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Der Pharmacia Lettre, 2016, 8 (2):169-179

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ISSN 0975-5071
USA CODEN: DPLEB4

Relative antioxidant efficacy of α-tocopherol and ascorbic acid on

blood lead, hemoglobin and hematocrit level of lead-exposed
Rattus norvegicus (albino rat)
Xhyrel June Jimenez Tagayloa, Nikko Laurenz Guarde Franciaa and Marlon C. Parejab
a
De La Salle University – Dasmariñas, Dasmariñas City, Cavite, Philippines
b
College of Science and Computer Studies, Biological Sciences Department, De La Salle University – Dasmariñas,
Dasmariñas City, Cavite, Philippines
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ABSTRACT

The relative efficacy of water soluble ascorbic acid and lipid soluble α-tocopherol on hematoxicity caused by lead
exposure were observed in rats. The experimental groups were given orally of 3IU antioxidant treatment as vitamin
C, vitamin E plus C, and vitamin E, while lead was injected subcutaneously. Blood lead, hemoglobin, and
hematocrit level with the supporting red blood cell count were measured as indicators to assess the efficacy and
synergistic competence of the antioxidants against lead. Solution containing 1/40 sub-lethal dose lead acetate
induced subcutaneously to rats for four wks produced harmful changes in the blood. These harmful effects, however,
were lessened by antioxidant treatments. The results showed that vitamin E has better potency as compared to
vitamin C as well as there is an apparent evidence of synergism between the vitamins. Although the doses were
halved on vitamin E plus C treatment, still, the combination was successful in stabilizing the hemoglobin and
hematocrit levels to normal with similar efficacy as the complete 3IU vitamin E treatment. The suggested
reinforcing antioxidant effect of ascorbic acid is the capacity to regenerate the active form of Vitamin E after it has
reacted with lead.

Key words: α-Tocopherol, Ascorbic acid, Antioxidant efficacy, Hematoxicity, Synergism

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INTRODUCTION

Greek physicians made the first clinical manifestation of lead poisoning in the first century B.C.E., and it is
controversially the earliest known industrial pollutant [1]. Today, lead is known worldwide because of its toxic
property, that its effect is primarily marked in the blood vessels which can cause secondary defects on organs [2].
Previously, in the year 2007, China’s toy products with lead containing paint were recalled and had been a global
issue [3]. Consequently, many researches open making lead as one of the highlighted heavy metal since its toxicity
bounds no age or gender [4]. In addition, many recent interpretations [5-8] show the protective function of
antioxidants against lead toxicity but, up to date, there is still no concrete study regarding what type of vitamin is the
most potent antioxidant in the blood and which antioxidant property is the best aid factor for heavy metal chelation
and detoxification.

In the Philippines, susceptible sources of lead include gasoline, soil, paint, house materials, and even canned goods
[9]. Though the leaded gasoline was phased out in the year 2000, still children have considerable blood lead levels

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and such is considered a public health concern [10]. Not all have the capacity to tolerate toxicity at higher amount
of exposure especially children below 6 years of age as their bodies absorb lead at a higher rate [11]. Other risks for
lead poisoning include pregnant women, people who work with lead either in their job or as a hobby (metal smelters,
pottery makers, and stained glass artists) and people live in communities with high industrial pollution [12]. Lead
can be transferred from the maternal to the infant through milk when there is maternal exposure to lead [13].

Lead is known to heighten lipid peroxidation and induces excess production of reactive oxygen species (ROS) in
different cells [2]. Detailed studies in the past decade [14-16] showed that lead possesses the ability to start chain
reaction resulting in oxidation of lipids, proteins and nucleic acids causing membrane damage, protein dysfunction,
cell death, mutagenicity and carcinogenesis. In addition, a past study indicated that lead alters various hematologic
parameters [8] other than simply RBC count. These include blood glucose and hemoglobin level which can now be
potentially addressed by antioxidant treatments.

α-tocopherol and ascorbic acid are the most common naturally occurring antioxidants. Lipid-soluble α-tocopherolis
a common form of vitamin E. Unlike ascorbic acid which is primarily transported through the blood and readily
excreted by the body, Vitamin E is accumulated in tissues [17]. Vitamin E has an important role in scavenging free
oxygen radicals, stabilizing the cell membranes and maintaining its permeability [18]. Vitamin E efficacy lies on the
fact that it needs not to be taken daily and can perform antioxidant activity longer than Vitamin C [19]. On the other
hand, although ascorbic acid is not storable in the body, it is known to be readily absorbed in the fluids and easily
transported. Adequate intake of vitamin C helps in the treatment of many cardiovascular diseases due to its water-
soluble nature. Vitamin C has no risk of vitaminosis or vitamin poisoning and is readily excreted during urination
[20].

This study determined the relative antioxidant efficacy of lipid-soluble α-tocopherol and water-soluble ascorbic acid
on the blood of lead-exposed albino rats. Hematological parameters like blood lead level (BLL), hemoglobin (Hb)
and Hematocrit (Hct) level plus the supporting red blood cell count (RBC) served as indicators under study. Actions
like presenting and commending the use of drugs with the best antioxidant property as an aid factor for chelation and
detoxification were recommended for evaluation.

MATERIALS AND METHODS

Research Design
The entire study covered a randomized complete block design. The design was used to discover if there is variance
on the BLL (O1), Hb (O2), Ht level (O3) and RBC count (O4) of normal rats, lead exposed rats (T1) and those
antioxidant-treated rats (T2, T3 and T4). This design assessed if there is significant difference on the antioxidant
activities of Vitamin C (T2), E+C (T3) and E (T4) on the blood of albino rats.

Preparation of the animal cage

The animal cage was accomplished April 2013. A cage with enough dimensions to bear five rooms was used. Each
room has 22”x14” area with 8” height. Its construction initiated right after the proposal defense and research
approval of the panel members.

Acquisition of specimen, lead and antioxidants

Thirty male Albino rats of Sprague dawley strain weighing about 120g -150g (2 month old) was obtained from Jing
Sea petshop and animal house Pasay, Metro Manila; lead acetate from Reman hospital, medical and laboratory depot
Rizal Avenue Sta. Cruz Manila; and vitamin supply directly from the nearest generic Skymed pharmacy distributor.
Two-week time was allotted for the acquisition of specimen and experimental supplies.

Acclimatization
The animals were randomly divided into five groups (six members each) and were kept under normal health
laboratory conditions upon adaptation for 2 wks. During this week, all necessary research materials were also
prepared and reviewed. The experimentation proper began right after the approval of the Animal Ethics Committee
and after the 2 wks adaptation.

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Treatment of antioxidants
The first group served as negative control (T0) which was not exposed to lead, no antioxidant treatment, and fed on
standard basal diet up to the end of the experiment. The second group served as positive control (T1), exposed to
lead but no antioxidant treatment. Antioxidant treatments were given daily for four wks. The third group (T2) was
given orally of ascorbic acid (0.15mg vit C = 3IU). The fourth group (T3) was given orally of both vitamins (1.00mg
vit E + 0.075mg vit C = 3IU). The fifth group (T4) was given orally of α-tocopherol (2.00mg vit E = 3IU).

Administration of lead acetate

The second, third, fourth and fifth group were given subcutaneously of 1/40 sub-lethal dose lead acetate dissolved in
0.5mL water on the second day and every other day of the experimental period (four wks). The dose and frequency
were patterned from a recent lead toxicity study [2].

Hematological studies
After the last day of experimental period, blood samples were collected early morning the following day through
saphenous venepuncture for blood count examination and hematological studies of hemoglobin and hematocrit using
cyanmethemoglobin method and microhematocrit centrifugation at Medico Medical Clinic Poblacion I-A, Imus
Cavite. Lastly, the hematological parameters gathered were tabulated using the preferred designs and analyzed using
prescribed statistical methods.

Blood lead level determination

Blood samples amounting to 5mL were collected through terminal cardiac puncture as advised by the consultant
veterinarian for blood-lead level analysis using Atomic absorption spectrometry (AAS) at Chempro Analytical
Services Laboratories, Inc. Shaw Blvd Pasig City.

Data Gathering and Statistical Analysis

All decisions were data driven based on hematological studies of the blood collected after the last day of the
experiment. Each unit of the control groups and experimental groups yielded 4 sets of data namely blood lead,
hemoglobin, and hematocrit level plus the supporting red blood cell count. The data were organized under the
treatment structure of randomized complete block design (RCBD). Analysis of Variance (ANOVA) was the
statistical tool used and all statements of differences at p<0.05 were considered significant. Ultimately, Tukey
method was used after ANOVA as post statistical test since ANOVA could only tell whether there was or there was
no significant difference between T0, T1, T2, T3 and T4 on RCBD. The post statistical test was used to determine
where exactly the difference among the groups is.

RESULTS

The following hematologic parameters were obtained by microhematocrit centrifugation and analysis. Blood lead
levels were obtained by spectrometry.

Table 1. Average Hb level, Hct level, blood lead level and RBC count of lead-exposed rats treated in different vitamin supplementation

Average values of hematologic parameters

Treatments BLL(ug/dL) Hb (g/dL) RBC(x1012/L) Hct(%)
T0 38.92 A 17.67 A 6.19 A 53.33 A
T1 158.32B 13.43B 4.73 B 40.67 B
C C C
T2 103.85 15.53 5.46 47.00 C
T3 84.79 C 16.33 AC 5.75 AC 49.50 AC
T4 98.36 C 16.75AC 5.89 AC 50.67 AC
Letters A and B and C compare the different treatments (rows). Means on the same column followed by a common letter are not significantly
different (p<0.05), ANOVA.

The mean BLL of lead exposed rats (T1, T2, T3, T4): 111.33ug/dL is significantly higher compared to the mean BLL
of normal rats: 38.92ug/dL. Based on post statistical analysis determining the 95% confidence limits, BLLs above
79.38ug/dL are already considered significantly higher to the predetermined normal value of 38.92ug/dL. Six out of
eight lead exposed rats tested for blood lead regardless of vitamin treatment gave results that are considered above
the normal value. Only two gave results still within the normal range. These two values are 56.71ug/dL (T3) and
70.17ug/dL (T4) respectively.

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Hemoglobin level
There were significant differences in the Hb level of lead exposed albino rats treated with different vitamin
supplement. The negative control (T0) mean of 17.67g/dL was significantly higher compared to the positive control
(T1) mean of 13.43g/dL which shows that lead exposure lowered the level of Hb. Lead exposed rats that received
vitamin C treatment gave a resulting mean of 15.53g/dL which was significantly higher to the lead exposed group
but still significantly lower to the normal group. Only T3 and T4 mean values were not significant different to the
normal mean value which showed that both vitamin E+C and vitamin E treatments were successful in stabilizing the
Hb level of lead exposed rats to normal.
Table 2.Blood lead levels of normal rats and lead exposed rats treated in different vitamin supplementation

BLL (ug/dL)
Treatments T0 T1 T2 T3 T4
49.41 A 139.23 C 108.66 C 56.71 A 70.17 A
28.42 A 177.41 B 99.03 C 112.87 C 126.54 C
158.32 B 103.85 C 84.79 C 98.36 C
Means 38.92 A 111.33 C
95% Confidence limits C: 79.38 -- 143.28
BLL followed by a common letter are not significant different. BLL followed by letter C are within the 95% confidence-limit-range above the
normal mean value. (38.92+40.46; 38.92+143.28)
Blood lead level

Red Blood Cell count and Hematocrit level

There was significant difference in the RBC count and Hct level of lead exposed albino rats treated with different
vitamin supplement. The mean values of the negative control (T0) (RBC=6.19x1012/L, Hct=53.33%) were
significantly higher compared to the mean values of the positive control (T1) (RBC=4.37x1012/L, Hct=40.67%)
which showed that lead exposure lowered RBC count and thus also the level of hematocrit. Lead exposed rats that
received vitamin C treatment gave mean values of 5.46x1012/L on RBC and 47.00% on Hct which were
significantly higher to the mean values of the lead exposed group but still significantly lower to the mean values of
the normal group. These results on T2 showed that, although Vitamin C affected the variables positively, the
treatment was not enough to normalize RBC count and Hct level of lead exposed rats. Only T3 and T4 mean values
were not significant different to their normal mean equivalents which showed that both vitamin E+C and vitamin E
treatments were successful in stabilizing the RBC count and Hct level of lead exposed rats to normal. Treatments
with vitamin E showed the most remarkable results.

DISCUSSION

Solution containing 1/40 sub-lethal dose lead acetate induced subcutaneously to albino rats for four wks produced
harmful changes in the studied blood parameters. However, most of the effects of lead in the blood were lessened by
the treatment of antioxidants.

Lead is a heavy metal which known to deplete cells’ major antioxidants, particularly thiol-containing antioxidants
and enzymes. Oxidative stress can result from enhance generation of ROS brought by lead exposure. Cells in
oxidative stress show various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA [14]. The
hallmark of lead exposure that results from ROS generation and subsequent erythrocyte hemolysis is hypochromic
anemia [21]. Along with silver, mercury and copper, lead is considered to be a strong hemolytic agent that causes
erythrocyte destruction through the formation of lipid peroxides in cell membranes [22].

The most common accepted and provable biomarker for lead exposure is BLL measurement. Once lead is absorbed,
99% of it is bound to RBC for about 30-35 days which indeed were reflected by the BLL results of this study.
However, BLL cannot be used to diagnose evidence of exposure that happened more than 6 wks prior to the
measurement due to the short half-life [23].

Lead causes hemolysis where lead-induced lipid peroxidation was suggested to be the possible mechanism for it.
But because lead cannot initiate lipid peroxidation directly, indirect mechanisms were considered. Lead interaction
with oxyhemoglobin has been suggested as a relevant cause of superoxide radical expansion in RBCs. An early
study in an in vitro liposome model [24], found that lead substantially increased the auto-oxidation of Hb whereas in
this present study, it was proven that lead exposure lowered the level of Hb thus attested the interaction.

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In addition, lead also obstructs hemoglobin synthesis. Lead inactivates δ-aminolevulinic acid dehydratase (ALAD),
an enzyme necessary for heme synthesis, by binding to its thiol group [25]. BBLs exceeding 20µg/dL can inhibit
ALAD activities by 50% [23]. ALAD inhibition results to increased circulating aminolevulinic acid (ALA) which
leads to decreased γ-aminobutyric acid (GABA) release in the central nervous system (CNS). This may explain
some behavioral disorders seen in both porphyria and lead toxicity. Ultimately, as more hemoglobin undergone
auto-oxidation and less hemoglobin produced, hemoglobin level will eventually dropped below its normal value and
the accumulating ALA also auto-oxidizes resulting to more generated ROS comprising hydrogen peroxide (H2O2),
superoxide radical (O2-) and hydroxyl radical (OH) [26]. Hemoglobin levels start to decrease as a result of lead
exposure when BLLs are 50µg/dL for adults and 40µg/dL for children [11]. As ALA is further oxidized, it becomes
4,5-dioxovaleric acid. The generation of this potentially genotoxic compound is a possible mechanism for the metal-
dependent DNA carcinogenicity of lead. ALA may be responsible for the increased frequency of liver cancer in
acute intermittent porphyria, another condition where elevated levels of ALA occur [27].

Anemia is one of the early manifestations of lead poisoning that result from reduction of the life span of circulating
RBCs by constraining Na-K-ATPase, lowering membrane integrity and interfering with several enzymatic steps in
hem pathway [28,29]. Lead can initiate lipid peroxidation that can cause hemolysis through various mechanisms.
One is that lead exposure increases the rate of auto-oxidation of Hb which makes antioxidant enzymes preoccupied
in inhibiting the process and leaving other ROS which can contribute to peroxidative hemolysis if left unneutralized.
The result of this present study on RBC count and Hct showed that lead exposure lowered the number of RBCs and
thus also the level of Hct. With these parameters affected, it was suggested that the rate of peroxidative hemolysis
was heightened by lead. Osmotic and mechanic susceptibilities of RBCs are proposed to worsen in lead toxicity.

The toxic effects of lead on membrane components were investigated by several studies and they found a direct
correlation between these effects with lead-induced oxidative damage. When lead was incubated with various
polyunsaturated fatty acids [30], an increase in oxidative stress biomarker, malondialdehyde (MDA), was observed.
It was earlier found that the increase on MDA was brought by the increase of double bonds in fatty acids [31].
Conversely, there was a study on the effects of lead on fatty acid metabolism which reflected that arachidonic acid
and arachidonate ratio in RBC membranes of chicks were increased by lead exposure [32]. Since fatty acid chain
length and unsaturation are important determinants of membrane susceptibility to peroxidation, it was suggested that
arachidonic acid augmentation might be responsible for the enhanced lipid peroxidation, increased hemolysis and
decreased fluidity in RBC membranes [33].

Lead is also known to bind to phosphatidylcholine membranes in vitro, thereby decreasing the levels of
phospholipid [34]. The data on their study suggested that lead-induced alteration on lipid composition of RBC may
alter its membrane integrity, permeability and function, thereby increasing susceptibility to lipid peroxidation thus
also to hemolysis.

Lead intoxication is as well accompanied by an acquired deficiency of erythrocyte pyrimidine-specific, 5'-

nucleotidase. Genetically determined deficiency of this enzyme is associated with chronic hemolysis, marked
basophilic stippling of erythrocytes on stained blood films, and unique intraerythrocytic accumulations of
pyrimidine-containing nucleotides. Increasing the pyrimidine nucleotides in RBCs and preventing the maturation of
erythroid elements leads to decreased red blood cell counts and eventually anemia [35]. A report documented that
lead-induced deficiency when severe gives rise to findings similar to hereditary disorder [36]. Basophilic stippling
and hypochromic anemia occur after significant levels of exposure at BLL over 50µg/dL in adults and 25-40µg/dL
in children.

Although there are various mechanisms which lead contributes to lipid peroxidation and hemolysis, its main
consequence lies on the body’s antioxidant defense system. Antioxidant enzymes neutralize free radicals and ROS
and prevent possible cell damage. Glutamyl-cysteinyl-glysine (GSH) plays a major role in protecting cells against
oxidative stress. Gluthathione reductase (GR) reduces gluthathione disulfide to GSH, thereby supporting the
antioxidant defense system. Lead may inhibit GR by interfering with the disulfide bond of its active site. Other
antioxidant enzymes, which remove peroxides and superoxide radicals including gluthathione peroxidase (GPx),
catalase (CAT), superoxide dismutase (SOD) are also targeted by lead. GPx requires selenium which lead forms a
complex with. Catalase contains heme but lead inhibits heme synthesis. Several studies [37-40] showed decrease
RBC SOD activity in lead exposed subjects including rats. In summary, lead impairs antioxidant defenses and
renders cells including RBC more susceptible to oxidative damage.

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Based on the results of this present study, the effects of lead in all of the studied blood parameters were lessened by
the treatment of antioxidants. Vitamin E plus C (T3) and Vitamin E (T4) were successful in giving exceptional
results similar to values of the normal group. Although Vitamin C (T2) gave the least beneficial outcome, the results
were considered positive since it lessed the effects of lead in the blood, only that it failed stabilizing the values to
normal as compared to other antioxidant-treated groups.

The beneficial role of vitamin C was previously presented by a study in 1988 which attributed to its ability to
complex with lead [41]. In their study, ascorbic acid was administered alone or in combination with thiamine to
lead-exposed rats. Ascorbic acid was found to be effective by means of increasing urinary elimination of lead,
reducing hepatic and renal lead burden and reversing lead-induced inhibition of the activity of blood ALAD.
Thiamine, however, did not show any beneficial effects. Another study in support to vitamin C treatment found that
it was effective in reducing lead levels in blood, liver, and kidney [42]. Also, the results showed both lead-induced
ALAD inhibition and elevation in zinc protoporphyrin in the blood were reversed by the treatment. Before such
studies about vitamin C, there was an early report found that vitamin C acted with similar potency with ethylene
diamine tetra acetic acid (EDTA) and thus it was then been proposed that it could be used as a possible chelator for
lead [43].

In this present study, vitamin C treated lead-exposed rats (T2) gave a lower blood lead level, higher hemoglobin,
more RBC, and higher hematocrit as compared with lead-exposed rats alone. These beneficial outputs on the blood
are the same with other studies that indicate vitamin C has significant chelation capacity for lead. In a male rat study
in 1998, the researchers attested that toxic effects of lead on heme production were reversed by a vitamin C dose of
100mg/kg. Conversely, the treatment normalized blood ALAD levels and resulted in a significant decrease in BLL
[44]. Another rat pharmacokinetic study found intravenously administered vitamin C lowered lead tissue levels in
rats that were continuously administered lead [45]. On the other hand, a human study, evaluating BLLs in pregnant
women, found that 1g vitamin C per day, in addition to a prenatal multivitamin supplement, significantly lowered
BLLs during the course of pregnancy [46]. The safety of chelating pregnant women, however, and potential
exposure of the fetus to lead, was not addressed in that study. In 1999, a study reported a population-based claim
that indicates an inverse relation between serum ascorbic acid and BLLs among Americans [47]. The authors
suggest that higher intake of ascorbic acid may be effective in preventing lead toxicity if a causal relationship is
confirmed.

Although improvement by different means of antioxidant administration to lead-exposed animals was reported, there
were not enough studies in the literature where the effectiveness of an antioxidant in counteracting lead-induced
oxidative damage is extensively investigated. It was only recently that the correlation between those beneficial
effects of antioxidants and other oxidative stress-related parameters has been investigated. A study in 2001 of silver
refining (involving lead smelting) workers with substantial BLLs and symptoms of lead toxicity (anemia, muscle
wasting, abdominal colic) were given vitamin C to evaluate the ability of the supplements to affect lead exposure.
With continuous lead exposure and 250 mg vitamin C twice daily for 30 days significantly lowered BLLs.
Conversely, vitamin C was effective in reversing ALAD inhibition, indicating an important antioxidant effect as
well [48].

Although there were correlations between vitamin C and BLLs, still, numerous studies done recently supported that
normal dose vitamin C supplementation produced only a small reduction in lead retention in the liver, kidney and
blood [49, 50]. It is biologically possible that vitamin C may affect lead absorption and excretion but based on many
recent studies, the effect is more obvious in low-exposed subjects with higher vitamin C intake. In this present study,
the results showed that 3IU dose vitamin C produced only a small reduction in lead retention in the blood. The
optimum effect of vitamin C on BLLs has been clarified by studies that showed ascorbic acid decreases intestinal
absorption of lead [51,52] by reducing ferric iron to ferrous iron in the duodenum thereby increasing iron absorption.
Vitamin C enhances the availability of iron which competes with lead for intestinal absorption. Since the present
experiment involved subcutaneous injection of lead and not through enteral route, vitamin C efficacy on decreasing
lead level was not fully detected.

Lead-lowering effect of vitamin C has not been proven effective in exposure studies where occupationally-exposed
workers had higher BLLs. Workers with long-term lead exposure and higher BLLs were given 1g vitamin C orally
once daily for 20 wks. The treatment did not affect BLLs and lead metabolism significantly [53] whereas in a study
assessing the mechanism of vitamin C’s lead-lowering capacity, male smokers with no known occupational or

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residential lead exposure were given 1g vitamin C daily for 30 days the BLLs were effectively lowered from a mean
of 1.8µg/dL to 0.4µg/dL within one week and remained at that level for the rest of the experiment [54]. This really
showed that the effect of vitamin C on reducing BLL was only observable in low-exposed subjects that urine lead
levels did not change during the length of the study, regardless of the vitamin C dosage. Its authors concluded that
vitamin C was working to inhibit intestinal absorption of lead. This would involve the entero-hepatic recycling of
lead through erythrocyte catabolism rather than increasing lead excretion through renal elimination.

In addition to acting as an antioxidant and anti-absorption agent, vitamin C also has an inhibiting effect on lead
uptake on a cellular level. Mammalian cell culture studies using a methodology that assessed lead uptake, lead
toxicity, and lead release by chelating agents and nutrients determined the capacity of these agents to modify the
way cells absorb and are damaged by lead [55]. Vitamin C was effective at inhibiting lead uptake and reducing lead
cytotoxicity just like known lead chelators as previously conferred [43].

In this present experiment, vitamin E, another well-known antioxidant, was proven effective in normalizing the
studied lead-induced changes in the blood. It was earlier confirmed that the said lipid soluble vitamin was effective
at decreasing lipoperoxidation in the liver, brain and kidney of lead-exposed rats when given 100 IU/Kg body
weight [50]. Vitamin E is a chain-breaking free radical-trapping antioxidant in cell membranes and plasma
lipoproteins. It reacts with lipid peroxide radicals formed by peroxidation of polyunsaturated fatty acids before they
can establish chain reactions [56]. But it was only in this present study that vitamin E efficacy on hematotoxicity
was indeed very well represented. That based on results, it acted with improved potency compared to vitamin C.

Vitamin E was evaluated by several groups regarding its remedial effect on lead toxicity. One early study done by
Levander and his colleagues determined the filterability of RBC as well as RBC lipid peroxidation from vitamin E-
deficient and vitamin E-supplemented lead-exposed rats. Lead was shown to increase the mechanical fragility of
RBC, which makes the RBC less deformable and more susceptible to oxidative stress. Filterability of lead-exposed
RBC was estimated by determining the time required for RBC to pass through a polycarbonate filter by visual
inspection. The filtration time for RBC from vitamin E-deficient rats was much greater than that of RBC from
vitamin E-supplemented rats. Furthermore, a strong correlation was found between increased lipid peroxidation and
decreased filterability of red cells from vitamin E-deficient lead-exposed rats [57].

The preventive role for vitamin E in lead toxicity, implied by Levander and his colleagues was confirmed by other
groups who found that in animal models, simultaneous supplementation of vitamin E is more effective than giving it
as a treatment after lead exposure [58]. Such preventive effect, reported to be due to the inhibition of lipoprotein
oxidation by Dhawan and his colleagues, was likewise understandable in the results of this present experiment.The
results on vitamin E-treated groups reflected normal RBC counts and Hct levels thus simultaneous vitamin E
supplementation indeed prevented lead-induced oxidation and destruction of RBCs. Conversely, other similar
studies done both in vivo and in vitro suggested that simultaneous supplementation of the vitamin to lead-exposed
rat erythrocytes prevents RBC membrane damage, ALAD inhibition and lipid oxidation [59, 60]. It increased SOD
and CAT activity as well. It was concluded in their studies that vitamin E could be useful in order to protect
membrane-lipids and, notably, to prevent protein oxidation produced by lead-intoxication. Vitamin E also played a
role in the protecting tissues at the early stages of life, it can be transported to the infant during the lactation and
pregnancy [61].

Vitamin E is a fat-soluble vitamin known to be one of the most potent endogenous antioxidants. α-tocopherol is the
term that encompasses a group of potent, lipid soluble, chain-breaking antioxidants that prevents the propagation of
free radical reactions. Vitamin C is a water-soluble antioxidant occurring in the organism as an ascorbic anion. It
also acts as a scavenger of free radicals and plays an important role in regeneration of α-tocopherol [62]. Evidence
of their synergistic effect is one of the factors that inspired the methods of this present research. Previously,
supplementation of ascorbic acid and α-tocopherol has already been known to alter the extent of DNA damage by
reducing TNF-α level and inhibiting the activation of caspase cascade in intoxicated animals [63]. Their study
strongly believed that vitamin supplementation perspective, though observed in animal model, will have sustainable
curative value among the already afflicted populations, neutralizing impact on freshly emerging metal poisoning
scenario and possible proactive protection to those potentially susceptible to heavy metal exposure.

Granting there were numerous studies about the synergism of various antioxidants like vitamin C + thiamine
combination [41, 42] and vitamin E + calcium disodium versenate [50], their relative effects were not fully founded.

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Flora and Tandon only alleged that both ascorbic acid and ascorbic acid in combination with thiamine were found to
be effective by means of reversing blood ALAD inhibition but did not mention about their comparative efficacy.
Patra and his colleagues only found that vitamin E alone or in combination with conventional chelator, calcium
disodium versenate (CaNa2EDTA), decreased lead-induced lipid peroxide levels in rats but did not discuss which of
the used chelating agent was more effective.

Also, there was only a little established relation of the synergistic action of vitamin C and E when it comes to the
circulation. One study on the protective action and synergistic action of vitamin C and E against lead was on
genotoxicity but not on hematoxicity. The study showed that co-administration of both vitamins at added doses to
lead-treated mice led to a significant decline in MDA content, lipid peroxidation in the testicular tissue along with
elevated sperm count and reduction in the percentage of abnormal sperm population [64]. Variations of efficacies
and difficulties on establishing comparison between antioxidants were due to the underlying pharmacologic factors
of dose, route of administration, duration of treatment, plasma half-life, organ system under study and individual
differences between animals.

Nevertheless, the synergistic efficacy of vitamin E and C was very well presented by the results of this present
experiment. That although the doses were halved (1.5 IU vitamin E + 1.5 IU vitamin C), still, the combination was
successful in stabilizing the levels to normal with improved potency than complete 3 IU vitamin C and similar
efficacy as the complete 3 IU vitamin E treatment. It was suggested that vitamin C reinforces the antioxidant effect
of vitamin E by regenerating the active form of vitamin E after it has reacted with a free radical as well as protecting
it, together with folate and polyunsaturated substances, from destruction by oxygen as they move throughout the
body [65]. This beneficial interaction has been demonstrated in biological fluids as well as in model systems. That in
animals, supplementation with one of these nutrients can relieve symptoms caused by a deficiency of the other [66].
However, they cannot fully replace each other, like vitamin E, through its presence in glutathione peroxidase
enzymes, functions to protect cell membranes against the action of lipid hydroperoxides [67]. As a result of
interactions such as these, combinations of antioxidants may be more effective than larger quantities of single
antioxidant [68, 69].

The study proved that with increased BLL, the red blood cell count, Hct and Hb level of Rattus norvegicus (albino
rats) decreased. It was evidently shown that lead exposure produced harmful changes in the studied parameters.
However, most of the effects of lead in the blood were lessened by the treatment of Vitamin C, Vitamin E+C and
Vitamin E as compared to the lead exposed rats alone. The synergistic competence of both vitamins was also well
presented by the results. The E+C treatment, like the E treatment, was successful in increasing the hemoglobin and
hematocrit to normal. Conversely, rats that received such synergism gave the lowest blood lead and proven more
efficient than vitamin C in all of the studied parameters despite of the same total IU dose used. Therefore
consumption of diet containing vitamins E and C could help to ameliorate the oxidation effect caused by heavy
metals by reversing hematoxicity.

It is worth recommending to further study other agents that are known to deplete cells’ major antioxidants (e.g.
silver, nickel and copper). Studies may also be done in a different mode of exposure to see whether the method can
affect the results. Further study on the comparison of vitamins in a different pre-set dosage, animal model, body-
system under study, route of administration, and duration of treatment to likewise establish differences in terms of
efficacy that are not limited by pharmacologic factors. Complementary examination of the synergistic competence
of other antioxidant-rich vitamins for chelation and detoxification is also recommended. Equally, advance analysisof
the protective function of antioxidants by giving it either as treatment preventing lead toxic effect or a remedy for
oxidative intoxication is suggested. Future analysis can collect information to develop new actions for cases of
"heavy metal poisoning" (an indirect cause of protein dysfunction, cell death, mutagenicity, and cancer). Actions
like commending the use of drugs with "the best" antioxidant property as an aid factor forchelation and
detoxification is strongly recommended for evaluation.

Acknowledgements
The authors wish to thank the faculty of Biological Sciences Department for their valuable contribution to the
content of the researchers’ study and the technicians in the Biology Research Laboratory of De La Salle University –
Dasmariñas for their support during the data collection and analysis stage. The researchers also express their
gratitude to Medico-medical Laboratory and Chempro Analytical Services Laboratories, Inc. for their chemical
supplies and technical assistance.

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______________________________________________________________________________
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