Over the past decade, anemia has emerged as a risk factor that is associated with a variety of adverse outcomes in humans, including hospitalization, disability, and mortality⁴. Anemia is essentially a homeostatic imbalance in the blood concentration of hemoglobin whereby the production of erythrocytes is outplaced by the destruction or loss of erythrocytes. The epidemiology of anemia is actually challenging because of increased heterogeneity in the distribution of social and biological risk factors^2,3. Given that anemia is multifactorial condition, the increased co-morbidity makes it difficult to establish whether anemia is a marker of disease burden or a mediator in the causal pathway leading to adverse events. Anemia is essentially a homeostatic imbalance in the blood concentration of hemoglobin whereby the production of erythrocytes is outplaced by the destruction or loss of erythrocytes ^1,3.

Iron is essential for all eukaryotes and most prokaryotes, where it is used in the synthesis of heme, iron-sulfur (Fe-S), and other cofactors. Fe-S proteins are involved in catalysis, redox reactions, respiration, DNA replication, and transcription. Hemes are found in hemoglobin, myoglobin and cytochromes, and recent studies have implicated heme proteins in the regulation of circadian rhythmicity and micro-RNA processing. Iron homeostasis is tightly regulated to avoid iron toxicity or iron deficiency in normal condition^4,6-8.

Oxidative stress is associated with increased mortality and morbidity particularly in patients with IDA. The disturbance in the prooxidant and antioxidant balance leads to potential damage producing oxidative stress. Numerous types of free radicals can be formed within the body because of increased lipid peroxidation^4,9,10. Oxidative stress has long been demonstrated, but the factors influencing their oxidative status have not been characterized extensively in IDA. Therefore, the present study was designed to investigate the influence of factors known to be associated with oxidative stress.

Materials and Methods:

Study design and subject recruitment- A total of eighty (80) individuals were selected for the study. Among them forty were iron deficient anemic individuals and rest forty was healthy controls. Blood was collected from antecubital vein from both groups in an aseptic condition. A written consent was taken from all the individuals including both patients and healthy controls. Healthy controls were selected on the basis of their non smoking, non alcoholic habit and without symptom of any diseases and sickness. Height and weight was measured to determine respective body mass index and surface area. Control group as well as case, were devoid of family history of iron deficiency anemia. The study group subjects age ranged from 15 to 45 years. Description criteria for IDA are hemoglobin concentration < 11.5 g/dl in women and <13 g/dl in men, iron concentration <45 μg/dl and total iron binding capacity >60 μmol/l.

Blood samples and lysates- Blood (5 ml) was collected in tubes containing ethylenediamine tetraacetic acid (EDTA), centrifuged at 4000 rpm for 10 min and the plasma was carefully separated and stored in a clean and dry vial. After the erythrocyte pellet was washed thrice with chilled physiological saline; 0.5 ml of cell suspension was diluted with 2 ml cold distilled water to lyse the erythrocytes. Glass-wares used for the experiment were acid washed and all the reading were measured by Shimadzu spectrophotometer. The hemoglobin was determined by the Beacon Diagnostics and iron and total iron binding capacity was assayed by Coral Clinical Systems kit method. The chemicals used were of analytical grade.

Determination of lipid peroxidation- The total amount of lipid peroxidation products in the plasma of healthy volunteers and patients was estimated using thiobarbituric acid reactive substances (TBARS) methods, which measures the MDA reactive products (Buege and Aust method, 1978)¹¹. In brief, 0.8 ml of plasma and 1.2 ml of TBA-reagent was mixed properly and heated in a boiling water bath for 10 min. after cooling it, the tubes were centrifuged at 3000 rpm after addition of 2 ml of 0.2 N NaOH to obtain a pink colored adduct formed at 535 nm. The malondialdehyde levels were expressed as nmol/ml.

Determination of lipid hydroperoxides- (FOX assay) – The lipid hydroperoxide (LOOH) was determined according to Jiang (1992)¹²using FOX reagent (7.6 mg xylenol orange, 88 mg butylated hydroxytoluene, 9.8 mg ferrous ammonium sulphate to 90 ml of methanol and 10 ml of 0.25 M H₂SO₄). To 0.1 ml of sample (plasma and RBC lysate), 0.9 ml of FOX reagent was added followed by incubation at 37^oC for 30 min. The tubes were centrifuged at 3000 rpm for 10 min. The absorbance of purple colored supernatant was measured at 560 nm against distilled water. The lipid hydroperoxide was calculated using molar extinction coefficient of 4.52 × 10⁴ M^-1 cm^-1_.The lipid hydroperoxide was expressed in nmol/ml of lipid hydroperoxides.

Enzymatic determination of superoxide dismutase (SOD) - CuZn-SOD activity in hemolysed RBC was determined by Kakkar et al method (1984)¹³ based on the 50% inhibition of the formation of nicotinamide adenine dinucleotide (NADH)-phenazine methosulfate-nitroblue tetrazolium formazan at 560 nm. One unit of CuZn-SOD activity is defined as the amount of the enzyme required to inhibit the rate of NADH autooxidation by 50 %. The superoxide dismutase activity was expressed in units per gram of hemoglobin.

Enzymatic determination of catalase (CAT) activity- CAT activity in the whole blood and hemolysed RBC (erythrocytes) were assessed in the erythrocyte lysates by the method as described by Sinha (1972)¹⁴. Briefly, hydrogen peroxide (0.2 M) was used as a substrate and the decrease in H₂O₂ concentration at 22 ^oC in phosphate buffer (0.05 M, pH 7.0) was followed spectrophotometrically at 240 nm. One unit of catalase is presented as units per gram hemoglobin (U/g Hb).

Enzymatic determination of Glutathione peroxidase (GPx) activity- GPx activity also analyzed in hemolysed RBC lysates by the method of Rotruck et al (1973)¹⁵ with modifications. 100 μl of enzyme preparation was allowed to react with H₂O₂ in the presence of reduced glutathione. After a specified period of enzyme action; the remaining reduced glutathione content was measured by the method of Beutler and Kelley (1963)¹⁶. The glutathione peroxidase activity was expressed in units per milligrams of hemoglobin.

Determination of Vitamin A- Vitamin A was estimated by the method of Bessey OA et al (1946)¹⁷. A mixture of 2 ml of plasma, 1ml of distilled water, 4 ml of absolute ethanol and 4 ml of heptane were mixed in cyclomixer and centrifuged at 3000 rpm for 15 min to get protein free filtrate. The concentration of the retinol in the protein free filtrate was determined by measuring the absorbance at 327 nm against heptane as a blank. A standard calibration curve of retinol palmitate (100 µg/dl) was also plotted. Vitamin A was expressed in µg/dl.

Determination of Vitamin C (Ascorbic acid) - Vitamin C in plasma is estimated by Natelson (1971)¹⁸2,4-dinitrophenyl hydrazine (DNPH) method where vitamin C is oxidized to diketogluconic acid which reacts DNPH with to form diphenylhydrazone. The hydrazone dissolves in strong acid solution to form orange-red colored complex. The absorbance was read at 520 nm and vitamin C was expressed in mg/dl.

Determination of Vitamin E- The vitamin E was measured by the method Baker and Frank (1980)¹⁹ by the reduction of ferric to ferrous ion which then forms a red colored complex with α, α’-bipyridyl. The absorbance was measured at 490 nm and 520 nm. Vitamin E was expressed in mg/dl.

Statistical methods- The packaged program SPSS (Statistical package for social sciences) for windows version 13.0 (SPSS, Chicago, Il, USA) was used for statistical analysis. The results are reported as means ± standard deviation (SD) for the patients and the controls group. Differences between the groups were determined by means of a Student’s t-test and ANOVA. Significance level was set at p less than 0.05.

Results and Discussion:

On analysis we found significantly decreased body mass index (BMI) and surface area in IDA patient (both in male and female) when compared to healthy individuals (Table: I and II). There was no family history of iron deficiency anemia in our study group- both case and control. Both the study groups were not supplemented with drug and vitamins (Table: I)

Table I: Demographic data of IDA- patients and controls.

Parameter	IDA case	Control
Number	40	40
Mean Age (yr)	33.611±6.12	38.841±2.83
BMI (kg/m²)	17.21±1.43	22.64±2.38
Surface area (m²)	1.48±0.21	1.72±0.38
Family History of IDA	No	No
Fruits	occasional	occasional
Vegetables	daily	daily
Diet-Veg (%)	58	32
Non-veg occasional (%)	35	48
Non-veg regular (%)	7	20
Drug supplementation	No	No
Vitamin supplementation	No	No

Table II: Biochemical parameters of antioxidants – oxidants in case and control group.

Characteristics

Iron

Deficiency anemia

(Case)

Control

Male

Female

Male

Female

Number

MeanAge

35.06±

5.97**

32.17±

6.27**

34.33±

2.30

33.35±

3.66

BMI

17.44±

1.28**

17.05±

1.55**

23.74±

0.73

22.54±

2.44

9.51±

1.21**

8.89±

1.53**

15.29±

1.91

13.61±

0.56

TBARS-WB

4.56±

1.85

5.26±

2.01**

2.32±

1.36

2.94±

1.77

TBARS-RBC

2.61±

1.16*

3.48±

1.94**

1.58±

0.45

1.75±

0.67

TBARS-plasma

5.69±

3.10*

5.2±

2.83**

1.33±

0.46

1.46±

0.65

LOOH-RBC

6.04±

1.75*

6.24±

1.79**

2.92±

1.64

2.98±

1.03

LOOH-plasma

3.72±

2.0*

3.52±

2.5**

0.8±

0.26

0.55±

0.25

CAT-WB

1444.58±

3.90**

1412.99±

2.58**

307.22±

15.87

833.26±

39.04

CAT-RBC

466.20±

19.08*

645.89±

40.025

643.77±

48.73

528.57±

23.83

SOD

20.71±

3.73**

28.81±

8.2**

36.14±

1.56

35.60±

1.8

GPx

1.69±

0.46

2.61±

0.91*

2.09±

0.25

3.56±

1.9

Vitamin A

55.97±

9.14*

53.83±

8.25**

79.86±

25.28

79.0±

21.14

Vitamin C

0.56±

0.04

0.43±

0.02*

0.91±

0.59

0.58±

0.24

Vitamin E

0.48±

0.12**

0.46±

0.13**

1.99±

0.71

1.38±

0.51

*p< 0.05, **p<0.005 (level of significance, with respect to Independent t-test), WB-whole blood, LOOH- lipid hydroperoxide

The oxidant parameters; thiobarbituric acid reactive substances and lipid hydroperoxides (in whole blood, erythrocyte and plasma) were significantly increased both in male and female IDA cases. Likewise, the decrease in enzymatic and non- enzymatic antioxidant (were statistically significant (Table: II).

The hemoglobin levels were divided into four different groups; 5-7, 7-9, 9-11 and 11-13 g/dl in IDA patients. Various parameters like oxidants, enzymatic and non-enzymatic antioxidants were analyzed in all four Hb groups. The lipid peroxidation was comparatively increased in the Hb 11-13 g/dl than other group. The lipid hydroperoxide in RBC was gradually decreased from 5-7 to 11-13 g/dl whereas lipid hydroperoxide in plasma was increased from 5-7 to 11-13 g/dl. Similarly, the non enzymatic antioxidants were also decreased from Hb level 5-7 to 11-13 g/dl group showing increased oxidants levels deteriorating IDA (Table: III).

Table III: The oxidant and antioxidant levels in IDA case according to Hb-code.

Hb-code

5-7g/dl

7-9g/dl

9-11g/dl

11-13g/dl

(N=10)

BMI

16.67±

0.57

17.46±

1.43

16.88±

0.74

17.58±

2.16

Hb**

6.28±

0.65

8.18±

0.49

9.99±

0.56

11.76±

1.45

TBARS-WB

4.57±

1.34

4.59±

2.32

4.95±

1.78

5.45±

2.06

TBARS-RBC*

2.56±

1.82

2.48±

1.25

2.81±

1.13

4.31±

2.21

TBARS-plasma

4.92±

1.62

4.97±

2.40

6.6±

3.54

4.38±

2.41

LOOH-RBC

7.34±

1.33

6.81±

2.04

6.09±

1.77

5.28±

1.15

LOOH-plasma

2.12±

0.18

3.60±

1.71

3.31±

0.21

4.43±

0.31

CAT-WB

1544.23±

25.96

1289.86±

43.4

1440.69±

25.16

1511.38±

26.05

CAT-RBC*

1087.57±

77.04

473.84±

31.34

534.29±

17.59

571.95±

30.54

SOD**

60.52±

1.89

46.53±

1.26

32.07±

1.43

23.52±

9.46

GPx**

2.98±

4.76

0.22±

0.14

2.17±

0.96

4.08±

1.96

Vitamin A

54.86±

1.21

53.41±

6.49

55.91±

1.07

54.44±

8.95

Vitamin C

0.57±

0.06

0.56±

0.03

0.49±

0.04

0.40±

0.009

Vitamin E

0.49±

0.07

0.48±

0.14

0.48±

0.10

0.45±

0.14

*p< 0.05, **p<0.005 (level of significance, with respect to ANOVA test), WB-whole blood, LOOH- lipid hydroperoxide

Anemia associated with iron deficiency has clinical and public health importance. Maintaining normal iron homeostasis is essential for the organism since both iron deficiency and iron excess are associated with the cellular dysfunction^2,4. The maintenance of optimal health requires an adequate supply of carbohydrates, proteins, lipids and macronutrients, micronutrients and trace elements. Red blood cell homeostasis is an excellent example of redox balance: erythroid progenitors accumulate hemoglobin during development, and erythrocytes continuously transport large amounts of oxygen over the course of their approximately 120 day lifespan resulting high level of oxidative stress⁷. Fully matured red blood cells, lacking a nucleus, cannot produce new proteins in response to stress- they have to rely on proteins synthesized earlier in development to protect themselves from damage by reactive oxygen species (ROS) and thus ensure their own survival. RBCs have thus evolved to have an extensive array of antioxidants to counter the level of stress, including membrane oxidative stress, cellular antioxidants such as catalase and superoxide dismutase, and enzymes that continuously produce reducing agents through the glutathione systems^20,21.

Defects in enzymes critical to the oxidative stress (OS) response have been implicated in human diseases ranging from mild chronic hemolysis to severe acute hemolysis. Because of a concomitant reduction in the normal red blood cell life span, these disease states are characterized by a compensatory increase in erythropoiesis. The deleterious effects of OS such as damage to cellular proteins, DNA and lipids are well characterized^2,4. However, the factors that regulate the oxidative stress response and the life span of erythrocytes are less clear. Iron deficiency is the most common nutritional deficiency encountered in surveys of diverse populations in industrialized countries and it is said to be the most common cause of anemia in the world. Iron needs are greatest during infancy because of rapid growth, expansion of the blood volumes and lack of reserve of iron in infants below 6 months of age. The risk of developing iron deficiency is greatest after the first two months in full term infants^10,20,21.

Iron deficiency has been associated with various disorders in the human body. There is altered immune response, limitations in physical performance and neurological dysfunction. Biological interactions among trace elements have been reported to alter the metabolism of other nutrients and metabolites^20-22. In the blood and or tissues of numerous organisms any or all of the following can contribute to protection against ROS: water soluble radical scavengers including GSH, ascorbate or urate; urate lipid soluble scavengers, a-tocopherol, g-tocopherol, flavonoids, carotenoids, ubiquinol; enzymatic scavengers such as superoxide dismutase, catalase and glutathione peroxidase and some glutathione-S-transferases; small molecule thiol-rich antioxidants such as thioredoxin and metallothionein; the enzymes that maintain small molecule antioxidants in a reduced state, thioredoxin reductase, GR, dehydroascorbate reductase, the glyoxalase system; the complement of enzymes that maintain a reduced cellular environment including glucose-6-phosphate dehydrogenase, in part responsible for maintaining levels of NADPH. Dietary supplements frequently claim to be enriched in antioxidants and free radical scavengers²².

DNA synthesis and ribonucleotides reductase require iron. This is evidenced in early S-phase of rapidly growing cells, not only by their expression of tranferrin receptor, but also by that of heavy subunit ferritin receptors. Lipid soluble iron chelators have a strong anti-malarial effect, as well as an effect on the erythrocyte pool of free iron. Iron treatment of patients with anemia of chronic diseases has been shown to lead to relapse of tuberculosis, brucellosis and malaria. However, on-going iron supplementation of the diet is needed in the minority of women in the fertile age group who are at risk of developing chronic iron deficiency because of large menstrual blood losses or frequent childbirth, in blood donors, and perhaps in some children. Over-prescription of iron tablets to patients should be avoided because it facilitates the progress of tumors or infections. Since both the risk of iron intoxication and that of free radical production is limited to the administration of ionizable iron, the possibility should be examined of giving supplementation in the form of heme iron, which does not increase the free iron concentration in the intestinal lumen, and which cannot be absorbed in excess²³.

Vitamin E and Vitamin A are most important chain breaking antioxidants and they protect polyunsaturated fatty acids from peroxidative damage by donating hydrogen to the lipid peroxyl radical. Because of the lipophilic property of the tocopherol molecule vitamin E is the major free radical chain terminator in the lipophilic environment. Vitamin C as a reducing and antioxidant agent directly reacts with superoxide, hydroxyl radical, and various lipid hydroperoxides. In addition it can also restore the antioxidant properties of vitamin E²⁴.

Anemia is one of the most frequent causes of medical visits because of the high incidence in children, young women and elderly people, especially if malnutrition is present. Moreover, anemia is one of the leading signs in many diseases or is the first evidence of disease observed in routine blood cell enumeration. Anemia is usually prevalent in developing countries because of malnutrition, and genetic, parasitic or infectious diseases. IDA is the most prevalent form of anemia worldwide, especially in women and children. Thirty-percent of the world’s population, some 1300 million people suffer from anemia. However, the prevalence of anemia worldwide is unequal (36 % in underdeveloped and 8 % in developed countries). The most likely cause of IDA is malnutrition in children, bleeding in adult males (especially gastrointestinal), menstruation or lactation in fertile women, and bleeding in the elderly. The distribution of nutrient-deficiency anemia in the elderly is: 48 % iron alone, 19 % folate alone, 17 % vitamin B₁₂ alone, and the rest have combined deficiencies. Therefore, in young male adults and in both sexes older than 65 years, the most likely cause of IDA is chronic bleeding, especially from gastrointestinal lesions. More than 100 diseases may cause anemia, but 90 % belong or three groups: nutritional deficiencies (iron, vitamin B12 and folic acid), chronic inflammation, bleeding (excluding chronic bleeding, which produces iron deficiency)²⁵.

Oxidative stress is shown to play an important role in the pathogenesis of IDA. The results of Tekin et al 2001 study showed that antioxidant enzymes activity like glutathione peroxidase, superoxide dismutase and catalase is decreased in IDA indicating increased lipid peroxidation²⁶^-27. Furthermore, it has been shown that the addition of synthetic antioxidants in the treatment of children with IDA results in decrease of lipid peroxidation, prevention of pathologic progression and rapid improvement of clinical manifestations. This confirms iron deficiency anemia is a state of oxidative stress. The effects of antioxidants with oral iron to combat the stress and side effects have been tried in both human subjects and animals. Commonly used antioxidants are vitamin C and vitamin E. vitamin E is the most potent liposoluble antioxidant and has the potential to improve tolerance of iron supplementation and prevent further tissue damage. Vitamin C helps in the absorption of iron by reducing non heme ferric to ferrous iron. By facilitating iron absorption, vitamin C makes more ferrous iron available to participate in Fenton reaction leading to oxidative damage. Addition of vitamin C with iron has proved to be toxic cocktail rather than being an advantage as an antioxidant or pro-oxidant is still not clear²⁶.

Conclusion:

Lipid peroxidation is one of the major outcomes of free radical-mediated injury to tissue. Peroxidation of lipids can greatly alter the physicochemical properties of membrane lipid bilayer resulting in severe cellular dysfunction and causes injure to biomolecules such as nucleic acids, proteins, structural carbohydrates, and lipids, changes fluidity and permeability alters ion transport and inhibition of metabolic processes.

The finding of our study signifies the increased lipid peroxidation and decreased antioxidant status in iron deficiency anemia both in male and female cases. The impaired antioxidant system may favor accumulation of free radical, which may induce the iron deficiency anemia process. Our study was limited to few patients, the vitamins and iron supplementation was not performed. Further research is recommended to identify the specific risk factors for IDA. A diet containing high amount of vitamin A, C and E and heme iron is recommended during the treatment course of IDA. Further studies on lipid peroxidation and antioxidant status after vitamin and iron supplementation is being carried out in the laboratory.

ACKNOWLEDGEMENT:

We are highly indebted to Central College Campus family, Bangalore University, postgraduate students and Bangalore University Ladies Hostel students who kindly agreed to draw blood for control samples. We would also like to thank K. C. General Hospital and anemic patients who kindly permitted us to draw blood to carry out this experiment. We would also like to thank Indian Council for Cultural Relations (Bangalore and New Delhi) and Indian Embassy (Nepal) for providing scholarship under Silver Jubilee Scheme.

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Received on 28.06.2012

Modified on 20.07.2012

Accepted on 24.07.2012

Research J. Science and Tech. 4(4): July-August. 2012: 162-167