International Journal of Analytical Mass Spectrometry and Chromatography, 2013, 1, 22-30
http://dx.doi.org/10.4236/ijamsc.2013.11004 Published Online September 2013 (http://www.scirp.org/journal/ijamsc)
Measurement of Glutathio n yl ated Haemog lobin by
MALDI-ToF Mass Spectrometry as a Biomarker of
Oxidative Stress in Heavy Smokers and in Occupational
Obese Subjects
Federico Maria Rubino1*, Cinzia Della Noce2, Luisella Vigna3, Rachele De Giuseppe4,
Cristina Novembrino5, Federica de Liso5, Rita Maiavacca5, Lorenzo Patrini3, Luciano Riboldi3,
Fabrizia Bamonti4
1Laboratorio di Tossicologia e Metabonomica Analitica (LaTMA), Dipartimento di Scienze della Salute, Università degli
Studi di Milano, Milano, Italy
2Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica, Dipartimento CardioToracico e Vascolare,
Ospedale Niguarda Ca’ Granda, Milano, Italy
3Dipartimento di Medicina Preventiva, U.O. Medicina del Lavoro, “Clinica del Lavoro L Devoto”, Fondazione IRCCS Ca’
Granda Ospedale Maggiore Policlinico, Milano, Italy
4Dipartimento di Scienze Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Unità Operativa di
Ematologia e CTMO, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico via F. Sforza 35, Milano, Italy
5Laboratorio Centrale di Analisi Chimico Cliniche e Microbiologia, Fondazione IRCCS Cà Granda Ospedale
Maggiore Policlinico, via F. Sforza 35, Milano, Italy
Received July 10, 2013; revised August 16, 2013; accepted September 7, 2013
Copyright © 2013 Federico Maria Rubino et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Glutathionyl-haemoglobin (Hb-SSG) is a minor form of haemoglobin characterized by the presence of a disulfide bond
between the β-93 cysteine residue and the thiol group of glutathione. Hb-SSG is naturally present in the erythrocytes at
levels comparable to those of glycated haemoglobin and can be measured by MALDI mass spectrometry on very small
samples of erythrocytes from peripheral blood. Since Hb-SSG has been recognized as a sensitive biomarker of oxidative
stress in several degenerative diseases (diabetes, hyperlipidemia, kidney disease) and in healthy workers exposed to
glutathione-depleting toxic agents such as butadiene, we have measured for the first time the levels of Hb-SSG in two
groups: healthy heavy cigarette smokers and overweight-obese. For both classes of subjects, the measured levels (6.4%
± 1.7%, n = 30 for smokers; 3.0% ± 0.8%, n = 20 for overweight-obese) are in the upper 97th percentile of those meas-
ured in the Italian general population. Levels in smokers show a small, yet statistically significant dependence on the
level of smoking addiction (>20 cig./day vs. 20 cig./day: 7.0% ± 1.4% vs. 5.7% ± 1.1%; p < 0.05). This biomarker thus
adds to those presently available to rationally assess the extent of biological damage caused by tobacco smoking.
Keywords: Biomarker; Glutathionyl-Haemoglobin; Oxidative Stress; Erythrocyte; Mass Spectrometry
1. Introduction
Tobacco smoking is a long known major cause of mor-
bidity and premature death, mainly due to cardiovascular
disease and lung cancer. Among the toxic effects of in-
halation of the combustion products of tobacco, a main
one is the induction of local and systemic oxidative stress.
This condition occurs when the excessive production of,
or exposure to, reactive chemical substances (e.g. metal
ions, unsaturated aldehydes, quinones) overwhelms the
level of natural defence mechanisms, (e.g. free radical
quenchers, antioxidant enzymes, electrophile scavengers).
As a consequence, key enzymes can be inactivated and
cellular functions impaired to the point that diseases such
as atherosclerosis and cancer can be triggered [1].
Overweight or obesity is a common condition in the
population of the “western” world and can lead to long-
term health impairment and risk of myocardial infarction,
brain stroke and other life-threatening complications [2].
*Corresponding author.
C
opyright © 2013 SciRes. IJAMSC

F. M. RUBINO ET AL. 23
One condition which may hamper weight loss notwith-
standing interventions on diet and physical exercise is
possibly an underlying condition of systemic oxidative
stress [3-5].
It has been very recently claimed that several compo-
nents of the atherosclerotic plaque are themselves able to
generate reactive oxygen species thus sustaining chronic
oxidative damage to biological structures [6].
The reversible oxidation of the thiol function of cys-
teine into the disulfide plays a key role as a buffer of
oxidation. The pool of cysteine-containing natural anti-
oxidants collectively is referred to as the glutathione me-
taboloma scavenges noxious agents through the forma-
tion of the respective thioethers, while the transcription
and the activity of detoxifying enzymes are modulated
through the action of molecular switches based on the
dynamic formation and dissociation of conformation-
crucial disulfide bonds in the catalytic or allosteric do-
mains of key proteins.
Smoking has been recognized as one of the major
source of an enhanced oxidation of the circulating pools
of glutathione and cysteine to the corresponding disul-
fides. The measurement of the concentrations of thiol and
disulfide forms of plasma and erythrocyte thiols has been
employed to calculate the electrochemical potential as a
synthetic bioindicator of the redox state of tissues of non-
smokers and smoking subjects [7].
Mixed disulfides of soluble thiols such as glutathione
and cysteine with proteins also contribute to the pool of
oxidized thiols. In particular, glutathionyl haemoglobin
(HbSSG) is the mixed disulfide generated by reaction of
erythrocyte haemoglobin with glutathione disulfide [8]
(Scheme 1(a)) or with the sulphinic acid derivative of
glutathione [9] (Scheme 1(b)) to yield a disulfide bond
between the thiol group of glutathione and that of the
side chain of the β-93 residue of cysteine.
This form of Hb is naturally present in erythrocytes as
a fraction of total haemoglobin and is thus a viable can-
didate as an innovative biomarker of cellular oxidative
stress [10] (both in vitro; [11] and in vivo, see Table 1)
as well as glycated Hb which is a marker of glycemic
status. HbSSG has been measured in micro-samples of
peripheral blood with several instrumental techniques
among which ion-exchange chromatography [12] and
mass spectrometry [13,14].
HbSSG levels have been so far measured in the control
population and in selected patient populations in a few
Scheme 1.Mechanisms for production of S-glutathionylated
haemoglobin in oxidatively stressed erythr ocytes.
countries differing from one another for ethnicity of
population, for lifestyle and for the ease of access to
high-quality health service. A total number of subjects
approaching one thousand have been examined. The re-
sults of the studies published in the literature are col-
lected in Table 1 (see references [12-23] therein).
As a biomarker of oxidative stress, HbSSG has been
found to increase in a number of different conditions,
including pathological states such as uncontrolled diabe-
tes, nephropathy, dislipidemia, Down’s syndrome and
Friedreich’s ataxia. It has also been found that clinical
interventions such as haemodialysis in nephropathic pa-
tients and administration of dietary supplements in dia-
betic and dislipemic patients are able to lower its concen-
tration, while patients under chronic treatment with an-
tiepileptic drugs show increased levels. HbSSG is also in-
creased in a dose-dependent way in healthy subjects who
are occupationally exposed to butadiene in chemical plants.
To investigate whether HbSSG can add up to the array
of useful biomarkers for assessing the extent of oxidative
stress in two conditions, smoking and obesity, which are
common in the general population, we have measured for
the first time the levels of HbSSG in a group of selected
heavy smokers and in one of clinically obese patients.
2. Materials and Methods
2.1. Investigated Subjects
Thirty healthy (no signs of respiratory impairment) heavy
smokers, defined as declaring to smoke >20 cig/day for
at least 10 years, were randomly selected from the popu-
lation of a study the main results of which were reported
previously [24,25]. Twenty otherwise healthy subjects,
defined as obese on then basis of their Body Mass Index
(BMI) exceeding the cut-off value of 25 were enrolled
from our outpatient clinic [26].
The main characteristics of the fifty subjects are
shown in Table 2. Blood samples were obtained upon
enrolment and after an informed consent were signed by
each, as part of their routine clinical assessment.
This study was conducted according to the Declaration
of Helsinki guidelines for Research on Human Subjects
and was approved by Human Ethic Committees of Fon-
dazione IRCCS Ca’ Granda Ospedale Maggiore Poli-
clinico (Registration number: 1370).
2.2. Instrumentation
HbSSG was measured by Matrix-Assisted Laser-De-
sorption Time-of-Flight (MALDI-ToF) mass spectrome-
try, essentially following the method of Biroccio et al.
(2005). Measurements were performed on an Autoflex
III (Bruker Daltonics) instrument equipped with a 355
nm Smart Beam solid state Nd:YAG UV laser and oper-
ated according to the manufacturer’s directions in the

F. M. RUBINO ET AL. 24
linear positive ion mode at a calculated resolution of ap-
proximately 5000. External calibration of the m/z scale
was accomplished with the manufacturer’s custom pro-
tein mixture. Automated routines for the analysis of a
large number of samples were established with the Flex
Control™ software.
2.3. Measurement Techniques
For the measurement of HbSSG a 2 mL blood sample
was withdrawn (EDTA anticoagulant) and RBC were
immediately separated by centrifugation, washed with
isotonic NaCl solution and aliquoted in 0.1 mL portions
of lysate which were immediately stored at −20˚C prior
to analysis.
The MALDI matrix was composed of an acetonitrile-
water 1:1 mixture saturated at room temperature with
solid sinapinic acid (approximately 10 mg/mL) which was
freshly prepared for each measurement session.
Table 1. Levels of glutathionyl-haemoglobin measured in the control population and in selected patient populations in differ-
ent countries.
Cohort country NaFbage GSSG (%)Techniquedetails Ref.
Normal population Jap 2010 3.7 ± 0.3 ESI-MS [13] Naito , 1999
Normal population I 2010 8 ± 1.8 MALDI-ToFage-matched [15] Piemonte, 2001
Normal population USA 9 1.21 ± 0.14IEX; ESI-MS [16] Al-Abed, 2001
Normal population I 64345.1 ± 2.32.65 ± 1.1MALDI-ToF [12] Pastore, 2003
Normal population I 7 > 5.2 MALDI-ToF101 M, 83 F [14] Biroccio, 2005
Normal population I 57 1.6 - 5.2 MALDI-ToF102 M, 83 F [14] Biroccio, 2005
Normal population I 120 <0.5 (65.2%)MALDI-ToF103 M, 83 F [14] Biroccio, 2005
Normal population In 3054 ± 6.74.85 ± 1.12ESI [17] Sampathkumar, 2005
Control group In 1549 ± 7.63.15±1.99ESI [18] Mandal, 2007
Foresters I 7641 ± 9 2.1 ± 1.7 MALDI-ToF [19] Primavera, 2008
Workers not exposed to butadiene I 4142 ± 7 6.3 ± 2.0 MALDI-ToF [19] Primavera, 2008
Diabetes mellitus Jap 10 10.2 ± 0.8ESI Basal value [20] Naito, 2000
Diabetes mellitus Jap 10 4.1 ± 0.4 ESI After Vit. E [20] Naito, 2000
Diabetes type 2 Jap 3713 7.9 ± 0.5 ESI-MS [21] Niwa, 2000
Diabetes mellitus USA 20 2.26 ± 0.29IEX; ESI-MS [16] Al-Abed, 2001
Diabetes mellitus w/o microangiopathy In 4756 ± 6.88.69 ± 1.24ESI [17] Sampathkumar, 2005
Diabetes mellitus w/ microangiopathy In 5357 ± 8.410.94 ± 0.81ESI [17] Sampathkumar, 2005
Hyperlipemic subjects Jap 1711 8.1 ± 0.8 ESI [21] Niwa, 2000
Haemodialysis patients Jap 10 18.6 ± 0.9ESI Pre-dialysis [13] Naito, 1999
Haemodialysis patients Jap 10 20.8 ± 0.9ESI Post-dialysis [13] Naito, 1999
Hemodialysis group In 1550.1 ± 8.54.69±3.19ESI [18] Mandal, 2007
Peritoneal dialysis group In 1554.2 ± 7.19.99±2.97ESI [18] Mandal, 2007
Transplant group In 1249.1 ± 6.87.63±1.81ESI [18] Mandal, 2007
Heavy smokers I 3045.5 ± 86.4 ± 1.7%MALDI-ToF this study
Obese I 201549.8 ± 13.33.0 ± 0.7%MALDI-ToF this study
Chemical workers exposed to butadiene I 4134 ± 7 7.2 ± 2.8 MALDI-ToF [19] Primavera, 2008
Epileptic patients (PHE-CBZ) Yu 23 2-7 IEX [22] Niketic, 1992
Friedreich’s ataxia I 14913.9 ± 4.115 ± 1.5 MALDI-ToF [15] Piemonte, 2001
Down’s syndrome I 46266.7 ± 2.71.47 ± 0.6MALDI-ToF [12] Pastore, 2003
Carotid stenosis surgery patients I 659 - 83 < 0.5 MALDI-ToFNo HbSSG [23] Ghilardi, 2013
Carotid stenosis surgery patients I 359 - 83 0.5 - 10 MALDI-ToFConstant level [23] Ghilardi, 2013
Carotid stenosis surgery patients I 359 - 83 2 - 9 MALDI-ToFRise and fall [23] Ghilardi, 2013
Notes. a total number of subjects investigated in the study; b number of female subjects. Abbreviations: ESI: electrospray ionization mass spectrometry; IEX:
ion-exchange chromatography; MALDI-ToF: matrix-Assisted Laser Desorption Ionization – Time-of-Flight mass spectrometry.

F. M. RUBINO ET AL. 25
Table 2. Main characteristics of the examined subjects.
smokers N Age
a Smoking (cig/day) a >20 cig/day
M 15 46 (28 - 70) 24 (10 - 40) 5/15
F 15 45 (35 - 59) 23 (20 - 40) 6/15
total 30 45.5 ± 8 (28 - 70) 23.7 ± 6.7 (10 - 50) 11/30
obese Body Mass Index
a
M 5 53 (37 - 67) None 34.5 (33.0 - 37.6)
F 15 44 (26 - 71) 3/15 current 4/15 former 32.9 (27.3 - 44.3)
total 20 51 (26 - 71)
a
median (min-Max).
For sample preparation one portion of frozen separated
RBC was thawed and a 1:100 haemolysate was prepared.
10 microliters of the hemolysate were mixed to an equal
volume of a matrix solution, thoroughly mixed and four
1-microliter portions from the same mixtures were ap-
plied each one in the centre of a different circular spot of
a standard 384-sample ground steel target (Bruker
MTP384 S/N 03229). Evaporation of the solvents was
accomplished in the free air at room temperature. The
manual preparation of one plate with approximately 250
spots (240 sample spots and a number of protein calibra-
tion samples) took approximately 2 hours. Sample plates
were inserted into the mass spectrometer for analysis
within 4 hours from preparation.
For analysis, desorption ionization of sample spots
was accomplished by firing the laser beam according to
an automated routine and accumulating valid spectra
until a suitable signal-to-noise ratio was obtained. Analy-
sis of an individual spot took approximately 2 - 3 min.
Unattended analysis of the whole plate containing the
samples of this study (approx. 250 spots) took approxi-
mately eight hours. After acquisition, MALDI spectra
were analyzed off-site. Spectra annotation and manual
integration was performed by a ‘blind’ operator with the
FlexAnalysis™ software and compared to that obtained
with the operator-independent routine.
Analysis and integration parameters are collected in
Table 3. Results were exported to a custom Microsoft
Excel® worksheet for further elaboration.
2.4. Statistical Elaboration
All calculations, statistical elaborations and graphics
were performed in custom Microsoft Excel® worksheets.
Statistical significance of differences was demonstrated
by two-tailed t-test at a significance level of 5%.
3. Results
3.1. Method Establishment
Due to the large number of samples which need to be
measured in clinically-oriented studies and to the ne-
cessity to perform an operator-independent unbiased
measurement, all stages of the analytical process, start-
ing from sample preparation and deposition, laser fir-
ing and mass spectrometer focussing, automation of
measurement and integration were the object of an op-
timization study and were modified with reference to
the published method by Biroccio et al. [14].
3.2. Instrumental Routine Procedures.
To implement automated spectra processing with the
FlexControl™ software, external calibration of the m/z
scale was accomplished at the beginning of each mea-
surement session with the manufacturer’s custom pro-
tein mixture and internal calibration of the measured
haemoglobin samples was accomplished in each meas-
ured sample by using the signals of doubly- and singly-
protonated homozigous
 - and  -haemoglobin (m/z
7564.2; 7934.2; 15128.4 and 15867.5 respectively).
3.3. Sample Preparation and Measurement
Main modifications to the published procedure in-
cluded using a single-layer of sinapinic acid matrix-
sample mixture, in order (a) to achieve a faster sample
preparation and deposition, (b) to avoid the formation
of a “crusty” matrix lump, thus allowing to use a much
lower laser power, which generates a lower contamina-
tion of the ion source and results in a lower laser con-
sumption. The best compromise was reached by using a
total number of 2500 shots in 50-shot groups, each
fired in a different position of the sample shot, selected
by the automated sample plate movement routine in the
“random walk” mode. As for mass spectrometer focu-
sing, using a very low Post-Ionization Extraction Delay
(PIE) time proved beneficial especially in terms of
resolution of the peak of protonated HbSSG from near-
by “shoulders”, thus allowing to appreciate abundances
lower than 1%.
3.4. Analytical Performance
Following optimization of all parameters, analytical
precision of each sample was calculated on the basis of
four replicate measurements recorded on four different
spot depositions and automatically measured within the
same analytical run. For samples with HbSSG/HbSH

F. M. RUBINO ET AL. 26
Table 3. Parameters for the measurement of glutathionyl haemoglobin with the automated technique in the bruker autoflex
instrument.
AutoXecute method
Laser: Fuzzy control – Parent mode ON Weight 1.00
Use initial laser power on new laser spot Initial laser power 35%
Max laser power 45%
Evaluation: Use masses from 5000 Da to 15000 Da for evaluation and processing.
Accumulation: Fuzzy control: OFF Sum up: 2000 shots in in 100-shot steps
Allow only 100 satisfactory shots per raster spot
Movement: Measure raster: spiral very small
Maximal allowed shot number at one raster position – Parent mode 100
Processing: DEFAULT
MS/MS: DEFAULT
FlexAnalysis - Processing method
Peak detection algorithm Centroid
Signal to noise threshold 6
Relative intensity threshold 0 DEFAULT
Minimum intensity threshold 0 DEFAULT
Maximum number of peaks 50
Peak width 10
Height 80% DEFAULT
Smoothing algorithm Savitzky-Golay
Width 5
Cycles 3
Baseline subtraction TopHat DEFAULT
ratios in the range of 3.5% - 11.7% was usually of
0.4% (<0.1% - 1.1%).
3.5. Reliability of Glutathionyl-Haemoglobin
Measurement
Two possible causes of measurement bias were evalu-
ated: (a) generation of HbSSG by RBC autoxidation
after blood withdrawal, especially under conditions
when separation of RBCs from whole blood had to be
delayed by as long as 6 hours or strict temperature
control at +4˚C could not be warranted over the same
time and (b) decomposition of HbSSG following sam-
ple storage.
To check whether HbSSG could be generated after
blood sampling from the investigated subjects and prior
to analysis, new aliquots of washed and packed RBCs
of some control subjects coming from other similar
studies, which had yielded no detectable levels of
HbSSG, were re-analyzed for confirmation immedi-
ately after preparation and after being kept at ambient
temperature for 12 - 16 hours. None of the examined
samples showed formation of HbSSG either in the first
or in the second analytical run, thus out-ruling its arte-
fact formation as a consequence of sample processing
and storage.
To check whether HbSSG may decompose upon
sample storage, washed and packed RBC samples with
initial HbSSG levels between 2 and 8% coming from
this and from other similar studies were prepared and
re-analyzed along with the samples of the current study.
HbSSG levels in washed and packed RBCs do not
change significantly when samples are stored at −20 to
−80˚C for at least six months. These results, as a whole,
demonstrate that measurement of HbSSG in RBC
haemolysates by MALDI-ToF mass spectrometry is
able to reliably detect event-related changes of 0.5 to
0.8% of absolute values.
The MALDI mass spectra of haemoglobin samples
obtained from subjects with no detectable (upper panel)
and high (lower panel) levels of haemoglobin glu-
tathionylation are shown in Figure 1.

F. M. RUBINO ET AL. 27
The upper spectrum, with no detectable HbSSG sig-
nal (subject not included in this study), is only shown
to the purpose of exemplifying the detection level of
the employed analytical technique. The subject yield-
ing the lower spectrum exemplifies the levels measured
in the investigated population. Analytical precision
(SD of four replicate measurements recorded on four
different spot depositions of the same sample and
automatically measured within the same analytical run)
was of 0.9% (0.1% - 4.5%) for samples with HbSSG/
HbSH ratios in the range of 3.5% - 11.7%.
3.6. Glutathionyl-Haemoglobin Levels in
Smokers
Mean HbSSG of the entire group of the 30 investigated
subjects is 6.2% ± 1.4% (3.5% - 9.1%), with no appar-
ent difference between genders and no significant sta-
tistical correlation with the declared number of ciga-
rettes smoked in the day. However subjects who de-
clared smoking up to 20 cig/day had statistically lower
HbSSG levels than those smoking >20 to 40 cig/day
(5.7% ± 1.1% vs. 7.0% ± 1.4%; p < 0.05; Figure 2).
HbSSG levels showed no correlation with haemo-
globin levels, measured either as concentration (in g/dL)
or as haematocrit.
3.7. Glutathionyl-Haemoglobin Levels in
Overweight-Obese Subjects
Mean HbSSG of the entire group of 20 investigated
subjects is 3.0% ± 0.8% (1.9% - 4.6%), with no appar-
ent difference between genders and no significant sta-
tistical correlation with the BMI calculated from height
and weight recorded at the time of first presentation to
the outpatient clinic. HbSSG levels showed no correla-
tion with haemoglobin levels, measured either as con-
centration (in g/dL) or as haematocrit.
4. Discussion and Conclusions
To carry large studies on oxidative stress which involve
the measurement of glutathionyl-haemoglobin in human
red blood cells, a method that uses MALDI-ToF mass
spectrometry is set up and the main factors of analytical
variability are studied. By fine-tuning sample depo-
Figure 1. MALDI-ToF mass spectra of 1:100 haemolysates of human subjects (for measurement conditions, see Experimen-
al). t

F. M. RUBINO ET AL. 28
sition on the MALDI target and ionization and detection for human transfusion is tested by measuring HbSSG on
conditions, analytical precision reached 0.9% (0.1% -
4.5%) for samples with HbSSG/HbSH ratios in the range
of 3.5% - 11.7% and sensitivity allowed to detect a frac-
tion of HbSSG as low as 0.5%. The stability of HbSSG
in red blood cells demonstrated to be sufficiently high for
all our study purposes. Neither generation of HbSSG by
autoxidation of RBC content, nor its degradation oc-
curred even in not-so-strict sample preparation and stor-
age conditions, in order that only standard separation and
washing of RBC was necessary. Further confirmation of
this observation comes from a contemporary running
study where the stability of red blood cell concentrates
the day of blood donation and after 42 days of storage at
+4˚C [27] (Carpani et al., 2011).
To better frame within the body of existing data the
results from our investigated population of otherwise
healthy subjects, the literature data which have been col-
lected in Table 1 have been used to generate the histo-
gram of Figure 3. Bars show the variability (min-Max)
of the levels of HbSSG measured for different examined
subpopulations (healthy or control groups of clinical stu-
dies; subjects with different conditions or diseases), in-
cluding those of this study.
The levels measured in our study of otherwise healthy
cig / day
0%
2%
4%
6%
8%
10%
5 10152025303540 cig/day
HbSSG
(%)
45
>20
=20
5. 7 ?1.1 %
7.0 ?1.4 %
5.7 ± 1.1%
7.0 ± 1.4%
Figure 2. Levels of glutathionyl haemoglobin in smoking subjects with different self-assessed mean daily cigarette consump-
tion.
0
5
10
15
20
25
30
I I I I I I IInInInJapUSAI I IJapJapJapUSAInInJapJapJapInInInInYuI I
control groups
of clinical and
population studies
HbSSG
Hb (%)
heavy
smokers
(this study)
obese
subjects
(this study)
Chemical
workers
exposed to
butadiene
diabetes
kidney
disease
hyperlipaemia
others
clinical conditions
Figure 3. Comparison of HbSSG levels measured in the literature studies (interval as determined by the reported results o
Table 1). The Country where the studies were performed are reported in the horizontal axis. f

F. M. RUBINO ET AL. 29
groups of heavy smokers and of obese subjects are best
ompared with those measured in the general Italian
Wesseling and E. F. M. Wouters, “Systemic Effects of
Smoking,” Chest, Vol. 1
c
population by Biroccio et al. [14]. The mean values
measured in our subjects place in the upper 3% of the
investigated group (two-thirds of the population <0.5%;
10th - 90th percentile of the remaining 1.6% - 5.2%). It is
also apparent that the values measured in the Italian
population are much lower than those reported as be-
longing to the “control groups” of the several studies
performed in Indian and Far-Eastern populations.
The values measured are also lower than those meas-
ured in dialyzed (5% - 20%; weighted mean 11.4%) [13,
18] and diabetic (5% - 11%; weighted mean 8.3%) [16,
17,20] subjects and, for the group of smokers, approxi-
mately in the same range (7.2% ± 2.8%) of healthy sub-
jects who are occupationally exposed to butadiene in
chemical plants at airborne concentrations in the range
0.1 - 200 μg/m3 [19].
To the best of our knowledge, this is the first study
measuring HbSSG in a group of otherwise healthy sub-
jects such as heavy smokers and obese, showing that
these subjects have higher levels than those reported by
others in a healthy population [14]. In particular, the ef-
fect of heavy tobacco smoking in generating systemic
oxidative stress as measured by glutathionyl haemoglo-
bin is similar to that of a health-threatening degenerative
disease such as diabetes and only two-fold less than that
experienced by kidney patients undergoing life-saving
haemo- or peritoneal dialysis. Glutathionyl-haemoglobin
thus shows the effect of oxidative stress associated to
tobacco smoking, and it is likely that this innovative
biomarker may be useful to assess the extent of damage
caused by the harmful effects of tobacco addiction and to
monitor the health improvement associated to smoke
cessation interventions. As for the effect on oxidative
stress of overweight and treatment-refractory obesity, a
low yet fairly constant level of HbSSG may strengthen
the hypothesis that atherosclerotic plaque is itself a pro-
ducer of diffusible oxidant species [6] which are able to
cross the erythrocyte membrane and oxidize the red
blood cell thiolome [28], thus causing the production of
disulfide and mixed disulfide species.
5. Acknowledgements
We are grateful to the Director and Staff of the Centro
[1] D. G. Yanbaeva, M. A. Dentener, E. C. Creutzberg,
31, No. 5, 2007, pp. 1557-1566.
doi:10.1378/chest.06-2179
[2] L. F. Van GalE. De Block, “Me-
chanisms Linkvascular Disease,”
Interdipartimentale Grandi Apparecchiature (CIGA) of
Università degli Studi di Milano for access to the Bruker
Autoflex III MALDI mass spectrometer. The assistance
of Marco Pitton, BSc, in performing measurements and
data analysis is gratefully acknowledged.
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