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Acta Tropica 126 (2013) 127– 131

Contents lists available at SciVerse ScienceDirect

Acta Tropica

journa l h o me pa g e: www.elsev ier .com/ locate /ac ta t ropica

aptoglobin phenotypes and iron status in children living in a malariandemic area of Kenyan coast

lice M. Nyakerigaa,b,c,∗, Marita Troye-Blombergb

Texas Tech University, Department of Biomedical Sciences, El Paso, TX, USAStockholm University, Department of Immunology Wenner-Gren Institute, Stockholm, SwedenKEMRI/Wellcome Trust Programme, Centre for Geographic Medicine Research, Coast, Kilifi District Hospital, Kilifi, Kenya

r t i c l e i n f o

rticle history:eceived 27 September 2012eceived in revised form 31 January 2013ccepted 1 February 2013vailable online xxx

eywords:alaria

ron status

a b s t r a c t

Malaria infection may be affected by host genetic factors as well as nutritional status. Iron status andthe phenotype of haptoglobin, a heme-binding acute phase reactant may be determinants of malariaparasitemia. A combination of cross sectional studies and longitudinal follow-up were used to describe theassociation between iron status, C-reactive protein, malaria infections and host genetic factors including;haptoglobin (Hp) phenotypes, in children below 9 years in a malaria endemic area in Coastal Kenya. Theprevalence of 0.45 and 0.41, respectively for Hp 1-1 and Hp 2-1 phenotypes was significantly higher than0.14 for Hp 2-2 phenotype (n = 162). Children with Hp 2-2 phenotype showed significantly higher ironstorage compared to those with Hp 1-1 and Hp 2-1 phenotypes when children with malaria parasites and

aptoglobinenetic factors

high C-reactive protein (>9 mg/L) were excluded from the analysis. There were no significant differencesin malaria parasite densities among Hp phenotypes but children with Hp 2-2 had lower number ofclinical malaria episodes (P = 0.045). Taken together, this study shows that the presence of malaria maycomplicate the interpretation of iron status in children based on their Hp-phenotypes. Further studieswill be required to address possible interactions among the various genetic factors and iron status in amalaria endemic setting.

. Introduction

Iron status in man is influenced by both environmental andenetic factors. Molecular variation of haptoglobin (Hp), a plasmarotein, is one of the genetic factors known to influence iron statusLanglois et al., 2000). Hp is a type II acute phase protein secretedy hepatocytes during an inflammatory reaction. In humans, Hp isharacterized by a genetic polymorphism with three structurallyifferent phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2) which are theesult of the expression of two different alleles; Hp1 and Hp2, of thep gene located on chromosome 16q22 (Langlois and Delanghe,996). Hp is responsible for the removal of free hemoglobin (Hb) inhe form of Hp–Hb complexes from the circulation with subsequenteduction in Hb loss through the kidney. Consequently, the kidneys protected against damage and iron recycling is permitted. Hp 2-2s associated with higher serum iron and transferrin saturation lev-

ls as compared to other phenotypes reflecting a relative increasen the iron transport (Delanghe and Langlois, 2002). For example,p 2-2 male Caucasians have been characterized with larger iron

∗ Corresponding author at: Department of Biomedical Sciences, El Paso, TX, USA.el.: +1 915 783 5227; fax: +1 915 783 5223.

E-mail address: alice.nyakeriga@ttuhsc.edu (A.M. Nyakeriga).

001-706X/$ – see front matter. Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.actatropica.2013.02.004

Published by Elsevier B.V.

stores as evidenced by higher serum ferritin levels compared totheir Hp 1-1 and Hp 2-1 counterparts (Langlois et al., 2000).

Haptoglobin phenotypes differ in their biological activities andhave been associated with resistance or susceptibility to infec-tious diseases (Langlois and Delanghe, 1996). Iron withholdingis an important example of nutritional immunity playing a rolein the defense against infectious diseases (Pradines et al., 2003;Weiss et al., 1994, reviewed in Gordeuk et al., 2001; Weinberg,2009). Haptoglobin acts as a natural bacteriostatic agent by pre-venting the utilization of hemoglobin by pathogenic bacteria, whichrequire iron for their growth. In malaria endemic areas, Hp 1-1but not Hp 2-2 with disease severity (Elagib et al., 1998), per-haps through iron withholding related mechanisms. While the roleof iron in malaria susceptibility remains controversial, there isevidence that iron deficiency protects against disease (reviewedin Oppenheimer, 2001). We previously observed an associationbetween iron deficiency and protection against malaria infectionin a cohort of children living on the coast of Kenya (Nyakerigaet al., 2004). Given the existing association between haptoglobinpolymorphism and iron status or malaria susceptibility, there was

need to investigate the interaction of these three conditions in ourcohort of children. We subsequently investigated the effect of hap-toglobin polymorphism on biochemical markers of iron and malariainfection.

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28 A.M. Nyakeriga, M. Troye-Blombe

. Methodology

.1. Study population

The study was conducted in Ngerenya area of Kilifi Districtocated in the north-coast of Kenya. The study population consistedf a cohort of children <9 years old, belonging to the Mgiriama ethnoinguistic group. Malaria transmission in this area occurs through-ut the year, although the majority of clinically evident infectionsccur following short and long rains that generally occur during theonths of October–November and March–July respectively (Snow

t al., 1997). Residents of Ngerenya experience an average of 10alaria infective bites/person/year (Mbogo et al., 1993). Our cohortas under weekly surveillance for clinical malaria events betweenov 2001 and May 2003. In addition, we conducted six-monthlyross-sectional surveys in May and October, 2002. At each survey,outine data were collected regarding a range of clinical parametersncluding axillary temperature, clinical symptoms of fever and theresence of malaria parasites on a finger-prick blood film. In addi-ion, a venous blood sample, collected during the survey in May,as used to determine markers of iron status (including serum

ron, transferrin, ferritin and sTfR) and, total as well as malaria-pecific immunoglobulins (total IgG and IgE, and malaria–specificmmunoglobulin subclasses including IgG1, IgG2, IgG3, and IgG4)n plasma samples.

.2. Laboratory assays

Plasma samples obtained in May 2002 were used for iron assaysnd immunoglobulin measurements. Ferritin and transferrin wereeasured by turbidimetry and plasma iron by Ferrozine®-based

hotometry using an automated analyzer (Hitachi 917, Hitachiorp., Japan). Transferrin saturations (TFS; %) were calculated fromhe plasma iron and transferrin measurements by standard meth-ds (Kasvosve et al., 2000; Langlois et al., 2000). We measuredoluble transferrin receptor (sTfR) concentration by enzyme-linkedmmunosorbent assay (ELISA) using a commercially available kitR&D Systems, Minneapolis). Plasma ferritin is an acute phase reac-ant and is unreliable in the presence of a range of infectionsnd inflammatory conditions. We therefore measured C-reactiverotein (CRP) (by turbidimetry) as an independent measure of

nflammation in order to aid interpretation. The hemoglobin typingas done by agarose gel electrophoresis and anti-malarial antibod-

es were measured against crude parasite sonicate antigen usingLISA as previously described (Nyakeriga et al., 2004). The alpha-halassaemia genotyping was done according to Chong and othersChong et al., 2000).

The haptoglobin phenotype was determined using starch gellectrophoresis of hemoglobin-supplemented serum. Visualizationf Hp–Hb complex bands was achieved by staining the gel withetal-enhanced peroxide reagents as described by Delanghe and

thers (Delanghe et al., 2000).Blood films were stained with Giemsa and examined for malaria

arasites by standard microscopy. Parasite densities were recordeds a ratio of parasites to white blood cells (read from thick smears),r to red blood cells (from thin smears) for heavier infections. Den-ities (parasites/�l whole blood) were then calculated assuming ahite blood count of 8 × 103/�l or a red cell count of 5 × 106/�l.

.3. Statistical analysis

Continuous data (CRP, markers of iron status, parasite den-

ity and age) were checked for normality and homogeneity ofariance respectively by Kolmogorov–Smirnov and Levene tests.ll variables, except for immunglobulins levels, failed to meet

he assumptions of normality even after log and square-root

ta Tropica 126 (2013) 127– 131

transformation. Consequently, the effects of Hp phenotypes onthese variables were determined using a non-parametric Kruskal-Wallis test. Chi square test was used to determine the associationbetween Hp phenotypes and host gender, malaria prevalence,iron deficiency, and sickle cell and �-thalassaemia traits. Analy-sis of variance (ANOVA) was used to analyze the differences inimmunoglobulin levels among Hp groups. The nutritional statuswas presented as height-for-age (HAZ), weight-for-age (WAZ) oras weight-for-height (WHZ) z-scores. Iron deficiency was definedaccording to low plasma ferritin in combination with low satura-tion transferrin as we previously described (Nyakeriga et al., 2004).Clinical malaria episode was defined as presence of blood smearparasite positive and an axillary temperature of >37 ◦C. All analy-sis was performed using SAS version 9.2 (SAS Institute, Cary, NC).Statistical significance was set at P value ≤0.05.

The Kenya Medical Research Institute National Ethical ReviewCommittee granted ethical permission for this study. Individualwritten informed consent was obtained from the parents of allstudy participants.

3. Results

The prevalence of 0.14 for Hp 2-2 phenotype was significantlylower than 0.45 and 0.41, respectively for Hp 1-1 and Hp 2-1 phen-otypes (n = 162, � = 27.44, df = 2, P < 0.0001; Table 1). The observedHp phenotype distribution was in agreement with the Hardy-Weinberg equilibrium.

The anthropometric measures (data not shown), age and pro-portion of parasite positive blood smears as well as the female tomale ratio were comparable among the three phenotypes. The bio-chemical markers of iron status were comparable when childrenwith blood smears parasite positive or with high CRP (>9 mg/L)were included in the analysis (data not shown). However, whenthese children (with inflammation or malaria parasites) wereexcluded from analysis there was a trend towards iron sufficiencyin children with Hp 2-2 phenotype compared to those with otherHp phenotype, with transferrin and sTfR reaching statistical signifi-cance (Table 1). While remaining relatively higher in children withHp 2-2 phenotype, differences in plasma ferritin, iron and trans-ferrin saturation levels did not reach significance. Similarly, theproportion of children that were defined as being iron deficientwas highest in children with Hp 1-1 phenotype and lowest in thosewith Hp 2-2 phenotypes, but the differences did not reach signif-icance. Further analysis revealed no significant differences in theproportion of children with sickle cell trait or alpha-thalassaemiaamong the Hp phenotype, as shown in the table.

We further investigated the level of anti-malarial antibody lev-els (Fig. 1). There was no difference in the antibody levels amongthe Hp groups. Similarly, we compared the prevalence of clinicalmalaria episodes in the children by their haptoglobin phenotypesas summarized in Table 2. In comparison to children with Hp 1-1,and Hp 2-1, children with Hp 2-2 phenotype had lower incidencesof clinical malaria. There was no child with more than one clinicalmalaria episode in the six months of follow-up preceding assess-ment of iron indices (P = 0.045).

4. Discussion

In this study we assessed the association of haptoglobin pheno-types, iron status and susceptibility/resistance to malaria infection.We have shown that there was a significant association between

biochemical markers of iron and haptoglobin polymorphism. Com-pared to the other phenotypes Hp 2-2 demonstrated lower levelsof plasma sTfR and higher plasma transferrin, raised ferritin butlower levels of plasma iron. Overall, these markers of iron indicated

A.M. Nyakeriga, M. Troye-Blomberg / Acta Tropica 126 (2013) 127– 131 129

Table 1Biological and biochemical characteristics by Hp phenotypes.

Hp 1-1 Hp 2-1 Hp 2-2 P value

N (% prevalence) 66 (40.7) 73 (45.1) 23 (14.2)Age (months) 41.44 ± 22.55 43.35 ± 24.30 38.83 ± 29.59 0.728F/M ratioa 0.74 0.62 1.1 0.502% + ve BSa 11/65 (16.9) 10/69 (14.5) 2/20 (10.0) 0.421Pfmcl min–max (median)-only + ve BSc 240–38000 (1080) 40–8200 (840) 200–2400 (1300) 0.322CRP min–max (median)c 0–70 (0.00) 0–25 (0.00) 0–16 (2.50) 0.029With CRP > 9 mg/L 6 3 2Iron (�mol/L)b 5.40 (3.10–10.90) 5.30 (4.05–10.50) 8.20 (4.65–10.70) 0.556Ferritin (�g/mL)b 11.00 (5.50–19.00) 12.00 (6.00–23.25) 21.00 (8.50–34.25) 0.084Transferrin (g/L)b 2.89 (2.62–3.45) 2.75 (2.56–3.03) 2.63 (2.41–2.82) 0.016Saturation transferrin (%)b 8.80 (4.01–15.08) 9.68 (5.93–17.53) 13.27 (7.41–16.08) 0.128sTfR(nmol/L)b 50.83 (37.48–65.90) 44.32 (35.39–57.00) 35.05 (25.80–42.88) 0.006% iron deficienta 21/40 (52.5) 17/37 (45.89) 4/11 (36.4) 0.612% ��-/��- thalasaemia 15/66 (22.7) 11/70 (15.7) 1/23 (0.04) 0.08% �-/�- thalassaemia 36/66 (54.5) 33/70 (47.1) 11/23 (47.8)% HbAS 11/66 (16.7) 10/73 (13.7) 0/23 (0.00) 0.134

a Proportions were compared by chi-Square, F/M: female:male ratio, + ve B/S: blood smear parasite positive at cross-sectional survey, sTfr: soluble transferrin receptor,CRP: C-reactive protein, pfmcl: malaria parasite densities.

b Children with high CRP (CRP > 9 mg/ml) or those with B/S positive were excluded, lrespectively. All iron values are represented as median (interquartile ranges).

c Comparison of mean ranks by Kruskal–Wallis test. The markers of iron reported here

Fig. 1. Immunoglobulin levels by haptoglobin phenotypes: Malaria specific antibod-ies, total IgG and its subclasses 1, 2, 3 and 4 and IgE against Plasmodium falciparumcrude extract were measured in plasma samples. All antibody concentrations werelog-transformed before analysis. The means from each group (n: Hp 1-1 = 66; Hp 1-2 = 73 and Hp 2-2 = 23) were compared using ANOVA. The error bars are mean ± SE.Tn

adosce

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and mediates endocytosis of Hp–Hb complexes (Graversen et al.,

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here were no significant differences in each antibody isotype among the Hp phe-otype groups.

trend towards iron sufficiency rather than iron deficiency in chil-ren with Hp 2-2. However, there were no differences in prevalencef blood smear parasite positive, parasite density or in the malariapecific antibody levels among Hp groups, but there was an asso-iation between Hp 2-2 and lower incidences of clinical malaria

pisodes.

Biochemical markers of iron especially plasma ferritin, whichs often used in conjunction with other markers of iron to define

able 2alaria episodes by haptoglobin phenotypes in six months of follow-up preceding the as

Hp 1-1

Number with an episode (%) 21/56 (37.5)Occurrence of a malaria episode:With 1 episode 17

With > 1 episodes 4

roportions among groups were compared by Chi square analysis.

eaving a balance of 49, 54, and 14 children in Hp 1-1, Hp 2-1 and Hp 2-2 groups

were measured in blood obtained in May 2002.

iron status, are affected by either inflammation and/or presence ofchronic infection such as malaria (Adelekan and Thurnham, 1990;Das et al., 1997). Even soluble transferrin receptor which was ini-tially thought to be stable in the presence of inflammation, andis used, singly, as a definitive marker for iron status (Kuvibidilaet al., 1994), has been shown to be affected by presence of malariainfection (Beesley et al., 2000; Williams et al., 1999). We thereforeexamined the effects of inflammation and/or malaria on plasmaindices of iron among children based on their Hp phenotype. Itis interesting to note that while there was no apparent differ-ence in the proportion of children with malaria parasite positiveblood smears or parasite density, their inclusion in the analysisrevealed no significant difference in markers of iron, among theHp groups (data not shown). However, when these children (withblood smear parasite positive) as well as those with high CRP(>9 mg/L) were excluded from a repeat analysis, significant differ-ences were achieved (Table 1). This implies that the presence ofmalaria infection and/or inflammation may affect the interpreta-tion of iron status by Hp phenotypes.

The association between iron status and haptoglobin typesremains controversial, with some studies indicating an associa-tion between iron loading and Hp 2-2 (Langlois et al., 2000; VanVlierberghe et al., 2001) while others do not (Atkinson et al., 2006;Beutler et al., 2002; Kasvosve et al., 2002). Here, we showed anassociation between Hp 2-2 and high markers of iron. This maybe indicative of high iron transport or storage in this phenotypeconsistent with previous reports (Delanghe and Langlois, 2002;Langlois et al., 2000). In humans it is thought that macromoleculecomplexes of Hb–Hp 2-2 as compared to Hb–Hp 1-1, exhibit ahigher affinity for CD163, a receptor that is present on macrophages

2002; Kristiansen et al., 2001). Also, a recent report has attributedthe higher iron indices in Hp 2-2 to increased presence of non-transferrin bound iron within the Hp–Hb complex (Viener and

sessment of iron indices.

Hp 2-1 Hp 2-2 P

16/69 (23.2) 7/17 (41.2) NS

9 77 0 0.045

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30 A.M. Nyakeriga, M. Troye-Blombe

evy, 2011). Consequently, Hp 2-2 subjects present with betterron indices than other phenotypes and this may partly explain thessociation observed in this study.

The role of haptoglobin in either resistance or susceptibility tonfectious diseases and immunity appears to be phenotype depend-nt (reviewed in (Kasvosve et al., 2010; Langlois and Delanghe,996)). Hp 2-2 is associated with poor prognosis in patients withIV (Delanghe et al., 1998; Friis et al., 2003) and mycobacterium

uberculosis (Kasvosve et al., 2000). Hp 1-1 phenotype has beenssociated with increased risk of chronic hepatitis C (Louagie et al.,996) while in malaria endemic areas the role of Hp 1-1 in dis-ase remains controversial. Some studies from Sudan (Elagib et al.,998), Ghana (Quaye et al., 2000) and Cameroon (Minang et al.,004) have reported association between Hp 1-1 and susceptibil-

ty to severe or uncomplicated malaria while other studies failedo detect any association (Aucan et al., 2002). Yet in another study,here was no obvious association with Hp type, but rather a slightncrease in severe malaria in Hp 2-2 (Bienzle et al., 2005). Some ofhe protection already described for Hp 2-2, may be explained by

single nucleotide polymorphism (SNP) promoter in the Hp gene,-61C. This SNP is highly prevalent in Hp 2-2 allele, and has beenssociated with protection from clinical malaria although this wasestricted to children between 3 and 6 years of age and not thoseounger (Cox et al., 2007).

While Hp 1-1 prevalence in sub-Saharan Africa is linked to selec-ion pressure due to Plasmodium falciparum malaria (Rougemontt al., 1980; Trape and Fribourg-Blanc, 1988), our observations werenconclusive. We were unable to demonstrate a significant associ-tion between Hp type and malaria parasite blood smears and/orensities, but children with Hp 2-2 had fewer episodes of clinicalalaria, which is in agreement with a previous observation in this

rea (Atkinson et al., 2007). Similarly there was no difference innti-malaria antibodies against parasite crude antigen among thehree groups of Hp phenotypes. We have previously shown that

alaria specific IgG is significantly associated with exposure toalaria (Nyakeriga et al., 2004). Therefore, we expected differences

n levels of these antibodies if Hp polymorphism had a direct effectn malaria outcome in our cohort. Together, this might be explainedy geographical differences and/or associated malaria endemicity,hanges in seasonal malaria transmission or malaria interventionrograms, or the fact that our sample size may not have had theower needed to detect the influence of Hp phenotype on malariaarasite prevalence, or misclassification of Hp 2-2 phenotype dueo low Hp concentration (Cox et al., 2007). In addition, other hostenetic factors that are associated with protection against malariauch as thalassemia and sickle trait, may have contributed to theack of expected relationship between malaria and malaria-specificntibodies or Hp phenotypes (Allen et al., 1997). In our study, thisas exemplified by a lack of sickle cell trait and the lowest propor-

ion of children with homozygous alpha thalassemia, in the Hp 2-2roup. This implies that the interpretation of data on interactionf malaria, Hp and iron status may require consideration of otherenetic factors in a larger cohort. Taken together, there is need forurther studies to comprehensively address possible interactionsmong Hp- phenotypes, iron status and other genetic factors inalaria endemic areas. In addition, further studies are required

o define the possible mechanisms underlying Hp 2-2 protectiongainst clinical malaria.

cknowledgements

We thank Rune Back, Margareta Hagstedt of Sweden and col-

eagues at the KEMRI Centre for Geographic Medicine Research,oast, including Moses Kortok and Drs Kevin Marsh and Thomasilliams for their support, the study participants and other mem-

ers of the field and laboratory staff for their help with this

ta Tropica 126 (2013) 127– 131

study. We thank Dr Ephantus Muturi for critical review of thismanuscript and help with statistics. This work was supported byfunds from UNDP/World Bank/WHO Special Program for Researchand Training in Tropical Diseases (TDR) to AMN, the SwedishAgency (SIDA/SAREC) for Research Cooperation with developingcountries to MTB. The CGMR-C receives financial support from theWellcome Trust. This study is published with permission from theDirector of KEMRI.

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