Vitamins and Micronutrients level among Transfusion-dependent Subjects in Maiduguri, Nigeria.

Waziri Gimba, Bukar Alhaji, Obi Simon Osita, Medugu Jessy Thomus, Bukar Abdullahi Audu,
Department of Haematology, University of Maiduguri Teaching Hospital, Maiduguri
Osaro Erhabor
Department of Medical Laboratory Science, Usmanu Danfodiyo University, Sokoto.
Imoru Momodu
Department of Haematology, Aminu Kano University Teaching Hospital, Kano.
Digban Kestar Awharentomah, Jeremiah Zaccheaus Awortu.
Department of Chemical Pathology, Igbinedion University Teaching Hospital, Okada.
Olaniyan Matthew Folaranmi
Department of Medical Laboratory Science, Achievers University, Owo
All correspondence to:


Background: Blood transfusion has been very beneficial in relieving symptoms of anaemia but is associated with the risk of iron accumulation in tissues. Excess iron, by Fenton chemistry enhances oxidative stress. Antioxidant vitamins and micronutrients supplementation quenches the oxidative stress and obviate tissue damage. Objectives: The aim of the study is to evaluate some antioxidant vitamins and micronutrients level and determine whether supplementation is necessary in the management of transfusion-dependent patients. Methods: Blood samples were collected from 101 patients with ?8 units’ transfusion per year and its equivalent with no history of transfusion as controls. Serum vitamins (A and E), micronutrients (Zn and Cu), serum iron, ferritin and some haematological parameters (Haemoglobin and Haematocrit) were measured from the blood samples. Result: The underlying condition for repeated transfusion was largely SCA (96%) with male preponderance. Antioxidants, vitamins A and E were significantly decreased (p=0.000) compared to the controls, whereas micronutrients, zinc and copper levels were elevated (p>0.05 and 0.05 respectively). There was significant increase in both serum iron and ferritin in the case subjects (p 0.000). Haematocrit and haemoglobin levels was also significantly reduced in the study subjects (P<0.001). Conclusions: Our study demonstrates that the level of antioxidant vitamins was low in the transfusion-dependent subjects. Therefore, its supplementation is necessary to improve the antioxidant defence system and obviate possible tissue damage.

Blood transfusion is an important therapeutic approach especially in patients with refractory anaemia (Khaled et al., 2013, Antoine et al., 2015). One unit of red cell transfused provides more than 200 times the normal iron intake through intestinal absorption (Crichton and Ward, 2003) and there is no major mechanism of its excretion from the body (Hoffbrand et al., 2006, Kohgoet al., 2008). Hence, in transfusion dependence, iron released from transfused red cells inevitably saturate storage sites (Salibaet al., 2015) and the unbound (free) iron molecules are available to catalysethe conversion of molecular oxygen to highly reactive oxygen species (ROS) by Fenton chemistry (Adhamet al., 2014).In addition, ROS arealso generated from the normal metabolic processes, but neutralized by the body’s antioxidant defence (Bilbiset al., 2010, Mahdi, 2014). The rate of generation of ROS and its clearance by the antioxidant defence is delicately balanced (Pamplona, 2008) and If the production exceeds the capacity of the antioxidant systems to scavenge these species, then oxidative stress ensues (McCord, 2000).Withoxidative stress resulting from antioxidant depletion, peroxidative damage to cells is evident especially in tissues that stores iron such as liver, pituitary gland, pancreas and heart (Brissotet al., 2011).Antioxidantdefence systems are intimately involved in the prevention of cellular damage (Asare et al., 2009, Lobo et al., 2010). There are several enzymes and non-enzymatic defence such as vitamins and micronutrients (Simseket al., 2005) that scavenge free radicals and greatly enhance resistance to oxidative insult (Okochi and Okpuzor, 2005). This defence is significantly diminished in oxidative stress (Dhawan et al., 2005, Rashidiet al., 2011) due to increased consumption of the antioxidants (Mainasaraet al., 2016). However, antioxidants supplementation has been reported to improve antioxidant status and precludeoxidative damage by eliminating ROS (Prasad, 2009; Arruda et al., 2013;Tukuret al., 2015).Since transfusion-dependence is prone to oxidative stress and data concerning antioxidant vitamins and micronutrients in transfusion-dependent subjects in this environment are quite limited,it is imperative to measure the exogenous non-enzymatic antioxidant status and determine whether supplementation may be necessary.

This cross-sectionalstudy was conducted at the University of Maiduguri Teaching Hospital (UMTH) and State Specialist Hospital Maiduguri, Borno State. The ethical committees of the hospitals approved the study protocol and written informed consent was sought for and obtained from all participants. A total of 101 subjects age 20-36 years who had =8 units of red blood cells transfusions per year; and an equivalent number of age and sex unmatched normal subjects who had no history of blood transfusion were recruited for the study. Hospital records were used to determine patient’s eligibility for participation. The case subjects had history of continuous treatment with blood transfusionfor the pass at least two years.
Demographic characteristics of the patients were obtained usinga structured questionnaire. About 5 mL of blood was aseptically collected from each subject and, 3 mL and 2 mL were separately dispensed into plain and EDTA bottles respectively. The serum was harvested by centrifugation of the samples at 4000 RPM for 5 minutes and stored in cyro-vial at -20 0C until analysis.
Serum ferritin was measured using immune-enzymatic assay kit from Monobind Inc. Lake Forest, C 92630, USA while serum Iron was analysed by NITRO-PAS colorimetric method as described by Makino and colleagues (1988) using Iron Nitro-Pas mono-reagent, Centronic GmbH Am Kleinfeld 11, 85456 Wartenberg, Germany.The anticoagulated blood samples collected were tested some haematological parameters using Auto Haematology Analyser LW D3600, Shenzhen Landwind Industry Co., Ltd. Spectrophotometric methods described by Hashim and Schuttringer, (1966) and Bassey et al.,(1946) were used to analysed serum vitamin E and vitamin A respectively, while serum zinc and copper were colorimetrically estimated as described by Knoellet al. (2009) and Bartnikas, (2012) respectively using Quanti-ChromTM Assay Kits from BioAssay Systems, Corporate Place, Hayward, USA.
Statistical Package for Social Sciences (SPSS) for windows, Version 22.0 (SPSS Inc., Chicago, IL, USA) was used to express data generated as Mean ± SD. Statistical comparison of observed variables were made using student’s t-test. The level of significance (P-value) was set at less than 0.05.

Demographic and clinical characteristics of the study subjects are presented in table 1. Demographic information showed that the age of the case subjects was not significantly different (P>0.05) compared to that of the controls. The subjects were gender unmatched but predominantly male in both case and control participants (79.2% and 56.4% respectively). The underlying clinical condition for transfusion dependence was largely sickle cell anaemia (96%). Despite the regular transfusion therapy, significant decrease in haematocrit and haemoglobin levels (20.17± 4.17 and 6.56± 1.24 respectively) was noticed in the case subjects compared to the control participants (P0.000). Although iron overload was not reported in the case subjects, serum iron indices (serum iron and ferritin) were significantly (P 0.000) elevated compared to the controls. Serum antioxidant vitamins A, E and copper were significantly lower (P0.000 and P0.05 respectively) in the case subjects compared to the controls. However, serum zinc was moderatelyincreased but there was no statistically significant difference compared with the controls (P >0.05).

Disorders with refractory anaemia are managed with repeated blood transfusion and dependence on itis associated with iron accumulation that may leads to iron overload (Cid et al., 2014). Excess iron accumulation is accompanied by increased generation of reactive oxygen species (ROS) which may outweigh body’s antioxidant defence. Disruption of pro- and anti-oxidant balance is inevitably followed by oxidative stress (Simseket al., 2005;Vaculinet al., (2010)). Non-enzymatic antioxidant supplementation has been found to be beneficial in the prevention of oxidative stress (Rashidiet al., 2011).
The present study revealed that the serum iron indices (serum iron and ferritin)were significantly elevated(P <0.05)compared to the controls, which is consistent with several other studies (Rahu et al., 2008;Simseket al., 2005;Attiaet al, 2011). Higher ferritin content was directly linked to the accumulation of iron in the tissues of the patients. However, iron overload was not observed in the case subjects largely due to the low transfusion rate. This finding is inconsistent with Cid et al., (2014);Jokhioet al., (2009) and Gao et al.,(2014)who showed many patients with iron overloaded due to much higher transfusion rate.

The result of the current study indicate that the subjects were significantly deficient of serum antioxidant vitamins A and E.Similar results have been described in other studies by Tesoriereet al., (2001); Das et al.,(2004); Dhawan et al.,(2005) in patients with thalassemia and other transfusion-dependent entities. Ironically, non-transfused (non-iron overloaded) patients with SCD have also demonstrated low levels vitamins A and E due to chronic inflammation (Buisonet al., 2004).Similarly, Arruda et al.,(2013) reported deficiency of vitamin E in70% of non-transfusion dependent SCA patients. There was no iron overload in the present study that could promote iron-induced oxidative stress that consumes the antioxidant vitamins. However, given that 96% of the case subjects were SCA patients, the vitamin deficiency observed could be pursuant to increased pro-oxidant production in the sickle red blood cells (Titus et al., 2004).
In addition to the vitamins, abundant evidence demonstrates the role of micronutrients in protecting against free radical-induced oxidative damage (Rostanet al., 2002). Although Zincdeficiency is not relatively rare in patients with SCD (Prasad, 2002), our findings revealed that serum zinc is elevated but not significant (P>0.05) compared to the controls. Similarly, Mansi et al., (2009); Rashidiet al.,(2011);Mehdizadehet al., (2008) documented increased zinc level in transfusion-dependent thalassemia patients. Regular blood transfusion which predisposes to iron overload has been suggested to increase zinc level and prevent the zinc deficiency(Alexander et al., 2000). However, the subjects in the current study were regularly transfused but there was no iron overload noticed. Nile et al.,(2008) documented that zinc deficiency induces iron accumulation or conversely increase in zinc level lessen iron accumulation. Therefore, elevated zinc level observed in the current study might have been induced by decreased iron accumulation (absence of iron overload) orabnormal glomerular filtration of zinc as seen in chronic haemolysis (Mansi et al., 2009), obvious symptom of most underlying clinical entities of the present study. This phenomenon can be exploited in transfusion-dependent subjects to prevent iron overload and its attendant consequences by maintaining normal zinc level. Copper level was significantly high (P0.05)compared the control group and this agrees with some studies (Irshaidet al., 2009; Malakaret al., 2014). Copper has an antioxidant property (Shaziaet al., 2012) and its deficiency is quite rare (Turnlundet al., 1998).
This study revealed that haemoglobin and haematocrit were significantly (p<0.05) lower compared to the controls and is in accordance with the outcome of studies by Mansi et al., (2009)and Karim et al., (2016).Anaemia is the hallmark of transfusion dependency and is reported even in non-chronically transfused subjects with SCD (Fung et al., 2007).

Conclusion and Recommendations
Our findings indicated that there was no iron overload that may induce oxidative stress which could consume the antioxidants vitamins. However, the antioxidant vitamins were deficient. Therefore,administration of antioxidants vitamins may help strengthen the antioxidant defencesystem and obviate possible tissue damagearising from pro-oxidants.Further study is suggested on the status of free radicals and endogenous antioxidants to determine whether the deficiency is coupled with enhanced free radicals’ generation and reduced endogenous antioxidants.

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