Miss.Shuchismita Behera Miss.Sujata Dixit Dr.G.Bulliyya Dr.S.K.Kar Status of Vitamin A (Retinol) and its Relation with Hematological Parameters in Sickle Cell Cases of Orissa Miss.Shuchismita Behera Miss.Sujata Dixit Dr.G.Bulliyya Dr.S.K.Kar Regional Medical Research Centre Indian Council of Medical Research Bhubaneswar

INTRODUCTION Sickle cell anaemia is a hereditary blood disorder of abnormal haemoglobin (HbS) caused by the substitution of valine for glutamic acid in the sixth position of β-chain. Individuals homozygous for sickle cell mutation have a high proportion of sickle hemoglobin (HbSS). However, in the heterozygous individuals upto 40% is sickle Hb (HbAS) Sickle cell anemia have high potential for oxidative damage due to a chronic redox imbalance in red blood cells resulting continuous generation of reactive oxygen species and clinical manifestations of mild to severe hemolysis. Sickling of RBCs lead to two major consequences, a chronic haemolytic anemia and occlusion of small blood vessels resulting in ischemic tissue damage.

To protect against oxidative damage, the body has endogenous defense mechanisms that are supported by dietary antioxidants. The lipid-soluble vitamin A is one among important antioxidants are better known as oxygen radical scavengers (Walter et al 2006). Children with sickle-cell disease have some a combination of micronutrient deficiencies of zinc, and vitamins A, B6, and D (Nelson et al 2002). Prevalence of sickle cell anaemia is alarmingly high in Orissa with sickle cell trait (46.4%) and homozygous sickle cell anemia (6.4%) (Balgir 2008). No data available on the role of anti-oxidant status of in sickle cell anaemia patients.

Objective The present study aimed to assess the antioxidant status particularly sub-clinical vitamin A (retinol) status in sickle cell anaemia cases and to identify whether these parameters could be in any way associated with the patho physiology of the disease.

Materials &Methods Blood samples collected from 130 referred cases (male 64 , female 66) for sickle cell disorders Sickling test conducted with sealed metabisulphite solution by microscopy (Daland &Casttle 1948). Haemoglobin types (HbA2, HbS and HbF) detected by Hb variant (Bio-rad, USA). Haemoglobin and other hematological parameters estimated by MS4 Cell Counter. Plasma retinol levels assayed by HPLC method (Biery et al 1979). Data analysis done by MS excel 2007 and SPSS package.

Mean (+SD) concentrations of hemoglobin and serum retinol Study variable Male (64) Female (66) Total (130) Haemoglobin (g/dl) 11.71±3.78 10.89±2.85 11.3±3.35 Serum retinol (ug/dl) 37.3±24.9 27.4±14.03 32.3±20.58

Mean levels of haemoglobin and retinol in sickled cell cases Groups Sex (n) Haemoglobin (g/dl) Serum retinol (µg/dl) Normal Male (23) 11.48±0.953 35.53±5.64 Female (21) 12.4±0.54 30.42±2.74 Total (44) 11.94±0.541 33.78±3.12 Sickle cell trait (HbAS) Male (14) 14.21±0.70 54.72±8.93 Female (10) 12.16±0.52 30.13±6.27 Total (24) 13.35±0.5 42.76±6.25 Sickle cell disease (HbSS) Male (4) 9.55±1.1 29.8±2.74 Female (9) 8.59±0.90 19.41±3.68 Total (13) 8.89±0.67 22.6±2.92

Status of Haemoglobinopathies Haemoglobinopathy Male in % (n) Female in % Total Normal 17.6 (23) 16.2 (21) 33.8 (44) Sickle cell trait (Hb AS) 10.8 (14) 7.7 (10) 18.5 (24) Sickle cell disease (Hb SS) 3.1 (4) 6.9 (9) 10.0 (13) Beta thalessemic trait 13.1 (17) 15.4 (20) 28.5 (37) Beta thalessemic disease 4.6 (6) 9.2 (12) 49.2 (64) 50.8 (66) 100 (130) Prevelance of different types of hemoglobinopathy from reffered cases

Association of hemoglobinopathies with vitamin A deficiency (retinol) Normal In % (n) Sickle cell trait (Hb AS) disease (Hb SS) β – thallessemic trait β – thallessemic disease in % (n) Total In %(n) Normal (>20 µg/dl) 25.4(33) 13.1(17) 5.3 (7) 19.2 (25) 3.1 (4) 66.1 (86) Vitamin A deficient (10-20μg/dl) 5.4(7) 4.6(6) 3.8(5) 9.2(12) 2.3 (3) 25.3(33) Severely vitamin A deficient (<10µg/dl) 3.0(4 ) 0.8(1) 0.8 (1 ) 0 (0) 3.8 (5) 8.4(11) 33.8 (44) 18.5 (24) 10 (13) 28.5 (37) 9.2 (12) 100 (130)

Comparison of Mean serum retinol in control and sickle cell cases Age Retinol Significance Control (normal) 13 15.3±10.86 33.7±16.12 2.098 <0.05 Group of sickle cell disease 15.8 ±10.47 22.6±10.13 Control group for sct 23 32.7±10.58 42.7±29.7 0.0011 NS Group of sickle cell trait 24 33.0±11.33 42.8±29.4

T = 2.3805 P < 0.05 Type of sickling Mean retinol±SD Number (N) Sickle cell trait 42.76±29.43 24 Sickle cell disease 22.6±10.13 13

With increase in sickling percentage there was a significant decrease in the levels of retinol (r = -0.381, P< 0.05).

Sickling percent has significant effect on decreasing haemoglobin levels (r = -0.575, p<0.001).

A significant positive correlation was found between hemoglobin and retinol level (r = 0.592, p< 0.001).

DISCUSSION The sickled erythrocyte has been shown to be susceptible to lipid peroxidation and a role of antioxidants has been suggested for this process.Essien (1995), Adelekan (1989), Hasanato (2006) also found that There was a significant decrease in plasma levels of vitamin A in patients with SCA as compared with controls and sickle cell trait cases.Since patients with sickle cell anemia are under continuous oxidative stress due to sickle cell redox imbalance, antioxidant vitamins may contribute to the severity of sickle cell manifestations. Although the retinol level of sickle cell trait cases does not vary significantly from the control group, statistically significant negative correlation between percentage of sickling points that sickling reduces body’s vitamin A level. It may be due to the fact that the sickling in heterozygous case is maximum upto 34% in this study. Another possible reason for reduction of vitamin A is that influence of anemia which can lead to a variety of health problems since the oxygen required by the body is carried by RBCs and because oxidative stress can be generated by the iron released from damaged RBCs. Oxidative stress, overproduction of reactive oxygen species and impaired antioxidant potential are associated with anemia (Grune et al 2000).

Conclusion Sickling influences vitamin A (retinol) status in the body, may be due to the cause that sickling cells are more susceptible to oxidative stress which utilizes more amount of vitamin A as an antioxidant to neutralize ROS. Besides, severity of anemia influences depletion of vitamin A in the body. Therefore supplementation of antioxidants vitamins may ameliorate some of the sickle cell symptoms and improve the quality of life. Further studies are needed on other antioxidant vitamins and traces elements to elucidate the clinical manifestations of sickle cell anemia patients.

CONCLUSION Sickling influences plasma retinol concentration in the body this may be due to the cause that sickling cells are more susceptible to oxidative stress which utilizes more amount of vitamin A as an antioxidant to neutralize ROS. Besides that anemia also influences depletion of vitamin A in the body. Therefore supplementation of antioxidants vitamins may ameliorate some of the sickle cell symptoms and improve the quality of life. Further studies are needed on other antioxidants, vitamins and traces elements to elucidate the clinical manifestations of sickle cell anemia of Odisha s patients.

DISCUSSION The sickled erythrocytes shown to have lipid peroxidation and a role of antioxidants has been suggested for this process. Retinol, fat-soluble vitamin A content in sickle cell anaemia cases are shown to significantly low as compared with controls and sickle cell trait cases. Since patients with sickle cell anemia are under continuous oxidative stress due to sickle cell redox imbalance, antioxidant vitamins may contribute to the management of severity of sickle cell manifestations. Although the retinol level of sickle cell trait cases does not vary significantly from the control group, significant negative correlation observed between percentage of sickling points that reduces body’s vitamin A level. It may be due to the fact that the sickling in heterozygous cases are maximum upto 40%. Another possible reason for reduction of vitamin A is that influence of anemia which can lead to a variety of health problems since the oxygen required by the body is carried by RBCs and because oxidative stress can be generated by the iron released from damaged RBCs. Oxidative stress, overproduction of reactive oxygen species and impaired antioxidant potential are associated with anemia (Grune et al 2000).