J. Human Ergol.

, 38: 51-65, 2009

G-6-PD LEVEL AND SURFACE NANOSCOPY: A NOVEL

APPROACH IN ERGONOMIC STRESS MANAGEMENT OF
FEMALE LABOURS IN BENGAL SUBURBS PERFORMING
MANUAL MATERIAL HANDLING
SUBRATA GHOSH1*, MUKTISH ACHARYYA2 AND ANANDI BAGCHI3
1
Department of Physiology Presidency College, 86/1, College Street, Kolkata 700073, West Bengal, India
*E-mail: subgh64@yahoo.co.in
2
Department of Physics, Presidency College, 86/1, College Street, Kolkata 700073, West Bengal, India
3
Department of Physiology, Hooghly Mohsin College, Chinsurah 712101, West Bengal, India

Strenous physical exercise like professional load bearing often produces oxidative stress,
increasing post exercise Malondialdehyde (MDA) levels. To quantify the cellular dimen-
sion/profile of the said stress, nanoscopic observation of the erythrocyte surface was made
by Atomic Forced Microscopy (AFM)/Lateral Forced Microscopy (LFM) and correspond-
ingly the average roughness of the surface was measured. An attempt has been made to
correlate the antioxidant vitamin mixture supplementation, endurance capacity, allied phys-
iological parameters and blood glucose-6-phosphate dehydrogenase (G-6-PD) level and
roughness-MDA correlation and thereby the deduced regression equation as crucial mark-
ers of performance related oxidative stress management in professional female load bear-
ers. Three experimental groups A, B and placebo, each consisting of ten female workers
(18-21 years old), were chosen. Group A was given 400 mg of vitamin E supplementation
daily, while Group B was given a clinical mixture of vitamin E, vitamin C and β-carotene
daily in capsular form for a period of 28 days. The exercise- induced hike in the status of
serum MDA was found to rise less significantly with vitamin supplementation. Further
study showed that the supplementation was instrumental in reducing the basal MDA level.
Endurance capacity, determined by bicycle ergometric method, was increased more signifi-
cantly (p<0.001) in group B than in group A (p<0.01), and first minute recovery heart rate
decreased significantly (p<0.05) in both groups. G-6-PD level was shown to increase more
significantly (p<0.01) with antioxidant vitamin mixture supplementation than with vitamin
E supplementation singly (p<0.05) in professional female load bearers. The regression
equation might be instrumental in early detection of oxidative damage in strenuous exercise
in manual material handling.
Key words: Antioxidant; G-6-PD; stress; MDA; RBC surface roughness; female load-bearers

INTRODUCTION

Strenous exercise is a stress situation itself; this oxidative stress produces reactive oxygen
species (ROS) in the body, which ignite the antioxidant defence within itself (Jenkins, 1993; Sen et
al., 1994). Earlier reports proved that even sub-maximal exercise may elevate the stress indices like
plasma lipid peroxides and higher blood glutathione oxidation (Sen et al., 1994). ROS are known to
have a wide variety of patho-physiological characteristics (Kehrer, 1993). ROS is responsible for
lipid, protein and DNA damage in a major way. Lipid peroxidation produces conjugated dienes, lipid
hydroperoxides and malondialdehyde (MDA). Oxidative modification of DNA and protein also
pathophysiologically alter enzyme and other active protein activity as well as abnormal gene modifi-
cation. All these lead to diseases. However, MDA is often assessed clinically as an indicator parame-
Received 24 July 2008; accepted 17 December 2009.
52 S. GHOSH et al.

ter or marker of oxidative stress. In almost all studies of oxidative stress analysis, MDA status, lipid
peroxide (LOOH ) status, serum superoxide dismutase (SOD), catalase and peroxidase levels are
assessed and it is almost established that higher stress indicates higher MDA status and conditional
alterations in all the antioxidant enzymes (Halliwell and Gutteridge, 1989; Jenkins, 1993; Sen et al.,
1994).
Moreover, ROS may also contribute to fatigue in oxidative skeletal muscles (Barclay and
Hansel, 1991). Recently, a number of studies have been directed towards an exercise-induced oxida-
tive stress management programme. Antioxidant supplementation studies have revealed a beneficial
trend (Jenkins, 1993; Sen et al., 1994; Bagchi et al., 2001).
Earlier studies showed that specific pharmacological stress (cyclosporin administration) resulted
in increased methemoglobin, increased antioxidant enzyme activities and decreased “reduced glu-
tathione”(GSH) concentration and G-6-PD activity in a rat model (Lexis et al., 2005). Earlier find-
ings indicate that oxidative damage (marked by increased MDA level) and loss of membrane asym-
metry in RBC are related to the development of anemia in Theileria sergenti infection (Shiono et al.,
2003). Again, another study indicates that oxidative damage accumulation in plasma has been consid-
ered as a cause of RBC damage and it is reflected in biochemical alterations in MDA level, GSH
level, etc. (Gil et al., 2006).
The specific subjects of the study (i.e. the female load-bearers in the brick field), specifically lift
10–12 bricks (each weighing 5 kg), sequentially on their head, carry them for at least 300 metres and
then unload all the bricks for burning and solidification, in an equally sequential way. The duration of
this job cycle of manual material handling (MMH) is generally 9–10 hours. They generally do not
receive adequate nutrition, with a dull look, working at 36°–42°C, with a relative humidity of
75–80% on an average, and are seriously underpaid. They generally consume 250–300 ml of home-
made alcohol from rice (Hadia) per day, during rest period in the evening (Ghosh et al., 2008).
This study attempts to quantify the “exercise induced oxidative stress” production in the body,
by virtue of assessing “serum malon-dialdehyde level” (MDA) which is an index of stress-induced
lipid peroxidation (Ji, 2000). Moreover, in this particular study, female subjects were only chosen
where estrogen plays a very crucial role as an antioxidant hormone (Halliwell and Gutteridge, 1989).
Serum status of SOD, catalase and peroxidase are already established markers for oxidative damage
(Kasapoglu and Ozben, 2001; Abraham et al., 2005; Halliwell and Gutteridge, 1989). In search of
newer indicators, this study purposefully avoided these enzymatic assays.
Another unique feature of this specific study is observation of RBC surface roughness of every
subject by AFM and LFM. Both the microscopic techniques used belong to scanning probe
microscopy (SPM). SPM technologies share the concept of scanning an extremely sharp tip (3-50 nm
radius of curvature) across the object surface. The atomic force microscope (AFM) or scanning force
microscope (SFM) is a very high-resolution type of scanning probe microscope, with demonstrated
resolution of fractions of a nanometer, more than 1000 times better than the optical diffraction limit.
AFM measures the interaction force between the tip and surface. The tip may be dragged across the
surface, or may vibrate as it moves. The interaction force will depend on the nature of the sample,
the probe tip and the distance between them.
A lateral force microscopy (LFM) is an atomic force microscope with a four quadrant photo
detector, which allows measuring the frictional force acting on the probing tip sliding on a surface
from the torsion of the cantilever beam where the tip is attached. LFM studies are useful for imaging
variations in surface friction that can arise from in homogeneity in surface material, and also for
obtaining edge-enhanced images of any surface.
Since MMH directly demands excess use of oxygen by the body, oxygen carriage and thereby
erythrocyte health/status is compelled to be changed. Additionally, G-6-PD itself is housed within the
RBC. Serum MDA level is also supposed to be an important factor in altering the RBC surface mor-
phodynamics. Thereby quantification of three-dimensional surface roughness in nanometric scale is
instrumental and indicates another parameter of oxidative damage in cellular dimension. Therefore,
the study aims in revealing the effects of antioxidant vitamin supplementation on exhaustive exer-
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 53

cise-induced oxidative stress in females and demands that the deduced regression equation between
serum MDA and RBC surface roughness may be treated as an early detector of damage profile, par-
ticularly in strenous exercise like MMH.
Therefore the study aims to quantify and correlate the stress experienced by the female load
bearers biochemically and nanoscopically. Further the study attempts to judge the effectiveness of
vitamin mixture supplementation in mitigating induced stress and finally to hypothesize the develop-
ment of a regression equation to be used as an early detector of stress.

MATERIALS AND METHODS

Thirty young, physically fit, female load bearers (brick field workers from Nadia District, West
Bengal) coming from comparable socio-economic background, with ages ranging from 18 to 21
years, volunteered for this study. They were debarred from taking extra vitamin supplementation
three months before and during the study.
The individuals selected in the study had normal diet, which consisted of optimum/normal levels
of vitamin E, vitamin C and beta carotene, prior to the survey (about 3 months). So it was assumed
that they had optimum amount of vitamin C uptake in their diet. Moreover, they did not report any
vitamin C deficiency at any stage of their life and Vitamin E deficiency is quite rare in humans
(Moore et al., 1960; Brody, 1999). Further, it may be recapitulated that there are neither any suitable
practical bio-markers to accurately reflect dietary intake or body stores of vitamin E (Gibson, 2005)
nor is there a generally accepted method for determining the vitamin E status in a human being
(Friedrich, 1988). Additionally vitamin E largely in the form of _-tocopherol circulates in the blood
mainly with LDL fraction (Gibson, 2005). On the other hand, it is already established that vitamin E
and vitamin C recycle each other and this characteristic may explain their synergism (Tappel, 1962;
Njus and Kelley, 1991).
Under the circumstances, all the subjects were considered to possess normal levels of vitamin E
and vitamin C. Accordingly, this particular study skipped the assessment of blood vitamin levels of
the subjects. The major objective was to see whether additional antioxidant supplementation would
be capable of restoring RBC surface health or not. None of these subjects had any history of chronic
cardiac and respiratory diseases, abnormal lipid profile or correlated ailments.
Before starting the experiment, they were familiarized with the bicycle ergometer in the labora-
tory. The entire study was performed with the permission of the government authority, brick field
owner and all the subjects, explaining the objectives and probable impact of the work. All the ethical
formalities were performed in writing and written consent of all the subjects was also obtained. All
the subjects were brought to the laboratory to explain the total experimental procedure, and they were
informed about the objectives of the study and about the probable outcomes of the vitamin supple-
mentation in a lucid way and thereby they unanimously agreed to volunteer for the study. All the
workers were supplied with the required amount of vitamin capsules during the entire experimental
tenure.
Bicycle ergometers have distinct advantages for exercise stress testing among the various stress
test protocols. Power output on the ergometer is independent of the person’s body mass and is easily
calculated and regulated. With the weight-loaded friction-type magnetic brake bicycle ergometer
power output is directly related to the frictional resistance and rate of pedalling. Moreover, from two
different points of view, namely “economy of movement” and “mechanical efficiency”, the opti-
mized stress test protocol always prefers bicycle ergometry amongst others.
In the present study, daily load work of subjects in stressful environment was observed by
“polar heartbeat monitor”. The subjects were then asked to exercise on the bicycle ergometer in the
same environment within the laboratory and with adjustable work load, so as to produce the standard-
ised heart rate picture as observed in the field by polar monitor earlier. The process of work load
standardization is best possible by bicycle ergometry (at least in this part of the world).
54 S. GHOSH et al.

A double-blind study was carried out whereby the subjects were divided into three groups. Two
groups each of ten subjects were randomly assigned for experimental group A and experimental
group B, who were given 400 mg of pure vitamin E only and a mixture of vitamin E, vitamin C and
β-carotene respectively for a period of 21 days. Experimental group C consisting of 10 female sub-
jects of the placebo group has also been studied. Peppermint lozenges were given to the placebo
group daily for 21 days. All the subjects continued to work as usual during the entire experimental
period of 21 days.
Each of the subjects was allowed to take rest for a minimum of half an hour after coming to the
laboratory, i.e. before the onset of any exercise or supplementation; at the end of which pre-exercise
heart rate and physical parameters (height, weight, etc.) were measured.
Endurance capacity (min) of each subject was determined by exercising them on a magnetic
brake bicycle ergometer with a moderate workload of 720 kgm.min-1, until exhaustion. At the end of
the exercise, peak heart rate, recovery heart rate up to 30 min of recovery period were recorded. The
entire procedure was repeated on each subject before and after vitamin supplementation.
Blood was taken before and after the endurance exercise for the determination of haemoglobin
(gm %) by cyanomethemoglobin method (Harold et al., 1980). The level of serum lipid peroxidation
was estimated in terms of serum MDA by the method of Yagi, (1984), using thiobarbituric acid
(TBA). Assessment of glucose-6-phosphate dehydrogenase was also carried out with the blood
drawn after exercise by UK-Kinetic Method (Kachmar and Moss, 1976), which is a reagent kit for
the quantitative estimation of G-6-PD in erythrocytes. The enzyme G-6-PD present in the RBC is
extracted by lysing the cells using a natural detergent. The extracted enzyme oxidises glucose-6-
phosphate to 6-phosphogluconate and simultaneously reduces co-enzyme NADP to NADPH giving
increase in absorbance at 340 nm.

The general parameters for estimation of G-6-PD include:

Type of reaction Kinetic
Wavelength 340 nm
Flowcell temperature 30°C
Delay time 180 seconds
Interval 60 seconds
Number of reading 4
Sample volume 25 microlitres (0.025 ml)
Working reagent volume 1.0 ml
Zero setting with Distilled Water
Light path 1.0 cm

The results can be calculated as follows:

G-6-PD Activity (U/g Hb) = ∆ A/min × 224×100

6.22×Hb
The results can be calculated as follows:

G-6-PD Activity (U/g Hb) = ∆ A/min × 224×100

6.22×Hb
where, ∆ A/min = Change in absorbance per minute
100 = Factor to convert to 100ml
224 = Total assay volume to sample volume
6.22 = Millimolar absorptivity of NADPH at 340 nm
Hb = Haemoglobin concentration (g/dl)

A thin blood film of every subject at resting condition was prepared on 20 square mm clean
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 55

glass slide for AFM. The RBCs of every individual were categorically observed in LFM as well, by
di Nanoscope, Version 4.10.01. The contact mode AFM in the digital instrument nanoscope SPM
system was employed. All the images captured were thereby analysed by di Nanoscope Command
Reference Manual, Version 4.10.01, for surface roughness (in root mean square (RMS) and average
scale). The image statistics are also being given in performing roughness analysis by the specific soft-
ware system mentioned. From the AFM Reference Manual the RMS (Rq) is the standard deviation of
the Z-values (the vertical range of the image corresponding to the full extent of the colour table)
within the given area and is calculated as:

Rq = R (F (Σ (Zi-Zave)2,N)). (1)

where, Zave is the average of the Z values within the given area
Zi is the current Z value
N is the number of points within the given area.

Earlier, structure of the erythrocyte membrane and its cytoskeleton by atomic force microscopy
had been studied by many scientists (Nowakowski et al., 2001; Takeuchi et al., 1998; Iwamoto and
Wakayama, 1997), either from the points of membrane-protein deformation, cytoskeletal deformity,
immobilized membranes or from viscoelastic nature and behaviour of the cell membrane. Model
dependence of AFM simulations in non-contact mode AFM and structural studies of ordered mono-
layers of RBC using atomic force microscopy had also been performed (Sokolov et al., 2000; Peachy
et. al,1994.). The present study, however, reports for the first time, the correlation between stress pro-
file and erythrocyte surface roughness as observed and calculated by AFM and corresponding
nanoscopic software system.
The variation of MDA with the damage of an RBC was studied. The damage of an RBC is
defined in a tricky way. From the microscopic observation it was noticed that an RBC surface
becomes more corrugated or rough when the cell experiences stressful situations. This prompted us to
measure the damage of an RBC by its surface roughness. This measure automatically provides the
zero roughness for a smooth surface of an RBC. The normal Height, hi, at any point on the surface of
an RBC is measured from any arbitrary level. Collecting all data of hi (depending on the location on
–
the surface), the average of these heights is calculated, h. Now the RMS (Root Mean Square) rough-
ness, r, is defined as follows:

(2)

where, N is the total number of data and i runs from 1 to N

This r now gives the right choice of the variables. The MDA status was studied as a function of r
and shown in fig 3. From a very careful inspection, we decided to fit the variations by least square fit,
which gives the linear variations like y=mx+c. where slope, m and intercept, c are given as:

(3)

and (4)

where, i runs from 1 to N.

The measurement of roughness (RMS) is invariant under choice of arbitrary level. So we

believe this is a very robust quantity. That is why this parameter is chosen for analysis (Gerald and
Wheatley, 2006).The correlation between the erythrocyte surface roughness and MDA status of the
56 S. GHOSH et al.

blood was plotted in best square fit curve and the regression equation was thereby developed. The
room temperature varied between 20°C-24°C and the relative humidity was about 78%.
Statistical analysis was done using a two-tail‘t’ test by difference method. The Student’s t-test
for independent sample was used to test differences between different groups regarding serum MDA
level, blood G-6-PD level and endurance capacity. For these variables within-group differences from
baseline to week 3 were evaluated using Student’s t-test for matched-pair samples. When associa-
tions between antioxidant supplementation and the variables were evaluated, we used the baseline
levels of all the study subjects.The statistical analyses were performed using SPSS for Windows, ver-
sion 11.0.1. A p-value of <0.05 was considered statistically significant.

RESULTS

Studies on thirty female load bearers had shown that after 400 mg of vitamin E supplementation
(Group A), the mean endurance capacity (min) rose from 13.05 ± 4.37 to 20.01 ± 4.60, while after
supplementation of a mixture of vitamin E, vitamin C and β-carotene (Group B), the value increased
from 13.86 ± 3.30 to 23.27 ± 1.91 (Table 1, Table 2). It is thus seen that the antioxidant mixture pro-
duces a much more significant (p<0.001) enhancement in endurance capacity as compared to vitamin
E supplementation singly (p<0.01). In group C (placebo group) there was, however, no change.

Table 1. Physical characteristics of female participants.

Body surface area

Group Age (years) Height (cm) Weight (Kg)
(square metres)
Group A (N=10) 19.05 ± 0.48 152.13 ± 7.32 47.16 ± 8.20 1.34 ± 0.10
Group B (N=10) 19.61 ± 0.31 156.28 ± 6.67 48.23 ± 6.41 1.48 ± 0.36
Placebo (N=10) 19.98 ± 0.22 160.00 ± 15.38 45.65 ± 7.20 1.41 ± 0.63
Va lues are mean ± SD

Inter-group comparison with respect to all the four parameters, viz., Age, Height, Weight and
body surface area have been made (by t-test) and each was found to be statistically insignificant. The
differences between the mean values were not statistically significant.

Table 2. Endurance capacity (mins) of females before and after antioxidant vitamin supplementation.
Endurance capacity (mins)
Experimental group Before After Level of significance
supplementation supplementation
Group A 13.05 ± 4.37 20.01 ± 4.60 Significant (p<0.01)
Group B 13.86 ± 3.30 23.27 ± 1.91 Highly significant (p<0.00l)
Placebo 13.02 ± 1.03 13.04 ± 0.91 Not significant
Va lues are mean ± SD

Recovery heart rate recorded at the first minute of recovery period showed significant (p<0.05)
decrease in both experimental groups A and B (Table 3). Recovery heart rate after exercise serves as
an effective indicator of physical fitness; faster the recovery rate, higher the physical fitness.
Antioxidant vitamin supplementation produces a faster recovery immediately after exercise. Thus
antioxidant vitamin supplementation increases the physical fitness of the subjects. Antioxidant vita-
min supplementation showed non-significant effects on resting heart rate, peak heart rate and haemo-
globin concentration (Table 4).
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 57

Table 3. First minute recovery heart rate (beats min-1) of females before and after antioxidant vitamin supple-
mentation.
First minute recovery heart rate (beats min 1)
Experimental
Before Level of significance
group After supplementation
supplementation
Group A 157 ± 2 15 2 ± 3 Significant (p<0.05)
Group B 153 ± 3 14 8 ± 7 Significant (p<0.05)
Placebo 153 ± 1 15 5 ± 1 Not significant
Va lues are mean ± SD

Table 4. Haemoglobin concentration [gm %] of females before and after antioxidant vitamin supplementation.
Haemoglobin concentration (gm %)
Experimental group Before After Level of significance
supplementation supplementation
Group A 11.6 ± 0.6 11.9 ± 0.8 Not significant
Group B 12.7 ± 0.4 12.1 ± 0.7 Not significant
Placebo 12.0 ± 0.3 11.9 ± 0.6 Not significant
Va lues are mean ± SD

Serum MDA level, which is a marker of lipid peroxidation, showed a significant rise after every
bout of exercise (p<0.001). However, antioxidant vitamin mixture supplementation reduced the basal
status of “serum lipid peroxidation” (p<0.001). On the other hand, it is observed that vitamin E sup-
plementation alone failed to reduce the MDA status. Another noticeable observation of the study is
that the effect of antioxidant vitamin supplementation (both mixture and vitamin E alone) ameliorat-
ed the exercise induced increase in serum MDA level (Table 5, Fig. 1).
In placebo group C, however, no such post supplemental changes were observed. G-6-PD level
showed a more significant (p<0.001) increase after antioxidant vitamin mixture supplementation as
compared to supplementation of vitamin E alone (p<0.05) G-6-PD level increased from 9.36 ± 0.74
to 10.36 ± 0.82 in group A while the value increased from 9.63 ± 0.78 to 10.64 ± 0.54 in experimen-
tal group B. In placebo group C, however, there is no change (Figure 2).
Since all the values of group A, group B and placebo group C are found to be comparable and
are not suffering from group-specific bias after statistical treatment, as depicted from Table 1 to
Table 5, it may be inferred that the differences observed after supplementation are exclusively due to
the vitamin management.
The average RBC surface roughness of every individual was also quantitatively measured using
the drawn blood at pre-exercise level in nanometric scale, irrespective of any group. The roughness
data was correlated with the respective MDA level and the regression equation thus derived has been
graphically represented in Figure 3. The equation developed is Y=0.1016X + 30.4617.

Table 5. Level of significance of changes in serum lipid peroxidation in females.

BS AS
Pre-exercise Post-exercise
Experimental group Pre-exercise vs Post- Pre-exercise vs
BS vs AS BS vs AS
exercise Post-exercise
Group A NS p<0.001 p<0.001 NS
Group B p<0.01 p<0.001 p<0.001 NS
Placebo NS NS p<0.001 p<0.001
The values of mean ± SD for each of the three groups in four different conditions have been mentioned
in the Table 5. BS; Before supplementation, AS; After supplementation, NS; Not significant.
58 S. GHOSH et al.

Fig. 1. Serum lipid peroxidation levels (K × 10-1 nmol/ml of serum)

of females before and after antioxidant vitamin supplementation.
BS1 : Before Supplementation Pre-Exercise Values,
BS2 : Before Supplementation Post-Exercise Values,
AS1 : After Supplementation Pre-Exercise Values,
AS2 : After Supplementation Post-Exercise Values,
Vertical bar : SD

Fig. 2. Glucose-6-phosphate dehydrogenase (G-6-PD) (U/gm Hb)

of females before and after antioxidant vitamin supplementation.
BS : Before Supplementation values,
AS : After Supplementation values,
Vertical bar : SD
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 59

Regression Analysis: MDA versus RMS Roughness

The regression equation is
MDA = 30.5 + 0.102 RMS Roughness

Predictor Coef SE Coef T P

Constant 30.4617 0.4968 61.31 0.000
RMS Roug 0.10160 0.01084 9.37 0.000
S = 0.9250 R-Sq = 88.0% R-Sq(adj) = 87.0%

Apparently the regression equation deducted, signifies that MDA level increases with RMS
roughness of the RBC surface linearly with a slope of 0.102 and intercept 30.5. For zero RMS rough-
ness value, MDA has a residual value of 30.5 and for unit RMS roughness value, the value of MDA
becomes 30.5 + 0.102 = 30.602. However, the clinical significance of this equation is instrumental.
Whenever the average RMS roughness value of a person is evaluated, immediately the average blood
MDA level – the stress parameter, may be predicted from this equation. This is highly significant for
the early detection of stress related diseases.

Fig. 3. The graphical representation of the linear relationship

between the RBC roughness (RMS) and the respective MDA sta-
tus. The black bullets represent the actual experimental data
(n=14). The Continuous straight line represents the least square
straight line fit of RMS roughness versus the MDA-level data.
r=0.938 (p<0.01).

In Figure 4, the image of an apparently healthy RBC with lesser damage profile has been moni-
tored in AFM and by means of LFM the quantitative surface roughness of the cell has been analysed
with a specific analytical software system. In Figure 5, the image of an apparently damaged RBC has
been monitored in AFM and by means of LFM the quantitative surface roughness of the cell has been
analysed with a specific analytical software system.

DISCUSSION

Mitochondria, golgi apparatus and RNA are extruded from the reticulocyte in the process of
maturation to form the erythrocyte and therefore erythrcytes are not living cells in the strict sense
(Nan Pilsum, 1986). RBC cannot synthesize protein, glycogen and lipids from glucose and are inca-
60 S. GHOSH et al.

Image Statistics: Roughness Analysis;

Image Z range : 161.12 nm
Image Rms (Rq): 29.567 nm

Fig. 4. AFM (left) and its corresponding LFM (right) image of the RBC
showing lesser surface damage, where the roughness of the cell has been
analysed by di Nanoscope Command Reference Manual, Version 4.10.01
software. Scale bar = 1µm.

Image Statistics: Roughness Analysis;

Image Z range : 166.1 nm
Image Rms (Rq): 301.50 nm

Fig. 5. AFM (left) and its corresponding LFM (right) image of the RBC
showing greater surface damage, where the roughness of the cell has been
analysed by di Nanoscope Command Reference Manual, Version 4.10.01
software. Scale bar = 1µm.

pable of oxidative metabolism, but it can act as a perfect indicator of oxidative damage of the body as
a whole (Sotirakopoulos et al., 2004). Contact mode AFM and LFM analysis for surface roughness
assessment is thereby considered as an essential eye-opener of stress-related damages, particularly
reflected by deterioration of performance.
The ability of the AFM to create three-dimensional micrographs with resolution down to the
nanometre and Angstrom scales has made it an essential tool for imaging surfaces in applications
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 61

ranging from semiconductor processing to cell biology (Tao et al., 1992). In addition to this topo-
graphical imaging, however, the AFM can also probe nanomechanical and other fundamental proper-
ties of sample surfaces.
Earlier Erythrocyte membrane skeleton were studied and observed by AFM (Takeuchi et al.,
1998) where ghost membrane skeleton were extensively studied. Further, we would like to mention
that no such correlation has been made earlier; however, holes on erythrocyte membrane and its
roughness contour imaged by atomic forced microscopy and lateral forced microscopy – have been
performed elsewhere (Guha et al., 2002). Structure of RBC membrane skeleton with pits of depres-
sions was also observed by AFM. But on the same group of subjects, average RBC surface roughness
analysis before and after vitamin supplementation and their comparison in-between, may be a newer
mode of observation bearing clinical significance.
Structural studies of human erythrocytes and corresponding membranes (erythrocyte ghosts and
inside-out vesicles) immobilized by centrifugation under aqueous/physiological conditions using
atomic force microscopy (AFM) were described (Iwamoto and Wakayama, 1997). Erythrocytes were
imaged under physiological conditions by atomic force microscopy where differences in both the
global properties of the cells and in the local features in cytoskeleton structure had been observed
(Nowakowski et al., 2001). Calcium-dependent human erythrocyte cytoskeleton stability analysis
through atomic force microscopy has also been performed where cytoskeleton morphology were
studied (Liu et al., 2005).
The measured surface roughness from the study is thereby considered as a probable impact of
multitudes of stress profiles and so its correlation with MDA, showed a significantly linear status as
evidenced by the specific regression equation (Figure 3). This may be considered as instrumental in
early detection of stress on individual for taking an immediate preventive measure. The detailed
mathematical modelling of all other relevant biochemical parameters and their variations with surface
roughness is under study and will be published elsewhere.
We hypothesize that AFM image of average RBC (more than 50%) is a powerful technique/pro-
tocol for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.
The entire study and the result are in tune with the finding of recent neurological study (D’Agostino
et al.,2009). In Figure 4 and Figure 5, the surface roughness of one healthier RBC and one compara-
tively damaged RBC are highlighted.
Antioxidants like vitamin E, vitamin C and glutathione (GSH) are known to act synergistically
(Constantinescu et al., 1993; Sen et al., 1994) in the form of antioxidant chain reaction (Sen et al.,
1994). Lipophilic vitamin E is a major lipid peroxidation chain breaking antioxidant. The water-solu-
ble antioxidants like ascorbate and GSH may be involved in regenerating α-tocopherol from its radi-
cal by-products like tocopherol 0, etc. (Constantinescu et al., 1993). In plasma, ascorbate (vitamin C)
is a potent antioxidant, the first to be depleted on exposure to peroxyl radicals and other types of
oxidative stress (Frei et al., 1988). The study is a unique one for supplementing β-carotene, along
with vitamin E and vitamin C, where β-carotene actively scavenges and deactivates free radicals both
in vitro and in vivo. Additionally it acts as a single oxygen quencher.
We would like to highlight that as it has been proved that vitamin E supplementation prevents
membrane lipid peroxidation (Halliwell and Gutteridge, 1989); in the study situation, the vitamin E
and Vitamin C mixture may produce some changes in the extracellular as well as intracellular envi-
ronment in RBC, so that early deformed membranes are being recovered/protected from further
oxidative deformations. We hypothesize that the specific regression line showing the correlation
would only be applicable in the specific class of subjects studied and not on other communities.
Regarding the exercise-induced oxidative stress management, damage-profile assessed at pre-
and post-supplemented levels in the form of serum MDA level, the study showed that vitamin sup-
plementation significantly decreased the resting MDA levels as well as post exercise MDA levels.
Vitamin mixture supplementation showed that there is no significant rise in post-exercise level com-
pared to its pre-exercise value. The mixture supplementation is found to decrease the exercise-
induced lipid peroxidation better than vitamin E supplementation alone.
62 S. GHOSH et al.

G-6-PD is the rate-limiting enzyme in the first step of the Pentose Phosphate Pathway, which is
essential for the production of NADPH in the red blood cells for the recycling of endogenous antioxi-
dant GSH (Jacobasch and Rapoport, 1996). Some of those NADPH importantly neutralise hydrogen
peroxide, glutathione and other organic peroxides produced in the oxidative stress (Stryer, 1995).
This information makes the G-6-PD level an even more important marker for post-exercise oxidative
stress management system, particularly for females, where the natural antioxidant hormone, estrogen,
is acting. This is the first time that such an intervention has been used for the study of exercise-
induced-oxidative stress. The head is held high even with a 50 kg load after the intervention with
vitamin supplementation. The summary of the cumulative impact of additional burden is highlighted
from the following figure (Figure 6).

RWL: Recommended Weight Limit,

NIOSH: National Institute for Occupational Safety and Health.

Fig. 6. The probable stress factors and its early management. The cumulative
impact of the various socio-economic stressors, malnutrition, environmental
stressors and occupational hazards lead to additional strain and early fatigue,
which is probably reflected by an increased surface roughness of the RBC.
 ERGONOMIC STRESS OF FEMALES MEASURED BY BLOOD G-6-PD AND SURFACE ROUGHNESS 63

Our hypothesis that endogenous whole blood G-6-PD status is crucially important in protecting
against exercise-induced oxidative stress, at least in females and thus in influencing endurance to
exhaustive exercise, was tested using the comparative data between the pre-supplementation and
post-supplementation of vitamin mixture. These experiments show that dietary supplementation with
the antioxidant vitamin mixture enhanced the erythrocyte antioxidant defence and thus may be bene-
ficial in oxidative stress management (Lexis et al., 2006).

CONCLUSIONS

Antioxidant vitamins help to act as an effective protocol in “exercise-induced oxidative stress

management” on working females, specifically by increasing G-6-PD level and thus play a permis-
sive role in the enhancement of endurance of trained females. Antioxidant vitamin mixture (vitamin
E, vitamin C and β-carotene) appears to be more effective over vitamin E supplementation singly in
increasing G-6-PD level.
It may be hypothesized that the antioxidant activity within the erythrocytes may be related to
changing level of oxidative stress within the RBC, as evident from the levels of G-6-PD itself.
Malnutrition and vitamin supplementation may be the regulating feature for such activity. Further
research is necessary in this exciting area. Antioxidant vitamins, especially antioxidant vitamin mix-
ture, effectively reduces the stress level of the female load bearers as evidenced by a reduction in the
basal status of lipid peroxidation (serum MDA level) and thus indicating a novel correlation between
antioxidants, G-6-PD status and serum MDA level – to be considered as a new horizon in the field of
“Stress Management”. However, the clinical setting of these beneficial findings has not yet been
established. Nanoscopic analysis of RBC surface roughness of all the subjects and formulation of
regression equation correlating with the corresponding MDA status may be treated as a unique model
for early detection of stress related damages. The linear relation would be highly significant from the
application point of view.
As a recommendation, the probable beneficial effect of antioxidant vitamin supplementation at a
regular basis has been recommended for the studied female brick field workers, to the employer and
Government authorities.

ACKNOWLEDGEMENTS

This work would not be possible without the active co-operation of Prof. H.N.Bhattacharya,
Head, Department of Applied Geology and Environmental System Management, Presidency College,
Kolkata, Dr. S. Chakraborty, Co-ordinator, Environmental System Management, Presidency College,
Kolkata for providing infra-structural facility and of Sri Biswajit Roy, Department of Statistics,
Presidency College, Kolkata, for statistical analysis. We sincerely acknowledge Dr. V.Ganeshan and
Ms. Deepti Jain, Low Temperature Laboratory, UGC-DAE Consortium, Indore, for providing AFM
and analytical facilities.

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