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A Study on the Presence of Ferritin-binding Proteins in Fetal Horse Plasma

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.

Ferritin is an iron storage protein with a molecular mass of 500,000 Da to accommodate up to maximum 4,500 iron atoms and is found in all tissues [6,21,27]. It has dual function to store iron and to segregate iron from iron-catalyzed reactive oxygen species production [6,21]. Tissue ferritin is a 24-mer protein composed of various proportions of two types of subunits termed H (heart type) and L (liver type) chains [6,21,27]. H and L chains have distinct physiological properties [6,11,12,21]; the H chain has ferroxidase essential for iron uptake, while the L chain does not have ferroxidase, but promotes iron core growth inside the ferritin shell by providing iron nucleation sites [6,17,21].
In commercial fetal bovine sera, high levels of serum ferritin and it's iron content were measured, these values being much higher than those of adult bovine serum and same level as bovine tissue ferritin, respectively [8]. Fetal bovine serum ferritin may play a physiological role as iron transporter [8], although the existence of FBPs in fetal bovine serum has been not revealed yet. However, there has been no report on fetal mammal FBPs because of the difficulties to get fetal animal blood.
Fortunately, we obtained an opportunity to get six fetal horse plasma samples between 130 and 315 days of gestation. The purpose of our study is to reveal the presence of FBPs in fetal horse plasma and examine their biochemical properties.

Blood
Peripheral blood samples were collected from the jugular vein of Thoroughbred horses kept at Kitasato University. Six male horses (age 12-23 years, body weight 480-550 kg) used in this study were housed in individual stables with grass supplemented by highquality hay and concentrated supplement. Plasma was obtained by centrifuging heparinized blood and was kept at 4°C in the presence of 0.1% sodium azide until analysis. Fetal blood samples from 130 to 315 days of gestation were available from previous research project which was conducted from 1995 to 2000 at the Equine Research Institute, JRA. Pregnant mares were killed by intravenous administration of a normal dose of xylazine followed by an overdose of a mixture of thiopental sodium and suxamethonium chloride. The fetuses were removed after euthanasia. The fetal blood samples were collected by cardiac puncture, and infused into plastic tubes containing heparin (20 IU ml -1 ), then kept in ice and centrifuged at 1,800 × g for 10 min at 4°C. Plasma obtained was stored at -20°C until measured. All procedures were performed with permission of the experimental animal management of the Equine Research Institute.

Horse spleen ferritin
Ferritin monomers were purified from commercial horse spleen ferritin (Sigma, St Louis, MO, USA) as described previously [19].

Protein measurement
Protein concentration was measured according to the method of Lowry et al. [13] using bovine serum albumin (Boeringer Mannheim, Germany) as the standard.

Plasma iron and ferritin concentrations
P l a s m a i r o n c o n c e n t r a t i o n w a s spectrophotometrically determined using ferrozine according to the method described previously [1]. The determination of ferritin was carried out by sandwich ELISA to according to the procedure described previously [19]. Briefly, the concentrations of the affinity purified anti-horse spleen ferritin antibody and the alkaline-phosphatase (ALP) labeled anti-horse spleen ferritin antibody were 100 ng ml -1 and 250 ng ml -1 , respectively. To eliminate inhibitory effect of horse plasma on ferritin immunoassay [19], adult or fetal plasma diluted 11-fold with phosphate-buffered saline (PBS: 20 mM sodium phosphate, 150 mM NaCl, pH 7.2) containing 0.1% Tween 20 and 0.1% gelatin wa s hea t -trea t ed (7 5°C, 15 min) follow ed by centrifugation (24,000 × g, 20 min), and resultant supernatant was subjected to sandwich ELISA.

Ferritin-binding activity
Plasma ferritin-binding activity was measured essentially according to the method described previously [20]. Briefly, 100 μl of horse plasma diluted 10,000-fold with PBS was added to each well of an Immuno Plate Maxisorp F96 microtiter plate (Nunc, Roskilde, Denmark) and the plate was kept overnight at 4°C. The horse plasma protein-coated plate was incubated with horse spleen ferritin (500 ng/well) in PBS containing 0.1% gelatin and 0.1% Tween 20 followed by ALP-rabbit anti-horse spleen ferritin antibody (250 ng/well) [19]. The enzyme reaction was performed as described previously [19].

Purification of plasma FBPs by affinity chromatography
Adult or fetal horse plasma (1-2 ml) was applied to a column (2 × 3.5 cm) prepared by coupling the horse spleen ferritin (10 mg) to 10 ml of CNBr-activated Sepharose 4B (Pharmacia Biotech, Tokyo, Japan) equilibrated with PBS. The column was washed with PBS until the absorbance of the effluent at 280 nm was less than 0.01. PBS containing 3 M KSCN (pH 7.0) was used as the elution buffer and the absorbance of each fraction was measured at 280 nm. Peak fractions were dialyzed by PBS. Pooled fractions collected from fetal or adult horse plasma samples were concentrated with Centriplus YM-50 (Millipore Corp., Billerica, MA, USA) and used as partially affinity-purified samples.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting
Affinity-purified adult and fetal horse FBPs were separated by SDS-PAGE using a 4. Immunoblot analysis was carried out to detect horse immunoglobulin classes according to a previously described procedure [16]. ALP conjugated polyclonal antibodies specific for horse IgM and IgA heavy chains (Bethyl Laboratories, Montgomery, TX, USA) and horse IgG Fc fragment (Rockland Immunochemicals, Gilbertsville, PA, USA) were used as probe. ALPlabeled antibodies bound on the membrane were detected using 100 mM Tris/HCl (pH 9.5) containing 5 mM MgCl 2 , 0.4 mM nitro blue tetrazolium, and 0.4 mM 5-bromo-4-chloro-3-indolylphosphate disodium salt 1,5-hydrate as described previously [16].

Iron and ferritin concentrations and ferritin-binding activity in adult and fetal horse plasma
Iron and ferritin concentrations of six fetal horse plasmas were measured in the gestation age ranged from 130 to 315 days (Table 1). Iron concentrations in six fetal plasmas (Mean ± SD: 2.34 ± 0.67 μg ml -1 ) were higher than those of six adult horse plasmas (1.9 ± 0.30 μg ml -1 ) (Fig. 1A). Remarkable lower plasma ferritin concentrations were found in fetal (24 ± 10 ng ml -1 ) than in adult horse (239 ± 90 ng ml -1 ) (Fig. 1B). Ferritin-binding activity was also found in both of fetal and adult horse plasma, and ferritin-binding activities of fetal plasmas were significantly lower than those of adult plasmas (Fig. 1C).

Characterization of fetal horse FBPs
For structural characterization of horse plasma FBPs, partially affinity-purified adult and fetal horse plasma FBPs were run against commercial horse fibrinogen as plasma specific FBP [20] and maker proteins on SDS-PAGE under reducing condition ( Fig. 2A). Partially purified fetal FBPs were separated into 65 and 41 kDa bands in addition to several bands with higher molecular masses ranged from 102 to 140 kDa, whereas partially affinity-purified adult horse plasma FBPs were separated into 74, 54 and 28 kDa under the same condition. Horse fibrinogen was separated into 74, 60 and 54 kDa bands as Aα, Bβ and γ chains, respectively. Immunoblotting analyses showed that 74 and 54 kDa bands detected in the adult horse plasma FBPs reacted with antibodies to horse IgM and IgG heavy chains, respectively, although antibody to IgM heavy chain cross-reacted with IgG heavy chain (Fig. 2B). Anti-IgA antibody did not detect any band in adult horse FBPs in contrary to previous data [16]. On the other hand, antibodies to horse immunoglobulin classes did not react with any band of fetal plasma FBPs. In addition, the corresponding bands with all three bands (Aα, Bβ and γ chains) detected in horse plasma specific fibrinogen were not observed in fetal and adult horse plasma FBPs, suggesting that the purified fetal and adult horse plasma FBPs did not contain fibrinogen.

Discussion
Because of high levels of ferritin and it's iron content in fetal bovine serum, fetal ferritin may be involved in the iron transporter [8]. On the other hand, various FBPs identified in mammalians provide additional means for the receptor-mediated uptake of circulating ferritin [21,24]. However, any fetal FBP as well as fetal bovine serum and/or plasma has not been purified yet.
Although it is very difficult to get fetal blood samples of large animals such as cattle and horse, we got a good opportunity to obtain valuable fetal horse plasma samples. The purpose of this study is to reveal the presence of fetal horse FBPs and their biochemical properties.
Although plasma iron concentrations of six fetal horse plasmas between 130 and 315 days of gestation age were higher than those of adult horse plasmas, the differences were not significant. This may explain that iron is actively transported from the mother to the fetus [9]. Although serum iron concentration was markedly higher at birth, but rapidly decreases at normal level of adult at 3 days old [7]. However, plasma ferritin concentrations in fetal horses were much lower than those in adult horses ranged from 141 to 340 ng ml -1 (239 ± 90 ng ml -1 ). Serum ferritin concentrations (85 ± 8 ng ml -1 ) for foals were lower than those for adult at birth, but transiently increases near normal levels (159 ± 11 ng ml -1 ) of adult after taking colostrums with high levels of ferritin (354 ± 44 ng ml -1 ) [7], although biochemical characterization of colostrum ferritin has not been performed yet. The lack of correlation between serum iron concentration and storage iron was observed in foal and adult horses [7], and serum ferritin level can be good indicator of iron stores from fetal stage to adult stage including foal development.
We developed ferritin-binding assay using plasma protein-coated microtiter plate as described previously [20]. Ferritin-binding activity was observed in both of fetal and adult horse plasmas, and the binding activities were significantly lower in fetal than in adult. The levels of plasma iron and ferritin concentrations and the ferritin-binding activities showed similar predictive biphasic changes between 130 and 315 days of gestation with concomitant sharp drop in around 250 days of gestation (data not shown), suggesting increasing growth of horse fetus. Eventually, fetal growth in maternal thoroughbred horse is about double size between 220 and 279 days of gestation [22]. However, this study provides only preliminary data, and further study is needed on larger group to examine correlation between fetal development and growth and iron metabolism.
To our knowledge, although it is the first report that horse plasma fibrinogen was identified as an FBP which bound ferritin and inhibit ferritin immunoassay, neither affinity-purified fetal nor adult horse plasma FBPs contain fibrinogen, probably due to it's lower affinity to affinity column as in alpha-2-macroglobulin [16]. However, further study need to examine whether fetal fibrinogen has ferritin-binding activity and inhibitory effect of ferritin immunoassay or not, using the alternative method. Additionally, immunoblot analysis showed that adult horse plasma FBPs contained IgM and IgG autoanibodies to ferritin, but not IgA contrary to previous data [16]. Eventually, IgA heavy chain as anti-ferritin autoantibody was only faintly detected by immunoblot analysis as described before [16] because of the low content in horse serum. This can explain differences of IgA content depending on individual condition. In this study, the attempt to identify fetal horse FBPs was unsuccessful, and antiferritin autoantibodies were not detected in fetal horse plasma FBPs although anti-ferritin autoantibodies are thought to be mammalian common FBP [21]. However, some FBPs are species-specific as in Hkininogen, fibrinogen, and unidentified FBP found in human [28], horse [21], and feline [23], respectively. Fetal horse FBPs might be species-specific and fetal specific FBP. It also remains to elucidate physiological role of FBSs in fetal circulation.