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Department of Clinical Chemistry, Immunology and Microbiology, University Hospital Gent, De Pintelaan 185, B-9000 Gent, Belgium.
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Génétique et Pathologie Moléculaire de
l’Hématopoièse, INSERM U409, Faculté Xavier Bichat,
16 rue Henri Huchard, BP416, 75870 Paris Cedex 18, France.
3
Unit for Renal and Infectious Diseases and Laboratory of
Clinical Chemistry, General Hospital St.-Jan, Ruddershove 10, B-8000
Brugge, Belgium.
4
Laboratory of Clinical Chemistry, General Hospital
Middelheim, Lindendreef 1, B-2020 Antwerpen, Belgium.
a Address correspondence to: Laboratory of Clinical Chemistry, University Hospital Gent, De Pintelaan 185, B-9000 Gent, Belgium. Fax 32-9-2404985; e-mail michel.langlois{at}rug.ac.be
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Reference values of serum iron status markers were compared according to Hp phenotypes (Hp 1-1, Hp 2-1, Hp 2-2; determined by starch gel electrophoresis) in 717 healthy adults. Iron storage was investigated in peripheral blood monocyte-macrophages by measuring cytosolic L- and H-ferritins and by in vitro uptake of radiolabeled (125I) hemoglobin-haptoglobin complexes.
Results: In males but not in females, the Hp 2-2 phenotype was associated with higher serum iron (P <0.05), transferrin saturation (P <0.05), and ferritin (P <0.01) concentrations than Hp 1-1 and 2-1, whereas soluble transferrin receptor concentrations were lower (P <0.05). Moreover, serum ferritin correlated with monocyte L-ferritin content (r = 0.699), which was also highest in the male Hp 2-2 subgroup (P <0.01). In vitro, monocyte-macrophages took up a small fraction of 125I-labeled hemoglobin complexed to Hp 2-2 but not to Hp 1-1 or 2-1.
Conclusions: The Hp 2-2 phenotype affects serum iron status markers in healthy males and is associated with higher L-ferritin concentrations in monocyte-macrophages because of a yet undescribed iron delocalization pathway, selectively occurring in Hp 2-2 subjects.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In humans, Hp is characterized by a genetic polymorphism with three structurally different phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), which result from the expression of two different alleles (Hp1 and Hp2) of the Hp gene located on chromosome 16q22. The phenotypes show an important molecular heterogeneity; Hp 1-1 is a small molecule (86 kDa) of well-defined structure, whereas Hp 2-1 is characterized by heteropolymers (86–300 kDa), and Hp 2-2 forms large macromolecular complexes (170–1000 kDa) (3).
The physiological importance of Hp-Hb complex formation has become evident by the increased susceptibility to Hb-driven lipid peroxidation demonstrated in conditions of hypo- or anhaptoglobinemia (Hp0 phenotype) (6)(7)(8)(9) or in Hp-deficient mice (10). Furthermore, functional differences between the various Hp phenotypes have important biological consequences (3). The hepatic "clearance" of free Hb in plasma appears to be less efficient for Hp 2-2 than for the other Hp phenotypes, producing some degree of iron-driven oxidative stress that is reflected by a lower stability of serum vitamin C in healthy Hp 2-2 subjects (11)(12).
From these observations, we postulated that functional differences between the Hp phenotypes might affect human iron status. Body iron stores are found associated with ferritin mainly in the hepatocytes and the mononuclear phagocytic system (13). We derived a working hypothesis that a less efficient transport of free plasma Hb to the liver could result in "delocalization" of iron into monocyte-macrophages.
In this study, we wanted to investigate the influence of Hp polymorphism on iron status in healthy subjects. For this purpose, we measured serum indicators of body iron compartments: iron and transferrin saturation (iron transport compartment), ferritin (iron storage compartment), and soluble transferrin receptors (sTfR; functional iron compartment) (14)(15)(16). Intracellular iron status in human monocyte-macrophages was studied by measuring cytosolic L- and H-ferritin concentrations (13). In vitro, interactions of Hp-Hb complexes with monocyte-macrophages were investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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analysis of serum and plasma samples
Venous blood was drawn between 0900 and 1000, allowed to clot, and
centrifuged (1000g for 10 min at room temperature). The
supernatant serum was collected for analysis. The Hp phenotype was
determined using starch gel electrophoresis of Hb-supplemented serum,
followed by visualization of Hp-Hb bands by staining the gel with
metal-enhanced peroxidase reagents (Pierce Corp.) as described
previously (17). Serum Hp, CRP, ferritin, and sTfR were
assayed by fixed-time immunonephelometry using commercial rabbit
anti-human antisera on a BN II nephelometer (Dade Behring), calibrated
against the CRM 470 reference material (18). Transferrin
saturation was calculated from measurements of serum iron concentration
by spectrophotometry (ferrozine method) and serum transferrin
concentration by immunoturbidimetry using commercial reagents on a
Hitachi 747 analyzer (Roche Diagnostics). Assuming that two iron atoms
bind to one molecule of transferrin, serum transferrin saturation (%)
was calculated according to the equation (19):
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Blood was simultaneously collected in tubes containing EDTA for determination of plasma Hb and hematocrit (Sysmex SE-9500; Toa Medical Electronics).
l- and h-ferritin in monocyte-macrophages
Heparinized venous blood (50 mL) was obtained from 44 healthy
males (ages 25–46 years) with known Hp phenotype. Peripheral blood
mononuclear cells were isolated by centrifugation (400g for
30 min at 20 °C) through Ficoll-Isopaque (Nycomed), followed by
purification of the monocytes by density gradient centrifugation using
Percoll (Pharmacia; 600g for 30 min at 20 °C)
(20). Monocytes (purity, 85–90%) were harvested from the
interface, and aliquots of 1–2 x 106 cells
were washed in phosphate-buffered saline (PBS), pH 7.4 (Unipath). The
cells were lysed by sonication in cold hypotonic buffer (20 mmol/L
Tris, 40 mmol/L KCl, 10 mL/L Triton, 1 mmol/L phenylmethylsulfonyl
fluoride) containing a protease inhibitor mixture (Roche). The
resulting lysates were then centrifuged (14 000g for 30 min
at 4 °C), and the total protein content of the supernatants was
determined spectrophotometrically by the Bradford method (Bio-Rad) in
triplicate, calibrated using bovine serum albumin.
Cytosolic L-ferritin and H-ferritin contents were measured by enzyme-linked immunosorbent assays based on monoclonal antibodies specific for L-ferritin (LFO3) and H-ferritin (rHO2) and calibrated using the corresponding recombinant homopolymers expressed in Escherichia coli (21). The bound ferritin was detected with the same antibodies conjugated to horseradish peroxidase (colorimetric reaction with o-phenylenediamine dihydrochloride). All measurements were performed in triplicate, and results are expressed as µg of ferritin/g of total proteins.
preparation of radiolabeled Hp-Hb complexes
Radioactive iodine (from Na125I) was
incorporated into human Hb (Sigma) by the chloramine-T method making
use of a radioiodination method (ICN) according to the manufacturer’s
protocol. Afterward, free 125I was removed by gel
filtration over a Sephadex G10 column. Specific activity of
125I-labeled Hb, measured in a gamma counter (LKB
Wallac), ranged between 0.9 and 1.6 x 106
cpm/mg protein. Before use in a cellular uptake assay,
125I-labeled Hb was diluted in 1 mL of PBS
containing 50 mL/L fetal calf serum to a final concentration of 1.0
g/L. Hp-Hb complexes were prepared by identical dilution of
125I-labeled Hb in 1 mL of PBS containing 50 mL/L
fetal calf serum and purified human Hp 1-1, Hp 2-1, and Hp 2-2
phenotypes (Sigma; final Hp concentrations, 0.5 and 1.0 g/L). Binding
of 125I-labeled Hb to Hp phenotypes was checked
electrophoretically by comparison with the typical migration patterns
of unlabeled Hp-Hb complexes on starch gel electrophoresis and by
detection of radioactivity in Hp-Hb bands on the gel.
125I-Hb uptake in monocyte-macrophages
Peripheral blood monocytes (2 x 107
cells), isolated by two-step density gradient centrifugation as
described above, were suspended in 1 mL of prewarmed (37 °C)
125I-labeled Hb preparations or phenotype-matched
125I-labeled Hb-Hp complexes. After 20 min
preincubation at 37 °C (to account for Hp binding to adhesion
molecules) (22)(23), the cell suspensions were
centrifuged (1000g for 10 min at 20 °C), the supernatants
were transferred to another tube, the cells were washed in PBS, and
baseline radioactivity in the cell pellets was measured (LKB Wallac).
The cells were then resuspended in the supernatants, and measurements
of cell-associated radioactivity were repeated at 1-h intervals using
the same procedure, during an incubation period of 3 h at 37 °C
under gentle shaking. The rate of cellular accumulation of
radioactivity was calculated as:
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where t0 = 20 min. The values were corrected for radioisotope decay. Identical experiments were performed in presence of the macrophage-activator zymosan A (Sigma; final concentration, 0.1 g/mL) (24). To distinguish intracellular uptake from cell surface binding, monocytes were incubated at 4 °C (25). 125I-labeled Hb uptake was also studied in monocytes pretreated with 0.1 g/L pronase E (protease type XIV from Streptomyces griseus; Sigma) for 30 min at 37 °C and washed with PBS. As a negative control, additional experiments were carried out in presence of a 10-fold excess of unlabeled Hb (added to 125I-labeled Hb before preparation of Hp-Hb complexes).
statistics
Results are given as mean ± SD or median and interquartile
range, where appropriate. The Wilcoxon test was used for evaluation of
differences between subgroups. Correlations between data were examined
using regression analysis. Statistical significance was considered at
the level of P <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Serum Hp concentrations corresponded with the Hp
phenotype-adjusted reference values in Caucasians and were 
30%
lower in Hp 2-2 than in Hp 1-1 individuals (3). Serum CRP,
plasma Hb, and hematocrit were comparable between Hp phenotypes in both
sexes (Table 1
).
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serum markers of iron status
Table 2
compares the serum iron status according to gender and Hp
phenotype. In all subgroups, serum concentrations of the various iron
status markers were within the expected reference intervals. However,
reference values of serum iron and transferrin saturation among males
differed significantly among Hp phenotypes, showing highest
concentrations in the Hp 2-2 subgroup (P <0.05), whereas
serum transferrin concentrations were comparable. More significantly,
serum ferritin concentrations in males were higher in the Hp 2-2
subgroup than in the Hp 1-1 and 2-1 subgroups (P <0.01).
Fig. 1
illustrates this Hp phenotype-dependent variation in ferritin
concentrations within the expected reference interval. In contrast,
serum sTfR concentrations were significantly (P <0.05)
lower in Hp 2-2 males than in those carrying another Hp phenotype. The
observed differences were not statistically significant among females.
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ferritin content in monocyte-macrophages
In peripheral blood monocytes from 44 healthy males, L- and
H-ferritin contents (measured in triplicate) were compared between the
Hp phenotypes (Table 3
). L-Ferritin concentrations in monocyte cell lysates from Hp
2-2 subjects (n = 17) were approximately twofold higher than in
monocytes from subjects carrying Hp 1-1 (n = 9) or Hp 2-1 (n
= 18; P <0.01). In contrast, cytosolic H-ferritin contents
in monocytes did not differ between the Hp phenotypes.
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There was a positive correlation between monocyte L-ferritin
concentrations and serum ferritin concentrations in the 44 subjects
(r = 0.699; P <0.001; Fig. 2
). This correlation was better in the Hp 2-2 subgroup
(r = 0.589) than in the Hp 1-1 and 2-1 subgroups
(r = 0.357 and r = 0.362,
respectively).
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Hb uptake in monocyte-macrophages
Peripheral blood monocytes were incubated (37 °C) with
125I-labeled Hb and phenotype-matched
125I-labeled Hb-Hp complexes, and cell-associated
radioactivity was measured at 1-h intervals during 3 h starting
from an initial baseline measurement after 20 min
(t0). The rate of cellular
accumulation of radioactivity in the interval t -
t0 = 3 h was used for comparison
(Fig. 3
). Incubation of monocytes with
125I-labeled Hb (1.0 g/L) produced a higher
baseline radioactivity in the presence of Hp than in the absence of Hp,
which can be attributed to earlier reported Hp binding to surface
membrane molecules on mononuclear cells
(22)(23). In the absence of Hp and in the
presence of Hp 1-1 or Hp 2-1 (0.5 and 1.0 g/L), cell-associated
(baseline) radioactivity changed very slowly during the next 3 h.
In contrast, loading of monocytes with
125I-labeled Hb complexed to Hp 2-2 (1.0 g/L)
produced higher baseline concentrations of cell-associated
radioactivity, which continued to increase at a constant rate during
the full incubation period. However, this cellular accumulation of
radioactivity represented only a small fraction of the total amount of
Hb-Hp 2-2 complexes added to monocytes (0.2% after 3 h). The
cellular uptake of 125I-labeled Hb was linear and
occurred at a lower rate when less Hp 2-2 (0.5 g/L) was added (Fig. 3A
).
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Activation of monocytes with zymosan A produced a moderate cellular
accumulation of 125I-labeled Hb in the absence of
Hp and in the presence of Hp 1-1 and 2-1 because of fluid phase uptake,
but this occurred at a higher rate in the presence of Hp 2-2 (Fig. 3B
).
Approximately 0.4% of the Hb-Hp 2-2 complexes added to
zymosan-activated monocytes was internalized after 3 h. On the
other hand, incubation experiments performed at 4 °C did not
demonstrate cellular uptake of Hb-Hp 2-2 complexes, whereas baseline
radioactivity attributable to cell surface binding was comparable
between the three Hp phenotypes (data not shown). The effect of Hp 2-2
on cellular accumulation of 125I-labeled Hb was
also not detected when a 10-fold excess of unlabeled Hb was added or
after pronase pretreatment of the monocytes (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum ferritin is a positive acute phase reactant, and inflammatory cytokines and immune activation are known to affect iron metabolism (26)(27). However, serum CRP concentrations were not different between Hp phenotypes, indicating that the influence of Hp 2-2 on the serum iron status is not biased by some degree of subclinical inflammation. The observed effect of Hp polymorphism on circulating sTfR concentrations cannot be attributed to different degrees of erythropoiesis because plasma Hb and hematocrit values were comparable between Hp phenotypes (14). Among females, the lack of correlation between Hp polymorphism and iron status could be explained by menstrual bleeding.
The observed association of Hp 2-2 with higher iron stores, at least in males, can be explained by a degree of iron delocalization in monocyte-macrophages. Indeed, we found a more pronounced iron storage in monocyte-macrophages of Hp 2-2 subjects, as evidenced by higher cytosolic L-ferritin concentrations in peripheral blood monocytes. Remarkably, cytosolic H-ferritin contents in monocytes were not different among the three Hp phenotypes, similarly to what is observed in iron-loaded livers where only the L-ferritin is up-regulated (28). The positive correlation we found between serum and monocyte ferritin concentrations suggests that increased L-ferritin synthesis in monocyte-macrophages produces higher ferritin concentrations in the circulation (14).
Earlier studies suggested that, in intravascular hemolysis, parts of Hb-derived iron are incorporated ("delocalized") into slowly turned over, poorly accessible iron storage compartments of the mononuclear phagocytic system, largely bypassed in the normal endogenous cycling of iron (29). In vitro experiments in our study showed that human monocytes take up a small fraction of Hb-Hp 2-2 complexes at 37 °C but not at 4 °C. In contrast, free Hb and Hb bound to Hp 1-1 or 2-1 are not internalized in monocytes. However, because even in zymosan-activated monocytes (24) only a small fraction of Hb-Hp 2-2 is internalized, these experiments only explain in part the iron delocalization observed in Hp 2-2 subjects.
In vivo, monocyte-macrophages can take up Hb via phagocytosis of
senescent erythrocytes, after which iron is released from heme
intracellularly and stimulates ferritin mRNA translation
(30)(31). Hepatocytes usually take up 2 mg of
iron/day (the typical plasma iron turnover is 30 mg/day) in the form
of Hb-Hp complexes, present in the circulation after intravascular
disintegration of 10% of erythrocytes, which occurs even in healthy
subjects (29). High-affinity receptors
(Kd, 7.4 nmol/L) expressed on
hepatocytes are involved in binding and subsequent internalization of
Hb-Hp complexes (5). In contrast, only a low affinity
(Kd, 6.5 µmol/L) Hp binding on
monocytes has been reported in the literature (22). If
hepatocytes, in vivo, represent the major target cells for the Hb-Hp
complexes, it remains unclear how monocyte-macrophages (without
receptors or with only low-affinity receptors) could acquire Hb-Hp 2-2
complexes. However, nothing is known on the relative affinities of
these receptors toward Hp 2-2 vs other Hp phenotypes. The Hp 2-2
molecule differs from the other phenotypes by its larger size because
of polymerization (up to 1000 kDa), and our present findings suggest
that it is taken up more efficiently than Hp 1-1 or 2-1 complexes,
presumably by a pinocytosis-like phenomenon (24). Although
only a small proportion of the added Hb-Hp 2-2 complexes is taken up in
our experiments, this does not necessarily reflect the in vivo
situation where tissue macrophages might have a more active pinocytotic
activity. To support our hypothesis, further basic research is
necessary to demonstrate the iron delocalization pathway.
The Hp polymorphism has been associated with the prevalence and the clinical outcome of many pathologies with altered iron metabolism such as cancer, infections, atherosclerosis, and neurological disorders (3). Males carrying the Hp 2-2 phenotype are at risk for premature atherosclerotic disease, a condition where iron-driven lipid oxidation is known to play a role (32)(33). The data presented in this report support the observation made by our group in a large cohort of HIV-infected patients, where Hp 2-2 was associated with iron-driven oxidative stress (vitamin C depletion), enhanced HIV-1 replication, and higher mortality (34)(35)(36). However, the involvement of Hp in pathological processes might be even more complex because the protein participates in various immunoinflammatory functions of lymphocytes, granulocytes, and monocytes (37)(38)(39).
Important sources of variability in the laboratory-based assessment of iron status include differences between subjects (e.g., age, sex, race, ethnicity, geographical region, or altitude of residence) and within-subject variations (e.g., circadian change and inflammation). In the present study, we identified a genetic variability arising from the Hp polymorphism in healthy males. In interpreting serum markers of iron status, this Hp phenotype-dependent variation in reference values may increase the risk of either unnecessary additional investigations (e.g., bone marrow biopsy) or failure to detect subclinical disease accurately, particularly in some cases of (pre-) latent iron deficiency or iron overload.
In conclusion, delocalization of iron into poorly exchangeable storage compartments of the mononuclear phagocytic system affects the iron status in males carrying the Hp 2-2 phenotype. This effect is a potential cause of over- or underestimation of serum iron status markers.
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Acknowledgments |
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Footnotes |
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), or
Hp 2-2 (n = 17, 
). Shown are data points and the linear
regression curve (r = 0.699; P
< 0.001).
, P <0.01; 
,
P <0.005 (comparison with 125I-labeled Hb
in the absence of Hp 2-2).