It is a ubiquitous molecule and provides defence against free radicals, hydroperoxides and other harmful oxidants. However, its production declines with age, and is often given orally or administered intravenously [24][25][26] , which causes its increased plasma concentration. Our studies show that incubation of α2M with GSH (20-100 µM) results in loss of functional status of this antiproteinase. ...
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Background: Glutathione (GSH) is a principle thiol-containing tripeptide (cysteine, glutamic acid and glycine) antioxidant against free radicals and other harmful oxidants in cellular defence. The alpha-2-macroglobulin (α2M) is large tetrameric zinc-binding glycoprotein which inhibits proteinases regardless of their specificity and catalytic mechanism. Materials and Methods: The interaction of GSH was analyzed with α2M including the structural and functional alterations in α2M using various biochemical and biophysical methods. UV-visible and fluorescence spectroscopy were used to study the binding of α2M with GSH and Fourier transform infrared (FT-IR) spectroscopy was explored to study the structural change induced in α2M. Results: The results suggest that exposure of α2M to GSH decreases the antiproteolytic potential as suggested by the amidase assay. The UV-spectroscopic study showed the formation of α2M-GSH complex and fluorescence analysis showed significant quenching in fluorescence intensity of α2M suggesting GSH binding and structural alteration in the protein. FT-IR spectroscopy was explored to study the structural change induced in α2M which suggest that the secondary structure of α2M changes upon complex formation. Conclusion: Our studies show that interaction of α2M with photoilluminated GSH results in functional and conformational changes of the protein. Keywords: glutathione, GSH, alpha-2-macroglobulin, photo-illumination, ITC, FTIR
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One of the growing global health problems are vector-borne diseases, including tick-borne diseases. The most common tick-borne diseases include Lyme disease, tick-borne encephalitis, human granulocytic anaplasmosis, and babesiosis. Taking into account the metabolic effects in the patient’s body, tick-borne diseases are a significant problem from an epidemiological and clinical point of view. Inflammation and oxidative stress are key elements in the pathogenesis of infectious diseases, including tick-borne diseases. In consequence, this leads to oxidative modifications of the structure and function of phospholipids and proteins and results in qualitative and quantitative changes at the level of lipid mediators arising in both reactive oxygen species (ROS) and ROS enzyme–dependent reactions. These types of metabolic modifications affect the functioning of the cells and the host organism. Therefore, links between the severity of the disease state and redox imbalance and the level of phospholipid metabolites are being searched, hoping to find unambiguous diagnostic biomarkers. Assessment of molecular effects of oxidative stress may also enable the monitoring of the disease process and treatment efficacy.
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Overwhelming evidence shows the quality of reporting of randomised controlled trials (RCTs) is not optimal. Without transparent reporting, readers cannot judge the reliability and validity of trial findings nor extract information for systematic reviews. Recent methodological analyses indicate that inadequate reporting and design are associated with biased estimates of treatment effects. Such systematic error is seriously damaging to RCTs, which are considered the gold standard for evaluating interventions because of their ability to minimise or avoid bias.
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The tripeptide glutathione (GSH) is the most abundant free radical scavenger synthesized endogenously in humans. Increasing mechanistic, clinical, and epidemiological evidence demonstrates that GSH status is significant in acute and chronic diseases. Despite ease of delivery, little controlled clinical research data exist evaluating the effects of oral GSH supplementation. The study objectives were to determine the effect of oral GSH supplementation on biomarkers of systemic oxidative stress in human volunteers. This was a randomized, double-blind, placebo-controlled clinical trial. The study was conducted at Bastyr University Research Institute, Kenmore, WA and the Bastyr Center for Natural Health, Seattle, WA. Forty (40) adult volunteers without acute or chronic disease participated in this study. Intervention: Oral GSH supplementation (500 mg twice daily) was given to the volunteers for 4 weeks. Primary outcome measures included change in creatinine-standardized, urinary F2-isoprostanes (F2-isoP) and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). Changes in erythrocyte GSH concentrations, including total reduced glutathione (GSH), oxidized glutathione (GSSG), and their ratio (GSH:GSSG) were also measured by tandem liquid chromatography/mass spectrometry. Analysis of variance was used to evaluate differences between groups. There were no differences in oxidative stress biomarkers between treatment groups at baseline. Thirty-nine (39) participants completed the study per protocol. Changes in creatinine standardized F2-isoP (ng/mg creatinine) (0.0±0.1 versus 0.0±0.1, p=0.38) and 8-OHdG (μg/g creatinine) (-0.2±3.3 versus 1.0±3.2, p=0.27) were nonsignificant between groups at week 4. Total reduced, oxidized, and ratio measures of GSH status were also unchanged. No significant changes were observed in biomarkers of oxidative stress, including glutathione status, in this clinical trial of oral glutathione supplementation in healthy adults.
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Cellular glutathione levels may exceed vitamin C levels by 10-fold, generating the question about the real antioxidant role that low intracellular concentrations of vitamin C can play in the presence of a vast molar excess of glutathione. We characterized the metabolism of vitamin C and its relationship with glutathione in primary cultures of human endothelial cells oxidatively challenged by treatment with hydrogen peroxide or with activated cells undergoing the respiratory burst, and analyzed the manner in which vitamin C interacts with glutathione to increase the antioxidant capacity of cells. Our data indicate that: (i) endothelial cells express transporters for reduced and oxidized vitamin C and accumulate ascorbic acid with participation of glutathione-dependent dehydroascorbic acid reductases, (ii) although increased intracellular levels of vitamin C or glutathione caused augmented resistance to oxidative stress, 10-times more glutathione than vitamin C was required, (iii) full antioxidant protection required the simultaneous presence of intracellular and extracellular vitamin C at concentrations normally found in vivo, and (iv) intracellular vitamin C cooperated in enhancing glutathione recovery after oxidative challenge thus providing cells with enhanced survival potential, while extracellular vitamin C was recycled through a mechanism involving the simultaneous neutralization of oxidant species. Therefore, in endothelial cells under oxidative challenge, vitamin C functions as an essential cellular antioxidant even in the presence of a vast molar excess of glutathione.
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Glutathione (GSH) is the most abundant nonprotein thiol in cells and has multiple biological functions. Glutathione biosynthesis by way of the γ‐glutamyl cycle is important for maintaining GSH homeostasis and normal redox status. As the only enzyme of the cycle located on the outer surface of plasma membrane, γ‐glutamyl transpeptidase (GGT) plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate‐limiting substrate, for intracellular de novo synthesis of GSH. GGT also initiates the metabolism of glutathione S‐conjugates to mercapturic acids by transferring the γ‐glutamyl moiety to an acceptor amino acid and releasing cysteinylglycine. GGT is expressed in a tissue‐, developmental phase‐, and cell‐specific manner that may be related to its complex gene structure. In rodents, there is a single GGT gene, and several promoters that generate different mRNA subtypes and regulate its expression. In contrast, several GGT genes have been found in humans. During oxidative stress, GGT gene expression is increased, and this is believed to constitute an adaptation to stress. Interestingly, only certain mRNA subtypes are increased, suggesting a specific mode of regulation of GGT gene expression by oxidants. Here, protocols to measure GGT activity, relative levels of total and specific GGT mRNA subtypes, and GSH concentration are described.
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It has been established that oxidative stress, defined as the condition when the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson's disease. Glutathione is a ubiquitous thiol tripeptide that acts alone, or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals and peroxynitrites. In this review, we examine the synthesis, metabolism and functional interactions of glutathione, and discuss how this relates to protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson's disease.