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Biochemical and Molecular Actions of Nutrients

Low Vitamin A Intake Affects Milk Iron Level and Iron Transporters in Rat Mammary Gland and Liver¹

Department of Nutrition, University of California-Davis, Davis, CA 95616

²To whom correspondence should be addressed. E-mail: slkelleher{at}ucdavis.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Marginal vitamin A deficiency is common and can result in a secondary iron (Fe) deficiency. A positive correlation between maternal Fe status and milk Fe was observed in lactating women supplemented with both vitamin A and Fe but not with Fe alone, suggesting effects of vitamin A on mammary gland Fe transport. We hypothesized that low vitamin A intake during lactation elicits differential effects on mammary gland and liver Fe transport and storage proteins, thus affecting milk Fe concentration but not maternal Fe status. We fed rats a control (CON, 4 RE/g) or a marginal vitamin A diet (AD, 0.4 RE/g) through midlactation. Effects on plasma, milk, liver and mammary gland Fe and vitamin A concentrations, and divalent metal transporter-1 (DMT1), ferroportin (FPN), ferritin (Ft), and transferrin receptor (TfR) expression were determined. Dams fed AD were not vitamin A or Fe deficient. Milk and liver vitamin A and Fe and mammary gland Fe concentrations were lower in rats fed AD compared with rats fed CON. Liver TfR expression was higher, whereas mammary gland TfR expression was lower in rats fed AD compared with rats fed CON. Liver Ft was unaffected, whereas mammary gland Ft was lower in rats fed AD compared with rats fed CON. Liver and mammary gland DMT1 and FPN protein levels were lower in rats fed AD compared with rats fed CON. Our results indicate that the mammary gland and liver respond differently to marginal vitamin A intake during lactation and that milk Fe is significantly decreased due to effects on mammary gland Fe transporters, putting the nursing offspring at risk for Fe deficiency.

KEY WORDS: • divalent metal transporter-1 • ferroportin • iron • retinol • lactation

Iron (Fe) deficiency anemia is the most common nutrient deficiency and is estimated to affect 1–2 billion people worldwide (1). Although maternal Fe deficiency in humans has not been associated with neonatal Fe deficiency anemia, neonatal Fe stores are decreased (2), leaving the newborn at increased risk for Fe deficiency if Fe intake is inadequate. In addition to low Fe intake, Fe deficiency anemia can be caused by a variety of factors including vitamin A deficiency (3). Many studies showed a positive effect of vitamin A supplementation on Fe status in humans and animal models (4–6); however, the interaction between vitamin A deficiency and Fe metabolism is complex. Vitamin A deficiency has been associated with multiple anomalies such as reduced incorporation of Fe into erythrocytes (7), altered RBC morphology (8), mild anemia (9), lower plasma total iron-binding capacity, increased Fe absorption, and Fe accumulation in spleen and bone (10). The mechanisms through which vitamin A deficiency affects whole-body Fe homeostasis are unknown; however, retinoic acid redistributes intracellular Fe pool(s), resulting in decreased transferrin receptor (TfR)³ expression in promonocytic U937 cells (11) and increased ferritin expression in rodent brain and neuronal cells in culture (12). Conversely, retinoic acid exposure increased TfR mRNA levels in keratinocytes (13), indicating that the effects of retinoic acid on the Fe transport machinery are cell specific.

The relatively high prevalence of marginal vitamin A status among pregnant and lactating women has raised the concern that it is a contributing factor in morbidity, mortality, and the etiology of anemia among women and their infants (4). Muslimatun et al. (5) observed that milk Fe concentration was lower in women supplemented with Fe alone compared with women supplemented with Fe and vitamin A, suggesting a positive effect of increased vitamin A intake on milk Fe concentration. Interestingly, vitamin A supplementation was not associated with improved maternal Fe status, which suggests that Fe metabolism in the liver and mammary gland may respond differently to inadequate vitamin A intake during lactation.

Cellular Fe transport is a tightly regulated process consisting of Fe uptake across the plasma membrane, partitioning of Fe into specific intracellular pools, and Fe export across the plasma membrane in some cell types. In both the liver and mammary gland, cellular Fe uptake is facilitated by TfR. Diferric transferrin binds to TfR at the cell surface (14). The transferrin-TfR complex is internalized in clathrin-coated vesicles that fuse with acidic endosomes, which have a pH 5.5, through the action of an ATP-dependent proton pump in the endosomal membrane. The acidic environment facilitates the release of Fe from the transferrin-TfR complex within the endosomal vesicle. In the liver, Fe is transported out of the endosome by divalent metal transporter 1 (DMT1) (15,16). We determined previously that DMT1 is also expressed in the mammary gland (17) and hypothesize that mammary gland DMT1 is similarly responsible for endosomal Fe export in the mammary epithelial cell; however, its localization and the role it plays in mammary gland Fe metabolism have not yet been characterized. Once Fe has entered the cytoplasm, it may partition into a chelatable Fe pool and participate in a multitude of cellular processes such as sequestration into ferritin (Ft) for storage, incorporation into essential Fe-containing proteins in the endoplasmic reticulum (ER), or transport to the basolateral membrane for export. A final step in cellular Fe metabolism in some specialized cell types is export across the plasma membrane back into circulation (liver) or into milk (mammary gland). Ferroportin (FPN) or iron-responsive element (IRE) G1 mediates Fe export across the enterocyte basolateral membrane and its expression is reciprocally regulated by Fe status via hepcidin (18). Although FPN was also identified in the plasma membrane of hepatocytes, its expression is downregulated in response to Fe deficiency; thus, its contribution toward maintaining Fe homeostasis is currently unknown (19). Alternatively, FPN is localized to the endoplasmic reticulum in reticuloendothelial cells where it is assumed to facilitate Fe transport into an intracellular vesicle before secretion (19). We determined previously that FPN is expressed in the mammary gland (17) and speculate that mammary gland FPN similarly transports Fe into secretory vesicles destined for export into milk.

In this study, we hypothesized that marginal vitamin A intake during lactation results in low milk Fe concentration through alterations in the expression of known Fe transport proteins. Because the liver and mammary gland play unique roles in Fe metabolism during lactation (i.e., maintaining maternal Fe homeostasis or providing Fe for secretion into milk, respectively), we further hypothesized that liver and mammary gland Fe transporters would be differentially affected, thus reflecting their unique contributions to Fe metabolism.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Diets. Rats were fed a casein-based semipurified experimental diet based on the AIN-93 recommendation (15). The diet composition differed only in vitamin A content with the control diet containing 4 RE/g (CON) and the diet marginally low in vitamin A containing 0.4 RE/g (AD), as previously described (20).

    Animals. This study was approved by Animal Research Services at the University of California, Davis, which is accredited by the American Association for the Accreditation of Laboratory Animal Care. Virgin Sprague-Dawley rats (n = 24) were obtained commercially (Simonsen) and maintained in stainless steel hanging cages. Rats (n = 12/diet) were randomly assigned to consume a control semipurified diet (CON) or a diet marginally low in vitamin A (AD) and deionized water ad libitum. To deplete liver vitamin A stores, rats were fed diets for 70 d before mating, through gestation, until midlactation (lactation d 10). On lactation d 2, litters were culled to 6–8 pups/dam. On lactation d 10, dams were removed from pups for 4 h, anesthetized (intraperitoneal, 1.6 mg xylazine/kg and 33 mg ketamine/kg), and milk was manually expressed after s.c. oxytocin injection (10 U/dam). Blood was collected by cardiac puncture into heparinized vials and rats were killed by asphyxiation with CO₂. Hemoglobin concentration and packed cell volume (hematocrit) were measured. Plasma was separated by centrifugation at 2000 x g for 15 min at 4°C and frozen at –80°C until analysis. Liver and mammary gland were dissected and stored in RNA Later (Ambion) for measurement of TfR, Ft, DMT1, and FPN mRNA levels using real-time RT-PCR or immediately snap-frozen in liquid nitrogen for determination of Fe and retinol (ROH) concentration and TfR, Ft, DMT1, and FPN protein levels using relative Western blot analysis.

    Measurement of hemoglobin, hematocrit, and transferrin saturation. Whole-blood hemoglobin was measured after conversion to cyanomethemoglobin using a commercially available colorimetric assay (Sigma). Packed RBC volume (hematocrit) was determined after whole blood was drawn into a microcapillary tube, sealed, and centrifuged for 10 min at 2000 x g. Hematocrit was expressed as a percentage of packed RBC volume. Transferrin saturation was measured using a commercially available kit (Total Iron Binding Capacity, Sigma).

    Iron and ROH analysis. Plasma was digested at room temperature with 0.1 mol/L trace mineral–free nitric acid (Fisher Scientific). Mammary glands were minced and rinsed 3 times in fresh isotonic saline at room temperature for 10 min each to remove sequestered milk. Whole milk, blot-dried, minced mammary gland, and liver were digested with concentrated nitric acid and wet-ashed using a modification of the method of Clegg et al. (21). Iron was analyzed by flame atomic absorption spectroscopy (Model Smith-Heifjie 4000, Thermo Jarrell Ash). Plasma, milk, liver and mammary gland ROH was quantified by reversed-phase HPLC as previously described (20).

    Quantification of TfR, Ft, DMT1, and FPN mRNA levels by real-time RT-PCR. Total RNA was isolated using TriZOL (Invitrogen) as previously described (22) and diluted (1 g/L) in RNAse-free water. RNA integrity was evaluated after electrophoresis through agarose and staining with ethidium bromide (Sigma). cDNA was generated from 1 µg RNA using a reverse transcription kit (Perkin Elmer Applied Biosystems) following the manufacturer’s instruction; the reaction was performed at 48°C for 30 min followed by 95°C for 5 min. Gene-specific primers to rat TfR (NM_011638, Forward: 5'-TCG GCT ACC TGG GCT ATT GT-3'; Reverse: 5'-CCG CCT CTT CCG CTT CA-3'); Ft (NM_012848, Forward: 5'-CCC TGG GAC ACG GTG ATG-3'; Reverse: 5'-GCC TCA GTG ACC AGT AAA GTC ACA-3'); DMT1 (NM_013173, Forward: 5'-CAT CCC TAT CCT CAC CTT CAC AAG-3'; Reverse: 5'-GCG ATC CTC CAG CCT ATT CC-3'); FPN (NM_133315, Forward: 5'-TGC CAC TGC AAT TAC AAT CCA-3'; Reverse: 5'-TCT GCT AAT CTG CTC CTG TTT TCT C-3') and GAPDH (NM_017008, Forward: 5'-TGC CAA GTA TGA TGA CAT CAA GAA G-3'; Reverse: 5'-AGC CCA GGA TGC CCT TTA GT-3') were chosen using Primer Express Software (Perkin Elmer Applied Biosystems) and purchased from Qiagen. Real-time PCR was performed on 4 µL of the cDNA reaction mixture using the ABI 7900HT real-time thermocycler (Perkin Elmer Applied Biosystems) coupled with SYBR Green technology (Perkin Elmer Applied Biosystems) and the following cycling parameters: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s; 60°C for 1 min; 95°C for 15 s; 60°C for 15 s; 95°C for 15 s.

The linearity of the dissociation curve was analyzed using the ABI 7900HT software and the mean cycle time of the linear part of the curve was designated Ct. Each sample was analyzed in duplicate and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the following equation: Ct_GENE = Ct_GENE –Ct_GAPDH. The fold-change relative to rats fed the control diet was calculated using the following equation: where Ct_GENE = mean Ct_GENE of the control rats –Ct_GENE of each AD rat. Values represent mean fold-change ± SD.

    Detection of liver and mammary gland TfR, Ft, DMT1, and FPN by Western blot. Tissue (500 mg) was homogenized in 10 mL Hepes-EDTA buffer as previously described (22), and protein concentration was determined by the Bradford protein assay (BioRad). An equal amount of total protein (100 µg) was separated by electrophoresis under reducing conditions and transferred to nitrocellulose for 1 h at 350 mA (Biorad). Membranes were blocked for 1 h in nonfat milk (0.5 g/L) in PBS/Tween-20 (0.01 g/L, PBS-T) and washed 3 times in PBS-T, followed by incubation with primary antibody (DMT1, 1:1,000; TfR, 1:5,000; Ft, 1:500; FPN, 1:1,000) for 45 min and washed 3 times in PBS-T. DMT1 and FPN antiserum was previously characterized by Leong et al. (23). Affinity purified mouse anti-rat TfR and rabbit anti-rat Ft were purchased from Pharmingen and Biogenesis, respectively. Fe transport proteins were detected after incubation with donkey anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Amersham Pharmacia Biotech) or anti-mouse IgG-HRP (Pharmingen), visualized with SuperSignal Femto Chemiluminescent Detection System (Pierce), and exposed to autoradiography film. Relative band density was quantified using the Chemi-doc Gel Quantification System (Biorad). Blots were stripped (Western Stripping Buffer, Pierce) and immunodetection of ß-actin was used as a loading control.

    Localization of DMT1 and FPN in the mammary gland. Mammary gland from control rats was fixed in paraformaldehyde (0.4 g/L in PBS) for 24 h at 4°C, washed extensively with PBS and sequentially dehydrated in ethanol as previously described (24). The paraffin-embedded mammary gland was sectioned (4 µm) and immunostained with affinity-purified antibody (DMT1, 1 mg/L) (25) or polyclonal antiserum (FPN, 1:1,000) for 1 h at room temperature. The localization of DMT1 and FPN was detected with 3,3'-diaminobenzidine tetrahydrochloride after incubation with anti-rabbit IgG conjugated to HRP (Vector Labs) and counterstained with hematoxylin.

    Statistical analysis. Results are presented as means ± SD, n = 6 rats/group for mRNA analysis and n = 12 rats/group for all other variables. Statistical comparisons were made using an unpaired t test (Prism Graph Pad) and differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Effect of marginal maternal vitamin A intake on vitamin A and iron status. Rats fed AD were not vitamin A deficient as determined by a plasma ROH concentration > 0.7 µmol/L, although liver ROH concentration was lower in rats fed AD (P < 0.05, Table 1) compared with CON-fed rats. The level of dietary vitamin A deficiency used in this study was not severe enough to significantly affect hemoglobin, hematocrit, and plasma Fe; however, rats fed AD had lower transferrin saturation and lower liver Fe concentration than CON rats (P < 0.05, Table 1). Although mammary gland ROH concentration was not significantly affected, milk ROH concentration was lower in rats fed AD compared with CON-fed rats (P < 0.05, Table 1). Rats fed AD had lower mammary gland and milk Fe concentration compared with control rats (P < 0.05, Table 1).

View larger version (150K): [in this window] [in a new window]   FIGURE 1 The localization of DMT1 and FPN in the mammary gland of control rats during lactation. Representative immunostains of rat mammary gland at d 10 of lactation using preimmune serum (1:1,000, Control) (A); FPN antiserum (1:1,000) (B); or affinity-purified DMT1 antibody (1 mg/L) (C). (D) Localization of DMT1 (1 mg/L) in the small intestine for comparison of cellular localization between tissues. Scale bar represents 10 µm. Images are magnified X40 and the asterisk (*) identifies the lumen of the alveoli (A, B, C) or gastrointestinal tract (D) for orientation. Arrows illustrate areas of enriched immunostained protein.

    Effect of marginal maternal vitamin A intake on mammary gland and liver iron transporter protein levels. We detected 2 DMT1 proteins of similar abundance at 66 and 85 kDa in the mammary gland; however, only the protein at 66 kDa was routinely detected in the liver (Fig. 2). Additionally, we detected 2 TfR proteins in the liver, a minor protein at the expected molecular weight of 98 and a major protein at 22 kDa; however, in the mammary gland, only the 98-kDa protein was detected. We determined that rats fed low vitamin A during lactation had lower DMT1 protein levels in the liver and mammary gland compared with control rats (P < 0.05, Fig. 2 and Table 2). We detected FPN protein at the expected molecular weight of 68 kDa, and FPN protein levels were lower in both the liver and the mammary gland in rats fed the marginal vitamin A diet compared with control rats (P < 0.05, Fig. 2 and Table 2). TfR protein levels were higher in the liver of rats fed the marginal vitamin A diet but lower in the mammary gland (P < 0.05, Fig. 2 and Table 2) compared with control rats. Ft protein levels were not affected in the liver, but mammary gland ferritin level was lower in rats fed the marginal vitamin A diet (P < 0.05, Fig. 2 and Table 2).

View larger version (76K): [in this window] [in a new window]   FIGURE 2 Effects on mammary gland and liver DMT1, FPN, TfR, and Ft protein levels in lactating rats fed the control (CON) or marginal vitamin A (AD) diet. Representative Western blots of rat mammary gland and liver (n = 3 rats for each diet/tissue) illustrating changes in DMT1, FPN, TfR, and Ft protein levels in response to the AD. ß-Actin was used as a loading control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The marginal level of vitamin A intake in our study did not reduce maternal Fe status (6,10,27) or plasma ROH concentration, most likely a consequence of the modest vitamin A deficiency used (6). However, in contrast to results in weanling rats, liver Fe was mobilized to maintain Fe homeostasis (10,11), suggesting a unique sensitivity of liver Fe metabolism to marginal vitamin A intake during reproduction as a result of the intensified demand to mobilize Fe from the liver to provide adequate Fe transfer (4,28). Interestingly, although the liver responded to marginal vitamin A deficiency by increasing TfR mRNA expression, abundant expression of a small TfR protein was detected. This smaller protein may functionally reduce liver Fe uptake, thus diverting transferrin-Fe to other maternal tissues during reproduction.

The mammary gland has a remarkable capacity for Fe transfer from plasma to milk (29,30) and although rat milk Fe concentration is 25 times higher than that of human milk, the amount of Fe transferred across the mammary gland per day is approximately equal. Although we determined previously that marginal Fe deficiency does not affect milk Fe concentration in rats (17), our results indicate that marginal vitamin A deficiency reduced milk Fe concentration despite having little effect on maternal Fe status. In contrast to results in the liver, both mammary gland Fe concentration and TfR expression were significantly lower in marginally vitamin A–deficient rats, likely reducing mammary gland Fe uptake. Many studies demonstrated reciprocal regulation of TfR protein levels by tissue Fe levels through alterations in TfR mRNA stability (31) due to the presence of an IRE on the 3'-UTR. However, our results indicate that this regulation is uncoupled in the mammary gland because both tissue Fe concentration and mammary gland TfR expression are reduced during both marginal vitamin A and Fe deficiency (17), thus contributing to the reduction in milk Fe concentration.

Although tissue Fe and ROH reductions were not severe enough to elicit a significant effect on liver Fe storage mechanisms (32), mammary gland Ft expression was lower, indicating that mammary gland Fe depletion was severe enough to deplete cellular Fe storage mechanisms similar to effects of Fe deficiency (17). Ferritin functions exclusively in cellular Fe homeostasis because it is not secreted into milk, and cellular Fe pools do not provide Fe for secretion into milk (27,28). However, the contribution of cellular Fe stores toward the regulation of Fe transporters is unknown because we determined previously that although mammary gland Fe level is maintained throughout lactation, mammary gland Fe transporter expression and milk Fe concentration decrease significantly (17).

DMT1 is localized to both the plasma membrane and an intracellular compartment (33) in hepatocytes. In addition to its role in endosomal Fe export, the presence of DMT1 on the microvillous membrane of hepatocytes suggests that the liver absorbs nonheme, nontransferrin-bound Fe from the circulation (33). Unlike intestine DMT1, hepatic DMT1 expression parallels Fe status (33), suggesting that DMT1 plays more of a role in cellular protection from excess Fe as opposed to regulating Fe homeostasis in the liver. Therefore, the decreased liver DMT1 protein levels observed in this study may reflect reduced liver Fe or ROH levels or both. The mechanisms behind the disparate regulation of intestine and liver DMT1 are currently unknown although recently 4 DMT1 variants resulting from alternative promoters and splicing were characterized (34). Interestingly, the expression pattern of these variants indicates that the less Fe-responsive variants (DMT1-exon 1B and DMT1-IRE) are expressed in the liver, whereas the classic Fe-responsive variant (DMT1-exon 1A) is not. Nevertheless, a low vitamin A diet reduced liver DMT1 protein expression by post-transcriptional regulation via decreased protein translation or increased degradation similar to observations in the yeast DMT1 homologue Smp1 (35); however, the role ROH may play in this regulation is unknown.

The localization of DMT1 in the mammary gland suggests that it plays a similar role in endosomal Fe export in the mammary epithelial cell and we speculate that a decrease in DMT1 would reduce vesicular Fe export, presumably reducing intracellular Fe pools. The molecular weight of DMT1 is tissue specific and was observed to be 66 and 85 kDa (23,25,28,36), which may reflect the expression of DMT1 variants, cell-specific post-translational modifications, localization, and/or function. Although there is no direct evidence indicating which DMT1 variants are expressed in the mammary gland, a search of the Expressed Sequence Tags database (37) using the exon 1A mRNA sequence (34) revealed that the reciprocally regulated Fe-responsive variant exon 1A is expressed in human and mouse mammary gland although no clones could be identified in rats. This species-specific difference may help to explain why milk Fe concentration is generally more refractory to maternal Fe status in humans, as DMTI provides reciprocal Fe regulation in the mammary gland of lactating women but not in the mammary gland of rats.

Although routinely referred to as a "basolateral Fe efflux protein," in tissues other than the intestine, FPN exports Fe from the cytosol into a vesicle or through the secretory pathway (19). Within the mammary epithelial cell, FPN is localized to both intracellular- and plasma membrane-associated compartments, similar to observations in Kupffer cells (intracellular) and hepatocytes (plasma membrane) (19), suggesting that FPN contributes either directly or indirectly to Fe secretion into milk. Fe in milk is bound to lactoferrin in mice and humans (38) and transferrin in rats (39); it is also associated with the casein and lipid fractions that are secreted in vesicles via the ER-Golgi pathway. Thus, the mammary cells must have an efficient mechanism for transporting Fe into the ER, Golgi, or secretory vesicles as they transit to the luminal membrane for membrane fusion and secretion into milk. FPN appears to be regulated by post-transcriptional mechanisms in both tissues, perhaps through reduced tissue Fe levels; in the mammary gland, its functional effect is to reduce Fe secretion into milk.

In conclusion, this study demonstrated that low vitamin A intake during lactation reduces milk Fe concentration, which is correlated with decreased DMT1 transcription and a post-translational decrease in FPN protein levels in the mammary gland. We speculate that DMT1 exports Fe from intracellular vesicles into the cytosol, possibly targeted toward cellular Fe pools, whereas FPN may import Fe from the cytosol into secretory vesicles, thus more directly participating in Fe secretion into milk. Although these protein levels were also reduced in the liver, the mechanisms through which these changes were elicited appear to be post-transcriptional (DMT1) and post-translational (FPN), respectively, suggesting that these differential responses to reduced tissue Fe levels reflect the disparate roles played by the liver and mammary gland in Fe and ROH homeostasis. Finally, the effect of low vitamin A intake on DMT1 and FPN expression may not be confined to the liver and mammary gland, and we speculate that it may be one of the primary effectors of a low vitamin A diet on Fe metabolism in other tissues such as the intestine.

	ACKNOWLEDGMENTS

 
The authors gratefully acknowledge the technical assistance of Mohammad Bakhtiar Hossain and Maggie Chiu.

	FOOTNOTES

 
¹ Funded in part by the University of California Davis Clinical Nutrition Research Unit, National Institutes of Health DK 35747 and faculty research grants to B.L.

³ Abbreviations used: AD, marginal vitamin A diet; CON, control diet; DMT1, divalent metal transporter-1; ER, endoplasmic reticulum; FPN, ferroportin; Ft, ferritin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; IRE, iron-responsive element; ROH, retinol; TfR, transferrin receptor.

Manuscript received 10 August 2004. Initial review completed 22 September 2004. Revision accepted 20 October 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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