Hepatic transferrin plays a role in systemic iron homeostasis and liver ferroptosis.

Although the serum-abundant metal-binding protein transferrin (encoded by the Trf gene) is synthesized primarily in the liver, its function in the liver is largely unknown. Here, we generated hepatocyte-specific Trf knockout mice (Trf-LKO), which are viable and fertile but have impaired erythropoiesis and altered iron metabolism. Moreover, feeding Trf-LKO mice a high-iron diet increased their susceptibility to developing ferroptosis-induced liver fibrosis. Importantly, we found that treating Trf-LKO mice with the ferroptosis inhibitor ferrostatin-1 potently rescued liver fibrosis induced by either high dietary iron or carbon tetrachloride (CCl4) injections. In addition, deleting hepatic Slc39a14 expression in Trf-LKO mice significantly reduced hepatic iron accumulation, thereby reducing ferroptosis-mediated liver fibrosis induced by either a high-iron diet or CCl4 injections. Finally, we found that patients with liver cirrhosis have significantly lower levels of serum transferrin and hepatic transferrin, as well as higher levels of hepatic iron and lipid peroxidation, compared with healthy control subjects. Taken together, these data indicate that hepatic transferrin plays a protective role in maintaining liver function, providing a possible therapeutic target for preventing ferroptosis-induced liver fibrosis.

Iron homeostasis: An anthropocentric perspective.

The regulation of iron metabolism in biological systems centers on providing adequate iron for cellular function while limiting iron toxicity. Because mammals cannot excrete iron, mechanisms have evolved to control iron acquisition, storage, and distribution at both systemic and cellular levels. Hepcidin, the master regulator of iron homeostasis, controls iron flows into plasma through inhibition of the only known mammalian cellular iron exporter ferroportin. Hepcidin is feedback-regulated by iron status and strongly modulated by inflammation and erythropoietic demand. This review highlights recent advances that have changed our understanding of iron metabolism and its regulation.

Role of hemoglobin and transferrin in multi-wall carbon nanotube-induced mesothelial injury and carcinogenesis.

Multi-wall carbon nanotubes (MWCNT) are a form of flexible fibrous nanomaterial with high electrical and thermal conductivity. However, 50-nm MWCNT in diameter causes malignant mesothelioma (MM) in rodents and, thus, the International Agency of Research on Cancer has designated them as a possible human carcinogen. Little is known about the molecular mechanism through which MWCNT causes MM. To elucidate the carcinogenic mechanisms of MWCNT in mesothelial cells, we used a variety of lysates to comprehensively identify proteins specifically adsorbed on pristine MWCNT of different diameters (50 nm, NT50; 100 nm, NT100; 150 nm, NT150; and 15 nm/tangled, NTtngl) using mass spectrometry. We identified >400 proteins, which included hemoglobin, histone, transferrin and various proteins associated with oxidative stress, among which we selected hemoglobin and transferrin for coating MWCNT to further evaluate cytotoxicity, wound healing, intracellular catalytic ferrous iron and oxidative stress in rat peritoneal mesothelial cells (RPMC). Cytotoxicity to RPMC was observed with pristine NT50 but not with NTtngl. Coating NT50 with hemoglobin or transferrin significantly aggravated cytotoxicity to RPMC, with an increase in cellular catalytic ferrous iron and DNA damage also observed. Knockdown of transferrin receptor with ferristatin II decreased not only NT50 uptake but also cellular catalytic ferrous iron. Our results suggest that adsorption of hemoglobin and transferrin on the surface of NT50 play a role in causing mesothelial iron overload, contributing to oxidative damage and possibly subsequent carcinogenesis in mesothelial cells. Uptake of NT50 at least partially depends on transferrin receptor 1. Modifications of NT50 surface may decrease this human risk.

Recombinant human erythropoietin-induced erythropoiesis regulates hepcidin expression over iron status in the rat.

The crosstalk between several factors controlling hepcidin synthesis is poorly clarified for different physiological and pathological conditions. Our aim was to study the impact of increasing recombinant human erythropoietin (rHuEPO) doses on erythropoiesis, iron metabolism and hepcidin, using a rat model. Male Wistar rats were divided in 5 groups: control (vehicle) and rHuEPO-treated groups (100, 200, 400 and 600IU/kgbody weight/week), 3 times per week, during 3weeks. Hematological and iron data were evaluated. The expression of several genes involved in iron metabolism was analyzed by qPCR. Liver hepcidin protein was evaluated by Western Blot. The rHuEPO treatment induced erythropoiesis and increased transferrin saturation (TSAT) in a dose dependent manner. Tf receptor 2 (TfR2), hemojuvelin (HJV) and bone morphogenetic protein 6 (BMP6) were up-regulated in rHuEPO200 group. Matriptase-2 was down-regulated in rHuEPO200 group, and up-regulated in the other rHuEPO-treated groups. Hepcidin synthesis was increased in rHuEPO200 group, and repressed in the rHuEPO400 and rHuEPO600 groups. Our study showed that when a high erythropoietic stimulus occurs, hepcidin synthesis is mainly regulated by TSAT; however, when the erythropoiesis rate reaches a specific threshold, extramedullary hematopoiesis is triggered, and the control of hepcidin synthesis is switched to matriptase-2, thus inhibiting hepcidin synthesis.

The glycation site specificity of human serum transferrin is a determinant for transferrin's functional impairment under elevated glycaemic conditions.

The mechanisms involving iron toxicity in diabetes mellitus are not completely understood. However, the spontaneous reaction of reducing sugars with protein amino groups, known as glycation, has been shown to compromise the action of Tf (transferrin), the systemic iron transporter. In order to understand the structural alterations that impair its function, Tf was glycated in vitro and the modification sites were determined by MS. Iron binding to glycated Tf was assessed and a computational approach was conducted to study how glycation influences the iron-binding capacity of this protein. Glycated Tf samples were found to bind iron less avidly than non-modified Tf and MS results revealed 12 glycation sites, allowing the establishment of Lys534 and Lys206 as the most vulnerable residues to this modification. Their increased susceptibility to glycation was found to relate to their low side-chain pKa values. Lys534 and Lys206 participate in hydrogen bonding crucial for iron stabilization in the C- and N-lobes of the protein respectively, and their modification is bound to influence iron binding. Furthermore, the orientation of the glucose residues at these sites blocks the entrance to the iron-binding pocket. Molecular dynamics simulations also suggested that additional loss of iron binding capacity may result from the stereochemical effects induced by the glycation of lysine residues that prevent the conformational changes (from open to closed Tf forms) required for metal binding. Altogether, the results indicate that Tf is particularly vulnerable to glycation and that this modification targets spots that are particularly relevant to its function.

Transferrin-a modulates hepcidin expression in zebrafish embryos.

The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo.

Role of transferrin, transferrin receptors, and iron in macrophage listericidal activity.

It is not yet known what properties distinguish macrophages which can kill facultative intracellular bacteria, such as Listeria monocytogenes, from those which cannot. Listeria is an organism which requires iron for growth, yet macrophage listericidal mechanisms are also likely to be iron dependent. We show here that resident peritoneal macrophages and thioglycollate-elicited macrophages cannot kill listeria, but proteose peptone-elicited and FCS-elicited macrophages can. All these cell populations phagocytose listeria. Transferrin receptor expression is low on resident cells, intermediate on peptone- and FCS-elicited cells, and high on thioglycollate-elicited cells. Transferrin transports iron into cells via the transferrin receptor: thus, iron content of resident cells is low, of peptone- and FCS-elicited cells is intermediate, and of thioglycollate-elicited cells is high. Moreover, antibody to transferrin, which prevents it binding its receptor, inhibits listericidal macrophages from killing this bacterium. Finally, nonlistericidal cells with high transferrin receptor expression and high intracellular iron become listericidal if they are incubated with apotransferrin, an iron-free ligand which prevents iron uptake by cells. These data suggest that macrophages must have enough available intracellular iron to support listericidal mechanisms, but too much iron favors growth of the bacterium, which no longer can be killed by the macrophage.

The role of transferrin in actinide(IV) uptake: comparison with iron(III).

The impact of actinides on living organisms has been the subject of numerous studies since the 1950s. From a general point of view, these studies show that actinides are chemical poisons as well as radiological hazards. Actinides in plasma are assumed to be mainly complexed to transferrin, the iron carrier protein. This paper casts light on the uptake of actinides(IV) (thorium, neptunium, plutonium) by transferrin, focusing on the pH dependence of the interaction and on a molecular description of the cation binding site in the protein. Their behavior is compared with that of iron(III), the endogenous transferrin cation, from a structural point of view. Complementary spectroscopic techniques (UV/Vis spectrophotometry, microfiltration coupled with gamma spectrometry, and X-ray absorption fine structure) have been combined in order to propose a structural model for the actinide-binding site in transferrin. Comparison of our results with data available on holotransferrin suggests some similarities between the behavior of Fe(III) and Np(IV)/Pu(IV)/ Np(IV) is not complexed at pH <7, whereas at pH approximately 7.4 complexation can be regarded as quantitative. This pH effect is consistent with the in vivo transferrin "cycle". Pu(IV) also appears to be quantitatively bound by apotransferrin at around pH approximately 7.5, whereas Th(IV) was never complexed under our experimental conditions. EXAFS data at the actinide edge have allowed a structural model of the actinide binding site to be elaborated: at least one tyrosine residue could participate in the actinide coordination sphere (two for iron), forming a mixed hydroxo-transferrin complex in which actinides are bound with transferrin both through An-tyrosine and through An--OH bonds. A description of interatomic distances is provided.

Advancement of the study on iron metabolism and regulation in tumor cells.

As an essential metal for sustaining life, iron is involved in a number of metabolic processes, including DNA synthesis, electron transport, oxygen delivery, and so on. Iron metabolism involves the absorption, transport, and use of iron and is strictly regulated. Numerous studies have found a positive correlation between iron storage and the risk of tumors, such as colorectal carcinoma, hepatic cancer, renal carcinoma, lung cancer, and gastric cancer. In tumor cells, iron metabolism changes by several mechanisms, such as regulating the growth of tumor cells by transferrin, accelerating the uptake of iron by the overexpressions of transferrin receptors 1 and 2 (TfR1 and TfR2), synthesizing or secreting ferritin by some malignant tumor cells, and upregulating the level of hepcidin in patients with cancer. Some advances on diagnosis and treatment based on iron metabolism have been achieved, such as increasing the transfection and target efficiency of transferrin-polyethylenimine (PEI), inducing cell apoptosis by beta-guttiferin through interacting with TfR1.

[Iron and age-related macular degeneration: a new track]. / La dégénérescence maculaire liée à l'âge: La piste du fer.

Iron has a fundamental role for cell physiology and especially in retina as a cofactor of many pathways of the visual transduction. A tightly regulated homeostasis avoids the accumulation of prooxidant and proinflammatory free iron. A dysfunction of iron retinal homeostasis is associated with many genetic or age-related degenerative diseases such as age-related macular degeneration (AMD). Here, we describe various mechanisms reported during AMD, enhanced by iron accumulation and its homeostasis dysregulation. We have investigated a local treatment with transferrin, the natural iron carrier, to control these pathological pathways and iron dysfunction, without side effects. Iron has a central role in pathogenesis of AMD and is a target for futures therapies.

TITLE: La dégénérescence maculaire liée à l'âge: La piste du fer. ABSTRACT: En raison de l'intense activité physiologique de la fonction visuelle, l'homéostasie du fer dans la rétine y est contrôlée localement. Sous l'effet de sa dérégulation (qui a des origines génétiques, environnementales, ou due au vieillissement), le fer libre s'accumule et devient, par ses propriétés oxydantes et inflammatoires, toxique, comme cela est observé au cours de la dégénérescence maculaire liée à l'âge (DMLA). Le rétablissement d'un métabolisme du fer équilibré est donc une possibilité thérapeutique. Néanmoins, la toxicité oculaire des chélateurs chimiques oriente les recherches vers des chélateurs biologiques naturels. Nos travaux montrent que la transferrine, le transporteur du fer, préserve la rétine des mécanismes associés à la DMLA.

Structural biology of bacterial iron uptake.

To fulfill their nutritional requirement for iron, bacteria utilize various iron sources which include the host proteins transferrin and lactoferrin, heme, and low molecular weight iron chelators termed siderophores. The iron sources are transported into the Gram-negative bacterial cell via specific uptake pathways which include an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. Over the past two decades, structures for the proteins involved in bacterial iron uptake have not only been solved, but their functions have begun to be understood at the molecular level. However, the elucidation of the three dimensional structures of all components of the iron uptake pathways is currently limited. Despite the low sequence homology between different bacterial species, the available three-dimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria.

Cell proliferation and expression of the transferrin receptor gene: promoter sequence homologies and protein interactions.

A 365-bp fragment from the 5' region of the human transferrin receptor gene has been subcloned and sequenced. This fragment contains 115 bp of flanking sequence, the first exon, and a portion of the first intron. It contains a TATA box, several GC-rich regions, and is able to efficiently promote expression of the bacterial CAT gene in mouse 3T3 cells. Sequence comparisons demonstrate that this DNA segment has homology to the promoter regions of the human dihydrofolate reductase gene and the mouse interleukin 3 gene, as well as to a monkey DNA sequence that has homology to the SV40 origin and promotes expression of an unidentified gene product. Several high molecular mass proteins that interact with the transferrin receptor gene promoter have been identified. The activity of these proteins is transiently increased in 3T3 cells that have been stimulated by serum addition. This increase precedes a rise in transferrin receptor mRNA levels in the cytoplasm, which in turn precedes entry of the cells into S phase. DNase I footprinting of the transferrin receptor promoter reveals several protein binding sites. Two of the sites are within the conserved GC-rich region of the promoter. One of these binding sites probably interacts with Spl, while the second interacts with an uncharacterized protein.

A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells.

A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy of ultracryosections of mucosal tissue, the protein was localized to the apical surface of the enterocytes, whereas it was absent from the basolateral plasma membrane. Interestingly, it was mainly found in patches of flat or invaginated apical membrane domains rather than at the surface of microvilli. Caveolae were not found in association with these labeled microdomains. In addition, the 80-kD protein was seen in apical endocytic vacuoles and in tubulo-vesicular structures, suggesting that the apical microdomains are involved in endocytosis of the 80-kD protein. By its NH2-terminal amino acid sequence, iron-binding capacity and partial immunological cross-reactivity with serum transferrin, the 80-kD protein was shown to belong to the transferrin family, and it is probably homologous to melanotransferrin, a human melanoma-associated antigen. The 80-kD iron-binding protein was fully detergent-soluble immediately after synthesis and only became insoluble after gaining resistance to endo H, supporting a mechanism for exocytic delivery to the apical cell surface by way of detergent-insoluble glycolipid "rafts" that fuse with the plasmalemma at restricted sites devoid of microvilli.

Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium.

Mature adult parenchymal hepatocytes, typically of restricted capacity to proliferate in culture, can now enter into clonal growth under the influence of hepatocyte growth factor (scatter factor) (HGF/SF), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in the presence of a new chemically defined medium (HGM). The expanding populations of hepatocytes lose expression of hepatocyte specific genes (albumin, cytochrome P450 IIB1), acquire expression of markers expressed by bile duct epithelium (cytokeratin 19), produce TGFalpha and acidic FGF and assume a very simplified morphologic phenotype by electron microscopy. A major change associated with this transition is the decrease in ratio between transcription factors C/EBPalpha and C/EBPbeta, as well as the emergence in the proliferating hepatocytes of transcription factors AP1, NFkappaB. The liver associated transcription factors HNFI, HNF3, and HNF4 are preserved throughout this process. After population expansion and clonal growth, the proliferating hepatocytes can return to mature hepatocyte phenotype in the presence of EHS gel (Matrigel). This includes complete restoration of electron microscopic structure and albumin expression. The hepatocyte cultures however can instead be induced to form acinar/ductular structures akin to bile ductules (in the presence of HGF/SF and type I collagen). These transformations affect the entire population of the hepatocytes and occur even when DNA synthesis is inhibited. Similar acinar/ductular structures are seen in embryonic liver when HGF/SF and its receptor are expressed at high levels. These findings strongly support the hypothesis that mature hepatocytes can function as or be a source of bipotential facultative hepatic stem cells (hepatoblasts). These studies also provide evidence for the growth factor and matrix signals that govern these complex phenotypic transitions of facultative stem cells which are crucial for recovery from acute and chronic liver injury.

Separate endocytic pathways regulate IL-5 receptor internalization and signaling.

Eosinophils are critically dependent on IL-5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, betac, which is shared by IL-5, IL-3, and GM-CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2-dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of betac-sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL-5Rs reside in and are internalized by clathrin- and lipid raft-dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of betac, IL-5Ralpha, and Cy3-labeled IL-5 with transferrin- (clathrin) and cholera toxin-B- (lipid raft) positive vesicles. Moreover, whereas internalized IL-5Rs were detected in both clathrin- and lipid raft-positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome-degraded IL-5Rs partitioned to the soluble, nonraft fractions (clathrin-containing). Lastly, we show that optimal IL-5-induced signaling requires entry of activated IL-5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL-5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL-5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL-5R endocytosis regulates signaling both positively and negatively.

Roles of insulin and transferrin in neural progenitor survival and proliferation.

Multipotent neural progenitor cells or neural stem cells (NSC) can be propagated in vitro from a variety of sources and have great potential for neural repair. Although it is well known that NSC divide in response to basic fibroblast growth factor (FGF-2) and epidermal growth factor (EGF), cofactors necessary for survival and maintenance of a multipotent potential are still a matter of debate. In the current study, we examined the requirements for NSC proliferation and survival in vitro using the neurosphere culture system. Apotransferrin (TF), along with EGF and FGF-2, was sufficient for the formation of primary neurospheres derived from embryonic rat cortices. The addition of low concentrations of insulin or insulin-like growth factor-1 (IGF-1) enhanced neurosphere size and number and was necessary for continued passaging. Both insulin and IGF-1 acted at low concentrations, suggesting that their effects were mediated by their cognate receptors, both of which were expressed by neurosphere cultures. Sphere-forming progenitors survived for long periods in culture without EGF or FGF-2 when either insulin or IGF-1 was added to the media. Cell cycle analysis determined that surviving progenitors were relatively quiescent during the period without mitogens. Upon the reintroduction of EGF and FGF-2, surviving progenitors gave rise to new spheres that produced largely glial-restricted progeny compared with sister cultures. These data indicate that the neurogenic potential of NSC may be intimately linked to a continuous exposure to mitogens.

Insect transferrin functions as an antioxidant protein in a beetle larva.

In insects transferrin is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone regulated protein. Here, a novel functional role for insect transferrin as an antioxidant protein is demonstrated. Stressors, such as heat shock, fungal challenge, and H(2)O(2) exposure, cause upregulation of the white-spotted flower chafer Protaetia brevitarsis (Coleoptera: Scarabaeidae) transferrin (PbTf) mRNA in the fat body and increases PbTf protein levels in the hemolymph. RNA interference (RNAi) treated PbTf reduction causes increased iron and H(2)O(2) levels in the hemolymph and results in induction of apoptotic cell death in the fat body during exposure to stress. The observed effects of PbTf RNAi suggest that PbTf inhibits stress-induced apoptosis by diminishing the Fenton reaction via the binding of iron, thus supporting an antioxidant role for PbTf in stress responses.

The role of transferrin in atherosclerosis.

Transferrin is a plasma protein with the primary role of transporting iron through the body and delivering it to the cells that utilize it. Because free ionic iron is very toxic by creating free radicals, the importance of transferrin lies in its antioxidant properties. Atherosclerosis, a pathological process affecting arterial walls, is a chronic inflammatory response in which oxidative stress caused by free radicals is a key factor in its pathogenesis. We postulate therefore that the plasma protein transferrin acts protectively in these events, by holding iron in containment and reducing oxidative stress. Furthermore, it is possible that a disturbance in transferrin function and homeostasis is a direct factor triggering and exacerbating atherosclerosis. Decreased transferrin levels, increased transferrin saturation, defective transferrin binding of iron, or other disorders may lead to increased oxidative stress and lipid peroxidation involved in the pathology of atherosclerosis. Some oxidative stress-related diseases have been linked to such systemic transferrin abnormalities, and we hypothesize that similar disruptions could account for an unfavorable microenviroment in the evolvement of atherosclerotic plaques. If confirmed, this proposed mechanism would significantly improve our understanding of the disease.

Iron trafficking inside the brain.

Iron, an essential element for all cells of the body, including those of the brain, is transported bound to transferrin in the blood and the general extracellular fluid of the body. The demonstration of transferrin receptors on brain capillary endothelial cells (BCECs) more than 20 years ago provided the evidence for the now accepted view that the first step in blood to brain transport of iron is receptor-mediated endocytosis of transferrin. Subsequent steps are less clear. However, recent investigations which form the basis of this review have shed some light on them and also indicate possible fruitful avenues for future research. They provide new evidence on how iron is released from transferrin on the abluminal surface of BCECs, including the role of astrocytes in this process, how iron is transported in brain extracellular fluid, and how iron is taken up by neurons and glial cells. We propose that the divalent metal transporter 1 is not involved in iron transport through the BCECs. Instead, iron is probably released from transferrin on the abluminal surface of these cells by the action of citrate and ATP that are released by astrocytes, which form a very close relationship with BCECs. Complexes of iron with citrate and ATP can then circulate in brain extracellular fluid and may be taken up in these low-molecular weight forms by all types of brain cells or be bound by transferrin and taken up by cells which express transferrin receptors. Some iron most likely also circulates bound to transferrin, as neurons contain both transferrin receptors and divalent metal transporter 1 and can take up transferrin-bound iron. The most likely source for transferrin in the brain interstitium derives from diffusion from the ventricles. Neurons express the iron exporting carrier, ferroportin, which probably allows them to excrete unneeded iron. Astrocytes lack transferrin receptors. Their source of iron is probably that released from transferrin on the abluminal surface of BCECs. They probably to export iron by a mechanism involving a membrane-bound form of the ferroxidase, ceruloplasmin. Oligodendrocytes also lack transferrin receptors. They probably take up non-transferrin bound iron that gets incorporated in newly synthesized transferrin, which may play an important role for intracellular iron transport.

Human suction blister interstitial fluid prevents metal ion-dependent oxidation of low density lipoprotein by macrophages and in cell-free systems.

LDL in the circulation is well protected against oxidation by the highly efficient antioxidant defense mechanisms of human plasma. LDL oxidation contributing to atherosclerosis, therefore, has been hypothesized to take place in the interstitial fluid of the arterial wall. We investigated the antioxidant composition and the capacity to inhibit LDL oxidation of human suction blister interstitial fluid (SBIF), a suitable representative of interstitial fluid. We found that the concentrations in SBIF of the aqueous small-molecule antioxidants ascorbate and urate were, respectively, significantly higher (P < 0.05) and identical to plasma concentrations. In contrast, lipoprotein-associated lipids and lipid-soluble antioxidants (alpha-tocopherol, ubiquinol-10, lycopene, and beta-carotene) were present at only 8-23% of the concentrations in plasma. No lipid hydroperoxides could be detected ( < 5 nM) in either fluid. The capacity of serum and SBIF to protect LDL from oxidation was investigated in three metal ion-dependent systems: copper, iron, and murine macrophages in Ham's F-10 medium. In all three systems, addition of > or = 6% (vol/vol) of either serum or SBIF inhibited LDL oxidation by > 90%. The concentration that inhibited macrophage-mediated LDL oxidation by 50% was as low as 0.3% serum and 0.7% SBIF. The enzymatic or physical removal of ascorbate or urate and other low molecular weight components did not affect the ability of either fluid to prevent LDL oxidation, and the high molecular weight fraction was as protective as whole serum or SBIF. These data demonstrate that both serum and SBIF very effectively protect LDL from metal ion-dependent oxidation, most probably because of a cumulative metal-binding effect of several proteins. Our data suggest that LDL in the interstitial fluid of the arterial wall is very unlikely to get modified by metal ion-mediated oxidation.