|Year : 2016 | Volume
| Issue : 1 | Page : 8-12
The human immunodeficiency virus infection is associated with positive iron balance among subjects in Nnewi, South East Nigeria
Patrick O Manafa1, John C Aneke2, Chide E Okocha2, Stella-Maris C Okeke1, Nancy C Ibeh1, George O Chukwuma1, Ejike K Nwene3
1 Department of Medical Laboratory Science, Nnamdi Azikiwe University, Nnewi, Anambra State, Nigeria
2 Department of Haematology, College of Health Sciences, Nnamdi Azikiwe University, Nnewi, Anambra State, Nigeria
3 Clinical Services, Initiative for Good Health in , Enugu, Nigeria
|Date of Web Publication||13-Jul-2017|
John C Aneke
Department of Haematology, College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, PMB 5001, Nnewi, Anambra State
Source of Support: None, Conflict of Interest: None
BACKGROUND: The human immunodeficiency virus (HIV) infection is associated with a number of metabolic derangements which have a remarkable impact on disease mortality and morbidity.
OBJECTIVE: To evaluate the effect of HIV infection on iron status in apparently healthy seropositive adult subjects seen at a tertiary hospital in South East Nigeria.
SUBJECTS AND METHODS: A total of eighty subjects were recruited for the study which comprised of randomly selected forty HIV seropositive and seronegative individuals, respectively. Each participant had 5 ml of blood collected for serum ferritin, iron, total binding capacity (TIBC), and percentage saturation of transferrin estimation. Ferritin was determined using the enzyme-linked immunosorbent assay technique while iron and TIBC were measured by spectrophotometry; percentage transferrin saturation was calculated using the standard formula. Statistical analysis was performed using SPSS (version 20), the Student's t-test was used for the comparison of means while the level of statistical significance was set at P< 0.05.
RESULTS: The means of serum ferritin and transferrin saturation were significantly higher (164.30 ± 138.52 ng/ml vs. 88.10 ± 57.75 ng/ml; P= 0.002 and 44.67 ± 12.95% vs. 32.42 ± 4.67%; P< 0.001, respectively) while the mean of serum TIBC was significantly lower (346.25 ± 81.83 μg/dl vs. 395.20 ± 66.70 μg/dl; P= 0.004, respectively) in test subjects compared with controls. Serum iron was not significantly different in the two populations of study subjects (P = 0.30).
CONCLUSION: Infection with the HIV is associated with significant tissue iron loading; this could have important implications on disease course and morbidity.
Keywords: Chronic inflammation, human immunodeficiency virus infection, iron status
|How to cite this article:|
Manafa PO, Aneke JC, Okocha CE, Okeke SMC, Ibeh NC, Chukwuma GO, Nwene EK. The human immunodeficiency virus infection is associated with positive iron balance among subjects in Nnewi, South East Nigeria. J HIV Hum Reprod 2016;4:8-12
|How to cite this URL:|
Manafa PO, Aneke JC, Okocha CE, Okeke SMC, Ibeh NC, Chukwuma GO, Nwene EK. The human immunodeficiency virus infection is associated with positive iron balance among subjects in Nnewi, South East Nigeria. J HIV Hum Reprod [serial online] 2016 [cited 2018 Aug 17];4:8-12. Available from: http://www.j-hhr.org/text.asp?2016/4/1/8/210592
| Introduction|| |
The human immunodeficiency virus (HIV) infection remains a major global health challenge and has recently assumed a pandemic dimension, particularly in poorer regions of the world. Globally, up to 33.2–37.2 million people were estimated to be living with the virus by the end of 2013, Nigeria has an infection incidence of 9%. Infection with the HIV has been documented to be pantropic, affects virtually all the organs of the body and causes diverse metabolic derangements in addition to the well-characterized immune dysfunction. Some of these metabolic derangements range from oxidative stress (due to persistent immune activation associated with uncontrolled viral replication, leading to excessive reactive oxygen species generation) to derangement in iron metabolism, resulting in excess accumulation of tissue iron.,,
Iron is an essential trace element which could be present either in the ferrous (Fe 2+) or ferric (Fe 3+) state in a number of biological systems, whereas transferrin is a glycoprotein receptor that transports iron from storage to sites of utilization in the body. Ferritin is a globular protein complex (consisting of 24 protein subunits) which is the primary storage form of iron; in so doing it is critical in protecting cells from iron-induced oxidation-reduction reactions. It is equally an acute phase protein, as such serum levels have been found to be raised in a number of acute and chronic inflammatory (including infectious and malignant) conditions. Increased serum ferritin levels have been associated with disease progression in individuals with HIV infection.
Progressive accumulation of iron in macrophages and endothelial cells have been documented to occur in HIV infection (particularly in subjects with advanced disease) resulting in excess storage iron in the bone marrow, brain and other vital organs and increase in serum ferritin levels., Excess tissue iron in subjects with HIV infection is thought to occur secondary to the chronic inflammatory response associated with the disease, this leads to iron retention in the reticuloendothelial system, with paradoxical low serum concentration. Sequel to the fact that iron is needed for lymphocyte activation and proliferation, imbalances in its metabolism has been linked with altered immune function and disease progression in patients with HIV infection. This effect appears to be mediated through iron-induced alteration in T-and B-lymphocytes proliferation and function; both cells are important “players” in the pathogenesis of HIV infection. It is thus based on the fact that iron homoeostasis is affected by multiple physiologic and pathological mechanisms (including immunologic, infectious, clinical, and nutritional variables) that make it an appropriate barometer of overall health status.,
Only a few studies had attempted to assess iron status in individuals with HIV infection in Nigeria (particularly in South-Eastern part of the country), this study was therefore designed to bridge this knowledge gap by evaluating serum ferritin, serum iron, total iron binding capacity (TIBC) and percentage transferrin saturation in adult seropositive subjects, seen at the Nnamdi Azikiwe University Teaching Hospital, Nnewi, South-East Nigeria.
| Subjects and Methods|| |
This research was carried out in Nnamdi Azikiwe University Teaching Hospital, a tertiary hospital located in Nnewi, Anambra State in South-East Nigeria. The hospital equally receives referrals from neighboring states of Enugu, Delta and Imo states.
This was a case-control study designed to assess serum ferritin, serum iron, TIBC and percentage transferrin saturation in HIV seropositive subjects attending the HIV outpatient clinic of Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State, Nigeria. A total of eighty subjects were randomly recruited for this study, comprising forty HIV seropositive subjects and forty HIV seronegative individuals (as the control group).
Inclusion criteria included:
- Confirmed HIV seropositivity for at least 1 year before recruitment for the study
- Subjects and controls must be above 18 years of age at the time of recruitment.
Exclusion criteria were as follows:
- Subjects with symptomatic anemia or who has had blood transfusion in the preceding 3 months
- Subjects with a history suggestive of hemoglobinopathy, on dialysis or had clinical evidence of sepsis (including fever or any other feeling of unwell).
The ethical approval for this research was obtained from the Ethics Committee of Nnamdi Azikiwe University Teaching Hospital, Nnewi, Anambra State and each participant gave informed consent.
Five milliliters of venous blood was collected aseptically from each subject and dispensed into a plain container, allowed to clot and centrifuged at 5000 rpm for 5 min. Serum was extracted for serum ferritin, iron and percentage of transferrin saturation estimation as well as confirmation of HIV status, and stored at −70°C for batch testing.
Human immunodeficiency virus testing
This was performed using the National algorithm as has been previously described, which involved serial testing by determine, Uni-Gold and Stat-Pak test kits.,, These are all rapid immunochromatographic test kits for the qualitative detection of antibodies to HIV-1 and -2.
Estimation of ferritin level
The levels of ferritin were estimated by solid phase enzyme-linked immunosorbent assay described by Uotila et al. The testing involved the addition of 25 μL each of dilutions of standard, controls and samples which were dispensed into appropriately coated wells. Ferritin biotin reagent was added to each well and swirled for 30 s to mix, followed by 30 min incubation at room temperature. The reaction mix was washed three times, followed by the addition of 100 μl of ferritin enzyme conjugate to each well and further incubation for 30 min at room temperature. Working substrate solution (100 μl) was added to each well followed by 50 μl of stop solution after which the liquid turned yellow, the absorbance of the reaction mix was read using the microplate reader at 450 nm immediately.
Estimation of serum iron, percentage transferrin saturation and total iron binding capacity
The protocol for estimating these were adapted from the methods earlier described by Sharma et al.
Serum iron estimation
Test tubes were selected and labeled for standard, blank, control and samples. Into each tube, 2.5 ml of Iron buffer reagent was added, followed by 0.5 ml of sample. For the reagent blank, 0.5 ml of iron-free water was used.
The contents of the tubes were mixed and the first absorbance of all the tubes was read against the reagent blank at 560 nm and recorded as A1. To all the tubes, 0.05 ml of Iron color reagent was added and incubated at 37°C. At exactly 10 min, the second absorbance was read and recorded as A2.
The change in absorbance (A2–A1) was determined and the serum iron concentration calculated using the formula below;
Unsaturated iron-binding capacity estimation
Test tubes were selected and labeled for standard, blank, control and Sample and 2 ml of UIBC buffer reagent was added into all the tubes. To the blank, standard and test were added 1 ml of iron-free water, 0.5 ml of iron-free water plus 0.5 ml standard and 0.5 ml of sample plus 0.5 ml of standard respectively.
The contents of the tubes were mixed, and the first absorbance read against the reagent blank at 560 nm and recorded as A1. Into all the tubes, 0.05 ml of Iron color reagent was added and incubated at 37°C. At exactly 10 min, the second absorbance was read and recorded as A2.
The change in absorbance (A2–A1) was determined and unsaturated iron-binding capacity concentration calculated using the formula below;
TIBC = Iron level + UIBC
Percentage transferrin saturation (%) = (100 × serum iron)/TIBC.
Statistical package for social science (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA) was used for all data analysis and presented as mean ± standard deviation. The Student's t-test was used to compare two means in test subjects and controls and the level of significance was set at P< 0.05.
| Results|| |
The means of serum ferritin and transferrin saturation were significantly higher in HIV subjects compared with controls (164.30 ± 138.52 ng/ml vs. 88.10 ± 57.75 ng/ml; P= 0.002 and 44.67 ± 12.95% vs. 32.42 ± 4.67%; P< 0.001, respectively), [Table 1].
|Table 1: Comparison of means of iron parameters in test and control subjects|
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Correspondingly, the mean of serum TIBC was significantly lower in test subjects compared with controls (346.25 ± 81.83 μg/dl vs. 395.20 ± 66.70 μg/dl; P= 0.004, respectively), [Table 1]. Serum iron was not significantly different in test subjects and controls (149.87 ± 36.97 μg/dl vs. 142.62 ± 24.13 μg/dl, P= 0.30), [Table 1].
| Discussion|| |
Abnormal iron distribution as well as low hematocrit has increasingly been linked with higher morbidity and mortality in HIV-infected subjects., This is thought to be sequel to widespread chronic immune activation and inflammation both of which are known to exacerbate anemia of inflammation (AI).,
Systemic inflammatory response that follow HIV infection has been shown to block dietary iron release from enterocyte and equally causes iron redistribution into cellular storage sites, including tissue macrophages. This affects iron availability for erythropoiesis and disrupts iron homeostasis, in addition, the direct effect of the HIV on erythroid precursors as well as the blunting of erythropoietin response to anemia are thought to impair iron balance and may contribute to development of clinical anemia in subjects infected with HIV infection. It is therefore on this basis that clinical anemia has been reported as one of the most common presentation of HIV infection, particularly in treatment naïve subjects. Even though, clinical anemia tends to resolve following the commencement of highly active antiretroviral therapy (HAART), a significant proportion of patients have unresolved anemia or it may develop after the initiation of therapy.
Findings from this study among our population of HIV-infected subjects appear congruous with earlier assertions to the effect that HIV seropositivity is significantly associated with positive iron balance. In this study, the mean serum ferritin levels and transferrin saturation were significantly higher in HIV-seropositive subjects compared with the controls (P = 0.002 and P< 0.001, respectively), [Table 1]. Similarly, the mean serum TIBC was significantly lower in seropositive subjects in comparison with the control group (346.25 ± 81.83 ug/dl vs. 395.20 ± 66.70 ug/dl, P= 0.004), [Table 1].
The earlier report of Banjoko et al. showed a significant positive iron balance in eighty treatment naïve HIV seropositive individuals in Ile-Ife, South-West Nigeria. The study equally emphasized that increased iron load in these subjects was associated with significant depletion in CD4 T-lymphocyte count and disease-related mortality. Similarly, McDermid et al. followed up 1362 HIV-seropositive subjects in the Gambia over a period of 11.5 years and showed that excess tissue iron in HIV-infected individuals was predictive of more severe clinical HIV infection and mortality. Other studies have equally emphasized that HIV infection is associated with a progressive accumulation of iron in macrophages, endothelial and other cells, with consequent deposition in the bone marrow, brain and other organs, particularly in patients with advanced disease.,
Central to iron accumulation in a number of disease conditions associated with significant inflammation is the protein hepcidin., Under inflammatory conditions, hepcidin production is greatly induced through both the interleukin-6 and bone morphogenetic protein-dependent pathways, this leads to the inhibition of ferroportin-mediated iron transport from the gastrointestinal enterocytes and intracellular iron retention in iron-recycling macrophages., In fact, it is increasingly becoming clear that increased serum hepcidin levels (and positive tissue iron balance) may be related to higher rates of viral transcription and greater immunosuppression in HIV-infected individuals.
The possible implications of the increased body iron status in HIV subjects are protean. Both increased plasma and body iron stores have been shown to significantly potentiate the generation of free radicals (oxidative stress) via the Fenton/Haber–Weiss reaction., Excess tissue iron is believed to react with hydroxyl ion and other free radicals leading to profound inflammatory tissue damage. In addition, iron potentially leads to the activation of a number of oncogenes; therefore derangement in iron homeostasis in HIV infection may be related to the pathogenesis of HIV-associated cancers like Kaposi's sarcoma and lymphomas. Importantly, it has equally been observed that the iron-rich environment found in conditions associated with positive iron balance could predispose to a number of microbial infections such as Mycobacterium tuberculosis, with remarkable impact of disease-related morbidity and mortality.
| Conclusion|| |
Significant iron loading occurred in our population of apparently healthy adult subjects with HIV infection. While this is in keeping with previous reports, it further underscores the importance of iron status in the pathogenesis of HIV infection. Routine evaluation of iron status in our patients could become a tool for risk stratification, prognostication and appropriate management.
Future line of research
The influence of iron loading on treatment outcome and overall survival in HIV-seropositive individuals in our center will be an interesting topic for future research.
Limitations of the study
- The duration and type of HAART in study subjects were not captured in our data set
- The immune status of test subjects as well as other markers of acute phase reaction was not evaluated in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Arindam P, Maitreyee B, Shantasil P, Biswadip G, Sandip S, Anirban G, et al
. Anemia in antiretroviral naïve HIV/AIDS patients: A study from Eastern India. Online J Health Allied Sci 2011;10:4. Available from: http://www.ojhas.org/issue40/2011-4-4.htm
. [Last accessed on 2017 Jan 03].
Martínez E, Gatell JM. Metabolic abnormalities and body fat redistribution in HIV-1 infected patients: The lipodystrophy syndrome. Curr Opin Infect Dis 1999;12:13-9.
Salman S, Berrula L. Immune modulators of HIV infection: The role of reactive oxygen species. J Clin Cell Immunol 2012;3:121-4.
Doherty CP. Host-pathogen interactions: The role of iron. J Nutr 2007;137:1341-4.
Drakesmith H, Prentice A. Viral infection and iron metabolism. Nat Rev Microbiol 2008;6:541-52.
Jain S, Gautam V, Naseem S. Acute-phase proteins: As diagnostic tool. J Pharm Bioallied Sci 2011;3:118-27.
Meyron-Holtz EG, Moshe-Belizowski S, Cohen LA. A possible role for secreted ferritin in tissue iron distribution. J Neural Transm (Vienna) 2011;118:337-47.
Okocha CE, Aneke CJ, Ibeh N, Onah CE, Okafor N. Evaluation of serum ferritin levels and blood counts in apparently normal individuals in Nnewi, South-east Nigeria. Niger J Med 2015;24:64-70.
López-Calderón C, Palacios R, Cobo A, Nuño E, Ruiz J, Márquez M, et al
. Serum ferritin in HIV-positive patients is related to immune deficiency and inflammatory activity. Int J STD AIDS 2015;26:393-7.
Boelaert JR, Piette J. The Potential Role of Iron in HIV Infection and its Implications. 6th
Internet World Congress for Biomedical Sciences. (Presentation # 142); 2000. Available from: Ibrarian.net/…/The_potential_negative_role_of_iron_in_HIV_infect.pdf? [Last accessed on 2017 Jan 03].
Melchior JC, Niyongabo T, Henzel D, Durack-Bown I, Henri SC, Boulier A. Malnutrition and wasting, immune-depression, and chronic inflammation as independent predictors of survival in HIV-infected patients. Nutrition 1999;15:865-9.
Gordeuk VR, Delanghe JR, Langlois MR, Boelaert JR. Iron status and the outcome of HIV infection: An overview. J Clin Virol 2001;20:111-5.
Minchella PA, Armitage AE, Darboe B, Jallow MW, Drakesmith H, Jaye A, et al.
Elevated hepcidin is part of a complex relation that links mortality with Iron homeostasis and anemia in men and women with HIV infection. J Nutr 2015;145:1194-201.
Obeagu EI, Aneke J, Okafor CN, Essein UC, Ochei KC, Obeagu GU. Assessment of Serum Iron Status of Malnourished Infants in Umuahia, Abia State, Nigeria. Sch J App Med Sci 2016;4:4384-7.
Manafa P, Chukwuanukwu R, Iloghalu E, Onyenekwe C, Ifeanyichukwu M, Mbamalu C. Cryptococcus neoformans antigenemia in HIV positive pregnant women attending a PMTCT clinic in South-East Nigeria. J Biol Agric Healthc 2013;3:2224-3208.
Okocha EC, Aneke JC, Ezeh TU, Ibeh NC, Nwosu GA, Okorie IO, et al
. The epidemiology of transfusion-transmissible infections among blood donors in Nnewi, South-East Nigeria. Afr J Med Health Sci 2015;14:125-9. [Full text]
Chukwuma GO, Ahaneku GI, Chukwuma OM, Nwaokorie FO, Faneye AO, Manafa PO, et al
. Discordant responses to highly active antiretroviral therapy in HIV-I infected subjects at the Nnamdi Azikiwe University Teaching Hospital Nnewi. Sky J Med Med Sci 2016;4:60-8.
Uotila M, Ruoslahti E, Engvall E. Two-site sandwich enzyme immunoassay with monoclonal antibodies to human alpha-fetoprotein. J Immunol Methods 1981;42:11-5.
Sharma D, Mathur R, Singh P. Iron metabolism: A review. Indian J Clin Biochem 1993;8:80-101.
May M, Boulle A, Phiri S, Messou E, Myer L, Wood R, et al
. Prognosis of patients with HIV-1 infection starting antiretroviral therapy in sub-Saharan Africa: A collaborative analysis of scale-up programmes. Lancet 2010;376:449-57.
O'Brien ME, Kupka R, Msamanga GI, Saathoff E, Hunter DJ, Fawzi WW. Anemia is an independent predictor of mortality and immunologic progression of disease among women with HIV in Tanzania. J Acquir Immune Defic Syndr 2005;40:219-25.
Kassebaum NJ, Jasrasaria R, Naghavi M, Wulf SK, Johns N, Lozano R, et al.
A systematic analysis of global anemia burden from 1990 to 2010. Blood 2014;123:615-24.
Ganz T, Nemeth E. Hepcidin and iron homeostasis. Biochim Biophys Acta 2012;1823:1434-43.
Belperio PS, Rhew DC. Prevalence and outcomes of anemia in individuals with human immunodeficiency virus: A systematic review of the literature. Am J Med 2004;116 Suppl 7A:27S-43S.
Banjoko SO, Oseni FA, Togun RA, Onayemi O, Emma-Okon BO, Fakunle JB. Iron status in HIV-1 infection: Implications in disease pathology. BMC Clin Pathol 2012;12:26.
McDermid JM, Jaye A, Schim van der Loeff MF, Todd J, Bates C, Austin S, et al.
Elevated iron status strongly predicts mortality in West African adults with HIV infection. J Acquir Immune Defic Syndr 2007;46:498-507.
Usanga EA, Okafor AO, Akwiwu EC. Iron-related parameters of HIV-infected patients attending University of Calabar Teaching Hospital, Nigeria. J Dent Med Sci 2016;15:65-8.
Gkouvatsos K, Papanikolaou G, Pantopoulos K. Regulation of iron transport and the role of transferrin. Biochim Biophys Acta 2012;1820:188-202.
Steinbicker AU, Muckenthaler MU. Out of balance-systemic iron homeostasis in iron-related disorders. Nutrients 2013;5:3034-61.
Xu M, Kashanchi F, Foster A, Rotimi J, Turner W, Gordeuk VR, et al
. Hepcidin induces HIV-1 transcription inhibited by ferroportin. Retrovirology 2010;7:104-8.
Salman S, Berrula L. Immune modulators of HIV infection: The role of reactive oxygen species. J Clin Cell Immunol 2012;3:121-5.
Kell DB. Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson's, Huntington's, Alzheimer's, prions, bactericides, chemical toxicology and others as examples. Arch Toxicol 2010;84:825-89.
Reizenstein P. Iron, free radicals and cancer. Med Oncol 1991;8:229-33.
McDermid JM, Hennig BJ, van der Sande M, Hill AV, Whittle HC, Jaye A, et al.
Host iron redistribution as a risk factor for incident tuberculosis in HIV infection: An 11-year retrospective cohort study. BMC Infect Dis 2013;13:48.