Background: Hepcidin is the principal regulator of iron absorption and its tissue distribution. Its correlation with iron homeostasis in individuals infected with human immunodeficiency virus type-1 (HIV-1) treated with different regimens of highly active antiretroviral therapy (HAART) was investigated. Methods: Serum hepcidin levels were determined in 448 volunteers. Of these, 372 were HIV-1-infected individuals, and 93 did not receive HAART (ART-na ïve) while 279 received HAART consisting of a non-nucleoside reverse transcriptase inhibitor (NNRTI-based) and protease inhibitors (PI-based); both were used in association with a nucleoside reverse transcriptase inhibitor (NRTI). Seventy-six additional HIV-1 seronegative individuals were enrolled in the study. The following parameters were quantified: hematological parameters, iron biomarkers and markers of infection (CD4+ and CD8+ T-cells), and HIV-1 RNA (viral load). Results: Serum hepcidin, iron and ferritin levels, as well as the marker of infection, CD4+ T-cells, were significantly lower in the ART-na ïve group compared with other groups. Additionally, transferrin saturation, iron binding capacity, hemoglobin level and erythrocyte level were not significantly different, and anemia was not observed in the different groups. Conclusions: HIV-1 infection affected serum hepcidin, iron and ferritin levels in the ART-na ïve group, and the different HAART regimens restored the levels of hepcidin and iron homeostasis in HIV-1-infected individuals who have undetectable HIV-1 RNA levels.
The control of absorption, storage and circulation of iron in the body is regulated by complex mechanisms to maintain an appropriate amount of iron in the circulation and within tissues and avoid deficiency or overload [
This cross-sectional study was conducted from November 2012 to December 2013 with 448 volunteers (age ≥ 18 years). Of these, 372 HIV-1-infected individuals were at the University Hospital of the Federal University of Santa Catarina-HU/UFSC, and the Clinical Hospital-University of São Paulo Medical School-HCFMUSP. Seventy-six additional HIV-1 seronegative individuals from the blood bank at HU/UFSC were also enrolled in the study (
Blood samples (5 µL) with ethylenediaminetetraacetic acid (EDTA) were obtained by antecubital venous puncture using a vacuum system (VacutainerÒ, Becton/Dickinson Co., NJ, USA) in the early morning after subjects had fasted for 12 to 14 hours. Serum samples were obtained by centrifuging the blood in a CELM model LS-II centrifuge (CELM Co., SP, BRA) at 2500 rpm (1050× g) at 4˚C for10 minutes. The samples were then divided into 300 μL aliquots, transferred to cryogenic tubes and stored in liquid nitrogen at −180˚C until testing.
HIV-1 RNA was quantified from plasma using the commercially available Nucleic Acid Sequence Based
HIV-1-infected individuals (n = 372) | |||||
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Parameters(1) | Seronegative (n = 76) | ART-naïve (n = 93) | NNRTI-based (n = 151) | PI-based (n = 128) | P value(2) |
Gender (M/F) | 48/28 | 55/38 | 98/53 40 (21, 57) 21 (19, 32) | 71/57 | - |
Age (years) | 36 (18, 54) | 38 (18, 56) | 41 (18, 57) | ns | |
BMI (kg/m2) | 21 (20, 24) | 18 (16, 23) | 22 (18, 33) | ns |
Note: NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; M, male; F, female; BMI, body mass index [weight/height2 (kg/m2)]; ns, no significant difference. (1)Median [interquartile range (IQR): 2 % - 75%; 95% confidence interval (CI)]. (2)P value: comparison between groups by univariate analysis of variance (ANOVA) for multiple comparisons and Tukey’s honest significant difference (HSD) test.
Antiretroviral therapy, n (%)(1) | (n = 279) |
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NNRTI-based, n = 151 (54) | |
Efavirenz (EFV) 600 mg (qd) + zidovudine (AZT) 300 mg + lamivudine (3TC) 150 mg (bid) | 78 (52) |
Efavirenz (EFV) 600 mg (qd) + estavudine (d4T) 40 mg + lamivudine (3TC) 150 mg (bid) | 11 (7) |
Efavirenz (EFV) 600 mg (qd) + tenofovir (TDF) 300 mg + lamivudine (3TC) 150 mg (bid) | 8 (5) |
Nevirapine (NVP) 200 mg (bid) + zidovudine (AZT) 300 mg + lamivudine (3TC) 150 mg (bid) | 28 (19) |
Nevirapine (NVP) 200 mg (bid) + estavudine (d4T) 40 mg + lamivudine (3TC) 150 mg (bid) | 15 (10) |
Nevirapine (NVP) 200 mg (bid) + tenofovir (TDF) 300 mg + lamivudine (3TC) 150 mg (bid) | 11 (7) |
PI-based, n = 128 (46) | |
Lopinavir/ritonavir (LOP/r) 400 mg/100 mg (bid) + zidovudine (AZT) 300 mg + lamivudine (3TC) 150 mg (bid) | 60 (46) |
Lopinavir/ritonavir (LOP/r) 400 mg/100 mg (bid) + estavudine (d4T) 40 mg + lamivudine (3TC) 150 mg (bid) | 68 (54) |
Note: NNRTI, non-nucleoside reverse transcriptase inhibitor; qd, once daily; bid, twice daily; PI, protease inhibitor. (1)Therapy with oral administration.
Amplification kit (NASBA®, Organon Teknika, Boxtel, the Netherlands). The minimum detection limit indicated by the manufacturer is 50 copies/mL of HIV-1 RNA. Subpopulations of CD4+ T-cells, CD8+ T-cells, and CD3+ T-cells as well as CD4+:CD3+ and CD8+:CD3+ cells were quantified by three-color flow cytometry using monoclonal antibodies and a Becton/Dickinson FACS count® flow cytometer (Becton/Dickinson Co., San Jose, CA, USA).
The serum hepcidin levels were determined by an enzyme-linked immunosorbent assay (ELISA) kit for hepcidin (ELISA-Hepc®; order no. E1979Hu, 96tests) following the manufacturer’s instructions (Uscn Life Science Inc., Wuhan, China). Blood in EDTA-containing vacutainers was analyzed using an automatic cell counter, Coulter® LH 750 Hematology Analyzer (Beckman Coulter Inc., CA, USA), to determine the complete blood count, including erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet and reticulocytes. Regarding iron metabolism, briefly, we quantified serumiron, ferritin, transferrin saturation and total iron binding capacity (TIBC) using the Siemens Dimension® Clinical Chemistry Systemand Siemens Immulite 2000 system (Siemens Medical Solutions Diagnostics, LA, USA). All of these parameters were measured during routine clinical screening.
The results are expressed as the arithmetic mean plus standard deviation (mean ± SD) and median [interquartile range (IQR): 95% confidence interval (CI)]. Calculations to determine whether differences between the various groups were significant were carried out using the non-parametric Kruskal-Wallistest or Student’s t-test. We also used univariate analysis of variance (ANOVA) for multiple comparisons followed by Tukey’ shonest significant difference (HSD) test. Correlation analysis between parameters was performed using Spearman’s test. All descriptive and statistical analyses were performed using the Statistical Package for Social Sciences Software version 12.0® (SPSS Inc., Chicago, IL, USA) and SAS®8e. Charts were constructed using Graph Pad Prism® version 5.0 (Graph Pad Software Inc., La Jolla, USA). Statistical significance was set at P < 0.05.
Participants were informed of all procedures in the study, and their consent to participate in the study was confirmed in writing according to the guidelines of the Bioethical Committee. Ethical approval for this study was granted by the ethical committees of the Human Research of Clinical Hospital-University of São Paulo Medical School-HCFMUSP and HU/UFSC, CAAE 07288912.6.0000.0065, number 141.739, November 25, 2012.
In the study population, the parameters age (years) and body mass index [BMI; weight/height2 (kg/m2)] were homogeneous, with no differences between the groups of seronegative and HIV-1-infected individuals. In both groups, the number of males was higher than females (
The markers of infection by HIV-1, such as CD4+ T-cells and CD8+ T-cells, showed suitable means in the treated groups (
Serum hepcidin levels were significantly lower in the ART-naïve group compared with the NNRTI-based, PI-based and seronegative groups. However, there were no significant differences among the other groups (
Hematological screening tests showed no occurrence of anemia in the study groups. Hemoglobin levels and erythrocyte counts showed no significant differences when compared (
HIV-1-infected individuals (n = 372) | ||||
---|---|---|---|---|
Parameters(1) | ART-naïve (n = 93) | NNRTI-based (n = 151) | PI-based (n = 128) | P value(2) |
CD4+ T-cell (cells/mm3) | 374 (217, 483)* | 477 (353, 695) | 497 (388, 679) | <0.05 |
CD8+ T-cell (cells/mm3) | 958 (641, 1287) | 940 (671, 1201) | 1.075 (732, 1223) | ns |
CD4+: CD8+ ratio | 0.4 (0.3, 0.6) | 0.5 (0.3, 0.6) | 0.5 (0.3, 0.5) | ns |
HIV-1 RNA (copies/mL)(3) | 98.340 ± 96.270* | <50 | <50 | <0.05 |
Note: NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; ns, no significant difference. (1)Median [interquartile range (IQR): 25% - 75%; 95% confidence interval (CI)]. (2)P value: comparison between groups by univariate analysis of variance (ANOVA) for multiple comparisons and Tukey’s honest significant difference (HSD) test. (3)Mean values (m ± SD). *P < 0.05 when compared with other groups.
HIV-1-infected individuals (n = 372) | |||||
---|---|---|---|---|---|
Parameters(1) | Seronegative (n = 76) | ART-naïve (n = 93) | NNRTI-based (n = 151) | PI-based (n = 128) | P value(2) |
Hepcidin (µg/L) | 352 ± 119 | 261 ± 137* | 388 ± 184 | 406 ± 205 | <0.05 |
Hemoglobin (g/dL) | |||||
Male | 15.9 ± 1.4 | 14.3 ± 1.9 | 15.3 ± 1.8 | 16.0 ± 1.9 | ns |
Female | 14.4 ± 1.3 | 13.0 ± 1.4 | 14.0 ± 1.6 | 14.2 ± 1.5 | ns |
Erythrocytes (cells/mm3) | |||||
Male | 5.2 ± 0.6 | 4.5 ± 0.5 | 5.1 ± 0.5 | 5.3 ± 0.6 | ns |
Female | 4.6 ± 0.3 | 4.0 ± 0.4 | 4.5 ± 0.3 | 4.8 ± 0.4 | ns |
Iron biomarkers | |||||
Iron (µg/dL) | 86 ± 18 | 77 ± 25* | 88 ± 25 | 85 ± 23 | <0.05 |
Ferritin (ng/dL) | 212 ± 57 | 151 ± 41* | 185 ± 54 | 181 ± 62 | <0.05 |
Transferrin saturation (%) | 27 ± 4 | 25 ± 6 | 27 ± 6 | 26 ± 10 | ns |
TIBC (µg/dL) | 306 ± 37 | 308 ± 63 | 317 ± 49 | 322 ± 84 | ns |
Note: NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; TIBC, total iron binding capacity; ns, no significant difference. (1)Mean values (m ± SD). (2)P value: comparison between groups by ANOVA and Tukey’s honest significant difference (HSD) test. *P < 0.05 when compared with other groups.
parameters did not show significant changes as characterized by anemia (data not shown). These data suggest that even in the ART-naïve group, HIV-1 infection associated with a reduction inCD4+ T-cells did not alter the physiology and morphology of erythrocytes.
Serum iron and ferritin levels were significantly different only in the ART-naïve group, which showed lower values compared with the NNRTI-based, PI-based and seronegative groups. The values for transferrin saturation and TIBC showed no significant differences between groups (
We demonstrated that serum hepcidin levels, as well as iron and ferritin, were reduced in the ART-naïve group and showed a positive correlation with the number of CD4+ T-cells, but not with HIV-1 RNA levels. In the groups receiving different HAART regimens, these same parameters were within the normal range and/or represented values that are considered indicators of immune recovery, suggesting that immune status in HIV-1- infected individuals may directly affect serum hepcidin levels and iron metabolism.
In this study, HIV-1-infected individuals who received different regimens of HAART had undetectable HIV-1 RNA levels, and the number of CD4+ T-cells was within the normal range, while the ART-naïve group had reduced levels of HIV-1 RNA and CD4+ T-cells. HIV-1 RNA levels and CD4+ T-cell numbers are indicators of the clinical stage of infection and are considered prognostic factors for the evaluation of the course of HIV-1 infection [
The observed positive correlation between serum hepcidin levels and CD4+ T-cells in the ART-naive group, but not in the groups receiving HAART, is different than the results obtained by Wisaksana and colleagues (2013) [
In HIV-1 infection, in addition to macrophages and CD4+ T-cells, there is a direct infection of progenitor cells in the bone marrow, which in turn can induce bone marrow suppression with a consequent increase in hepcidin expression, especially upon high concentrations of viral replication common in acute or advanced HIV-1 infection [
The possibility that HAART affects the metabolism of iron and, consequently, hepcidin levels, can alsobe considered because this effect has been documented, for example, with zidovudine (AZT)-induced suppression of bone marrow [
In acute and/or advanced infectious processes, inflammation is known to be an aggravating factor in iron metabolism because iron [
Previous studies on the relationship between iron status and hepcidin levels showed a positive association between hepcidin and serum ferritin and iron levels [
It is important to consider that the survival of individuals who are seropositive for HIV-1 is accompanied by a chronic infectious process and continuous treatments with side effects, exposing these individuals to a number of complex situations that are multifactorial and interrelated, including changes in iron metabolism [
Our results showed that reduced levels of hepcidin, iron and ferritin were associated with a reduction in the number of CD4+ T-cells in HIV-1-infected individuals with no treatment. In the groups receiving different regimens of HAART, which exhibited a restored immune system as characterized by the recovery of CD4+ T-cells and undetectable levels of HIV-1 RNA, the same parameters were within the normal range. These results suggest that HIV-1 infection affects the levels of serum hepcidin, the main regulator of iron metabolism, with subsequent changes in iron levels in the circulation and within deposits. Moreover, different HAART regimens can adequately reverse this condition.
The authors would like to thank Professor Manoel Rosa de Oliveira Lino (PhD) from the Department of Informatics and Statistics at the Federal University of Santa Catarina for his contributions to the statistical analyses. This study was financially supported by resources from The National Council for Scientific and Technological Development (CNPq), the State of São Paulo Research Foundation (FAPESP), and the National Institute of Science and Technology of Complex Fluids (INCT-FCx).
The authors state that there are no conflicts of interest regarding the publication of this paper.
Joel daCunha,Luciana Morganti FerreiraMaselli,Jovino dos SantosFerreira,CelsoSpada,Sérgio PauloBydlowski, (2015) The Effects of Treatment on Serum Hepcidin and Iron Homeostasis in HIV-1-Infected In-dividuals. World Journal of AIDS,05,151-160. doi: 10.4236/wja.2015.53018
HIV-1: Human immunodeficiency virus type1
HAART: Highly active antiretroviral therapy
NNRTI: Non-nucleoside reverse transcriptase inhibitor
NRTI: Nucleoside reverse transcriptase inhibitor
PI: Protease inhibitors
ANOVA: Analysis of variance (univariate variance for multiple comparisons)
TIBC: Total iron binding capacity