Characterization of transferrin receptors on plasma membranes of lactating rat mammary tissue

Little is known about the transport of iron into the mammary secretory cell and the process of milk iron secretion. The concentration of iron in milk is remarkably unaffected by maternal iron status, suggesting that the uptake of iron into the mammary gland is regulated. It is known that iron enters other cells via transferrin receptor-mediated endocytosis. This study was designed to isolate and characterize the mammary gland transferrin receptor in lactating rat mammary tissue using immunochemical techniques. The existence of functional mammary gland transferrin receptors in lactating rodents was demonstrated using radiolabel-binding techniques. Isolation of mammary transferrin receptors by affinity chromatography was confirmed using immunoelectrophoresis and slot blot analysis. The intact transferrin receptor was found to have a molecular weight of 176 kd as determined by Western blotting followed by scanning densitometry. Reduction of the receptor with beta-mercaptoethanol gave a molecular weight of 98 kd. An additional immunoreactive band of 135 kd was observed. The presence of transferrin receptors in normal lactating rat mammary tissue is likely to explain iron transport into mammary tissue for both cellular metabolism and milk iron secretion.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Influence of ashing techniques on the analysis of trace elements in biological samples

Dry ashing and wet ashing are two commonly used methods for the preparation of biological materials for trace element analysis by atomic absorption spectrophotometry. In this paper, National Bureau of Standards (NBS) bovine liver was dry ashed at 450°C for 24 h in silica glass (Vycor) or procelain crucibles; the resulting ash was dissolved in either concentrated nitric or hydrochloric acid. Dry ashing efficiency was evaluated by comparing iron, copper, zinc, and manganese concentrations of the samples with the values certified by NBS. Highest recoveries were obtained by dry ashing in silica glass (Vycor) crucibles. Dissolving the resultant ash in either hydrochloric or nitric acids did not significantly alter the results. A comparison between dry and wet ashing shows the latter method to be superior for the preparation of biological tissues for analysis of iron, copper, zinc, and manganese.     

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system

AIMS: The lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS. METHODS AND RESULTS: Exposure of E. coli to the LPAS, upon which 99.6% of the bacteria were killed, resulted in the following percentage of inactivation of substrate-specific membrane oxidases: succinate (94.2%) > NADH (84.6%) > glycerol-3-phosphate (67.8%) > DL-lactate (64.1%). With the same treatment, substrate-specific membrane dehydrogenases were inactivated as follows: succinate (99.1%) > DL-lactate (53.8%) > glycerol-3-phosphate (45.0%) > NADH (36.8%). Terminal oxidase, however, measured using a ubiquinone analogue (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) after reduction, was only 21.4% inactivated by the LPAS. CONCLUSION: These data suggest that dehydrogenases are the primary targets of the LPAS in the respiratory chain of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has determined for the first time the primary targets of LPAS in the bacterial respiratory chain.     

Proteomic characterization of human milk whey proteins during a twelve-month lactation period

Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breast-fed infant.     

Ontogenic changes in lactoferrin receptor and DMT1 in mouse small intestine: implications for iron absorption during early life.

It has been proposed that lactoferrin receptor (LfR) may be involved in intestinal iron transport during early life. However, it is known that iron homeostasis is regulated by divalent metal transporter 1 (DMT1; Nramp2/DCT1) in the adult small intestine. To address the hypothesis that LfR may play a role as an alternative iron transport pathway during early life, we used immunohistochemistry (IHC) to examine the localization of mouse LfR (mLfR) and DMT1. In addition to studying the localization and abundance of LfR and DMT1 on the apical membrane, intestinal brush-border membrane vesicles (BBMV) were isolated during the first 3 postnatal weeks (postnatal day (PD) 0, 5, 10, and 20). We found that mLfR is expressed in fetal mice as early as gestational days (GD) 13.5, 15.5, and 18.5. A 34 kD band for mLfR was detected at PD 0 through PD 20 in total intestine homogenate. However, mLfR protein did not appear in the BBMV preparations until PD 5 and was highly expressed at PD 10. By IHC, DMT1 protein was minimally observed at PD 0 and PD 5, but by PD 10 DMT1 was predominantly localized in the apical membrane of the maturing intestine. BBMV fractionation revealed 50-120 kD protein bands for DMT1. In these BBMV preparations, the apical-membrane-associated 120 kD band for DMT1 increased in abundance with age. However, in the corresponding total homogenates, only the deglycosylated form of DMT1 (50 kD) was identified. These results indicate that DMT1 is mislocalized during late gestation, minimally expressed during early life, and predominantly expressed in its deglycosylated form until PD 20. The immunolocalization and abundant protein expression of mLfR suggest that accrual of iron from Lf may be the principal iron uptake pathway at this age. In conclusion, our findings support the notion that until the development-dependent expression of DMT1 in the intestine is induced, mLfR may serve as an alternative iron uptake pathway.     

Bioavailability of zinc and its binding to casein in milks and formulas

Differences in zinc bioavailability among milk and formulas may be attributed to binding of zinc to various ligands. We determined the distribution of zinc and protein at different pHs and zinc and calcium concentrations. We used radiolabelled cow's milk, human milk, whey-predominant (WPF) and casein-predominant (CPF) infant formula. Lowering the pH changed zinc and protein distribution: zinc shifted from pellet (casein) to whey in cow's milk, from fat to whey in human milk and from fat and pellet to whey in formulas. Protein shifted from whey to pellet in human milk and from whey and pellet to fat in formulas. Increasing zinc and calcium concentrations shifted protein and zinc from pellet to whey for cow's milk and from whey and pellet to fat for the formulas. Protein distribution was not affected by calcium or zinc addition in human milk or CPF, while zinc shifted from whey to fat in human milk and from fat and pellet to whey in CPF. Zinc and calcium binding to isolated bovine or human casein increased with pH. At 500 mg/L of zinc, bovine casein bound 32.0 +/- 1.8 and human casein 10.0 +/- 0.9 mg zinc/g protein. At 500 mg/L of calcium, calcium was preferentially bound over zinc. Adding calcium and zinc resulted in 32.0 +/- 1.8 mg zinc/g bound to bovine casein and 17.0 +/- 0.8 mg zinc/g to human casein, while calcium binding was low. Suckling rat pups dosed with 65Zn labelled infant diets were killed and individual tissues were gamma counted. Lower zinc bioavailability was found for bovine milk at pH = 4.0 (%65Zn in liver = 18.7+1.4) when compared to WPF (22.8 +/- 1.6) or human milk (26.9 +/- 0.8). Lowering the pH further decreased zinc bioavailability from human milk, but not from cow's milk or WPF. Knowledge of the compounds binding minerals and trace elements in infant formulas is essential for optimizing zinc bioavailability.     

Non-heme Iron as Ferrous Sulfate Does Not Interact with Heme Iron Absorption in Humans

The absorption of heme iron has been described as distinctly different from that of non-heme iron. Moreover, whether heme and non-heme iron compete for absorption has not been well established. Our objective was to investigate the potential competition between heme and non-heme iron as ferrous sulfate for absorption, when both iron forms are ingested on an empty stomach. Twenty-six healthy nonpregnant women were selected to participate in two iron absorption studies using iron radioactive tracers. We obtained the dose?Cresponse curve for absorption of 0.5, 10, 20, and 50?mg heme iron doses, as concentrated red blood cells. Then, we evaluated the absorption of the same doses, but additionally we added non-heme iron, as ferrous sulfate, at constant heme/non-heme iron molar ratio (1:1). Finally, we compare the two curves by a two-way ANOVA. Iron sources were administered on an empty stomach. One factor analysis showed that heme iron absorption was diminished just by increasing total heme iron (P?<?0.0001). The addition of non-heme iron as ferrous sulfate did not have any effect on heme iron absorption (P?=?NS). We reported evidence that heme and non-heme iron as ferrous sulfate does not compete for absorption. The mechanism behind the absorption of these iron sources is not clear.     

Baculovirus expression of mouse lactoferrin receptor and tissue distribution in the mouse

Lactoferrin (Lf) has been shown to have a role in the immune system and in early development of the mouse embryo. A specific receptor for Lf has been suggested to mediate the functions of Lf. We have recently identified a Lf receptor (LfR) in human fetal small intestine. We therefore hypothesized that the mouse homologue of this protein functions as a LfR. We expressed mouse Lf (MLf) and the mouse homologue (MLfR) in a baculovirus-insect cell system. The recombinant MLfR (rMLfR) was purified by immobilized recombinant MLf (rMLf) affinity chromatography, demonstrating an interaction between rMLf and the rMLfR. RT-PCR revealed that MLfR was expressed in various tissues and during embryonic development. Immunohistochemical analysis revealed that the MLfR was localized in various tissues including small intestinal epithelium, stomach, kidney, ovary, and various regions of brain. In summary, the MLfR functions as a receptor for MLf, is expressed and localized in various tissues, and may be involved in the indispensable function of MLf during early embryonic development.